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US20020103147A1 - Gene therapy for congestive heart failure - Google Patents

Gene therapy for congestive heart failure Download PDF

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US20020103147A1
US20020103147A1 US09/750,240 US75024000A US2002103147A1 US 20020103147 A1 US20020103147 A1 US 20020103147A1 US 75024000 A US75024000 A US 75024000A US 2002103147 A1 US2002103147 A1 US 2002103147A1
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beta
asp
vector
viral
gene
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H. Hammond
Paul Insel
Peipei Ping
Steven Post
Meihua Gao
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Priority claimed from PCT/US1997/015610 external-priority patent/WO1998010085A2/fr
Priority claimed from PCT/US1999/002702 external-priority patent/WO1999040945A2/fr
Priority claimed from US09/472,667 external-priority patent/US6752987B1/en
Application filed by Individual filed Critical Individual
Priority to US09/750,240 priority Critical patent/US20020103147A1/en
Publication of US20020103147A1 publication Critical patent/US20020103147A1/en
Priority to US10/942,072 priority patent/US7235236B2/en
Assigned to NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT reassignment NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT EXECUTIVE ORDER 9424, CONFIRMATORY LICENSE Assignors: UNIVERSITY OF CALIFORNIA, SAN DIEGO
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    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • CHF congestive heart failure
  • CHF is defined as abnormal heart function resulting in inadequate cardiac output for metabolic needs (Braunwald, E. (ed), In: Heart Disease, W. B. Saunders, Philadelphia, page 426, 1988). Symptoms include breathlessness, fatigue, weakness, leg swelling, and exercise intolerance. On physical examination, patients with heart failure tend to have elevations in heart and respiratory rates, rates (an indication of fluid in the lungs), edema, jugular venous distension, and, in general, enlarged hearts. The most common cause of CHF is atherosclerosis which causes blockages in the blood vessels (coronary arteries) that provide blood flow to the heart muscle.
  • CHF myocardial infarction
  • Other causes of CHF include valvular heart disease, hypertension, viral infections of the heart, alcohol, and diabetes.
  • Some cases of heart failure occur without clear etiology and are called idiopathic.
  • CHF is also typically accompanied by alterations in one or more aspects of beta-adrenergic neurohumoral function; see, e.g., Bristow M R, et al., N Engl J Med 307:205-211, 1982; Bristow M R, et al., Circ Res 59:297-309, 1986; Ungerer M, et al., Circulation 87:454-461, 1993; Feldman A M, et al., J Clin Invest 82:189-197, 1988; Bristow M R, et al., J Clin Invest 92:2737-2745, 1993; Calderone A, et al., Circ Res 69:332-343.
  • pharmacological therapies have been directed toward increasing the force of contraction of the heart (by using inotropic agents such as digitalis and beta-adrenergic receptor agonists), reducing fluid accumulation in the lungs and elsewhere (by using diuretics), and reducing the work of the heart (by using agents that decrease systemic vascular resistance such as angiotensin converting enzyme inhibitors).
  • inotropic agents such as digitalis and beta-adrenergic receptor agonists
  • reducing fluid accumulation in the lungs and elsewhere by using diuretics
  • reducing the work of the heart by using agents that decrease systemic vascular resistance such as angiotensin converting enzyme inhibitors.
  • Beta-adrenergic receptor antagonists have also been tested. While such pharmacological agents can improve symptoms, and potentially prolong life, the prognosis in most cases remains dismal.
  • Some patients with heart failure due to associated coronary artery disease can benefit, at least temporarily, by revascularization procedures such as coronary artery bypass surgery and angioplasty. Such procedures are of potential benefit when the heart muscle is not dead but may be dysfunctional because of inadequate blood flow. If normal coronary blood flow is restored, viable dysfunctional myocardium may contract more normally, and heart function may improve. However, revascularization rarely restores cardiac function to normal or near-normal levels in patients with CHF, even though mild improvements are sometimes noted.
  • heart transplantation can be a suitable option for patients who have no other confounding diseases and are relatively young, but this is an option for only a small number of patients with heart failure, and only at great expense.
  • CHF has a very poor prognosis and responds poorly to current therapies.
  • fluid retention tends to result in edema and retained fluid in the lungs that impairs breathing; heart enlargement can lead to deleterious left ventricular remodeling with subsequent severe dilation and increased wall tension, thus exacerbating CHF; and long-term exposure of the heart to norepinephrine tends to make the heart unresponsive to adrenergic stimulation and is linked with poor prognosis.
  • cloned DNA encoding two different isoforms of adenylylcyclase (AC V and AC VI ) that are known to be predominant in mammalian cardiac tissue, and proposed using the DNA and/or recombinant protein to identify new classes of drugs that might stimulate adrenergic pathways (See, e.g., American Cyanamid, WO 93/05061, Mar. 18, 1993, and EP 0 529 662, Mar. 03, 1993; and Ishikawa U.S. Pat. No. 5,334,521, issued Aug. 02, 1994).
  • AC V and AC VI adenylylcyclase
  • beta-adrenergic agonists such as dopamine and dobutamine
  • beta-receptor “blockers” or antagonists may be more useful for improving morbidity and mortality rates (see, e.g., Baughman, K., Cardiology Clinics 13:27-34, 1995). While some agents may improve symptoms, the prognosis for patients receiving such pharmacological agents remains dismal.
  • the invention described and claimed herein addresses and overcomes these and other problems associated with the prior art by providing techniques by which cardiac function can be effectively enhanced in vivo without the administration of beta-adrenergic-agonist drugs.
  • FIG. 1A shows a schematic of the construction of an exemplary replication-defective recombinant adenovirus vector useful for gene transfer into cells and into the heart, as described in Example 5-1 below.
  • FIG. 1B shows a schematic of a clone used in the construction of an exemplary replication-defective recombinant adenovirus vector useful for gene transfer into cells and into the heart, as described in Example 5-2 below.
  • FIG. 2 shows a schematic of the cell surface beta-adrenergic-Gs-adenylylcyclase pathway. It is through this pathway that beta-adrenergic stimulation increases intracellular cAMP thereby influencing heart rate responsiveness and force of contraction.
  • the pathway includes a beta-adrenergic receptor, a stimulatory GTP-binding protein (Gs) linking receptor occupation with cAMP production, and an adenylylcyclase, as described in more detail below.
  • Gs stimulatory GTP-binding protein
  • FIG. 3 shows data from experiments using forskolin-stimulated cAMP production to assess the function of adenylylcyclase in left ventricular membranes from normal pigs and from pigs with severe heart failure, using a model of heart failure with very high fidelity to human clinical dilated heart failure, as described in Example 3.
  • FIG. 4 shows data indicating that AC content sets a limit upon beta-AR-mediated signal transduction in cardiac myocytes, as described in Example 8-1.
  • FIG. 5 shows data indicating that gene transfer of an AC VI transgene to cultured neonatal rat ventricular myocytes increased the levels of cAMP obtained after stimulation with either isoproterenol (10 micromolar) or forskolin (3 micromolar), as described in Example 8-1.
  • FIG. 6 shows data from a Northern analysis indicating the presence of transgene mRNA in cardiac myocytes, as described in Example 8-2.
  • FIG. 7 shows data from a Western analysis indicating the presence of transgene protein in cardiac myocytes, as described in Example 8-2.
  • FIG. 8A shows data from a forskolin binding study indicating that net GTP gamma-stimulated forskolin binding was increased after AC VI gene transfer (data are mean values from three experiments), as described in Example 8-2.
  • FIG. 8B shows data from a cAMP production study indicating that cardiac myocytes expressing transgene AC VI have increased adrenergic responsiveness not only to forskolin stimulation, reflecting increased amounts of AC, but to isoproterenol, suggesting that newly synthesized AC is functionally coupled and recruitable through beta-AR stimulation, as described in Example 8-2. Shown are mean values from three experiments.
  • FIG. 8C shows the observed relationship between AC VI content and cAMP production, as described in Example 8-2.
  • the graph displays three measure of altered adrenergic signaling (forskolin binding, and isoproterenol- and forskolin-stimulated cAMP production). These data indicate that a proportional increase in AC content and enhanced adrenergic signaling has occurred.
  • FIG. 9 shows the results of an isoproterenol stimulation study as described in Example 8-2.
  • Neonatal rat cardiac myocytes underwent gene transfer using recombinant adenovirus expressing lacZ or AC VI .
  • AC VI vs lacZ
  • the EC50 for isoproterenol-stimulated cAMP production was unchanged.
  • FIG. 10 shows data summarizing the effects of in vivo gene transfer of AC VI on heart rate in pigs, as described in Example 13. These data demonstrate, for the first time, that in vivo gene transfer can effectively increase adrenergic responsiveness in a large mammal heart.
  • FIG. 11 shows results of in vivo gene transfer of AC VI on left ventricular (LV) dP/dt in a normal pig, as described in Example 13.
  • the present invention relates to methods and compositions for enhancing cardiac function in mammalian hearts by inserting transgenes that increase beta-adrenergic responsiveness within the myocardium.
  • the present invention can thus be used in the treatment of heart disease, especially congestive heart failure.
  • a method of enhancing cardiac function in a mammal comprising delivering a vector to the heart of said mammal, the vector comprising a gene encoding a beta-adrenergic signaling protein (beta-ASP) operably linked to a promoter.
  • the vector is introduced into a blood vessel supplying blood to the myocardium of the heart, so as to deliver the vector to cardiac myocytes; more preferably the vector is introduced into the lumen of a coronary artery, a saphenous vein graft, or an internal mammary artery graft.
  • the vector is introduced into the lumen of both the left and right coronary arteries.
  • the mammal is a human.
  • the vector comprises at least one gene encoding a beta-ASP selected from the group consisting of a beta-adrenergic receptor (beta-AR), a G-protein receptor kinase inhibitor (GRK inhibitor) and an adenylylcyclase (AC), each operably linked to a promoter.
  • the method can also comprise introducing a vector encoding two different beta-adrenergic signaling proteins (beta-ASPs), each operably linked to a promoter, or introducing a second vector comprising a second beta-ASP gene operably linked to a promoter.
  • the vector comprises a gene encoding an adenylylcyclase (AC), preferably a cardiac AC such as AC isoform II, AC isoform V or AC isoform VI, more preferably AC isoform VI.
  • AC adenylylcyclase
  • the human AC isoform VI of SEQ ID No. 10 is employed.
  • a modified (i.e., chimeric) AC isoform VI is employed.
  • the modified AC isoform VI of SEQ ID NO. 13 is employed in the vector in accordance herewith.
  • the vector comprises a gene encoding a beta-AR, preferably a beta 1 -adrenergic receptor (beta 1 -AR) or a beta 2 -adrenergic receptor (beta 2 -AR), more preferably a beta 1 -AR.
  • the vector comprises a gene encoding a GRK inhibitor, which is preferably a gene encoding a GRK protein having a mutation that impairs kinase activity without eliminating receptor binding activity, more preferably the mutation is a truncation deleting the kinase domain.
  • the vector comprises a gene encoding a beta-ASP operably linked to a heterologous constitutive promoter or a heterologous inducible promoter.
  • a preferred heterologous constitutive promoter is a CMV promoter which also includes an enhancer.
  • the promoter is a tissue-specific promoter, preferably a cardiac-specific promoter, more preferably, a ventricular myocyte-specific promoter.
  • Preferred examples of ventricular myocyte-specific promoters include a ventricular myosin light chain 2 promoter and a ventricular myosin heavy chain promoter.
  • the gene encoding a beta-ASP can also be operably linked to a heterologous enhancer, such as the CMV enhancer.
  • the gene encoding a beta-ASP is also operably linked to a polyadenylation signal.
  • the vector is a viral vector or a lipid-based vector, preferably a viral vector.
  • the vector can be a targeted vector, especially a targeted vector that preferentially binds to ventricular myocytes.
  • Presently preferred viral vectors are derived from adenovirus (Ad) or adeno-associated virus (AAV). Both human and non-human viral vectors can be used but preferably the recombinant viral vector is replication-defective in humans.
  • the vector is an adenovirus
  • it preferably comprises a polynucleotide having a promoter operably linked to a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase), and is replication-defective in humans.
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • replication-defective adenoviral vector have deletions that remove the E1A and E1B genes, or have deletions that remove the E1A, E1B and E4 genes.
  • Preferably about 10 7 to 10 13 adenovirus vector particles, more preferably about 10 9 to 10 12 vector particles, are introduced into a blood vessel, preferably a blood vessel supplying the myocardium as described above.
  • the vector preferably comprises a polynucleotide having a promoter operably linked to a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase) and, preferably, the gene encoding a beta-ASP is flanked by AAV inverted terminal repeats (ITRs).
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • ITRs AAV inverted terminal repeats
  • the AAV vector is replication-defective in humans.
  • the vector can be a lipid-based vector comprising a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase) as described herein.
  • a recombinant replication-defective viral particle comprising a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase) operably linked to a promoter.
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • the promoter is a heterologous constitutive or inducible promoter.
  • the vector can also comprise genes encoding more than one beta-ASP.
  • Preferred viral vectors are derived from adenovirus (Ad) or adeno-associated virus (AAV). Both human and non-human viral vectors can be used but preferably the recombinant viral vector is replication-defective in humans.
  • the vector is an adenovirus
  • it preferably comprises a polynucleotide having a promoter operably linked to a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase), and is replication-defective in humans.
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • the vector preferably comprises a polynucleotide having a promoter operably linked to a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase) and, preferably, the gene encoding a beta-ASP is flanked by AAV inverted terminal repeats (ITRs).
  • a beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • ITRs AAV inverted terminal repeats
  • the AAV vector is replication-defective in humans.
  • Presently preferred replication-defective AAV vectors have deletions affecting one or more AAV replication or encapsidation sequences.
  • lipid-based vectors such as liposomes
  • lipid-based vectors comprising one or more genes encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase), as described herein.
  • beta-ASP such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase
  • a filtered injectable adenovirus particle preparation comprising: (i) a recombinant replication-defective adenovirus particle as described above, and (ii) a carrier.
  • the carrier is preferably a pharmaceutically-acceptable carrier.
  • the adenovirus vector has been filtered through a 0.1-0.5 micron filter.
