Title of Invention: Recombinant beta2-adrenergic receptor delivery and use in treating airway and vascular diseases Abstract: A method of treating a human subject having airway or vascular disease is disclosed that comprises administering to at least one cell type selected from the group consisting of airway epithelial cells, airway smooth muscle cells, blood vessel endothelial cells, and blood vessel smooth muscle cells, a first composition comprising a vector comprising a DNA sequence encoding a &bgr;2AR (p2-adrenergic receptor) or a mutant thereof, operably linked to a promoter that is functional in at least one of said cells said subject, under conditions whereby the DNA sequence encoding the &bgr;2AR is expressed in at least one of the cells; and optionally administering a second composition comprising at least one &bgr;2-adrenergic agonist into the cells of said subject. A further pharmaceutical composition may be administered comprising a hormone or pharmacological agent that induces the promoter to express the &bgr;2AR in at least one of the target cells. Pharmaceutical compositions comprising the vector comprising a DNA sequence encoding a &bgr;2AR or a mutant thereof, operably linked to a promoter that is functional in at least one of the cells of the subject and kits containing these compositions are also disclosed. An in vitro method of expressing the &bgr;2AR gene in mammalian cells and a method of evaluating the effect of pharmacological compounds on the expression of the &bgr;2AR gene is disclosed. Summary of Invention: BACKGROUND OF THE INVENTION &lsqb;0002&rsqb; The present invention relates to a novel treatment of airway and vascular diseases in which dilation of the affected airway or blood vessel would be of benefit to relieve symptoms, in diseases, such as asthma, pulmonary hypertension and systemic hypertension. The present invention provides a method for increasing the &bgr;2-adrenergic receptors (MAR) in the airway epithelial cells and smooth muscle cells and blood vessel endothelial cells and smooth muscle cells resulting in the dilation of the airways and blood vessels, thus relieving symptoms of airway and vascular diseases. The present invention particularly relates to a novel adjunctive treatment of airway and vascular diseases by delivering a first composition comprising a recombinant vector that carries the nucleic acid sequence encoding the native &bgr;2AR or a modified &bgr;2AR, that is delivered into the airways and blood vessels, allowing infection or transduction of at least one type of cell selected from epithelial cells lining the airways, endothelial cells lining the blood vessels and from smooth muscle cells composing the airways and blood vessels. &lsqb;0003&rsqb; The present invention more particularly relates to a novel adjunctive treatment of severe asthma in patients who have become hypo-responsive to the bronchodilatory effect of &bgr;2-adrenergic agonists (&bgr;2-agonists) or who may benefit from increasing the bronchodilatory effect of &bgr;2-agonists. The present invention employs a recombinant vector that carries the nucleic acid sequence encoding the native &bgr;2AR or a modification thereof, that is delivered into the airways via bronchoscopy, allowing infection or transduction of at least one type of cell from the epithelial cells lining the airways and from the smooth muscle cells composing the airways and expression of the &bgr;2ARs in these cells. The present invention relates to the delivery of the recombinant vector simultaneously or sequentially with &bgr;2-agonists that are administered to treat acute symptoms by producing rapid bronchodilation. &lsqb;0004&rsqb; The present method relates to an in vitro method of expressing the &bgr;2AR gene and evaluating the effect of pharmacological compositions on the expression of &bgr;2ARs in mammalian cells that are transduced with a recombinant vector that carries the nucleic acid sequence encoding the native &bgr;2AR or a modified &bgr;2AR. &lsqb;0005&rsqb; Asthma is a collection of symptoms that produces an airway state that causes excessive airway narrowing in response to stimuli that typically do not produce the same effect on the nonasthmatic airway.151 In the past, asthma's etiology was ascribed principally to airway smooth muscle spasm and bronchodilator therapy was the front-line approach for managing asthmatic patients. It is now evident that asthma is a disease of chronic inflammation involving inflammatory cells that release numerous mediators. These mediators initiate airway hyperresponsiveness to various stimuli and lead to the clinical endpoint of bronchoconstriction.6 The majority of asthmatic patients display bronchospasm, airway inflammation and mucous plugging. Asthmatics experience recurrent episodes of wheezing, chest tightness, and coughing, particularly at night and in the early morning. In the United States, asthma affects 14 to 15 million individuals and causes more than 5,000 deaths annually. Two general classes of medication are currently available for treatment: long-term control medications (e.g., corticosteroids, cromolyn sodium, methylxanthines, leukotriene modifiers) aimed at obtaining control of the persistent, inflammatory component of asthma and quick relief medications (e.g., short-acting &bgr;2-agonists, anti-cholinergics) used to treat acute symptoms, such as wheezing and shortness of breath. &lsqb;0006&rsqb; In the mature airway, the epithelium forms a continuous lining whose cellular composition varies with anatomical location. 14,112 From these studies, at least 12 types of airway epithelial cells have been identified, although the proportions of the various cell types vary from species to species. 14,112 Within the proximal airway, the epithelial cell layer is thicker and assumes a pseudostratified appearance. 14 Basal, ciliated, intermediate, and goblet cells are present in the proximal airway and typically there is an absence of Clara cells. In the distal airways, the epithelium becomes thinner. Within the bronchioles, the epithelium is composed of low cuboidal cells and Clara cells become more prevalent. Airway epithelium is generally considered to perform five primary functions:116 1) acts as a barrier to the diffusion of particles from the airway to underlying lung structures, 2) secretes mucin-like materials, 3) clears the airway of debris through ciliary action, 4) repairs damaged tissue following injury in the airway, and 5) modulates the response of other airway cells particularly to environmental agents that enter the airways. Airway epithelial cells regulate bronchoconstriction,42 bronchial vascular responses,66 and inflammatory cell recruitment into the airway.75,118 Because of easy accessibility, airway epithelium is an attractive target for delivering genetic material to treat both acquired and inherited lung diseases. A prominent example involves the cystic fibrosis transmembrane conductance regulator (CFTR) gene, in which mutations cause the disease cystic fibrosis. 