Patent Application
20140221463
The Sequence Listing written in file 92150—846394_ST25.TXT, created on Oct. 11, 2013, 7,821,616 bytes, machine format IBM-PC, MS Windows operating system, is hereby incorporated by reference.
Heart failure remains one of the leading causes of mortality in the developed world. Whereas the mammalian heart is endowed with certain regenerative potential, endogenous cardiomyocyte proliferation is insufficient for functional heart repair upon injury. Interestingly, non-mammalian vertebrates, such as the zebrafish, can regenerate the damaged heart by inducing cardiomyocyte dedifferentiation and proliferation. By screening regenerating zebrafish hearts Applicants identified miR-99/100 down-regulation as a key process driving cardiomyocyte dedifferentiation. Experimental down-regulation of miR-99/100 in primary adult murine and human cardiomyocytes led to an increase in the number of proliferating cardiomyocytes. AAV-mediated in vivo down-regulation of miR-99/100 after acute myocardial injury in mice induced mature cardiomyocyte proliferation, diminished infarct size and improved heart function. Applicants' study unveils conserved regenerative mechanisms between zebrafish and mammalian cardiomyocytes and represents a proof-of-concept on the suitability of activating pro-regenerative responses for healing the diseased mammalian heart.
In one aspect, a method of modulating proliferation of a cardiomyocyte is provided. The method includes (i) transfecting a cardiomyocyte with a nucleic acid encoding a micro RNA modulator, thereby forming a transfected cardiomyocyte; and (ii) allowing the transfected cardiomyocyte to divide, thereby modulating proliferation of the cardiomyocyte.
In another aspect, a method of modulating proliferation of a cardiomyocyte is provided. The method includes (i) contacting a cardiomyocyte with a small molecule, thereby forming a treated cardiomyocyte; and (ii) allowing the treated cardiomyocyte to divide, thereby modulating proliferation of the cardiomyocyte.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a nucleic acid encoding a micro RNA modulator, wherein the RNA modulator increases cardiomyocyte proliferation thereby treating the myocardial infarction.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a nucleic acid encoding an antagonist of a mir 99 micro RNA and a nucleic acid encoding an antagonist of a let-7a micro RNA, thereby treating the myocardial infarction.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a small molecule, wherein the small molecule increases cardiomyocyte proliferation thereby treating the myocardial infarction.
While various embodiments and aspects of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in the application including, without limitation, patents, patent applications, articles, books, manuals, and treatises are hereby expressly incorporated by reference in their entirety for any purpose.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. See, e.g., Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term “polynucleotide” refers to a linear sequence of nucleotides. The term “nucleotide” typically refers to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of polynucleotides contemplated herein include single and double stranded DNA, single and double stranded RNA (including siRNA), and hybrid molecules having mixtures of single and double stranded DNA and RNA. The terms also encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, and 2-O-methyl ribonucleotides.
A “miRNA” or “microRNA” as provided herein refers to a nucleic acid that forms a double stranded RNA, which double stranded RNA has the ability to reduce or inhibit expression of a gene or target gene when expressed in the same cell as the gene or target gene. The complementary portions of the nucleic acid that hybridize to form the double stranded molecule typically have substantial or complete identity. In one embodiment, a microRNA refers to a nucleic acid that has substantial or complete identity to a target gene and forms a double stranded miRNA. In embodiments, the miRNA inhibits gene expression by interacting with a complementary cellular mRNA thereby interfering with the expression of the complementary mRNA. Typically, the nucleic acid is at least about 15-50 nucleotides in length (e.g., each complementary sequence of the double stranded miRNA is 15-50 nucleotides in length, and the double stranded miRNA is about 15-50 base pairs in length). In other embodiments, the length is 20-30 base nucleotides, preferably about 20-25 or about 24-29 nucleotides in length, e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.
The words “complementary” or “complementarity” refer to the ability of a nucleic acid in a polynucleotide to form a base pair with another nucleic acid in a second polynucleotide. For example, the sequence A-G-T is complementary to the sequence T-C-A. Complementarity may be partial, in which only some of the nucleic acids match according to base pairing, or complete, where all the nucleic acids match according to base pairing.
The terms “protein”, “peptide”, and “polypeptide” are used interchangeably to denote an amino acid polymer or a set of two or more interacting or bound amino acid polymers. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. The terms “non-naturally occurring amino acid” and “unnatural amino acid” refer to amino acid analogs, synthetic amino acids, and amino acid mimetics which are not found in nature.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids or proteins, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (i.e., about 60% identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov/BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. Identity typically exists over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 50-100 amino acids or nucleotides in length, or over the entire length of a given sequence.
The term “gene” means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a “protein gene product” is a protein expressed from a particular gene.
