Lipid nanoparticle, and methods of use thereof

Abstract

The present disclosure provides lipid nanoparticles for delivery of a payload (e.g., messenger RNA (mRNA)) to the heart. For example, lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da. In some embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids. The disclosure also encompasses methods for delivery of messenger RNA (mRNA) for transfection of cells, methods of treatment, as well as kits thereof.

Description

LIPID NANOPARTICLE, AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 543,689, filed October 11, 2023, the disclosure of which is incorporated herein by reference in its entirety. INTRODUCTION

[0002] Heart failure (HF) has been defined as a global pandemic (A systematic analysis for the Global Burden of Disease Study 2017. Lancet 392, 1789-1858 (2018)) and the leading cause of death worldwide (Tsao, C. W. et al. Heart Disease and Stroke Statistics-2022 Update: A Report From the American Heart Association. Circulation 145, e153-e639 (2022)). HF can arise from a variety of sources, most often from coronary artery disease and myocardial infarction (MI) (Gabriel-Costa, D. The pathophysiology of myocardial infarction-induced heart failure. Pathophysiology 25, 277-284 (2018)). In past decades, the HF prognosis has slightly improved; however, due to sparse innovation in HF therapeutics, mortality remains high (Savarese, G. et al. Global burden of heart failure: a comprehensive and updated review of epidemiology. Cardiovasc Res 118, 3272-3287 (2023); Yang, Q., Fang, J., Lei, Z., Sluijter, J. P. G. & Schiffelers, R. Repairing the heart: State-of the art delivery strategies for biological therapeutics. Adv Drug Deliv Rev 160, 1-18 (2020)). A primary reason behind the lack of progress in heart therapeutics is the inability to use phenotypic human tissue-level approaches to discover novel therapies. In recent years, there have been significant advances in the development of non-animal models (NAMs) such as organ- on-a-chip microfluidic culture devices (i.e., microphysiological systems (MPS)), which recapitulate organ-level and even organism-level functions (Ingber, D. E. Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat Rev Genet 23, 467-491 (2022); Cho, S., Discher, D. E., Leong, K. W., Vunjak-Novakovic, G. & Wu, J. C. Challenges and opportunities for the next generation of cardiovascular tissue engineering. Nat Methods 19, 1064- 1071 (2022); Mathur, A. et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci Rep 5, 8883 (2015)). Furthermore, since the FDA no longer requires the use of animals in master files for new drug applications, MPS are quickly becoming representative of the future of disease modeling and drug screening, therefore paving the way for complex in vitro models to dominate the preclinical drug discovery landscape (Cho, S., Discher,D. E., Leong, K. W., Vunjak-Novakovic, G. & Wu, J. C. Challenges and opportunities for the next generation of cardiovascular tissue engineering. Nat Methods 19, 1064-1071 (2022); Wadman, M. FDA no longer has to require animal testing for new drugs. Science 379, 127-128 (2023); Charwat V. et al. Validating the Arrhythmogenic Potential of High-, Intermediate-, and Low-Risk Drugs in a Human-Induced Pluripotent Stem Cell-Derived Cardiac Microphysiological System, ACS Pharmacol. Transl. Sci. 2022, 5, 652-667; Charrez B. et. al, In vitro safety “clinical trial” of the cardiac liability of drug polytherapy, Clin Transl Sci.2021; 00; 1-11).

[0003] An additional obstacle in developing therapeutics for the heart is that dense cardiac tissue suffers from low transfection efficiency of non-proliferative cardiomyocytes, as well as diffusional barriers posed by the extracellular matrix of 3D cardiac muscle. Consequently, the development of successful cardiac therapies is dependent on the efficient delivery of biological macromolecules such as proteins, DNA, and mRNAs into the heart (ang, Q., Fang, J., Lei, Z., Sluijter, J. P. G. & Schiffelers, R. Repairing the heart: State-of the art delivery strategies for biological therapeutics. Adv Drug Deliv Rev 160, 1-18 (2020); van Haasteren, J., Li, J., Scheideler, O. J., Murthy, N. & Schaffer, D. V. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat Biotechnol 38, 845-855 (2020)). Recent innovation in developing non-viral vectors like solid lipid nanoparticles (LNPs) holds great promise in overcoming these limitations and can make a breakthrough in cardiovascular medicine due to the transient nature of mRNA transfection. Compared to viral delivery systems, LNPs exhibit advantages such as lower immunogenicity and toxicity, better cell specificity, high modifiability, and improved productivity (Park, H. J., Yang, F. & Cho, S. W. Nonviral delivery of genetic medicine for therapeutic angiogenesis. Adv Drug Deliv Rev 64, 40-52 (2012)). However, there has yet to be an effective LNP formulation for therapeutic mRNA delivery to the heart. Therefore, despite progress in this area, one of the remaining challenges is to develop a LNP formulation capable of diffusing within human cardiac muscle, transfecting cardiomyocytes, and escaping the endo-lysosome before degradation more efficiently than current strategies (Yang, Q., Fang, J., Lei, Z., Sluijter, J. P. G. & Schiffelers, R. Repairing the heart: State-of the art delivery strategies for biological therapeutics. Adv Drug Deliv Rev 160, 1-18 (2020)).SUMMARY

[0004] The present disclosure provides lipid nanoparticles for delivery of a payload (e.g., messenger RNA (mRNA)) to the heart. The disclosure also provides methods for delivery of messenger RNA (mRNA) for transfection of cells, methods of treatment, as well as kits thereof.

[0005] Aspects of the present disclosure include a lipid nanoparticle for messenger RNA (mRNA) delivery to the heart, comprising: an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids.

[0006] In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is represented by formula (I):R1comprises an acyl moiety comprising one or more oxyanions, each complexed with a metal cation; R2comprises a hydrophobic group or lipid that can strengthen the rigidity of the lipid nanoparticle; L1and L2are linkers selected from a bond, an optionally substituted heteroatom and an optionally substituted C1-18hydrocarbyl or heterohydrocarbyl, providing acid degradable linkages.

[0007] In certain embodiments, the oxyanions are selected from carbonic acid, phosphoric acid, or sulfonic acid thereof.

[0008] In certain embodiments, the metal cation is selected from sodium, potassium, or lithium thereof.

[0009] In certain embodiments, the hydrophobic group or lipid is a steroid, fatty acid or fatty acid ester.

[0010] In certain embodiments, the lipid nanoparticle further comprises ionizable cationic lipids, helper lipids, phospholipids and cholesterol.

[0011] In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 10% of the total lipids.

[0012] In certain embodiments, the nanoparticle encapsulates molecules selected from nucleic acids, proteins, polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, nutritional supplements, biomarkers, or combinations thereof.

[0013] In certain embodiments, the encapsulated molecules comprise a nucleic acid selected from plasmid DNAs, antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or combinations thereof.

[0014] In certain embodiments, the encapsulated molecule is mRNA.

[0015] In certain embodiments, the lipid nanoparticle has a size of less than 125 nm. In certain embodiments, the lipid nanoparticle has a size of less than 100 nm.

[0016] Aspects of the present disclosure include for delivery of messenger RNA (mRNA) for transfection of cells, comprising administering to a subject a composition comprising an mRNA, wherein the mRNA is encapsulated within a lipid nanoparticle of the present disclosure and wherein the administering of the composition results in the transfection of cells by the mRNA.

[0017] In certain embodiments, the transfection of cells is in vivo. In certain embodiments, the transfection of cells is in vitro.

[0018] In certain embodiments, the cells are cardiomyocytes, cardiac fibroblasts, pericytes, Natural Killer cells, T cells endothelial cells, tissue resident macrophages, smooth muscle cells, nerve cells, stem cells, progenitor cells, interstitial cells, adipose / fat cells, or other immune cells such as lymphocytes, neutrophils, or dendritic cells.

[0019] In certain embodiments, the mRNA is endothelial nitric oxide synthase (eNOS) mRNA.

[0020] In certain embodiments, the administering comprises administering the composition systemically.

[0021] In certain embodiments, the administering comprises administering the composition intramuscularly.

[0022] In certain embodiments, the administering comprises administering the composition subcutaneously.

[0023] Aspects of the present disclosure include a kit comprising a lipid nanoparticle of the present disclosure; and a syringe.

[0024] Aspects of the present disclosure include a method of treating a subject for a heart condition, comprising: administering to a subject in need thereof a therapeutically effective amount of an mRNA within a lipid nanoparticle that provides release of mRNA in the heart, wherein the lipid nanoparticle is of the present disclosure.

[0025] In certain embodiments, the heart condition is heart failure, coronary artery disease, cardiovascular disease, or myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIG. 1 shows schematic of therapeutic candidate product profile. (Left to right) Lipid nanoparticle (LNP) containing endothelial nitric oxide synthase (eNOS) mRNA are introduced into a syringe of an injection catheter. Percutaneous minimally invasive approach into a patient with an myocardial infarction (MI) delivers the LNP to the infarcted area. (Right) Either a transendocardial or intracoronary approach is possible, with the latter favored due to simplicity, cost, and widespread clinical access. Yellow dots represent LNPs delivered to the heart by either method, keeping the LNPs located in the infarct border zone, or infarcted vascular bed.

[0027] FIG. 2 shows cardiac microphysiological system (MPS) features and function. (a) SEM image of the decellularized human myocardial extracellular matrix (ECM) with collagen fibers surrounding individual myocytes in a thin endomysial sheet. Cardiac myocytes are organized into an aligned structure, typically ± 15º within main axis, by the large coiled perimysial fibers (yellow arrow) that are separated by 150 µm. Bar = 100 µm. (b) Brightfield image of the cardiac MPS with nutrient / media channels (yellow arrows) and central tissue chamber. Cardiac MPS enables real time measurements of beating physiology (optical), electrophysiology (e.g., voltage and Ca dyes), and force of contraction (PDMS pillars). (c) SEM micrograph of the MPS showing the 2µm endothelial-like barriers connecting the nutrient channel and the tissue chamber which allows for diffusive transport of drugs and molecules. Simulated velocity profile of flow in the nutrient channel and barrier interface, demonstrating mass transport to the tissue is exclusively diffusive. (d) Confocal image of the microtissue reveals aligned sarcomeric α-actinin (green) and 3D tissue with 5-7 cell layers in the z-axis (55µm ZY x-section). (e) Magnitude of contraction (1stpeak) and relaxation (2ndpeak) velocities in x / y directions. (f) Heat map of the time-averagedbeating motion, capturing motion along the x and y axes. Bar = 100µm. (g) Histogram shows ~ 70% of the contraction displacement is within ± 25° deviation of the x-axis.

[0028] FIG. 3A shows a schematic illustration of LNP production containing an acid degradable PEG-lipid which improves the transfection efficiency and endosomal escape of LNP / mRNA complex. FIG. 3B shows transmission electron microscopy (TEM) images of LNPs with different ADP-lipid mole percentages.

[0029] FIG. 4 shows synthesis of acid-degradable PEG-cholesterol. Schematic of the synthetic strategy used to generate PEG-cholesterol (1), the PEG molecular weight was either 1000 or 2000 Da.