1,150 &lsqb;0007&rsqb; The bronchodilatory effects of &bgr;2-adrenergic agonists are exerted by stimulating airway smooth muscle relaxation directly and secondarily by causing release of an airway smooth muscle relaxing factor from epithelial cells. FIG. 1 provides a diagram of the &bgr;2AR life cycle following agonist binding to the receptor. Despite their widespread use, &bgr;2-agonists have several shortcomings. For maximum symptom control, frequent dosing is required. This can lead to development of subsensitivity or tolerance, which blunts their effectiveness. Development of tolerance to inhaled &bgr;2-agonists is due to either uncoupling of &bgr;2ARs from downstream effector proteins that produce bronchodilation or the actual loss of &bgr;2ARs from the cell surface. &lsqb;0008&rsqb; Although beneficial in the short-term, prolonged use or overuse of &bgr;2-agonists has been associated with reduced &bgr;2AR responsiveness. The phenomenon of reduced &bgr;2AR responsiveness, also known as tachyphylaxis or tolerance, results from a culmination of events, which include desensitization, sequestration, and down-regulation. Desensitization occurs as a consequence of receptor phosphorylation by either protein kinase A (PKA) or a G protein coupled receptor kinases (GRK). Phosphorylation of the receptor causes the receptor to uncouple from downstream effector proteins and also promotes binding of high-affinity arresting proteins, known as arrestins. Binding of arrestins to the &bgr;2AR also targets the receptor for sequestration and down-regulation. Sequestration (or internalization) moves the &bgr;2AR from the cell surface to endosomes located in the cell's interior. Since the native hormones/neurotransmitters epinephrine and norepinephrine cannot cross the cell membrane, internalized receptors are no longer able to trigger a physiological response. Once in endosomes, receptors reach a sorting point in the pathway. The degradation pathway results in a net loss of receptors, also called down-regulation. Internalized &bgr;2ARs can also be recycled back to the cell surface. It is not known what determines whether an internalized &bgr;2AR will be degraded or recycled. Thus, when a severe asthmatic uses his/her &bgr;2-agonist inhaler too many times, levels of &bgr;2ARs decrease in their airways to a point where no matter how much &bgr;-agonist they use there are not enough receptors available to bind the drug and produce airway relaxation. &lsqb;0009&rsqb; The present invention overcomes this loss of native &bgr;2ARs in asthma patients by the expression of recombinant &bgr;2ARs in airway epithelial and smooth muscle cells to keep &bgr;2AR levels at normal or even at higher than normal levels on the cell surface. A recombinant &bgr;2AR gene, under the control of a very active viral promoter, transcribes high levels of &bgr;2AR MRNA, augmenting the &bgr;2AR mRNA that simultaneously is made by the native &bgr;2AR gene. The niRNA is translated into &bgr;2AR protein and is delivered to the cell membrane at a rate that is faster than the rate at which &bgr;2ARs are removed from the membrane via the desensitization/sequestration/down-regulation pathway. &lsqb;0010&rsqb; The &bgr;2AR is a member of a superfamily of membrane assoicated receptors coupled to guanine nucleotide regulatory proteins (G-proteins) and produces its effects by activating intracellular signal transduction pathways.136 Results from radioligand assays and receptor autoradiography studies have documented the presence of &bgr;2ARs on a variety of cell types within the mammalian lung.8,98 Airway epithelial cells in human,72 murine,106 bovine,101 and rat125 lungs express &bgr;2ARs. Several aspects of airway epithelial function are under the control of &bgr;2ARs. &bgr;2-agonists increase bronchial epithelial chloride and mucus secretion, and increase ciliary activity.99 The effects of inhaled &bgr;2-agonists on mucociliary clearance in humans seem to be variable. In one study, the &bgr;2-agonist, procaterol, had no effect in both controls and in asthmatic individuals.68 However, inhaled terbutaline increases mucociliary clearance in both controls and in patients with asthma.93 Results from several studies have suggested that the relaxation of airway smooth muscle that occurs in response to &bgr;2-agonists is reduced if the bronchial epithelium is removed.42,155 This finding could be explained by the release of a smooth muscle relaxing factor from bronchial epithelial cells in response to stimulation with &bgr;2-agonists. This factor, designated epithelium-derived relaxant factor (EpDRF),42,155 could either act directly on smooth muscle to cause relaxation or alternatively enhance the effect of &bgr;2-agonists on bronchial smooth muscle (See a schematic of the proposed mechanism in FIG. 2). &bgr;2-agonists and glucocorticoids are the two most effective treatments available for asthma therapy and frequently are used in combination. Glucocorticoids are used principally because of their anti-inflammatory properties,7 although additional beneficial effects of glucocorticoids in the asthmatic lung have been observed. 135 Synthetic glucocorticoids are efficacious for treating asthma and other diseases with associated inflammatory processes because they mimic glucocorticoids produced endogenously by the adrenal cortex. The cellular and molecular mechanisms of action of the glucocorticoids have been extensively studied. 135 154 The current model of glucocorticoid action postulates intracellular glucocorticoid receptors, which in the absence of ligand, are complexed with heat shock proteins (hsp90, hsp56, hsp70 and an acidic 23 kD protein) (See FIG. 3). Glucocorticoids are found in the blood bound to transcortin, albumin, and other serum proteins. Free glucocorticoids enter target cells, by still unidentified mechanisms 49,152 and bind to glucocorticoid receptors causing dissociation of the associated heat shock proteins. Association of hsp90 with glucocorticoid receptors appears to maintain the hormone binding domain in its high affinity conformation.113 The functional roles of the other associated heat shock proteins are not as well understood, but may include trafficking of the receptor within the cell.113 The glucocorticoid receptor is a member of a superfamily of transcriptional regulators that include receptors for estrogens, progesterones, androgens, vitamin D, thyroid hormones, and retinoic acid.83 Members of the superfamily share a similar structure with functional domains for binding of hormone, binding to DNA, and transcriptional activation. Hormone-receptor complex translocates from the cytoplasm to the nucleus. Activated glucocorticoid receptor with hormone bound has an increased affinity for binding to specific DNA sites termed glucocorticoid response elements (GRE) found within glucocorticoid reponsive genes. GREs can either be simple or composite.48 Most simple GREs consist of two half-site hexamers separated by three nucleotides with resemblance to the consensus sequence GTCACAnnnTGTTCT. Association of glucocorticoid receptor, typically as a homodimer, to simple GREs results in enhanced