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  • C12N2501/999—Small molecules not provided for elsewhere

Definitions

• Heart disease is the number one killer of men and women worldwide.
• heart tissue has a limited capacity for regeneration or self-renewal. After a patient recovers from a myocardial infarction, the organ bears a scar and heart function is diminished. The ability to regenerate damaged organs such as the heart remains elusive. As such, there is a pressing need in the art to develop new strategies for the regeneration of damaged organs.
• compositions and methods for cellular reprogramming comprising one or more miRs comprising a nucleotide sequence having at least 80% sequence identity to miR-l, miR-l26, miR-l33, miR- l33a, mir-206, miR-208, miR-499, mir-499-5p, and combinations thereof; and an activator of NFKB.
• compositions including an effective amount of the composition of claim 1 and one or more pharmaceutically acceptable carriers, excipients, or diluents.
• Another aspect of the invention is a method for enhancing or upregulating cardiomyocyte maturation in a cell comprising contacting the cell with an effective amount of any of the compositions described herein for a sufficient time such that the cell is reprogrammed into a cardiomyocyte.
• Another aspect of the invention is a method of enhancing or upregulating cardiomyocyte maturation in a subject comprising administering (i) an effective amount of any of the compositions described or (ii) any of the pharmaceutical compositions comprising the effective amount of any of the compositions described and one or more pharmaceutically acceptable carriers, excipients, or diluents.
• Another aspect of the invention is a method for inhibiting or downregulating cardiomyocyte maturation in a cell comprising contacting the cell with an effective amount of a composition comprising a TLR3 inhibitor, a NFKB inhibitor, a ikbkb inhibitor, or a combination thereof for a sufficient time such that cardiomyocyte maturation is inhibited or down-regulated in the cell.
• FIGS lA-lEiii show TLR3 inhibition inhibits maturation of reprogrammed fibroblasts into cardiomyocytes.
• Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with either vehicle or the TLR3 pharmacological inhibitor CU-CPT-4a ( 1 OmM) for a further 4 days. After incubation with the TLR3 pharmacological inhibitor, cells were cultured in normal growth media for a further 6 days. Quantitative PCR was used to analyze mRNA levels of 13 components of the cardiomyocyte sarcomere.
• Figures IBi-lCiii show neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with either vehicle or the TLR3 pharmacological inhibitor CU-CPT-4a (IOmM) for a further 4 days. After incubation with the TLR3 pharmacological inhibitor, cells were cultured in normal growth media for a further 10 days.
• CU-CPT-4a ILR3 pharmacological inhibitor
• Figures ICi and ICii show cells provided miR combo (Fig. lCi) or miR combo and TLR3 antagonist (Fig. lCii) fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar 50 microns.
• Figure ICiii shows quantification of immunostaining.
• Cells expressing Actn2 were counted and expressed as a percentage of the total cell population.
• N 6 independent experiments.
• Figures ID -lEiii show neonatal cardiac fibroblasts were first transfected with either a control siRNA or a siRNA that targeted TLR3. Two days later, the cells were transfected again with either the negative control miR negmiR or miR combo. The day after transfection with miRNAs the media was replaced and the cells cultured in normal growth media for 14 days.
• Figures 2Ai-2Biv show neither TLR3 inhibition nor TLR3 activation affects early stage cardiac reprogramming.
• Figures 3Ai-3Diii show NFKB is important for miR combo reprogramming.
• Figures 3Ai-3Biii show neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with vehicle or the NFKB antagonist Bay 11-7085. After one day of treatment, the media was replaced with normal growth media and cells cultured for a further 12 days.
• FIG. 3Ai-3Aiii RNA levels of the cardiomyocyte structural proteins Myh6 (amyosin heavy chain) (Fig. 3Ai), Actn2 (asarcomeric actinin) (Fig. 3 Aii), and Tnni3 (cardiac troponin-I) (Fig. 3 Aiii) following treatment with the NFKB antagonist Bay 11-7085 was determined by qPCR. N 3 independent experiments.
• Figures 3Bi-3Bii shows cells provided miR combo (Fig. 3Bi) or miR combo and NFKB inhibitor (Fig. 3Bii) were fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar 100 microns. Inset pictures are at 5x magnification.
• Figures 3Biii shows quantification of immunostaining.
• Cells expressing Actn2 were counted and expressed as a percentage of the total cell population.
• N 3 independent experiments.
• Figures 3C-3Diii show neonatal cardiac fibroblasts were transfected with microRNAs (negmiR or miR combo) and siRNA (control siRNA or a siRNA that targeted Ikbkb). The day after transfection with miRNAs the media was replaced and the cells cultured in normal growth media for either 4 days (to assess knockdown efficiency) or 14 days (to assess RNA levels of cardiomyocyte structural proteins).
• FIGS 3Di-3Diii show RNA levels of the cardiomyocyte structural proteins Myh6
• Figures 4A-4DU show RelA mediates the effects of NFKB.
• Neonatal cardiac fibroblasts were first transfected with either a control siRNA or a siRNA that targeted the NFKB subunit RelA. Two days later, the cells were transfected again with either the negative control miR negmiR or miR combo. The day after transfection with miRNAs, the media was replaced and the cells cultured in normal growth media for 13 days.
• Figure 4A shows quantification of RelA knockdown by qPCR.
• Figures 4Bi-4Bii show cells provided miR combo and control siRNA (Fig. 4Bi) and miR combo + RelA siRNA (Fig. 4Bii) fixed and stained with anti-Actn2 antibodies (red). Nuclei were stained with DAPI (blue). Scale bar 100 microns. Inset pictures are at 5x magnification.
• Figure 4Biii shows quantification of immunostaining.
• Cells expressing Actn2 were counted and expressed as a percentage of the total cell population.
• N 3 independent experiments.
• Figures 5Ai-5Aviii show neonatal cardiac fibroblasts were transfected with negmiR or miR combo. After 7 days, chromatin DNA was subjected to ChIP analysis. Primers were designed to target the first lKb of the indicated cardiomyocyte sarcomere genes Actn2 (Fig. 5Ai), Myh6 (Fig. 5 Aii), Mypn (Fig. 5 Aiii), Tnni3 (Fig. 5Aiv), Ttn (Fig. 5Av), Myoz2 (Fig. 5Avi), Tnncl (Fig. 5Avii), and Tnnt2 (Fig. 5Aviii), (represented by -OlKb).
• FIGS 6A-6B show microRNAs activate TLR3.