  • first and second plasmids into a replication-permissive mammalian cell expressing one or more adenovirus genes conferring replication competence
  • said first plasmid comprises a gene encoding a beta-ASP (such as a beta-AR, a GRK inhibitor, and/or an adenylylcyclase) operably linked to a promoter and further comprises a replication-defective adenovirus genome
  • said second plasmid comprises a replication-proficient adenovirus genome and further comprises an additional polynucleotide sequence making the second plasmid too large to be encapsidated in an adenovirus particle, whereby rescue recombination takes place between the first plasmid and the second plasmid to generate a recombinant adenoviral genome comprising the gene encoding a beta-ASP but lacking one or more adenoviral replication genes, said recombinant a
  • a beta-ASP such as a
  • the introducing step can be accomplished by co-transfection of the first and second plasmids into the permissive mammalian cell.
  • the method can also comprise, prior to said step of introducing first and second plasmids, the step of cloning a gene encoding a beta-ASP into a plasmid containing a promoter and partial adenovirus sequences of the left end of a replication-defective adenovirus genome such that the gene encoding the beta-ASP is operably linked to said promoter.
  • the method further comprises, after said propagation step, the step of purifying the propagated viral particles, which can include filtering the purified viral particles through a 0.1-0.5 micron filter.
  • An exemplary first plasmid as described above is plasmid pAC1 or plasmid ACCMVPLPA comprising a gene encoding a beta-ASP.
  • the identification step described above preferably comprises the steps of: (i) monitoring transfected cells for evidence of cytopathic effect; (ii) isolating viral nucleic acid from the cell supernatant of cultures of the transfected cells showing a cytopathic effect (by treating the cell supernatant from cell cultures showing a cytopathic effect with a proteinase (such as proteinase K), followed by phenol/chloroform extraction and ethanol precipitation); (iii) identifying successful recombinants with PCR using primers complementary to the promoter operably linked to the beta-ASP gene and primers complementary to adenovirus sequences; and (iv) purifying the recombinant viral particles by plaque purification (preferably for at least two rounds).
  • a proteinase such as proteinase K
  • Viral nucleic acid can be isolated by treating the cell culture supernatant suspected of containing recombinant viral particles with a proteinase (such as proteinase K), followed by phenol/chloroform extraction of the proteinase-treated supernatant to remove proteins, and finally, ethanol precipitation of the lysate to obtain viral DNA.
  • a proteinase such as proteinase K
  • the purification step as described above preferably comprises the steps of: (i) propagating the resulting recombinants in cells transformed with the replication competence conferring genes to titers in the 10 10 -10 12 viral particles range; and (ii) purifying the propagated recombinants (preferably by double CsCl gradient ultracentrifugation).
  • a recombinant pro-viral plasmid comprising a gene encoding a beta-ASP operably linked to a promoter and further comprising a replication-defective viral genome.
  • the beta-ASP is a beta-AR, a GRK inhibitor or an adenylylcyclase, more preferably, adenylylcyclase isoform VI.
  • Exemplary replication-defective viral genomes include an adenovirus genome and an AAV genome.
  • the adenovirus may be either a human or a non-human mammalian adenovirus (preferably non-human mammalian), but in either case is preferably replication-defective in humans.
  • the recombinant replication-defective adenovirus genome has deletions removing the E1A and E1B genes, or deletions removing the E1A, E1B and E4 genes.
  • the recombinant replication-defective viral genome is an AAV genome
  • the AAV genome preferably has deletions affecting one or more AAV replication or encapsidation sequences.
  • a cell comprising a recombinant pro-viral plasmid according to one of the preceding embodiments.
  • a “polynucleotide” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. This term refers to the primary structure of the molecule, and thus includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified polynucleotides such as methylated and/or capped polynucleotides.
  • Recombinant as applied to a polynucleotide, means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a “gene” refers to a polynucleotide or portion of a polynucleotide comprising a sequence that encodes a protein. For most situations, it is desirable for the gene to also comprise a promoter operably linked to the coding sequence in order to effectively promote transcription. Enhancers, repressors and other regulatory sequences may also be included in order to modulate activity of the gene, as is well known in the art. (See, e.g., the references cited below).
  • polypeptide “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include glycosylation, acetylation and phosphorylation.
  • a “heterologous” component refers to a component that is introduced into or produced within a different entity from that in which it is naturally located.
  • a polynucleotide derived from one organism and introduced by genetic engineering techniques into a different organism is a heterologous polynucleotide which, if expressed, can encode a heterologous polypeptide.
  • a promoter or enhancer that is removed from its native coding sequence and operably linked to a different coding sequence is a heterologous promoter or enhancer.
  • a “promoter,” as used herein, refers to a polynucleotide sequence that controls transcription of a gene or coding sequence to which it is operably linked.
  • a large number of promoters, including constitutive, inducible and repressible promoters, from a variety of different sources, are well known in the art and are available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • an “enhancer,” as used herein, refers to a polynucleotide sequence that enhances transcription of a gene or coding sequence to which it is operably linked.
  • enhancers from a variety of different sources are well known in the art and available as or within cloned polynucleotide sequences (from, e.g., depositories such as the ATCC as well as other commercial or individual sources).
  • a number of polynucleotides comprising promoter sequences (such as the commonly-used CMV promoter) also comprise enhancer sequences.
  • operably linked refers to a juxtaposition, wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter is operably linked to a coding sequence if the promoter controls transcription of the coding sequence. Although an operably linked promoter is generally located upstream of the coding sequence, it is not necessarily contiguous with it.
  • An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within or downstream of coding sequences.
  • a polyadenylation sequence is operably linked to a coding sequence if it is located at the downstream end of the coding sequence such that transcription proceeds through the coding sequence into the polyadenylation sequence.
  • a “replicon” refers to a polynucleotide comprising an origin of replication which allows for replication of the polynucleotide in an appropriate host cell.
  • examples include replicons of a target cell into which a heterologous nucleic acid might be integrated (e.g., nuclear and mitochondrial chromosomes), as well as extrachromosomal replicons (such as replicating plasmids and episomes).
  • Gene delivery are terms referring to the introduction of an exogenous polynucleotide (sometimes referred to as a “transgene”) into a host cell, irrespective of the method used for the introduction.
  • exogenous polynucleotide sometimes referred to as a “transgene”
  • Such methods include a variety of well-known techniques such as vector-mediated gene transfer (by, e.g., viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) as well as techniques facilitating the delivery of “naked” polynucleotides (such as electroporation, “gene gun” delivery and various other techniques used for the introduction of polynucleotides).
  • the introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are known to be capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • “In vivo” gene delivery, gene transfer, gene therapy and the like as used herein, are terms referring to the introduction of a vector comprising an exogenous polynucleotide directly into the body of an organism, such as a human or non-human mammal, whereby the exogenous polynucleotide is introduced to a cell of such organism in vivo.
  • a “vector” refers to a macromolecule or complex of molecules comprising a polynucleotide to be delivered to a host cell, either in vitro or in vivo.
  • the polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy.
  • Vectors include, for example, viral vectors (such as adenoviruses (“Ad”), adeno-associated viruses (AAV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • vectors can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • a large variety of such vectors are known in the art and are generally available (see, e.g., the various references cited below).
  • a “recombinant viral vector” refers to a viral vector comprising one or more heterologous genes or sequences. Since many viral vectors exhibit size-constraints associated with packaging, the heterologous genes or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-defective, requiring the deleted function(s) to be provided in trans during viral replication and encapsidation (by using, e.g., a helper virus or a packaging cell line carrying genes necessary for replication and/or encapsidation) (see, e.g., the references and illustrations below).
  • Viral “packaging” as used herein refers to a series of intracellular events that results in the synthesis and assembly of a viral vector.
  • Packaging typically involves the replication of the “pro-viral genome”, or a recombinant pro-vector typically referred to as a “vector plasmid” (which is a recombinant polynucleotide than can be packaged in an manner analogous to a viral genome, typically as a result of being flanked by appropriate viral “packaging sequences”), followed by encapsidation or other coating of the nucleic acid.
  • vector plasmid which is a recombinant polynucleotide than can be packaged in an manner analogous to a viral genome, typically as a result of being flanked by appropriate viral “packaging sequences”
  • Viral “rep” and “cap” genes found in many viral genomes, are genes encoding replication and encapsidation proteins, respectively.
  • a “replication-defective” or “replication-incompetent” viral vector refers to a viral vector in which one or more functions necessary for replication and/or packaging are missing or altered, rendering the viral vector incapable of initiating viral replication following uptake by a host cell.
  • the virus or pro-viral nucleic acid can be introduced into a “packaging cell line” that has been modified to contain genes encoding the missing functions which can be supplied in trans).
  • packaging genes can be stably integrated into a replicon of the packaging cell line or they can be introduced by transfection with a “packaging plasmid” or helper virus carrying genes encoding the missing functions.
  • a “detectable marker gene” is a gene that allows cells carrying the gene to be specifically detected (e.g., distinguished from cells which do not carry the marker gene).
  • a large variety of such marker genes are known in the art. Preferred examples thereof include detectable marker genes which encode proteins appearing on cellular surfaces, thereby facilitating simplified and rapid detection and/or cellular sorting.
  • the lacZ gene encoding beta-galactosidase can be used as a detectable marker, allowing cells transduced with a vector carrying the lacZ gene to be detected by staining, as described below.
  • a “selectable marker gene” is a gene that allows cells carrying the gene to be specifically selected for or against, in the presence of a corresponding selective agent.
  • an antibiotic resistance gene can be used as a positive selectable marker gene that allows a host cell to be positively selected for in the presence of the corresponding antibiotic.
  • Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated.
  • a variety of such marker genes have been described, including bifunctional (i.e. positive/negative) markers (see, e.g., WO 92/08796, published May 29, 1992, and WO 94/28143, published Dec. 8, 1994). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts.
  • Beta-adrenergic signaling refers to beta-adrenergic receptor-mediated signaling which is mediated via beta-adrenergic receptors (“beta-ARs”) present on cellular surfaces.
  • beta-ARs beta-adrenergic receptors
  • beta-ARs beta-adrenergic receptors
  • Activated AC then catalyzes the synthesis of cyclic AMP (cAMP), and increased intracellular concentrations of cAMP mediate increased cytosolic calcium transients which enhance both the rate and force of cardiac contraction (referred to as positive chronotrophy and positive inotrophy, respectively).
  • cAMP cyclic AMP
  • increased intracellular concentrations of cAMP mediate increased cytosolic calcium transients which enhance both the rate and force of cardiac contraction (referred to as positive chronotrophy and positive inotrophy, respectively).
  • cAMP cyclic AMP
  • cytosolic calcium transients which enhance both the rate and force of cardiac contraction
  • a “beta-adrenergic signaling protein” (sometimes abbreviated “beta-ASP” herein) or “beta-adrenergic signaling element” refers to a protein that is capable of enhancing beta-adrenergic receptor-mediated signaling when expressed in mammalian tissue, preferably (for purposes of the present invention) when expressed in mammalian myocardial tissue.
  • Beta-adrenergic signaling proteins thus include “beta-adrenergic signal transducer” proteins that mediate or transduce beta-adrenergic signaling, preferably in mammalian myocardial cells, as well as proteins which can either stimulate such transducer proteins or which can inactivate or compete with inhibitors of such transducer proteins (thereby indirectly enhancing signal transduction).
  • beta-adrenergic signal transducer proteins that mediate or transduce beta-adrenergic signaling, preferably in mammalian myocardial cells, as well as proteins which can either stimulate such transducer proteins or which can inactivate or compete with inhibitors of such transducer proteins (thereby indirectly enhancing signal transduction).
  • beta-adrenergic signal transducer proteins proteins that are associated with beta-adrenergic receptor-mediated signaling in mammalian cardiac tissue have been identified (see, e.g., the various references regarding beta-adrenergic responsiveness cited above) and are illustrated herein.
  • beta-ASPs for use in the present invention are those that are known to play a role in beta-adrenergic receptor-mediated signal transduction in mammalian heart tissue, such as the various proteins associated with the “beta-AR-Gs-AC” pathway, comprising a beta-adrenergic receptor (“beta-AR”), a G s protein transducer and an adenylylcyclase (“AC”) effector, as well as proteins enhancing the activity of such beta-AR-Gs-AC proteins, as described in more detail herein and in the cited art. Recent data have demonstrated that G s protein is generally present at a much higher molar proportion than either beta-AR or AC.
  • beta-adrenergic receptors such as beta 1 -adrenergic receptors or beta 2 -adrenergic receptors, more preferably beta 1 -adrenergic receptors
  • adenylylcyclases preferably a cardiac AC such as AC V or AC VI , more preferably AC VI
  • inhibitors of the function of G-protein receptor kinases which are generally referred to herein as “GRK” inhibitors.
  • Beta-adrenergic receptors are the cell-surface receptors involved in beta-adrenergic receptor-mediated signaling via the beta-AR-Gs-AC pathway.
  • beta-ARs are the principal receptors for norepinephrine (the sympathetic neurotransmitter) and for epinephrine (the adrenal hormone).
  • Human myocardium contains both beta 1 -adrenergic receptors and beta 2 -adrenergic receptors, but beta 1 -ARs are predominant and are most closely associated with the altered beta-adrenergic signaling that is observed with heart failure, as described below.
  • G s protein is a GTP-binding regulatory protein that effectively couples activation of a variety of cell-surface receptors (including beta-adrenergic receptors) to the activation of adenylylcyclase, as described in the art and herein.
  • Adenylylcyclase (EC 4.6.1.1, also referred to as “adenylcyclase”, “adenylate cyclase”, and “cAMP synthetase”) is an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to 3′.5′-cyclic adenosine monophosphate (cAMP).
  • Adenylylcyclase (abbreviated herein as “AC”) is known to exist in a number of different isoforms that are found in varying levels in most all mammalian tissues.
  • the most preferred adenylylcyclases of the present invention are “cardiac adenylylcyclases” which are isoforms found to be predominant in mammalian heart tissue, particularly in cardiac myocytes; as described in more detail below.
  • G-protein receptor kinases (abbreviated “GRK”, but also referred to in the art as “beta-adrenergic receptor kinases” or “beta-ARK”), are kinase proteins that catalyze phosphorylation of G-protein-coupled receptor proteins including beta-adrenergic receptors (“beta-ARs”). Phosphorylation of beta-ARs by GRK proteins leads to uncoupling of the receptors and a concomitant decrease in responsiveness to beta-adrenergic signaling.
  • GRK G-protein receptor kinases
  • GRK inhibitors refer to proteins that inhibit the function of G-protein receptor kinases. Such inhibitors of GRK include modified GRK proteins in which receptor-binding activity has been uncoupled from kinase activity. Exemplary GRK inhibitors thus include modified GRKs that have been truncated (typically by deletions beginning at the amino-terminus) to remove kinase function while retaining the ability to bind to G-protein-coupled receptor proteins such as beta-ARs. Such truncated GRK proteins can thus effectively compete with or prevent normal GRK from binding to beta-AR but do not cause subsequent inhibition of receptor activity (since they lack kinase activity). Examples of GRK inhibitors that can be used in the present invention are described below.