transcription of the target gene. A second type of DNA sequence that binds glucocorticoid receptors, termed composite GREs, has been found in certain glucocorticoid-responsive genes.31 At composite GREs, the hormone receptor complex interacts with both specific DNA sequences and other transcription factors to regulate transcription.31,47,91 The first demonstrated composite GRE was shown to have binding sites for both the glucocorticoid receptor and activating protein-1 (AP-1).31 AP-1 is a dimer of the oncogene products c-fos and c-jun. Since glucocorticoid receptors are expressed in many cell types, composite GREs may explain how signal specificity can be achieved in a system with an apparent common fmal pathway.48 &lsqb;0011&rsqb; For many G-protein coupled receptors, modulation of receptor number is an established mechanism controlling responsiveness to hormones and neurotransmitters. Heterologous regulation of &bgr;2AR levels by glucocorticoids is a physiologically important example of such control.23 Numerous in vitro and in vivo studies in a variety of cell types have shown that &bgr;2AR levels and &bgr;-agonist-stimulated adenylyl cyclase activity are increased by glucocorticoids.19,28,46,104,140,157 Glucocorticoids increase EAR levels in the lung of several species including rat, rabbit and human. 19 84,87 The increase in &bgr;2AR number results from an increase in rate of new receptor synthesis,104 which is preceded by increased steady-state levels of &bgr;2AR MRNA 23,28,58,59,81,90,140 and increased transcription rate of the &bgr;2AR gene.23,59,81 The increase in EAR density induced by glucocorticoids is blocked by the transcriptional inhibitor actinomycin D.95 Taken together, these findings suggest that enhanced &bgr;2AR gene transcription is a principal mechanism underlying glucocorticoid mediated increases in &bgr;2AR levels. The &bgr;2AR genes from several mammalian species contain GRE-like sequences in both coding and non-coding regions.16,39,70,73,88 Although indirect evidence suggests that the 5′-noncoding region of the &bgr;2AR gene is involved,82 data discussed below identifies specific genetic elements responsible for the functional effect of glucocorticoids on &bgr;2AR gene transcription. &lsqb;0012&rsqb; Since the development of &bgr;2-adrenergic selective drugs and metered-dose inhaler delivery systems, agents that target the &bgr;2ARs have become the most commonly prescribed medications for asthma.34 The principal beneficial effects of &bgr;2-agonists are on bronchomotor tone and airway patency. Agonist stimulation of &bgr;2ARs in airway smooth muscle inhibits contractile processes, resulting in bronchodilation.115 This is an important property of &bgr;2ARs because several bronchoconstricters (histamine, bradykinin, acetylcholine, LTD4, and PGD2) are elevated in the asthmatic lung.5 &lsqb;0013&rsqb; &bgr;2-agonists are effective bronchodilators in large and small airways.97 &bgr;2AR-mediated processes in airway epithelial cells (e.g., mucous clearance, production of bronchoactive factors) may also either directly or indirectly affect the contractile state of airway smooth muscle,92 &bgr;2-agonists have been shown to decrease mediator release from basophils and mast cells.22,109 Administration of &bgr;2-agonists reduces vascular leakage caused by inflammatory mediators including histamine, platelet activating factor, and certain prostaglandins.105,111 Finally, &bgr;2ARs are expressed on the surface of potential inflammatory cells, including eosinophils, alveolar macrophages, lymphocytes and polymorphonuclear leukocytes 24,33,78,156 While the role of the &bgr;2AR on proinflammatory cells in the asthmatic lung is currently unclear, some physiological effects have been reported. &bgr;2-agonist treatment in vitro is associated with decreased proliferation of human T lymphocytes in response to mitogenic stimuli24 and inhibition of lysosomal enzyme release from granulocytes.65 &lsqb;0014&rsqb; Despite their well-documented clinical efficacy, regular use of &bgr;2-agonists and glucocorticoids can lead to several clinical problems. Doubts concerning the safety of &bgr;2-agonists arose from epidemiologic studies conducted in New Zealand25,55,110 and Canada133 that demonstrated an association between the regular use of &bgr;2-agonists, particularly fenoterol, and the risk of dying from bronchial asthma. The conclusions drawn from these studies are controversial and do not necessarily prove a cause-effect relationship since patients with more severe asthma, and therefore a higher risk of fatal attacks, are more likely to use &bgr;2-agonists more frequently or at higher doses.5 A balanced review on the safety of &bgr;2-agonists also can be found.141 Overuse of inhaled &bgr;2-agonists may also increase asthma morbidity. Separate studies have shown that regular &bgr;2-agonist use can be associated with poorer control of symptoms and increased airway hyper-responsiveness compared to asthmatics who use the same medications “as needed”.128 142 These observations are not surprising since it is well-known that &bgr;2ARs are prone to the process of desensitization and down-regulation resulting in loss of receptor function following prolonged &bgr;2-agonist exposure.57,74 The present invention circumvents the adverse effects associated with overuse of &bgr;-agonists. &lsqb;0015&rsqb; Because all cells express a common glucocorticoid receptor, every cell and organ system can be affected by administration of exogeneous glucocorticoids. Numerous potential adverse effects that can arise from systemic glucocorticoid use,132 and include effects that can occur immediately (e.g., hypokalemia and hyperglycemia), those that develop over a longer period of time (e.g., osteoporosis and cataracts), and those that are limited to children (e.g., growth suppression). Chronic glucocorticoid use results in development of posterior subcapsular cataracts with a prevalence of up to 29% in adults and children.120, 145 Patients on >7.5 mg/day prednisone for longer than 6 months are at risk for developing osteoporosis.79 Inhibition of linear growth in children has been observed with regular daily therapy, frequent short course therapy and high-dose alternate day therapy with glucocorticoids. 