• Figure 6A shows neonatal cardiac fibroblasts were transfected with negmiR or miR combo.
• FIGS 7Ai-7Dii show TLR3 agonists enhance maturation of miR combo reprogrammed cardiomyocytes.
• Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with vehicle or the TLR3 agonist Poly(LC) LMW (low molecular weight Poly(LC)) for a further 4 days. After incubation with the TLR3 agonist, cells were cultured in normal growth media for a further 10 days.
• TLR3 agonists enhance maturation of miR combo reprogrammed cardiomyocytes.
• Neonatal cardiac fibroblasts were transfected with negative control miR (negmiR) or miR combo. The day after transfection media was replaced and the cells incubated with vehicle or the TLR3 agonist Poly(LC) LMW (low molecular weight Poly(LC)) for a further 4 days. After incubation with the TLR3 agonist, cells
• Figure 7C show quantification of immunostaining. Cells expressing Actn2 were counted and expressed as a percentage of the total cell population.
• Figures 8A-8D show ICR2-activated cardiomyocyte maturation evaluated via qPCR by measuring the expression of Actn2 (Fig. 8 A), Myh6 (Fig. 8B), Tnni3 (Fig. 8C), and Cacnalc (Fig. 8D).
• Figures 9A-9D shows ICR4-activated cardiomyocyte maturation evaluated via qPCR by measuring the expression of Actn2 (Fig. 9A), Myh6 (Fig. 9B), Tnni3 (Fig. 9C), and Cacnalc (Fig. 9D).
• Figure 10 shows high doses of miR combo impair normal cellular functions by measuring the change in cell number.
• Figures 11A-11B show miR combo mediated maturation with ICR2 (Fig. 11 A) and PolylC (Fig. 11B) evaluated by antibody staining for a-sarcomeric actinin.
• Cardiomyocyte maturation may be enhanced or upregulated by agonists of the innate immune system, such as pattern recognition receptor agonists, or inhibited or downregulated by antagonists. As demonstrated in the Examples, cardiomyocyte maturation may be effectively controlled in committed cellular precursors to accelerate or retard maturation via pattern recognition receptors and associated signaling pathways.
• PRRs Protein recognition receptors
• PAMPs pathogen-associated molecular patterns
• DAMPs damage-associated molecular patterns
• PAMPs activate immune responses by identifying exogenous molecules.
• exemplary PAMPs include, without limitation, nucleic acids, bacterial lipopolysaccharides, endotoxins, bacterial flagellin, lipoteichoic acid, peptidoglycan, and unmethylated CpG motifs. Induction of the immune response to one or more exogenous molecules assists with the prevention or recovery from infection.
• “Damage-associated molecular patterns” or“DAMPs” activate immune responses by identifying host molecules.
• Exemplary DAMPs include, without limitation, nuclear or cytosolic proteins released outside the cell and nucleic acids.
• Toll-like receptors are a subset of PRRs.“Toll-like receptors” or“TLRs” are a class of extracellular, membrane-bound PPRs that share a common structural motif of a leucine-rich repeat. TLRs interacting with PAMPs or DAMPs trigger signaling through NFKB resulting in the increase of inflammatory cytokines.
• the TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13.“Cytokines” include broad category of proteins, typically between about 5 to about 20 kDa, that are involved with cell signaling, such as chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors.
• NFKB or“nuclear factor kappa-light-chain-enhancer of activated B cells” is a protein complex that controls transcription of DNA, cytokine production, and cell survival. NFKB is important in regulating cellular responses because it belongs to the category of "rapid-acting" primary transcription factors, i.e., transcription factors that are present in cells in an inactive state and do not require new protein synthesis in order to become activated. Proteins of the NFKB family share a Rel homology domain in their N-terminus. A subfamily of NF-kB proteins, including RelA, RelB, and c-Rel, have a transactivation domain in their C-termini. In contrast, the NF-KB1 and NF-KB2 proteins are synthesized as large precursors, pl05, and plOO, which undergo processing to generate the mature NF-kB subunits, p50 and p52, respectively.
• NFKB may be activated by PAMPs and DAMPs as well as other heterologous compounds, including heterologous nucleic acids.
• An“activator” is a substance that increases the activity of an enzyme.
• An“activator of NFKB” is a substance that increase the activity of NFKB.
• Activators of NFKB may interact with cells to increase the activity of NFKB through various mechanisms, including, but limited to interaction with various PPRs such as TLRs or, more specifically, TLR3.“TLR3” or“Toll-like receptor 3” is a transmembrane protein encoded by the TLR3 gene that is a member of the toll-like receptor family of PRRs of the innate immune system.
• TLR3 recognizes nucleic acids, such as dsRNA associated with viral infections, and induces the activation of NFKB. AS demonstrated in the Examples that follow, NFKB activation or inhibition may be effectively used to accelerate or retard cardiomyocyte maturation.
• a first aspect of the invention is compositions and methods for enhancing or upregulating cardiomyocyte maturation via the direct reprogramming of precursor cells.
• Reprogramming compositions for enhancing or upregulating cardiomyocyte maturation comprise (i) one or more miRs comprising a nucleotide sequence having at least 80% sequence identity to miR-l, miR- 126, miR-l 33, miR-l 33a, mir-206, miR-208, miR-499, mir-4995p, and combinations thereof and (ii) an activator of NFKB.
• the activator of NFKB may be a TLR-pathway agonist, suitably a TLR3-pathway agonist.
• A“TLR-pathway agonist” is a composition or substance capable of interacting a TLR receptor or a substance associated with a TLR pathway that induces a biological response.
• A“TLR3-pathway agonist” is a TLR-pathway agonist that is capable of interacting with TLR3 or a substance associated with a TLR3 pathway that induces a biological response.
• A“miR”, also known as“miRNA” or“microRNA”, is a small non-coding RNA typically comprising RNA having between about 15 to about 25 nucleotides. Some miRs are capable of folding back onto themselves to resemble dsRNA. miR-l, miR- 126, miR- 133, miR-l33a, mir- 206, miR-208, miR-499, mir-499-5p may be capable activating NFKB.
• miR-l, miR- 126, miR- 133, miR-l 33a, mir-206, miR-208, miR-499, mir-499-5p, or combinations thereof may be suitable for use activating NFKB
• activator of NFKB excludes miR-l, miR- 126, miR- 133, miR-l 33a, mir-206, miR-208, miR-499, mir-499-5p, or any combination thereof.