  • Vasculature or “vascular” are terms referring to the system of vessels carrying blood (as well as lymph fluids) throughout the mammalian body.
  • Blood vessel refers to any of the vessels of the mammalian vascular system, including arteries, arterioles, capillaries, venules, veins, sinuses, and vasa vasorum.
  • Coronary arteries supply the tissues of the heart itself, while other arteries supply the remaining organs of the body.
  • the general structure of an artery consists of a lumen surrounded by a multi-layered arterial wall.
  • An “individual” as used herein refers to a large mammal, most preferably a human.
  • Treatment refers to administering, to an individual patient, agents that are capable of eliciting a prophylactic, curative or other beneficial effect in the individual.
  • Gene therapy refers to administering, to an individual patient, vectors comprising a therapeutic gene.
  • a “therapeutic polynucleotide” or “therapeutic gene” refers to a nucleotide sequence that is capable, when transferred to an individual, of eliciting a prophylactic, curative or other beneficial effect in the individual.
  • Additional references describing delivery and logistics of surgery which may be used in the methods of the present invention include the following: Topol, E J (ed.), The Textbook of Interventional Cardiology, 2nd Ed. ( W.B. Saunders Co. 1994); Rutherford, R B, Vascular Surgery, 3rd Ed. (W.B. Saunders Co. 1989); Wyngaarden J B et al. (eds.), The Cecil Textbook of Medicine, 19th Ed. (W. B. Saunders, 1992); and Sabiston, D, The Textbook of Surgery, 14th Ed. (W.B. Saunders Co. 1991).
  • One preferred aspect of the present invention is to provide methods for treating heart disease (especially CHF), in which one or more beta-adrenergic signaling elements is synthesized in vivo in a patient by targeting the myocardium with a vector construct containing a gene encoding a beta-adrenergic signaling element.
  • the preferred methods employ vector constructs and/or delivery methods that result in localized expression of the beta-adrenergic signaling element that is relatively restricted to the myocardium of the patient.
  • the presently preferred beta-adrenergic signaling proteins include adenylylcyclases (“AC”s) (preferably a cardiac AC such as AC II , AC V or AC VI , more preferably AC VI ), beta-adrenergic receptors (such as beta 1 -adrenergic receptors or beta 2 -adrenergic receptors, preferably beta 1 -adrenergic receptors), and inhibitors of the function of G-protein receptor kinases “GRK inhibitors”). Examples of such preferred beta-ASPs are described and illustrated below.
  • AC adenylylcyclases
  • beta-adrenergic receptors such as beta 1 -adrenergic receptors or beta 2 -adrenergic receptors, preferably beta 1 -adrenergic receptors
  • GRK inhibitors inhibitors of the function of G-protein receptor kinases
  • Preferred vectors for use in the present invention include viral vectors, lipid-based vectors and other vectors that are capable of delivering DNA to non-dividing cells in vivo.
  • viral vectors particularly replication-defective viral vectors (including, for example replication-defective adenovirus vectors and adeno-associated virus (AAV) vectors.
  • replication-defective adenovirus vectors are presently most preferred.
  • the presently preferred means of in vivo delivery is by injection of the vector into a blood vessel directly supplying the myocardium, preferably by injection into a coronary artery.
  • Such injection is preferably achieved by catheter introduced substantially (typically at least about 1 cm) within the ostium of one or both coronary arteries or one or more saphenous veins or internal mammary artery grafts or other conduits delivering blood to the myocardium.
  • the vector stock preferably containing no wild-type virus, deeply into the lumen of one or both coronary arteries (or grafts and other vascular conduits), preferably into both the right and left coronary arteries (or grafts and other vascular conduits), and preferably in an amount of 10 7 -10 13 viral particles as determined by optical densitometry (more preferably 10 9 -10 11 viral particles), it is possible to locally transfect a desired number of cells, especially cardiac myocytes, with genes that encode proteins that increase beta-adrenergic signal transduction in the affected myocardium, thereby maximizing therapeutic efficacy of gene transfer, and minimizing undesirable effects at extracardiac sites and the possibility of an inflammatory response to viral proteins.
  • At least one injection is made into the right coronary circulation and at least two injections are made into the left coronary circulation (i.e. one via the left anterior descending and one via the left circumflex).
  • Vector constructs that are specifically targeted to the myocardium such as vectors incorporating myocardial-specific binding or uptake components, and/or which incorporate beta-adrenergic signaling transgenes that are under the control of myocardial-specific transcriptional regulatory sequences (e.g., ventricular myocyte-specific promoters) can be used in place of or, preferably, in addition to such directed injection techniques as a means of further restricting expression to the myocardium, especially the ventricular myocytes.
  • the present invention provides a filtered, injectable adenovirus vector preparation, comprising a recombinant adenovirus vector, preferably in a final viral titer of 10 7 -10 14 viral particles, said vector containing no wild-type virus and comprising a partial adenovirus sequence from which one or more required adenovirus genes conferring replication competence, for example, the E1A/E1B genes have been deleted, and a transgene coding for a beta-adrenergic signaling element such as AC VI , AC V , other adenylylcyclases, beta 1 -adrenergic receptors, beta 2 -adrenergic receptors, or inhibitors of the function of G-protein receptor kinases, driven by a promoter flanked by the partial adenovirus sequence; and a pharmaceutically acceptable carrier.
  • a beta-adrenergic signaling element such as AC VI , AC V , other adenylylcyclases, beta
  • the present invention provides methods for the generation of recombinant viral stocks capable of effecting expression of a beta-adrenergic signaling element in vivo in the myocardium, comprising the steps of cloning a transgene coding for a beta-adrenergic signaling element (such as AC VI , AC V , other adenylylcyclases, beta 1 -adrenergic receptors, beta 2 -adrenergic receptors, or inhibitors of the function of G-protein receptor kinases) into a plasmid containing a promoter and a polylinker flanked by partial adenovirus sequences of an adenovirus genome from which one or more adenovirus genes required for replication competence (generically referred to as viral replication or “rep” genes), such as the E1A/E1B genes of the human adenovirus 5 genome, have been deleted; co-transfecting said plasmid into a transgene coding for a
  • the present invention employs genes encoding protein or peptide elements that increase beta-adrenergic signaling and are therefore capable of enhancing responsiveness to endogenous beta-adrenergic stimulation within dysfunctional regions of a mammalian heart.
  • Such proteins are referred to herein as “beta-adrenergic signaling proteins” (or “beta-ASPs”).
  • beta-ASP refers to a protein that is capable of enhancing beta-adrenergic signaling when expressed in mammalian tissue, preferably (for purposes of the present invention) when expressed in mammalian myocardial tissue.
  • Beta-adrenergic signaling proteins include beta-adrenergic signal transducer proteins that mediate or transduce beta-adrenergic signaling, preferably in mammalian myocardial cells, as well as proteins which can either stimulate such transducer proteins or which can inactivate or compete with inhibitors of such transducer proteins (thereby indirectly enhancing signal transduction).
  • beta-adrenergic signal transducer proteins that mediate or transduce beta-adrenergic signaling, preferably in mammalian myocardial cells, as well as proteins which can either stimulate such transducer proteins or which can inactivate or compete with inhibitors of such transducer proteins (thereby indirectly enhancing signal transduction).
  • a variety of such proteins that are associated with beta-adrenergic signaling in mammalian cardiac tissue have been identified (see, e.g., the various references regarding beta-adrenergic responsiveness cited above) and are illustrated herein.
  • Preferred beta-ASPs for use in the present invention are those that are known to play a role in beta-adrenergic signal transduction in mammalian heart tissue, such as the various proteins associated with the “beta-AR-Gs-AC” pathway, comprising a beta-adrenergic receptor (“beta-AR”), a G s protein transducer and an adenylylcyclase (“AC”) effector, as described in more detail herein and in the cited art.
  • beta-AR beta-adrenergic receptor
  • G s protein transducer a G s protein transducer
  • AC adenylylcyclase
  • Recent data have demonstrated that G s protein is generally present at a much higher molar proportion than either beta-AR or AC.
  • the latter two proteins (beta-AR and AC), as well as inhibitors of G-protein receptor kinases (which affect beta-AR activity) are more preferred beta-adrenergic signaling components for use in the present
  • Beta-adrenergic signaling within myocardial tissue is initially mediated by agonist binding to beta-AR, followed by G s -mediated signal transduction to AC.
  • Activated AC then catalyzes the synthesis of cyclic AMP, and increased intracellular concentrations of cAMP mediate increased cytosolic calcium transients which enhance both the rate and force of cardiac contraction (referred to as positive “chronotrophy” and positive “inotrophy,” respectively).
  • beta-adrenergic receptors such as beta 1 -adrenergic receptors or beta 2 -adrenergic receptors, preferably beta 1 -adrenergic receptors
  • adenylylcyclases preferably a cardiac AC such as AC V or AC VI , more preferably AC VI
  • inhibitors of the function of G-protein receptor kinases which are generally referred to herein as “GRK” inhibitors.
  • Beta-adrenergic receptors are cell-surface receptors involved in beta-adrenergic signaling via the beta-AR-Gs-AC pathway.
  • beta-ARs are the principal receptors for norepinephrine (the sympathetic neurotransmitter) and for epinephrine (the adrenal hormone).
  • Human myocardium contains both beta 1 -adrenergic receptors and beta 2 -adrenergic receptors, but beta 1 -ARs are predominant and are most closely associated with the altered beta-adrenergic signaling that is observed with heart failure.
  • G s protein is a GTP-binding regulatory protein that effectively couples activation of a variety of cell-surface receptors (including beta-adrenergic receptors) to the activation of adenylylcyclase, as described in the art and herein.
  • Adenylylcyclase (also referred to as “adenylylcyclase,” and abbreviated “AC”) is an enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to 3′:5′-cyclic adenosine monophosphate (cAMP).
  • Adenylylcyclase is known to exist in a number of different isoforms that are found in varying levels in most all mammalian tissues.
  • the most preferred adenylylcyclases of the present invention are “cardiac adenylylcyclases” which are isoforms found to be predominant in mammalian heart tissue, particularly in cardiac myocytes; as described in more detail below.
  • G-protein receptor kinases (abbreviated “GRK”, but also referred to in the art as “beta-adrenergic receptor kinases” or “beta-ARK”), are kinase proteins that catalyze phosphorylation of G-protein-coupled receptor proteins including beta-adrenergic receptors (“beta-ARs”). Phosphorylation of beta-ARs by GRK proteins leads to inactivation of the receptors and a concomitant decrease in responsiveness to beta-adrenergic signaling.
  • GRK G-protein receptor kinases
  • GRK inhibitors refer to peptide inhibitors of the function of G-protein receptor kinases.
  • Peptide inhibitors of GRK include modified GRK proteins in which receptor-binding activity has been uncoupled from kinase activity.
  • Exemplary GRK inhibitors thus include modified GRKs that have been truncated (typically by deletions beginning at the amino-terminus) to remove kinase function while retaining the ability to bind to G-protein-coupled receptor proteins such as beta-ARs.
  • Such truncated GRK proteins can thus effectively compete with or prevent normal GRK from binding to beta-AR but without causing subsequent inhibition of receptor activity.
  • beta-adrenergic signaling proteins including preferred genes encoding beta-ARs, AC isoforms and inhibitors of GRK proteins are known in the art and generally available (see, e.g., the references cited above regarding beta-adrenergic signaling components).
  • beta-adrenergic signaling components tend to be relatively highly conserved, new homologs (or isoforms) of known genes can generally be readily obtained by screening a cDNA or genomic library of interest (e.g., a tissue-specific cDNA library), using techniques that are now quite well known in the art (see, e.g., the molecular biology references cited herein).
  • the most preferred adenylylcyclases of the present invention are “cardiac adenylylcyclases” which are isoforms found to be predominant in mammalian heart tissue, particularly in cardiac myocytes.
  • Presently preferred cardiac ACs include AC isoform V (abbreviated “AC V ”) and AC isoform VI (abbreviated “AC VI ”), with AC VI being presently most preferred for reasons described herein.
  • AC isoforms are distinct in terms of DNA and protein sequence, and are typically expressed in a tissue-specific manner, certain of the isoforms are closely homologous to each other and the mammalian isoforms generally have a common topographical feature comprising transmembrane spanning regions that are associated with large cytoplasmic loops. In addition, the amino acid composition of the cytoplasmic loops tends to be conserved among isoforms.
  • telomeres DNA sequences
  • polymerase chain reaction PCR
  • detection, purification, and characterization of adenylylcyclases including assays for identifying and characterizing new adenylylcyclases effective in a given cell type, have also been described in a number of publications (see, e.g., the references cited herein by Ishikawa et al. and Krupinski et al., regarding AC isoforms).
  • modified or chimeric adenylylcyclases wherein portions of the adenylylcyclase gene are derived from at least two different species.
  • a transmembrane and/or cytoplasmic region of an AC gene from one species, such as murine may be replaced by the corresponding region of an AC gene from a second species, such as human.
  • modified adenylylcyclases are readily constructed using standard methods known to those of skill in the art. Illustrative modified adenylylcyclases are exemplified herein.
  • the gene of interest is transferred to the heart, including cardiac myocytes, in vivo and directs production of the encoded protein. Preferably such production is relatively constitutive.
  • a variety of different gene transfer vectors including viral as well as non-viral systems, can be employed to deliver transgenes for use in the present invention (see, e.g., the references cited above).
  • the helper-independent replication-defective human adenovirus 5 system can be used effectively transfect a large percentage of myocardial cells in vivo by a single intracoronary injection.
  • Such a delivery technique can be used to effectively target vectors to the myocardium of a large mammal heart. Additional means of targeting vectors to particular cells or tissue types are described below and in the art.
  • adenovirus vectors based on the human adenovirus 5 (as described by McGrory W J, et al., Virology 163:614-617, 1988) which are missing essential early genes from the adenovirus genome (usually E1A/E1B), and are therefore unable to replicate unless grown in permissive cell lines that provide the missing gene products in trans.
  • a transgene of interest can be cloned and expressed in tissue/cells infected with the replication-defective adenovirus.
