Patent Application
20190185852
The subject technology generally relates to methods of altering the expression of proteins involved in lipid transport and metabolism, for example, to prevent and treat cardiovascular diseases and risk factors such as atherosclerosis and hyperlipidemia.
All publications, patents, and patent applications cited herein are incorporated by reference in their entirety. U.S. application Ser. No. 14/370,846, filed Jan. 9, 2013, also relates to methods of treating atherosclerosis and hyperlipidemia with a microRNA, the content of which is hereby incorporated by reference in its entirety.
High plasma concentrations of plasma low density lipoprotein (LDL) and low plasma concentrations of high density lipoprotein (HDL) cholesterol levels are risk factors for cardiovascular diseases. Thus, an ideal treatment goal is to simultaneously decrease LDL and increase HDL.
The subject technology provides methods of administering a microRNA (miR) comprising SEQ ID NO:1, wherein the miR simultaneously reduces plasma LDL, increases plasma HDL, and enhances hepatic fatty acid oxidation (FAO) and reverse cholesterol transport. In some embodiments, the methods of the subject technology reduce hepatic very low density lipoprotein (VLDL) production.
In some embodiments of the subject technology, the miR further comprises a sequence with at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO:2. In another embodiment, the miR is hsa-miR-1200 (Dharmacon) (referred to herein as “miR-1200”), and has the sequence of SEQ ID NO:2. See Table 1.
In some aspects of the subject technology, a miR comprising SEQ ID NO:1 is administered to a mammal. In some embodiments, the mammal is a mouse. In yet another embodiment, the mammal is an Apoe−/− mouse. In some embodiments, the mammal is a human. In yet another embodiment, the methods of the subject technology provide for the administration of a therapeutically effective amount of a miR comprising SEQ ID NO:1 to a human in need thereof, wherein the treatment prevents or reduces hyperlipidemia or atherosclerosis.
In some embodiments of the subject technology, a therapeutically effective amount of miR comprising SEQ ID NO:1 for treatment of a human is 0.1-2 mg/kg/week. In some of these embodiments, the therapeutically effective amount is 0.1-0.5 mg/kg/week, 0.5-1 mg/kg/week, 1-1.5 mg/kg/week, 1.5-2 mg/kg/week, 0.1 mg/kg/week, 1 mg/kg/week, 1.5 mg/kg/week or 2 mg/kg/week. A person of ordinary skill in the art would understand that this initial dose can be adjusted based on the severity and type of condition being treated, the mode of administration and the response of the individual patient. The dose may also be administered twice a week as a divided dose, biweekly, or as an extended release formulation.
In some embodiments of the subject technology, apoAI expression is increased by contacting a cell with an inhibitor of BCL11B. In one aspect of the subject technology, a miR comprising SEQ ID NO:1 increases apoAI transcription by reducing the expression and/or activity of its repressor, BCL11B. In another aspect of the subject technology, a miR comprising SEQ ID NO:1 reduces apoB expression by targeting the 3′-untranslated region of mRNA and enhancing posttranscriptional degradation. In yet another aspect of the subject technology, a miR comprising SEQ ID NO:1 increases hepatic fatty acid oxidation by repressing NCOR1.
In some embodiments of the subject technology, apoAI expression is increased by contacting a cell with an inhibitor of NRIP1. The inhibitor may be a nucleic acid inhibitor, such as an siRNA, or it may be a small molecule, peptide or protein inhibitor, such as an antibody or a fusion protein. Inhibitors of NRIP1 may be administered in combination with another inhibitor, such as an inhibitor of BCL11B or apoB expression. In one aspect of the subject technology, an NRIP1 inhibitor is administered to an animal or human in an amount sufficient to increase apoAI expression, thereby causing a therapeutically desirable effect, such as preventing or treating atherosclerosis and/or hyperlipidemia.
In some of the methods of the subject technology, a miR comprising SEQ ID NO:1 is administered to prevent, mitigate or reduce atherosclerosis, hyperlipidemia, dyslipidemia, cardiovascular disease. In other methods of the subject technology, a miR comprising SEQ ID NO:1 is administered to prevent, mitigate or reduce insulin resistance, type II diabetes, schizophrenia, fatty liver disease, inflammation, hepatitis C, familial hypercholesterolemia, multiple sclerosis and rheumatoid arthritis.
