Patent Application
20230364267
This application contains a sequence listing filed in ST.26 format entitled “222119-1160_Sequence_Listing.xml” created on May 5, 2023, and having a file size of 12 kB. The content of the sequence listing is incorporated herein in its entirety.
Patients with severe acute myocardial infarction (AMI) often progress to end-stage congestive heart failure (CHF), which is one of the most significant problems in public health. From the molecular and cellular perspective, heart failure occurs due to the loss of the contractile unit of the left ventricle: cardiomyocytes (CMs). Mammalian CMs exit the cell cycle shortly after birth, but the results from previous studies with neonatal pig indicated that when AMI was induced on postnatal day 1 (P1), CMs re-entered the cell cycle and proliferated, leading to the complete restoration of cardiac function with a little evidence of scarring by P30. Some anecdotal evidence in pediatric patient suggests that the regenerative capacity of newborn infant hearts is similar. However, mammalian CM exit the cell cycle within a few days after birth, so less than 1% of the CM in adult human hearts are replaced each year. Studies in adult mice suggest that cardiomyocyte proliferation increases only marginally in response to cardiac injury. This meager proliferative capacity cannot repair the damage caused by AMI in adult mammals.
Investigations in mouse, rat, and pig MI models have targeted many of the pathways that regulate the cell cycle of CM in an attempt to promote cardiomyocyte proliferation and improve recovery from myocardial injury. For example, Cyclin D2 (CCND2) in humans controls the G1-to-S phase transition in CM, and targeted CM cyclin D2 expression has been associated with improvements in infarct size and cardiac performance. Furthermore, when human induced-pluripotent stem cells (hiPSCs) were engineered to MHC driven overexpress CCND2 CM (CCND2-OEhiPSC-CMs), and the CCND2-OEhiPSC-CMs were transplanted into infarcted mouse hearts, the small number of hiPSC-CMs that were engrafted at the site of administration proliferated and repopulated the myocardial scar, thereby reducing infarct size and improving cardiac performance. However, the methods used to manipulate the expression of regulatory molecules are often accompanied by safety concerns that impede their translation to clinical use. Viral-based therapies, especially those that can become integrated into the genome of the host cell (e.g., adeno-associated virus [AAV]), may promote excessive and enduring expression of the gene of interest, which could result in cardiac hypertrophy and induce uncontrolled cardiomyocyte proliferation, increasing the risk of arrhythmia. Although nonintegrating lentiviral vectors (NILV) are expressed only transiently in vivo, their off the target side-effects, and therefore, the safety in humans remains largely unknown.
Despite advances in cardiac regeneration and repair research, there is still a scarcity of gene delivery systems and methods that are both potent and efficacious at achieving selective repair of cardiac tissue without overexpressing proteins in non-target tissue. These needs and other needs are satisfied by the present disclosure.
In accordance with the purpose(s) of the present disclosure, as embodied and broadly described herein, the disclosure, in one aspect, relates to a synthetic modified mRNA (modRNA) and a novel gene therapy approach using the same that offers efficient, transient, safe, nonimmunogenic, and controlled mRNA delivery to the heart tissue without any risk of genomic integration. In a further aspect, the disclosure relates to methods of using the modRNA to achieve transient and exclusive overexpression of CCND2 only in cardiomyocytes, increasing cell cycle markers, enhancing cardiac function, and promoting myocardial repair.
Other systems, methods, features, and advantages of the present disclosure will be or become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be protected by the accompanying claims. In addition, all optional and preferred features and modifications of the described embodiments are usable in all aspects of the disclosure taught herein. Furthermore, the individual features of the dependent claims, as well as all optional and preferred features and modifications of the described embodiments are combinable and interchangeable with one another.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Moreover, in the drawings, like reference numerals designate corresponding parts throughout the several views.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
modRNA technology provides an alternative strategy for transiently inducing gene expression that is efficient, titratable, and minimally immunogenic with bell-shaped pharmacokinetics and no risk of genomic integration. Furthermore, the safety profile of modRNA was thoroughly studied during the development of mRNA-based vaccines against SARS-CoV-2. Disclosed herein is a Cardiomyocyte Specific Modified mRNA Translation System (CM SMRTs) that allows gene expression exclusively in cardiomyocytes (CM). In one aspect, the disclosed CM SMRTs have been used to drive CCND2 transient expression in endogenous CM in both mouse and pig acute myocardial infarction (AMI) models and has been shown to activate the cell cycle of CMs and improve the recovery of left ventricular (LV) injury in hearts with AMI.