[0030] FIG.5, panel A, shows brightfield and fluorescent microscopy images of 2D XR1- iCMs exposed overnight to different LNP formulations containing eGFP mRNA. FIG.5, panel B. Flow cytometry analysis of 2D XR1 iCM monolayers transfected with LNPs / eGFP mRNA. FIG. 5, panel C. Quantitative analysis of B (n=3-5). FIG.5, panel D. Fluorescent microscopy images of 2D XR1-iCMs exposed overnight to different LNP formulations containing CRE rec mRNA. FIG. 5, panel E. Flow cytometry analysis of 2D XR1 iCM monolayers transfected with LNPs / Cre mRNA. FIG.5, panel F. Quantitative analysis of E (n=3-5)

[0031] FIG. 6, panel A, shows transfection (eGFP+ cells) of XR1-iCMs in the MPS exposed to the different LNP formulations. FIG. 6, panel B. Volumetric nuclear segmentation of the cardiac micromuscle showing eGFP+ cell population in the XY and ZX plane (inset). FIG. 6, panel C. Representative dot plot showing the intensity distribution of eGFP+ cells after LNP / Cre mRNA delivery into the cardiac MPS. The dotted line represents the cutoff for eGFP+ cells in 3D cardiac micromuscle. FIG. 6, panel D. Quantitative analysis of eGFP+ cells in the cardiac micromuscle after LNP delivery into the MPS (n=3-5). FIG.6, panel E. Representative distribution of eGFP+ cells within the center of the cardiac MPS (dotted vertical lines). Each point represents an individual cell. The green line is the cutoff for eGFP+ cells. Every condition was normalized to 1100 iCMs. FIG.6, panel F. Quantitative analysis of E (n=3-4).

[0032] FIG.7A shows in vivo Luciferase activity in anesthetized mice showing decreased liver uptake of 2kDa 10% ADP-LNP when compared to LNP1, with a concomitant increase in cardiac uptake after direct intracardiac injection. FIG. 7B. Quantification of luciferase activity in isolated heart and liver confirms that 2kDa 10% ADP-LNP displays a much higher cardiac specificity and lower off target activity when compared to LNP-1 (n=4).

[0033] FIG. 8, panel A, shows brightfield (left) and fluorescence (right) whole-mount images showing ZsGreen1 activity upon intracardiac delivery of 2kDa 10% ADP-LNP in MI- induced hearts. FIG. 8, panel B. Cryosections and immunofluorescence show the spatial distribution of ZsGreen1 activity in the left ventricle of the heart in homeostasis induced by CRE recombination of the stop cassette. Inset shows cardiac delivery highlighted by presence of sarcomeres in ZsGreen1 positive cells. FIG. 8, panel C. Immunofluorescence for cardiac (Tnni, left), endothelial cells (IB4, middle), and smooth muscle / pericyte cells (aSMA, right). scale bars- 20mM.

[0034] FIG. 9 shows synthesis of acid-degradable lipids that generate either endosomal disruptive molecules or are converted into neutral lipids in the endosome. A library of ionizable lipids will be synthesized, from either primary amine or secondary amine azido acetal intermediates. The library will be synthesized via a Michael addition reaction. These new lipids are designed to hydrolyze into membrane disruptive surfactants in the endosome or generate non- toxic neutral lipids after endocytosis.

[0035] FIG.10, panel A, shows fluorescent image of a 3D cardiac micromuscle projection upon exposure of ADP-LNPs containing CRE mRNA. Bi-cellular cardiac micromuscle composition is 80% iCMs-20% hcFb. iCMs are identified based on the Cardiac Troponin T (cTnT) positive expression and hcFb based on the Vimentin positive expression. FIG.10, panel B. Section of the cardiac micromuscle exhibiting the tissue height consisting of 5-6 layers of iCMs. FIG.10, panel C. XY section of the cardiac micromuscle showing eGFP+ iCMs and hcFb upon exposure of ADP-LNPs containing CRE mRNA. FIG. 10, panel D. Representative dot plot showing the intensity distribution of eGFP+ cells after 2 kDA 10% ADP-LNPs / Cre delivery vs. naked CRE mRNA into the cardiac MPS. The dotted line shows the cutoff for eGFP+ cells in 3D cardiac micromuscle. FIG. 10, panel E. Quantitative analysis of D (n=3). FIG. 10, panel F. Motion contraction analysis over time of the cardiac micromuscle before and after LNP exposure (n=6-9). Representative brightfield images of the cardiac MPS showing the displacement vectors used to estimate the peak twitch amplitude. FIG. 10, panel G. APD analysis over time of the cardiac micromuscle before and after LNP exposure (n=5-9).

[0036] FIG.11, panel A, shows a photograph of a cardiac MPS under fluorescent lighting with fluidic tubing. FIG. 11, panel B. Layout of a multiplexed cardiac MPS device comprising four parallel tissue chambers with individual cell loading ports and a common media inlet (in) andoutlet (out). The media channels run parallel to each tissue chamber, and the fenestration barrier provides protection from fluid shear stress while allowing for the exchange of media via diffusion. Anchor pillars located at both extremities of the tissue chamber provide attachment points to keep the cardiac muscle elongated and prevent collapsing. FIG. 11, panel C. Brightfield images of the MPS with cardiac tissues formed using XR1 WTC iCMs. FIG. 11, panel D. SEM micrograph of the MPS showing the 2μm endothelial-like barriers connecting the nutrient channel and the tissue chamber which allows for diffusive transport of drugs and molecules. Simulated velocity profile of flow in the nutrient channel and barrier interface, demonstrating mass transport to the tissue is exclusively diffusive.

[0037] FIG. 12A shows time-course experiment showing the diffusion of 2kDa PEG conjugated to FITC over time. Images were captured every 15 mins. FIG. 12B. FRAP experiment was performed after the PEG and Dextran polymers reached a steady state. Finitial is the time regime that corresponds to the initial fluorescence before bleaching; F0 is the fluorescence measurement immediately after photobleaching; Ft>0 corresponds to the recovery of fluorescence after photobleaching; Ft>>0 corresponds to the maximal recovery of fluorescence at the end of the experiment. FIG. 12C. Half-time of recovery (sec) and diffusion coefficient (μm2 / s) were estimated for the different polymers in the FRAP assay.

[0038] FIG.13, panel A, shows a schematic illustration of the development of a genetically modified hiPSC Cre-reporter line by TALEN-Mediated homologous recombination. FIG. 13, panel B. eGFP expression in the XR1 hiPSC upon Cre recombinase plasmid transfection.

[0039] FIG. 14A shows flow cytometry analysis of 2D iCMs after RNA iMAX and LNP- 1 / eGFP mRNA transfection (controls). FIG.14B. Flow cytometry analysis of 2D iCMs after RNA iMAX, LNP-1 / CRE rec mRNA and Tat-Cre Rec protein transfection (controls).

[0040] FIG. 15, panel A, shows fluorescent microscopy images showing the eGFP+ cells within the XR1 cardiac micromuscles after exposure to naked CRE rec mRNA, Tat-CRE Rec protein, and LNP-1 / CRE rec mRNA. FIG.15, panel B. Representative distribution of eGFP+ cells within the center of the cardiac MPS (dotted vertical lines). Each point represents an individual cell. Green line is the cutoff for eGFP+ cells. Every condition was normalized to 1100 cell.

[0041] FIG. 16, panel A, shows a schematic diagram showing the protocol for small molecule-directed differentiation to hiPSC-derived cardiomyocytes and cardiac fibroblasts. FIG. 16, panel B. Representative flow cytometry analysis showing protein levels of hcFb markers, suchas CD90, DDR2, and Vimentin. The protein level for the endothelial marker CD31 was also assessed. FIG. 16, panel C. Representative brightfield image of the hcFbs in culture and representative immunofluorescence images showing the positive expression for hcFB markers, such as Collagen Type I, CD90, DDR2 and TE-7. FIG. 16, panel D. mRNA expression analysis of the expression levels of genes that are highly expressed in cardiac fibroblasts, such as COL1A1, GATA4, POSTN, and TBX20 relative to their pluripotent state. Direct comparisons were made using an unpaired Student’s t-test (*p < 0.05, **p < 0.001).

[0042] FIG.17, panel A, shows schematic illustration describing transfection of Ai6 mouse with either Luc mRNA or CRE mRNA delivered by 2 kDa 10% ADP-LNPs and LNP-1. FIG.17, panel B. Characterization of the region used in quantification presented in FIG. 5, panel B. upon 15 seconds of exposure. FIG.17, panel C. Normalized images of Liver (top, exposure 30 seconds) and hearts (bottom, exposure 300 seconds) show clear increase in heart luciferase activity in 2 kDa 10% ADP-LNP when compared to LNP-1 with a small decrease in overall off-target liver expression. DEFINITIONS

[0043] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0044] “Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms and such as 1 to 6 carbon atoms, or 1 to 5, or 1 to 4, or 1 to 3 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3CH2-), n-propyl (CH3CH2CH2-), isopropyl ((CH3)2CH-), n-butyl (CH3CH2CH2CH2-), isobutyl ((CH3)2CHCH2-), sec-butyl ((CH3)(CH3CH2)CH-), t-butyl ((CH3)3C-), n-pentyl (CH3CH2CH2CH2CH2-), and neopentyl ((CH3)3CCH2-).

[0045] The term “substituted alkyl” refers to an alkyl group as defined herein wherein one or more carbon atoms in the alkyl chain (except the C1carbon atom) have been optionally replaced with a heteroatom such as -O-, -N-, -S-, -S(O)n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-aryl, -SO2-heteroaryl, and -NRaRb, wherein R’and R”may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0046] “Alkylene” refers to divalent aliphatic hydrocarbyl groups preferably having from 1 to 6 and more preferably 1 to 3 carbon atoms that are either straight-chained or branched, and which are optionally interrupted with one or more groups selected from -O-, -NR10-, -NR10C(O)-, - C(O)NR10- and the like. This term includes, by way of example, methylene (-CH2-), ethylene (-CH2CH2-), n-propylene (-CH2CH2CH2-), iso-propylene (-CH2CH(CH3)-), (-C(CH3)2CH2CH2-), (-C(CH3)2CH2C(O)-), (-C(CH3)2CH2C(O)NH-), (-CH(CH3)CH2-), and the like.

[0047] “Substituted alkylene” refers to an alkylene group having from 1 to 3 hydrogens replaced with substituents as described for carbons in the definition of “substituted” below.

[0048] The term “alkane” refers to alkyl group and alkylene group, as defined herein.

[0049] The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R’NHR”- where R’ is alkyl group as defined herein and R” is alkylene, alkenylene or alkynylene group as defined herein.

[0050] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0051] “Alkoxy” refers to the group –O-alkyl, wherein alkyl is as defined herein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec- butoxy, n-pentoxy, and the like. The term “alkoxy” also refers to the groups alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0052] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0053] The term “alkoxyamino” refers to the group –NH-alkoxy, wherein alkoxy is defined herein.

[0054] The term “haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

[0055] The term “haloalkyl” refers to a substituted alkyl group as described above, wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group. Examples of such groups include, without limitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl, trifluoroethyl and the like.

[0056] The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0057] The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S- substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0058] “Alkenyl” refers to straight chain or branched hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and having at least 1 and preferably from 1 to 2 sites of double bond unsaturation. This term includes, by way of example, bi-vinyl, allyl, and but-3-en-1-yl. Included within this term are the cis and trans isomers or mixtures of these isomers.

[0059] The term “substituted alkenyl” refers to an alkenyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0060] “Alkynyl” refers to straight or branched monovalent hydrocarbyl groups having from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms and having at least 1 and preferably from 1 to 2 sites of triple bond unsaturation. Examples of such alkynyl groups include acetylenyl (-C≡CH), and propargyl (-CH2C≡CH).

[0061] The term “substituted alkynyl” refers to an alkynyl group as defined herein having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0062] “Alkynyloxy” refers to the group –O-alkynyl, wherein alkynyl is as defined herein. Alkynyloxy includes, by way of example, ethynyloxy, propynyloxy, and the like.