The subject technology provides methods of reducing plasma LDL and increasing plasma HDL without causing liver injury. In one aspect provided herein, miR-1200 significantly reduced plasma LDL- and increased HDL-cholesterol in diet-induced hyperlipidemic mice. In another embodiment, an miR comprising SEQ ID NO:1 reduces plasma LDL and increases plasma HDL in a hyperlipidemic human.
Despite significant advances in lowering risk factors, cardiovascular diseases (CVD) accounted for 30.8% of deaths in 2003-2013 in the United States, and the estimated annual cost of CVD and stroke for 2011-2012 was about $316.6 billion. Most of the risk factors for CVD are controllable, especially plasma cholesterol, which is carried in the blood by apolipoprotein B (apoB)-containing lipoproteins, such as low-density lipoproteins (LDLs), and non-apoB-containing high-density lipoproteins (HDLs). apoB-containing lipoproteins are primarily synthesized and secreted by the liver and small intestine to transport lipids to other peripheral tissues. Excess accumulation of these lipoproteins and their modifications in the plasma contribute to atherosclerosis as these modified lipoproteins are taken up by macrophages. apoAI interacts with ATP-binding cassette transporter family A and protein 1 (ABCA1) present on the plasma membrane of different cells, especially macrophages, extracts cholesterol and transports it back to the liver for excretion from the body. This reverse cholesterol transport (RCT) is believed to be anti-atherogenic. For these reasons, elevated LDL and low HDL are two well-established risk factors for atherosclerosis.
Statins lower plasma LDL-cholesterol by reducing hepatic cholesterol synthesis and increasing LDL clearance. However, these drugs only decrease the incidence of cholesterol related diseases by 30-40%, and almost 20% of the population fails to respond to or cannot tolerate statins. Further, high doses of statins sometimes cause muscle pain, elevations in plasma levels of liver and muscle enzymes, and new onset of diabetes mellitus.
While PCSK9 inhibitors have been shown to lower plasma cholesterol, PCSK9 inhibitors have also been associated with neurocognitive side effects. Because the target of both statins and PCSK9 inhibitors is the LDL receptor, these drug classes are not useful in the treatment of homozygous familial hypercholesterolemia subjects that are deficient in this receptor. Prior to the subject technology, no effective therapeutic methods were available to increase functional HDL to prevent CVD. Thus, a need remains for novel therapeutic agents that modulate plasma LDL and HDL to achieve therapeutically beneficial outcomes.
Other known methods for reducing LDL include total plasma exchange (TPE) and LDL apheresis. TPE replaces all plasma every 7-14 days and can reduce plasma LDL to below target levels. HDL levels are also severely reduced however, and the sharp decrease in LDL is followed by a rebound phase as new VLDL is synthesized and secreted. LDL apheresis is similar in that it selectively removes apoB containing lipoproteins, but unlike TPE, LDL apheresis spares HDL. The side effects for both procedures, however, include hypotension, anemia, and hypocalcaemia. Moreover, these treatments are time consuming, invasive and not universally available.
In severe cases, liver transplantation may also be a viable option to lower lipid levels and prevent early onset cardiac events. Liver transplantation is however costly, not readily available globally, and limited by the availability of suitable donors.
MicroRNAs (miRs) are small (˜22 nucleotides) non-coding RNAs that target multiple genes and affect multiple pathways by interacting with the 3′-untranslated region (3′-UTR) of mRNA and destabilizing mRNA or blocking translation. In >70% of cases, miRs mediate regulation by mRNA degradation. MiRs bind to the target mRNA via seed and supplementary sequences. A seed sequence (2-7 nucleotides from the 5′-end of the miR) forms perfect complementary base pairs, while the supplementary site in the 3′-region may or may not form perfect base pairs with the target mRNA. MiRs with the same seed sequence belong to the same family. MiR-30c and miR-33 have been identified to decrease LDL and HDL, respectively, and MiR-148a consistently decreased HDL but had variable effects on plasma LDL levels. However, no MiR has previously been shown to both decrease LDL and increase HDL.