In one aspect, disclosed herein is a synthetic modified mRNA (modRNA) system for delivery of at least one cell cycle regulator gene to cardiac cells, wherein the modRNA system includes a composition including at least a first modRNA and a second modRNA. Further in this aspect, the first modRNA includes an mRNA sequence complementary to SEQ ID NO. 3 and recognition sequences for the microRNAs miR-1 and miR-208. In an aspect, miR-1 and miR-208 are cardiomyocyte (CM)-specific microRNAs and the combination of miR-1 and miR-208 is not found in non-cardiac cells. In one aspect, the recognition sequences for miR-1 and miR-208 can be located anywhere in the first modRNA but, in some aspects, they are located 3′ to the mRNA sequence complementary to SEQ ID NO. 3. In an aspect, SEQ ID NO. 3 is a DNA sequence encoding the archaeal large ribosomal subunit. Thus, in this aspect, targeting CM-specific microRNAs allows the disclosed systems and methods to be active in target cardiac cells and not in other cell types. In another aspect, the second modRNA includes a kink-turn motif and an mRNA sequence complementary to SEQ ID NO. 1. In an aspect, the kink-turn motif is a common structural motif in RNA that introduces a tight kink into the helical axis. In a further aspect, kink-turns play an important role in RNA structures and can serve as binding sites for a number of proteins, including, but not limited to, L7Ae. In an aspect, any kink-turn motif capable of serving as a binding or recognition site for L7Ae is contemplated in the systems and methods described herein and should be considered disclosed. In another aspect, the kink-turn motif can be located anywhere in the second modRNA but, in some aspects, is located 5′ to the mRNA sequence complementary to SEQ ID NO. 1. In one aspect, SEQ ID NO. 1 is a DNA sequence encoding cell cycle regulator CCND2. In any of these aspects, the disclosed modRNA system is inactive in non-cardiac cells.
In any of these aspects, the first and/or second modRNA can include N1-methylpseudouridine residues in place of one or more uridine residues, or in place of all uridine residues. Further in this aspect, N1-methylpseudouridine as a component of an exogenously-administered RNA stimulates less of an innate immune response to the RNA than use of uridine residues at the same location. In another aspect, N1-methylpseudouridine can be incorporated into the modRNAs disclosed herein by any method known in the art including, but not limited to, incorporation of N1-methylpseudouridine triphosphate in a mixture of nucleotides used for standard molecular biological methods of RNA synthesis. In an aspect, the first and second modRNAs disclosed herein are nonimmunogenic.
In one aspect, the cardiac cells can be cardiac fibroblasts, cardiomyocytes, endothelial cells, other cardiac cells, or any combination thereof. In an aspect, the composition can include about 1.5 μg of the first modRNA and about 3 μg of the second modRNA.
Also disclosed herein is a method for improving at least one measure of cardiac health in a subject, the method including administering the disclosed modRNA system to the subject. In some aspects, the method can be performed after myocardial infarction (MI) or another source of cardiac damage. In an aspect, in the disclosed method, the modRNA system can be administered to the subject via intromyocardial injection. In any of these aspects, neither the first modRNA nor the second modRNA is genomically integrated into the subject.
In one aspect, performing the method induces transient overexpression of cell cycle regulator CCND2 in at least one cardiac cell type such as, for example, cardiac fibroblasts, cardiomyocytes, endothelial cells, other cardiac cells, or any combination thereof. In an aspect, the transient expression may peak at about 2 days after performing the method. Further in this aspect, the transient overexpression declines to substantially background levels about 14 days after performing the method.