[0063] “Acyl” refers to the groups H-C(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, alkenyl- C(O)-, substituted alkenyl-C(O)-, alkynyl-C(O)-, substituted alkynyl-C(O)-, cycloalkyl-C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, substituted aryl-C(O)-, heteroaryl-C(O)-, substituted heteroaryl-C(O)-, heterocyclyl-C(O)-, and substituted heterocyclyl-C(O)-, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. For example, acyl includes the “acetyl” group CH3C(O)-

[0064] “Acylamino” refers to the groups –NR20C(O)alkyl, -NR20C(O)substituted alkyl, N R20C(O)cycloalkyl, -NR20C(O)substituted cycloalkyl, - NR20C(O)cycloalkenyl, -NR20C(O)substituted cycloalkenyl, -NR20C(O)alkenyl, - NR20C(O)substituted alkenyl, -NR20C(O)alkynyl, -NR20C(O)substituted alkynyl, -NR20C(O)aryl, -NR20C(O)substituted aryl, -NR20C(O)heteroaryl, -NR20C(O)substituted heteroaryl, -NR20C(O)heterocyclic, and -NR20C(O)substituted heterocyclic, wherein R20is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0065] “Aminocarbonyl” or the term “aminoacyl” refers to the group -C(O)NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined togetherwith the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0066] “Aminocarbonylamino” refers to the group –NR21C(O)NR22R23where R21, R22, and R23are independently selected from hydrogen, alkyl, aryl or cycloalkyl, or where two R groups are joined to form a heterocyclyl group.

[0067] The term “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0068] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl-C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclyl-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclyl are as defined herein.

[0069] “Aminosulfonyl” refers to the group –SO2NR21R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic and where R21and R22are optionally joined together with the nitrogen bound thereto to form a heterocyclic or substituted heterocyclic group and alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic and substituted heterocyclic are as defined herein.

[0070] “Sulfonylamino” refers to the group –NR21SO2R22, wherein R21and R22independently are selected from the group consisting of hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic and where R21and R22are optionally joined together with the atoms bound thereto to form a heterocyclic or substituted heterocyclic group, and wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0071] “Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from 6 to 18 carbon atoms having a single ring (such as is present in a phenyl group) or a ring system having multiple condensed rings (examples of such aromatic ring systems include naphthyl, anthryl and indanyl) which condensed rings may or may not be aromatic, provided that the point of attachment is through an atom of an aromatic ring. This term includes, by way of example, phenyl and naphthyl. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2- alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl and trihalomethyl.

[0072] “Aryloxy” refers to the group –O-aryl, wherein aryl is as defined herein, including, by way of example, phenoxy, naphthoxy, and the like, including optionally substituted aryl groups as also defined herein.

[0073] “Amino” refers to the group –NH2.

[0074] The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that at least one R is not hydrogen.

[0075] The term “azido” refers to the group –N3.

[0076] “Carboxyl,” “carboxy” or “carboxylate” refers to –CO2H or salts thereof.

[0077] “Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or “carboxylalkyl” refers to the groups -C(O)O-alkyl, -C(O)O-substituted alkyl, -C(O)O-alkenyl, -C(O)O-substituted alkenyl, -C(O)O-alkynyl, -C(O)O-substituted alkynyl, -C(O)O-aryl, -C(O)O-substituted aryl, -C(O)O-cycloalkyl, -C(O)O-substituted cycloalkyl, -C(O)O-cycloalkenyl, -C(O)O-substitutedcycloalkenyl, -C(O)O-heteroaryl, -C(O)O-substituted heteroaryl, -C(O)O-heterocyclic, and -C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0078] “(Carboxyl ester)oxy” or “carbonate” refers to the groups –O-C(O)O- alkyl, -O-C(O)O-substituted alkyl, -O-C(O)O-alkenyl, -O-C(O)O-substituted alkenyl, -O-C(O)O- alkynyl, -O-C(O)O-substituted alkynyl, -O-C(O)O-aryl, -O-C(O)O-substituted aryl, -O-C(O)O- cycloalkyl, -O-C(O)O-substituted cycloalkyl, -O-C(O)O-cycloalkenyl, -O-C(O)O-substituted cycloalkenyl, -O-C(O)O-heteroaryl, -O-C(O)O-substituted heteroaryl, -O-C(O)O-heterocyclic, and -O-C(O)O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0079] “Cyano” or “nitrile” refers to the group –CN.

[0080] “Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems. Examples of suitable cycloalkyl groups include, for instance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl and the like. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0081] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and - SO2-heteroaryl.

[0082] “Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to 10 carbon atoms having single or multiple rings and having at least one double bond and preferably from 1 to 2 double bonds.

[0083] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, - SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0084] “Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10 carbon atoms having single or multiple rings and having at least one triple bond.

[0085] “Cycloalkoxy” refers to –O-cycloalkyl.

[0086] “Cycloalkenyloxy” refers to –O-cycloalkenyl.

[0087] “Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

[0088] “Hydroxy” or “hydroxyl” refers to the group –OH.

[0089] “Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms, such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (such as, pyridinyl, imidazolyl or furyl) or multiple condensed rings in a ring system (for example as in groups such as, indolizinyl, quinolinyl, benzofuran, benzimidazolyl or benzothienyl), wherein at least one ring within the ring system is aromatic. To satisfy valence requirements, any heteroatoms in such heteroaryl rings may or may not be bonded to H or a substituent group, e.g., an alkyl group or other substituent as described herein. In certain embodiments, the nitrogen and / or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes, by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, and furanyl. Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2-heteroaryl, and trihalomethyl.

[0090] The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. This term includes, by way of example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0091] “Heteroaryloxy” refers to –O-heteroaryl.

[0092] “Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl” refer to a saturated or unsaturated group having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 20 ring atoms, including 1 to 10 hetero atoms. These ring atoms are selected from nitrogen, sulfur, or oxygen, where, in fused ring systems, one or more of the rings can be cycloalkyl, aryl, or heteroaryl, provided that the point of attachment is through the non-aromatic ring. In certain embodiments, the nitrogen and / or sulfur atom(s) of the heterocyclic group are optionally oxidized to provide for the N-oxide, -S(O)-, or –SO2- moieties. To satisfy valence requirements, any heteroatoms in such heterocyclic rings may or may not be bonded to one or more H or one or more substituent group(s), e.g., an alkyl group or other substituent as described herein.

[0093] Examples of heterocycles and heteroaryls include, but are not limited to, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4- tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to as thiamorpholinyl), 1,1- dioxothiomorpholinyl, piperidinyl, pyrrolidine, tetrahydrofuranyl, and the like.

[0094] Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selectedfrom alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl, -SO2-heteroaryl, and fused heterocycle.

[0095] “Heterocyclyloxy” refers to the group –O-heterocyclyl.

[0096] The term “heterocyclylthio” refers to the group heterocyclic-S-.

[0097] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein.

[0098] The term “hydroxyamino” refers to the group -NHOH.

[0099] “Nitro” refers to the group –NO2.

[0100] “Oxo” refers to the atom (=O).

[0101] “Sulfonyl” refers to the group -SO2-alkyl, -SO2-substituted alkyl, -SO2-alkenyl, -SO2- substituted alkenyl, -SO2-cycloalkyl, -SO2-substituted cylcoalkyl, -SO2-cycloalkenyl, -SO2- substituted cylcoalkenyl, -SO2-aryl, -SO2-substituted aryl, -SO2-heteroaryl, -SO2-substituted heteroaryl, -SO2-heterocyclic, and -SO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein. Sulfonyl includes, by way of example, methyl-SO2-, phenyl-SO2-, and 4-methylphenyl-SO2-.

[0102] “Sulfonyloxy” refers to the group -OSO2-alkyl, -OSO2-substituted alkyl, -OSO2- alkenyl, -OSO2-substituted alkenyl, -OSO2-cycloalkyl, -OSO2-substituted cylcoalkyl, -OSO2- cycloalkenyl, -OSO2-substituted cylcoalkenyl, -OSO2-aryl, -OSO2-substituted aryl, -OSO2- heteroaryl, -OSO2-substituted heteroaryl, -OSO2-heterocyclic, and -OSO2-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0103] “Sulfate” or “sulfate ester” refers the group -O-SO2-OH, -O-SO2-O-alkyl, -O-SO2-O- substituted alkyl, -O-SO2-O-alkenyl, -O-SO2-O-substituted alkenyl, -O-SO2-O-cycloalkyl, -O- SO2-O-substituted cylcoalkyl, -O-SO2-O-cycloalkenyl, -O-SO2-O-substituted cylcoalkenyl, -O- SO2-O-aryl, -O-SO2-O-substituted aryl, -O-SO2-O-heteroaryl, -O-SO2-O-substituted heteroaryl, - O-SO2-O-heterocyclic, and -O-SO2-O-substituted heterocyclic, wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, and substituted heterocyclic are as defined herein.

[0104] The term “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0105] “Thiol” refers to the group -SH.

[0106] “Thioxo” or the term “thioketo” refers to the atom (=S).

[0107] “Alkylthio” or the term “thioalkoxy” refers to the group -S-alkyl, wherein alkyl is as defined herein. In certain embodiments, sulfur may be oxidized to -S(O)-. The sulfoxide may exist as one or more stereoisomers.

[0108] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl.

[0109] The term “thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined herein including optionally substituted aryl groups also defined herein.

[0110] The term “thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined herein including optionally substituted aryl groups as also defined herein.

[0111] The term “thioheterocyclooxy” refers to the group heterocyclyl-S- wherein the heterocyclyl group is as defined herein including optionally substituted heterocyclyl groups as also defined herein.

[0112] In addition to the disclosure herein, the term “substituted,” when used to modify a specified group or radical, can also mean that one or more hydrogen atoms of the specified group or radical are each, independently of one another, replaced with the same or different substituent groups as defined below.

[0113] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for substituting for one or more hydrogens (any two hydrogens on a single carbon can be replaced with =O, =NR70, =N-OR70, =N2or =S) on saturated carbon atoms in thespecified group or radical are, unless otherwise specified, -R60, halo, =O, -OR70, -SR70, -NR80R80, trihalomethyl, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -SO2R70, -SO2O– M+, -SO2OR70, -OSO2R70, -OSO2O–M+, -OSO2OR70, -P(O)(O–)2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)O–M+, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)O -M+, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2– M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60is selected from the group consisting of optionally substituted alkyl, cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl, each R70is independently hydrogen or R60; each R80is independently R70or alternatively, two R80’s, taken together with the nitrogen atom to which they are bonded, form a 5-, 6- or 7-membered heterocycloalkyl which may optionally include from 1 to 4 of the same or different additional heteroatoms selected from the group consisting of O, N and S, of which N may have -H or C1-C3alkyl substitution; and each M+is a counter ion with a net single positive charge. Each M+may independently be, for example, an alkali ion, such as K+, Na+, Li+; an ammonium ion, such as+N(R60)4; or an alkaline earth ion, such as [Ca2+]0.5, [Mg2+]0.5, or [Ba2+]0.5(“subscript 0.5 means that one of the counter ions for such divalent alkali earth ions can be an ionized form of a compound of the invention and the other a typical counter ion such as chloride, or two ionized compounds disclosed herein can serve as counter ions for such divalent alkali earth ions, or a doubly ionized compound of the invention can serve as the counter ion for such divalent alkali earth ions). As specific examples, -NR80R80is meant to include -NH2, -NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl and N- morpholinyl.

[0114] In addition to the disclosure herein, substituent groups for hydrogens on unsaturated carbon atoms in “substituted” alkene, alkyne, aryl and heteroaryl groups are, unless otherwise specified, -R60, halo, -O-M+, -OR70, -SR70, -S–M+, -NR80R80, trihalomethyl, -CF3, -CN, -OCN, -SCN, -NO, -NO2, -N3, -SO2R70, -SO3– M+, -SO3R70, -OSO2R70, -OSO3–M+, -OSO3R70, -PO3-2(M+)2, -P(O)(OR70)O–M+, -P(O)(OR70)2, -C(O)R70, -C(S)R70, -C(NR70)R70, -CO2– M+, -CO2R70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OCO2– M+, -OCO2R70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70CO2–M+, -NR70CO2R70, -NR70C(S)OR70, -NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined, provided that in case of substituted alkene or alkyne, the substituents are not -O-M+, -OR70, -SR70, or -S–M+.