The word “expression” or “expressed” as used herein in reference to a gene means the transcriptional and/or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
The term “heterologous” when used with reference to portions of a nucleic acid or protein indicates that the nucleic acid or protein comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
The term “exogenous” refers to a molecule or substance (e.g., nucleic acid or protein) that originates from outside a given cell or organism. Conversely, the term “endogenous” refers to a molecule or substance that is native to, or originates within, a given cell or organism.
A “vector” is a nucleic acid that is capable of transporting another nucleic acid into a cell. A vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment.
A “viral vector” is a viral-derived nucleic acid that is capable of transporting another nucleic acid into a cell. A viral vector is capable of directing expression of a protein or proteins encoded by one or more genes carried by the vector when it is present in the appropriate environment. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors.
A “cell culture” is an in vitro population of cells residing outside of an organism. The cell culture can be established from primary cells isolated from a cell bank or animal, or secondary cells that are derived from one of these sources and immortalized for long-term in vitro cultures.
The terms “culture,” “culturing,” “grow,” “growing,” “maintain,” “maintaining,” “expand,” “expanding,” etc., when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell is maintained outside the body (e.g., ex vivo) under conditions suitable for survival. Cultured cells are allowed to survive, and culturing can result in cell growth, differentiation, or division. The term does not imply that all cells in the culture survive or grow or divide, as some may naturally senesce, etc. Cells are typically cultured in media, which can be changed during the course of the culture.
The terms “media” and “culture solution” refer to the cell culture milieu. Media is typically an isotonic solution, and can be liquid, gelatinous, or semi-solid, e.g., to provide a matrix for cell adhesion or support. Media, as used herein, can include the components for nutritional, chemical, and structural support necessary for culturing a cell.
The term “derived from,” when referring to cells or a biological sample, indicates that the cell or sample was obtained from the stated source at some point in time. For example, a cell derived from an individual can represent a primary cell obtained directly from the individual (i.e., unmodified), or can be modified, e.g., by introduction of a recombinant vector, by culturing under particular conditions, or immortalization. In some cases, a cell derived from a given source will undergo cell division and/or differentiation such that the original cell is no longer exists, but the continuing cells will be understood to derive from the same source.
The term “transfection” or “transfecting” is defined as a process of introducing a nucleic acid molecule to a cell using non-viral or viral-based methods. The nucleic acid molecule can be a sequence encoding complete proteins or functional portions thereof. Typically, a nucleic acid vector, comprising the elements necessary for protein expression (e.g., a promoter, transcription start site, etc.). Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. For viral-based methods, any useful viral vector can be used in the methods described herein. Examples of viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some aspects, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art.
Expression of a transfected gene can occur transiently or stably in a host cell. During “transient expression” the transfected nucleic acid is not integrated into the host cell genome, and is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is co-transfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Expression of a transfected gene can further be accomplished by transposon-mediated insertion into to the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision.
The terms “inhibitor,” “repressor” or “antagonist” or “downregulator” interchangeably refer to a substance that results in a detectably lower expression or activity level as compared to a control. The inhibited expression or activity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In certain instances, the inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to a control.
The terms “therapy,” “treatment,” and “amelioration” refer to any reduction in the severity of symptoms, e.g., of a neurodegenerative disorder or neuronal injury. As used herein, the terms “treat” and “prevent” are not intended to be absolute terms. Treatment can refer to any delay in onset, amelioration of symptoms, improvement in patient survival, increase in survival time or rate, etc. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. In some aspects, the severity of disease is reduced by at least 10%, as compared, e.g., to the individual before administration or to a control individual not undergoing treatment. In some aspects the severity of disease is reduced by at least 25%, 50%, 75%, 80%, or 90%, or in some cases, no longer detectable using standard diagnostic techniques.
The term “therapeutically effective amount,” as used herein, refers to that amount of the therapeutic agent sufficient to ameliorate a given disorder or symptoms. For example, for the given parameter, a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as “-fold” increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control.
“Subject,” “patient,” “individual in need of treatment” and like terms are used interchangeably and refer to, except where indicated, mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but typically refers to an individual under medical supervision.
Provided herein are methods of modulating proliferation of a cardiomyocyte using micro RNA modulators. A micro RNA modulator as used herein refers to an agent capable of modulating the level of expression of a micro RNA (e.g. let-7a, mir 100). In some embodiments, the micro RNA modulator is encoded by a nucleic acid. In other embodiments, the micro RNA modulator is a small molecule (e.g. a chemical compound, synthetic micro RNA molecule). In some embodiments, the micro RNA modulator decreases the level of expression of a micro RNA compared to the level of expression in the absence of the micro RNA modulator. Where the micro RNA modulator decreases the level of expression of a micro RNA relative to the absence of the modulator, the micro RNA modulator is an antagonist of said micro RNA. In other embodiments, the micro RNA modulator increases the level expression of a micro RNA compared to the level of expression in the absence of the micro RNA modulator. Where the micro RNA modulator increases the level of expression of a micro RNA relative to the absence of the modulator, the micro RNA modulator is an agonist of the micro RNA.