10,96,129 Inhaled glucocorticoids, used as long-term management by many asthmatics, generally display fewer risks than oral glucocortoids.53 At doses up to 800 &mgr;g/day, few clinically important adverse effects are observed with oral glucocorticoids; however, with long-term use of doses greater than 800 &mgr;g/day, osteoporosis, suppressed linear growth in children, adrenal-pituitary axis suppression, and cataracts are the most likely adverse effects to develop.15,53,143 Although inhaled corticosteroids have significantly less potential for causing adverse effects compared to oral systemic corticosteroids, current recommendations are to use the lowest possible dose to maintain control of symptoms.40 Some of the beneficial effects of glucocorticoids are the result of increased &bgr;2AR numbers. The present invention provides a method whereby &bgr;2AR overexpression in airway epithelial cells and smooth muscle cells leads to decreased glucocorticoid requirements in severe asthmatics. &lsqb;0016&rsqb; Shortcomings in safety and persistently delivering effective quantities of specific proteins to patients using recombinantly produced proteins has led to the development of gene therapy methods for delivering sustained levels of specific proteins into patients. Gene therapy is defined as the insertion into a patient of DNA that codes for either normal or altered genes,in order to correct a genetic or acquired disorder. The normal or altered gene that is inserted corrects the disorder via production in the patient of either missing, defective, or insufficient gene products. The DNA may be introduced by known cell transfection methods, but viral-mediated gene delivery methods, such as retrovirus, adenovirus, herpes virus, pox virus, and adeno-associated virus (AAV) are used in more than 95% of gene therapy trials conducted. 159 &lsqb;0017&rsqb; Gene therapy techniques utilize various vehicles for gene transfer and Table 1 of Kay et al.160 disclose retroviruses, adenoviruses and AAV as viral vehicles and liposomes as nonviral vehicles. Each one of these vehicles has limitations in gene therapy applications (discussed in Stone et al.161). Nonviral vechicles, such as liposomes presently lack target cell specificity. Widespread expression of transgenes can have deleterious outcomes. The disadvantages of retroviral vectors include random insertion into the host genome, inactivation by human complement, inability to transduce non-dividing cells, and possible decreased transgene expression over time. Additionally and very importantly, the retroviruses and retroviral vectors have been banned for use in gene therapy by the Food and Drug Administration. The principal drawback of adenoviruses is a significant host immune response against the viral vector, vector-encoding proteins, and the cells expressing these proteins. This leads to inflanunation and elimination of transduced cells by the immune system, requiring frequent re-administration of the transgene. Because adenovirus does not incorporate into the host genome, duration of transgene expression is limited. AAV possesses a number of features not possessed by the other viruses, such as: wide host range, ability to infect different species, no known association with any human or animal disease; does not appear to alter the biological properties of the host cell when it integrates, its stability over a wide range of physical and chemical conditions, its small size, and less complicated epitopes presented to a patient than adenoviruses. For all of these reasons, a more suitable choice of a vector for transducing epithelial cells, is the AAV vector as originally suggested by Hermonat et al.62 &lsqb;0018&rsqb; The AAV genome is a linear, single-stranded DNA molecule containing 4681 nucleotide and an internal non-repeating genome flanked on each end by inverted terminal repeats (ITRs). The ITRs are approximately 145 base pairs in length and have multiple functions. The internal non-repeated portion of the genome includes two large open reading frames, known as the AAV replication (rep) and capsid (cap) genes, code for viral proteins that allow the virus to replicate and package the viral genome into a virion. &lsqb;0019&rsqb; AAV is a helper-dependent virus that requires co-infections with a helper virus, such as adenovirus, herpesvirus or vaccinia) in order to form AAV virions. AAV has been engineered to contain heterologous genes by deleting the internal non-repeating portion of the AAV genome (i.e., the rep and cap genes) and inserting the heterologous gene linked to a promoter between the ITRs. The ITRs are the only viral elements necessary for efficient encapsidation and integration of the viral genome of the host cell. 162 The AAV genome stably integrates into a specific site in human chromosome 19163 and AAV is able to transduce both mitotic and post-mitotic cells.164 In order to prepare AAV a helper virus (usually adenovirus) is required. Although high titer AAV preparations result, the preparations are contaminated with helper virus that must be removed by tedious purification steps. Alternative strategies to prepare recombinant AAV have been developed165 (See also U.S. Pat. No. 6,004,797, U.S. Pat. No. 6,001,650, U.S. Pat. No. 5,945,335) that produce high AAV titers with no helper virus contamination. &lsqb;0020&rsqb; In regard to the usefulness of gene therapy methods for the treatment of asthma, a number of research groups have looked at new treatments for asthma. Demoly et al. (1997) 166 considered the possibility of a gene therapy based strategy for the treatment of asthma. This publication admits that in the treatment of asthma, there are no deficient genes to replace and no protein is truly repressed but rather there is a hypersecretion. These authors consider blocking the expression of an inflammatory or immunoregulatory protein but admit that sophisticated gene therapy techniques would be required. Further, a publication by Rogers et al. (1998)167, considers gene therapy as a new approach to treat lung disease because the lung provides an accessible target through the airways or the vasculature. This publication considers the lung diseases of &agr;1-antitrypsin deficiency and cystic fibrosis as good candidates for gene therapy because the genetic defect in each disease is well characterized. This publication also considers the different vectors and the advantages and disadvantages of using them. &lsqb;0021&rsqb; McGraw et al. reported at the 1998 ALA/ATS International Conference89 and in a later publication168 that their group had generated transgenic mice lines using the human surfactant protein C promoter and the rat CC10 promoter to overexpress the human &bgr;2AR in distal alveolar epithelium (type II cells) and bronchial tracheal epithelium (Clara cells). Epithelial cell &bgr;2AR overexpression was verified by receptor autoradiography. These transgenic mice lines may be useful in selectively isolating the effect of proximal and distal epithelial cell &bgr;2ARs upon airway physiology and pathophysiology. In 1999, McGraw et al. 169 reported that transgenic mice expressed &bgr;2ARs in airway smooth muscle using a mouse smooth muscle &agr;-actin promoter as compared to normal mice. &lsqb;0022&rsqb; Another group looked at the overexpression of &bgr;2AR in adenoviral transduced cultured adult rabbit ventricular myocytes,170 and concluded that recombinant adenoviral gene transfer of EAR or an inhibitor of &bgr;-adrenergic receptor kinase (&bgr;ARK)-mediated desensitization can potentiate &bgr;-adrenergic signaling. Recently another publication by some of the members of the previous group, delivered adenoviral transgenes including the human &bgr;2AR gene to the myocardium of rabbits using catheter-mediated delivery. This study demonstrated global myocardial in vivo gene delivery and that genetic manipulation of &bgr;2AR density can result in enhanced cardiac