[0115] In addition to the groups disclosed with respect to the individual terms herein, substituent groups for hydrogens on nitrogen atoms in “substituted” heteroalkyl and cycloheteroalkyl groups are, unless otherwise specified, -R60, -O-M+, -OR70, -SR70, -S-M+, -NR80R80, trihalomethyl, -CF3, -CN, -NO, -NO2, -S(O)2R70, -S(O)2O-M+, -S(O)2OR70, -OS(O)2R70, -OS(O)2O-M+, -OS(O)2OR70, -P(O)(O-)2(M+)2, -P(O)(OR70)O-M+, -P(O)(OR70)(OR70), -C(O)R70, -C(S)R70, -C(NR70)R70, -C(O)OR70, -C(S)OR70, -C(O)NR80R80, -C(NR70)NR80R80, -OC(O)R70, -OC(S)R70, -OC(O)OR70, -OC(S)OR70, -NR70C(O)R70, -NR70C(S)R70, -NR70C(O)OR70, -NR70C(S)OR70, - NR70C(O)NR80R80, -NR70C(NR70)R70and -NR70C(NR70)NR80R80, where R60, R70, R80and M+are as previously defined.

[0116] In addition to the disclosure herein, in a certain embodiment, a group that is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3 substituents, 1 or 2 substituents, or 1 substituent.

[0117] It is understood that in all substituted groups defined above, polymers arrived at by defining substituents with further substituents to themselves (e.g., substituted aryl having a substituted aryl group as a substituent which is itself substituted with a substituted aryl group, which is further substituted by a substituted aryl group, etc.) are not intended for inclusion herein. In such cases, the maximum number of such substitutions is three. For example, serial substitutions of substituted aryl groups specifically contemplated herein are limited to substituted aryl-(substituted aryl)-substituted aryl.

[0118] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. For example, the substituent “arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O-C(O)-.

[0119] As to any of the groups disclosed herein which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and / or synthetically non-feasible. In addition, the subject compounds include all stereochemical isomers arising from the substitution of these compounds.

[0120] “Pharmaceutically effective amount” and “therapeutically effective amount” refer to an amount of a compound sufficient to treat a specified disorder or disease or one or more of its symptoms and / or to prevent the occurrence of the disease or disorder. In reference to tumorigenic proliferative disorders, a pharmaceutically or therapeutically effective amount comprises an amount sufficient to, among other things, cause the tumor to shrink or decrease the growth rate of the tumor.

[0121] “Patient” refers to human and non-human subjects, especially mammalian subjects.

[0122] The term “treating” or “treatment” as used herein means the treating or treatment of a disease or medical condition in a patient, such as a mammal (particularly a human) that includes: (a) preventing the disease or medical condition from occurring, such as, prophylactic treatment of a subject; (b) ameliorating the disease or medical condition, such as, eliminating or causing regression of the disease or medical condition in a patient; (c) suppressing the disease or medical condition, for example by, slowing or arresting the development of the disease or medical condition in a patient; or (d) alleviating a symptom of the disease or medical condition in a patient.

[0123] As used herein the term “isolated” is meant to describe a compound of interest that is in an environment different from that in which the compound naturally occurs. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and / or in which the compound of interest is partially or substantially purified.

[0124] As used herein, the term “substantially purified” refers to a compound that is removed from its natural environment and is at least 60% free, at least 75% free, at least 80% free, at least 85% free, at least 90% free, at least 95% free, at least 98% free, or more than 98% free, from other components with which it is naturally associated.

[0125] The term “physiological conditions” is meant to encompass those conditions compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.

[0126] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0127] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. 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 invention.

[0128] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace subject matter that are, for example, compounds that are stable compounds (i.e., compounds that can be made, isolated, characterized, and tested for biological activity). In addition, all sub-combinations of the various embodiments and elements thereof (e.g., elements of the chemical groups listed in the embodiments describing such variables) are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0129] 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 this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. 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.

[0130] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, thisstatement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0131] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination.

[0132] 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 may be different from the actual publication dates which may need to be independently confirmed. DETAILED DESCRIPTION

[0001] The present disclosure provides lipid nanoparticles for delivery of a payload (e.g., messenger RNA (mRNA)) to the heart. For example, a lipid nanoparticle of the present disclosure may comprise an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da. In some embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids. The disclosure also encompasses methods for delivery of messenger RNA (mRNA) for transfection of cells, methods of treatment, as well as kits thereof. LIPIDNANOPARTICLES

[0133] The present disclosure provides lipid nanoparticles for delivery of a payload (e.g., messenger RNA (mRNA)) to the heart. For example, a lipid nanoparticle according to the present disclosure comprises an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da. In some embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids.

[0134] As used herein, the phrase “lipid nanoparticle” refers to a transfer vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids). The lipid nanoparticles can be formulated to deliver one or more mRNA to one or more targetcells. Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides). In one embodiment, the lipid nanoparticle is selected based upon its ability to facilitate the transfection of mRNA to a target cell.

[0135] As used herein, the term “target cell” refers to a cell or tissue to which a lipid nanoparticle of the present disclosure is to be directed or targeted. The lipid nanoparticles of the present disclosure may be prepared to target a variety of target cells, which include, but are not limited to, cardiomyocytes, cardiac fibroblasts, endothelial cells, tissue resident macrophages, smooth muscle cells, nerve cells, stem and progenitor cells, pericytes, interstitial cells, adipose / fat cells, and other immune cells such as lymphocytes, neutrophils, and dendritic cells.

[0136] The invention contemplates the use of lipid nanoparticles as transfer vehicles to encapsulate and / or enhance the delivery of mRNA into the target cell.

[0137] In certain embodiments, compounds comprising an oxyanion azide-benzaldehyde acetal acid-degradable lipid that can be incorporated in the lipid nanoparticle (LNP) and used to transfect cells.

[0138] In some embodiments, the polyethylene glycol (PEG)-lipid is represented by formula (I):R1comprises an acyl moiety comprising one or more oxyanions, each complexed with a metal cation; R2comprises a hydrophobic group or lipid that can strengthen the rigidity of the lipid nanoparticle; L1and L2are linkers selected from a bond, an optionally substituted heteroatom and an optionally substituted C1-18hydrocarbyl or heterohydrocarbyl, providing acid degradable linkages.

[0139] In some embodiments, the oxyanions are selected from carbonic acid, phosphoric acid, or sulfonic acid thereof.

[0140] In some embodiments, the metal cation is selected from sodium, potassium, or lithium thereof.

[0141] In some embodiments, the hydrophobic group or lipid is a steroid, fatty acid or fatty acid ester.

[0142] In some embodiments, R1comprises a framework of a: C1-C6alkyl-substituted heteroatom (N, O or S), C1-C18linear or branched alkyl or hetero alkyl, C3-C6cycloalkyl or cyclo hetero alkyl, or C5-C6aryl or heteroaryl.

[0143] In some embodiments, R1comprises one or more acetate groups (e.g., of structur-COR(COOM)n).

[0144] In some embodiments, R1comprises C1-C6alkyl-substituted heteroatom (N, O oS) such as:

[0145] In some embodiments, n = 1 to 4, X = NH, O, S, such as:

[0146] In some embodiments, R1comprises C1-C18linear or branched alkyl or hetero alkyl such as:

[0147] In some embodiments, n = 1 to 4, x = 1-3, X = NH, O, S, such as:

[0148] or

[0149] In some embodiments, m = 1 to 5, such as:

[0150] or

[0151] In some embodiments, m = 1 to 5, y = 2 to 3, X = NH, O, S, such as:30

[0152] In some embodiments, R1comprises C3-C6cycloalkyl or cyclo hetero alkyl such as cyclohexyl:

[0153] In some embodiments, R1comprises C5-C6aryl or heteroaryl such as phenyl, pyridinyl, diazine (e.g.2, 3 or 4-pyrindinyl, 3,5 or 3,6-diazinyl):

[0154] In some embodiments, R1comprises:

[0155] In some embodiments, R1comprises:

[0156] In some embodiments, R1comprises:

[0157] In some embodiments, R2comprises steroid selected from:

[0158] In some embodiments, R2comprises an alkyl chain, such as in:

[0159] In some embodiments, L1and L2comprise linkers independently optionally hetero- , optionally substituted linear C1-C12alkyl; or L1and L2comprise linkers independently selected from:

[0160] In some embodiments, L1and L2are –CH2CH2NH-R1and –CH2CH2NHCO-R2.

[0161] In some embodiments, the lipid nanoparticle of the present disclosure further comprises ionizable cationic lipids, helper lipids, phospholipids, cholesterol or combination thereof. The contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non-cationic lipids and helper lipids.

[0162] As used herein, the phrase “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Several cationic lipids have been described in the literature, many of which are commercially available. For example, cationic lipids include, but are not limited to, C12-200; 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1- yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin-KC2-DMA); 1,2-Dioleoyl-3- Dimethylammonium-Propane (DODAP); HGT4003; (15Z,18Z)-N,N-dimethyl-6-(9Z,12Z)- octadeca-9,12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000); (15Z,18Z)-N,N-dimethyl-6- ((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001); N,N-dioleyl- N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N— (N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3- yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). The cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40% of the total lipids.

[0163] In some cases, the lipid nanoparticle include an ionizable lipid. As used herein, the term “ionizable lipid” refers to a lipid that becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged polynucleic acids (e.g., oligonucleotides). In one embodiment, the ionizable lipid is an amino lipid. Representative amino lipids include 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin- DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3- trimethylaminopropane chloride salt (DLin-TMA·Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP·Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3- (N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2- dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0164] As used herein, the phrase “neutral lipid” or “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Several non-cationic lipids have been described in the literature, many of which are commercially available.

[0165] Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipid present in the transfer vehicle. For example, non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl- ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2- oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids. When used in combination with a cationic lipid, the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10% to about 70% of the total lipids.

[0166] Preferably, the lipid nanoparticle is prepared by combining multiple lipids and / or polymer components. The selection of cationic lipids, non-cationic lipids and / or helper lipidswhich comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus, the molar ratios may be adjusted accordingly.

[0167] In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 4500 Da (e.g., about 1500 Da, about 2000Da, about 2500 Da, about 3000 Da, about 3500 Da, or about 4000 Da). In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 4000 Da. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 3000 Da. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 2000 Da.

[0168] In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids (e.g., about 2%, about 5%, about 10%, about 12%, about 15%, or about 20%). In certain embodiments, the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 20% of the total lipids. In certain embodiments, the acid-degradable polyethylene glycol (PEG)- lipid is present in a mole percentage of about 1% to about 15% of the total lipids. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 10% of the total lipids.

[0169] In some embodiments, the lipid nanoparticle encapsulates molecules selected from nucleic acids, proteins, polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, nutritional supplements, biomarkers, or combinations thereof.

[0170] As used herein, the term “nucleic acid” is meant to include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer fragments are called polynucleotides. In particular embodiments, oligonucleotides of the present invention are 20-50 nucleotides in length. In the context of this invention, the terms “polynucleotide” and “oligonucleotide” refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The terms “polynucleotide” and “oligonucleotide” also includes polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5′ and 3′ carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose. The nucleic acid that is present in a lipid particle according to this invention includes any form of nucleic acid that is known. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNA, and triplex-forming oligonucleotides.

[0171] In one embodiment, the polynucleic acid is an antisense oligonucleotide. In certain embodiments, the nucleic acid is an antisense nucleic acid, a ribozyme, tRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, pre-condensed DNA, or an aptamer.

[0172] In some embodiments, the encapsulated molecules comprise a nucleic acid selected from plasmid DNAs, antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or combinations thereof. In some embodiments, the encapsulated molecule is mRNA.