• the use of miRs for direct reprogramming of cells to cardiomyoctes and cardiomyocytic tissue is described in US Patent Pub. No. 2014/0011281, published Jan. 9, 2014, and US Patent Pub. No. 2018/0042969, the contents of which are incorporated herein by reference in its entirety.
• oligomeric compounds range in size from 50-90 nucleotides in length (or any length within that range, with an average length of approximately 70 nucleotides), and exemplary mature oligonucleotide compounds are 17 to 25 subunits in length, e.g., oligomeric compounds are 17, 18, 19, 20, 21, 22, 23, 24 or 25 subunits in length.
• a stem-loop precursor is approximately 70 nucleotides and the mature nucleotide product is approximately 22 nucleotides in length.
• the uncapitalized“mir-” refers to the pre-miRNA, while a capitalized“miR-” refers to the mature form.
• a pre-microRNA comprises a stem-loop secondary structure.
• STEM-LOOP SEQ ID NO: 3
• STEM-LOOP SEQ ID NO: 5
• STEM-LOOP SEQ ID NO: 7
• STEM-LOOP SEQ ID NO: 9
• the one or more miRs contacting cells are present in an amount less than about 0.30 mM, suitably less than or equal to about 0.28 mM, 0.26 mM, 0.24 nM, 0.22 mM, 0.20 mM, 1.8 mM, 1.6 mM, 0.14 mM, 0.12 mM, or 0.10 mM.
• the one or more miRs may be suitably selected from a variety of miRs, including one or more nucleotide sequences having at least 80% sequence identity to miR-l, miRl26, miR-l33, miR-l33a, mir206, miR-208, miR-499, and mir-499-5p.
• the one or more MiRs may comprise a nucleotide sequence having at least 85%, 90%, 95% or more sequence identity to miR-l, miRl26, miR-l33, miR-l33a, mir206, miR-208, miR-499, and mir-499-5p.
• a combination more than one miR may include any two, any three, or any four miRs having a nucleotide sequences having at least 80%, 85%, 90%, 95% or more sequence identity to miR-l, miRl26, miR-l33, miR-l33a, mir206, miR-208, miR-499, and mir-499-5p.
• the combination may include four miRs having at least 80%, 85%, 90%, 95% or more sequence identity to miR-l, miR-l33a, miR208, and mir-499-5p.
• the combination may include four miRs consisting essentially of miR-l, miR-l33a, miR208, and mir-499-5p.
• the one or more miRs comprise mirl; mirl, mirl33a, and mir208; mirl, mirl33a, and mir206; mirl, mirl33a, mir208, and mir499-Sp; mirl, mirl33a, mir206, and mir499-Sp; mirl and mirl33; mirl and mirl38; mirl and mir206; mirl and mir208; mirl33 and mirl38; mirl33 and mir206; mirl33 and mir208; mirl38 and mir206; mirl38 and mir208; mir206 and mir208; mirl, mirl38, and mir208; mirl, mir206, and mir208; mirl38, mir206, and mir208; mirl, mirl33, and mir206; mirl, mirl33, and mir208; mirl, mirl33, and mir208; mirl, mirl38, and mir206; mirl, mirl33, and mir206; mirl, mirl33, and mir208; mirl, mirl38, and mir206; mirl33,
• the one or more miRs consist essentially of mirl; mirl, mirl33a, and mir208; mirl, mirl33a, and mir206; mirl, mirl33a, mir208, and mir499-Sp; mirl, mirl33a, mir206, and mir499-Sp; mirl and mirl33; mirl and mirl38; mirl and mir206; mirl and mir208; mirl33 and mirl38; mirl33 and mir206; mirl33 and mir208; mirl38 and mir206; mirl38 and mir208; mir206 and mir208; mirl, mirl 38, and mir208; mirl, mir206, and mir208; mirl38, mir206, and mir208; mirl, mirl33, and mir206; mirl, mirl33, and mir206; mirl, mirl33, and mir208; mirl, mirl38, and mir206; mirl, mirl33, and mir206; mirl, mirl33, and mir208; mirl, mirl38, and mir206; mir
• “miR combo” is a combination of mirl, mirl33a, mir208, and mir499-5p while“negmiR” is a miRNA that does not target TLR3 and used as a negative control.
• the composition comprises an activator of NFKB such as a TLR agonist or TLR3 agonist.
• the TLR3 agonist may comprise an RNA composition such as a 5’-triphospate, 2’-fluoro modified non-linear RNA.
• the 5’-triphospate, 2’-fluoro modified non-linear RNA comprises 2’-fluoro modified pyrimidines or 2’-fluoro modified purines.
• the 2’-fluoro modification may be present on at least one pyrimidine or purine, and may be present on any number of pyrimidines or purines, including all of the pyrimidines, all of the purines, or all of the pyrimidines and purines.
• the 2’-fluoro-modification is present in 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the pyrimidines and/or purines or any range therebetween.
• the 2-fluoro modification may be present on a uridine, a cytidine, a guanine, an adenine, or any combination thereof. In some embodiments, only uridines are 2’-fluoro modified.
• all of the uridines in the RNA are 2’-fluoro-modified, all of the cyti dines in the RNA are 2’-fluoro-modified, all of the guanines in the RNA are 2’-fluoro-modified, all of the adenines in the RNA are 2’-fluoro-modified, or any combination thereof.
• 5’-triphospate, T - fluoro modified non-linear RNA is described in International Pub. No. 2018/187328, published Oct. 11, 2013, the contents of which are incorporated herein by reference in its entirety.
• the RNA compositions may comprise phosphorothioate modified nucleotides where a sulfur atom is substituted for a non-bridging oxygen of the phosphate.
• the phosphorothioate modification is present in 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the nucleotides or any range therebetween.
• the last 3 to 5 nucleotides at the 5'- and/or 3'-end of the oligonucleotide are phosphorothioate modified.
• all of the nucleotides of the oligonucleotide are phosphorothioate modified.
• RNA compositions may comprise a blunt-end stem loop, a stem-loop having a 5’- overhang, a stem -loop having a 3’ -overhang, or both a 5’ -overhang and a 3’ -overhang.
• Blunt-end stem loops comprise a 5’-terminal nucleotide and its 3’-terminal complement that are capable of hybridizing with each other, forming the stem-loop.
• Stem-loops having only a 5’-overhang comprise a 3’-terminal nucleotide capable of hybridizing with its complement to form the stem loop.
• Stem-loops having only a 3’-overhang comprise a 5’-terminal nucleotide capable of hybridizing with its complement to form the stem loop.