In one aspect, the at least one measure of cardiac health includes increased expression of cell cycle markers, enhancement of cardiac function, promotion of myocardial repair, or any combination thereof. In another aspect, the subject can be a mammal such as, for example, a human, mouse, or pig.
Many modifications and other embodiments disclosed herein will come to mind to one skilled in the art to which the disclosed compositions and methods pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the disclosures are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. The skilled artisan will recognize many variants and adaptations of the aspects described herein. These variants and adaptations are intended to be included in the teachings of this disclosure and to be encompassed by the claims herein.
Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure.
Any recited method can be carried out in the order of events recited or in any other order that is logically possible. That is, unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.
While aspects of the present disclosure can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present disclosure can be described and claimed in any statutory class.
It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods belong. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly defined herein.
Prior to describing the various aspects of the present disclosure, the following definitions are provided and should be used unless otherwise indicated. Additional terms may be defined elsewhere in the present disclosure.
As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cardiomyocyte,” “a cyclin D protein,” or “an mRNA,” include, but are not limited to, mixtures or combinations of two or more such cardiomyocytes, cyclin D proteins, or mRNAs, and the like.
It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.
When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y′, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y′, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
Unless otherwise specified, temperatures referred to herein are based on atmospheric pressure (i.e. one atmosphere).
Now having described the aspects of the present disclosure, in general, the following Examples describe some additional aspects of the present disclosure. While aspects of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit aspects of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
All experiments and procedures involving animals were approved by the Institutional Animal Care and Use Committee (Animal Protocol Number 20216) of the University of Alabama at Birmingham, School of Medicine and were consistent with the Guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (2011). The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
modRNA Synthesis and Transfection
modRNAs were synthesized from plasmid templates containing open reading frame sequences of the gene of interest (Table 1) via in vitro transcription (IVT) as described previously. IVT was performed with a customized ribonucleotide blend of CleanCap Reagent AG, m7G(5′)ppp(5′)(2′OMeA)pG (TriLink Biotechnologies), guanosine triphosphate (GTP; Invitrogen), adenosine triphosphate (ATP; Invitrogen), cytidine triphosphate (CTP; Invitrogen), N1-methylpseudouridine-5′-triphosphate (TriLink Biotechnologies), T7 enzyme, a tailed DNA template containing the T7 promoter, and a buffer system. mRNA was purified with a Megaclear kit (Life Technology), characterized using Agilent 2100 bioanalyzer at Heflin Center for Genomic Science at the University of Alabama at Birmingham, and concentrated with Amicon Ultra-2 30 k Centrifugal Filters (UFC203024, Millipore). In vitro transfection of modRNA was performed with Lipofectamine MessengerMax Reagent (Invitrogen, LMRNA015) by the manufacturer's instructions, and a biocompatible sucrose-citrate buffer was used for in vivo transfection.
Human cardiac fibroblasts induced pluripotent stem cells (hiPSCs) were maintained in mTeSR Plus medium (STEMCELL Technologies). The α-myosin heavy chain (α-MHC) promoter—driven CCND2 CMs (CCND2-OEhiPSC-CMs) were derived as previously described in details using GiWi protocol for cardiomyocyte differentiation. Both wildtype hiPSC-CMs and CCND2-OEhiPSC-CMs were cultured for 60 days after differentiation, which time point has minimal mitotic activity.
To calculate the CMs cell proliferation, 2×104 hiPSC-CMs were seeded in 12-well plate and treated with CCND2-CM SMRTs or Luc-CM SMRTs. Cell numbers from day 0 to day 7 were quantified by an automatic cell counter (Countess 3, Invitrogen). Time lapse movies showing CCND2-CM SMRTs—treated hiPSC-CMs dividing were taken by Lionheart FX automated microscope (Agilent, Santa Clara, CA, USA) for 48 hours.
