Patent Grant
11760987
This invention generally relates to gene therapy and medicine. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for treating a heart failure in a subject in need thereof comprising administering to the subject an isolated or recombinant nucleic acid, an isolated or recombinant or chimeric polypeptide, or an engineered cell, as provided herein, thereby treating the subject. In alternative embodiments, the administration reduces left ventricular (LV) hypertrophy, increases LV peak pressure development, reduced cAMP production and/or improves LV peak pressure decay in a pressure-overload in the subject. In alternative embodiments, provided are compositions and methods for: treating, ameliorating, or slowing the progress of, or protecting (preventing) an individual or a patient against heart failure; or, reducing LV hypertrophy, increasing LV peak pressure development, and/or improving LV peak pressure decay in a pressure-overload in an individual in need thereof.
Adenylyl cyclase (AC) is a transmembrane protein in cardiac myocytes and other cells, the effector molecule for β-adrenergic receptor (bAR) and other G protein-coupled receptors, which regulates the conversion of adenosine triphosphate (ATP) to 30,50-cyclic adenosine monophosphate (cAMP) and thereby initiates a variety of intracellular signaling cascades that influence heart function and additional physiological events.
There are 9 membrane-bound isoforms of mammalian ACs, each consisting of 2 transmembrane domains and 2 cytoplasmic domains (C1 and C2). The C1 and C2 domains form the catalytic core of AC (
C1C2 contains binding sites for Gas, Gai, forskolin, ATP, Mg2+, the regulator of G protein signaling (RGAS2), protein associated with Myc (PAM), Snapin, Ric8a, A-kinase-anchoring protein (AKAP79), protein kinase C, PH domain leucine-rich protein phosphatase 2 (PHLPP2) and phosphorylation and dephosphorylation sites for protein kinase A. Interactions of these factors alters the conformation of C1C2 and regulates AC activity (2).
Increased cardiac expression of AC type 6 (AC6), a dominant AC isoform expressed in mammalian cardiac myocytes (3), has protean beneficial effects on the failing left ventricle (LV), including: 1) increased survival in genetically-induced cardiomyopathy (4) and in acute myocardial infarction (5); 2) reduced action potential duration (6), facilitated atrioventricular (AV) conduction (7), and reduced AV block (5); 3) reductions in both LV dilation and pathological hypertrophy (4,8); 4) beneficial effects on Ca2+ handling via altered activity of SERCA2a and phospholamban (PLB) (9,10); and 5) increased cardiac troponin I phosphorylation (11).
These beneficial effects, consistent in several species and models, appear in large part to not depend upon increased cAMP generation. A phase 2 randomized clinical trial in patients with symptomatic heart failure (HF) and reduced ejection fractions showed that intracoronary AC6 gene transfer appears to be safe and potentially effective, and not associated with increased cardiac arrhythmias (12). Even so, there may be advantages in selecting a transgene that attenuates bAR responsiveness when treating HF.
We generated a catalytically inactive AC6 mutant (AC6mut) molecule by replacing Ala with Asp at position 426 in AC6's catalytic core. This AC6mut is catalytically inactive (does not generate cAMP) but retains the cellular distribution pattern and favorable signaling effects associated with normal AC6, thereby providing compelling evidence that the beneficial effects of AC6 do not require increased cAMP generation (13). AC6mut seemed an ideal candidate for the treatment of HF, retaining the beneficial effects of the parent AC6 while circumventing the potential deleterious effects of sustained cAMP generation. However, a shortcoming of both AC6mut and AC6 is that the molecules are too large to insert into an adeno-associated virus (AAV) with regulated expression, therefore only constitutive expression would be possible, a potential limitation.
Pharmacological inotropes, which increase LV function, for example, dobutamine, dopamine, milrinone, have consistently failed to enhance calcium handling in cardiac myocytes because of adverse effects of increased cardiac cAMP levels.
In alternative embodiments, provided are isolated or recombinant, or chimeric, polypeptides comprising a first peptide sequence and a second peptide sequence, wherein the first peptide sequence comprises or consists of a sequence as set forth in SEQ ID NO:1 and the second peptide sequence comprises or consists of a sequence as set forth in SEQ ID NO:2, wherein the first peptide sequence and the second peptide sequence are operably linked. In alternative embodiments, of the isolated or recombinant, or chimeric, polypeptides, the first peptide sequence and the second peptide sequence are operably linked via a fusion or a linker peptide, or amino acid sequence.
In alternative embodiments, provided are isolated or recombinant nucleic acids (polynucleotides) or nucleic acid sequences encoding an isolated or recombinant, or chimeric, polypeptide as provided herein.
In alternative embodiments, provided are isolated or recombinant nucleic acids or nucleic acid sequences encoding the first peptide sequence as provided herein, or an isolated or recombinant nucleic acid nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO:1, wherein optionally the first peptide is encoded by a nucleic acid sequence comprising or consisting of a sequence as set forth in SEQ ID NO:6, or having a sequence as set forth in SEQ ID NO:6.
In alternative embodiments, provided are isolated or recombinant nucleic acids or nucleic acid sequences encoding the second peptide sequence as provided herein, or an isolated or recombinant nucleic acid nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO:2, wherein optionally the first peptide is encoded by a nucleic acid sequence comprising or consisting of a sequence as set forth in SEQ ID NO:7, or having a sequence as set forth in SEQ ID NO:7.
In alternative embodiments of an isolated or recombinant nucleic acid or nucleic acid sequence as provided herein:
In alternative embodiments, provided are vectors or expression constructs (e.g., recombinant viruses) comprising or having contained therein an isolated or recombinant nucleic acid, or nucleic acid sequence, as provided herein. In alternative embodiments, the vector is selected from the group consisting of an adeno-associated virus (AAV), a lentivirus, an adenovirus, and a plasmid. In alternative embodiments, the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, pseudotyped AAV, a rhesus-derived AAV, AAVrh8, AAVrh10 and AAV-DJan AAV capsid mutant, an AAV hybrid serotype, an organ-tropic AAV, a cardiotropic AAV, and a cardiotropic AAVM41 mutant.
In alternative embodiments, provided are isolated, recombinant or engineered cells or host cells comprising a vector or expression construct as provided herein, or an isolated or recombinant nucleic acid, or nucleic acid sequence, as provided herein, wherein optionally the isolated, recombinant or engineered cell or host cell is or is derived from a mammalian, a human, a non-human primate, a monkey, a mouse, a rat, a guinea pig, a rabbit, a hamster, a goat, a bovine, an equine, an ovine, a canine or a feline cell.
In alternative embodiments, provided are non-human transgenic animals comprising or having contained therein the host cell as provided herein, or as a heterologous sequence, the non-human transgenic animal comprises or has contained therein an isolated or recombinant nucleic acid, or nucleic acid sequence, as provided herein.
In alternative embodiments, provided are methods for:
the method comprising:
thereby:
In alternative embodiments of methods as provided herein the administration to the subject in need thereof:
In alternative embodiments, provided are methods for enhancing calcium handling in a cardiac myocyte without increasing cAMP generation, comprising administering to or implanting or transfecting into to the myocyte: an isolated or recombinant nucleic acid, or nucleic acid sequence, as provided herein, a vector or expression construct as provided herein, or an isolated or recombinant, or chimeric, polypeptide as provided herein, wherein expression of the nucleic acid sequence enhances calcium handling. In alternative embodiments, calcium handling is measured by uptake and release of calcium.
In alternative embodiments of methods as provided herein, administration is by intravenous (IV) injection, intra-arterial (IA) injection, or intracoronary injection.
In alternative embodiments, provided are methods the isolated or recombinant nucleic acid, or isolated or recombinant or chimeric polypeptide, is formulated in a liquid, a gel, a hydrogel, a vesicle, a liposome, a nanoparticle, a nanolipid particle, a powder or an aqueous or a saline formulation and wherein the nucleic acid is optionally in a vector or an expression construct.
In alternative embodiments of methods as provided herein, the cardiac myocyte is selected, derived from or isolated from a mammalian, a human, a non-human primate, a monkey, a mouse, a rat, a guinea pig, a rabbit, a hamster, a goat, a bovine, an equine, an ovine, a canine or a feline subject or host.
In alternative embodiments, provided are uses of:
in the manufacture of a medicament for treating, ameliorating or protecting (preventing) an individual or a patient against heart failure, or reducing left ventricular (LV) hypertrophy, increasing LV peak pressure development, and/or increasing that rate of LV peak pressure decay in an individual in need thereof.
In alternative embodiments, provided are isolated or recombinant nucleic acids, or nucleic acid sequences as provided herein, for use in treating, ameliorating or protecting (preventing) an individual or a patient against heart failure, or reducing left ventricular (LV) hypertrophy, increasing LV peak pressure development, and/or increasing that rate of LV peak pressure decay in an individual in need thereof.
In alternative embodiments, provided are vectors or expression constructs as provided herein, or a host cell as provided herein, for use in treating, ameliorating or protecting (preventing) an individual or a patient against heart failure, or reducing left ventricular (LV) hypertrophy, increasing LV peak pressure development, and/or increasing that rate of LV peak pressure decay in an individual in need thereof.
In alternative embodiments, provided are isolated or recombinant, or chimeric, polypeptides as provided herein, for use in treating, ameliorating or protecting (preventing) an individual or a patient against heart failure, or reducing left ventricular (LV) hypertrophy, increasing LV peak pressure development, and/or increasing that rate of LV peak pressure decay in an individual in need thereof.
The details of one or more exemplary embodiments as provided herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
The drawings set forth herein are illustrative of exemplary embodiments provided herein and are not meant to limit the scope of the invention as encompassed by the claims.
as described in detail in Example 1, below.
as described in detail in Example 1, below.
as described in detail in Example 1, below.
as described in detail in Example 1, below.