[0173] The lipid nanoparticle has a size (mean particle diameter) from about 15 nm to about 200 nm. In some embodiments, the lipid nanoparticle is having a size of less than 175 nm, or less than 150 nm, or less than125 nm, or less than 115 nm. In some embodiments, the lipid nanoparticle is having a size of less than 100 nm. METHODS

[0174] The present disclosure provides a method for delivery of messenger RNA (mRNA) for transfection of cells. For example, a method according to the present disclosure comprises administering a composition comprising an mRNA to a subject, wherein the mRNA is encapsulated within a lipid nanoparticle and wherein the administering of the composition results in the transfection of cells by the mRNA, wherein the lipid nanoparticle comprises an acid- degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000to 4500 Da; wherein the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

[0175] The acid-degradable polyethylene glycol (PEG)-lipid is as described in the above paragraphs. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 4500 Da (e.g., about 1500 Da, about 2000Da, about 2500 Da, about 3000 Da, about 3500 Da, or about 4000 Da). In certain embodiments, the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids (e.g., about 2%, about 5%, about 10%, about 12%, about 15%, or about 20%).

[0176] The lipid nanoparticle is as described in the above paragraphs. In some embodiments, the lipid nanoparticle further comprises ionizable cationic lipids, helper lipids, phospholipids, cholesterol or combination thereof. The cationic lipids and the non-cationic lipids are as described in the above paragraphs.

[0177] In some embodiments, the transfection of cells is in vivo.

[0178] In some embodiments, the cells are cardiomyocytes, cardiac fibroblasts, pericytes, Natural Killer cells, T cells endothelial cells, tissue resident macrophages, smooth muscle cells, nerve cells, stem cells, progenitor cells, interstitial cells, adipose / fat cells, or other immune cells such as lymphocytes, neutrophils, or dendritic cells.

[0179] In some embodiments, the mRNA is endothelial nitric oxide synthase (eNOS) mRNA.

[0180] In some embodiments, the administering comprises administering the composition systemically. In some embodiments, the administering comprises administering the composition intramuscularly. In some embodiments, the administering comprises administering the composition subcutaneously. Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. KITS

[0181] The present disclosure provides a kit for delivery of messenger RNA (mRNA) for transfection of cells. For example, a kit according to the present disclosure comprises alipid nanoparticle and a syringe, wherein the lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

[0182] The acid-degradable polyethylene glycol (PEG)-lipid is as described in the above paragraphs. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 4500 Da (e.g., about 1500 Da, about 2000Da, about 2500 Da, about 3000 Da, about 3500 Da, or about 4000 Da). In certain embodiments, the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids (e.g., about 2%, about 5%, about 10%, about 12%, about 15%, or about 20%).

[0183] FIG.1 shows schematic of therapeutic candidate product profile. Lipid nanoparticle (LNP) containing endothelial nitric oxide synthase (eNOS) mRNA are introduced into a syringe of an injection catheter. Percutaneous minimally invasive approach into a patient with an myocardial infarction (MI) delivers the LNP to the infarcted area. Either a transendocardial or intracoronary approach is possible, with the latter favored due to simplicity, cost, and widespread clinical access. Yellow dots represent LNPs delivered to the heart by either method, keeping the LNPs located in the infarct border zone, or infarcted vascular bed. METHOD OFTREATMENT

[0184] The present disclosure provides a method of treating a subject for a heart condition. For example, a method of treatment according to the present disclosure comprises administering to a subject in need thereof a therapeutically effective amount of an mRNA within a lipid nanoparticle that provides release of mRNA in the heart; wherein the lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

[0185] The acid-degradable polyethylene glycol (PEG)-lipid is as described in the above paragraphs. In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 4500 Da (e.g., about 1500 Da, about 2000Da, about 2500 Da, about 3000 Da, about 3500 Da, or about 4000 Da). In certain embodiments, the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids (e.g., about 2%, about 5%, about 10%, about 12%, about 15%, or about 20%).

[0186] As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the compositions and methods of the present invention are administered. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0187] In some embodiments, the mRNA is endothelial nitric oxide synthase (eNOS) mRNA.

[0188] In some embodiments, the heart condition is heart failure, coronary artery disease, cardiovascular disease, or myocardial infarction. EXAMPLES

[0189] 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 to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. By “average” means the arithmetic mean. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like. EXAMPLE1: Synthesis of acid-degradable lipid nanoparticles

[0190] FIG. 9 shows synthesis of acid-degradable lipids that generate either endosomal disruptive molecules or are converted into neutral lipids in the endosome. A library of ionizable lipids will be synthesized, from either primary amine or secondary amine azido acetal intermediates. The library will be synthesized via a Michael addition reaction. These new lipidsare designed to hydrolyze into membrane disruptive surfactants in the endosome or generate non- toxic neutral lipids after endocytosis. EXAMPLE 2: LNP chemistry and characterization

[0191] The contemplated lipid nanoparticles incorporate acid degradable analogs of PEG- lipids using a linker based on an azide-acetal that solves the stability problems associated with developing rapidly hydrolyzing linkages. These ADP-LNPs show rapid hydrolysis in endosomes and the possibility of combining high levels of PEGylation with efficient mRNA delivery (Fig. 3A, B)..

[0192] FIG. 4 shows synthesis of acid-degradable PEG-cholesterol. Schematic of the synthetic strategy used to generate PEG-cholesterol (1), the PEG molecular weight was either 1000 or 2000 Da.

[0193] The LNPs are comprised of 5 main lipids: 1) the ionizable lipid (10Z,13Z)-1- (9Z,12Z)-9,12-octadecadien-1-yl-10,13-nonadecadien-1-yl ester (D-Lin-MC3-DMG); 2) the cationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl (DOTAP); 3) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); 4) cholesterol; and, 5) the acid- degradable PEG-cholesterol (1) (Fig.3). The benefits of using acid cleavable PEG chains in LNPs is widely known. However synthesizing acid-degradable lipids has been challenging due to their instability. The azido acetal linkage shown in Fig. 3 is designed to overcome the challenges associated with making acid- degradable lipids, due to its two-step mechanism of degradation. The azido acetal is a very stable acetal, due to the weak electron donating character of its azide (hammett sigma value = 0.1). This high stability allowed synthesis of the acid-degradable PEG- cholesterol (1) on a multigram scale. Two variants of PEG-cholesterol were synthesized, which contained either a 1kDa PEG or a 2kDa PEG.

[0194] Lipids stock solutions of DOTAP, D-Lin, DOPE, Cholesterol, and DMG-PEG2K at a concentration of 10 mg / mL were separately dissolved in ethanol. Commercially available DOTAP was in a solution of chloroform (25 mg / mL), and the solvent was removed under reduced pressure before use. It was then dissolved in the ethanol for storage. A stock solution of ADP (1) at 40 mg / mL was dissolved in DMSO. All stock solutions were stored at -30 °C. Before LNP formation, lipids were allowed to warm to room temperature or heated up to 37 °C to ensure complete dissolution, and vortexed whenever necessary. Lipids in the stock solutions were addedto a 200 μL PCR tubes, following the details in Tables 1 and 2, to generate a 10 μL solution of lipids. 1 μL of the mixed lipids solution was mixed with 1 μL of eGFP mRNA or CRE mRNA (1 mg / mL) and pipetted for mixing. For the ADP-LNPs, 2 μL of DTT (10 mM in PBS pH 7.4) was added to the mixture and mixed again by pipetting. The LNP solutions were incubated at room temperature for 15 min.

[0195] Development of XR1 WTC human iPSC line: WTC-hNIL transgenic iPSC were further modified by integrating the 8.4kb XR1 (eXcision Reporter 1) transgene into the CLYBL safe harbor locus. The XR1 donor plasmid was derived from pC13N-dCas9-BFP-KRAB and a gift from Martin Kampmann by replacing the dCas9-BFP-KRAB coding sequence with a lox-STOP- lox-eGFP-P2A-mTagRFP coding sequence. The construct is designed such that CAG driven expression of the fluorescent reporter is prevented until the STOP terminator sequence is removed, either by Cre-Lox recombination or nuclease excision, similar to the Ai9 murine reporter. To generate the cell line, 1 x 106WTC-hNIL iPSC were added to 100 μL P3 buffer with 6 µg of the XR1 donor plasmid, and 3 µg of each paired TALEN (Addgene, 62196 / 62197) targeting the CLYBL locus (Cerbini et al., 2015). Cells were transfected using the P3 Primary Cell 4D- Nucleofector X Kit L (Lonza, V4XP-3024) with pulse code DS138. After nucleofection, cells incubated for 5 minutes at room temperature and were then plated in Stemfit media with 10 µm Y-27632 in a serial dilution into 5 wells of a 6-well plate. Non-transfected cells were plated in the sixth well. Two days post-nucleofection media was exchanged for Stemfit containing G418 and 10 µM Y-27632. Cells were kept on G418 selection until all cells in the control wells were dead and only red fluorescent colonies remained in the experimental wells. Fluorescent colonies were manually picked and transferred to a new culture plate for expansion. Individual clones were screened for on-target integration via junction PCR using a vector-specific primer paired with a flanking primer outside of the CLYBL homology arms in the donor vector (Table 1 – listed below). The final clone (clone 4) was selected based on single copy integration confirmed using a ddPCR assay with a FAM-labeled probe targeting the Neomycin resistance gene and a HEX-labelled RPP30 reference probe (BioRad).

[0196] Table 1:47

[0197] hiPSC line culture: XR1 WTC-11 human induced pluripotent stem cells (XR1 WTC-11 line) were obtained through material transfer agreements from B. Conklin, Gladstone Institute and routinely checked for mycoplasma contamination. The XR1 WTC-11 hiPSC line harbors an eGFP gene downstream of a STOP cassette, which was integrated by nucleofection using TALEN and a donor plasmid. Cells were expanded on growth factor-reduced Matrigel- coated plates (Corning, 354248) in mTeSR1 Plus medium (Stemcell Technologies, 100-0276) that was changed daily, passaged at 80% confluency using Accutase (Thermo Fisher Scientific, A1110501), and plated at a density of 12,000 cells / cm2. For the first 24 h after passaging, the culture medium was supplemented with 5 μM Y-27632 dihydrochloride (Biogems, 1293823).

[0198] Differentiation of hiPSC into cardiomyocytes (iCMs): Once confluent, the XR1 hiPSC cells were differentiated into human CMs utilizing a chemically defined cardiomyocyte differentiation protocol with some modifications. Briefly, hiPSCs were treated with 6 μM CHIR99021 (Biogems, 2520691) for 1 day in RPMI 1640 (Thermo Fisher Scientific, 11875119) with B27-insulin (Thermo Fisher Scientific, A1895601). The cells were subsequently treated with Wnt inhibitor IWP4 (Biogems, 6861787) in RPMI / B27- for another 2 days. Between 5–11 days of differentiation, RPMI / B27- medium was used and changed every other day. Robust spontaneous contractile activity was typically observed on day 8-10 of differentiation, at that point medium was switched to RPMI / B27+insulin (Thermo Fisher Scientific, 17504044). Cardiomyocyte purity was characterized using flow cytometry for cardiac troponin T (cTnT).

[0199] Isogenic differentiation of hiPSC into human cardiac fibroblasts (hcFb): The cardiac fibroblast differentiation was optimized for the XR1 hiPSC line following a previously published protocol (Fig.16, panel A). (Zhang, H., Shen, M. & Wu, J. C. Generation of Quiescent Cardiac Fibroblasts Derived from Human Induced Pluripotent Stem Cells. Methods Mol Biol 2454, 109-115 (2022)). A confluent hiPSC monolayer was induced with 6 μM of CHIR99021 for 2 days, recovered in RPMI+B27- for 24 hr, and treated with 5 μM of IWP4 for 2 days. On day 5, hiPSC-derived cardiac progenitor cells (hiPSC-CPCs) were re-plated at a density of 20,000 cells / cm2in advanced DMEM medium (Thermo Fisher Scientific, 12634028). On days 5 to day 8, cells were treated with 5 μM of CHIR99021 and 2 μM of retinoic acid (Sigma-Aldrich, R2625)for 3 days and recovered in advanced DMEM for 4 days. By this time, cells were differentiated into hiPSC-derived epicardial cells (hiPSC-EPCs). Next, these cells were re-plated and treated with 10 μM of FGF2 (PeproTech, 100-18B) and 10 μM of SB431542 (Selleck chemicals, S1067) in Fibroblast Growth Medium 3 (FGM3, Promocell, c-23025) for another 6 days. The cardiac fibroblasts were either passaged or frozen down for long-term storage. An XR1 cardiac fibroblast mRNA and protein characterization was performed. It was observed that icFbs expressed multiple fibroblast genes at the mRNA and protein level such as DDR2, CD90, Vimentin, TE-7, Periostin, Collagen Type I, and there was a significant mRNA upregulation of cardiac-specific markers like GATA4 and TBX20 (Fig.16, panels B-D).