Flow cytometry was performed as described previously to detect GFP-positive hiPSC-CM ratio in dose-dependent study and time-course study. To determine the optimized dosage for modRNA transfection, different dosages of GFP-modRNA were transfected to 5×104 hiPSC-CMs seeded in 24-well plate 24 hours prior to flow cytometry analysis. Time-course study was performed by transfecting 3 μg GFP-modRNA to 5×104 hiPSC-CMs seeded in 24-well plate. Then, hiPSC-CMs were trypsinized into individual cells, fixed in fixation and permeabilization solution (51-2090KZ, BD Biosciences) for 30 minutes at 4° C., blocked in Human BD Fc Block (564219, BD Biosciences) at room temperature for 10 minutes, incubated with fluorescent conjugated antibodies (Table 2) or isotype control antibodies at room temperature for 40 minutes, resuspended in wash buffer (554723, BD Biosciences), and evaluated with an LSR Fortessa instrument (BD Biosciences, USA).
After the mouse or pig hearts were explanted, they were briefly washed by natural saline and weighted. Then, the pig cardiac tissue (infarct zone and remote zone) and mouse hearts were dehydrated by 30% sucrose buffer at 4° C. overnight, embedded in O.C.T. compound, sectioned into 10 μm slides using a cryostat (Leica), and processed for histology or immunofluorescence. Cells were seeded in chamber slides, treated with modRNA or CM SMRTs, then processed for immunofluorescent staining.
For immunofluorescent staining, cells or frozen sections were tripled washed by PBS, fixed in 4% paraformaldehyde (PFA) for 10 minutes at room temperature, permeabilized with 90% acetone for 3 minutes at −20° C., blocked with 10% donkey serum for 20 minutes, incubated with primary antibodies at 4° C. overnight, then incubated with fluorescently labeled secondary antibodies for 30 minutes at room temperature in dark. The primary and secondary antibodies are listed in Table 2. Lastly, slides are mounted with Antifade Mounting Medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories), and imaged with a confocal microscope (Olympus, Japan).
BrdU incorporation analysis was performed using 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit I (Roche). Briefly, 10 μM BrdU labeling solution was added to culture medium 12 hours prior to fixation and permeabilization. Next, cells were incubated with Anti-BrdU working solution for 30 minutes at 37° C. before subjected to immunofluorescent staining as described above.
Cell apoptosis was evaluated with an In-situ Cell Death Detection Kit (12156792910; Roche Applied Science, Germany) as described previously. Briefly, after fixation and permeabilization, cells were stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) solution (Roche) at 37° C. for 1 hour, then subjected to the following primary and secondary staining to identify cTnT. Apoptosis was quantified as the ratio of the number of TUNEL positive nuclei to the total number of nuclei.
To measure CM cross-sectional area and CM nucleation, tissue sections were incubated with WGA conjugated to Alexa Fluor® 488 (Molecular Probes, Invitrogen) diluted in PBST at room temperature for 15 minutes before they were subjected to primary/secondary antibody staining as described above.
Immunofluorescence staining was quantified in every tenth serial section from the region of interest for each heart; mouse data were collected for 25-30 short axis sections out of total 250-300 sections per entire heart, and pig data were collected from at least 60 sections from two blocks each of 300 sections in the border zone containing each side of the anterior-septal LV scar perfused by left anterior descending coronary artery (i.e. border zone); and 30 sections from the region perfused by the left circumflex coronary artery (i.e., the remote zone). Five randomly selected high-resolution (40× magnification) images were evaluated for each section, and the results were quantified with Image J software.
The infarct size was evaluated by Picro-Sirius Red/Fast Green staining as previously described56. Tissue sections were fixed in Bouin's solution and stained with Fast Green dye to identify functional myocardial tissue and with Picrosirius Red to identify scar tissue. Sections were photographed with an Olympus light microscope and analyzed with Image J software. In mouse study, infarct size was calculated in every 100th serial section from apex to base (usually 8-10 sections per heart). In pig study, fibrotic area was calculated from ring 2, ring 3 and ring 4.