Bars ¼ mean values; SE ¼ error bars; number in bars ¼ numbers of cells (B to D) or mice (E, F); numbers above bars ¼ p values from Student t-test (unpaired, 2-tailed); Abbreviations as in
Data are mean +SE. Numbers in bars denote the number of cardiomyocytes per group, obtained from 6 mice per group (2-5 cells per animal, blinded to group identity); P values are from Student's t-test (unpaired, two tails) comparing TG− vs C1C2 4 w after TAC;
as discussed in detail in Example 3, below.
Data are mean +SE. Numbers in bars denote the number of cardiomyocytes, obtained from 6 mice per group (4-6 cells per mouse, blinded to group identity); P values are from Student's t-test (unpaired, two tails) comparing TG− vs C1C2 4 w after TAC;
as discussed in detail in Example 3, below.
Error bars denote mean±SE from 19-24 cardiomyocytes per group, isolated from 6 mice per group (3-4 cells per mouse, blinded to group identity); P value is from Student's t-test (unpaired, two-tailed);
as discussed in detail in Example 3, below.
as discussed in detail in Example 3, below.
as discussed in detail in Example 3, below.
Like reference symbols in the various drawings indicate like elements.
In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for treating a heart failure in a subject in need thereof comprising administering to the subject a nucleic acid sequence as provided herein, thereby treating the subject. In alternative embodiments, the administration reduces left ventricular (LV) hypertrophy, increases LV peak pressure development, reduced cAMP production and/or improves LV peak pressure decay in a pressure-overload in the subject. In alternative embodiments, provided are compositions and methods for: treating, ameliorating or protecting (preventing) an individual or a patient against heart failure; or, reducing LV hypertrophy, increasing LV peak pressure development, and/or improving LV peak pressure decay in a pressure-overload in an individual in need thereof.
In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for altering intracellular signaling in a manner that enhances calcium (Ca2+) handling in cardiac myocytes, and does so without being linked to cell surface beta-adrenergic receptors. In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for treating, ameliorating or protecting (preventing) an individual or a patient against heart failure. In alternative embodiments, provided are chimeric or synthetic polypeptide comprising: a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, and the C1 and the C2 domains have an AC catalytic activity, and nucleic acids encoding them, and expression vehicles, vectors, recombinant viruses, or equivalents expressing them, and engineered or recombinant expressing them.
In alternative embodiments, provided are truncated AC6 polypeptides made by eliminating the amino terminus and the 2 trans-membrane domains of AC6 and subsequently fusing the 2 cytoplasmic domains (C1 and C2) with a 12-amino acid linker yields a C1C2 protein (
We speculated that elimination of the trans-membrane regions of AC6 (
Eliminating the two transmembrane domains of AC6 yields a protein with an intact catalytic domain that is disengaged from membrane-associated J3-adrenergic receptor stimulation, but with enhanced propensity for intracellular interactions with other proteins, which, ultimately, enhance Ca2+ handling and thus systolic and diastolic heart function. Transgenic mice with cardiac-directed expression of C1C2 exhibit resistance to sustained adrenergic stimulation compared with TG negative (control) siblings (JACC Basic & Translational Science 2016; 1:617-629). C1C2 gene transfer promotes enhanced Ca2+ handling, which is not offset by the potentially deleterious effects of increased cAMP levels. C1C2 molecule is sufficiently small that its coding sequence can be expresses in an adeno-associated virus vector, unlike AC6 or AC6 mutant.
Transgenic mice with cardiac-directed C1C2, a fusion protein of the intracellular C1 and C2 segments of adenylyl cyclase type 6, had normal left ventricular (LV) function, but diminished cAMP generation. Cardiac myocytes from C1C2 mice showed increased Ca2+ release. Mice underwent continuous isoproterenol infusion to stress the heart. In C1C2 mice, sustained isoproterenol infusion increased rather than decreased LV function. LV SERCA2a and Ca2+ release was increased. Reduced cAMP generation and resistance to catecholamine cardiomyopathy are attractive features of this potential heart failure therapeutic.
In alternative embodiments, a virus vector encoding C1C2 (AAV9.C1C2) as provided herein is suitable to use either intravenously or by intra-coronary infusion in patients with heart failure. In alternative embodiments, C1C2 gene transfer has beneficial effects on the failing human heart, and is effective regardless of ejection fraction (preserved or reduced). Thus, this embodiment can be used to treat individuals with heart failure, including the 6 million patients with heart failure in US, with 400,000 new cases diagnosed each year.
Generating and Manipulating Nucleic Acids
In alternative embodiments, provided are nucleic acids encoding polypeptides comprising a first peptide sequence and a second peptide sequence, wherein the first peptide sequence comprises or consists of a sequence as set forth in SEQ ID NO:1 and the second peptide sequence comprises or consists of a sequence as set forth in SEQ ID NO:2, wherein the first peptide sequence and the second peptide sequence are operably linked. In alternative embodiments, the isolated or recombinant nucleic acid or nucleic acid sequence encodes a first peptide sequence, and optionally the first peptide is encoded by a nucleic acid sequence comprising or consisting of a sequence as set forth in SEQ ID NO:6. In alternative embodiments, the isolated or recombinant nucleic acid or nucleic acid sequence encodes a second peptide, or an isolated or recombinant nucleic acid nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO:2, wherein optionally the first peptide is encoded by a nucleic acid sequence comprising SEQ ID NO:7.
C1-Linker-C2 DNA Sequence (SEQ ID NO: 4)
For purposes of illustration and clarity only, the separate domains of this single exemplary polypeptide-encoding nucleic acid are separated, below:
GTCTCTCCTCCCAGCACGTTGCC
ATG
GCTGCAGCGGGTGGAATTCCTCCTGCGGCGGCAATG
LSSQHVA
MEM KEDINTKKED MMFHKIYIQK HDNVSILFAD IEGFTSLASQ
In alternative embodiments, to practice the methods and compositions (e.g., kits, products of manufacture) as provided herein, provided are isolated, synthetic and/or recombinant nucleic acids or genes encoding, e.g., a C1 and a C2 domain of an adenylyl cylase (AC) (optionally a human or a murine AC) and optionally lacking all or substantially most AC transmembrane domains. In alternative embodiments, to practice the methods as provided herein, the invention provides nucleic acids or genes in recombinant form in an (e.g., spliced into) an expression vehicle for in vivo expression, e.g., in a vector or a recombinant virus. In other alternative embodiments, provided are, e.g., isolated, synthetic and/or recombinant nucleic acids encoding inhibitory nucleic acids (e.g., siRNA, microRNA, antisense, ribozyme) that can inhibit the expression of genes or messages (mRNAs) that inhibit the expression of the desired gene or nucleic acid, e.g., encoding a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains.
In alternative embodiments, nucleic acids as provided herein are made, isolated and/or manipulated by, e.g., cloning and expression of cDNA libraries, amplification of message or genomic DNA by PCR, and the like. The nucleic acids and genes used to practice compositions and methods as provided herein, including DNA, RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof, can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides (e.g., chimeric proteins used to practice compositions and methods as provided herein) generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system or gene therapy delivery vehicle can be used, including e.g., viral (e.g., AAV constructs or hybrids) bacterial, fungal, mammalian, yeast, insect or plant cell expression systems or expression vehicles.
Alternatively, nucleic acids used to practice compositions and methods as provided herein can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.
Techniques for the manipulation of nucleic acids used to practice compositions and methods as provided herein, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).
Another useful means of obtaining and manipulating nucleic acids used to practice the methods as provided herein is to clone from genomic samples, and, if desired, screen and re-clone inserts isolated or amplified from, e.g., genomic clones or cDNA clones. Sources of nucleic acid used in the methods as provided herein include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld (1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC); bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see, e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see, e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinant viruses, phages or plasmids.
In alternative embodiments, to practice the methods as provided herein, chimeric or fusion proteins and nucleic acids encoding them are used. In alternative embodiments, the chimeric or fusion protein is fused to a heterologous peptide or polypeptide, such as a peptide for targeting the polypeptide to a desired cell type, such a cardiac myocytes, or a lung cell.
In alternative embodiments, a heterologous peptide or polypeptide joined or fused to a protein used to practice compositions and methods as provided herein can be an N-terminal identification peptide which imparts a desired characteristic, such as fluorescent detection, increased stability and/or simplified purification. Peptides and polypeptides used to practice compositions and methods as provided herein can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr. Purif. 12:404-414). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.
Nucleic acids or nucleic acid sequences used to practice compositions and methods as provided herein can be an oligonucleotide, nucleotide, polynucleotide, or to a fragment of any of these, to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material, natural or synthetic in origin. Compounds use to practice compositions and methods as provided herein include “nucleic acids” or “nucleic acid sequences” including oligonucleotide, nucleotide, polynucleotide, or any fragment of any of these; and include DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic or synthetic origin which may be single-stranded or double-stranded; and can be a sense or antisense strand, or a peptide nucleic acid (PNA), or any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double stranded iRNAs, e.g., iRNPs). Compounds use to practice compositions and methods as provided herein include nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides. Compounds use to practice compositions and methods as provided herein include nucleic-acid-like structures with synthetic backbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Dev 6:153-156. Compounds use to practice compositions and methods as provided herein include “oligonucleotides” including a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that may be chemically synthesized. Compounds use to practice compositions and methods as provided herein include synthetic oligonucleotides having no 5′ phosphate, and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide can ligate to a fragment that has not been dephosphorylated.