High plasma LDL and low HDL cholesterol levels are risk factors for cardiovascular diseases. Although therapeutics would ideally both lower LDL and increase HDL, there were no known drug therapies that concomitantly mitigate these risk factors prior to the subject technology. Moreover, existing therapeutics such as statins and PCSK9 inhibitors are only partially effective and can cause serious adverse effects.
The subject technology provides methods of administering a miR comprising SEQ ID NO:1, wherein the miR decreases apoB and increases apoAI in a mammal, resulting in lower levels of LDL and higher levels of HDL in plasma.
In some embodiments of the subject technology, the miR comprises a sequence with at least 70%, 75%, 80%, 85%, 90% or 95% identity to SEQ ID NO:2. In certain embodiments, the miR is miR-1200, and has the sequence of SEQ ID NO:2. (See Table 1.)
The subject technology provides methods of simultaneously lowering plasma LDL and increasing plasma HDL. In some embodiments of the subject technology, a microRNA comprising SEQ ID NO:1 is administered to a mammal, wherein the microRNA reduces plasma LDL and increases plasma HDL via different mechanisms, thus mitigating dyslipidemia and atherosclerosis. In some embodiments, the microRNA is miR-1200.
The subject technology includes methods of significantly reducing apoB (an LDL structural protein) while increasing apoAI (main HDL protein) secretion. In some embodiment, the methods reduce apoB while increasing apoAI in cell culture. In some embodiments, the methods reduce apoB while increasing apoAI in hepatic or hepatoma cells. In some embodiments, the methods reduce apoB while increasing apoAI in the liver of a human or other mammal. In some aspects of the subject technology, apoB expression is decreased by an inhibitor that causes degradation of mRNA encoding apoB, e.g. the human apoB mRNA (Gene accession NM_000384, Appendix A). In other aspects of the subject technology, apoAI expression is increased by an inhibitor that causes degradation of mRNA encoding a repressor of ApoAI, such as NRIP1, e.g. human NRIP1 mRNA (Gene accession NM_003489, Appendix A) and/or BCL11B, e.g. human BCL11B mRNA (Gene accession NM_022898, Appendix A).
In some embodiments of the subject technology, apoAI is increased by inhibiting its repressor, BCL11B. In some embodiments, BCL11B expression is inhibited by a miR. In yet another embodiment, BCL11B is inhibited by an RNA longer than 20 nucleotides, such as an RNA that is longer than 30, 50, 75, 100, 125 or 200 nucleotides. In another embodiment, BCL11B is inhibited by a nucleic acid comprising modified nucleotides, a double-stranded nucleic acid inhibitor, a protein inhibitor or a small molecule inhibitor.
In some embodiments of the subject technology, apoAI is increased by inhibiting its transcriptional repressor, NRIP1. In some embodiments, NRIP1 expression is inhibited by an siRNA. In yet another embodiment, NRIP1 is inhibited by an RNA longer than 20 nucleotides, such as an RNA that is longer than 30, 50, 75, 100, 125 or 200 nucleotides. In another embodiment, NRIP1 is inhibited by a nucleic acid comprising modified nucleotides, a double-stranded nucleic acid inhibitor, a protein inhibitor or by a small molecule inhibitor. In some embodiments, NRIP1 inhibitors are administered to an animal or human, alone or in combination with inhibitors of BCL11B and/or apoB, to achieve a therapeutically effective result, such as treating or preventing hyperlipidemia and/or atherosclerosis.
A microRNA is a short RNA. MicroRNAs may also be denoted miRNA or miR herein. Preferably a miRNA to be used with the subject technology is 19-25 nucleotides in length and consists of non-protein-coding RNA. Mature miRNAs may exert, together with the RNA-induced silencing complex, a regulatory effect on protein synthesis at the post-transcriptional level. More than 1500 human miRNA sequences have been discovered to date and their names and sequences are available from the miRBase database (http://www.mirbase.org).
A miRNA of the subject technology can be synthesized, altered, or removed from the natural state using a number of standard techniques known in the art. A synthetic miRNA, or a miRNA partially or completely separated from its coexisting materials is considered isolated. An isolated miRNA can exist in substantially purified form, or can exist in a cell into which the miRNA has been delivered. A miRNA can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Rosetta Genomics (North Brunswick, N.J.), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Ambion (Foster City, Calif., USA), and Cruachem (Glasgow, UK).