• stem-loops having both a 5’- overhang and a 3’ -overhang neither the 5’ -terminal nucleotide nor the 3’ -terminal nucleotide form a part of the stem-loop.
• a 5’- or 3’ -overhang may be any length that allows for the RNA composition to inhibit cell growth or induce cell death.
• the 5’- and/or 3’-overhang may be about 1 to about 50 nucleotides in length.
• the 5’- and/or 3’ -overhang is about 1 to about 10 nucleotides in length, including lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides or any range of lengths therebetween.
• the 5’- and/or 3’-overhang is about 10 to about 50 nucleotides in length, including lengths of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
• the RNA composition comprises multiple stem-loops.
• RNA compositions having multiple stem-loops minimally comprise a first stem-loop, a second stem- loop, and a spacer between the stem-loops.
• the RNA composition may comprise a nucleotide sequence allowing for a terminal nucleotide to hybridize with it complement to form either the first stem-loop, the second stem- loop, or both.
• the RNA composition comprises a 5’ -triphosphate modified terminal nucleotide capable of hybridizing with its complementary nucleotide to form either the first or second stem-loop.
• the RNA composition comprises a 3’ -terminal nucleotide capable of hybridizing with its complementary nucleotide to form either stem-loop.
• the RNA composition may comprise a 5’- or 3’-overhang associated with either or both of the first stem-loop and the second stem-loop.
• the 5’- or 3’ -overhang associated with either the first stem-loop or the second stem-loop may be any length that allows for the RNA composition to inhibit cell growth or induce cell death.
• the 5’- and/or 3’-overhang may be about 1 to about 50 nucleotides in length.
• the 5’- and/or 3’ -overhang is about 1 to about 10 nucleotides in length, including lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, of 10 nucleotides or any range of lengths therebetween.
• the 5’- and/or 3’-overhang is about 10 to about 50 nucleotides in length, including lengths of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
• the spacer connects the stem loops in a multi-stem loop composition.
• the spacer comprises a segment of ssRNA, a segment of dsRNA, or a combination thereof.
• a dsRNA segment may comprise a completely or partially hybridized segment of a segment of a first nucleotide sequence with a second nucleotide sequence.
• Spacers having only partial hybridization may have any number of nucleotide-pair mismatches that prevent nucleotide pairing between complementary nucleotides along the spacer.
• the spacer remains thermodynamically or kinetically stable under physiological conditions.
• the stem-loop has 1, 2, 3, 4, 5, or more nucleotide-pair mismatches.
• the spacer may be any suitable length to provide the benefit of cytotoxicity without substantially inducing IFN production.
• the length of the spacer may include between about 5 to about 100 nucleotides along a ssRNA segment, about 5 to about 100 hybridized or mismatched nucleotide pairs along a dsRNA segment, or a combination thereof.
• the length of the spacer is about 5 to about 50 nucleotides, including lengths of 5,
• the spacer is not associated with secondary structure. In other embodiments, the spacer is associated with secondary structure. Structured spacers may comprise a stem-loop, resulting in RNA compositions comprising at least a third stem-loop.
• the third stem-loops may be formed from the complete or partial hybridization of nucleotides and result in a hair-pin structural motif.
• the stem-loop may be formed from any suitable number of nucleotide pairings, including any number of nucleotide pairings between about 5 and about 30 or about 8 to about 25.
• the stem-loop comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotide pairings or any number of nucleotide pairings therebetween.
• Stem-loops having only partial hybridization may have any number of nucleotide-pair mismatches that prevent nucleotide pairing between complementary nucleotides along the stem so long as the stem-loop remains stable under physiological conditions.
• the stem-loop has 1, 2, 3, 4, or 5 nucleotide-pair mismatches or any range of nucleotide-pair mismatches therebetween.
• RNA oligonucleotides are provided in Table 2.
• the RNA compositions referred to as Immunogenic Cancer cell-killing RNAs (ICRs), comprising 2’F pyrimidine- incorporated 5’ppp RNAs were designed and generated to contain 5’ppp and various predicted secondary structures including 3’-overhanged hairpin (ICR1, ICR1A, ICR1B, ICR1C), blunt- ended hairpin (ICR2-3, ICR2, ICR2A, ICR2B), 5’overhanged hairpin (ICR3, ICR3A, ICR3B, ICR3C), ssRNA comprising multiple stem-loops (ICR4, ICR4A) and dsRNA comprising multiple stem-loops (ICR5, which is formed from the hybridization of ICR5X and ICR5Y) at various lengths.
• ICRs Immunogenic Cancer cell-killing RNAs
• ICR-L Linear 5’ppp ssRNA
• pIC long dsRNA
• ICR1, ICR1A, ICR1B, ICR1C, ICR2A, ICR2B, ICR3, ICR3A, ICR3B, ICR3C, ICR4, ICR4A, ICR5X, and ICR5Y comprise the oligonucleotide sequence of ICR2.
• the RNA composition comprises an oligonucleotide capable of forming a stem-loop.
• the RNA composition comprises one or more stem- loops formed from the complete or partial hybridization of an oligonucleotide having at least 50% sequence identity to ICR2.
• the RNA composition comprises one or more stem-loops formed from the complete or partial hybridization of an oligonucleotide having at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to ICR2.
• the RNA composition may also consist essentially of one or more stem-loops formed from the complete or partial hybridization of an oligonucleotide having at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%.
• the RNA composition comprises one or more oligonucleotides having at least 50% sequence identity to ICR1, ICR1A, ICR1B, ICR1C, ICR2A, ICR2B, ICR3, ICR3A, ICR3B, ICR3C, ICR4, ICR4A, ICR5X, or ICR5Y.
• the RNA composition comprises one or more oligonucleotides having at least 60%, 70%, 80%, 81%, 82%,
• RNA composition may also consist essentially of one or more oligonucleotides having at least 50%, 60%, 70%, 80%, 81%,
• the activator of NFKB may be a substance other than an RNA product.
• the activator of NFKB may be a microbe (e.g., a bacteria or a virus), a microbial product (e.g., a bacterial product or a viral product), an cytokine, a oxidative stressor, a physical stressor, a therapeutically used drug, a modified protein, an overexpressed protein, an overexpressed ligand, a apoptic mediator, a mitogen, a growth factor, a hormone, a physiological mediator, a chemical agent.
• Exemplary activators of NFKB are described in Pahl, H. L., Oncogene (1999) 18, 6853-6866, which is incorporated herein by reference in its entirety.