Mouse MI Model and modRNA Injection
MI was induced in female and male C57BL/6J mice (stock #000664; The Jackson Laboratory) as previously described. Briefly, 8-12-week-old mice were anesthetized with inhaled 2% isoflurane, intubated, and ventilated with a small animal respirator. A left thoracotomy was performed to expose the heart, and the left anterior descending (LAD) coronary artery was permanently ligated with an 8-0 non-absorbable suture; mice in the Sham group underwent all surgical procedures for MI induction except for the ligation step. Treatments (GFP-CM SMRTs, CCND2-CM SMRTs, Luc-CM SMRTs, L7Ae modRNA, or Vehicle [sucrose-citrate buffer]) were administered to three sites around the infarcted zone immediately after LAD ligation. Mice were administered buprenorphine (0.1 mg/kg every 12 hours for 3 consecutive days) and carprofen (5 mg/kg every 12 hours for 1 day) after surgery for pain control during recovery.
Pig MI Model and modRNA Injection
MI was experimentally induced in female and male Yorkshire swine (˜15 kg, Snyder Farms, Birmingham) as previously described. Briefly, pigs were anesthetized with inhaled 2% isoflurane, intubated, and ventilated with a respirator to maintain anesthesia. A center thoracotomy was performed, and the roots of the first and second diagonal coronary arteries from the LAD coronary artery were ligated with 4.0 polypropylene sutures for 60 minutes before reperfusion. Treatments (CCND2-nGFP-CM SMRTs, CCND2-CM SMRTs, nGFP-CM SMRTs, or the delivery Vehicle) were injected immediately after reperfusion into five sites in the border zone of the injured myocardium. After surgery, the chest was closed in layers, and the animals were allowed to recover. Animals received subcutaneous injections of buprenorphine SR (0.24 mg/kg; Buprenex, Rupkitt Benckiser Pharmaceuticals Inc) every 12 hours for up to 3 days and intramuscular injections of carprofen (4 mg/kg; Rimadyl, Zoetis) every 24 hours for up to 2 days after surgery.
Cell sample or tissue collected from mice or pigs were lysed with T-PER™ Tissue Protein Extraction Reagent (Thermo Scientific) containing protease-inhibitor and phosphatase-inhibitor cocktails (Sigma-Aldrich); then, protein extracts were quantified with a Pierce™ BCA Protein Assay Kit (Thermo Scientific), run on Mini-PROTEANN TGX Stain-Free Gels (Bio-Rad), and transferred to polyvinylidene difluoride (PVDF) membranes using semi-dry transfer method with Trans-Blot Turbo RTA Transfer Kit (Bio-Rad). The membranes were blocked by 5% non-fat milk for 1 hour at room temperature, incubated with primary antibodies overnight at 4° C. and with secondary antibodies for 2 hours at room temperature, and then with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore Sigma) before imaged using Bio-Rad ChemiDoc Imager. The protein signal was digitized and quantified with ImageJ software.
Animals were lightly anesthetized with 1-2% inhaled isoflurane, and heart rates were stable at 400-500 bpm for mice or 60-80 bpm for pigs; then, B-mode and two-dimensional M-mode images of the heart were acquired from both the long-axis and short-axis views with high-resolution micro-ultrasound systems (for mice: Vevo 2100, VisualSonics, Inc.; for pigs: GE LOGIQ V2 Ultrasound, GE Healthcare). Data were analyzed to calculate left ventricular ejection fraction and left ventricular fractional shortening.
Cardiac Magnetic Resonance Imaging (cMRI)
cMRI was performed with a 1.5-Tesla clinical scanner (GE signa horizon software 9.1) and a phased-array 4-channel surface coil with electrocardiogram (ECG) gating as previously described. Pigs were anesthetized with 2% inhaled isoflurane and positioned in a spine position within the scanner with ECG, respiratory, and cutaneous temperature monitoring. The heart was scanned along vertical and horizontal long axis views and with a set of short axis views covering the entire LV from atrioventricular valve plane to the apex. Cine imaging was performed with the following parameters: TR=3.1 ms, TE=1.6 ms, flip angle=45°, matrix size=224×128, field of view=340×265 mm2, slice thickness=8 mm (with no gap between slices); 20 phases were acquired across the cardiac cycle. Infarct size was measured via late gadolinium enhancement (LGE) cMRI (0.20 mmol/kg gadopentetate dimeglumine, intravenous bolus) with the following parameters: TR=16 ms, TE=4 ms, TI=150-300 ms (TI depends on how fast the contrast washes out of the myocardium), flip angle=20°, matrix size=256×148, field of view=320×185 mm2, slice thickness=8 mm (with no gap between slices).