In some embodiments, the miRs of the invention are delivered to target cells using an expression vector encoding the miR. A variety of suitable vectors are known in the art, including plasmids, viruses, and linear polynucleotides. Plasmids suitable for expressing any of the miRs of the subject technology, methods for inserting nucleic acid sequences into the plasmid to express the miR of interest, and methods of delivering the recombinant plasmid to cells of interest are well established and practiced in the art. Examples of suitable plasmids and methods of expression and delivery can be found in Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.
In other embodiments, the miRs of the subject technology are expressed from recombinant viral vectors. Non-limiting examples of viral vectors include retroviral vectors, adenoviral vectors (AV), adeno-associated virus vectors (AAV), herpes virus vectors, and the like. Recombinant viral vectors suitable for expressing miRs of the subject technology, methods for inserting nucleic acid sequences for expressing RNA in the vector, methods of delivering the viral vector to cells of interest, and recovery of the expressed RNA molecules are within the skill in the art. Examples include Dornburg (1995), Gene Therap. 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum. Gene Therap. 1:5-14; and Anderson (1998), Nature 392:25-30, the entire disclosures of which are herein incorporated by reference.
Various modifications to the miRs of the subject technology can be introduced as a means of increasing intracellular stability, therapeutic efficacy, and shelf life. Some modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
In yet other embodiments, the miRs of the subject technology are expressed from recombinant circular or linear plasmids using any suitable promoter. Selection of suitable promoters is within the skill in the art. Suitable promoters include but are not limited to U6 or H1 RNA pol III promoter sequences or cytomegalovirus promoters. Recombinant plasmids can also comprise inducible or regulatable promoters for miRNA expression in cells. For example, the CMV intermediate-early promoter may be used with the miRNAs of the subject technology to initiate transcription of the miRNA gene product coding sequences.
A further embodiment of the subject technology provides a method of preventing or treating a disease associated with high apoB and/or low apoAI levels, including but not limited to insulin resistance, type II diabetes, schizophrenia, fatty liver disease, inflammation, hepatitis C, familial hypercholesterolemia, and rheumatoid arthritis.
An additional embodiment of the subject technology provides a method of preventing or treating a disease associated with reduced LDL and increased HDL, including but not limited to cardiovascular disease (coronary artery disease, peripheral arterial disease, cerebral vascular disease, cardiomyopathy, hypertensive heart disease, cardiac dysrhythmias, inflammatory heart disease, aortic aneurysm, renal artery stenosis, valvular heart disease), atherosclerosis, fatty liver disease, diabetic dyslipidemia, and hypocholesterolemia.
In one embodiment, the subject technology features changing levels of apoB, apoAI, HDL, and/or LDL with a microRNA administered with additional agents at a therapeutically effective amount. The term “therapeutically effective amount,” as used herein, refers to the total amount of microRNA and each additional agent that is sufficient to show a meaningful benefit to the subject.
Pharmaceutical compositions of the subject technology can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate).
Delivery of the compositions in the claimed methods may be facilitated by use of a biocompatible gel, a lipid-based delivery system, such as liposomes, polycationic liposome-hyaluronic acid (LPH) nanoparticles (Medina, 2004), LPH nanoparticle conjugated to a peptide, such as an integrin-binding peptide (Liu, 2011), cationic polyurethanes such as polyurethane-short branch-polyethylenimine (PU-PEI), a glycoprotein-disulfide linked nanocarrier (Chiou, 2012) or other known miR delivery systems including, but not limited to dendrimers, poly(lactide-co-glycolide) (PLGA) particles, protamine, naturally occurring polymers, (e.g. chitosan, protamine, atelocollagen), peptides derived from protein translocation domains, inorganic particles, such as gold particles, silica-based nanoparticles, or magnetic particles. (Zhang, 2013).
If desired, the miRs of the subject technology may be modified to protect against degradation, improve half-life, or to otherwise improve efficacy. Suitable modifications are described, e.g. in U.S. Patent Publication Nos. 20070213292, 20060287260, 20060035254, 20060008822, and 20050288244, each of which is hereby incorporated by reference in its entirety.