In alternative aspects, compounds used to practice compositions and methods as provided herein include genes or any segment of DNA involved in producing a chimeric or fusion polypeptide as provided herein; it can include regions preceding and following the coding region (leader and trailer) as well as, where applicable, intervening sequences (introns) between individual coding segments (exons). “Operably linked” can refer to a functional relationship between two or more nucleic acid (e.g., DNA) segments. In alternative aspects, it can refer to the functional relationship of transcriptional regulatory sequence to a transcribed sequence. For example, a promoter can be operably linked to a coding sequence, such as a nucleic acid used to practice compositions and methods as provided herein, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. In alternative aspects, promoter transcriptional regulatory sequences can be operably linked to a transcribed sequence where they can be physically contiguous to the transcribed sequence, i.e., they can be cis-acting. In alternative aspects, transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
In alternative aspects, the invention comprises use of “expression cassettes” comprising a nucleotide sequences used to practice compositions and methods as provided herein, which can be capable of affecting expression of the nucleic acid, e.g., a structural gene or a transcript (e.g., encoding a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains) in a host compatible with such sequences. Expression cassettes can include at least a promoter operably linked with the polypeptide coding sequence or inhibitory sequence; and, in one aspect, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression may also be used, e.g., enhancers.
In alternative aspects, expression cassettes used to practice compositions and methods as provided herein also include plasmids, expression vectors, recombinant viruses, any form of recombinant “naked DNA” vector, and the like. In alternative aspects, a “vector” used to practice compositions and methods as provided herein can comprise a nucleic acid that can infect, transfect, transiently or permanently transduce a cell. In alternative aspects, a vector used to practice compositions and methods as provided herein can be a naked nucleic acid, or a nucleic acid complexed with protein or lipid. In alternative aspects, vectors used to practice compositions and methods as provided herein can comprise viral or bacterial nucleic acids and/or proteins, and/or membranes (e.g., a cell membrane, a viral lipid envelope, etc.). In alternative aspects, vectors used to practice compositions and methods as provided herein can include, but are not limited to replicons (e.g., RNA replicons, bacteriophages) to which fragments of DNA may be attached and become replicated. Vectors thus include, but are not limited to RNA, autonomous self-replicating circular or linear DNA or RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No. 5,217,879), and can include both the expression and non-expression plasmids. In alternative aspects, the vector used to practice compositions and methods as provided herein can be stably replicated by the cells during mitosis as an autonomous structure, or can be incorporated within the host's genome.
In alternative aspects, “promoters” used to practice compositions and methods as provided herein include all sequences capable of driving transcription of a coding sequence in a cell, e.g., a mammalian cell such as a heart, lung, muscle, nerve or brain cell. Thus, promoters used in the constructs as provided herein include cis-acting transcriptional control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter used to practice compositions and methods as provided herein can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) transcription.
In alternative embodiments, “constitutive” promoters used to practice compositions and methods as provided herein can be those that drive expression continuously under most environmental conditions and states of development or cell differentiation. In alternative embodiments, “Inducible” or “regulatable” promoters used to practice compositions and methods as provided herein can direct expression of the nucleic acid as provided herein under the influence of environmental conditions, administered chemical agents, or developmental conditions.
Gene Therapy and Gene Delivery Vehicles
In alternative embodiments, methods as provided herein comprise use of nucleic acid (e.g., nucleic acid encoding a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains) delivery systems to deliver a payload of a nucleic acids or genes, or a C1C2-expressing nucleic acid, transcript or message, to a cell or cells in vitro, ex vivo, or in vivo, e.g., as gene therapy delivery vehicles.
In alternative embodiments, expression vehicle, vector, recombinant virus, or equivalents used to practice methods as provided herein are or comprise: an adeno-associated virus (AAV), a lentiviral vector or an adenovirus vector; an AAV serotype AAV5, AAV6, AAV8 or AAV9; a rhesus-derived AAV, or the rhesus-derived AAV AAVrh.10hCLN2; an organ-tropic AAV, or a cardiotropic AAV, or a cardiotropic AAVM41 mutant; and/or an AAV capsid mutant or AAV hybrid serotype. In alternative embodiments, the AAV is engineered to increase efficiency in targeting a specific cell type that is non-permissive to a wild type (wt) AAV and/or to improve efficacy in infecting only a cell type of interest. In alternative embodiments, the hybrid AAV is retargeted or engineered as a hybrid serotype by one or more modifications comprising: 1) a transcapsidation, 2) adsorption of a bi-specific antibody to a capsid surface, 3) engineering a mosaic capsid, and/or 4) engineering a chimeric capsid. It is well known in the art how to engineer an adeno-associated virus (AAV) capsid in order to increase efficiency in targeting specific cell types that are non-permissive to wild type (wt) viruses and to improve efficacy in infecting only the cell type of interest; see e.g., Wu et al., Mol. Ther. 2006 September; 14(3):316-27. Epub 2006 Jul. 7; Choi, et al., Curr. Gene Ther. 2005 June; 5(3):299-310.
For example, the rhesus-derived AAV AAVrh.10hCLN2 or equivalents thereof can be used, wherein the rhesus-derived AAV may not be inhibited by any pre-existing immunity in a human; see e.g., Sondhi, et al., Hum Gene Ther. Methods. 2012 October; 23(5):324-35, Epub 2012 Nov. 6; Sondhi, et al., Hum Gene Ther. Methods. 2012 Oct. 17; teaching that direct administration of AAVrh.10hCLN2 to the CNS of rats and non-human primates at doses scalable to humans has an acceptable safety profile and mediates significant payload expression in the CNS.
Also, for example, AAV vectors specifically designed for cardiac gene transfer (a cardiotropic AAV) can be used, e.g., the AAVM41 mutant having improved transduction efficiency and specificity in the myocardium, see, e.g., Yang, et al. Virol J. 2013 Feb. 11; 10(1):50.
Because adeno-associated viruses (AAVs) are common infective agents of primates, and as such, healthy primates carry a large pool of AAV-specific neutralizing antibodies (NAbs) which inhibit AAV-mediated gene transfer therapeutic strategies, the methods as provided herein comprise screening of patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, to facilitate individualized treatment design and enhance therapeutic efficacy; see, e.g., Sun, et al., J. Immunol. Methods. 2013 Jan. 31; 387(1-2):114-20, Epub 2012 Oct. 11.
Kits and Instructions
Provided are kits comprising compositions and methods as provided herein, including instructions for use thereof. As such, kits, cells, expression vehicles (e.g., recombinant viruses, vectors) and the like can also be provided.
For example, in alternative embodiments, provided are kits comprising compositions used to practice compositions and methods as provided herein, e.g., comprising a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, (b) a liquid or aqueous formulation as provided herein, or (c) the vesicle, liposome, nanoparticle or nanolipid particle as provided herein. In one aspect, the kit further comprising instructions for practicing any methods as provided herein, e.g., in vitro or ex vivo methods for enhancing calcium handling in a cardiac myocyte.
Formulations
In alternative embodiments, provided are compositions and methods for use in treating heart failure in vivo. In alternative embodiments, these compositions comprise nucleic acids formulated for these purposes, e.g., expression vehicles or nucleic acids formulated in a buffer, in a saline solution, in a powder, an emulsion, in a vesicle, in a liposome, in a nanoparticle, in a nanolipoparticle and the like.
In alternative embodiments, provided are methods comprising administration of nucleic acids encoding a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, to treat, ameliorate or prevent a heart failure. Accordingly, provided are the appropriate formulations and dosages of peptides or polypeptides as provided herein, or C1C2-encoding nucleic acids, for same.
In alternative embodiments, the compositions (including formulations of nucleic acids encoding a C1 and a C2 domain of an adenylyl cylase (AC)) can be formulated in any way and can be applied in a variety of concentrations and forms depending on the desired in vitro, in vivo or ex vivo conditions, including a desired in vivo or ex vivo method of administration and the like. Details on techniques for in vitro, in vivo or ex vivo formulations and administrations are well described in the scientific and patent literature.
Formulations and/or carriers used to practice compositions and methods as provided herein are well known in the art. Formulations and/or carriers used to practice compositions and methods as provided herein can be in forms such as tablets, pills, powders, capsules, liquids, gels, syrups, slurries, suspensions, etc., suitable for in vivo or ex vivo applications.
In alternative embodiments, peptides or polypeptides, or nucleic acids, used to practice compositions and methods as provided herein can be in admixture with an aqueous and/or buffer solution or as an aqueous and/or buffered suspension, e.g., including a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate. Formulations can be adjusted for osmolarity, e.g., by use of an appropriate buffer.
In practicing compositions and methods as provided herein, the compounds (e.g., formulations) as provided herein (e.g., nucleic acids encoding a C1 and a C2 domain of an adenylyl cylase (AC), comprise a solution dissolved in a pharmaceutically acceptable carrier, e.g., acceptable vehicles and solvents that can be employed include water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any fixed oil can be employed including synthetic mono- or diglycerides, or fatty acids such as oleic acid. In one embodiment, solutions and formulations used to practice the invention are sterile and can be manufactured to be generally free of undesirable matter. In one embodiment, these solutions and formulations are sterilized by conventional, well known sterilization techniques.
The solutions and formulations used to practice the invention can comprise auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent (e.g., C1C2-encoding nucleic acids or genes) in these formulations can vary widely, and can be selected primarily based on fluid volumes, viscosities and the like, in accordance with the particular mode of in vivo or ex vivo administration selected and the desired results, e.g., increasing in vivo C1C2 expression.
The solutions and formulations used to practice the invention can be lyophilized; for example, provided are a stable lyophilized formulation comprising C1C2-encoding nucleic acids or genes, or C1C2 peptides or polypeptides. In one aspect, this formulation is made by lyophilizing a solution comprising a C1C2-encoding nucleic acid or gene, or C1C2 peptides or polypeptides, and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL protein, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. patent app. no. 20040028670.
The compositions and formulations as provided herein can be delivered by the use of liposomes (see also discussion, below). By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific tissue or organ type, one can focus the delivery of the active agent into a target cells in an in vivo or ex vivo application.