In one aspect, a method of modulating proliferation of a cardiomyocyte is provided. The method includes (i) transfecting a cardiomyocyte with a nucleic acid encoding a micro RNA modulator, thereby forming a transfected cardiomyocyte; and (ii) allowing the transfected cardiomyocyte to divide, thereby modulating proliferation of the cardiomyocyte. In some embodiments, the nucleic acid is a lentiviral vector. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124 or SEQ ID NO:1125. In some embodiments, the nucleic acid is a lentiviral vector. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124 and SEQ ID NO:1125. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1125. In embodiments, the nucleic acid has the sequence as set forth in SEQ ID NO:1124 or SEQ ID NO:1125. In embodiments, the nucleic acid has the sequence as set forth in SEQ ID NO:1124. In embodiments, the nucleic acid has the sequence as set forth in SEQ ID NO:1125.
In other embodiments, the micro RNA modulator is an antagonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA. In some embodiments, the proliferation of the cardiomyocyte is increased compared to a control cardiomyocyte lacking the nucleic acid encoding said RNA modulator. In some embodiments, the micro RNA modulator is an agonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA.
In another aspect, a method of modulating proliferation of a cardiomyocyte is provided. The method includes (i) contacting a cardiomyocyte with a small molecule, thereby forming a treated cardiomyocyte; and (ii) allowing the treated cardiomyocyte to divide, thereby modulating proliferation of the cardiomyocyte. In some embodiments, the proliferation of the cardiomyocyte is increased compared to a control cardiomyocyte lacking the small molecule. In some further embodiment, the small molecule modulates expression of a mir 99 micro RNA-regulated protein, a let-7a micro RNA-regulated protein, a mir 100 micro RNA-regulated protein, a mir 4458 micro RNA-regulated protein, a mir 4500 micro RNA-regulated protein or a mir 89 micro RNA-regulated protein. In other embodiments, the small molecule is a chemical compound. In some embodiments, the small molecule is a synthetic micro RNA molecule. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth in SEQ ID NO:1124 or SEQ ID NO:1125. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth in SEQ ID NO:1124 and SEQ ID NO:1125. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth in SEQ ID NO:1124. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth in SEQ ID NO:1125. In embodiments, the synthetic micro RNA molecule has a nucleic acid sequence as set forth in SEQ ID NO:1124. In embodiments, the synthetic micro RNA molecule has a nucleic acid sequence as set forth in SEQ ID NO:1125.
In other embodiments, the proliferation of the cardiomyocyte is increased compared to a control cardiomyocyte lacking the synthetic micro RNA molecule. In some embodiments, the synthetic micro RNA molecule is an antagonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA. In other embodiments, the synthetic micro RNA molecule is an agonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a nucleic acid encoding a micro RNA modulator, wherein the RNA modulator increases cardiomyocyte proliferation thereby treating the myocardial infarction. In some embodiments, the micro RNA modulator is an antagonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA. In embodiments, the micro RNA modulator is an antagonist of a mir 99 micro RNA and an antagonist of a let-7a micro RNA. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124 or SEQ ID NO:1125. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1125. In embodiments, the nucleic acid includes a nucleic acid sequence as set forth in SEQ ID NO:1124 and SEQ ID NO:1125.
In embodiments, the method includes administering to the subject a therapeutically effective amount of a first nucleic acid and a second nucleic acid, wherein the first nucleic acid encodes an antagonist of a mir 99 micro RNA and the second nucleic acid encodes an antagonist of a let-7a micro RNA. In embodiments, the administering to the subject a therapeutically effective amount of a nucleic acid includes administering a first nucleic acid and a second nucleic acid, wherein the first nucleic acid encodes an antagonist of a mir 99 micro RNA and the second nucleic acid encodes an antagonist of a let-7a micro RNA.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a nucleic acid encoding an antagonist of a mir 99 micro RNA and a nucleic acid encoding an antagonist of a let-7a micro RNA, thereby treating the myocardial infarction.