Pharmaceutical compositions of the subject technology can be packaged for use in liquid or solid form, or can be lyophilized. Conventional nontoxic solid pharmaceutically-acceptable carriers can be used for solid pharmaceutical compositions of the subject technology. Examples of carriers include but are not limited to pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, and magnesium carbonate.
Pharmaceutical formulations may be adapted for administration by any appropriate route. For example, appropriate routes may include oral, nasal, topical (including buccal, sublingual, or transdermal), or parenteral (including subcutaneous, intrasternal, intracutaneous, intramuscular, intraarticular, intraperitoneal, intrasynovial, intrathecal, intralesional, intravenous, intradermal injections or infusions). For human administration, the formulations preferably meet sterility, pyrogenicity, general safety, and purity standards, as required by the offices of the Food and Drug Administration (FDA).
The therapeutically effective amount of microRNA varies depending on several factors, such as the condition being treated, the severity of the condition, the time of administration, the duration of treatment, the age, gender, weight, and condition of the subject. In some embodiments of the subject technology, a therapeutically effective amount of miR comprising SEQ ID NO:1 for treatment of a human is 0.1-2 mg/kg/week. In some of these embodiments, the therapeutically effective amount is 0.1-0.5 mg/kg/week, 0.5-1 mg/kg/week, 1-1.5 mg/kg/week, 1.5-2 mg/kg/week, 0.1 mg/kg/week, 1 mg/kg/week, 1.5 mg/kg/week or 2 mg/kg/week. A person of ordinary skill in the art would understand that this initial dose can be adjusted based on the severity and type of condition being treated, the mode of administration and the response of the individual patient. One of ordinary skill in the art may also modify the route of administration in order to obtain the maximal therapeutic effect. Where a dosage regimen comprises multiple administrations, the effective amount of the miRNA molecule administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
The microRNA in the subject technology can be administered with additional agents in combination therapy, either jointly or separately, or by combining the microRNA and additional agents(s) into one composition.
For example, the miRNA pharmaceutical compositions of the subject technology can be used to treat hypercholesterolemia or atherosclerosis, either alone or in combination with a statin. Examples of statins include Atorvastatin (Lipitor), Ezetimibe/Simvastatin (Vytorin), Lovastatin (Mevacor), Simvastatin (Zocor), Pravastatin (Pravachol), Fluvastatin (Lescol), and Rosuvastatin (Crestor), Fenofibrate (Lipofen), Gemfibrozol (Lopid) and/or Ezetimibe (Zetia).
In other embodiments, the pharmaceutical compositions of the subject technology are administered in combination with ACE inhibitors, aldosterone inhibitors, angiotensin II receptor blockers (ARBs), beta-blockers, calcium channel blockers, cholesterol lowering drugs, digoxin, diuretics, inotropic therapy, potassium or magnesium, PCSK9 inhibitors (otherwise known as monoclonal antibodies), vasodilators, or warfarin.
Examples of ACE inhibitors include but are not limited to Accupril (quinapril), Aceon (perindopril), Altace (ramipril), Capoten (captopril), Lotensin (benazepril), Mavik (trandolapril), Monopril (fosinopril), Prinivil, Zestril (lisinopril), Univasc (moexipril), and Vasotec (enalapril).
Examples of aldosterone inhibitors include but are not limited to eplernone (Inspra) and spironolactone (Aldoctone).
Examples of angiotensin II receptor blockers (ARBs) include but are not limited to candesartan (Atacand), eprosartan (Teventen), irbesartan (Avapro), Iosartan (Cozar), telmisartan (Micardis), valsartan (Diovan), and olmesartan (Benicar).
Examples of beta-blockers include acebutolol hydrochloride (Sectral), atenolol (Tenormin), betaxolol hydrochloride (Kerlone), bisoprolol fumarate (Zebeta), carteolol hydrochloride (Cartrol), esmolol hydrochloride (Brevibloc), metoprolol (Lopressor, Toprol XL), and penbutolol sulfate (Levatol).
Examples of calcium channel blockers include Amlodipine (Norvasc), Diltiazem (Cardizem, Tiazac), Felodipine, Isradipine, Nicardipine (Cardene SR), Nifedipine (Procardia) Nisoldipine (Sular), and Verapamil (Calan, Verelan, Covera-HS).