[0200] Fabrication of cardiac MPS: Microfluidic cardiac MPS were formed using an optimized version of the protocol described in previous work. (Mathur, A. et al. Human iPSC- based cardiac microphysiological system for drug screening applications. Sci Rep 5, 8883 (2015)). Each MPS consisted of four multiplexed individual cell chambers (1,300 μm × 130 μm) connected to their cell-loading ports, two media ports, one media inlet, and one media outlet per multiplexed MPS. The media channels run adjacent to each side of the four cell chambers, separated by an array of fenestrations (2 µm × 2 µm high / width) to deliver nutrients while protecting the tissue from media flow shear stress. Two step photolithography was used to fabricate the microdevice silicon wafer mold, which contains the features of multiple MPS. The first photolithography step was 2 µm high for the fenestration pattern: the piranha cleaned wafer was put onto a headway spinner where SU82002 (Kayaku Advanced Materials, Westborough, MA) was poured and spun (first 500 rpm for 10 s with 100 rpm / s acceleration; then 2000 rpm for 30 s with 300 rpm / s acceleration). The wafer was baked for 1 min at 95°C, exposed at 80 mJ / cm2UV exposure (Karl Suss MA6 mask aligner with i-line) and baked again for 2 min at 95°C. Non-crosslinked photoresist was developed in SU8 developer for 1 min 15 s, and the wafer was hard baked at 180°C for 30–45 min. The second layer for the cell chamber and media channel (55 μm high) could then be fabricated onto of the fenestration with a similar protocol. SU8 3050 (Kayaku Advanced Materials) was used and spun (first 500 rpm for 10 s with 100 rpm / s acceleration; then 3000 rpm for 30 s with 300 rpm / s acceleration). The wafer was baked slowly to allow for improved resolution by increasing the temperature from 30°C to 65°C at a rate of 10°C / min. The temperature was held at 65°C for 10 min before it was increased to 95°C at a rate of 5°C / 3 min. Wafers were kept at 95°C for 45 min. The wafers were then allowed to cool down slowly to room temperature beforebeing exposed to UV at 160 mJ / cm2. Alignment marks allowed for perfect overlay of both layers. Post exposure bake was the same as previously described, with only 2 min at 65°C and 10 min at 95°C. Finally, wafers were developed for 35 min in a shaker and sonicator before being hard baked at 180°C for 30–45 min. The cardiac MPS was formed by replica molding from the silicon wafer with polydimethylsiloxane (PDMS; Sylgard 184 kit, Dow Chemicals, Midland, MI) at a 10:1 ratio of Sylgard base to crosslinker. PDMS chips were then bonded to glass slides using oxygen plasma for 24 s, RF power at 21W, and 100% oxygen flow.

[0201] Diffusion characterization: FRAP experiments were performed on live tissue samples in the cardiac MPS. An inverted ZEISS LSM 710 confocal microscope, with an argon- ion laser (488 nm), and a 403, 1.1 NA water objective was used. The experimental setting was: frame size 512 3 256 pixels, zoom factor of 3, circular region of interest 30 mm in diameter, pinhole setting 1 a.u. For this assay, the following probes were used: 1-40 kDa FITC-PEG (Nanocs, PG1-FC) and 4,70 kDa Dextran-FITC (Nanocs, DX4-70-FC). The working concentration for all the polymers was 100 μg / ml in PBS. Experiments started with 10 pre-bleach scanned images at low laser intensity, bleached at scan speed of 12.6 msec. at double zoom at 100% laser intensity, and followed by fluorescence recovery at low intensity. Data was normalized by the prebleach fluorescence intensity. All experiments were performed at room temperature.

[0202] Heart micromuscle assembly within the MPS: LNP screening was performed in cardiac MPS directly loaded with XR1 iCMs dissociated from the differentiation plate. Only iCM differentiations with a cTnT+ population higher than 80% were used in the study. On days 8-10, the hiPSC-CMs were dissociated with TrypLE 10x (Thermo Fisher Scientific, A1217703) and suspended in EB20 medium supplemented with 10 µM Y27632. A cell suspension with a density of 5,000 cells / 3 μL was injected into the loading port of each MPS. After 3 min of centrifugation at 300g, MPS were inspected under the microscope. Chambers that were not filled at this point were discarded. MPS were fed with 200 μL EB20 medium supplemented with 10 µM Y27632 into the inlet tip, and gravity allowed for constant flow to the outlet until equilibrium was reached. The following day and every day from then on, the medium was changed to RPMI / B27+insulin. To create isogenic iCM-icFB micromuscles in the MPS, the iCMs was purified by replating at a density of 100,000 cells / cm2onto Matrigel, culturing in RPMI / B27+ without glucose (Thermo Fisher Scientific, 11879020) with 5 mM sodium Lactate (Sigma Aldrich, 71718) for 4 days. Cells were allowed to recover in RPMI / B27+ for 2 days. On days 9-10, iCMs were dissociated usingTrypLE10X, and hicFbs were dissociated using Accutase. An isogenic cardiac micromuscle was created by mixing 80% iCM-20% icFb EB20 and loaded as described above. On day 2 and every day after, heart micromuscles were fed with a medium composition of 80% RPMI / B27+, 20% FGM3 and 2 μM SB431542.

[0203] LNP preparation and delivery: For the 2D iCM screening, 1.5 x 105iCMs / cm2was seeded in each well of a 96-well plate (Corning, 3474). The cardiomyocytes were dissociated and plated following the same protocol previously described. On day 4, different mRNA-LNP formulations were added to the cells in the plate and incubated overnight. First, 1 μL LNP + 1 μL mRNA (Trilink Biotech) + 2 μL DTT was mixed in a tube and incubated for 1 hr at room temperature. Then, this was mixed solution with 300 μL of RPMI / B27+insulin and added 100 μL of the LNP / media mixture to each of the wells. The following day, the medium was replaced with RPMI / B27+, and the cells were dissociated 2 days later under the different treatments to analyze the percent eGFP+ cells using flow cytometry. For the 3D cardiac MPS screening, the cells were loaded as described above. On day 4, the LNP / mRNA mix was delivered in RPMI / B27+ medium to the microtissues through the media inlet and let it flow overnight through the endothelial-like barrier that connects the media channel and the tissue chamber (Fig. 11, panels A-D). The following day, the medium with RPMI / B27+ was replaced and let the cardiac MPS recover for 4 days until they were fixed for confocal image analysis. For 3D screening in the cardiac MPS, the LNP preparation protocol was the same as the one described for 2D iCMs, except the LNP / mRNA concentration delivered to the microtissues was doubled. For controls, the iCMs were treated overnight with 60 μg / ml Tat-CRE protein (Sigma Aldrich, SCR508), and Lipofectamine RNAiMAX (Thermo Fisher Scientific, 13778100) was also used.

[0204] Gene expression in monolayer cultures: Total RNA was purified from tissues according to the manufacturer’s instructions using RNA Clean & Concentrator-5 (Zymo Research, R1015). Reverse transcription was performed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, 4368814), following the manufacturer’s instructions. Expression profiles for genes of interest were determined by real-time PCR using primers (Life Technologies) and the iTaq Universal SYBR Green Supermix (Bio-Rad, 1725120) in a Bio-Rad CFBX384 real time system. Gene expression was normalized to the geometrical mean of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and Ribosomal Protein L37a (RPL37A) housekeepingvalues. The data was then log-transformed and relativized to the average of the biological replicates for XR1 hiPSC at day 0.

[0205] Immunofluorescent staining in monolayer cultures: hiPSC-derived cells were dissociated and seeded onto Matrigel-coated ultra-low plate bottom 96-well plates (Perkin Elmer, 6055302). On days 4-5, cells were fixed using 4% paraformaldehyde for 15 min, then permeabilized and blocked with blocking buffer (0.05% Saponin and 3% normal goat serum (NGS) in PBS 1x) for 30 min. The cells were incubated overnight at 4 °C with a 1:200 primary antibody dilution in blocking buffer. After washing them three times with PBS, cells were incubated with a 1:500 Alexa Fluor™ secondary antibody dilution at room temperature for 1 h, rinsed three times with PBS for 5 min, and stained with DAPI for 5 min at room temperature. Primary and secondary antibodies used for these studies are listed in Table 2 below. Labeled cells were imaged using a widefield microscope (Lionheart FX, Biotek) and post-processed using Gen5 imaging analysis software (Biotek) and Image J.

[0206] Flow cytometry: For the LNP screening with the 2D XR1 iCMs, cells were dissociated by incubation in TrypLE 1x, then resuspended in PBS. eGFP+ cell populations were quantified using the autosampler accessory for 96-well plates attached to the Attune NxT flow cytometer (Thermo Fisher Scientific). For iCM and icFb characterization, cells were fixed using 4% paraformaldehyde for 15 min, then permeabilized and washed using a Perm / Wash buffer (R&D, FC005). Cell suspensions were incubated with 1:200 primary antibodies in the same buffer for 30 min at room temperature. After washing, cells were incubated with 1:500 secondary antibodies in the Perm / Wash buffer for 30 minutes at room temperature. Stained cells were analyzed in the Attune NxT flow cytometer, and data was analyzed using the FlowJo software (Tree Star). The primary and secondary antibodies used for these studies are listed in Table 2 below.

[0207] Table 2:52

[0208] Image acquisition for action potential and beating physiology studies: During imaging, cardiac MPS were maintained at 37 °C on a stage equipped with a heating unit (Tokai Hit). Spontaneous recordings included 6-s voltage dye videos (BeRST-1) for action potential measurements, as well as 6-s brightfield videos for contractile activity studies. BeRST-1 dye was synthesized and purity-verified as previously described. (Huang, Y. L., Walker, A. S. & Miller, E. W. A Photostable Silicon Rhodamine Platform for Optical Voltage Sensing. J Am Chem Soc 137, 10767-10776 (2015)). For action potential recordings, MPS were first labeled overnight with 2 μM BeRST-1. After acquiring the videos, the post-experiment processing was performed with an in-house developed Python library that could analyze the fluorescence intensity over the time of the recording and the contractile activity from the brightfield videos. A NIKON TE300HEM microscope with a HAMAMATSU digital CMOS camera C11440 / ORCA-Flash 4.0 was used, which recorded 100 FPS videos. For fluorescence imaging, a Lumencor (Beaverton, OR) SpectraX Light Engine and filtered with a QUAD filter (Semrock (IDEX), Rochester, NY) was used. BeRST-1 videos utilized the Far-red LED, 640 ms (4 × 4 binning, 10 ms). Videos were acquired using Nikon’s NIS-Elements software.