Nanoparticles, Nanolipoparticles and Liposomes
The invention also provides nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds used to practice the methods of compositions and methods as provided herein, e.g., to deliver a nucleic acid encoding a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, to an individual, a patient or mammalian cells in vivo or ex vivo. In alternative embodiments, these compositions are designed to target specific molecules, including biologic molecules, such as polypeptides, including cell surface polypeptides, e.g., for targeting a desired cell type, e.g., a mammalian cardiac cell, a kidney cell, a lung cell, a nerve cell and the like.
Provided are multilayered liposomes comprising compounds used to practice compositions and methods as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, e.g., to entrap a C1C2-encoding nucleic acid or gene.
Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., C1C2-encoding nucleic acids or genes, or C1C2 peptides or polypeptides), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.
In one embodiment, liposome compositions used to practice compositions and methods as provided herein comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound (e.g., C1C2-encoding nucleic acids or genes) used to practice compositions and methods as provided herein to a desired cell type, as described e.g., in U.S. Pat. Pub. No. 20070110798.
The invention also provides nanoparticles comprising compounds used to practice compositions and methods as provided herein in the form of active agent-containing nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent of compositions and methods as provided herein or a fat-solubilized water-soluble active agent to act with a bivalent or trivalent metal salt.
In one embodiment, solid lipid suspensions can be used to formulate and to deliver C1C2-encoding nucleic acids or genes, or C1C2 peptides or polypeptides, used to practice the invention to a patient, an individual, or mammalian cell in vivo or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.
Delivery Vehicles
In alternative embodiments, any delivery vehicle can be used to practice the methods or compositions of compositions and methods as provided herein, e.g., to deliver C1C2 peptides or polypeptides, or to practice the methods as provided herein in vivo or ex vivo. For example, delivery vehicles comprising polycations, cationic polymers and/or cationic peptides, such as polyethyleneimine derivatives, can be used e.g. as described, e.g., in U.S. Pat. Pub. No. 20060083737.
In one embodiment, a dried polypeptide-surfactant complex is used to formulate a composition as provided herein, wherein a surfactant is associated with a nucleic acid via a non-covalent bond e.g. as described, e.g., in U.S. Pat. Pub. No. 20040151766.
In one embodiment, a nucleic acid or polypeptide used to practice compositions and methods as provided herein can be applied to cells as polymeric hydrogels or water-soluble copolymers, e.g., as described in U.S. Pat. No. 7,413,739; for example, a nucleic acid or protein can be polymerized through a reaction between a strong nucleophile and a conjugated unsaturated bond or a conjugated unsaturated group, by nucleophilic addition, wherein each precursor component comprises at least two strong nucleophiles or at least two conjugated unsaturated bonds or conjugated unsaturated groups.
In one embodiment, a nucleic acid or protein is applied to cells using vehicles with cell membrane-permeant peptide conjugates, e.g., as described in U.S. Pat. Nos. 7,306,783; 6,589,503. In one aspect, the nucleic acid itself is conjugated to a cell membrane-permeant peptide. In one embodiment, a nucleic acid, protein, and/or the delivery vehicle are conjugated to a transport-mediating peptide, e.g., as described in U.S. Pat. No. 5,846,743, describing transport-mediating peptides that are highly basic and bind to poly-phosphoinositides.
In one embodiment, electro-permeabilization is used as a primary or adjunctive means to deliver C1C2-encoding nucleic acids or genes to a cell, e.g., using any electroporation system as described e.g. in U.S. Pat. Nos. 7,109,034; 6,261,815; 5,874,268.
Products of Manufacture, Implants and Artificial Organs
The invention also provides products of manufacture comprising cells as provided herein (e.g., cells modified to express a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, to practice the methods as provided herein), and use of cells made by methods of compositions and methods as provided herein, including for example implants and artificial organs, bioreactor systems, cell culture systems, plates, dishes, tubes, bottles and flasks comprising cells modified to express C1C2 proteins to practice the methods as provided herein. Any implant, artificial organ, bioreactor systems, cell culture system, cell culture plate, dish (e.g., petri dish), cell culture tube and/or cell culture flask (e.g., a roller bottle) can be used to practice compositions and methods as provided herein.
In alternative embodiments provided are a bioreactor, implant, stent, artificial organ or similar device comprising cells modified to express C1C2 proteins to practice the methods as provided herein; for example, including implants as described in U.S. Pat. Nos. 7,388,042; 7,381,418; 7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; and U.S. Pat. App. Pub. Nos. 20040127987; 20080119909 (describing auricular implants); 20080118549 (describing ocular implants); 20080020015 (describing a bioactive wound dressing); 20070254005 (describing heart valve bio-prostheses, vascular grafts, meniscus implants); 20070059335; 20060128015 (describing liver implants).
Implanting Cells In Vivo
In alternative embodiments, the methods as provided herein also comprise implanting or engrafting cells, e.g., cardiac, lung or kidney cells, comprising or expressing a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, used to practice the invention; and in one aspect, methods as provided herein comprise implanting or engrafting the nucleic acids or genes (or cells expressing them) encoding or expressing a C1 and a C2 domain of an adenylyl cylase (AC), and lacking all AC transmembrane domains, in a vessel, tissue or organ ex vivo or in vivo, or implanting or engrafting the re-programmed differentiated cell in an individual in need thereof.
Cells can be removed from an individual, treated using the compositions and/or methods of compositions and methods as provided herein, and reinserted (e.g., injected or engrafted) into a tissue, organ or into the individual, using any known technique or protocol. For example, de-differentiated re-programmed cells, or re-programmed differentiated cells, can be re-implanted (e.g., injected or engrafted) using microspheres e.g., as described in U.S. Pat. No. 7,442,389; e.g., in one aspect, the cell carrier comprises a bulking agent comprising round and smooth polymethylmethacrylate microparticles preloaded within a mixing and delivery system and an autologous carrier comprising these cells. In another embodiment, the cells are re-administered to a tissue, an organ and/or an individual in need thereof in a biocompatible crosslinked matrix, as described e.g., in U.S. Pat. App. Pub. No. 20050027070.
In another embodiment, the cells as provided herein (e.g., cells made by practicing the methods of compositions and methods as provided herein) are re-administered (e.g., injected or engrafted) to a tissue, an organ and/or an individual in need thereof within, or protected by, a biocompatible, nonimmunogenic coating, e.g., as on the surface of a synthetic implant, e.g., as described in U.S. Pat. No. 6,969,400, describing e.g., a protocol where a cAMP-incompetent AC can be conjugated to a polyethylene glycol that has been modified to contain multiple nucleophilic groups, such as primary amino or thiol group.
In one embodiment, the cells as provided herein (e.g., cells made by practicing the methods of compositions and methods as provided herein) are re-administered (e.g., injected or engrafted) to a tissue, an organ and/or an individual in need thereof using grafting methods as described e.g. by U.S. Pat. Nos. 7,442,390; 5,733,542.
Any method for delivering polypeptides, nucleic acids and/or cells to a tissue or organ (e.g., a lung, kidney, heart) can be used, and these protocols are well known in the art, e.g., as described in U.S. Pat. No. 7,514,401, describing e.g., using intracoronary (IC), intravenous (IV), and/or local delivery (myocardial injection) of polypeptides, nucleic acids and/or cells to a heart in situ. For example, in alternative embodiments, aerosol drug particles into the lungs and into the bloodstream, gene therapy, continuous infusions, repeated injections and/or sustained release polymers can be used for delivering polypeptides, nucleic acids and/or cells to a tissue or organ (e.g., a lung, kidney, heart). In alternative embodiments, nucleic acids and/or cells can be given through a catheter into the coronary arteries or by direct injection into the left atrium or ventricular myocardium via a limited thoracotomy; or delivered into the myocardium via a catheter passed during cardiac catheterization; or delivered into the pericardial space.
In alternative embodiments, nucleic acids or proteins used to practice compositions and methods as provided herein, or a vector comprising a nucleic acid used to practice the invention (e.g., an AAV, or adenoviral gene therapy vector), or vesicle, liposome, nanoparticle or nanolipid particle (NLP) as provided herein, and the like, to a tissue or organ (e.g., a lung, kidney, heart); e.g. as described in U.S. Pat. No. 7,501,486, e.g., polypeptides as provided herein comprising an amino acid sequence CRPPR (SEQ ID NO:1), the amino acid sequence CARPAR (SEQ ID NO:2) or a peptidomimetic thereof, or amino acid sequence CPKRPR (SEQ ID NO:3) or a peptidomimetic thereof.
Compositions used to practice compositions and methods as provided herein can be used in combination with other therapeutic agents, e.g. angiogenic agents, anti-thrombotic agents, anti-inflammatory agents, immunosuppressive agents, anti-arrhythmic agents, tumor necrosis factor inhibitors, endothelin inhibitors, angiotensin-converting enzyme inhibitors, calcium antagonists, antibiotic agents, antiviral agents and viral vectors.
The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols, for example, as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
This example demonstrates that methods and compositions as provided herein including C1C2 gene transfer has beneficial effects on the failing human heart, and is effective regardless of ejection fraction (preserved or reduced), and can be used to treat individuals with heart failure.
Methods
GENERATION OF C1C2 FUSION PROTEIN. C1C2 peptide was generated by ligating the cytoplasmic domain 1 (C1, amino acids 349-576) with cytoplasmic domain 2 (C2, amino acids 939-1157) of mouse adenylyl cyclase type 6 (AC6) using a linker (12 amino acids: AAAGGIPPAAAM) (SEQ ID NO:8) as illustrated in
GENERATION OF C1C2 TRANSGENIC MICE. Animals were used in accordance with Association for Assessment and Accreditation of Laboratory Animal Care guidelines and approved by the Institutional Animal Care and Use Committee of VA San Diego Healthcare System. To generate mice with cardiac-directed expression of C1C2, the murine C1C2 cDNA with an AU1 tag at the C terminus was subcloned between the a-myosin heavy chain promoter and SV40 poly(A). A 7.1-kb fragment containing the expression cassette was used for pronuclear injection, carried out in the transgenic mouse facility at University of California, San Diego (inbred C57BL/6). Founder mice were identified by polymerase chain reaction (PCR) of genomic DNA prepared from tail tips. The C1C2 gene was detected using a primer homologous to the α-MHC promoter (forward: 5′-CACATAGAAGCCTAGCCCA CACC-3′) (SEQ ID NO:10) and another primer in the C1 region (5′-GTTAGCCAGGGTCA CATCGT-3′) (SEQ ID NO:11). C1C2 mRNA was detected in reverse transcription quantitative PCR, using the forward primer 5′-TGGGCCTCTCTACTCTGCAT-3′ (SEQ ID NO:12) and the reverse primer 5′-TGGATGTAACCTCGGG TCTC-3′ (SEQ ID NO:13), enabling quantification of fold increase of C1C2 mRNA relative to that of endogenous AC6 mRNA.