  • adenovirus-based gene transfer does not generally result in stable integration of the transgene into the host genome (less than 0.1% adenovirus-mediated transfections result in transgene incorporation into host DNA)
  • adenovirus vectors can be propagated in high titer and transfect non-replicating cells; and, although the transgene is not passed to daughter cells, this is suitable for gene transfer to adult cardiac myocytes, which do not actively divide.
  • Retrovirus vectors provide stable gene transfer, and high titers are now obtainable via retrovirus pseudotyping (Burns, et al., Proc Natl Acad Sci (USA) 90:8033-8037, 1993), but current retrovirus vectors are generally unable to efficiently transduce nonreplicating cells.
  • An advantage associated with nondividing cells such as myocytes is that the viral vector is not readily “diluted out” by host cell division.
  • various second generation adenovirus vectors that have both E1 and E4 deletions, which can be used in conjunction with cyclophosphamide administration (See, e.g., Dai et al., Proc. Nat'l Acad Sci. (USA) 92:1401-1405, 1995).
  • cyclophosphamide administration See, e.g., Dai et al., Proc. Nat'l Acad Sci. (USA) 92:1401-1405, 1995.
  • multiple infusions, or infusion in an isolated coronary circuit can also be employed.
  • Human 293 cells which are human embryonic kidney cells transformed with adenovirus E1A/E1B genes, typify useful permissive cell lines for the production of such replication-defective vectors.
  • adenovirus E1A/E1B genes typify useful permissive cell lines for the production of such replication-defective vectors.
  • other cell lines which allow replication-defective adenovirus vectors to propagate therein can also be used, such as HeLa cells.
  • vectors include, for example, other viral vectors (such as adeno-associated viruses (AAV), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • viral vectors such as adeno-associated viruses (AAV), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • AAV adeno-associated viruses
  • liposomes and other lipid-containing complexes such as liposomes and other lipid-containing complexes
  • macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell.
  • vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated.
  • a variety of such marker genes have been described, including bifunctional (i.e. positive/negative) markers (see, e.g., Lupton, S., WO 92/08796, published May 29, 1992; and Lupton, S., WO 94/28143, published Dec. 8, 1994).
  • Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts.
  • a large variety of such vectors are known in the art and are generally available (see, e.g., the various references cited above).
  • Additional references describing adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz, M. S., Adenoviridae and Their Replication, in Fields, B., et al. (eds.) Virology, Vol. 2, Raven Press New York, pp. 1679-1721, 1990); Graham, F., et al., pp. 109-128 in Methods in Molecular Biology, Vol. 7: Gene Transfer and Expression Protocols, Murray, E. (ed.), Humana Press, Clifton, N.J.
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996).
  • Additional references describing AAV vectors which could be used in the methods of the present invention include the following: Carter, B., Handbook of Parvoviruses, vol. I, pp. 169-228, 1990; Berns, Virology, pp. 1743-1764 (Raven Press 1990); Carter, B., Curr. Opin. Biotechnol., 3:533-539, 1992; Muzyczka, N., Current Topics in Microbiology and Immunology, 158:92-129, 1992; Flotte, T. R., et al., Am. J. Respir. Cell Mol. Biol. 7:349-356, 1992; Chatterjee et al., Ann. NY Acad.
  • first generation adenovirus vector that can be constructed by the rescue recombination technique as described in McGrory W J, et al., Virology 163:614-617, 1988. Briefly, the transgene of interest is cloned into a shuttle vector that contains a promoter, polylinker and partial flanking adenovirus sequences from which E1A/E1B genes have been deleted.
  • Illustrative shuttle vectors include, e.g., plasmid “pAC1” (Virology 163:614-617, 1988) (or an analog) which encodes portions of the left end of the human adenovirus 5 genome but lacks the early protein region comprising E1A and E1B sequences that are essential for viral replication; and plasmid “ACCMVPLPA” (J Biol Chem 267:25129-25134, 1992) which contains a polylinker, CMV promoter and SV40 polyadenylation signal flanked by partial adenovirus sequences from which the E1A/E1B genes have been deleted.
  • plasmids such as pAC1 or ACCMVPLA can thus facilitate the cloning process.
  • the shuttle vector can then be co-transfected, along with a plasmid comprising the entire human adenovirus 5 genome (but with a length too large to be encapsidated), into suitable host cells such as human 293 cells.
  • Co-transfection can be conducted by calcium phosphate precipitation or lipofection (see, e.g., Biotechniques 15:868-872, 1993).
  • JM17 encodes the entire human adenovirus 5 genome plus portions of the vector pBR322 including the gene for ampicillin resistance (4.3 kb) (Giordano, et al. Nature Medicine 2:534-539, 1996). Although JM17 encodes all of the adenovirus proteins necessary to make mature viral particles, it is too large to be encapsidated (40 kb versus 36 kb for wild type).
  • “rescue recombination” occurs between the transgene-containing shuttle vector (such as plasmid pAC1) and the plasmid having the entire adenovirus 5 genome (such as plasmid pJM17) which generates a recombinant genome that contains the transgene of interest in place of the deleted E1A/E1B sequences, and that secondarily loses the additional sequence (such as pBR322 sequences) during recombination, thereby being small enough to be encapsidated (see, e.g., Giordano, et al. Nature Medicine 2: 534-539, 1996).
  • the transgene-containing shuttle vector such as plasmid pAC1
  • the plasmid having the entire adenovirus 5 genome such as plasmid pJM17
  • FIG. 1 An illustration of such a vector is presented in FIG. 1.
  • the CMV driven beta-galactosidase gene in adenovirus HCMVSP1lacZ (Nature Medicine 2:534-539, 1996) can be used to evaluate the efficiency of gene transfer using X-gal treatment.
  • adenovirus vectors include the ability to effect high efficiency gene transfer (as many as 50% of target organ cells transfected in vivo), the ease of obtaining high titer viral stocks and the ability of these vectors to effect gene transfer into cells such as cardiac myocytes which do not divide.
  • a variety of other vectors suitable for in vivo gene therapy can also be readily employed to deliver beta-ASP transgenes in accordance with the present invention.
  • Such other vectors include, by way of illustration, other viral vectors such as adeno-associated virus (AAV) vectors; non-viral protein-based delivery platforms); as well as lipid-based vectors (including, e.g., cationic liposomes and analogous gene delivery complexes.
  • AAV adeno-associated virus
  • lipid-based vectors including, e.g., cationic liposomes and analogous gene delivery complexes.
  • the present invention contemplates the use of cell targeting not only by delivery of the transgene into the coronary artery, for example, but also by use of targeted vector constructs having features that tend to target gene delivery and/or gene expression to particular host cells or host cell types (such as the myocardium).
  • targeted vector constructs would thus include targeted delivery vectors and/or targeted vectors, as described in more detail below and in the published art.
  • Restricting delivery and/or expression can be beneficial as a means of further focusing the potential effects of gene therapy. The potential usefulness of further restricting delivery/expression depends in large part on the type of vector being used and the method and place of introduction of such vector.
  • Targeted delivery vectors include, for example, vectors (such as viruses, non-viral protein-based vectors and lipid-based vectors) having surface components (such as a member of a ligand-receptor pair, the other half of which is found on a host cell to be targeted) or other features that mediate preferential binding and/or gene delivery to particular host cells or host cell types.
  • vectors such as viruses, non-viral protein-based vectors and lipid-based vectors
  • surface components such as a member of a ligand-receptor pair, the other half of which is found on a host cell to be targeted
  • other features that mediate preferential binding and/or gene delivery to particular host cells or host cell types.
  • a number of vectors of both viral and non-viral origin have inherent properties facilitating such preferential binding and/or have been modified to effect preferential targeting (see, e.g., Miller, N., et al., FASEB Journal 9:190-199, 1995; Chonn,
  • Targeted vectors include vectors (such as viruses, non-viral protein-based vectors and lipid-based vectors) in which delivery results in transgene expression that is relatively limited to particular host cells or host cell types.
  • vectors such as viruses, non-viral protein-based vectors and lipid-based vectors
  • beta-ASP transgenes to be delivered according to the present invention can be operably linked to heterologous tissue-specific promoters thereby restricting expression to cells in that particular tissue.
  • tissue-specific transcriptional control sequences derived from a gene encoding left ventricular myosin light chain-2 (MLC 2V ) or myosin heavy chain (MHC) can be fused to a beta-ASP transgene (such as the AV VI gene) within a vector such as the adenovirus constructs described above. Expression of the transgene can therefore be relatively restricted to ventricular cardiac myocytes.
  • MLC 2V left ventricular myosin light chain-2
  • MHC myosin heavy chain
  • the MLC 2V promoter comprises only about 250 bp, it will fit easily within even size-restricted delivery vectors such as the adenovirus-5 packaging system exemplified herein.
  • the myosin heavy chain promoter known to be a vigorous promoter of transcription, provides another alternative cardiac-specific promoter and comprises less than 300 bp.
  • Recombinant viral vectors such as adenoviral vectors
  • adenoviral vectors can be plaque purified according to standard methods.
  • recombinant adenoviral viral vectors can be propagated in human 293 cells (which provide E1A and E1B functions in trans) to titers in the preferred range of about 10 10 -10 12 viral particles/ml.
  • Adenoviral vectors are exemplified herein but other viral vectors such as AAV can also be employed.
  • AAV adenovirus
  • cells can be infected at about 80% confluence and harvested 48 hours later. After 3 freeze-thaw cycles the cellular debris can be collected by centrifugation and the virus purified by CsCl gradient ultracentrifugation (double CsCl gradient ultracentrifugation is preferred).
  • the viral stocks Prior to in vivo injection, the viral stocks can be desalted by gel filtration through Sepharose columns such as G25 Sephadex. The product can then be filtered through a 30 micron filter, thereby reducing the potential for deleterious effects associated with intracoronary injection of unfiltered virus.
  • the resulting viral stock preferably has a final viral titer that is at least about 10 10 -10 12 viral particles/ml.
  • the recombinant adenovirus is highly purified, and is substantially free of wild-type (potentially replicative) virus.
  • propagation and purification can be conducted to exclude contaminants and wild-type virus by, for example, identifying successful recombinants with PCR using appropriate primers, conducting two rounds of plaque purification, and double CsCl gradient ultracentrifugation.
  • problems associated with cardiac arrhythmias that can be induced by adenovirus vector injections into patients can be essentially avoided by filtration of the recombinant adenovirus through an appropriately-sized filter prior to intracoronary injection. This strategy also appears to substantially improve gene transfer and expression.
  • a vector can be in the form of an injectable preparation containing pharmaceutically acceptable carrier/diluent such as saline, for example.
  • the final titer of the virus in the injectable preparation is preferably in the range of about 10 7 -10 13 viral particles which allows for effective gene transfer.
  • Other pharmaceutical carriers, formulations and dosages are described below.
  • Vectors comprising beta-ASP transgenes can be delivered to the myocardium by direct intracoronary (or graft vessel) injection using standard percutaneous catheter based methods under fluoroscopic guidance, in an amount sufficient for the transgene to be expressed and to provide a therapeutic benefit.
  • Such an injection is preferably made deeply into the lumen (about 1 cm within the arterial lumen) of the coronary arteries (or graft vessel), and preferably is made in both coronary arteries (to provide general distribution to all areas of the heart).
  • porcine model of heart failure that mimics human clinical congestive heart failure in a number of important ways, including the clinical abnormalities associated with beta-adrenergic signaling.
  • sustained rapid ventricular pacing in these large mammals results in left ventricular chamber enlargement, depressed systolic function, and hemodynamic abnormalities, all of which mimic clinical dilated heart failure in humans (see, e.g., Roth D A, et al., J Clin Invest 91:939-949, 1993).
  • beta-adrenergic signaling in the porcine model heart particularly plasma and myocardial catecholamine levels, beta-adrenergic receptor down-regulation and uncoupling, and alterations in adenylylcyclase function, likewise mimic conditions associated with heart failure in humans (Roth D A, et al., J Clin Invest 91:939-949, 1993).
  • the fundamental findings from studies conducted on myocardium from these animal models with heart failure vis-à-vis the current invention include the following. First, there is a 75% reduction in left ventricular beta 1 -adrenergic receptor number, with a similar decrease in beta 1 -adrenergic receptor mRNA; whereas beta 2 -adrenergic receptor number (and mRNA) do not change. This mirrors what is seen in human heart failure (see, e.g., Bristow M R, et al., J Clin Invest 92:2737-2745, 1993). Second, left ventricular beta-adrenergic receptors are uncoupled from Gs, and there is increased function and expression of G-protein receptor kinase.
  • a problem with the assessment of the catalytic subunit of AC is that there are currently only imperfect means to assess either its concentration or its function.
  • the diterpene forskolin is an activator of AC and, accordingly, forskolin-stimulated cAMP production has been the most commonly employed method for assaying its biological activity.
  • full response to forskolin involves interaction of Gs I and AC, and since activation of Gi I inhibits forskolin response (Darfler, F. J., et al., J. Biol. Chem. 257, 11901-11907, 1982), forskolin stimulation falls short of providing a precise measure of AC function. It is widely recognized that AC tends to be quite labile and is generally unstable out of its normal cellular environment.
  • Antibodies to AC are not widely available, and the protein is expressed in low abundance, exacerbating the problems of quantitation and functional assessment of this pivotal transducing element. Because of these problems, little is known about the precise alterations in quantity and function of AC in pathophysiological settings. Regulation of cellular function by AC is further complicated by the recent evidence that multiple isoforms of AC exist, at least two of which have been demonstrated to be expressed in the heart (Ishikawa, Y., et al., J. Clin. Invest. 93, 2224-2229, 1994; Iyengar R. FASEB J. 7, 768-775, 1993; Katsusshika, S., et al., Proc. Natl. Acad. Sci.
  • RNase protection assays are used to provide quantitative assessment of mRNA levels for AC isoforms (Ping P, et al., Circulation 90: I-I-580, 1994; Ping P and Hammond H K, Am J Physiol 267: H2079-H2085, 1994).
  • AC II cardiac myocyte origin
  • PCR was used to detect adenovirus DNA in myocardium from animals that had received gene transfer. Effective gene transfer was documented in the hearts of the pigs, and was not found in other tissues. As further confirmation of the success of these techniques, myocardial samples from lacZ-infected animals were found to exhibit substantial beta-galactosidase activity on histological inspection.
  • Beta-ASP AC
  • beta-adrenergic responsiveness and cardiac function can be increased using such a method.
  • Other preferred beta-ASP can be delivered in an analogous manner.
  • additional types of beta-ASPs including a beta-adrenergic receptor (beta 1 -AR) and a GRK inhibitor.