performance as compared to control rabbits. &lsqb;0023&rsqb; The human &bgr;2AR gene is known and sequenced. The first report of cloning and sequencing the human &bgr;2-AR was reported in. 171, 73 &lsqb;0024&rsqb; As discussed above, a significant problem associated with prolonged treatment of asthmatics with &bgr;2-agonist treatment is desensitization or tolerance. The loss of clinical responsiveness to &bgr;2-agonists has potentially serious outcomes including increased mortality, poor baseline asthma control, escalating medication usage, and increased cost of care. The present invention provides a recombinant vector and a method of using this vector to provide additional &bgr;2ARs to airway epithelial cells and smooth muscle cells, but particularly airway epithelial cells, to provide increased levels of &bgr;2ARs in these cells. The increased levels of &bgr;2ARs alone or with adjunct &bgr;2-agonists or controlled &bgr;2AR expression by endogenous or administered inducers of the promoter operably linked to the &bgr;2AR gene provide a method of treating airway diseases, such as asthma. The present method also is useful for providing treatment for other diseases that can benefit from increased levels of &bgr;2ARs, such as vascular diseases. SUMMARY OF THE INVENTION &lsqb;0025&rsqb; The present invention relates to vectors comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in at least one cell type of the airways and blood vessels of a human subject. In a preferred embodiment, the vectors are adeno-associated virus based. Adeno-associated virus has a natural tropism for airway epithelia. So these adeno-associated virus based vectors are particularly preferred for respiratory gene therapy applications. The present invention is directed to cells containing the vector. &lsqb;0026&rsqb; The present invention is further directed to a method for providing a &bgr;2AR to airway epithelial cells, airway smooth muscle cells, blood vessel endothelial cells, blood vessel smooth muscle cells or a combination thereof, of a human subject comprising administering to at least one of these enumerated cell types, a first composition comprising a vector comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in at least one of the cells, under conditions whereby the DNA sequence encoding said &bgr;2AR is expressed in at least one of these cells. In a further embodiment, the DNA sequence encodes a &bgr;2AR that is modified in its function as compared to the native &bgr;2AR. &lsqb;0027&rsqb; The present invention further is directed to an adjunct therapy for treating a human subject having airway or vascular disease comprising administering to at least one cell type selected from the group consisting of airway epithelial cells, airway smooth muscle cells, blood vessel endothelial cells, and blood vessel smooth muscle cells of the human subject, a first composition comprising a vector comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in at least one of these types of cells of the subject, under conditions whereby the DNA sequence encoding the &bgr;2AR is expressed in at least one of these types of cells; and administering a second composition comprising at least one &bgr;2-agonist to at least one cell type of the subject. &lsqb;0028&rsqb; The present invention additionally is directed to administering simultaneously or after the administeration of a vector comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in at least one of the types of cells of the subject enumberated above, a hormone or other pharmacological agent that induces the promoter to express &bgr;2ARs. &lsqb;0029&rsqb; The present invention is further directed to pharmaceutical compositions containing the vector comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in cells of the airway epithelium and smooth muscle cells and blood vessel endothelium and smooth muscles. &lsqb;0030&rsqb; The present invention additionally is directed to a kit that contains in separate containers at least one pharmaceutical composition comprising the vector comprising a DNA sequence encoding a &bgr;2AR operably linked to a promoter that is functional in cells of the airway epithelium and smooth muscles and blood vessel endothelium and smooth muscles, at least one additional pharmaceutical composition comprising a &bgr;2-agonist, and optionally at least one pharmacological agent that induces the promoter to express &bgr;2ARs in the target cells. The pharmaceutical compositions are in a formulation suitable for aerosol delivery or intravenous delivery. &lsqb;0031&rsqb; The present method further is directed to an in vitro method of expressing the &bgr;2AR gene and evaluating the effect of pharmacological compositions on the expression of &bgr;2ARs in mammalian cells that are transduced with a recombinant vector that carries the nucleic acid sequence encoding the native &bgr;2AR or a modified &bgr;2AR. Detailed Description: DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT &lsqb;0051&rsqb; The present invention is directed to a novel approach for adjunctive treatment of patients having airway and/or vascular diseases, in which dilation of the affected airways or blood vessels would be of benefit to relieve symptoms, in diseases, such as asthma, and pulmonary or systemic hypertension. The present treatment utilizes gene therapy to dilate the airways and blood vessels by providing increased levels of &bgr;2ARs for endogenous hormones, such as epinephrine or norepinephrine. Optionally the &bgr;2AR gene containing try vector is administered in conjunction with &bgr;2-agonists to enhance treatment by providing increased levels of &bgr;2ARs for the &bgr;2-agonist to bind. More specifically, a vector comprising a &bgr;2AR gene under the control of regulatory sequences that express the gene in the airway epithelial cells and/or airway smooth muscle cells, is delivered to the patient's airway, and optionally, either simultaneously or sequentially, a &bgr;2-adrenergic agonist is delivered to the patient's airway. Similarly, a vector comprising a &bgr;2-AR gene under the control of regulatory sequences that express the gene in the blood vessel endothelial cells and/or blood vessel smooth muscle cells, is delivered to the patient's blood stream, and optionally, either simultaneously or sequentially, a &bgr;2-agonist is delivered to the patient's blood stream. &lsqb;0052&rsqb; In a preferred embodiment, the present invention is directed to a novel adjunctive treatment for patients or human subjects with severe asthma who become hypo-responsive to the bronchodilatory effects of &bgr;2-agonists. The treatment method of the present invention comprises administering a vector comprising the &bgr;2AR gene, and optionally administering a &bgr;2-agonist into the airways of the patient. The vector and the &bgr;2-agonist can be administered simultaneously or sequentially, resulting in the airway epithelial cells being transduced by the vector with the resulting infection of airway epithelial cells lining the patients' airways, and also the infection of the smooth