cMRI images were analyzed using commercially available research software package (CAAS MRV 3.4, Pie Medical Imaging, Netherlands). Global LV functional parameters (end-diastolic volume [EDV] and end-systolic volume [ESV], ejection fraction [EF], stroke volume [SV]) and regional functional parameters (left ventricular regional wall thickening [LVWT] and radial strain [εRR]) were measured as previously described. LV endocardial and epicardial borders were manually contoured on all short-axis cine images at the end-diastolic and end-systolic frames to determine the EDV and ESV, respectively, as well as EF and SV. Indexes of EDV, ESV and SV were calculated by normalizing the individual values against body surface area of the pigs. To calculate regional LV function, the middle slices (area of interest), orthogonal to LV long axis, were divided into 6 circumferential segments according to the American Heart Association 17-segment model. The left-right ventricular (LRV) junction point was defined at the inferior portion of the interventricular septum. Six segments were plotted to generate the curve and subsequently calculate the area under curve (AUC).
Post-infarction fibrosis was measured in LGE-cMRI images of the LV short-axis using the same software. The quantification of LGE was performed by manually adjusting a greyscale threshold to define areas of visually identified LGE. The proportion of infarct size was calculated by dividing the total area of LGE versus total are of LV myocardium. The infarct core and the infarct gray zone (consisting necrotic and viable myocardium surrounding the infarct core) were analyzed using the full-width half-maximum method.
Infarct size was measured by Picro-Sirius Red/Fast Green staining in mouse model, and by LGE-cMRI in pig model using the following equation:
In pig model of AMI, infarct was also evaluated by quantifying the gadolinium-enhanced region (Scar mass) and the entire LV weight, to derive the Infarct Mass Percentage:
Programmed electrical stimulation (PES) was performed as previously described. Briefly, a 6F electrode catheter was advanced through the right femoral vein and into the right ventricular apex, and the heart was paced (51) at a cycle length of 400 ms with one to three additional stimuli (S2, S3, or S4) delivered at progressively shorter intervals. S1-S2 began at 10 ms and ended at 5 ms, and the protocol was repeated for S2-S3 and S3-S4. Programmed stimulation ceased immediately after an episode of ventricular arrhythmia (VA) began. VA episodes that lasted fewer than 15 heart beats were categorized as non-sustained VA, and episodes that lasted for 15 or more heart beats were categorized as sustained VA.
Data are presented as mean±SEM. Significance was determined via the Student's t-test for comparisons between two groups and via one-way analysis of variance for comparisons among three or more groups. A P-value of less than 0.05 was considered statistically significant.