The practice of aspects of the subject technology can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. These techniques are fully explained in literature. Examples of conventional techniques can be found in Molecular Cloning: A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated in their entirety by reference.
The following specific examples are to be construed as merely illustrative, and not limiting the remainder of the disclosure in any way. It is believed that one skilled in the art can, based on the description herein, utilize the subject technology to its fullest extent.
To identify miRs regulating apoB and apoAI secretion, human hepatoma Huh-7 cells were transfected with 1237 human miRs (human miRIDIAN Mimic 16.0 library, Dharmacon).
MiRs were suspended in RNase free water to obtain 2 μM stocks and 3 μL of each miR was added in duplicate wells to obtain a final concentration of 50 nM. 7 μl of Opti-MEM and 10 μl of lipofectamine RNAiMAX (Life technologies) diluted 1:20 in Serum Reduced Opti-MEM was added to each well. After 20 to 30 minutes, 25,000 cells in 100 μl of Opti-MEM were added to each well. After additional 24 hours, culture media were changed with fresh DMEM containing 10% fetal bovine serum. Media were changed 24 hours later and cells were incubated with DMEM containing oleic acid/BSA complex ((oleic acid (0.4 mM)/BSA (1.5%)) for 2 hours.
apoB and apoAI concentrations in medium were measured by ELISA (Hussain et al., 1995). Secreted apolipoproteins were quantified by ELISA as shown in
The first screening performed in duplicate plates showed high reproducibility (Spearman r=0.96 and 0.92;
Hsa-miR-1200 is located in the 6th intron of Engulfment and cell motility protein 1 (ELMO1) on human chromosome 7, and the precursor miR-1200 is conserved (
Lower cellular apoB protein levels could be due to reductions in mRNA or protein synthesis. Quantifications revealed that apoB mRNA levels were reduced in miR-1200 and increased in anti-1200 over-expressing cells, suggesting that miR-1200 modulates mRNA levels (
RNA isolation and qRT-PCR: Total RNA from tissues and cells was extracted using TRIzol (Invitrogen). RNA was reverse transcribed into cDNA with the Omniscript RT kit (QIAGEN). Expression levels of gene are quantified by qRT-PCR using SYBER Green qPCR Core Kit (Eurogentec), and data was analyzed with ΔΔCT method and normalized to 18S. Primers specific for miR-1200, miR-30c, snoRNA 202 were purchased from Life Technologies.
The mechanism by which miR-1200 regulates apoB mRNA degradation was further elucidated by in silico analysis using miRanda (http://www.microrna.org/microrna/home.do), showing that apoB mRNA contains a miR-1200 interacting site in its 3′-UTR (
The following example demonstrates that miR-1200 increases apoAI secretion by reducing BCL11B, a repressor of apoAI transcription. MiR-1200 dose-dependently enhanced apoAI secretion by ˜41% in Huh-7 cells compared to Scr (
Although miRs normally reduce gene expression (He and Hannon, 2004), they have been shown to activate transcription by interacting with promoter sequences involving complementary base pairing via RNA activation (Huang et al., 2012; Place et al., 2008). There were no miR-1200 complementary sequences in the 1.2-kb apoAI promoter. To determine whether miR-1200 may instead increase apoAI transcription by suppressing a transcriptional repressor(s), three transcriptional repressors were selected from a list of predicted miR-1200 target genes generated by TargetScan (http://www.targetscan.org/) as they had the potential to bind the apoAI promoter, and the target sites were conserved in human and mouse. Huh-7 cells were then transfected with siRNAs against NRIP1 (Nuclear Receptor Interacting Protein 1), BCL11B (B-Cell Lymphoma 11B), or ZBTB7A (Zinc Finger and BTB Domain Containing 7A) (
To test whether BCL11B is an intermediary in the regulation of apoAI by miR-1200, miR-1200 was co-transfected with siRNAs in Huh-7 cells (
Bioinformatics analyses showed that the 3′-UTR of the human BCL11B mRNA contained 4 miR-1200 binding sites and 3 of these sites were evolutionarily conserved (
To ascertain that the regulation of apoB and apoAI by miR-1200 is not specific to Huh-7 cells, its effects in other human hepatoma HepG2 cells were studied. MiR-1200 and anti-1200 decreased and increased media and cellular apoB, respectively (