Founder animals were crossbred with normal mice of the same strain, and selected animals were used for analysis of cardiac transgene expression. We documented variable transgene mRNA expression but similar levels of C1C2 protein expression in 2 lines and used the line with a 23-fold increase in C1C2 mRNA expression (vs. endogenous AC6) in this study. C1C2 protein was detected using anti-AU1 antibody, which has a low background in LV homogenates from transgene negative mice. C1C2 protein was 13-fold increased relative to that of the AU1 signal in transgene negative mice. The study included 114 mice (68 male, 46 female), 4.4±0.1 months of age, weighing 23.9±0.6 g.
ECHOCARDIOGRAPHY. Echocardiography was per-formed prior to isoproterenol (Iso) infusion and on day 6 or 7 of sustained Iso infusion, using methods previously described (15). Anesthesia was induced with 5% isoflurane (at a flow rate of 1 L/min oxygen) and maintained with 1% isoflurane in oxygen.
LV SYSTOLIC AND DIASTOLIC FUNCTION. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg), and a 1.4-F micromanometer catheter (Millar Instruments, Houston, Tex.) was advanced via the right carotid artery across the aortic valve and into the LV cavity. Left ventricular pressure was recorded and stored digitally for processing (IOX1.8; Emka Technologies, Christchurch, Va.) as previously reported (11). Data were acquired and analyzed without knowledge of group identity. Subsequently, tissue samples were obtained.
CARDIAC MYOCYTE ISOLATION. Cardiac myocytes were isolated as previously described (15,16).
Ca2+ TRANSIENTS. Cytosolic Ca2+ transients (Indo-1) were measured from cardiac myocytes isolated from transgenic mice as previously described (13,17).
CYCLIC AMP MEASUREMENT AND PKA ACTIVITY ASSAY. Isolated cardiac myocytes were stimulated with Iso (10 mM, 10 min) or the water-soluble forskolin analog NKH477 (10 mM, 10 min) Cyclic AMP was measured using the cAMP Biotrak EIA™ (GE Healthcare, Chalfont, United Kingdom) as previously reported (15). PKA activity was determined as previously described (17).
IMMUNOFLUORESCENCE. Isolated cardiac myocytes were attached to laminin-coated 2-well chamber slides for 1 h, washed, fixed (10% formalin, 15 min, 23° C.), blocked with normal goat serum (1 h), and incubated (4° C., overnight) with the following antibodies: anti-AU1 antibody (1:300 dilution; for detecting C1C2 transgene protein; Fitzgerald, Atlanta, Ga.) and anti-Cav3 antibody (1:100 dilution, for detecting caveolae; BD Pharmingen, San Jose, Calif.). Cardiac myocytes were washed with phosphate-buffered saline and then incubated with secondary antibodies (Alexa Fluor 488 or 594 conjugated; 1:1,000 dilution) for 1 h. To identify the nucleus, cells were incubated with Hoechst dye (1:1,000 dilution, 20 min) Then, cardiac myocytes were imaged as previously described (18).
ISOPROTERENOL INFUSION. Osmotic minipumps (Alzet; DuRECT Corp., Cupertino, Calif.) were filled with Iso or saline. The 7-day continuous infusion delivered 60 mg/d/kg as previously described (19). RT-PCR. Primers for detecting phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 2 (P-Rex2) included forward primer 5′-ATCATGTGCAGCAGTGGTGT-3′ (SEQ ID NO:14) and reverse primer 5-CCTTGGAGCTGACTGAGG AG-3′ (SEQ ID NO:15). The absence of suitable AC type-specific antibodies precluded quantitative assessment of LV AC isoform protein content. Instead, we used quantitative real-time RT-PCR to determine whether cardiac-directed C1C2 expression altered the cardiac expression of other AC isoforms (AC3, AC4, AC5, AC6, AC7, AC8, and AC9). Details of PCR conditions and primer sequences can be found in our previous studies (20).
MICROARRAY, ANTIBODIES. Expression of adenylyl cyclase-related proteins were analyzed using the Agilent gene expression microarray technique (PhalanxBio Inc., San Diego, Calif.). C1C2 protein was detected by anti-AU1 antibody (1:2,000 dilution: Fitzgerald) or anti-AC5/6 antibody (1:200 dilution; Santa Cruz Biotechnology, Dallas, Tex.). Additional antibodies included GAPDH (1:20,000 dilution; Fitzgerald); cMLCK (1:1,000 dilution; Abgent, San Diego, Calif.); MLC2v (1:1,000 dilution; Synaptic Systems, Gottingen, Germany), PKA catalytic subunit (1:1,000 dilution; BD Transduction, San Jose, Calif.); p-ERK1/2, p38, p-PKARII a and b (1:200 dilution; Santa Cruz Biotechnology); PLB (1:5,000 dilution; Affinity Bioreagents, Rockford, Ill.); phospho-16-PLB (1:3,000 dilution; Badrilla, Leeds, United Kingdom); S100A1 (1:1,000 dilution; Acris); SERCA2a antibody (1:1,000 dilution; Enzo, Exeter, United Kingdom); phospho-aB-crystallin (CryAB) and total CryAB antibodies (1:1,000 dilution; Enzo); P-308-Akt, P-473-Akt, T-Akt, p-GSK3a/b, p-MDM2, P-p70S6K, p70S6K, and phospho-S22/23 troponin I (TnI) (1:1,000 dilution each; Cell Signaling, Danvers, Mass.); vinculin (1:100,1000 dilution; Sigma, Darmstadt, Germany).
NECROPSY. Body, liver, lung, and LV weight (including interventricular septum) were recorded and a short axis midwall LV ring was fixed in formalin and embedded in paraffin. The remaining LV was quickly frozen in liquid nitrogen and stored at −80° C.
HISTOLOGY. Transmural sections of the LV were formalin-fixed and paraffin-embedded. Sections (5 mm) were mounted and counterstained with hematoxylin and eosin and with Masson's trichrome. For quantitative assessment of LV fibrosis, images of a short-axis midwall LV ring stained with Picro Sirius red stain were obtained by using a NanoZoomer™ digital slide scanner (Hamamatsu, Hamamatsu, Japan). Blinded analysis of the degree of fibrosis was conducted using ImageJ software (U.S. National Institutes of Health, Bethesda, Md.).
STATISTICAL ANALYSIS. Data are mean±SE. Between-group comparisons were made using Stu-dent t-test (unpaired, 2-tailed). Interactions of C1C2 expression and Iso infusion on outcomes were tested for statistical significance using 2-way ANOVA followed by Bonferroni t-test. The null hypothesis was rejected when p<0.05.
Results
C1C2 GENE TRANSFER IN CARDIAC MYOCYTES. Location and AC activity. Anti-AU1 antibody was used to detect C1C2 expression in neonatal rat cardiac myocytes after Ad5.C1C2 infection (200 vp/cell) (
Akt Signaling. Adenovirus-mediated gene transfer of C1C2 or AC6 into cultured cardiac myocytes was performed to compare signaling events evoked by C1C2 versus AC6. Both C1C2 and AC6 expression activated PI3K/Akt signaling pathways, increased phosphorylation of Akt at Ser308 and Thr473, and increased phosphorylation of the downstream Akt target proteins GASK3a/b, murine double mutant 2 (MDM2), and p70S6K (
C1C2 TRANSGENIC LINES. Cardiac-directed expression of C1C2 increased LV C1C2 mRNA: one line 14-fold greater than endogenous AC6 mRNA, a second line 23-fold greater (
LV SYSTOLIC AND DIASTOLIC FUNCTION. Echocardiography. C1C2 mice had normal LV dimensions and fractional shortening prior to 7 days of Iso infusion (Table 1, No Iso). Fractional shortening was reduced after 7 days of Iso infusion with no between-group difference (Table 1, 7 days of Iso).
LV function prior to sustained isoproterenol infusion. In vivo assessment of rates of LV pressure development (+dP/dt) and decline (−dP/dt) showed no group differences in peak +dP/dt (control [Con] mice: 5,070±553 mm Hg/s, n ¼ 12; C1C2 mice: 4,319±253 mm Hg/s, n ¼ 13; p ¼ 0.22) or in peak −dP/dt (Con: −5,003±527 mm Hg/s, n ¼ 12; C1C2 mice: −4,362±248 mm Hg/s, n ¼ 13; p ¼ 0.27) (Table 2). In addition, there were no differences in LV peak +dP/dt, LV peak −dP/dt, LV developed pressure or heart rate through a wide range of brief graded doses of infused Iso (
LV function after 7 days of continuous isoproterenol infusion. The response to sustained Iso infusion was critically influenced by the presence of C1C2 for LV peak +dP/dt (p ¼ 0.012), LV peak −dP/dt (p<0.001), and LV developed pressure (p<0.001), with no such interaction with heart rate (Table 2). In Con mice, 7 days of continuous Iso infusion tended to reduce LV peak +dP/dt (23% reduction; p ¼ 0.24) and LV peak −dP/dt (30% reduction; p ¼ 0.06) (
In contrast, continuous Iso infusion showed a directionally opposite effect on LV function in mice with cardiac-directed C1C2 expression, with increases in both LV peak +dP/dt (23% increase, p ¼ 0.026) and LV peak −dP/dt (27% increase; p ¼ 0.006) (
NECROPSY. Table 3 shows that sustained Iso infusion was associated with similar increases in LV and liver weight in both groups.