The CM SMRTs (
modRNAs for L7Ae, luciferase (Luc), CCND2, GFP, nuclear GFP (nGFP), and CCND2-nGFP were transcribed in vitro and validated with a bioanalyzer (
CCND2-CM SMRTs Promotes Cardiomyocyte Proliferation and Myocardial Regeneration and Improves Recovery from AMI in Mice
CCND2 was abundantly expressed in cultured, post-mitotic (60-day-old) hiPSC-CMs two days after transfection with CCND2-CM SMRTs (
AMI was induced via permanent ligation of the left anterior descending (LAD) coronary artery, and then the animals were randomly distributed into four treatment groups. The CCND2-CM SMRTs group received 100 μg CCND2-modRNA with 50 μg L7Ae-modRNA, the Luc-CM SMRTs group received 100 μg Luc-modRNA with 50 μg L7Ae-modRNA, the L7Ae-modRNA group received 50 μg-L7Ae modRNA, and the Vehicle group received an equivalent volume of the delivery vehicle; a fifth group of animals, the Sham group, underwent all surgical procedures for MI induction except for LAD artery ligation and recovered without any of the experimental treatments. Survival rates for mice in all four groups that underwent MI were similar (
Immunofluorescence images of sections collected from the border zone of the infarct on Day 7 after MI induction and treatment indicated that the proportion of CM expressing Ki67 (
Cardiac function was evaluated 1 day before MI induction and 0.5, 2, 4, 7, 14, and 28 days afterward via echocardiographic assessments (
This investigation of the potency of CCND2-CM SMRTs for improving recovery from myocardial injury continued with experiments in a more clinically relevant, large-mammalian model (
Western blot assessments conducted in organs from pigs sacrificed 3 days after MI induction and treatment confirmed that CCND2 protein levels were upregulated in the hearts of CCND2-CM SMRTs animals, but not in other major organs, and were unchanged in all organs (including the hearts) of animals in the nGFP-CM SMRTs and Vehicle groups (
CCND2-CM SMRTs Reduces Infarct Size and Improves Global and Regional Cardiac Function after AMI in Pigs
Cardiac function was evaluated before AMI induction (Day 0) and 10 and 28 days afterward via echocardiography (
Infarct size was evaluated on Day 28 via late gadolinium enhancement (LGE) cMRI. The gadolinium-retaining region (
CCND2-CM SMRTs does not Induce Long-Term Cardiomyocyte Proliferation or Increase the Risk of Arrythmia after Administration to Infarcted Pig Hearts
At 4 weeks after the surgery, cardiac hypertrophy was significantly reduced in CCND2-CM SMRTs group evidenced by decreased heart weight/body weight ratio and left ventricular weight/body weight ratio (
One of the primary safety concerns associated with the administration of CCND2-overexpressing hiPSC-CMs is that long-term, uncontrolled expression of cell-cycle regulatory molecules could lead to excessive cardiomyocyte proliferation and arrhythmia. However, Ki67- and PH3-positive border-zone and remote-zone CM were no more prevalent on Day 28 in CCND2-CM SMRTs-treated hearts than in hearts from animals in the nGFP-CM SMRTs- or Vehicle-treatment groups (
Despite current intensive treatment regimens, patients with severe AMI often progress to end stage CHF, which is one of the most significant problems in public health today. From the molecular and cellular perspective, heart failure occurs due to the loss of the contractile unit of the left ventricle: CM. However, the regenerative capacity of adult mammalian hearts is limited because the majority of CM exit the cell cycle shortly after birth and arrest at the G1/S transition, known as the restriction point (R-point) of cell cycle withdrawal. R-point transit is partially governed by the activity of cyclin-dependent kinase 4 (CDK4) and its obligate cofactors, the D-type cyclins. CDK4/cyclin D complex disrupts RB-E2F binding by phosphorylating the RB protein family, permitting E2F-mediated transcription of genes involved in activating DNA synthesis and cell cycle progression. Previous study on the cyclin D family using transgenic mouse model has demonstrated that cyclin D2 gene driven by a CM-restricted MHC promoter led to a robust nuclear CCND2 overexpression and preserved CM cell cycle activity in both healthy and myocardial hypertrophic mice. Whereas when hypertrophy was induced in cyclin D1 or D3 transgenic mice, the nuclear localization of cyclin D1 and D3 protein was compromised, impeding their association with CDK4 before nuclear translocation thus, failed to maintain DNA synthesis under myocardial hypertrophy.