Since mouse models are commonly used to evaluate the role of miRs in lipid metabolism and atherosclerosis, the effects of miR-1200 on apoB and apoAI in mouse hepatoma AML12 cells were examined. Expression of miR-1200 decreased apoB and increased apoAI but had no effect on MTTP and ABCA1 mRNA levels (
To investigate the physiological consequences of miR-1200 overexpression, a dose-escalation study in wild type C57BL/6J mice fed a Western diet for 6 weeks was performed (
Analysis of Plasma Constituents
Blood was collected in EDTA containing tubes from overnight fasted mice. Plasma was separated by centrifugation. Total plasma cholesterol, triglyceride, and phospholipid were measured using commercial kits (Thermo Fisher Scientific, Wako Diagnostic). To precipitate apoB-containing lipoproteins, 25 μL of 0.44 mM phosphotungstic acid and 20 mM MgCl2 were added to 10 μL of plasma, incubated for 5 min at room temperature, and centrifuged at 12,000*g. Supernatants were used to measure cholesterol in HDL. Cholesterol levels in non-HDL fractions were determined by subtracting HDL-cholesterol from total cholesterol. Lipids were extracted from liver homogenates using methanol/chloroform and quantified using kits. Plasma ALT, AST, glucose and CK were measured using commercial available kits (Pointe Scientific, Wako Diagnostic, and Thermo scientific) according to the manufacturer's instructions.
Analyses of plasma constituents revealed no significant changes in total plasma cholesterol (
The effects of miR-1200 on hepatic lipid metabolism were tested. Assimilation of miR-1200 in the liver had no effect on hepatic cholesterol and triglyceride levels, indicating that miR-1200 does not cause hepatic steatosis (
The effect of miR-1200 on lipid synthesis and FAO was assessed. In the livers of miR-1200 injected group, FAO was increased by >2-fold but had no effect on the synthesis of different lipids (
Fatty acid oxidation and synthesis of fatty acids, triglycerides, and phospholipids: For hepatic FAO, ˜100 mg fresh liver slices were incubated with 0.2 μCi of 14C-oleate for 2 h. Released 14C—CO2 was trapped in phenylethylamine soaked Whatman filter paper and counted (Khatun et al., 2012; Soh et al., 2013). To study FAO in cells, Huh-7 cells were plated in 12-well plates and incubated with DMEM containing 0.4 μCi/ml of 14C-oleate and covered with phenylethylamine soaked Whatman filter paper for 3 hours at 37° C. At the end of incubation, 200 μl of 1M perchloric acid was added to media and incubated for 1 h at room temperature to precipitate acid-insoluble metabolites, and centrifuged (10 min 12,000*g). The radioactivity in the supernatant and the filter paper was counted.
For fatty acid synthesis (de novo lipogenesis), about 50 mg fresh liver slices were incubated with 1 μCi 14C-acetate. After one hour, the liver slices were washed with PBS and subjected to fatty acids extraction using Petroleum Ether. The radioactivity in fatty acids was measured by scintillation counter. For triglyceride and phospholipid synthesis, 50 mg fresh liver slices were labeled with 1 μCi of 3H-glycerol for 1 hour. Total lipids were extracted by chloroform and methanol and separated on silica-60 Thin Layer Chromatography. The bands containing triglyceride or phospholipid were scraped off from the plates and counted in a scintillation counter.
MiR-1200 significantly reduced plasma LDL cholesterol levels (
MiR-1200 injected mice accumulated reduced amounts of triglyceride in plasma over time (
For in vivo RCT (McGillicuddy et al., 2009), mice were injected with 3H-cholesterol labeled macrophages. After 48 hours, miR-1200 treated mice had 13% more 3H-cholesterol in plasma, 22% more in feces, and 16% more in the liver compared with PBS controls (
For RCT (Rohatgi et al., 2014; Khera et al., 2011; McGillicuddy et al., 2009), J774A.1 cells (105/well) were plated in 6-well plates one day before loading. For loading, cells were incubated with Ac-LDL (50 μg/ml)+3H-cholesterol (5 μCi/ml) in DMEM containing 10% FBS for 48 hours. After washing with PBS three times, cells were incubated with 0.5% BSA containing DMEM for one hour. Cells were harvested, washed, and suspended in 0.5% BSA containing DMEM. A small aliquot of cells was counted in scintillation counter to measure the total injected dpm. 300 μl of cells were injected into each mouse. Samples were collected after 48 hours.