In another aspect, a method of treating myocardial infarction in a subject in need thereof is provided. The method includes administering to the subject a therapeutically effective amount of a small molecule, wherein the small molecule increases cardiomyocyte proliferation thereby treating the myocardial infarction. In some embodiments, the small molecule modulates expression of a mir 99 micro RNA-regulated protein, a let-7a micro RNA-regulated protein, a mir 100 micro RNA-regulated protein, a mir 4458 micro RNA-regulated protein, a mir 4500 micro RNA-regulated protein or a mir 89 micro RNA-regulated protein. In embodiments, the small molecule modulates expression of a mir 99 micro RNA-regulated protein and a let-7a micro RNA-regulated protein. In some other embodiments, the small molecule is a chemical compound. In other embodiments, the small molecule is a synthetic micro RNA molecule. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth by SEQ ID NO:1124 or SEQ ID NO:1125. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth by SEQ ID NO:1124 and SEQ ID NO:1125. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth by SEQ ID NO:1124. In embodiments, the synthetic micro RNA molecule includes a nucleic acid sequence as set forth by SEQ ID NO:1125. In some embodiments, the synthetic micro RNA molecule is an antagonist of a mir 99 micro RNA, a let-7a micro RNA, a mir 100 micro RNA, a mir 4458 micro RNA, a mir 4500 micro RNA or a mir 89 micro RNA.
Heart failure remains one of the leading causes of mortality in the developed world. Whereas the mammalian heart is endowed with certain regenerative potential, endogenous cardiomyocyte proliferation is insufficient for functional heart repair upon injury. Interestingly, non-mammalian vertebrates, such as the zebrafish, can regenerate the damaged heart by inducing cardiomyocyte dedifferentiation and proliferation. By screening regenerating zebrafish hearts Applicants identified miR-99/100 down-regulation as a key process driving cardiomyocyte dedifferentiation. Experimental down-regulation of miR-99/100 in primary adult murine and human cardiomyocytes led to an increase in the number of proliferating cardiomyocytes. AAV-mediated in vivo down-regulation of miR-99/100 after acute myocardial injury in mice induced mature cardiomyocyte proliferation, diminished infarct size and improved heart function. Applicants' study unveils conserved regenerative mechanisms between zebrafish and mammalian cardiomyocytes and represents a proof-of-concept on the suitability of activating pro-regenerative responses for healing the diseased mammalian heart.
Cardiovascular disease remains the leading cause of mortality in the developed world. Attempts at developing curative strategies have mainly focused on the activation of endogenous cardiac progenitor cells and the transplantation of in vitro-derived cardiomyocytes (A. Aguirre et al., Cell Stem Cell 12, 275-284 (2013); S. R. Braam et al., Trends in pharmacological sciences 30, 536-45 (2009)). More recently, in vivo reprogramming strategies have emerged as potential treatments for heart failure (L. Qian et al., Nature (2012), doi:10.1038/nature11044; K. Song et al., Nature 485, 599-604 (2012) A. Eulalio et al., Nature (2012), doi:10.1038/nature11739). Along this line, a recent report by Porrello et al. has highlighted a remarkable regenerative capacity in neonatal murine hearts upon injury (E. R. Porrello et al., Science (New York, N.Y.) 331, 1078-80 (2011)). Although adult mammalian cardiomyocytes retain a certain ability to proliferate (S. E. Senyo et al., Nature, 2-6 (2012)), endogenous regenerative responses during adulthood are largely insufficient for replenishing the lost cardiac tissue. Noticeably, heart repair can be induced upon manipulation of miRNA pathways identified as drivers of cardiomyocyte proliferation in neonatal murine models (A. Eulalio et al., Nature (2012), doi:10.1038/nature11739L; E. R. Porrello et al., Circulation research 109, 670-9 (2011)). This may suggest that the mechanisms underlying heart regeneration at birth are still present, yet dormant and/or repressed, in adult murine hearts. Other vertebrates, such as the zebrafish, are able, throughout their entire lifetime, to activate endogenous regenerative responses that lead to complete cardiomyocyte-mediated heart regeneration similar to that observed in neonatal mice (R. Zhang et al., Nature (2013), doi:10.1038/nature12322; K. D. Poss et al., Science (New York, N.Y.) 298, 2188-90 (2002); A. Raya et al., Proceedings of the National Academy of Sciences of the United States of America 100 Suppl, 11889-95 (2003); C. Jopling et al., Nature 464, 606-9 (2010); K. Kikuchi et al., Nature 464, 601-5 (2010)). Together, these observations led us to hypothesize on the existence of conserved pro-regenerative pathways between zebrafish and mammals (A. W. Seifert et al., Nature 489, 561-5 (2012)), and if present, whether they could be altered to drive terminally differentiated mammalian cardiomyocytes to a regeneration-competent state.