muscle composing the epithelial cell boundary, and possibly even entry into the bloodstream. The &bgr;2ARs are expressed in the transduced cells, providing additional &bgr;2ARs to which the administered &bgr;2-agonist binds. By the present method of treatment, the airway smooth muscles are relaxed and airway dilation is achieved. &lsqb;0053&rsqb; The present invention differs from standard gene therapy approach for treating diseases in that it is not utilized to replace a defective protein with a version of the protein that functions properly. The present invention provides increased numbers of &bgr;2ARs on the surface of the epithelial cells, in addition to the native &bgr;2ARs already present. In the treatment of asthma, there is no evidence that the &bgr;2AR is defective, and that this is the cause of the asthmatic condition. Yet, the &bgr;2AR is an important therapeutic target in the treatment of asthma. &lsqb;0054&rsqb; The principal clinical benefits anticipated in asthmatic patients receiving &bgr;2AR gene therapy are: 1) increased sensitivity to the airway relaxing effects of circulating epinephrine and norepinephrine, and inhaled &bgr;2-agonists resulting in decreased dependency upon &bgr;2-agonist medications; and 2) decreased susceptibility to the development of subsensitivity or tolerance to inhaled &bgr;2-agonists. The asthma treatment of the present invention is effective in decreasing the incidence of worsening asthma control and mortality that is associated with frequent use of &bgr;2-agonists in some patients who are frequent users of &bgr;2-agonists and suffer from the negative effects of tachyphylaxis. &lsqb;0055&rsqb; The present invention also is directed to the use of an inducible &bgr;2-AR gene that is targeted to airway epithelial and/or smooth muscle cells to dilate the airways by increasing the number of &bgr;2-ARs, but also is useful to improve airway responsiveness to &bgr;2-agonists. The use of gene therapy for the management of individuals, whose asthma is poorly controlled by current conventional treatment methods, provides a useful and innovative treatment to overcome desensitization to &bgr;2-agonists. The present invention is built upon the kinetic relationship between &bgr;2-agonists and &bgr;2-ARs to produce beneficial physiological responses (e.g., bronchodilation). &lsqb;0056&rsqb; The present invention discloses a new therapy that provides a means to increase &bgr;2-adrenergic responsiveness by increasing levels of &bgr;2AR in airway epithelial cells and/or airway smooth muscle cells. This is accomplished in a step-wise fashion, that in addition to creating a novel approach to treating asthma, advances basic knowledge in research in lung biology. Although the airway smooth muscle possibly represents a more physiologically significant target for gene therapy involving the &bgr;2AR, the present invention focuses on airway epithelium for several reasons. First, there is evidence that part of the airway relaxing effects of &bgr;2-agonists are mediated via interactions with epithelial cell &bgr;2ARs. Second, airway epithelial cells are easily targeted using vectors derived from either adenoviruses or adeno-associated viruses (AAV). Moreover, the feasibility of transducing epithelial cells with recombinant viruses carrying a mammalian gene and achieving expression of a functional protein has been demonstrated by numerous cystic fibrosis laboratories working with the CFTR protein.26 This is a procedure that has been used in humans. Third, the present invention provides a practical procedure for specifically targeting and/or expressing a recombinant gene in airway epithelial cells and smooth muscle cells that can be safely used in humans. &lsqb;0057&rsqb; The vector and the &bgr;2-agonist is administered simultaneously or sequentially, but preferably the vector comprising the &bgr;2AR gene is administered, and the airway epithelial cells and/or smooth muscle cells are transduced, resulting in the infection of airway epithelial cells and/or smooth muscle cells. The vector containing the &bgr;2AR gene is administered to the subset of asthmatic patients, who are difficult to manage with traditional therapies (e.g., the ones that end up in the emergency room). The recombinant &bgr;2AR vector is administered in aerosolized form, in the same manner that &bgr;2-agonists and glucocorticoids are taken by asthmatics. &lsqb;0058&rsqb; Vehicles for gene transfer to cells may be selected from retroviruses, adenoviruses, AAV or nonviral vehicles. But the present invention prefers AAV vectors as the vehicle for transduction. Examples of U.S. patents, disclosing viral transduction of genes using AAV based vectors are U.S. Pat. No. 5,670,488; U.S. Pat. No. 5,139,941 and U.S. Pat. No. 5,252,479. An example of non-viral transfection of lungs is disclosed in U.S. Pat. No. 6,022,737. &lsqb;0059&rsqb; After a sufficient period to allow for the transduction of the epithelial cells and the expression of the &bgr;2ARs in the epithelial cells, for example for approximately 1 to 2 days, the &bgr;2-agonist is optionally administered. During this period of time, there is episomal expression that is transient. Long-term expression occurs as the recombinant virus integrates into the host genome. This occurs in a specific location on human chromosome 19. Increased levels of &bgr;2AR are present in infected cells over the course of the cell's lifetime, approximately 180 days in human airways. During that time period, individuals administered the vector containing the &bgr;2AR gene are hyperresponsive to aerosolized &bgr;-adrenergic agonist, compared to individuals not given the vector. Eventually, the beneficial effect diminishes as the airway epithelial cells slough off, divide, and otherwise disappear from the airway. Thus, it is necessary to reinfect a human subject or patient with the recombinant &bgr;2AR vector if control of the patient's asthma becomes difficult again. The persistence of AAV vectors in animals has been studied.60,172 &lsqb;0060&rsqb; The vector may optionally contain a DNA sequence encoding a mutant or modified &bgr;2AR that is modified as compared to the native &bgr;2AR or wild-type &bgr;2AR. The modified &bgr;2AR possesses at least one property that is different from the native &bgr;2AR. For example, the modified &bgr;2AR may possess increased responsiveness to &bgr;2AR agonists, increased affinity to &bgr;2AR agonists, and/or the capability to increase the potency of &bgr;2AR agonists to simulate downstream signal transduction pathways, as compared to the native &bgr;2AR. The modified &bgr;2AR is modified from the native &bgr;2AR by any one of or a combination of the following modifications, to include deletion of amino acids, substitution of amino acids, and/or replacement of amino acids. The modified &bgr;2AR is produced by modifing the DNA sequence encoding the &bgr;2AR prior to inserting the sequence into the vector. &lsqb;0061&rsqb; For example, &bgr;2AR mutants within the scope of the present invention are mutant &bgr;2ARs with phosphorylation sites removed