HISTOLOGY. Histological inspection showed normal morphology without group differences in fibrosis of C1C2 mice compared to control mice at 8 months of age (
CYCLIC AMP GENERATION AND PKA ACTIVITY. Before sustained Iso infusion, transmural LV samples showed no group differences (C1C2 vs. control mice) in basal cAMP, but Iso-stimulated cAMP production was reduced in LV samples from C1C2 mice (
Ca2+ TRANSIENT AND Ca2+ HANDLING PROTEINS. Before sustained isoproterenol infusion. Basal Ca2+ released during contraction (systolic-diastolic Ca2+) was not affected by C1C2 expression (
C1C2 expression reduced time to peak Ca2+ release before (p ¼ 0.025) and during brief Iso stimulation (p ¼ 0.047) (
After sustained isoproterenol infusion. After 7 days of Iso infusion, C1C2 expression was associated with increased peak Ca2+ release (
Discussion
The most important finding of this study is that, in mice with cardiac-directed expression of C1C2, sustained Iso infusion increases LV function. Seven days of continuous Iso infusion increased Ca2+ release, reduced time to peak Ca2+ release, and reduced rate of Ca2+ decline (Tau) in cardiac myocytes from C1C2 mice, and LV SERCA2a content from C1C2 mice was increased. These favorable alterations in Ca2+ handling provide mechanistic underpinnings for this interesting and unexpected LV response to catecholamine infusion.
LV FUNCTION. There were 2 lines of evidence for C1C2's favorable effects on LV function. First, despite reduced Iso-stimulated cAMP generation in LV from C1C2 transgenic mice, LV systolic and diastolic function were preserved through a wide range of Iso concentrations (
For example, we saw increased Iso-stimulated Ca2+ release (
However, increased Ca2+ uptake and release were seen in cardiac myocytes from C1C2 animals prior to 7 days of Iso infusion (
Why was the increase in LV peak +dP/dt in the C1C2 mice after sustained Iso infusion (
The beneficial effects of C1C2 on LV SER-CA2a expression (
Typically, LV cAMP generating capacity is tightly linked with LV contractile function. In severe HF, LV cAMP production is 50% reduced, and LV contractile function is similarly reduced (3,8). Targeted deletion of AC6 is associated with a 60% reduction in LV cAMP generating capacity, and a proportional reduction in LV contractile function (20). In contrast, C1C2 expression was associated with preserved LV function in response to sustained bAR stimulation despite reduction in cAMP generation. In the present study, C1C2 had favorable effects on SERCA2a expression and Ca2+ release, effects that may have increased LV function, counterbalancing decreased cAMP generation. C1C2 expression did not increase phosphorylation of PLB, troponin I (
C1C2 AND INTRACELLULAR SIGNALING PATHWAYS. C1C2 influences other intracellular signaling path-ways such as CryAB, Akt, ERK1/2 and p38 MAPK pathways (
C1C2 expression was associated with increased LV P-Rex2 expression detected by gene array and confirmed by RT-PCR (
C1C2 expression increased phosphorylation of CryAB at S45 and S59 sites and C1C2 protein, based on co-immunoprecipitation, is physically associated with CryAB (
Because of the complexity of multiple signaling pathways that are relevant in the failing heart, no single study can establish precise molecular pathways for every favorable adaptation. Despite this generic limitation, it is encouraging that cardiac-directed expression of C1C2 has a favorable impact on function of the intact heart in the setting of conditions that mimic aspects of clinical heart failure. The present study examines Iso-stimulated cardio-myopathy only at 7 days, and it remains to be seen whether these protective effects of C1C2 will persist for longer periods and in other models of heart failure.
Conclusions
Increased expression of C1C2 in cardiac myocytes and heart mimics some of the beneficial cardiac effects of AC6. C1C2 enhances Ca2+ handling, which is of mechanistic importance in its beneficial effects. Sustained Iso infusion decreases LV function in control mice, as expected, but increases LV function in C1C2 mice. If these benefits of C1C2 expression translate to clinical settings, one would predict protection of the heart against catecholamine stimulation, a common feature in clinical HF.
Finally, C1C2 is sufficiently small to be inserted in an AAV vector with a regulated expression system. It will be interesting to determine whether transgene delivery of C1C2 via AAV can increase function of the failing heart.
This example demonstrates that methods and compositions as provided herein including C1C2 gene transfer has beneficial effects on the failing human heart, and is effective regardless of ejection fraction (preserved or reduced), and can be used to treat individuals with heart failure.
A fusion protein (C1C2) constructed by fusing the intracellular C1 and C2 segments of adenylyl cyclase type 6 (AC6), retains beneficial effects of AC6 expression, without increasing cAMP generation. For example, transgenic mice with cardiac-directed C1C2 expression have normal left ventricular (LV) function despite reduced cAMP generation. Sustained isoproterenol (Iso) infusion reduces LV function in normal mice. In contrast, C1C2 mice show increased LV function with sustained Iso infusion. The effects of C1C2 expression in pressure-overload is unknown.
METHODS: LV pressure overload was induced by transaortic constriction (TAC) in C1C2 mice and in transgene negative mice. Three weeks after TAC, LV systolic and diastolic function were measured, and Ca2+ handling was assessed in isolated cardiac myocytes.
RESULTS: C1C2 expression reduced LV hypertrophy (p=0.017), increased LV peak pressure development (+dP/dt, p=0.018), and improved LV peak pressure decay (−dP/dt, p=0.038) in the pressure-overload. Cytosolic peak Ca2+ concentration was increased (p=0.047), and time to peak Ca2+ transient and Tau were decreased (p=0.002 and p=0.003, respectively) in cardiac myocytes isolated from pressure-overloaded hearts.
CONCLUSIONS: Cardiac-directed expression of C1C2 improves Ca2+ handling and LV systolic and diastolic function in pressure-overload. These data provide a rationale for further exploration of C1C2 gene transfer as a potential treatment for heart failure.
This example demonstrates that methods and compositions as provided herein including C1C2 gene transfer has beneficial effects on the failing human heart, and cardiac-directed C1C2 expression improves Ca2+ handling and increases LV contractile function in pressure-overload.
Summary:
A fusion protein (C1C2) constructed by fusing the intracellular C1 and C2 segments of adenylyl cyclase type 6 (AC6) retained beneficial effects of AC6 expression, without increasing cAMP generation. The effects of cardiac-directed C1C2 expression in pressure-overload is unknown. LV pressure overload was induced by transverse aortic constriction (TAC) in C1C2 mice and in transgene negative (TG−) mice. Four weeks after TAC, LV systolic and diastolic function were measured, and Ca2+ handling was assessed. Four weeks after TAC, TG− animals showed reduced LV peak +dP/dt. LV peak +dP/dt in C1C2 mice was statistically indistinguishable from that of normal mice and was higher than that seen in TG negative mice 4 w after TAC (p=0.02), despite similar and substantial cardiac hypertrophy. In addition to higher LV peak +dP/dt in vivo, cardiac myocytes from C1C2 mice showed shorter time to peak Ca2+ transient (p=0.002) and a reduced time constant of cytosolic Ca2+ decline (Tau; p=0.003). Sarcomere shortening fraction (p=0.03) and the rate of sarcomere shortening (p<0.02) were increased in C1C2 cardiac myocytes. Myofilament sensitivity to Ca2+ was increased in systole (p=0.02) and diastole (p=0.04) in C1C2 myocytes. These findings indicate enhanced Ca2+ handling associated with C1C2 expression. Favorable effects on Ca2+ handling and LV function were associated with increased LV SERCA2a protein content (p<0.007) and reduced LV fibrosis (p=0.008). Cardiac-directed C1C2 expression improved Ca2+ handling and increased LV contractile function in pressure-overload.
Adenylyl cyclase (AC), a membrane protein in cardiac myocytes and other cells, is the effector molecule in the ß-adrenergic receptor signaling pathway, catalyzing conversion of ATP to cyclic adenosine monophosphate (cAMP). Mammalian AC consists of two transmembrane domains and two cytoplasmic domains (C1 and C2). The C1 and C2 domains form the catalytic core of AC. Many of the beneficial effects of AC6 that we previously have described are independent of cAMP generation and appear, instead, to involve intracellular AC6-protein interactions (3,4). By removal of the two transmembrane domains of AC6 and fusing the two cytoplasmic domains, as illustrated in
These characteristics of C1C2—small size, solubility, retention of regulatory binding sites, and disengagement from cell-surface ß-adrenergic receptor stimulation—make it an attractive way to safely and effectively increase function of the failing heart. A phase 2 randomized clinical trial in patients with symptomatic heart failure (HF) showed that AC6 gene transfer is safe and potentially effective, and not associated with increased cardiac arrhythmias (8). However, a modified AC6 that circumvents production of cAMP could have advantages and is a motivation for the current studies. Indeed, we recently showed that, in mice with cardiac-directed expression of C1C2, sustained isoproterenol infusion increases rather than decreases LV function despite attenuated cAMP generation (5).
Although the isoproterenol infusion model of cardiomyopathy is useful and mimics aspects of clinical HF, to obtain a more complete picture of the potential benefits of LV C1C2 expression, we conducted studies in pressure overload—a major cause of clinical HF. Indeed, before advancing a new therapy to clinical trials, it is imperative to be certain that the strategy is safe and effective in more than a single model of disease. There are striking differences between sustained isoproterenol infusion vs pressure overload—especially when using a transgene expected to reduce cAMP generation, which would not be expected to influence response to pressure stress. In the present study we tested the hypothesis that cardiac-directed expression of C1C2, through adaptations in cytosolic Ca2+ handling, would preserve LV contractile function in the setting of severe pressure stress.