Increases in RCT are due to augmentations in cholesterol efflux potential of HDL. Total plasma and HDL isolated from miR-1200 treated mice effluxed ˜26% more radiolabeled cholesterol than controls (
Cholesterol Efflux Assay
For cholesterol efflux (Khera et al., 2011), J774A.1 cells (1.2×104) were plated in each well of a 96-well plate one day before loading. For loading, cells were incubated with DMEM containing 50 μg/mL Ac-LDL, 0.2 μCi/mL 3H-cholesterol, 10% FBS and 0.5% BSA for 48 hours. Then cells were washed three times with PBS and equilibrated in serum free DMEM containing 2 μM of LXR agonist TO901317 and 0.5% BSA for 24 hours. HDL or whole plasma (5%, v/v) was used as cholesterol acceptor. DMEM containing 0.5% BSA was used as control. The efflux was performed in the presence of TO901317 and 0.5% BSA for 6 hours. After efflux, radiolabeled cholesterol in media and cells were counted separately. Percent Cholesterol efflux=media counts/(media counts+cell counts)*100% −% blank efflux.
This example demonstrates that miR-1200 can reduce atherosclerosis. Western diet fed Apoe−/− mice were injected with 1 mg/kg/week miR-1200 for 7 weeks (
In a second experiment, mice fed a Western diet were injected with 2 mg/kg/week of miR-1200 or PBS for 5 weeks. Again, miR-1200 accumulated in the liver, kidney, spleen and heart of these mice and hepatic accretions had no effect on miR-30c expression (
Cells used in the foregoing Examples including, Human hepatoma Huh-7 and HepG2; mouse hepatoma AML12; and mouse macrophage J774A.1 cells from American Type Culture Collection were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin and 1% L-glutamine in a 37° C., 5% CO2 cell culture incubator.
A therapeutically effective amount of a miR comprising SEQ ID NO:1 is administered to a human patient, wherein LDL is decreased and HDL is increased, without causing liver or muscle injury. The miR is administered at a dose of 0.1-2 mg/kg/week, with the specific dosage chosen based on the type and severity of the disease and patient response and characteristics. A dose as low as 0.1 mg/kg/week, i.e. a dose 10-fold lower than that used in mice, may be therapeutically effective, given the slower metabolic rate in humans. To achieve optimal response, the dose may be increased up to 1 mg/kg/week, the same dose as in mice. If needed, the dose may be further increased up to 2 mg/kg/week. Such dose optimization is within the skill of a person of ordinary skill in the art.
A therapeutically effective amount of an NRIP1 inhibitor is administered to cells in vitro or in vivo, thereby increasing the expression of apoAI. The inhibitor may be a nucleic acid inhibitor, such as an siRNA, e.g. with the sequence of SEQ ID NO:3, shown in Table 4. Alternatively, the NRIP1 inhibitor may be a small molecule or a protein, such as an antibody or a fusion protein. To further enhance apoAI expression, the NRIP1 inhibitor is optionally administered in combination with an inhibitor of BCL11B and/or an inhibitor of apoB expression or activity. When administered to an animal or a human patient, the specific dosage of each inhibitor is chosen and adjusted based on the type and severity of the disease, as well as the patient response and characteristics.
HOMO SAPIENS APOLIPOPROTEIN B (APOB), MRNA
HOMO SAPIENS NUCLEAR RECEPTOR
HOMO SAPIENS B-CELL CLL/LYMPHOMA 11B (BCL11B),
This application claims priority to U.S. Provisional Application No. 62/332,442, entitled “THERAPEUTICALLY MODULATING APOB AND APOAI,” filed May 5, 2016, the disclosure of which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant numbers 2R56DK046900-17A1 and 5R01DK46900-15 from the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/031240 | 5/5/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62332442 | May 2016 | US |