or a constitutively active mutant &bgr;2AR. &lsqb;0062&rsqb; A mutant &bgr;2AR with phosphorylation sites removed is useful in the present invention. After binding hormone, the &bgr;2AR rapidly loses its ability to respond to subsequently administered hormone. This process is commonly referred to as desensitization. Desensitization is mediated by G-protein-coupled receptor kinases (GRK) and arresting. Following hormone binding, the &bgr;2AR is phosphorylated by a GRK which in turn leads to binding of arrestin. The binding of arrestin has two effects on &bgr;2AR function, both of which diminish responsiveness to &bgr;2-agonists. First, arrestin associated with the &bgr;2AR prevents the &bgr;2AR from interacting with the stimulatory guanine nucleotide regulatory protein G, and activating downstream signaling events. Second, arrestin binding to the &bgr;2AR increases the likelihood that the receptor will be removed from the cell surface and internalized. This reduces the total number of &bgr;2AR on the cell surface and reduces the responsiveness of the cell to &bgr;2-agonist. Based on all of this information, the removal (or replacement) of the amino acids of the &bgr;2AR that are phosphorylated by GRK prevents the process of desensitization. This concept is similar to related receptors for other hormones.173 To prevent phosphorylation of the &bgr;2AR by GRK, the receptor is either truncated (e.g., remove a portion of the carboxy tail that contains the phosphorylation sites or replace the serine and theronine residues in the carboxy tail with alanine and glycine residues. The amino acids serine and theronine are phosphorylation substrates. 173, 174 &lsqb;0063&rsqb; Constitutively active mutant &bgr;2ARs are also useful in the present invention. Normally, in order to observe activation of downstream signal transduction pathways (activation of adenylyl cyclase and cyclic AMP production) by the &bgr;2AR, hormone must bind to the receptor. However, a report was published several years ago that reported a mutant &bgr;2AR that activated adenylyl cyclase constitutively.175 The mutant was created by replacing the carboxy terminal portion of the third intracellular loop of the &bgr;2AR with the corresponding region of the &agr;1B-adrenergic receptor, a related receptor subtype that also binds epinephrine and norepinephrine. The resulting mutant receptor was expressed in COS-7 and CHO cells in vitro and tested for activity. The constitutively active &bgr;2AR displayed the following characteristics: 1) an increased affinity for agonists, and 2) increased potency of agonists in stimulating adenylyl cyclase activity. Both effects are desirable in the present invention. &lsqb;0064&rsqb; The vector of the present invention contains a promoter that is operably linked to the &bgr;2AR gene and is functional in the cells to which the vector is administered. These cells include the airway epithelial cells and smooth muscle cells and the blood vessel endothelial cells and smooth muscle cells. Preferably, the promoter is a viral vector promoter or a mammalian cell specific promoter. If the promoter is a mammalian cell specific promoter, it is preferably is an epithelial cell specific promoter, an endothelial cell specific promoter, or a smooth muscle cell specific promoter. For example, a promoter was developed that directs expression of the human cystic fibrosis transmembrane conductance regulator gene to airway epithelial cells.21 &lsqb;0065&rsqb; The preferred promoter is a viral vector promoter, which is functional in mammalian cells including either epithelial, endothelial or smooth muscle cells. Examples of such viral promoters are a cytomegalovirus (CMV) promoter or an adeno-associated vector (AAV) promoter. More preferably the vector is an AAV vector containing a CMV promoter. The promoter is selected to obtain the maximum expression of the &bgr;2AR gene in the transfected cells. &lsqb;0066&rsqb; The promoter may alternatively be an inducible promoter that allows the regulation of the amount of receptor that is expressed. Such a promoter can be induced or upregulated by hormones or by other pharmacological agents. The inducible promoter preferably should be a weaker promoter, such as the endogenous &bgr;2AR gene promoter or a tissue-specific promoter. The present invention is intended to encompass the administration of the inducer of the promoter to a human subject to induce the expression of &bgr;2ARs in the target cells. The administration of the inducer in a pharmaceutical composition occurs at the same time that the vector comprising the DNA sequence encoding a &bgr;2AR operably linked to the &bgr;2AR endogenous promoter or a tissue-specific promoter so that the &bgr;2AR gene is expressed. However, after the target cells are transduced and stably carrying the &bgr;2AR gene, to obtain increased &bgr;2AR expression in the target cells, it may be necessary to administer only the inducer or the inducer in combination with the &bgr;2-agonist, to provide enhanced dilation of the airways or blood vessels of the subject. &lsqb;0067&rsqb; Suzuki et al.137 provide an example of a regulatable promoter that can be upregulated by exogenous agents that raise the intracellular levels of cAMP. In addition to a promoter, the vector optionally may contain at least one enhancer or regulatory element that allows the &bgr;2AR gene to be turned on or off in the target cells. Burcin et al. 176 discloses a regulator to a liver-specific promoter. &lsqb;0068&rsqb; The application of gene therapy to treat human disease, while relatively simple in concept, is composed of series of challenges and problems that must be solved before beneficial effects can be realized. Two forms of gene therapy are currently used, ex vivo and in vivo. In ex vivo gene therapy that has been used in cancer patients144, cells are removed from the patient, transfected with the transgene and then re-implanted into the patient. Gene therapy for tissues that cannot be easily removed from the patient and reimplanted requires using in vivo gene therapy.146 Airway epithelium falls under the second category making in vivo gene therapy the required route of administration. Because all viruses transfer and express genetic material during their life cycles, they have frequently been used as vectors in in vivo therapeutic gene transfer protocols. Using treatment of cystic fibrosis patients with the cystic fibrosis transmembane conductance regulator (CFTR) gene as a model, several human gene therapy trials have shown that is possible to obtain expression of transferred genes to humans by a number of in vivo strategies26. The three principle viral vectors used in cystic fibrosis therapy have been retroviruses, adenoviruses, and adeno-associated viruses. Each has its advantages and disadvantages.43,44 The present inventors have chosen an AAV vector because of 1) natural tropism towards airway epithelium, 2) efficient integration into the genome of nondividing cells, 3) lack of association with any known human disease, and 4) ability to express the transgene