Material and Methods
Generation of C1C2 Fusion Protein:
C1C2 peptide was generated as previously described (5). Briefly, cytoplasmic domain 1 (C1, amino acids 349-576) and cytoplasmic domain 2 (C2, amino acids 939-1157) of mouse adenylyl cyclase type 6 (AC6) were ligated using a linker (12 amino acids: AAAGGIPPAAAM) (SEQ ID NO:8). The C-terminus of the generated C1C2 peptide was linked with an AU1 epitope tag (DTYRYI) (SEQ ID NO:9) to facilitate detection.
Generation of C1C2 Transgenic Mice:
Animals were used with approval by the Institutional Animal Care and Use Committee of VA San Diego Healthcare System. The generation and characterization of the cardiac-directed C1C2 murine lines were described previously (5). We documented variable transgene mRNA expression in 2 lines and used a line with a 23-fold increase in C1C2 mRNA expression (vs. endogenous AC6) in this study. C1C2 protein was 13-fold increased relative to that of the AU1 signal in transgene negative mice. We used 84 mice (40M, 44F) mean age 9±0.1 months, mean weight 31±0.6 grams. To determine mortality, we set aside 34 mice (17 per group) and did no interventions after TAC.
Transverse Aortic Constriction (TAC):
Mice were anesthetized with 5% isoflurane in oxygen (1 L/min), intubated, and ventilated (pressure-controlled). Anesthesia was maintained with 1% isoflurane in oxygen. The chest was entered at the second intercostal space at the left upper sternal border, and a segment of the aortic arch between the innominate and left carotid arteries dissected. A 7-0 silk suture was tied against a 26-gauge needle, which yields a substantial aortic constriction. Collaborators at our institution using these methods documented mean pressure gradients across the constriction of >45 mmHg (9).
Study Design:
Three groups of mice were studied: 1) control mice that did not receive TAC; 2) mice with cardiac-directed C1C2 expression that were studied 4 w after TAC; 3) transgene negative mice that were studied 4 w after TAC.
Echocardiography:
Echocardiography was performed 28±1 days after TAC, using a Vevo 3100™ system with a MX550s (32-56 MHz) transducer (Visualsonics, Toronto, Canada), as previously described (5). Transgene negative mice with no TAC (n=7) served as controls. We previously showed no baseline echocardiographic differences in C1C2 vs transgene negative mice (5). For anesthesia we used 5% isoflurane at a flow rate of 1 L/min oxygen and maintained with 1% isoflurane in oxygen.
Left Ventricular Systolic and Diastolic Function:
Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg). A 1.4 F micromanometer catheter (SPR 839, Millar Instruments, Houston, Tex.) was advanced via the right carotid artery across the aortic valve and into the LV to measure the left ventricular pressure. The data were recorded and analyzed using software IOX 2.9 (Emka Technologies, Christchurch, Va.) as previously reported (6).
Cardiac Myocyte Isolation:
Cardiac myocytes were isolated with the use of modified methods as previously reported (6,11)
Ca2+ Transients and Sarcomere Shortening:
Cytosolic Ca2+ transients in isolated cardiac myocytes were measured using indo-1 AM, as previously described (2,7) with modifications. Cardiac myocytes were plated onto laminin pre-coated glass cover slips and loaded with indo-1 AM (5 μM, Life Technologies, Carlsbad, Calif.) and dispersing agent, pluronic F-127™ (0.02 mg/ml, Life Technologies, Carlsbad, Calif.) for 20 minutes. After dye loading, cover slips were placed in a field stimulation superfusion chamber, and then mounted on an inverted microscope (Nikon TMD, Japan) equipped with a photomultiplier tube-based fluorescence imaging system (PTI, Edison, N.J.). During the measurements, cardiac myocytes were continuously perfused with KRH solution (125 mM NaCl, 5 mM KCl, 1.2 mM NaH2PO4, 6 mM glucose, 1.2 mM MgCl2 and 25 mM HEPES, pH 7.37-7.38) containing 2 mM CaCl2). Cardiac myocytes were field-stimulated at 0.3 Hz with amplitude of 10 volts and pulse width of 10 msec. Excitation wave length was set to 365 nm via a monochromator. Fluorescence emission was slit and directed to two photomultiplier tubes through 20 nm band-pass filters centered at 405 and 485 nm, respectively. The ratio F405/485 represents a measure for [Ca2+]i, and EC50 for sarcomere shortening is calculated based on this ratio. Focusing on a region including several consecutive sarcomeres, simultaneous fractional shortening of sarcomere length was measured using a video edge camera (Flare 2M360-CL™, IO Industries, London, Canada) attached to the fluorescence imaging system. Ca2+ transients and sarcomere length shortening data were analyzed using FelixGX™ software (PTI, Edison, N.J.) and Clampfit™ software (Molecular Devices, Sunnyvale, Calif.).
RT-PCR:
Quantitative reverse transcription polymerase chain reaction (RT-PCR) was conducted to detect the expression level of mRNA of hypertrophy and fibrosis related proteins, as previously described (15).
Immunoblotting:
Western blots were performed to assess the expression of total or phosphorylated proteins from frozen LV samples. Antibodies were used including: SERCA2a (1:500 dilution; ENZO, Exeter, UK); p16-PLB (1:5000 dilution; Badrilla, Leeds, UK); p17-PLB (1:5000 dilution; Badrilla, Leeds, UK); p-TnI (1:500 dilution; Cell Signaling, Danvers, Mass.); p-CaMKII (1:200 dilution; Santa Cruz Biotechnology, Dallas, Tex.); p-PKA (1:1000 dilution; Cell Signaling, Danvers, Mass.); p-PKC (1:1000 dilution; Cell Signaling, Danvers, Mass.); p-Akt (1:1000 dilution; Cell Signaling, Danvers, Mass.); GAPDH (1:20000 dilution; Fitzgerald, Acton, Mass.). Protein quantification was conducted using Image Lab™ software (BIO-RAD, Hercules, Calif.).
Necropsy and Histology:
Body, liver, lung and LV weight (including interventricular septum) were recorded. Transmural sections of the LV were fixed in 10% formalin (Sigma-Aldrich), paraffin-embedded, sliced into 5-μm sections, mounted, and then counterstained with picrosirius red (to evaluate fibrosis) and hematoxylin and eosin. The slides were scanned using an Axio Scan Z1™ slide scanner (Zeiss, Oberkochen, Germany). Quantitative assessment of fibrosis, conducted on picrosirius red-stained sections, was performed using ImageJ™ software (NIH, Bethesda, Md.). The remaining LV was quickly frozen in liquid nitrogen and stored at −80° C.
Statistical Analysis:
Data acquisition and analysis were done without knowledge of group identity. Group sizes were determined by power calculations, and male and female mice were used in equal proportions. The hypothesis was that cardiac-directed expression of C1C2, through adaptations in cytosolic Ca2+ handling, would preserve LV contractile function in the setting of severe pressure stress. Therefore, the key statistical comparison was between C1C2 and TG− mice 4 weeks after TAC, and group differences were tested via Student's t-test (unpaired, two-tailed). In order to confirm that the intervention (TAC) was successful in inducing the expected alterations in LV structure and function, we also included an age- and sex-matched control group of TG− mice that did not undergo TAC. Analyses were performed using GraphPad Prism™ (GraphPad Software, Inc., San Diego, Calif.). The null hypothesis was rejected when p<0.05.
Results:
LV Function and Dimensions
Echocardiography (Table 1).
Ejection fraction fell and LV ESD increased 4 w after TAC to similar degrees in TG− and C1C2 mice. End-diastolic thickness of the posterior wall (p=0.0002) and the interventricular septum (p=0.06) were increased 4 w after TAC, with no between-group differences (TG− vs C1C2).
LV Pressure Development and Decay (Table 2)
It was important to obtain a measure of LV function less load-dependent than ejection fraction—especially in the setting of LV obstruction to ejection imposed by transverse aortic constriction. We therefore measured, using micromanometer catheters, peak LV pressure development, which is less load-sensitive than ejection phase indices of LV function such as ejection fraction. Four weeks after TAC, compared to control mice (no TAC) TG− animals showed reductions in LV peak +dP/dt (p=0.04), while C1C2 mice showed no such reductions (Table 2). These results indicate that C1C2 expression enabled the LV to maintain normal function despite marked pressure overload and LV hypertrophy. Consequently, LV peak +dP/dt was 19% higher than that in TG− mice (p=0.02; Table 2). LV peak −dP/dt showed no group differences, although it tended to be lower after TAC in TG− mice than in C1C2 mice (p=0.09). C1C2 mice tended to have higher LV developed pressure than TG− mice after TAC (p<0.06) (Table 2). There was no group difference in mortality 4 w after TAC placement: TG−: 3 of 17 died; C1C2: 4 of 17 died; p=0.76). The deaths were unwitnessed, and likely cardiac in nature, with pleural fluid observed in most mice.
Ca2+ Handling
Ca2+ transients. To investigate the underlying Ca2+ handling changes, we measured Ca2+ transients in cardiac myocytes isolated 4 w after TAC from C1C2 and TG− mice and from TG− mice that had not undergone TAC. Four weeks after TAC cardiac myocytes from C1C2 mice showed superior Ca2+ handling vs TG− mice. Ca2+ transient amplitude was higher (
Sarcomere shortening. As an additional means to assess cardiac function, we measured sarcomere shortening in cardiac myocytes 4 w after TAC (
Myofilament sensitivity to Ca2+. Using Ca2+ transient measurements and sarcomere shortening, we analyzed myofilament sensitivity to Ca2+. We used Indo-1 F405/485 ratio as an estimate of [Ca2+]i. Cardiac myocytes from mice with cardiac-directed C1C2 expression showed higher Ca2+ sensitivity, defined as reduced Ca2+ concentration required to attain 50% of maximal sarcomere shortening (EC50).