To first elucidate the presence of conserved regulatory pathways underlying regeneration Applicants decided to focus on microRNAs promoting cardiomyocyte dedifferentiation in the zebrafish. Expression of 90 microRNAs was significantly changed 3 days post amputation (dpa) of the ventricular apex (
Since regeneration might be considered to a large extent redolent of development (J. P. Brockes, A. Kumar, Annual review of cell and developmental biology 24, 525-49 (2008); J. L. Whited, C. J. Tabin, 2-4 (2010); D. Knapp, E. M. Tanaka, Current Opinion in Genetics & Development (2012), doi:10.1016/j.gde.2012.09.006) Applicants next decided to investigate the role of miR-99/100 and their target proteins Fntβ and Smarca5 during zebrafish heart development and maturation. qRT-PCR and immunofluorescence analyses demonstrated low levels of miR-99/100 expression during early heart development concomitantly with high levels of Smarca5 and Fntβ (
In light of the evolutionary conservation of their structures and downstream signaling pathways, Applicants wondered whether microRNA-99/100 and Let-7a/c functions would be similar between mammals and zebrafish. To this end, Applicants first analyzed microRNA-99/100 and FNTβ/SMARCA5 expression in developing and adult murine hearts. qRT-PCR and immunofluorescence analyses highlighted a progressive up-regulation of microRNA-99/100 paralleling cardiac maturation and FNTβ/SMARCA5 down-regulation (
Applicants next sought to evaluate the effects of microRNA down-regulation in adult murine cardiomyocytes. Seven days after shRNA-mediated microRNA silencing significant up-regulation of SMARCA5 and FNTβ was observed accompanied by an increased amount of cardiomyocytes with disorganized sarcomeric structures and immature morphology (
Lastly, Applicants decided to test the efficacy of anti-microRNA delivery for the induction of regeneration in a murine model of myocardial infarction. Following LAD artery ligation, anti-miR-99/100 and anti-Let-7 were administered by injection of serotype 9 adeno-associated viral (AAV) particles, specifically targeting the cardiomyocyte population, in the periphery of the infarcted area. 18 days after treatment, both ejection fraction and fractional shortening significantly improved in the treated group (
These observations constitute a proof-of-concept on how animal models naturally capable of regeneration can be used for the identification of regenerative factors that may, subsequently, be applied to mammals. Experimental manipulation of conserved microRNAs unveiled during adult zebrafish heart regeneration led to similar responses in mice after heart infarction (replenishment of the lost cardiac tissue and inhibition of scar formation). In vivo activation of conserved cardiac regenerative responses may help to circumvent many of the problems associated with heart cell transplantation as well as those associated with reprogramming technologies (A. Aguirre et al., Cell Stem Cell 12, 275-284 (2013), M. a. Laflamme, C. E. Murry, Nature 473, 326-335 (2011)), serving as an additional tool to the clinical armamentarium of regenerative medicine towards the treatment of human heart disease (K. R. Chien et al., Journal of molecular and cellular cardiology 53, 311-3 (2012)).
Detailed experimental procedures can be found in Supplementary information.
Animals.
Wild-type zebrafish (AB) and cmlc2:GFP were maintained at 28.5° C. by standard methods, unless otherwise indicated. All protocols were previously approved and performed under institutional guidelines.
Culture and isolation of adult mouse ventricular myocytes. Wild-type mice (C57B6/J) were sacrificed and hearts were quickly recovered and washed with ice-cold Ca2+-free ModifiedTyrode's Solution (MTS). Ventricles were dissected from the rest of the heart and subjected to enzymatic digestion (Liberase DH, Roche) for 10-15 min in a spinner flask at 37 C under continuous agitation. Afterwards cells were pelleted by short centrifugation, resuspended in KB solution and cardiomyocytes were left to sediment by gravity, thus greatly reducing the presence of other contaminating cell types. Calcium was restore to 1 mM in a step-wise fashion in three gradual steps and subsequently cardiomyocytes were centrifuged, resuspended in culture medium (IMDM 5%, 1% Pen/Strep, 0.1 ng/ml FGFb, 1 ng/ml TGF-β3) and seeded in laminin-coated tissue-culture plates. Cells were kept in culture for 1 week.
Lentiviral and AAV constructs. Anti-miR constructs, miRZip-99/100 and miRZip-let7 (SBI), were used according to the manufacturer instructions. As respective controls, the anti-miRs were removed from the parent vector by digesting with BamHI and EcoRI, end filled and re-ligated. Lentiviruses were packaged by transfecting in 293T cells followed by spinfection in the respective mouse or human ES derived cardiomyocytes. AAVs were generated as described before (Eulalio et al, 2012). Briefly, the antimiR constructs contained in the miRZip vectors were excised and ligated into pZacf-U6-luc-ZsGreen. Serotype 9 AAVs were packaged by transfection of 293T cells with the appropriate plasmids.
Organotypic Heart Slice Culture.