• the TLR-pathway agonist may further comprise reprogramming media.
• Reprogramming media comprises a base tissue culture media, insulin-transferrin-selenium (ITS) or ascorbic acid in a somatic cell-reprogramming, e.g., fibroblast-to-cardiomyocyte-reprogramming, amount.
• the media may further comprise bovine serum albumin (BSA) or L-glutamine.
• a somatic cell reprogramming amount of insulin-transferrin-selenium is characterized by insulin being present in an amount of 10 nanomolar to 10 micromolar (e.g., 100 nM), transferrin being present in an amount of 0.002 to 1 gram per liter (e.g., 0.055 g/l), and selenium being present in an amount of 1-100 pg per liter (e.g., 6.7 pg per liter).
• the media comprises 0.2 mM to 20 mM L- glutamine (e.g., 2 mM).
• the media may also optionally include 50 pM to 50 millimolar ascorbic acid such as 100-500 pM, e.g, 250 pM, of ascorbic acid.
• 50 pM to 50 millimolar ascorbic acid such as 100-500 pM, e.g, 250 pM, of ascorbic acid.
• the reprogramming composition may comprise one or more reprogramming efficiency- enhancing molecules.
• “Reprogramming efficiency-enhancing molecules” are molecules suitable for increasing the efficiency of conversion to cardiac myocytes.
• Exemplary reprogramming efficiency-enhancing molecules include valproic acid, bone morphogenetic protein 4 (BMP4), Janus protein tyrosine kinase (JAK) inhibitor 1, RG108, R(+) Bay K 8644, PS48, and A83-01. These agents are delivered (e.g., infused or injected) to the subject before, after, or together with the TLR-pathway agonist.
• the reprogramming composition may comprise a cytoplasmic delivery agent.
• a “cytoplasmic delivery agent” is an agent that transport of molecules, suitably nucleic acids, across membranes.
• exemplary cytoplasmic delivery agents include, without limitation, transfection agents such as DharmaFECT, liposomes, synthetic polymers, cell-penetrating peptides, nanoparticles, viral particles, electroporation buffers, nucleofection reagents, or any combination thereof
• Methods of enhancing or upregulating cardiomyocyte maturation comprise contacting a cell with an effective amount of any of the compositions described for a sufficient time such that the cell is reprogrammed into a cardiomyocyte.
• the cell is a fibroblast, e.g., a cardiofibroblast or a dermal fibroblast, and/or comprises cardiac fibrotic tissue.
• Methods of enhancing or upregulating cardiomyocyte maturation in a subject comprising administering an effective amount of any of the compositions described or any of the pharmaceutical compositions described.
• the subject is in need of enhancing or upregulating cardiomyocyte maturation to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of cardiac fibrotic tissue.
• the cell is a fibroblast, e.g., a cardiofibroblast or a dermal fibroblast, and/or comprises cardiac fibrotic tissue.
• compositions and methods for inhibiting or downregulating cardiomyocyte maturation comprise a TLR inhibitor, a NFKB inhibitor, a ikbkb inhibitor, or any combination thereof.
• A“TLR inhibitor” is a composition or substance capable of interacting specifically with a TLR receptor that blocks or dampens a biological response.
• the TLR inhibitor may be a TLR3 inhibit.
• a TLR inhibitor may be a TLR antagonist.
• A“TLR-pathway antagonist” is a composition or substance capable of interacting a TLR receptor or a substance associated with a TLR pathway that blocks or dampens a biological response.
• A“TLR3-pathway antagonist” is a TLR-pathway antagonist that is capable of interacting with TLR3 or a substance associated with a TLR3 pathway that blocks or dampens a biological response.
• Exemplary TLR or TLR3 inhibitors include, without limitation, CU-CPT-4a, or a siRNA that interferes with the translation of the TLR protein.
• A“NFKB inhibitor” is a composition or substance capable of interacting specifically with NFKB that blocks or dampens a biological response.
• Exemplary NFKB inhibitors include, without limitation, Bay 11-7085, or a siRNA that interferes with the translation of a NFKB protein such as RelA.
• A“ikbkb inhibitor” or“inhibitor of NFKB kinase subunit beta” is a composition or substance capable of interacting specifically with ikbkb that blocks or dampens a biological response or a siRNA that interferes with the translation of the ikbkb protein.
• the TLR inhibitor is a TLR3 inhibitor e.g., an antibody, shRNA small molecule or other competitive inhibitor capable of blocking TLR3 activation and/or signaling.
• the TLR inhibitor is a TLR3 inhibitor such as CU-CPT-4a used in the Examples.
• composition may further comprise a cytoplasmic delivery agent, cellular media, or any combination thereof.
• Methods of inhibiting or downregulating cardiomyocyte maturation comprise contacting a cell with an effective amount of any of the compositions described for a sufficient time such that cardiomyocyte maturation is inhibited or down-regulated in the cell.
• the cell is a fibroblast, e.g., a cardiofibroblast or a dermal fibroblast, and/or comprises cardiac fibrotic tissue.
• compositions utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents.
• the pharmaceutical composition may include one or more compounds as disclosed herein in a range of about 0.1 to 2000 mg, including about 0.5 to 500 mg or about 1 to 100 mg.
• the pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to 100 mg/kg body weight, including about 0.5 to 20 mg/kg body weight or about 0.1 to 10 mg/kg body weight.
• after the pharmaceutical composition is administered to a patient ( e.g ., after about 1, 2, 3, 4, 5, or 6 hours post- administration).
• the concentration of the compound at the site of action is an effective amount of a composition if at least some of the cells at the site of action have or will mature into a cardiomyocyte.
• the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier.
• the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste.
• the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents.
• Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing.
• the compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route.
• the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary route.
• the compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art.
• compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route.
• Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
• compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.
• compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
• the active ingredient may be delivered from the patch by iontophoresis.
• compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
• the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
• Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
• compositions and methods disclosed herein may be administered as pharmaceutical compositions and, therefore, pharmaceutical compositions incorporating the compounds are considered to be embodiments of the compositions disclosed herein.
• Such compositions may take any physical form which is pharmaceutically acceptable; illustratively, they can be orally administered pharmaceutical compositions.
• Such pharmaceutical compositions contain an effective amount of a disclosed compound, which effective amount is related to the daily dose of the compound to be administered.
• Each dosage unit may contain the daily dose of a given compound or each dosage unit may contain a fraction of the daily dose, such as one-half or one- third of the dose.