  • Beta 1 -AR is functionally upstream of AC in the beta-AR-G s -AC pathway, and like AC has been found to be down-regulated in association with heart failure.
  • the enhancement of beta-adrenergic responsiveness according to the present invention is expected to be beneficial for enhancing cardiac function in the numerous disease situations in which heart failure is associated with reductions in beta-adrenergic signaling.
  • it is important to distinguish etiology from putative intervention points, particularly in situations such as this in which a molecular pathway leads from upstream signaling events to downstream effector events, and in which signaling components tend to be decreased (in number or activity) without actually being eliminated (thereby making the dysfunction and potential treatment more quantitative in nature).
  • interventionary treatment such as that described herein can be directed at the principal molecular site of impact or potentially at a downstream site that tends to obviate or “by-pass” the principal limitation.
  • beta-AR beta-adrenergic receptor
  • an effective reduction in the level of a beta-adrenergic receptor may be, and preferably is, treated directly by increasing the level of that same protein; it may also be compensated for indirectly, for example by increasing the activity of the residual beta-AR proteins or relieving inhibition of such proteins (e.g. using GRK inhibitors), by increasing the activity of an analogous beta-AR, and/or by increasing the availability of downstream signal transducers (e.g. AC) to make it more likely that initial signaling events result in downstream stimulation.
  • downstream signal transducers e.g. AC
  • beta-AR number and/or activity are significantly affected in severe heart failure
  • our results involving gene therapy in vivo according to the present invention demonstrate that even intervention to increase a downstream component such as AC can substantially enhance cardiac function.
  • Therapies directed at upstream deficiencies employing, e.g., the delivery of beta-AR and/or GRK inhibitors
  • beta-ASP transgenes would also be expected to provide substantial benefit in terms of beta-adrenergic signaling and cardiac function.
  • a number of different vectors can be employed to deliver the beta-ASP transgene in vivo according to the present invention.
  • the replication-defective recombinant adenovirus vectors exemplified resulted in highly efficient gene transfer in vivo without cytopathic effect or inflammation in the areas of gene expression.
  • compositions or products of the invention may conveniently be provided in the form of formulations suitable for administration into the blood stream (e.g. in an intracoronary artery).
  • a suitable administration format may best be determined by a medical practitioner for each patient individually, according to standard procedures.
  • Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulations treatises, e.g., Remington's Pharmaceuticals Sciences by E. W. Martin. See also Wang, Y. J. and Hanson, M. A. “Parental Formulations of Proteins and Peptides: Stability and Stabilizers,” Journals of Parental Sciences and Technology, Technical Report No. 10, Supp. 42:2S (1988).
  • Vectors of the present invention should preferably be formulated in solution at neutral pH, for example, about pH 6.5 to about pH 8.5, more preferably from about pH 7 to 8, with an excipient to bring the solution to about isotonicity, for example, 4.5% mannitol or 0.9% sodium chloride, pH buffered with art-known buffer solutions, such as sodium phosphate, that are generally regarded as safe, together with an accepted preservative such as metacresol 0.1% to 0.75%, more preferably from 0.15% to 0.4% metacresol.
  • Obtaining a desired isotonicity can be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes.
  • Sodium chloride is preferred particularly for buffers containing sodium ions.
  • solutions of the above compositions can also be prepared to enhance shelf life and stability.
  • Therapeutically useful compositions of the invention can be prepared by mixing the ingredients following generally accepted procedures. For example, the selected components can be mixed to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water and/or a buffer to control pH or an additional solute to control tonicity.
  • compositions can be provided in dosage form containing an amount of a vector of the invention which will be effective in one or multiple doses to induce beta-ASP transgene delivery/expression at a desired level.
  • an effective amount of therapeutic agent will vary with many factors including the age and weight of the patient, the patient's physical condition, and the level of enhancement of cardiac function desired, and other factors.
  • the effective does of the compounds of this invention will typically be in the range of at least about 10 7 viral particles, preferably about 10 9 viral particles, and more preferably about 10 11 viral particles.
  • the number of viral particles may, but preferably does not exceed 10 14 .
  • the exact dose to be administered is determined by the attending clinician, but is preferably in 1 ml phosphate buffered saline.
  • the presently most preferred mode of administration in the case of heart disease is by intracoronary injection to one or both coronary arteries (or to one or more saphenous vein or internal mammary artery grafts or other conduits) using an appropriate coronary catheter.
  • a variety of catheters and delivery routes can be used to achieve intracoronary delivery, as is known in the art.
  • a variety of general purpose catheters, as well as modified catheters, suitable for use in the present invention are available from commercial suppliers such as Advanced Cardiovascular Systems (ACS), Target Therapeutics and Cordis.
  • a catheter can be conveniently introduced into a femoral artery and threaded retrograde through the iliac artery and abdominal aorta and into a coronary artery.
  • a catheter can be first introduced into a brachial or carotid artery and threaded retrograde to a coronary artery.
  • a Konigsberg micromanometer was placed into the left ventricular apex and an epicardial unipolar lead was placed 1.0 cm below the atrioventricular groove in the lateral wall of the left ventricle.
  • the power generator (Spectrax 5985; Medtronic Incorporated, Minneapolis, Minn.) was inserted into a subcutaneous pocket in the abdomen. The pericardium was loosely approximated and the chest closed.
  • Two-dimensional and M-mode images were obtained using a Hewlett Packard Sonos 1500 imaging system. Images were obtained from a right parasternal approach at the mid-papillary muscle level and recorded on VHS tape. Measurements were made using criteria from the American Society of Echocardiography (Sahn D J, et al., Circulation 58:1072-1083, 1978). End-diastolic dimension (EDD) and end-systolic dimension (ESD) were measured on at least 5 beats and averaged. End-diastolic dimension was obtained at the onset of the QRS complex. End-systolic dimension was taken at the instant of maximum lateral position of the interventricular septum, or at the end of the T wave.
  • pigs were anesthetized, and midline stemotomies made.
  • Hearts were removed, rinsed in sterile saline (4 degrees Celsius), and the coronary arteries perfused with sterile saline (4 degrees Celsius).
  • Transmural samples of the left ventricular free wall were taken mid-way from base to apex, near the midportion of the left anterior descending coronary artery. Myocardial samples were then frozen ( ⁇ 80 degrees Celsius). Time from heart removal to placing samples in liquid nitrogen was 5-10 min.
  • Frozen transmural samples ( ⁇ 80 degrees Celsius) were powdered in a stainless steel mortar and pestle (also ⁇ 80 degrees Celsius), placed in Tris buffer, glass-glass homogenized, and contractile proteins extracted (0.5 M KCl, 20 min, 4 degrees Celsius). The pellet of a 45,000 ⁇ g centrifugation was resuspended in buffer. Protein concentrations were determined by the method of Bradford (Bradford M M, Anal. Biochem, 72:248-254, 1976); the protein yield (mg protein/mg wet weight) was assessed in all preparations.
  • beta-ARs were identified using the radioligand [ 125 I]-iodocyanapindolol. Agonist affinity was determined by performing competitive binding assays using ( ⁇ )isoproterenol (Hammond H K, et al., Circulation 8:, 666-679, 1992).
  • Transfer efficiency was recorded by photocopies of membranes dyed with reversible Ponceau staining, and gel retention checked with Coomasie Blue staining. Background blocking was accomplished by incubating membranes in Tris-buffered saline (TBS, pH 7.5) with 2% non-fat dry milk, 2 hours, 25 degrees Celsius.
  • Purified primary polyclonal antibodies (NEN, Boston Mass., rabbit Anti-G-proteins: RM/1 Gs alpha; AS/7, transducin, Gi alpha 1 , Gi alpha 2 ) were diluted 1:600 in 15 ml of TBS with 0.05% Tween-20 (TTBS, pH 7.5) and 1% non-fat dry milk, and membranes incubated for 14 hours, 4 degrees Celsius.
  • Transmural left ventricular samples were placed in a lysis buffer containing 20 mM Tris HCl (pH 7.5), 2 mM EDTA, 10 micrograms/ml benzamidine, 10 micrograms/ml leupeptin, 100 micrograms/ml PMSF and 5 micrograms/ml pepstatin A. Powdered samples were homogenized then centrifuged and resuspended by sonication in lysis buffer. Eighty micrograms of protein from each left ventricular sample was mixed with Laemmli buffer and boiled, then electrophoresed on a 10% denaturing gel. Proteins were transferred to PVDF paper (Immobolin-P, Millipore); transfer efficiency was determined by Ponceau staining.
  • PVDF paper Immobolin-P, Millipore
  • the membrane was blocked for 2 hr in Tris-buffered saline containing 0.1% Tween-20 and 5% non-fat dry milk and developed by conventional methods using GRK 2 or GRK 5 antiserum followed by exposure to horseradish peroxidase-lined anti-rabbit immunoglobulin (1:1000 in TBS).
  • the blots were developed by the ECL method and bands were visualized after exposing blots to X-ray film. Densities of bands co-migrating with purified bovine GRK 2 were quantified by densitometric scanning; for GRK 5 , we quantified the GRK 5 -specific band migrating at approximately (68 kD).
  • GRK enzymatic activity was determined using light-dependent phosphorylation of rhodopsin (Benovic J L, Methods Enzymology, 200:351-363, 1991) as we have previously reported (Ping P, et al., J. Clin Invest, 95:1271-1280, 1995). No GRK subtype-specific activity assay is available so phosphorylation of rhodopsin reflects activity of GRK 2 (beta-ARK 1 ) and GRK 5 , the predominant GRK isoforms in the heart (Inglese J, et al., J Biol Chem, 268:23735-23738, 1993).
  • the pellet was resuspended in 4 ml of lysis buffer with 250 mM NaCl (used to dissociate membrane-associated GRK) and homogenized again in a power-driven glass rotor (4 degrees Celsius).
  • the pellet suspension was then re-centrifuged and ion exchange columns (Amicon) were used to remove NaCl in the supernatant of the pellet suspension.
  • Both the supernatant and the supernatant of the pellet suspension then underwent DEAE-Sephacel column purification to eliminate endogenous kinases that could contaminate GRK-dependent phosphorylation (Ping P, et al., J Clin Invest 95:1271-1280, 1995; Ungerer M, et al., Circulation, 87:454-461, 1993). Both supernatant and pellet fractions were independently column-purified.
  • GRK-dependent phosphorylation was measured by incubating 100 micrograms protein from either fraction with 250 pmol rhodopsin in buffer containing 18 mM Tris HCl, 1.8 mM EDTA, 4.8 mM MgCl 2 , 73 micromolar ATP, and 2.9 cpm/fmol [ 32 P]-ATP.
  • the GRK-dependent phosphorylation reaction was confirmed by adding protein kinase A inhibitor (1 micromolar) and heparin (10 micrograms/ml) into the reaction.
  • Protein concentration for both pellet and supernatant were determined before and after DEAE-Sephacel purification and the final enzyme activity was expressed as pmol phosphate/min/mg of protein as well as per gram of tissue.
  • 45,000 ⁇ g centrifugation (30 min) provides a supernatant which contains less than 1% of the total cellular activity of p-nitrophenylphosphatase (a sarcolemmal membrane-associated enzyme), suggesting excellent separation of cytosolic from membrane components (Roth D A, et al., FEBS Lett, 29:46-50, 1992).
  • the final pellet was washed with 70% ethanol, dissolved in diethylpyrocarbonate-treated water and stored at ⁇ 80 degrees Celsius.
  • the integrity and purity of the RNA were assessed by gel electrophoresis and the ultraviolet absorbance ratio (260 nm/280 nm); the sample was rejected if the ratio was less than 1.5, or if visual inspection of the gel photograph suggested degradation.
  • AC isoforms in porcine left ventricle were isolated by the polymerase chain reaction (PCR). Porcine heart RNA was isolated and reverse-transcribed into cDNA using AMV (avian myeloblastosis virus) reverse transcriptase (Life Science Incorporated, St. Louis, Fla.). Degenerate primers spanning a total of 207 bp of the putative nucleotide binding region of the AC gene family (Krupinski J, et al., J Biol Chem, 267:24858-24862, 1992) were used to amplify the porcine heart cDNA AC genes. The primer sequences used included:
  • RNA DNA derived from 5 micrograms of total RNA was amplified with 4 micromolar PCR primers in 10 mM Tris-HCl and 50 mM KCl reaction buffer (pH 8.3). Taq DNA polymerase (2.5 units) was used (Gibco BRL). The amplification reaction was run for 30 cycles at 95 degrees Celsius (2 min), 52 degrees Celsius (2 min), and 72 degrees Celsius (2 min), followed by extension at 72 degrees Celsius for 10 min.
  • the three sequenced AC isoform plasmids were linearized with either HindIII to generate the control RNA or with EcoR1 to generate the antisense riboprobes for AC types II, V, and VI. In vitro transcription was then performed using either SP6 or T7 polymerase (Promega) to generate the control RNA (214 bp) or antisense riboprobes (219 bp) for subsequent RNase protection assays. Riboprobes synthesized in vitro contain the complementary sequences of both the mRNA and the pGEM 4Z vector, and are therefore longer than the protected band fragment from the porcine RNA sample (mRNA only). The protected band from porcine heart is sized at 207 bp. The longer length of the control RNA, also derived from the extra sequence from the pGEM 4Z vector, protects the in vitro synthesized control mRNA from contamination by sample RNA.
  • RNA (20 micrograms) from tissue and various amounts of in vitro synthesized sense strand control RNA were hybridized with 2-8 ⁇ 10 4 cpm probe (in 5-8 fold excess of mRNA as determined in preliminary experiments) in 20 microliter of 80% formamide, 40 mM Hepes (pH 7.6), 400 mM NaCl, and 1.0 mM EDTA for 12-16 hours (45 degrees Celsius).
  • Digestion buffer 300 microliter
  • RNAse resistant hybrids were precipitated and run on a 6% polyacrylamide urea gel.
  • the AC II , AC V , and AC VI mRNA signals were quantitated by counting the excised gel band with a beta-counter. After counting, the cpm of control RNA could be expressed as cpm/micrograms of control RNA. These data then were used to quantitate mRNA levels in myocardial tissue (pmol specific mRNA/g total RNA).
  • the cardiac content of AC II , AC V , and AC VI mRNA were calculated from the ratio of their signal to the signal from their sense strand control RNA in the same hybridization reaction.
  • a mammalian 18S riboprobe 400 cpm; plasmid construct from Ambion was used together with AC II , AC V , and AC VI riboprobes in the hybridizations to assess the loading and hybridization conditions for each tissue sample.