Mice ventricles were washed in cold Modified Tyrode's Solution, embedded in 4% low melting point agarose and immediately cut into 300 μm slices using a vibratome (Leica). Heart slices were then maintained in complete IMDM 5%, 1% Pent/Strep in 12-well plates at the medium-air interface using 0.4 μm membrane transwells (Corning) at 37 C in a 5% CO2 incubator. For experimental hypoxia-like conditions, slices were kept in a hypoxia chamber incubator for 4 hours at 37 C, 5% O2. Lentiviral transduction was performed by immersion of the slices in virus-containing medium for 24 h.
Myocardial Infarction.
Myocardial infarction was induced CD1 mice (8-12 weeks old) by permanent left anterior descending (LAD) coronary artery ligation. Briefly, mice were anesthetized with an injection of ketamine and xylazine, intubated and placed on a rodent ventilator. Body temperature was maintained at 37° C. on a heating pad. After removing the pericardium, a descending branch of the LAD coronary artery was visualized with a stereomicroscope and occluded with a nylon suture. Ligation was confirmed by the whitening of a region of the left ventricle. Recombinant AAV vectors, at a dose of 1011 viral genome particles per animal, were injected immediately after LAD ligation into the myocardium bordering the infarct zone (single injection), using an insulin syringe with incorporated 30-gauge needle. Three groups of animals were studied, receiving AAV9-control (shRNA-Luc), AAV9-antimiR-99/100 or AAV9-anti-Let-7a/c. The chest was closed, and the animals moved to a prone position until the occurrence of spontaneous breathing. BrdU was administered intraperitoneally (500 μg per animal) every 2 days, for a period of ten days. Echocardiography analysis was performed at days 12, 30 and 60 after infarction, as described below, and hearts were collected at 12 (n=6 animals per group) and 60 (n=10 animals per group) days after infarction.
Echocardiography Analysis.
To evaluate left ventricular function and dimensions, transthoracic two-dimensional echocardiography was performed on mice sedated with 5% isoflurane at 12, 30 and 60 days after myocardial infarction, using a Visual Sonics Vevo 770 Ultrasound (Visual Sonics) equipped with a 30-MHz linear array transducer. M-mode tracings in parasternal short axis view were used to measure left ventricular anterior and posterior wall thickness and left ventricular internal diameter at end-systole and end-diastole, which were used to calculate left ventricular fractional shortening and ejection fraction.
Heart Collection and Histological Analysis.
At the end of the studies, animals were anaesthetized with 5% isoflurane and then killed by injection of 10% KCl, to stop the heart at diastole. The heart was excised, briefly washed in PBS, weighted, fixed in 10% formalin at room temperature, embedded in paraffin and further processed for histology or immunofluorescence. Haematoxylin-eosin and Masson's trichrome staining were performed according to standard procedures, and analysed for regular morphology and extent of fibrosis. Infarct size was measured as the percentage of the total left ventricular area showing fibrosis.
Zebrafish Heart Amputation.
Adult fish were anaesthetized in 0.4% Tricaine and secured, ventral side uppermost, in a slotted sponge. Watchmaker forceps were used to remove the surface scales and penetrate the skin, muscle and pericardial sac. Once exposed, the ventricle was gently pulled at the apex and cut with iridectomy scissors. After surgery, fish were immediately returned to system water.
Cryosectioning.
At the specified time points, hearts were removed, washed in PBS-EDTA 0.4% and fixed for 20 min in 4% paraformaldehyde at 4° C. Afterwards, they were washed several times in PBS, equilibrated in 30% sucrose, and then frozen for cryosectioning. 10 μm slices were obtained with a cryostat (Leica).
Real Time RT-PCR.
For RNA, tissue was obtained from adult heart ventricles from different time points and conditions, extensively washed in PBS-EDTA 0.4% to remove blood, and then mechanically homogenized and processed using RNeasy kit (Qiagen) as per manufacturer's instructions. RT and PCR were performed using Quantitect Reverse Transcription Kit (Qiagen) and Quantitect Primers for the following genes: Fntb, Fntα, Smarca5, myc-a, myc-b, H-rasa, H-rasb, N-ras, K-ras, tnnt2. For miRNAs, small RNA (<200 pb) was obtained employing the miRNeasy mini kit (Qiagen) using the same procedure as before. RT and PCR reactions were carried out employing miRCURY LNA RT and PCR kits (Exiqon) and stem-loop LNA primers (Exiqon).
MicroRNA microarrays. RNA was obtained as for PCR applications. GenechipmiRNA 2.0 microarrays were purchased from Affymetrix and small RNA labeling was performed using FlashTag HSR labeling kit (Genesphere). 200 ng of small RNA was polyA-tailed and biotin conjugated. After labelling, RNA was hybridized using GeneChip reagents (Affymetrix) and protocols as indicated by the manufacturer. The chip contains hybridization probes for the miRbase v15 annotations, including 248 zebrafishmiRNAs. MicroRNA data was analyzed by using the R package.