• the amount of each compound to be contained in each dosage unit can depend, in part, on the identity of the particular compound chosen for the therapy and other factors, such as the indication for which it is given.
• the pharmaceutical compositions disclosed herein may be formulated so as to provide quick, sustained, or delayed release of the active ingredient after administration to the patient by employing well known procedures.
• compositions for use according to the methods of disclosed herein may be administered as a single composition or a combination of compounds.
• a composition for cardiomyocyte maturation may be administered as a single compound or in combination with another compound for cardiomyocyte maturation or that has a different pharmacological activity.
• pharmaceutically acceptable salts of the compounds are contemplated and also may be utilized in the disclosed methods.
• pharmaceutically acceptable salt refers to salts of the compounds which are substantially non toxic to living organisms.
• Typical pharmaceutically acceptable salts include those salts prepared by reaction of the compounds as disclosed herein with a pharmaceutically acceptable mineral or organic acid or an organic or inorganic base. Such salts are known as acid addition and base addition salts. It will be appreciated by the skilled reader that most or all of the compounds as disclosed herein are capable of forming salts and that the salt forms of pharmaceuticals are commonly used, often because they are more readily crystallized and purified than are the free acids or bases.
• Acids commonly employed to form acid addition salts may include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like
• organic acids such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p- bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
• Suitable pharmaceutically acceptable salts may include the sulfate, pyrosulfate, bi sulfate, sulfite, bi sulfate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, hydrochloride, dihydrochloride, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleat-, butyne-.l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, hydroxybenzoate, methoxybenzoate, phthalate, xylenesulfonate, phenyl acetate, phenylpropionat
• Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like.
• Bases useful in preparing such salts include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide, calcium carbonate, and the like.
• the particular counter-ion forming a part of any salt of a compound disclosed herein is may not be critical to the activity of the compound, so long as the salt as a whole is pharmacologically acceptable and as long as the counterion does not contribute undesired qualities to the salt as a whole.
• Undesired qualities may include undesirably solubility or toxicity.
• esters and amides of the compounds can also be employed in the compositions and methods disclosed herein.
• suitable esters include alkyl, aryl, and aralkyl esters, such as methyl esters, ethyl esters, propyl esters, dodecyl esters, benzyl esters, and the like.
• suitable amides include unsubstituted amides, monosub stituted amides, and disubstituted amides, such as methyl amide, dimethyl amide, methyl ethyl amide, and the like.
• solvate forms of the compounds or salts, esters, and/or amides, thereof.
• Solvate forms may include ethanol solvates, hydrates, and the like.
• a“subject” may be interchangeable with“patient” or“individual” and means an animal, which may be a human or non-human animal, in need of treatment.
• A“subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with the compositions disclosed herein.
• a“subject in need of treatment” may include a subject having a cardiovascular disease, such as an atherosclerotic disease, or having suffered a myocardial infarction.
• the terms“treating” or“to treat” each mean to alleviate symptoms, eliminate the causation of resultant symptoms either on a temporary or permanent basis, and/or to prevent or slow the appearance or to reverse the progression or severity of resultant symptoms of the named disease or disorder.
• the methods disclosed herein encompass both therapeutic and prophylactic administration.
• the term “effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment.
• the disclosed methods may include administering an effective amount of the disclosed compositions (e.g ., as present in a pharmaceutical composition) for inducing cardiomyocyte maturation or inhibiting cardiomyocyte maturation.
• an effective amount can be readily determined by the attending diagnostician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
• determining the effective amount or dose of compound administered a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances.
• a typical daily dose may contain from about 0.01 mg/kg to about 100 mg/kg (such as from about 0.05 mg/kg to about 50 mg/kg and/or from about 0.1 mg/kg to about 25 mg/kg) of each compound used in the present method of treatment.
• compositions can be formulated in a unit dosage form, each dosage containing from about 1 to about 500 mg of each compound individually or in a single unit dosage form, such as from about 5 to about 300 mg, from about 10 to about 100 mg, and/or about 25 mg.
• unit dosage form refers to a physically discrete unit suitable as unitary dosages for a patient, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical carrier, diluent, or excipient.
• Cardiomyocytes are an essential component of the heart. Genetic manipulation can cause abnormal cardiac morphogenesis; typically leading to embryonic lethality. This embryonic lethality is difficult to diagnose prenatally and limits our understanding of the process by which precursors commit to the cardiac lineage and mature into fully functional cardiomyocytes. Due to this hurdle, many researchers have taken to replicating cardiomyocyte development in the culture dish. Various methods have been employed including reprogramming strategies [1-8] [9-14]
• Initiation phase of cardiomyocyte development the precursor cell initially expresses a number of so-called pioneer transcription factors. These pioneer transcription factors induce significant epigenetic remodeling; the precursor phenotype is silenced and various genes that are necessary for commitment to the cardiomyocyte lineage are activated.
• the pioneer transcription factors have been identified: combined expression of Gata4, Tbx5, Mef2C and Handl is necessary for the initial commitment to the cardiomyocyte lineage [16]
• the key epigenetic mechanism in the Initiation phase of cardiomyocyte development is histone methylation.
• H3K4 becomes methylated [19] whereas H3K27 is de-methylated [14]
• TLR3 is important for reprogramming fibroblasts to iPS [20] and endothelial cells [21] Specifically, activation of TLR3 causes global changes in the expression and activity of epigenetic modifiers that favor DNA accessibility, and phenotypic fluidity. Interestingly, TLR3 plays a role in the inflammatory response and it is known that inflammation plays a major role in the cardiac response to injury [22, 23]
• cardiomyocyte maturation requires TLR3 activated NFKB. Precursor cells that had committed to the cardiomyocyte lineage were prevented from maturing into cardiomyocytes by TLR3 inhibitors or TLR3 knockdown. Further experiments demonstrate that TLR3 controlled cardiomyocyte maturation via NFKB.
• NFKB Pharmacological inhibition of NFKB, as well as knockdown of Ikbkb (Inhibitor of Nuclear Factor Kappa B Kinase) which activates NFKB, prevented cardiomyocyte maturation. Moreover, conditions that induce cardiomyocyte formation induced NFKB binding to the promoters of cardiomyocyte maturation genes. Moreover, we found that microRNAs activate TLR3.
• Ikbkb Inhibitor of Nuclear Factor Kappa B Kinase
• TLR agonists were purchased from Invivogen (Mouse TLR1-9 agonist kit, tlrl-kitlmw).