  • the high yield and very low specific activity of the 18S riboprobe (5-8 ⁇ 10 4 cpm/micrograms; Megascript, Ambion) was obtained to assure accurate measurement of the 18S transcript from 20 micrograms of total RNA.
  • RNase resistance ( ⁇ 1%) and riboprobe specificity were confirmed by complete digestion of single stranded antisense riboprobe plus 40 micrograms of transfer RNA (yeast) with RNase A and T1.
  • a mouse GRK 2 (beta-ARK 1 ) cDNA fragment provided by Dr. P. A. Drei was used to assess GRK 2 mRNA content in left ventricular samples. Twenty micrograms of total RNA was gel denatured and blotted onto nylon membranes. The Northern blot was hybridized with a [ 32 P]-labeled random primer GRK 2 cDNA fragment (bp 1134-1688) for 24 h at 42 degrees Celsius in a buffer containing 5 ⁇ SSPE, 10 ⁇ Denhardt's solution, 100 micrograms/ml salmon sperm DNA, 50% formamide, and 2% SDS. The blot was washed with 2 ⁇ SSC/0.1% SDS at 27 degrees Celsius for 15 min.
  • a human GRK 5 cDNA probe (Marzo K P, et al., Circ Res, 69:1546-1556, 1991) provided by Dr. J. Benovic was used to assess GRK 5 mRNA content in left ventricular samples. Twenty micrograms of total RNA was gel denatured and blotted onto nylon membrane. The Northern blot was hybridized with a [ 32 P]-labeled random primer GRK 5 cDNA fragment (bp 383-1540) for 24 h at 42 degrees Celsius in a buffer containing 5 ⁇ SSPE, 10 ⁇ Denhardt's solution, 100 micrograms/ml salmon sperm DNA, 50% formamide, and 2% SDS. The blot was washed with 2 ⁇ SSC/0.1% SDS at 27 degrees Celsius for 30 min, followed by a high stringency wash with 0.12 ⁇ SSC/0.1% SDS at 42 degrees Celsius for 30 min.
  • beta-adrenergic receptor The transduction pathway by which a hormone or neurotransmitter interacting with a beta-adrenergic receptor on the cell surface alters intracellular behavior is known as the beta-adrenergic-Gs-adenylylcyclase (or “beta-AR:Gs:AC”) pathway as shown in FIG. 2. It is through this pathway that beta-adrenergic stimulation increases intracellular cAMP thereby influencing heart rate responsiveness and force of contraction.
  • the pathway includes three principle components: the beta-adrenergic receptor (beta-AR), the stimulatory GTP-binding protein (Gs) and adenylylcyclase (AC).
  • beta-AR beta-adrenergic receptor
  • Gs stimulatory GTP-binding protein
  • AC adenylylcyclase
  • Activated AC then catalyzes the synthesis of cyclic AMP, and increased intracellular concentrations of cAMP mediate increased cytosolic calcium transients which enhance both the rate and force of cardiac contraction (referred to as positive “chronotrophy” and positive “inotrophy,” respectively.
  • beta-AR:Gs:AC pathway as shown in FIG. 2
  • FIG. 2 We used a large animal model predictive of humans to examine the possibility that one or more components of the beta-AR:Gs:AC pathway (as shown in FIG. 2) might effectively limit transmembrane beta-adrenergic signaling in cardiac myocytes, and that elevating expression of one or more of the proteins in the pathway might lead to enhanced responsiveness to endogenous beta-adrenergic signaling.
  • beta-ASPs we have initially focused attention on AC, beta-AR, and GRK inhibitors (which indirectly enhance beta-AR activity), as described in more detail below.
  • Post hoc testing was performed using Student's t-test with the Bonferroni correction: a p ⁇ 0.05; b p ⁇ 0.01; c p ⁇ 0.001 (vs control); d p ⁇ 0.05; e p ⁇ 0.01; f p ⁇ 0.001 (4d vs 28d).
  • liver to body weight ratios were increased two-fold (p ⁇ 0.0001) and left ventricle to body weight ratios were unchanged, as previously reported (Roth D A, et al., J. Clin Invest, 91:939-949, 1993).
  • RT-PCR was used to amplify a DNA fragment corresponding to the expected size of AC from cardiac myocyte samples obtained from adult pigs. The DNA fragment was absent when reverse transcriptase was omitted from the reaction.
  • beta-AR and GRK Inhibitors as beta-Adrenergic Signaling Proteins in a Large Animal Model of Congestive Heart Failure
  • LV total GRK 2 protein content was unchanged by mild heart failure (4d pacing) but tended to decrease in LV from animals with severe heart failure (Control: 1.37 ⁇ 0.17 du/microgram; 4d: 1.58 ⁇ 0.34 du/microgram; 28d: 1.02 ⁇ 0.07 du/microgram; p ⁇ 0.02 by ANOVA).
  • post hoc analysis revealed that the only group mean comparisons that were statistically significant was 4d vs 28d (p ⁇ 0.03).
  • GRK 2 concentration was reduced in the supernatant (p ⁇ 0.04), but not the pellet fraction of the LV homogenate in severe heart failure.
  • a band migrating at 68 kD, identifying GRK 5 was detected using the GRK 5 antibody.
  • Beta-AR and GRK Inhibitors as beta-Adrenergic Signaling Proteins (beta-ASPs)
  • results obtained in our large animal model of heart failure confirmed that reduced beta-AR responsiveness in heart failure is associated with selective down-regulation of myocardial beta 1 -AR and beta 1 -AR mRNA levels; beta 2 -AR expression and mRNA content do not change (see, e.g., Bristow M R et al., J Clin Invest 92:2737-2745, 1993; Ping P, et al., Am J Physiol, 267:H2079-H2085, 1994; Ungerer M, et al., Circulation, 87:454-461, 1993); and that remaining beta-ARs are uncoupled from Gs (the stimulatory GTP-binding protein which links receptor activation with AC stimulation), as reflected by a reduction in high affinity agonist binding (see, e.g., Bristow M R, et al., Mol Pharm, 35:295-303, 1989; Bristow M R, et al., J Clin Invest, 92
  • GRK primarily studied in cells with beta 2 -ARs, phosphorylates the beta 2 -AR after adrenergic activation (Hausdorff W P, et al., FASEB J, 4:2881-2889, 1990; Inglese J, et al., J Biol Chem, 268:23735-23738, 1993), and may play a role in beta-AR desensitization in the setting of sustained sympathetic activation, as occurs in CHF.
  • GRK 2 translocates from cytosol to sarcolemma, phosphorylates the beta 2 -AR, and thereby uncouples the beta 2 -AR and Gs, thus attenuating the signal (Hausdorff W P, et al., FASEB J, 4:2881-2889, 1990; Inglese J, et al., J Biol Chem, 268:23735-23738, 1993).
  • a second new finding is that the increase in total GRK activity was an early event in heart failure, occurring prior to alterations in beta-AR number, G-protein content, or AC isoform expression or catalyst activity.
  • increased GRK activity may serve to phosphorylate and uncouple Gs and the beta-AR.
  • total GRK 5 protein and mRNA content were increased, also at this early time point, and these elevations persisted in severe heart failure.
  • beta-adrenergic signaling protein we initially selected an adenylylcyclase protein, in particular adenylylcyclase isoform VI (AC VI ).
  • AC VI adenylylcyclase isoform VI
  • lacZ an illustrative detectable marker gene which encodes beta-galactosidase.
  • the system used to generate recombinant adenoviruses imposes packaging constraints that are believed to increase as the size of the transgene insert exceeds about 5 kb.
  • transgene itself i.e. without additional control elements
  • the transgene itself would therefore preferably be less than about 4 kb.
  • smaller transgenes are therefore preferred, we have also shown that substantially larger transgenes can nevertheless be employed, even in these “first generation” vectors.
  • a murine adenylylcyclase gene approximately 5748 bp
  • a heterologous CMV promoter approximately 790 bp
  • an SV40 polyadenylation signal approximately 230 bp
  • beta-ASP transgene in view of the known packaging constraints of these particular vectors, and as another exemplary beta-ASP transgene, we constructed an altered adenylylcyclase gene in which an untranslated region of the transcript was deleted to generate a shorter beta-ASP transgene.
  • the 3′-untranslated region from the AC VI gene was removed, and the resulting construct was incorporated into a vector for delivery of the transgene to the heart, as described herein.
  • the desirability of truncating the transgene depends in part on the length of the native gene, as well as the choice of control elements used.
  • the transgene can be truncated, preferably in the 3′-untranslated region, to result in a shorter insert.
  • the preferred extent of truncation depends on the insert size, but is typically at least about 100 bp, more preferably at least about 500 bp, still more preferably at least about 1000 bp (particularly if the total insert size is still greater than about 5 kb).
  • First generation AAV vectors are also size constrained and efficiency decreases with inserts significantly greater than about 5 kb.
  • “second generation” derivatives can provide additional space for transgene packaging.
  • Other viral vectors, as well as various non-viral vectors can be used to accommodate substantially larger inserts.
  • Example 5-3 describes the identification of sequences encoding a human adenylylcyclase gene.
  • human beta-ASP transgenes can be obtained by screening of human DNA libraries (using probes from homologous mammalian genes), and such human beta-ASP transgenes can be usefully employed in the context of the present invention.
  • the therapeutic target is a human heart
  • it is expected that the use of human beta-ASP transgenes can provide additional advantages (including potentially closer coordination with other components of the beta-adrenergic signaling pathway, as well as further minimizing the possibility of a host response to non-human proteins).
  • the isolation and sequencing of an exemplary human beta-ASP transgene is described below in Example 5-3.
  • beta-ASP transgenes including genes encoding beta 1 -adrenergic receptors (beta 1 -AR) and GRK inhibitors are described in Examples 6 and 7.
  • pAC-V a Ca(2+)-inhibitable AC from murine NCB-20 cells as reported by Yoshimura M and Cooper D M, Proc Natl Acad Sci (USA) 89:6716-6720, 1992, referred to in the paper as “pAC-V”, now known as “pAC-VI”; see also Krupinski, J., et al., J. Biol. Chem. 267:24858-24862, 1992).
  • the full length AC VI cDNA was cloned into the polylinker of plasmid ACCMVPLPA (J Biol Chem 267:25129-25134, 1992) which contains the CMV promoter and the SV40 polyadenylation signal flanked by partial adenovirus sequences from which the E1A and E1B genes, which are essential for viral replication, had been deleted.
  • the resulting plasmid was co-transfected (by lipofection) into human 293 cells with plasmid JM17 (Giordano, et al. Nature Medicine 2:534-539, 1996) which contains the entire human adenovirus 5 genome as well as an additional 4.3 kb insert (thereby making pJM17 too large to be encapsidated).
  • Transfected human 293 cells were monitored for evidence of cytopathic effect which usually occurred 10-14 days after transfection.
  • cell supernatant from plates showing a cytopathic effect was treated with proteinase K (50 mg/ml with 0.5% sodium dodecyl sulfate and 20 mM EDTA) at 56 degrees Celsius for 60 minutes, followed by phenol/chloroform extraction and ethanol precipitation.
  • cells were infected at 80% confluence and harvested at 36-48 hours; and, after freeze-thaw cycles, the cellular debris was collected by standard centrifugation and the virus further purified by double CsCl gradient ultracentrifugation (discontinuous 1.33/1.45 CsCl gradient; cesium prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); 90,000 ⁇ g (2 hr), 105,000 ⁇ g (18 hr)).
  • the viral stocks Prior to in vivo injection, the viral stocks were typically desalted by gel filtration through Sepharose columns such as G25 Sephadex. The resulting viral stock had a final viral titer in the 10 10 -10 12 viral particles range. The viral preparation was found to be highly purified, with essentially no replicative virus present (as determined by an absence of cytopathic effect after transfection of the vector into human host 293 cells).
  • beta-ASP transgene As another illustrative beta-ASP transgene, we constructed an altered adenylylcyclase gene in which an untranslated region of the normal transcript was removed to generate a shorter beta-ASP transgene. In particular, we essentially removed the 3′-untranslated region from an AC VI construct, and incorporated the resulting truncated transgene into a viral vector for gene delivery.
  • a murine AC VI cDNA without the 3′-untranslated region was constructed as follows. A subfragment containing the 3′ portion of AC VI with an Xho I site at its 5′ end was generated using two PCR primers: AC VI PX, which anneals to AC VI cDNA at bases 2500-2521; and AC VI p3′HA which contains sequences from the 3′ end of AC VI and sequences from human influenza hemoagglutinin. The PCR product was digested with restriction enzymes Xho I and Xba I which were designed in the primers at the 3′ end.
  • the 5′ portion of the AC VI fragment (base pair ⁇ 92 to +2500) was obtained by Eco RI and Xho I digestion of the full length cDNA (as described above) and was isolated on an agarose gel. Two AC VI fragments were then subcloned into an Eco RI-Xba I digested adenovirus vector (pAd5CI, a gift of Dr. Swang Huang, The Scripps Research Institute, La Jolla, Calif.) by three molecule ligation.
  • pAd5CI Eco RI-Xba I digested adenovirus vector
  • the pAd5/CI vector contains a cytomegalovirus immediate-early enhancer/promoter region (CMV promoter), a chimeric intron, and a multicloning site derived from pCI plasmid DNA (Promega, Madison, Wis.). It also contains a bovine growth hormone polyadenylation (poly A) sequence and partial human adenovirus-5 sequences.
  • CMV promoter cytomegalovirus immediate-early enhancer/promoter region
  • poly A bovine growth hormone polyadenylation
  • AC VI Ad-Adenylylcyclase VI
  • CMVp Cytomegalovirus immediate-early enhancer/promoter region
  • HA-tag Hemagglutinin protein of influenza virus-tag
  • BGH-poly A Bovine growth hormone-poly A
  • Adv5 Addenovirus 5
  • the AC VI -containing vector was cotransfected (calcium phosphate) into a human embryonal kidney cell line (H293) with pJM17 which contains the adenovirus genome except the E1 region. After recombination, plaques were selected and expanded in H293 cells. H293 cells have been transformed with adenovirus E1, and therefore provide this viral transcription factor in trans. Expression of AC VI was examined by RT-PCR and Western blot analysis. Virus was purified by cesium chloride ultracentrifugation and desalted by column filtration through Sephadex G-25 equilibrated with PBS as described above. The viral concentration was determined by optical densitometry at OD 260 . Plaque-forming units (pfu) were assayed by plaque titration using H293 cells overlaid with agarose-DMEM medium.