In the present study, the immunostaining results from both the in vitro (
The increase in the proportion of cardiomyocytes that expressed PH3 and AuB in the hearts of animals treated with CCND2-CM SMRTs after MI is consistent, or even greater, than the increases reported in other studies of induced cardiomyocyte proliferation. Nevertheless, it is acknowledged that PH3+ and AuB+ cardiomyocytes may be undergoing either multinucleation events (i.e., karyokinesis) or cytokinesis, so the analysis has been refined by determining the proportion of cardiomyocytes in which AuB expression is localized asymmetrically, which identifies a karyokinesis event within a single cell, or symmetrically between two daughter nuclei. Mouse (
These findings of CCND2 preventing cardiomyocytes hypertrophy (
A major concern on some of the established approaches for CM regeneration is that excessive activation of CM and reduction in sarcomere stability may lead to lethal arrhythmia. For instance, a previous study offered an approach by transplanting CCND2 overexpressed hiPSC-CMs into myocardial infarcted porcine heart, which enhanced the proliferation of both the transplanted and endogenous CMs22. Although no arrhythmic incidence was observed, a concern has been raised that the lentiviral transduced CCND2 may lead to permanent and prolonged cell cycle activation and excessive CM proliferation. For another instance, lethal arrhythmic incidence is reported by a recent study where AAV-mediated delivery of miR-199a was evaluated in a pig MI model, the miR-199a treatment led to persistent miR-199a expression, and the majority of pigs died from sudden arrhythmia despite improvements in contractility and scar size. In order to minimize the arrhythmogenic complications caused by persistent CM proliferation, it is aimed to induce a transient cell cycle activation in CM by delivering the targeted gene, CCND2, using CM SMRTs technology, taking advantage of its controlled and bell-shaped expression profile.
The successful use of modRNA technology for the development of vaccines against SARS-CoV-2 demonstrates the feasibility of this platform for administration to patients, and because modRNA transcripts are expressed only transiently, they are unlikely to be associated with some of the long-term safety concerns that have limited the clinical translation of other delivery methods. Cell-type specificity, into CM, is also a key component of strategies for improving myocardial repair via the delivery of cell-cycle regulators to the heart, because off-target expression of these molecules in other cell types could promote fibrosis, inflammation, and other complications. Here, it is demonstrated that the CM SMRTs platform can be used to rapidly upregulate CCND2 expression in CM nuclear with exceptional cell-type specificity.
CCND2-CM SMRTs induced transient CM cell cycle activation, but did not increased the risk of arrhythmia. It was observed that CCND2 expression in cultured CM peaked 1 day after the cells were treated with CCND2-CM SMRTs and declined to nearly undetectable levels by Day 14 (
It was also observed that L7Ae-modRNA alone protected cardiomyocyte apoptosis. Both miR-1 and miR-208 contribute to cardiomyocyte apoptosis and were likely to be “sponged” by miR1 and miR208 recognition sites on the L7Ae modRNA, as shown previously. Thus, since modRNA transcripts appear to be expressed almost immediately after transfection, at least some of the improvements observed in CCND2-CM SMRTs—treated animals could depend on the time of treatment administration (i.e., immediately after LAD artery ligation or reperfusion), which likely reduced the number of CM that were lost to the infarct event.
This study has some limitations. It is noted that interventions immediately after the AMI has limitations from the perspectives of translatability and may have confounding factors such as post-conditioning effects. However, it is appreciated that almost all preclinical animal models have limitations considering extrapolating findings to patients with chronic postinfarction LV remodeling and heart failure. In the current study the main hypothesis of whether the modRNA technology, specifically the SMRTs intervention, can reactive CM cell-cycle in large mammal hearts with AMI, and consequently decrease of infarct size and prevent LV dilatation, is investigated.
In conclusion, the results presented in this report demonstrate that CM SMRTs could rapidly and transiently drive the expression of CCND2 in CM nuclear with high cell-type specificity, and that intramyocardial injections of CCND2-CM SMRTs activated cardiomyocyte cell cycle, reduced infarct size, and improved cardiac performance in both small and large mammalian models of myocardial injury. These findings demonstrate, for the first time, that an acute myocardial infarct of mammalian hearts could be remuscularized by the CM modRNA SMRTs technology.
It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. All such modifications and variations are intended to be included herein within the scope of this disclosure and protected by the following claims.
This application claims the benefit of U.S. Provisional Application No. 63/364,664, filed on May 13, 2022, which is incorporated herein by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63364664 | May 2022 | US |