• the TLR3 antagonist CU-CPT-4a, NFKB antagonist Bay 11-7085 and the AP1 antagonist SR 11302 were purchased from Tocris.
• MicroRNA transfection Mouse (C57BL/6) neonatal cardiac fibroblasts were isolated from 2 day old mouse neonates according to the method outlined in Jayawardena et al [10] Following isolation fibroblasts were cultured in growth media containing DMEM (ATCC, Catalogue number 30-2002) supplemented with 15%n/n FBS (Thermo Scientific Hyclone Fetal bovine serum, Catalogue number SH30071.03, Lot number AXK49952) and l%v/v penicillin/streptomycin (Gibco, Catalogue number 15140-122, lOOunits Penicillin, lOOug/ml Streptomycin).
• DMEM Adibco, Catalogue number 15140-122, lOOunits Penicillin, lOOug/ml Streptomycin.
• Fibroblasts were passaged once the cells had reached 70-80% confluence using 0.05% w/v trypsin (Gibco, Catalogue number 25300-054). Freshly isolated fibroblasts were labelled as Passage 0. Experiments were conducted with cells at passage 2. For all experiments, cells were seeded at 5000 cells/cm 2 in growth media.
• the cells were transfected with transfection reagent alone (Dharmafect-I, ThermoScientific), with transfection reagent plus non-targeting microRNAs (negmiR), or with transfection reagent plus our previously reported combination of cardiac reprogramming microRNAs[9] (miR combo, miR-l, miR-l33, miR-208, miR-499).
• Gapdh (Mm99999915_m 1 ), Tnni3 (Mm00437l64_ml), Actn2 (Mm00473657_ml), Myh6 (Mm00440359_m 1 ), Cacnalc (Mm004379l7_ml), Mef2C (Mm0l340482_ml), Tbx5 (Mm00803518_m 1 ), Gata4 (Mm00484689_ml) and Hand2 (Mm00439247_ml).
• Immunofluorescence Cells were fixed with 2%v/v paraformaldehyde (EMS) as described previously [24] Fixed cells were blocked in antibody buffer (5%w/v BSA, 0.l%v/v Tween-20, in PBS) for 1 hr at room temperature. Following blocking, cells were incubated overnight at 4°C with a-sarcomeric actinin antibody (Sigma, A7811, 1 : 100) in antibody buffer. After the overnight incubation, cells were washed three times in antibody buffer. Following washing, cells were incubated with Alexa-Fluor conjugated secondary antibodies (Invitrogen, Goat Anti-mouse 594nm) at a 1 :500 dilution in antibody buffer for lhr at room temperature. Nuclei were stained by DAPI at 1 pg/ml for 30 minutes at room temperature in antibody buffer. Following washing in PBS to remove unbound complexes, immunofluorescence was measured using a Zeiss Axiovert 200 inverted microscope.
• siRNA knockdown siRNA pools (four siRNAs targeting the gene) and a negative control siRNA were purchased from Dharmacon. siRNAs were made to 20mM in nuclease free water, aliquoted, and stored -80°C until use. Fibroblasts were seeded into 12 well plates at 20,000 cells per well one day prior to transfection. On the day of transfection siRNAs were diluted to 5mM in nuclease free water. For each well, 5m1 of the working siRNA solution was diluted with 95m1 Optimem-Serum Free media. In a separate tube 5m1 of Dharmafect-I (Dharmacon) was diluted with 95m1 Optimem-Serum Free media. After 5 minute incubation the two solutions were combined. After 20 minutes complete media lacking antibiotics was added (800m1) and the transfection complexes added to the cells.
• Dharmacon Dharmafect-I
• ChIP assays were performed according the manufacturer’s instructions (Cell Signaling, SimpleChIP Enzymatic Chromatin IP kit #9003). Neonatal cardiac fibroblast nuclei were digested with O. lul Micrococcal nuclease per 4xl0 6 cells (amount of Micrococcal nuclease was empirically determined according the manufacturer’s instructions). Immunoprecipitation was performed with ChIP validated antibodies: (1) rabbit IgG control (Cell Signaling, #2729); (2) Histone H3 (Cell Signaling, #4620); and (3) RelA (Cell Signaling, #8242).
• Immunoprecipitated DNA was quantified by qPCR (Therm oFisher, Power SYBR Green PCR Master Mix, #4367659) with primers for the promoters of Myh6 (Qiagen, EpiTect ChIP qPCR Primer Assay For Mouse Myh6, NM_0l0856.3 (-)08Kb #GPMl045733(-)08A and EpiTect ChIP qPCR Primer Assay For Mouse Myh6, NM_0l0856.3 (-)OlKb #GPMl045733(- )0lA), Actn2 (Qiagen, EpiTect ChIP qPCR Primer Assay For Mouse Actn2, NM_033268.3 (- )0lKb, #GPMl 04478 l(-)0l A) and Tnni3 (Qiagen, EpiTect ChIP qPCR Primer Assay For Mouse Tnni3, NM_009406.3 (-)OlKb, #
• PCR reactions included the positive control Histone H3 sample and the negative control rabbit IgG sample.
• a serial dilution of the 2% input chromatin DNA (undiluted, 1 :5, 1 :25, 1 : 125) was used to create a standard curve and determine efficiency of amplification. Percent input was calculated and negative control IgG values subtracted. Data is presented as the fold change of percent input between miR combo and negmiR treated samples.
• IL6 ELISA IL6 ELISA kits were from R&D Systems. Fresh media (lml) was added to the cells one day prior to assaying for IL6. Per manufacturer’s instructions 50ul of media was assayed and the amount of IL6 in pg/ml in the culture media was determined via a standard curve. The IL6 pg/ml value was then adjusted for the total volume of the media (lml) and the total cellular protein in each well to correct for differences in cell number[25].
• Generating beating reprogrammed cardiomyocytes Isolated mouse (C57BL/6) neonatal cardiac fibroblasts (passage 2) were seeded into l2-well dishes at 15000 cells/cm 2 in growth media. Twenty-four hours later growth media was removed and the cells transfected with negmiR or miR combo as described above. One day later, the transfection complexes were removed and media was replaced with a chemically defined reprogramming media[l2] that contained lug/ml Poly(TC) (LMW). For the next four days, cells received fresh chemically defined reprogramming media [12] containing lug/ml Poly(TC) (LMW) daily. After this period, the cells received chemically defined reprogramming media [12] without Poly(TC) (LMW) for a further 10 days. Media was replaced every other day. Beating colonies were identified with a Zeiss Axiovert 200 inverted microscope.