  • AC VI Expressing Clones To identify adenovirus clones that expressed AC VI , sixteen clones were screened by RT-PCR using a pair of specific primers (AC VI PX and AC VI p340 HA) which hybridize to transgene AC VI but not endogenous AC VI in H293 cells. The 512-bp RT-PCR product was confirmed by digestion with the restriction enzyme Apa I to produce 312-bp and 200-bp fragments. Three of sixteen clones expressed AC VI mRNA.
  • beta-ASP genes exist in many mammalian tissues, with different isoforms typically predominating in certain tissue types. In the case of adenylycyclases, as noted above, there tends to be a predominance of isoforms II, V and VI in cardiac tissues. Such genes also tend to exhibit a fairly significant degree of sequence conservation between different mammals, particularly with respect to the isoforms found in a specific tissue such as the heart. DNA hybridization and associated molecular biological techniques can thus be employed to identify additional beta-ASP transgenes for use in the context of the present invention.
  • clones 1, 4 and 5 Three of these clones (designated clones 1, 4 and 5) were sub-cloned into a vector for sequencing.
  • the “Bluescript” vector pBS-SK commercially available from Stratagene.
  • the first round of sequencing was carried out using T3 and T7 primers, and then internal primers were employed for subsequent sequencing. All three of the clones contained sequences that were highly homologous to AC VI genes of other species including the mouse.
  • These clones, and sub-fragments thereof, were used to identify overlapping clones containing the remaining sequence. From the overlapping clones we obtained the nucleotide sequences (SEQ ID NOS: 1 and 3), which correspond to more than 2 kb of the presumed 3.4 kb coding sequence of human AC VI .
  • SEQ ID NOS: 2 and 4 depict the amino acid sequences corresponding to the nucleotide sequences shown in SEQ ID NOS: 1 and 3, respectively. From the sequence information provided in SEQ ID NO: 1 or 3, the complete nucleotide sequence encoding the full length human AC VI and variants thereof can be readily obtained using standard recombinant DNA methodology. The complete nucleotide sequence of human AC VI as obtained is shown in SEQ ID NO: 5. The corresponding amino acid sequence is depicted in SEQ ID NO: 6.
  • Polynucleotides comprising closely related sequences can likewise be obtained, using techniques such as hybridization, as is known in the art. Such sequences would include, for example, those exhibiting at least about 80% overall sequence identity, preferably at least 90%, even more preferably at least 95% sequence identity with a nucleotide sequence comprising that shown in SEQ ID NO. 1 or 3 or 5. Isolated polynucleotides that hybridize at high stringency to a polynucleotide having the nucleotide sequence of SEQ ID NO. 1 or 3 or 5 can thus be readily obtained based on standard molecular biological techniques. These polynucleotides can also be used to obtain isolated polypeptides encoded by the polynucleotides.
  • an “isolated polypeptide” or protein is a polypeptide or protein which has been substantially separated from any cellular contaminants and components naturally associated with the protein in vivo.
  • the phrase embraces a polypeptide which has been removed from its naturally occurring environment, and includes recombinant polypeptide and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • An “isolated polynucleotide” is similarly defined.
  • the variants will include allelic variants.
  • An “allelic variant” in the context of a nucleic acid or a gene is an alternative form (allele) of a gene that exists in more than one form in the population.
  • allelic variants generally differ from one another by only one, or at most, a few amino acid substitutions.
  • the amino acid residue positions which are not identical in the variant differ by conservative amino acid substitutions.
  • Constant amino acid substitutions refers to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • the ACVI polynucleotides encoding variants can also comprise silent nucleotide subtitutions.
  • silent nucleotide subtitution is meant that the substituted nucleotide does not result in an amino acid change at the protein level. Even more substantial nucleotide changes can be introduced into regions that do not affect the enzymatic activity of the encoded adenycyclase polypeptide.
  • Many such polynucleotides will encode polypeptides that maintain adenycyclase enzymatic activity, which can be tested by routine methods as known in the art. “High stringency” conditions for polynucleotide hybridization are described, e.g., in J.
  • small fragments of the human AC VI polynucleotide sequence can be used as primers or probes to identify and isolate isoforms or variants of the native polypeptides.
  • Isolated polypeptides encoded by the polynucleotides of the preceding embodiments include, for example, polypeptides comprising a sequence in which at least about 300 amino acid residues is at least 80% (preferably 90%, even more preferably 95%, most preferably greater than 98%) identical with a sequence of comparable length within SEQ ID NO. 2 or 4 or 6.
  • a fourth beta-ASP transgene encoding a human AC VI polypeptide was obtained from RNA preparations derived from human cardiac tissues; the complete nucleotide and corresponding amino acid sequences of which are provided in SEQ ID NOS. 10 and 11, respectively.
  • DNA sequence can be incorporated into gene delivery vectors as described herein, for example in Example 5-1, and can be introduced to the myocardium to enhance beta-adrenergic signaling.
  • the resulting vector comprising this polynucleotide can then be incorporated into a method for treating congestive heart failure, as described in this and in predecessor applications.
  • beta-ASP transgene As yet another illustrative beta-ASP transgene, we constructed a modified AC VI gene in which the transmembrane-encoding portions of the AC VI gene of Example 5-1 were replaced with the corresponding regions of the AC VI gene of Example 5-4 (SEQ ID NO. 10).
  • the 5′ coding region of the AC VI gene of Example 5-4 up to about amino acid 400, and the 3′ coding region of that same gene, from about amino acid 630, were ligated into (and as replacement for) corresponding regions of the AC VI sequence of Example 5-1.
  • the cDNA and amino acid sequences of the resulting modified AC VI are provided in SEQ ID NOS: 12 and 13, respectively.
  • the modified AC VI polypeptide from an expression plasmid was confirmed and AC activity was demonstrated in a standard cAMP assay (Example 1-6 above).
  • the modified AC VI cDNA sequence was incorporated into an adenoviral gene delivery vector.
  • Such vector may be introduced into the myocardium of a patient, for example suffering from congestive failure, to enhance beta-adrenergic signaling in accordance with this and predecessor applications.
  • AC VI genes may likewise be made to AC VI genes and are equally contemplated herein.
  • silent nucleotide changes can be made to an AC VI gene, for example to increase expression efficiency while leaving unaltered the amino acid sequence and/or polypeptide structure.
  • beta-Adrenergic Receptor Protein (beta 1 -AR) as a Second Type of beta-ASP
  • beta-adrenergic signaling proteins for use in the present invention include beta-adrenergic receptor proteins, particularly beta 1 -adrenergic receptors (beta 1 -AR), and GRK inhibitors (which can indirectly enhance beta-AR activity as described above).
  • beta 1 -adrenergic receptors beta 1 -AR
  • GRK inhibitors which can indirectly enhance beta-AR activity as described above.
  • Gene delivery vectors comprising transgenes encoding such additional beta-ASPs can be readily generated using techniques such as those described in the preceding example.
  • beta 1 -AR cDNA fragment (which had been cloned into the EcoRI site of pSP65) was inserted into an E1 deleted recombinant human adenovirus-5 vector using the techniques described in the preceding example, thereby generating a recombinant vector for the delivery of a gene encoding a second preferred beta-ASP (i.e. a beta-adrenergic receptor protein).
  • a second preferred beta-ASP i.e. a beta-adrenergic receptor protein
  • beta-adrenergic signaling protein for use in the present invention is a G-protein receptor kinase inhibitor (GRK inhibitor), which can be used to indirectly enhance beta-AR activity and therefore beta-adrenergic responsiveness.
  • GPK inhibitor G-protein receptor kinase inhibitor
  • Gene delivery vectors comprising transgenes encoding such a beta-ASP can be readily generated using techniques such as those described in the preceding examples.
  • GRK inhibitors can be readily prepared.
  • a GRK inhibitor can be constructed as described for the “beta-ARK1-minigene” (Koch, et al., Science 268: 1350-1353, 1995), or an analogous construct (in which a GRK is mutated to effectively delete or impair kinase function without disrupting receptor binding activity) can be used.
  • the DNA fragment encoding the GRK inhibitor can be inserted into an E1-deleted recombinant human adenovirus-5 vector using the techniques described in the preceding examples, or using another vector as known in the art.
  • Beta-ASP gene transfer vectors can initially be tested by examining the ability of the vectors to deliver beta-adrenergic signaling proteins to ventricular cells maintained in cell culture. Such cell culture studies can thus be useful in screening putative gene transfer vectors (having, e.g., particular combinations of beta-ASP transgenes and promoters) for the ability to deliver expressible transgenes to cells of particular types, such as exemplified herein.
  • the first round of such screening can be conveniently accomplished using a standard detectable marker gene (such as lacZ) so that gene delivery and gene expression can be readily and rapidly quantified, as illustrated below.
  • a standard detectable marker gene such as lacZ
  • Vectors that effectively deliver and cause expression of the first round “test” transgene can then be subjected to a second screening round using a beta-ASP according to the present invention.
  • neonatal rat ventricular myocytes were prepared with a collagenase-containing perfusate according to standard methods.
  • Rod-shaped cells were cultured on laminin-coated plates and at 48 hours were infected with a vector comprising a detectable marker gene (viz. an adenovirus vector comprising a lacZ gene, as described in the examples above) at a multiplicity of infection of 1:1 (plaque forming units: cell). After a further 36 hour period, the cells were fixed with glutaraldehyde and incubated with X-gal.
  • vectors as described herein, including other vectors (e.g. other viral vectors such as AAV as well as non-viral vectors including lipid-based vectors and various non-viral delivery platforms), vectors in which transgenes are linked to different transcriptional control sequences (such as ventricular-specific promoters), as well as vectors encoding other beta-ASP transgenes, as described and illustrated herein.
  • other vectors e.g. other viral vectors such as AAV as well as non-viral vectors including lipid-based vectors and various non-viral delivery platforms
  • transgenes are linked to different transcriptional control sequences (such as ventricular-specific promoters)
  • vectors encoding other beta-ASP transgenes as described and illustrated herein.
  • Cardiac Myocyte Preparation and Gene Transfer Hearts from 1 to 2 day old Sprague-Dawley rats were removed, atria and great vessels discarded, and ventricles trisected. Myocardium was digested with collagenase II (Worthington) and pancreatin (GibcoBRL Life Technology, Gaithersburg, Md.), and the myocardial cell suspension was centrifuged through Percoll step gradients to separate cardiac myocytes from other cells. Cells then were plated (4 ⁇ 10 4 cells/cm 2 ) in plates precoated with gelatin, and incubated for 24 h.
  • Adenovirus-mediated gene transfer was performed 3d after initial isolation by adding recombinant adenovirus expressing AC VI or lacZ (10 pfu/cell), and incubating for 20 h in DMEM containing 2% fetal bovine serum. Adenovirus was removed and the cells were maintained for 24 h and then used for study. The extent of gene transfer was evaluated by X-gal staining of cells after gene transfer with lacZ.
  • RT-PCR was used to identify AC VI mRNA.
  • the reverse transcription reaction was performed (SuperScript II, GibcoBRL Life Technology). Briefly, 1 microgram of mRNA was mixed with 100 ng of the primer AC VI 3′pHA in 11 microliter, heated (70 degrees Celsius, 10 m) and quickly chilled on ice. Four microliters of 5 ⁇ first strain buffer, 2 microliter of 0.1 M DTT, and 1 microliter of 10 mM dNTP were added and the reaction mixture allowed to equilibrate (37 degrees Celsius, 2 m). Finally, 1 microliter (200 units) of SuperScript II Rnase H reverse transcriptase was added; reaction duration was 1 h (37 degrees Celsius).
  • Hybridization was carried out in Hood buffer (50% formamide, 5 ⁇ SSC, 20 mM NaHPO4, pH6.7, 7% SDS, 1% PEG 15,000-20,000, and 0.5% non-fat milk) at 42 degrees Celsius for 16 h.
  • the membrane was washed once with 2 ⁇ SSC-0.5% SDS for 30 m at RT, and 2-3 times with 0.1 ⁇ SSC-0.1% SDS for 30 m each (60-65 degrees Celsius) and exposed to X-ray film.
  • membranes were incubated with anti-ACV/VI antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) diluted 1:100 in blocking buffer for 1 h (RT).
  • anti-ACV/VI antibody Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.
  • Gs alpha and Gi alpha 2 membranes were incubated with anti-Gs alpha or anti-Gi alpha 2 antibodies as previously described (as in Ping et al., J. Clin. Invest. 95:1271-1280, 1995).
  • Primary antibodies were detected with goat anti-rabbit IgG horseradish peroxidase conjugate (GIBCO BRL Life Technology) in blocking buffer. The antigen then was visualized with chemiluminescent substrates A and B (Kirkegaard and Perry Laboratories, Gaithersburg, Md.) and exposed to X-ray film.
  • Binding assays were initiated by the addition of saponin (20 micrograms/ml final), 20 nM [ 3 H]forskolin, 1 micromolar 1,9-dideoxy-forskolin (to reduce association of radiolabel with non-adenylylcyclase molecules) and the additions indicated in the figure legends.
  • Cells were incubated in a final volume of 0.5 ml for 15 min at 25 degrees Celsius. Reactions were terminated by aspiration of media and cells were washed twice with ice-cold washing buffer (50 mM Tris, 10 mM MgCl 2 , pH 7.4).
  • the amount of [ 3 H]forskolin associated with cells was determined by extraction of cells in 0.2% Triton X-100 and scintillation counting of the soluble cell extract.
  • Cyclic AMP Measurements Prior to treatment of cells, growth medium was removed and cells were equilibrated for 30 m (RT) in serum- and sodium bicarbonate-free DMEM supplied with 20 mM HEPES pH 7.2. Subsequently, cells were incubated for 10 m (RT) in fresh DMEM containing either 10 micromolar isoproterenol, or 10 micromolar forskolin in the presence of 0.1 mM ascorbic acid (to prevent oxidization) and 250 micromolar IBMX, a phosphodiesterase inhibitor. The reaction was terminated by aspiration of medium and addition of 7.5% ice-cold trichloroacetic acid (TCA).
  • TCA 7.5% ice-cold trichloroacetic acid
  • TCA extracts were frozen ( ⁇ 20 degrees Celsius) until assayed.
  • Intracellular cAMP levels were determined by radioimmunoassay (Calbiochem, San Diego, Calif.) of TCA extracts following acetylation according to the protocol provided by the manufacturer. The sensitivity of this assay allowed for large dilution of TCA extracts such that ether extraction of TCA was unnecessary. Production of cAMP was normalized to the amount of acid-insoluble protein assayed by the BioRad protein assay.