In cardiac myocytes isolated from C1C2 mice 4 w after TAC, EC50 for Ca2+ was lower during contraction (
LV Ca2+ Handling Protein Expression
To determine potential mechanisms underlying the favorable effects of cardiac-directed C1C2 expression in the setting of pressure overload, we used immunoblotting to assess key signaling proteins. Four weeks after TAC, LV samples from C1C2 mice showed a 2.3-fold higher sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) protein content vs TG− mice (p<0.007;
In Table 4: TAC, transverse aortic constriction; No TAC, transgene negative mice; C1C2, transgenic mice with cardiac-directed C1C2 expression; TG−, transgene negative mice; 4 w, 4 weeks. Values are relative expression from LV samples. These data were normalized to expression of the housekeeping gene, hypoxanthine guanine phosphoribosyl transferase (HPRT). Values are mean±SE and reflect relative expression vs control gene; p values from Student's t-test (unpaired, two-tailed).
Histology: Transmural LV samples from C1C2 mice 4 w after TAC showed less fibrosis in C1C2 as compared with TG− mice (
Necropsy (see Table 5): Although there was marked LV hypertrophy associated with TAC (80% increases in LV weight; p<0.0002 vs No TAC), no differences were observed between C1C2 and TG− mice 4 w after TAC. There also was trend toward an increase in lung to body weight ratio of 52% (p=0.06 vs No TAC), but with no C1C2 vs TG− differences (see Table 5).
3 ± .1
6 ± .4
A. Representative sarcomere shortening traces recorded from cardiomyocytes from C1C2 and TG− mice 4 w after TAC. B. Fractional shortening (FS) showed overall differences (p=0.01) and was higher (p<0.03) in cardiac myocytes from C1C2 mice vs TG− mice 4 w after TAC. C. Peak rate of cardiac myocyte sarcomere shortening (+dL/dt) showed overall differences (p=0.002) and a between-group difference (p<0.02) favoring mice with cardiac-directed C1C2 expression. D. Peak rate of sarcomere relaxation (−dL/dt) showed an overall difference (p=0.009) and tended to be more rapid in cardiac myocytes from TG− vs C1C2 mice 4 w after TAC (p=0.06). Data are mean +SE. Numbers in bars denote the number of cardiomyocytes, obtained from 6 mice per group (4-6 cells per mouse, blinded to group identity). P value (upper left corner of each panel) is from 1-Way ANOVA; TG− vs C1C2 p value from Sidak's multiple comparison test with correction.
Discussion
The most important finding of this study is that cardiac-directed expression of C1C2 protects the heart against the deleterious effects of pressure stress. For example, 4 w after TAC, cardiac expression of C1C2 preserved LV systolic function as measured by LV peak +dP/dt. Four weeks after TAC, LV peak +dP/dt was statistically indistinguishable from that of normal mice without TAC and was 19% higher than that seen in TG negative mice 4 w after TAC (p=0.02; Table 2). Thus, despite severe mechanical hindrance to LV ejection, contractile function in C1C2 mice was preserved.
Measurement of the peak rate of pressure development is particularly germane in the setting of aortic constriction because the peak rate occurs prior to opening of the aortic valve and provides information on intrinsic contractile function relatively isolated from the effects of the mechanical obstruction—something that ejection fraction—an ejection-phase index of LV function—does not provide (10). These data suggest an inherent increase in LV contractile function that should be of utility in clinical settings associated with pressure overload such as systemic hypertension. We did not see (nor did we expect) reduction in LV hypertrophy following aortic constriction, because C1C2 expression is not known to influence hypertrophic signaling pathways.
The preservation of LV function in C1C2 mice revealed by the rate of LV pressure development was also demonstrated by other measures. For example, studies of isolated cardiac myocytes showed that the time to peak Ca2+ transient amplitude was shorter in cardiac myocytes from C1C2 mice (p=0.002;
As a further means to explore C1C2's effects, we measured sarcomere shortening in cardiac myocytes isolated 4 w after TAC. These data showed higher fractional shortening (p=0.03;
Previous studies indicated that C1C2 improves Ca2+ handling (5). However, no previous studies included measures of myofilament Ca2+ sensitivity. By examining the relationship between Ca2+ transients and sarcomere shortening at different cytoplasmic Ca2+ concentrations, we determined the effective concentration for 50% of maximal shortening and relaxation. These data show a leftward shift in C1C2 cardiac myocyte compared with cardiac myocytes from TG negative mice, indicating higher myofilament sensitivity to cytosolic Ca2+(
Regarding the molecular mechanism by which C1C2 affects Ca2+ handling, we found increased LV SERCA2a protein content in C1C2 vs TG negative mice (
SERCA2a activity can be regulated by phospholamban (PLB). However, we found no group differences in p16-PLB, p17-PLB, and no change in p-PKA. We previously have shown that cardiac-directed C1C2 increased LV SERCA2a content in the absence of increasing cAMP generation or PLB expression in the setting of sustained ß-adrenergic receptor stimulation (5).
The precise molecular pathway by which C1C2 expression regulates SERCA2a expression will require additional cell-based studies.
Cardiac-directed expression of AC6 in TAC (13), unlike C1C2, increased LV PLB phosphorylation at Ser16. This difference between C1C2 and AC6 expression in TAC may reflect the relative disengagement of C1C2 from the influence of cell-surface β-adrenergic receptor stimulation and reduced cAMP generation—a potential advantage conferred by C1C2 expression over AC6 expression. We previously reported that C1C2 expression reduces isoproterenol-stimulated cAMP generation in LV membranes and in cardiac myocytes (5). C1C2 is found predominantly in the cytoplasm of cardiac myocytes and is, therefore, less accessible to stimulation via cell surface βARs. It is noteworthy that even though LV AC6 expression (and other AC types) is unaltered in C1C2 mice (5), ß-adrenergic receptor mediated cAMP production is impaired, presumably through a dominant negative effect of C1C2 interacting with Gas to reduce endogenous AC responsiveness. An appealing profile for a heart failure therapeutic would include limitation of cAMP generation while promoting more efficient myofilament Ca2+ sensitivity, an effect that we have shown in the present studies (
We previously showed in cultured cardiac myocytes that C1C2 gene transfer affects intracellular signaling pathways such as CryAB, Akt, ERK1/2 and p38 MAPK (5). C1C2 may influence intracellular signaling by interacting with G-protein coupled receptors, Gαs, Gßγ and A-kinase anchoring proteins. Indeed, the C1C2 portion of AC6 is important in targeting AC6 to lipid rafts (17). We reported previously that C1C2 expression in isolated cardiac myocytes increases phosphorylation of Akt at Ser308 and Thr473 and phosphorylation of downstream Akt target proteins (5). However, we did not find increased Akt activation in LV homogenates from C1C2 mice in the present study, which may reflect the inherent limitations of defining signaling events in LV homogenates vs cells or may represent model differences (isoproterenol infusion vs TAC).
There were 10-fold increases compared with normal mice in molecular markers of hypertrophy 4 w after TAC (Table 4), but only ß-MHC showed a C1C2 vs TG− difference, with C1C2 LV samples showing 50% lower values than TG− (p=0.03). Necropsy revealed the expected severe LV hypertrophy (Table 5), similar in both experimental groups. However, LV samples from C1C2 mice 4 w after TAC showed less fibrosis than TG negative mice (p=0.008;
Although we found increased LV SERCA2a in C1C2 mice after TAC, SERCA2a may not be the sole molecular signaling event that gave rise to such striking increases in Ca2+ handling. The failing heart manifests multiple signaling abnormalities, and there is always unavoidable uncertainty using animal models to test therapies that will be useful in clinical settings. Despite these inescapable limitations, it is promising that cardiac-directed expression of C1C2 has such a favorable impact on function of the intact heart in pressure overload, a pathophysiological context frequently encountered in clinical HF. The present study examined pressure overload at 4 weeks. Whether the protective effects of C1C2 will persist for longer periods and in other models of heart failure remains to be seen.
Limitations.
Despite favorable alterations in Ca2+ handling and LV contractile function in C1C2 mice after TAC, no group difference was seen in ejection fraction. However, the model under study uses a mechanical obstruction to LV ejection (aortic constriction), so we did not expect to alter the degree of obstruction by the intervention. The rationale was to determine whether C1C2 expression would change Ca2+ handling and LV contractile function in the setting of pressure overload. Just as severe clinical aortic stenosis has no remedy other than replacement of the aortic valve (surgically or via transcatheter aortic valve replacement), it is not expected that other interventions would remedy a mechanical obstruction to LV outflow.
In conclusion, cardiac-directed C1C2 expression increases LV systolic function and measures of diastolic function in pressure-overload. The mechanism for these benefits resides in increases in many aspects of Ca2+ handling, which result in direct effects on sarcomere shortening and myofilament Ca2+ sensitivity, associated with increased expression of LV SERCA2a. These studies, taken together with the previous demonstration that C1C2 protects the heart from isoproterenol-induced cardiomyopathy, indicate that increasing cardiac content of C1C2 is likely to be of benefit in HF of diverse causes.
A number of embodiments as provided herein have been described. Nevertheless, it can be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application claims the benefit of priority to U.S. Provisional Patent Application Ser. No. 62/555,471, filed Sep. 7, 2017. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
This invention was made with government support under NIH, NHLBI grant no. P01HL066941; and Department of Veteran's Affairs grants: 1101 BX001515 and 1I01 BX003774. The government has certain rights in the invention.
| Number | Date | Country |
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| 2013123094 | Aug 2013 | WO |
| 2014197624 | Dec 2014 | WO |
| 2015150914 | Oct 2015 | WO |
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| 20190264189 A1 | Aug 2019 | US |
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| 62555471 | Sep 2017 | US |