[0209] MPS tissue isolation and immunofluorescence imaging: Tissues were flushed for 10 min with PBS at 25 °C. Following this wash, 4% paraformaldehyde was added to the media channel for 15 min to fix the tissues. MPS were then washed with PBS twice for 5 min and carefully cut with a clean scalpel to open the device and expose the tissue. At this point, the PDMS component had the tissue structure attached to it. The tissues were then stained by submerging the PDMS blocks in different staining solutions: first, tissues were blocked with blocking buffer (3% NGS, 0.05% Saponin in 1X PBS) for 1hr at 4 °C. The next day, they were submerged in primary antibodies at 1:100 concentration in blocking buffer for 24 hr at 4 °C. Tissues were then washed twice at 25 °C in blocking buffer for 2–3 hr and washed a third time at 4 °C overnight. The secondary antibodies, along with 1:1000 DAPI (Abcam, ab108410), were incubated in blockingbuffer for 24 h. Tissues were then washed twice at 25 °C in blocking buffer for 2–3 hr and a third time at 4 °C overnight before tissues were imaged. Then, the microtissues were reversibly mounted with 90% Glycerol (VWR, BDH1172) onto ultrathin microscope slides (Ibidi, 10812). Microtissues were imaged with the Opera Phenix high content screening system. All images were taken through proprietary Synchrony Optics ×63 water immersion lens. Images were acquired using Harmony software. Z-stacks over 60 μm with a step size of 0.5 μm were performed.

[0210] Spatial and quantitative eGFP expression analysis in the heart micromuscles: 3D quantification of eGFP+ iCMs in the heart micromuscles was performed using the surface detection function of the IMARIS software (Bitplane). Objects were identified in the pipeline using the surface tool to segment DAPI or DRAQ5 positive nuclei (Thermo Fisher Scientific, 62251). Then, the eGFP intensity in each of the segmented objects was analyzed, which allowed the percentage of eGFP+ cells per microtissue to be calculated. Finally, based on the xyz positions of each object, the percentage of eGFP+ cardiomyocytes was quantified along the width of the heart micromuscle. For the nuclear segmentation, the whole 3D heart micromuscle (1300 μm x 150 μm x 50 μm) was first defined as Region of Interest (ROI). A background subtraction was applied with local contrast, considering that 12-13 μm is the maximal diameter of the largest sphere which fits into the object. Then, the Split Touching Objects tool was applied using the morphological split option with a seed point size of 4-5 μm. Finally seed points were filtered based on a quality parameter to remove objects that would otherwise interfere with the interpretation of results. Once the nuclear segmentation was obtained, the image processing tool was used to adjust the eGFP intensity over the z-stack. First, a Normalize Layer tool was applied to adjust the brightness and contrast of individual z-slices to a uniform level. Then finally a Gaussian filter was applied to define the background and performed a baseline subtraction of this variable background.

[0211] Delivery of firefly luciferase mRNA after myocardial infarction. Male C57Bl6 / J mice, aged 8-10 weeks, were obtained from Jackson Laboratory (Stock No: 000664) and were positioned on a temperature-controlled small-animal surgical table to maintain body temperature (37°C) throughout the surgical procedure. Mice were anesthetized with 2-4% isoflurane, mechanically ventilated (Harvard Apparatus), and subjected to thoracotomy followed by direct intracardiac injection of 50 ml LNP containing 25 mg of modified RNA encoding for Luciferase was administrated at the heart apex using 31G insulin syringes. For luciferase analysis, i.p. injection of 150 mL of XenoLight RediJect D-Luciferin (Perkin Elmer) was performed 12-24hours after intracardiac injection of LNP-RNA. Light emission from the firefly luciferase- catalyzed luciferin in vivo and ex vivo was detected using IVIS-Lumina S5 (Perkin Elmer) and quantified using total counts on selected areas (Fig.17, panels B, C) and graphed using Prism 10. Student’s t-test analysis was performed in each sample tested (n=4).

[0212] Delivery of Cre recombinase mRNA in infarcted and non-infarcted mouse hearts. This study was performed by intramyocardial injection of the LNPs in the apex of the adult heart in control and MI-induced mice, as described above. The MI was performed in 10 week mice by permanent ligation of the left anterior descending (LAD) artery, as described previously (REF). After surgery, control or MI animals were subjected to intramyocardial injection of 50 ml LNP containing 25 mg of modified mRNA encoding Cre recombinase or Luciferase (control) at the heart apex. One week after intervention, the expression of ZsGreen1 was visualized after optical clearing with CUBIC solution. Briefly, adult hearts were perfused with 10 ml of HBSS, 10 ml of CUBIC-P, 10 ml of 4%PFA and dissected, proceeded to tissue clearing. The tissue-clearing steps include the modified CUBIC protocol that includes delipidation of hearts with CUBIC-L at 37o.C degrees with agitation for 3 days, washing with PBS for 1 day, and RI matching with 50% CUBIC- R+(M) / H2O for 1 day, followed by CUBIC-R+(M) incubation for 3 days. Images were captured on UltraMicroscope Blaze (Miltenyi Biotech) or SmartSPIM (LifeCanvas Technologies) Lightsheet microscopes and processed with Imaris (ver 10.0.1). Samples were processed for cryosectioning and either visualized directly or subjected to immunohistochemistry using antibodies for cTnI, Isolectin GS-IB4-conjugated to Alexa Fluor 568 and, alpha Smooth Muscle Actin (Table 2). Images were obtained using BZ-X810 fluorescent microscope (Keyence) and processed using Adobe Photoshop 2023 software (Adobe).

[0213] Statistical Analysis: The data is shown as Mean ± SEM. Graphs were plotted on GraphPad Prism 9.5 and RStudio. Statistical analysis was performed using GraphPad Prism 9.5. Results with p values p < 0.05 were considered statistically significant. Global one-way ANOVA was performed when comparisons were made across more than two groups followed by post-hoc Tukey analysis. Two-factor repeated measures ANOVA was performed with two groups / conditions, where one was measured at different time points. Direct comparisons were made using an unpaired Student’s t-test. Results:

[0214] High diffusivity of PEG-FITC polymers in the Cardiac MPS. Pegylated lipids have multiple functions in lipid nanoparticles, such as reducing cellular toxicity, quenching ROS, and enhancing the diffusion of LNPs within tissues. Therefore, to determine if PEG is a suitable candidate for enhancing LNP diffusion through cardiac tissue, its linear PEG diffusion through tissue in the MPS was measured and its diffusivity was compared against the hydrophilic polymer dextran. In addition, to determine the optimal size of PEG for incorporation into the LNPs, the diffusive transport of PEG-FITC polymers was assessed with varying molecular weights (ranging from 1 to 40 kDa) within the 3D cardiac micromuscle compared to 4 and 70kDa Dextran-FITC molecules. PEG-FITC polymers were infused through the media channels of the MPS, allowed the concentration within the microtissue to reach a steady-state overnight, and subsequently ran a fluorescence recovery after a photobleaching (FRAP) experiment (Fig.12A). It was observed that the half time to recovery was similar across the range of PEG molecular weights tested that had various hydrodynamic radii (Rh) (1.13-6.59 nm) (Fig. 12B); however, 70 kDa dextran recovery was significantly slower with a similar Rhto the high molecular weight PEGs (20-40kDa), suggesting some interaction with either the cardiac MPS or the microtissue. The diffusivity of PEG-FITC polymers ranged from 85-75 μm2 / s, with PEG 1kDa and 2kDa having the highest mean values (Fig. 12C). ADP-LNPs using 1 and 2kDa ADP-PEG were therefore created to promote LNP diffusion within the micromuscle.

[0215] Development and characterization of the acid degradable PEG-LNPs. Based on the previous FRAP results showing a higher diffusivity of 1-2kDa PEG polymers in the cardiac MPS and minimal interactions with the ECM, a LNPs with high levels of PEGylation were generated. High levels of PEGylation generated LNPs that could diffuse through tissue efficiently. Molecular weights of PEG of 1 and 2kDa were chosen for grafting onto LNPs based on their high diffusivity through cardiac tissue and their ability to efficiently generate LNPs. Conventional LNPs cannot tolerate high levels of PEGylation since it lowers cell uptake and endosomal disruption. To overcome this limitation, LNPs were formulated with the acid degradable PEG-lipid, which rapidly hydrolyzes in the acidic environment of the endosome. ADP-LNPs exhibited rapid hydrolysis in endosomes and can efficiently deliver mRNA in vitro and in vivo, despite their high levels of PEGylation (Fig.3A, B). Therefore, 2D and 3D cardiomyocyte screenings of ADP-LNP formulations was performed with different PEG molecular weights (1kDa and 2kDa) and various ADP-lipid mole percentages ranging from 1% to 40% (Table 3). The size of ADP-LNPs was ~100nm. Their efficiency was compared with a standard LNP formulation (LNP-1) made with a non- degradable 1.48% PEG-lipid conjugate whose size is 144nm (Table 4).

[0216] Table 3. Components of ADP-LNPs.

[0217] Table 4. Components of LNP-1 (DLin-MC3-DMG-LNP).

[0218] XR1 WTC hiPSC line development for gene editing efficiency quantification. To perform the ADP-LNP screening, a Cre-hiPSC reporter line was aimed to combine with cardiac MPS as a powerful tool for the optimization of molecular therapeutics by pursuing an approach similar to the Ai6 murine reporter. Therefore, a genetically modified hiPSC line (XR1 hiPSC) was generated containing an eGFP gene downstream of a STOP cassette that is removed upon Cre delivery, making it an excellent reporter for gene editing in hiPSC-derived cells and a novel tissue level in vitro approach (Fig.13, panels A-B).

[0219] High transfection efficiency was achieved in 2D iCM monolayers. To quantify transfection efficiency, 2D iCM monolayers were incubated overnight with the standard LNP-1 formulation and various ADP-LNP formulations containing eGFP mRNA.80% eGFP+ iCMs was obtained when they were incubated with 1-2kDa ADP-LNP with mole percentages of PEG-lipid ranging from 1% to 20% (Fig. 5, panels A-C). ADP-LNPs exhibited a significantly higher transfection efficiency compared to the RNA iMAX / eGFP mRNA control (Fig. 5, panel C, Fig. 14A). It was also observed that 2kDa 40% ADP-LNPs containing eGFP mRNA was highly cytotoxic, therefore not being able to quantify the transfection efficiency. Lastly, the standardLNP-1 formulation also showed an increased transfection efficiency when compared to the RNA iMAX control, but with a lower %eGFP+ cells when compared to the specific ADP-LNP formulations (Fig. 5, panel C, Fig. 14A).

[0220] Robust gene editing efficiency was achieved for specific ADP-LNP formulations in 2D XR1 iCM monolayers. 2D XR1 iCMs was exposed overnight to the various LNP formulations as described in the previous section, but now containing Cre mRNA. It was observed that 50-55% eGFP+ iCMs when cells were incubated with ADP-LNPs whose PEG-lipid mole percentages ranged from 5% to 20% regardless of the PEG molecular weight (Fig.5, panels D-F). These ADP-LNP formulations showed a significantly higher gene editing efficiency than XR1 iCM monolayers treated with either the standard LNP-1 formulation or the naked Cre mRNA without a delivery vehicle (Fig. 5, panel F, Fig. 14B). Lastly, it was observed the highest gene editing efficiency for the recombinant cell-permeant Tat-Cre recombinase protein, resulting in 70% eGFP+ iCMs. Of note, the gene editing efficiency upon Cre delivery was considerably lower compared to the eGFP mRNA transfection efficiency shown in the previous section, most likely due to a different mRNA encapsulation efficiency in the LNPs or to the more complex gene editing process involved after the LNP / Cre cell transfection.