  • FIG. 8C displays three measures of altered adrenergic signaling (forskolin binding, and isoproterenol- and forskolin-stimulated cAMP production). These data indicate that a proportional increase in AC content and enhanced adrenergic signaling have occurred.
  • FIG. 9 shows cAMP production when cardiac myocytes were stimulated by a range of isoproterenol concentrations. After gene transfer with AC VI (vs lacZ), there was an obvious increase in cAMP produced through a wide range of isoproterenol concentrations. The EC50 for isoproterenol-stimulated cAMP production was unchanged (lacZ: 16 ⁇ 13 nM; AC VI : 32 ⁇ 19 nM).
  • beta-Adrenergic Receptor Binding Studies were identified in radioligand binding experiments using [ 125 I]-iodocyanopindolol (ICYP; 30 240 pM); 10 ⁇ 4 M isoproterenol was used to define nonspecific binding.
  • Transfected cells (lacZ vs AC VI ) were lysed and membranes prepared for radioligand binding (as in Ping et al., J. Clin. Invest. 95: 1271-1280, 1995); experiments were performed with triplicate samples. Data are reported as specifically bound ICYP (fmol/mg).
  • Gs alpha and Gi alpha 2 Content To determine whether increased AC content affected G protein content, we performed immunoblotting studies with antibodies directed against Gs alpha and Gi alpha 2 . These assays identified similar amounts of Gs alpha and Gi alpha 2 . These data indicate that the content of Gs alpha and Gi alpha 2 were not changed by gene transfer of AC VI .
  • beta-ASP transgenes including variant transgenes in which untranslated regions have been altered
  • beta-ASP transgenes can be readily constructed and used to deliver beta-ASP gene products to cardiac cells, and further confirm that such transgenes can be used to alter the functional responsiveness of the cells.
  • the altered beta-ASP transgene resulted in an approximately 4-fold increase in forskolin-stimulated cAMP production, and an approximately 9-fold increase in isoproterenol-stimulated cAMP production.
  • the ability to employ genes exhibiting differing levels of expression thus provides an added tool that can be used to optimize the present invention in the context of varying therapeutic needs (depending on the desired level of expression in the cells and tissue to be treated).
  • the observed increases in effective expression levels using the altered transgene may be due to an increase in vector packaging efficiency and/or an increase in message stability.
  • the substantially reduced size of the transgene construct may allow it to be packaged more efficiently in the viral vector used in these experiments.
  • Our results also suggest that the resulting mRNA was more abundant in transfected cells (approximately 25-fold higher as compared to the parental-type construct), which may be the result of an increase in message stability.
  • a potential mRNA destabilizing element depicted in SEQ ID NO: 9 (UUAUUUA(UA)(UA)) in the 3′-untranslated region of the original ACVI construct. Removal of such message destabilizing elements may thus enhance the effective expression of other beta-ASP transgenes having such elements.
  • an adenoviral vector was propagated in permissive human 293 cells and purified by CsCl gradient ultracentrifugation (with a final viral titer of 1.5 ⁇ 10 10 viral particles), based on the procedures of Example 5.
  • this in vivo procedure for monitoring delivery and expression of transgenes in the porcine heart failure model can also be readily employed to test other in vivo gene delivery vectors according to the present invention, including, for example, other in vivo delivery vectors (e.g. other viral vectors such as AAV as well as non-viral vectors including lipid-based vectors and various non-viral delivery platforms), vectors in which transgenes are linked to different transcriptional control sequences (such as ventricular-specific promoters), as well as vectors encoding other beta-ASP transgenes, as described and illustrated herein.
  • other in vivo delivery vectors e.g. other viral vectors such as AAV as well as non-viral vectors including lipid-based vectors and various non-viral delivery platforms
  • vectors in which transgenes are linked to different transcriptional control sequences such as ventricular-specific promoters
  • vectors encoding other beta-ASP transgenes as described and illustrated herein.
  • vectors including various viral vectors (such as adeno-associated virus (AAV)), liposomes and other lipid-containing gene delivery complexes, and other macromolecular complexes (such as multifunctional gene delivery fusion proteins) that are capable of mediating delivery of a polynucleotide to a mammalian host cell in vivo can be used to deliver beta-ASP transgenes to the myocardium in accordance with present invention.
  • AAV can be used to deliver one or more beta-ASP transgenes to the myocardium in vivo.
  • one or more beta-ASP transgenes (preferably comprising less than about 5 kb) as described herein is cloned into a recombinant AAV vector from which some (preferably all) of the AAV coding sequences (i.e. AAV rep and cap genes) have been deleted, but which retains at least the AAV inverted terminal repeats (ITRs).
  • AAV rep and cap genes AAV inverted terminal repeats
  • the packaging cell line can then be used to replicate and encapsidate the recombinant AAV vector into infective (but replication-defective) AAV particles once the necessary AAV helper virus functions are provided, as described in the art.
  • the recombinant AAV vector can be introduced prior to or coincident with the introduction of the helper virus or helper virus functions.
  • helper viruses or genetic functions derived therefrom can be used to provide helper activity to AAV, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • helper virus is Adenovirus.
  • the deleted AAV packaging functions can be stably introduced into the genome of the packaging cell or they can be provided transiently (by, e.g., transfection with a helper plasmid or by inclusion within the helper virus, such as adenovirus). Recombinant AAV particles are then purified as described in the art (using, e.g., isopycnic ultracentrifugation).
  • beta-ASP transgenes are generated for cloning into recombinant vectors, in this case AAV.
  • the transgene or transgenes should comprise less than about 5 kb in order to be efficiently packaged.
  • different beta-ASP transgenes can also be placed into separate vectors.
  • Methods such as those illustrated in Example 8 can be used to select vector constructs mediating efficient delivery of beta-adrenergic signaling proteins to ventricular cells maintained in cell culture; and, as illustrated in Example 9, a detectable marker gene (such as lacZ) can be used to select vector constructs mediating efficient delivery of transgenes to the myocardium in vivo.
  • a detectable marker gene such as lacZ
  • Suitable beta-ASP vector constructs can then be used (as described in Examples 12-13 below) to deliver beta-ASP transgenes to the myocardium in vivo.
  • viral vectors as illustrative gene delivery vehicles can also be readily applied to the use of non-viral vectors, such as liposomes and other lipid-containing gene delivery complexes, and other macromolecular complexes (such as multifunctional gene delivery fusion proteins) that are capable of mediating delivery of a polynucleotide to a mammalian host cell in vivo.
  • non-viral vectors such as liposomes and other lipid-containing gene delivery complexes, and other macromolecular complexes (such as multifunctional gene delivery fusion proteins) that are capable of mediating delivery of a polynucleotide to a mammalian host cell in vivo.
  • liposomes and other lipid-containing gene delivery complexes can be used to deliver one or more beta-ASP transgenes.
  • the principles of the preparation and use of such complexes for gene delivery have been described in the art (see, e.g., Ledley, F D, Human Gene Therapy 6:1129-1144, 1995; Miller, N., et al., FASEB Journal 9:190-199, 1995; Chonn, A., et al., Curr. Opin. in Biotech. 6:698-708, 1995; Schofield, J P, et al., British Med. Bull. 51:56-71, 1995; Brigham, K. L., et al., J. Liposome Res. 3:31-49, 1993; and the other references cited above).
  • beta-ASP transgenes as described herein is introduced into a liposome or other lipid-containing gene delivery complex using techniques known in the art.
  • Various beta-ASP transgenes can be generated as described in Examples 5-7, and then introduced into liposomes or other lipid-based vectors.
  • Methods such as those illustrated in Example 8 can be used to select vectors mediating efficient delivery of beta-adrenergic signaling proteins to ventricular cells maintained in cell culture; and, as illustrated in Example 9, a detectable marker gene (such as lacZ) can be used to select vectors mediating efficient delivery of transgenes to the myocardium in vivo.
  • Suitable beta-ASP vector constructs can then be used (as described in Examples 12-13 below) to deliver beta-ASP transgenes to the myocardium in vivo.
  • Catheters were placed in the left atrium and aorta, providing a means to calibrate the left ventricular high fidelity pressure gauge used to measure pressure development, and to monitor pressures. Wires were sutured on the left atrium to permit ECG recording and atrial pacing.
  • the illustrative adenovirus vector system described above was used to deliver transgenes by in vivo gene delivery.
  • As an exemplary beta-ASP transgene we used the AC VI isoform referred to above.
  • the vector material injected in vivo was highly purified and contained no wild-type (replication competent) adenovirus. Thus adenovirus infection and inflammatory infiltration in the heart were minimized.
  • the vector preparation was injected into the lumen of the coronary artery by coronary catheters.
  • a vasoactive agent such as for example histamine, a histamine agonist or a vascular endothelial growth factor (VEGF)
  • VEGF vascular endothelial growth factor
  • vectors comprising an exemplary beta-ASP transgene (AC VI from Example 5-1 above), according to the methods of Hammond, et al., effectively increased left ventricular contractile function and cardiac output in pig models of ischemic heart failure; and, moreover, the administration of histamine within several minutes prior to addition of such vectors further enhanced cardiac function (both left ventricular (LV) peak dP/dt and cardiac output being increased by greater than 100% over controls (lacZ)).
  • LV left ventricular
  • lacZ left ventricular
  • Vectors comprising other beta-ASPs such as vectors encoding beta-ARs or GRK inhibitors as described in Examples 6 and 7, can be used to deliver other preferred beta-ASP transgenes using techniques as described in Example 12-1.
  • beta-ASP transgenes can be used in place of, or in addition to, delivery of an AC transgene.
  • the combination can be provided in a single vector (comprising two or more beta-ASP transgenes) or in separate vectors (each comprising a beta-ASP transgene).
  • size-constrained vectors such as adenovirus
  • an additional beta-ASP transgene such as a GRK inhibitor
  • E4 additional replication gene
  • newer generations of such viral vectors are being used in which size-constraints are relieved in additional ways.
  • a number of available non-viral vectors such as lipid-based vectors (including, e.g., cationic liposome complexes) do not exhibit such restrictive size constraints as observed with viral particle vectors.
  • Such additional beta-ASPs can also be delivered using separate vectors. Where separate vectors are used, the vectors can be introduced together in a single injection (such as illustrated above) or in separate injections. While such separate vectors providing different transgenes are most conveniently analogous vectors (in which one beta-ASP transgene is effectively replaced with another), different promoters can be employed as well as different base vectors.
  • Rate-pressure products and left atrial pressures were found to be similar before and after gene transfer, indicating similar myocardial oxygen demands and loading conditions. Echocardiographic measurements were made using standardized criteria (Sahn, et al. Circulation 58:1072, 1978). The left ventricular end-diastolic diameter (EDD) and end-systolic diameter (ESD) were measured from 5 continuous beats and averaged.
  • EDD left ventricular end-diastolic diameter
  • ESD end-systolic diameter
  • glycopyrrolate (a muscarinic cholinergic antagonist) was given (at 0.14 mg/kg, by i.v.) in order to block the effects of the parasympathetic nervous system on heart rate responses. Isoproterenol was then delivered in bolus doses into the pulmonary artery catheter as heart rate responses were recorded. These studies were conducted one day prior to gene transfer and then again 5-10 days after gene transfer in each pig. Data were then examined by paired analyses comparing each animal before and after gene transfer.
  • FIG. 10 shows data summarizing the effects of in vivo gene transfer of AC VI on heart rate.
  • Animals were studied before and 5-10 days after intracoronary delivery of 10 11 viral particles of an adenovirus expressing AC VI .
  • Glycopyrrolate was used to remove parasympathetic influences on heart rate, thereby optimally isolating the myocardial beta-AR pathway.
  • Basal heart rate was unchanged, but maximal (isoproterenol-stimulated) heart rate was increased significantly by in vivo delivery of a beta-ASP transgene (i.e. AC VI ) according to the present invention.
  • AC VI beta-ASP transgene
  • FIG. 11 shows results of in vivo gene transfer of AC VI on LV dP/dt in a normal pig.
  • the animal was studied before and 7 days after intracoronary delivery of 10 12 viral particles of an adenovirus carrying the beta-ASP transgene AC VI .
  • Glycopyrrolate was used to remove parasympathetic influences on contractile function, thereby optimally isolating the myocardial beta-AR pathway.
  • basal LV dP/dt was substantially increased at the same basal heart rate.
  • Response to isoproterenol was also increased after AC VI gene transfer.
  • beta-ASP gene constructs can also be modified to alter the effective levels of expression of the resulting transgene being employed in the context of the present invention.
  • expression of beta-ASP transgenes can be altered by placing the transgene under the control of a heterologous promoter (including, for example, various constitutive or inducible promoters, as well as tissue-specific promoters such as cardiac-specific promoters).
  • a heterologous promoter including, for example, various constitutive or inducible promoters, as well as tissue-specific promoters such as cardiac-specific promoters.
  • expression can be altered by changing other regions of the genes, including, for example, the untranslated regions of such genes.
  • AC VI constructs having deletions in the 3′-untranslated region can be generated (as described in Example 5-3), and tested for their relative ability to affect expression and functional responsiveness in cardiac cells (as illustrated in Example 8-2).
  • the ability to employ genes exhibiting differing levels of expression provides an added tool that can be used to readily tailor and optimize the present invention in the context of varying therapeutic needs (e.g., depending on the desired level of expression in the cells and tissue to be treated), and can also be used to reduce the amount of vector required to generate a given physiological effect.
  • the illustrations presented in Examples 9-13 provide additional guidance as to means for testing and using such beta-ASP transgenes in the context of the present invention.
  • beta-AR-G s -AC pathway the ability to deliver beta-ARs, GRK inhibitors or combinations of such beta-ASPs in accordance with the present invention (as described above) is expected to provide an even greater enhancement of endogenous beta-adrenergic responsiveness and cardiac function in such dysfunctional mammalian hearts.
  • beta-ARs and proteins affecting beta-ARs such as GRK
  • GRK beta-adrenergic agonists
  • the methods and compositions of the present invention thus provide greatly-needed alternatives to the use of exogenous pharmacological agonists and other methods for the treatment of congestive heart failure in humans.
  • n A,T,C or G 1 atgtcatggt ttagtggcct cctggtcct aaagtggatg aacggaaac agcctggggt 60 gaacgcaatg ggcagaagcg ttcgcggcgc cgtggcactc gggcaggtgg cttctgcacg 120 ccccgctata tgagctgcct cccgggatgca gagccaccca gcccacccc tgcgggccc 180 cctcggtgcc cctggcagga tgacgccttc atccggaggg gcggcccang caagggcaag 240 gaactggggc tgcgggcagt ggcccc

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