Bioinformatic Analysis of miRNA Targets.
Signaling pathways and downstream target prediction related to the identified miRNAs were determined by using DIANA, Miranda and TargetScan. Gene ontology analysis was performed with DAVID software.
Fluorescence In Situ Hybridization.
10 μm heart slices were further fixed in 4% PFA for 10 min at room temperature, washed in PBS and acetylated for 10 min in acetylation solution. After washing in PBS, samples were treated with proteinase K, prehybridized for 4 h and hybridized overnight at the appropriate temperature with LNA DIG-labeled probes for the corresponding miRNAs (Exiqon). The next day slides were washed and immunolabeled with anti-DIG-alkaline phosphatase antibodies (1:2,000) and antibodies against cardiomyocytic proteins of interest (1:100) overnight at 4° C. Secondary antibody incubation was performed as for immunofluorescence experiments. Alkaline phosphatase activity was detected by incubating samples in a Fast Red solution (Dako) for 2 hours. Samples were then washed, mounted in Vecta-shield and imaged in a confocal microscope. Fast Red fluorescence was detected with Cy3 settings.
Immunofluorescence.
Tissue slices were fixed for 15 min in 4% paraformaldehyde, washed in PBS-gly 0.3 M, and blocked in PBS-10% donkey serum, 0.5% TX-100, 0.5% BSA for 1 hour. Primary antibodies were diluted at the appropriate concentrations in PBS-1% donkey serum, 0.5% TX-100, 0.5% BSA and incubated overnight. After washing, slices were incubated overnight with secondary antibodies, washed and mounted in Vecta-shield. Antibodies employed are listed in table S3.
Cell Culture.
COS7 cells were maintained in DMEM (high glucose) supplemented with 10% FBS, L-Glutamine and non-essential amino acids (Invitrogen). Human ES cells, H1 and H9 (WA1 and WA9, WiCell), were cultured in chemically defined hES/hiPS growth media, mTeSR1 on growth factor reduced matrigel (BD biosciences) coated plates. Briefly, 70-80% confluent hES/iPS cells were treated with dispase (Invitrogen) for 7 minutes at 37° C. and the colonies were dispersed to small clusters and lifted carefully using a 5 ml glass pipette, at a ratio of ˜1:4.
Culture and Isolation of Adult Mouse Ventricular Myocytes.
Wild-type mice (C57B6/J) were sacrificed and hearts were quickly recovered and washed with ice-cold Ca2+-free ModifiedTyrode's Solution (MTS). Ventricles were dissected from the rest of the heart and subjected to enzymatic digestion (Liberase DH, Roche) for 10-15 min in a spinner flask at 37 C under continuous agitation. Afterwards cells were pelleted by short centrifugation, resuspended in KB solution and cardiomyocytes were left to sediment by gravity, thus greatly reducing the presence of other contaminating cell types. Calcium was restore to 1 mM in a step-wise fashion in three gradual steps and subsequently cardiomyocytes were centrifuged, resuspended in culture medium (IMDM 5%, 1% Pen/Strep, 0.1 ng/ml FGFb, 1 ng/ml TGF-β3) and seeded in laminin-coated tissue-culture plates. Cells were kept in culture for 1 week.
Differentiation of Human ES Cells to Immature Cardiomyocytes.
Human ES cells grown on matrigel dots (BD biosciences) were carefully dissociated using dispase and were plated on low attachment plates in EB media (IMDM, 20% FBS, 2.25 nM L-Glutamine and non-essential aminoacids). After 6 days of suspension in culture, the EBs were seeded on gelatin-coated plates in EB media. Spontaneously beating EBs were manually picked and used for further analysis. For directed differentiation, human ES cells grown in mTeSR on matrigel coated plates were treated with 12 μM GSK3β inhibitor CHIR 99021 (Stemgent) in cardiomyocyte differentiation base media (RPMI 1640 supplemented with 125 μg/ml human holo-transferrin (Sigma-Aldrich)) for 24 hours, followed by 24 hour of rest in the base media. On day 3, the cells were treated with 5 μM WNT inhibitor, IWP4 (Stemgent) for 48 hours, followed by treatment with Cardiac differentiation base media supplemented with 20 μg/ml human Insulin (SAFC) until colonies started beating.
Lentiviral Constructs.
Anti-miR constructs, miRZip-99/100 and miRZip-let7 (SBI), were used according to the manufacturer instructions. As respective controls, the anti-miRs were removed from the parent vector by digesting with BamHI and EcoRI, end filled and re-ligated. Lentiviruses were packaged by transfecting in 293T cells followed by spinfection in the respective mouse or human ES derived cardiomyocytes.