• TLR3 inhibition blocks the maturation phase of cardiac reprogramming.
• the mechanisms by which committed cells mature into cardiomyocytes are unclear.
• Two recent studies have shown that TLR3 is important for reprogramming fibroblasts to iPS [20] and endothelial cells [21]
• TLR3 induces inflammation and inflammation is known to be important in injury. Consequently, we asked our if TLR3 played a hitherto unknown role in the development of mature cardiomyocytes.
• We were interested in TLRs as these receptors are key mediators of the inflammatory responses in the heart.
• TLR3 inhibition nor TLR3 activation affects the initiation phase of cardiac reprogramming. Following these results we wanted to investigate the mechanism by which TLR3 influenced the maturation of reprogrammed cells in more detail.
• epigenetic processes act to turn on expression of the cardiomyocyte-lineage commitment factors Gata4, Hand2, Tbx5 and Mef2C are expressed[28].
• cardiomyocyte-lineage commitment factors Gata4, Hand2, Tbx5 and Mef2C that was induced by miR combo, was not affected by either TLR3 knockdown ( Figures 2Ai-2Aiv) or by TLR3 activation ( Figures 2Bi- 2Biv). This data indicates that the effects of TLR3 upon the cardiac reprogramming were not due to changes in the initiation phase of cardiac reprogramming.
• TLR3 controls the maturation phase of cardiac reprogramming via the RelA subunit of NFKB. TLR3 mediates the activation of a number of transcription factors[22]. Of these transcription factors, two mediate the vast majority of the effects of TLR3: AP1 and NFKB[22] Consequently, we hypothesized that TLR3 would influence maturation of reprogrammed cells via AP1 and/or NFKB. Pharmacological inhibition of AP1 had no effect on the ability of miR combo to reprogram fibroblasts (data not shown). In contrast, the pharmacological inhibition of NFKB completely inhibited miR combo reprogramming at both the RNA ( Figures 3 Ai-3 Aiii) and protein ( Figures 3Bi-3Biii) level.
• NF-KB1 NF-KB1 (pl05/p50); NF-KB2 (pl00/p52); RelA (p65); RelB; and c-Rel. Only RelA, RelB and c-Rel induce transcription. We focused on RelA as it is the most highly expressed Rel protein. Knockdown of RelA, which was found to be robust ( Figure 4A), prevented the appearance of Actn2(+) cells in miR combo transfected fibroblasts ( Figures 4Bi-4Biii).
• MicroRNAs activate TLR3 The pharmacological inhibitor and siRNA mediated knockdown experiments suggested that miR combo activated TLR3.
• TLR3 activity by measuring IL6 secretion into the media.
• IL6 secretion is an accepted measurement of the activity of TLRs, including TLR3 [30-37]
• both the control non-targeting miRNA (negmiR) and miR combo significantly induced IL6 secretion ( Figure 6A). Comparisons between negmiR and miR combo indicated that miR combo had the stronger effect.
• TLR3 Pharmacological activation of TLR3 enhances maturation of reprogrammed fibroblasts. Following the identification of the mechanism by which TLR3 controlled miR combo reprogramming, we next examined if stimulation of TLR3 could enhance the efficiency of miR combo. As expected, miR combo increased RNA levels of Myh6, Actn2 and Tnni3 ( Figures 7Ai-7Aiii). The effect of miR combo uoon Mvh6, Actn2 and Tnni3 expression was significantly enhanced by the addition of the TLR3 agonist Poly(LC) ( Figures 7Ai-7Aiii).
• TLR3 activated NFKB causes global changes in the expression and activity of epigenetic modifiers that favors increased DNA accessibility.
• the activation of the pluripotency program by the Yamanaka factors[20], or the induction of endothelial lineage by trans-differentiation factors[2l] is facilitated.
• the epigenetic plasticity that is induced by TLR3 activation is largely mediated by NFKB, as shown using pharmacological or molecular antagonists of NFKB.
• TLR3 activation increased the binding of NFKB directly to cardiomyocyte sarcomere genes.
• TLR3 inhibition or knockdown did not influence the expression of various transcription factors that are necessary for commitment into the cardiomyocyte lineage.
• TLR3 recognizes double-stranded (ds) RNA; whereas TLR7 and TLR8 bind to single-stranded RNA.
• TLR9 is activated by unmethylated CpG sequences in DNA molecules [47] Only a limited number of reports have demonstrated that microRNAs bind to TLRs.
• microRNAs are dsRNA molecules
• TLR3 TLR3
• this assumption is likely to need revision both in light of our results as well as the recent report that the plant derived microRNA FvmiRl68 binds to dendritic cell TLR3[5l]
• TLR3 activation by microRNAs is sequence dependent.
• siRNA mediated activation of TLRs has been shown to be sequence dependent [52]
• Cardiac fibroblasts were transfected with miR combo or negmiR (control) and incubated with various concentrations of ICR2 (Figs. 8A-8D) or ICR4 (Figs. 9A-9D) for 4 days. Reprograming was evaluated via qPCR, where we measure the expression of genes that are necessary for sarcomere function (e.g. Actn2, Myh6, Tnni3) as well as cardiac ion channels (Cacnalc). Gene expression was evaluated at day 14. To further verify that ICR2 induced maturation, cardiac fibroblasts were incubated with miR combo in addition to PolylC or ICR2 for 14 days. Sarcomeres were visualized by antibody staining for a-sarcomeric actinin (Figs 11A and 11B).
• Neonatal cardiac fibroblasts were transfected with either O. lmM or 0.3mM miR combo.
• Equivalent concentrations of a non-targeting miRNA were used as a control.
• Cell number is represented as a fold change derived from GAPDH expression at day 0 and day 14 (Fig. 10). The dotted line indicates the cell number at day 0.
• Cell number increased significantly in both concentrations of control miR and the standard concentration of miR combo.
• High concentrations of miR combo impaired normal cell number growth either by inhibiting cell proliferation or by increasing rate of cell death.
• N 2.
• Kis A Kis A, Yellon DM, Baxter GF. Role of nuclear factor-kappa B activation in acute ischaemia-reperfusion injury in myocardium. Br J Pharmacol. 2003;138:894-900.
• Ashbumer BP Westerheide SD
• the p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HD AC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol Cell Biol. 2001;21 :7065-7077.
• HD AC histone deacetylase
• TLR3-specific adjuvant that induces NK and CTL activation without significant cytokine production in vivo. Nat Commun. 20l5;6:6280.

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