longterm without inactivation in vivo. &lsqb;0069&rsqb; Recently, there has been increasing use of AAV in human gene therapy protocols. Currently, AAV is being tested in Phase I clinical trials with cystic fibrosis patients at the University of Florida and Johns Hopkins University. Initially, it appeared that there are no deleterious effects to overexpression of the CFTR protein in transgenic animals,137 and therefore in early gene transfer approaches to cystic fibrosis the prevailing strategy was to express as much CFTR as possible. Therefore many transgenes were typically driven by strong viral promoters to obtain maximal expression levels.137 More recently it has been recognized that the use of regulatable promoters may be a useful approach to increase safety and/or efficacy in human gene therapy protocols.51,61,137 Consequently, the present invention is intended to encompass the use of a regulatable promoter featuring a GRE to drive expression of the &bgr;2AR transgene. The &bgr;2AR gene is relatively small (˜2 kb) and has no introns. The entire transcription cassette that is inserted in AAV, including the &bgr;2AR gene and the neomyocin resistance gene, is approximately 3.6 kb, well under the packaging limit of 5.2 kb for efficient replication of AAV.63 &lsqb;0070&rsqb; The present vector and methods also are useful in treating vascular diseases. Vascular tone of the pulmonary arteries is a consequence of pulmonary vasorelaxation and vasoconstriction.177,178 The pulmonary vasculature expresses both &agr;-adrenergic receptors and &bgr;-adrenoreceptors, both of which help to regulate pulmonary vascular tone by producing vasoconstriction or vasodilation, respectively.179 Pulmonary hypertension starts out with vasoconstriction of the small and medium size pulmonary arteries. As pulmonary hypertension progresses, there is vascular remodeling as a result of smooth muscle hypertrophy. At the level of the pulmonary arteries, there are only two cell types that have any known effect on arterial diameter: smooth muscle cells and endothelial cells. Vasorelaxation is achieved through pathways that are dependent or independent of the endothelial cells. The &bgr;2-agonist, isoproterenol, produces vasorelaxation by interacting with &bgr;2ARs on pulmonary vascular smooth muscle cells. Other agents, such as acetylcholine, produce vasorelaxation by stimulating endothelial cells to produce nitric oxide, which in turns, causes vascular smooth muscle cells to relax. The reason typical vasodilators such as &bgr;2-agonists cannot be used to treat pulmonary hypertension is that they also cause arteries in the systemic circulation to dilate. This causes a precipitous drop in blood pressure and would lead to circulatory shock and death. The administration of &bgr;2AR containing vector to the smooth muscle cells of the pulmonary arteries is the appropriate method, which results in increased &bgr;2AR levels in these cells, making them more sensitive to circulating &bgr;2-agonists. Low doses of &bgr;2-agonists optionally are given, which cause pulmonary artery smooth muscle cells to relax, and thereby increase arterial diameter and reduce pulmonary arterial pressure. At this low dose of &bgr;2-agonist, the dose is too low to appreciably affect the &bgr;2ARs in the peripheral circulation detrimentally. &lsqb;0071&rsqb; To target the vector containing the &bgr;2AR gene to the pulmonary arterial smooth muscle cells, it is injected into a vein, for example the jugular vein. Venous blood returns to the right heart. This blood is then pumped to the lungs where it is oxygenated. Although the endothelial cells form a fairly tight barrier to the diffusion of anything including viruses, an appropriately designed gene delivery vehicle would transport the vector to the targeted cells. &lsqb;0072&rsqb; The examples set forth below serve to further illustrate the present invention in its preferred embodiments, and are not intended to limit the present invention. The examples utilize specific vectors and nucleic acid sequences but it is intended that the vectors and nucleic acid sequences with similar functional properties are also intended to be encompassed by the present invention. EXPERIMENTAL EXAMPLES Construction of AAV Vectors &lsqb;0073&rsqb; System for Producing Recombinant Adeno-Associated Virus &lsqb;0074&rsqb; Viral vectors and their application to gene therapy are known. The original gene mapping and phenotype determinations in AAV,64 were first published using recombinant AAV to transduce mammalian cells in vitro,62 and reported transduction of of hematopoietic stem cells with recombinant AAV,76 and reported the maximum packaging capacity of AAV.63 Several AAV genomes are useful as vectors to transfer genes into mammalian cells (FIG. 4). These include d13-94 which includes the terminal repeats, poly &lsqb;A&plus;&rsqb; motif, and a unique BglII cloning site,64 d16-95 which includes the same features as d13-94 plus the AAV P5 promoter64 which allows constitutive expression of inserted transgenes. d16-95/LacZ/Neo allows color selection and d16-95/GFP/Neo allows expression of green fluorescent protein for detection of infected living cells. The AAV terminal repeats are the only cis sequences in the AAV genome that are required for DNA replication, packaging and integration into the host genome. Approximately 5 kb of DNA can be packaged into d16-95 and d13-94.63 The Neo cassette can be removed and replaced with “filler DNA” for applications in which neomycin expression is not desirable (e.g., in vivo transduction of airway epithelial cells). &lsqb;0075&rsqb; For generating recombinant AAV stock, a system was developed by Hermonat and Muzcyzka62 which uses a “wild-type”, replication competent AAV genome called ins96-&lgr;-M (U.S. Pat. No. 5,139,941) (see FIG. 4 for structure). This AAV variant contains a 1.1 kb &lgr; phage DNA insert at map unit 96 and consequently is too large to be packaged effectively, but promotes packaging of recombinant AAV. This system produces consistently high titers of recombinant AAV (˜105-106 IU/ml compared to ˜103-105 IU/ml) when a non-replicating complementor AAV is used.122 Using this method, wild-type AAV is produced at a level that is ˜10-20% of that of recombinant AAV, an outcome that is not desirable in preparing recombinant AAV for human trials. However, in experiments presented herein, the presence of low levels of wild-type AAV will not affect the outcome of our experiments. &lsqb;0076&rsqb; Improvements in vector titer and purity are necessary for use in human gene therapy applications. A method of recombinant adeno-associated virus production has been described that is completely free of helper adenovirus.165 Use of this method to produce recombinant adeno-associated virus is advantageous in that a more defined reagent will result that is less likely to produce a host immune response. Further improvements in the methodology for recombinant adeno-associated virus preparation are likely as use of the vector in human gene therapy
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