Luciferase Constructs and microRNA Binding Validation.
3′ UTR of human and zebrafish FNTB and SMARCA5 were amplified with the indicated primers using genomic DNA as a template and were cloned into PGL3 vector (Promega) at the XhoI site downstream of luciferase gene. COS7 cells (seeded at 3×104 cells per well of a 12 well plate and grown for 24 hours) were transfected with 50 ng each of indicated luciferase reporter vectors, pRL TK (Renilla luciferase control vector, Promega) either in the presence or absence of 20 nM or 40 nM of double stranded DNA oligonucleotide mimics of miR-99 or miR-100 (Dharmacon) using Lipofectamine (Invitrogen) following manufacturer's protocol. 12-16 hours post-transfection, cells were lysed using passive lysis buffer (Promega). Luminescent signals arising from the cell lysates obtained 12 hours post transfection of COS7 cells with appropriate luciferase constructs were measured using the Dual Luciferase assay system (Promega) in a Synergy H1 hybrid reader (BioTek). The relative luminescence intensity of each sample was calculated after normalization with corresponding Renilla luciferase activity, and were represented as % values compared to the corresponding sample without the miR mimic.
Confocal Microscopy.
Samples were imaged using a Zeiss L710 confocal microscope. For every sample, at least two different fields were examined at two different magnifications (using a 20× objective and a 63× oil-immersion objective). Z-stacks were obtained for further analysis and 3D reconstruction. For intensity comparison purposes, images were taken with the same settings (pinhole size, laser intensity, etc).
Organotypic Heart Slice Culture.
Mice ventricles were washed in cold Modified Tyrode's Solution, embedded in 4% low melting point agarose and immediately cut into 300 μm slices using a vibratome (Leica). Heart slices were then maintained in complete IMDM 5%, 1% Pent/Strep in 12-well plates at the medium-air interface using 0.4 μm membrane transwells (Corning) at 37 C in a 5% CO2 incubator. For experimental hypoxia-like conditions, slices were kept in a hypoxia chamber incubator for 4 hours at 37 C, 5% O2. Lentiviral transduction was performed by immersion of the slices in virus-containing medium for 24 h.
Morpholino and microRNA Injections in Zebrafish Embryos.
Morpholinos (Gene Tools) were dissolved in water at a 2 mM stock concentration and diluted to a 2 ng/nl working concentration in PBS/phenol red solution. Embryo injections were performed by injecting ˜1 nl morpholino solution at the 1-cell stage using a FemtoJet (Eppendorf). For microRNA mimic injection, a miR-99/100 equimolar mixture at 2 ng/nl in PBS was employed. Morphants were evaluated at 24, 48 and 72 h in a StereoLumar stereoscope (Zeiss).
In Vivo microRNA Delivery.
MicroRNA siRNA mimics without chemical modifications were purchased from Life Technologies, dissolved in nuclease-free water and complexed to jetPEI (10 N/P ratio) for in vivo, intra-cardiac administration. 0.2 μg siRNA was injected per animal every 2 days. To determine the efficiency of the delivery, a control Cy5-labeled siRNA directed against GFP was used in cmlc2:GFP animals. MicroRNA inhibitors against the miR-99/100 family were purchased from Exiqon and used at 0.2 μg siRNA per animal every 2 days.
Tipifarnib Injections.
Tipifarnib was dissolved in DMSO at 10 mg/ml and 2 μl were administered by intraperitoneal injection (final concentration 0.02 mg/animal) every 2 days for 14 days. Control animals were administered DMSO.
BrdU Labeling.
Fish were anaesthetized in 0.4% Tricaine, and 10 μl of a 10 mg/ml solution of BrdU (in PBS) was injected into the abdominal cavity once every 2 days for 14 days. At that point, hearts were removed and fixed overnight in 4% paraformaldehyde at 4° C., washed in PBS, equilibrated in 30% sucrose in PBS and frozen for cryosectioning.
Histology and Histomorphometry.
Masson's trichrome staining was performed in 10 μm tissue slices by immersion in Bouin's fixative followed by sequential incubation in Weigert's hematoxylin, Acid Fuchsin, phosphotungstic/phosphomolybdic acid, Aniline Blue and acetic acid. After washes, slices were mounted for bright field observation. Histomorphometric measurements were performed with Fiji. Injured areas were quantified in four independent different slices per animal (four animals were used per condition) and normalized to whole tissue area.
Statistical Analysis.
Results are expressed as mean±SEM. Statistical significance was determined by Student's t-test. Results are representative of at least 3 independent experiments except when otherwise indicated.
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This application claims the benefit of U.S. Provisional Appl. No. 61/712,701, filed Oct. 11, 2012, the contents of which are incorporated herein by reference and for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61712701 | Oct 2012 | US |