[0221] 2kDa 10% ADP-LNP enhanced gene editing efficiency in the 3D cardiac micromuscle. Based on the 2D iCM monolayer results, the Cre mRNA ADP-LNP formulations were selected with the highest gene editing efficiency and performed a similar screening in dense 3D cardiac micromuscles within the MPS. To this end, a volumetric nuclear segmentation of the entire microtissue was optimized to assess the eGFP intensity distribution in the cardiac cells and the %eGFP+ iCMs was quantified for all the LNP formulations (Fig. 6, panels A-C). It was observed that when the 2kDa ADP-lipid molar percentage of the LNP formulations was increased from 1% to 10%, the gene editing efficiency improved significantly, resulting in ~25% eGFP+ iCMs in the micromuscle (Fig. 6, panel D, Fig. 15, panel A). Cardiac microtissues treated with 1kDa ADP-LNPs exhibited the same trend for the LNP formulations whose ADP-Lipid ranged from 5% to 10% (Fig.6, panel D). Interestingly, unlike what was observed in 2D iCM monolayers, 1kDa 10% ADP-LNP exhibited a significantly lower gene editing efficiency compared to 2kDa 10% ADP-LNP. Furthermore, Tat-Cre protein exhibited the highest gene editing efficiency in 2D cardiac monolayers but only 3% eGFP+ iCMs were found in the cardiac MPS (Fig.5 panels D-F, Fig.6, panel D, Fig.15, panel A), supporting the requirement to conduct screening studies in the3D micromuscle. In line with the previous results, the standard LNP-1 which contains 1.5% non- degradable PEG-lipid showed a significantly lower gene editing efficiency compared to 1-2kDa ADP-LNP with high PEG mole ratios.

[0222] A higher ADP-lipid molar percentage enhanced LNP diffusion in the 3D cardiac micromuscle. To assess the combined diffusion and gene editing behavior of LNPs in dense tissues, regiospecific LNP / Cre gene editing was analyzed from the edges of the tissue chamber adjacent to the media channel to the center of the cardiac microtissues, taking advantage of the volumetric nuclear quantification described above. The %eGFP+ cells were quantified in the center of the tissue based on the position of every nucleus in the cardiac micromuscle (Fig 6A, B). It was found that microtissues treated with 2kDa 10% ADP-LNP / Cre exhibited 18% eGFP+ iCMs in the center of the micromuscle, and this percentage significantly decreased for the other formulations (Fig. 6 panels E and F, Fig.15, panel B). This result clearly indicates that a higher ADP-lipid molar percentage in the LNPs enhanced their diffusivity, reaching the maximum for cardiac MPS where 2kDa 10% ADP-LNP / Cre was delivered. Surprisingly, it was observed a significantly lower gene editing efficiency for microtissues treated with 2kDa 20% ADP-LNP / Cre, most likely due to instability or aggregation. The same trend was observed in cardiac microtissues treated with LNP containing 1kDa ADP-lipid whose molar percentage ranged from 5 to 10%. These results highlight the relevance of taking into consideration how the cell-cell and cell-ECM interactions in 3D dense tissues produce a different outcome compared to what was observed in 2D monolayer screenings.

[0223] 2kDa 10% ADP-LNP / Cre edited isogenic bi-cellular cardiac micromuscles. Cardiac fibroblasts play an essential role in the heart by maintaining and remodeling the extracellular matrix. Therefore, more complex micromuscles were generated formed by isogenic XR1 hiPSC-derived cardiomyocytes and human cardiac fibroblasts (hicFb) (Fig.16, panels A-D) with an 8:2 ratio, respectively. Four days after generating the bi-cellular cardiac micromuscles within the MPS, 2kDa 10% ADP-LNP / Cre was delivered overnight and then, the microtissues were kept with flowing media for 4 more days. It was observed that both cell types were successfully edited with neither preference for a specific cell type nor XYZ spatial location within the micromuscle (Fig. 10, panels A-C). More importantly, the bi-cellular microtissues exhibited ~30% eGFP+ cardiac cells upon 2kDa 10% ADP-LNP / Cre delivery (Fig. 10, panels D and E). Therefore, it was demonstrated the potential of using the 2kDa 10% ADP-LNP formulation as anmRNA delivery carrier within the 3D cardiac micromuscle consisting of a composition more comparable to that of the heart.

[0224] Electrophysiology and contractility of the cardiac micromuscle were not affected upon LNP / Cre delivery. One of the main concerns for LNPs is their clinical safety since it is widely known that cationic lipids are inherently toxic, and in high concentrations, they can affect the normal function of the organ target for delivery. The action potential duration (APD) and contractile activity of the microtissues was therefore assessed over time for 4 days, right before (D0) and after LNP delivery (D1-3). Regarding the contractile activity, a significant difference in the peak twitch amplitude between LNP-treated microtissues and control was not observed (Fig. 10, panel F). Furthermore, the APD showed no difference compared to the controls (Fig.10, panel G). Interestingly, the peak twitch amplitude and the APD exhibited a slight increase over time in both conditions due to the ongoing remodeling of the micromuscles over time. Taken together, the cardiac MPS demonstrated that the 2kDa 10% ADP-LNP formulation is safe and effective for mRNA delivery to cardiomyocytes in vitro.

[0225] In vivo administration of 2kDa 10% ADP-LNP / Luc in the mouse heart showed increased accumulation in the heart and lower off target levels in the liver. Based on the best perfomring ADP-LNP formulation identified in the cardiac MPS screening, its effectiveness was tested for in vivo mRNA delivery by direct intracardiac injection of 2kDa 10% ADP-LNP and the standard LNP-1 containing firefly luciferase mRNA (Luc) (Fig. 7A, 17A). Analysis of luciferase activity in the heart and liver showed that 2kDa 10% ADP-LNP / Luc exhibited a significant fivefold increase in luciferase activity in the heart, in line with the results shown in the cardiac MPS and lower off target levels of liver uptake when compared to the standard LNP-1 formulation. Analysis of relative activity in isolated organs showed that 2kDa 10% ADP-LNP displays approximately 9 fold enrichment of its heart to liver ratio when compared to LNP1 (Fig.7B, 17B- C). Collectively, these results demonstrate the vital role of ADP-lipid in controlling LNP biodistribution.

[0226] Effective 2kDa 10% ADP-LNP delivery to cardiomyocytes of infarcted and non-infarcted mouse hearts. To explore the functional delivery of mRNA by ADP-LNPs and identify the cells targeted in the mouse heart in vivo, a similar approach was pursued to the one which was used for the cardiac MPS screening using the XR1 hiPSC reporter line (Fig. 17, panel A). For these experiments, Ai6 mice were used that harbor a silent GFP variant (ZsGreen1) cassettethat becomes activated upon Cre-mediated deletion of the stop cassette. Cre mRNA using 2kDa 10% ADP-LNP was injected into non-infarcted control and infarcted mice hearts, and ZsGreen1 expression was analyzed after one week of injection. High levels of ZsGreen1 signal were evident in the heart, with the strongest expression detected in the apex of the left ventricle (Fig.8, panels A and B). The same level of ZsGreen1 expression was observed in infarcted and non-infarcted Ai6 mouse hearts, suggesting that this LNP formulation would be equally efficient for developing new therapeutics in both cases. These results indicate effective delivery of Cre recombinase in vivo using 2kDa 10% ADP-LNP. To confirm these findings and to get a better understanding of the specificity of the ADP-LNP / Cre delivery, an immunofluorescence staining of different cell- specific markers was performed to identify ZsGreen1-expressing cell types in the heart. It was observed that most of the ZsGreen1+ cells were identified as cardiomyocytes, as indicated by the presence of cardiac troponin I (cTnI) and distinctive sarcomere bands. It was also observed a partial co-localization with smooth muscle and perycites cells (αSMA+) and endothelial cells (IB4+) (Fig. 8, panel C). Collectively, it was found an effective LNP for mRNA delivery into the heart where it was observed a significant number of edited cardiomyocytes as well as non-cardiomyocytes cells. Furthermore, the in vivo experiment allowed us to demonstrate that cardiac MPS is a useful preclinical model to predict which formulations will be effective and safe for the heart.

[0227] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A lipid nanoparticle for messenger RNA (mRNA) delivery to the heart, comprising: an acid-degradable polyethylene glycol (PEG)-lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid-degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids.

2. The lipid nanoparticle of claim 1, wherein the acid-degradable polyethylene glycol (PEG)- lipid is represented by formula (I):R1comprises an acyl moiety comprising one or more oxyanions, each complexed with a metal cation; R2comprises a hydrophobic group or lipid that can strengthen the rigidity of the lipid nanoparticle; L1and L2are linkers selected from a bond, an optionally substituted heteroatom and an optionally substituted C1-18hydrocarbyl or heterohydrocarbyl, providing acid degradable linkages.

3. The lipid nanoparticle of claim 2, wherein the oxyanions are selected from carbonic acid, phosphoric acid, sulfonic acid or combination thereof.

4. The lipid nanoparticle of claim 2, wherein the metal cation is selected from sodium, potassium, lithium or combination thereof.

5. The lipid nanoparticle of claim 2, wherein the hydrophobic group or lipid is a steroid, fatty acid or fatty acid ester.

6. The lipid nanoparticle of claim 1, wherein the acid- degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 2000 Da.

7. The lipid nanoparticle of claim 1, wherein the acid-degradable polyethylene glycol (PEG)- lipid is present in a mole percentage of about 1% to about 20% of the total lipids.

8. The lipid nanoparticle of claim 1, wherein the acid-degradable polyethylene glycol (PEG)- lipid is present in a mole percentage of about 1% to about 10% of the total lipids.

9. The lipid nanoparticle of claim 1, further comprises ionizable cationic lipids, helper lipids, phospholipids, cholesterol or combination thereof.

10. The lipid nanoparticle of claim 1, wherein the nanoparticle encapsulates molecules selected from nucleic acids, proteins, polysaccharides, lipids, radioactive substances, therapeutic agents, prodrugs, nutritional supplements, biomarkers, or combinations thereof.

11. The lipid nanoparticle of claim 10, wherein the encapsulated molecules comprise a nucleic acid selected from plasmid DNAs, antisense oligonucleotides, miRs, anti-miRs, shRNAs, siRNAs, or combinations thereof.

12. The lipid nanoparticle of claim 10, wherein the encapsulated molecule is mRNA.

13. The lipid nanoparticle of claim 1, having a size of less than 125 nm.

14. A method for delivery of messenger RNA (mRNA) for transfection of cells, comprising: administering a composition comprising an mRNA to a subject, wherein the mRNA is encapsulated within a lipid nanoparticle and wherein the administering of the composition results in the transfection of cells by the mRNA, wherein the lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)- lipid ( ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

15. The method of claim 14, wherein the acid-degradable polyethylene glycol (PEG)-lipid (ADP) has molecular weight in the range of 1000 to 2000 Da and is present in a mole percentage of about 1% to about 20% of the total lipids in the lipid nanoparticle.

16. The method of claim 14, wherein transfection of cells is in vivo.

17. The method of claim 14, wherein the cells are cardiomyocytes, cardiac fibroblasts, pericytes, Natural Killer cells, T cells endothelial cells, tissue resident macrophages,smooth muscle cells, nerve cells, stem cells, progenitor cells, interstitial cells, adipose / fat cells, or other immune cells such as lymphocytes, neutrophils, or dendritic cells.

18. The method of claim 14, wherein the mRNA is endothelial nitric oxide synthase (eNOS) mRNA.

19. The method of claim 14, wherein the administering comprises administering the composition systemically.

20. The method of claim 14, wherein the administering comprises administering the composition intramuscularly.

21. The method of claim 14, wherein the administering comprises administering the composition subcutaneously.

22. A kit comprising: a lipid nanoparticle; and a syringe, wherein the lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)- lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

23. A method of treating a subject for a heart condition, the method comprising: administering to a subject in need thereof a therapeutically effective amount of an mRNA within a lipid nanoparticle that provides release of mRNA in the heart; wherein the lipid nanoparticle comprises an acid-degradable polyethylene glycol (PEG)- lipid (ADP) having molecular weight in the range of 1000 to 4500 Da; wherein the acid- degradable polyethylene glycol (PEG)-lipid is present in a mole percentage of about 1% to about 25% of the total lipids in the lipid nanoparticle.

24. The method of claim 23, wherein the mRNA is endothelial nitric oxide synthase (eNOS) mRNA.

25. The method of claim 23, wherein the heart condition is heart failure, coronary artery disease, cardiovascular disease, or myocardial infarction.

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