WO2024238655A2 - Delivery vehicle for targeted delivery to cardiomyocytes - Google Patents

Abstract

The present invention relates to compositions and methods for effective delivery of a therapeutic agent to a cardiomyocyte in a subject having an ApoE deficiency or in combination with an ApoE inhibitor. The invention also relates to methods of use of the delivery methods of the invention for the treatment of diseases and disorders, including the treatment of cardiac diseases and disorders.

Description

DELIVERY VEHICLE FOR TARGETED DELIVERY TO

CARDIOMYOCYTES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/502,150, filed May 15, 2023, which is hereby incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] The heart is an important locus for pharmacological interventions, but it is difficult to target drug delivery to the heart. Currently, no drug or drug delivery system (DDS) enters and acts in the myocardium effectively and specifically.

[0003] The endothelium in the endocardium and main coronary arteries, exposed to violent hemodynamics, is tight and nearly impervious. This helps prevent heart edema, but limits drug delivery into cardiac tissue. The high interstitial pressure in the dense myocardial parenchyma formed by cardiomyocytes (CMCs) impedes flow and diffusion in the tissue. Elaborate lymphatics assisted by contraction/relaxation rapidly cleanse interstitial space in the heart. So even drugs injected locally via coronary catheters or directly into myocardium are rapidly eliminated from the beating heart (Evers et al., 2022, J Control Release 343, 207-216). Using ligands of extracellular matrix epitopes may enhance DDS retention in the interstitial heart, but this strategy does not achieve delivery to specific cells, including CMCs. Even when pathology renders the heart vasculature or tissue more permeable to passive drug delivery, the above factors limit drug delivery to the heart.

[0004] Targeting circulating agents may surpass passive delivery. For example, specific targeting of diverse agents and carriers to pulmonary, cerebral, and other targets has been reported using ligands of defined vascular epitopes (Massey et al., 2009, Proc Am Thorac Soc 6, 419-430; Marchio et al., 2016, EMBO Mol Med 8, 592-594, Glassman et al., 2020, Adv Drug Deliv Rev 157, 96-117). Fortuitous tropisms for different tissues or cells have been discovered using high-throughput screening of chemical, biological and hybrid libraries (Myerson et al., 2022, Nat Nanotechnol 17, 86-97; Cheng et al., 2020, Nat Nanotechnol 15, 313-320; Scalzo et al., 2022, Int J Nanomedicine 17, 2865-2881; Dahlman et al., 2014, Nat Nanotechnol 9, 648-655; Siegwart et al., 2011, Proc Natl Acad Sci U S A 108, 12996-13001; Zhang et al., 2022, J Biomed Mater Res A 1 10, 1101-1 108). Formulations including antibody-coated liposomes, tannic acid- linked protein complexes, and “cardiotropic exosomes” can bind, be taken up by, and exert effects in cardiac cells in vitro. Some of these formulations have elevated cardiac uptake compared to untargeted controls in vivo. However, even with targeting, cardiac uptake is dwarfed by hepatic uptake, usually by several orders of magnitude (Shin et al., 2018, Nat Biomed Eng 2, 304-317; Mentkowski et al., 2019, Sci Rep 9, 10041; Kim et al., 2018, Biochem Biophys Res Commun 499, 803-808). Exploitation of natural tropisms of viral and cellular carriers (e.g., AAV9 and leukocytes) has also failed to yield effective cardiac delivery. Exosomes with heart tropism had only a 15% increase in cardiac delivery (Mentkowski et al.,

2019, Sci Rep 9, 10041; Kim et al., 2018, Biochem Biophys Res Commun 499, 803-808; de Couto, 2019, Exp Mol Med 51, 1-10; Li et al., 2021, Theranostics 11, 3916-3931; Liu et al.,

2020, Drug Deliv 27, 599-606; Saludas et al., 2021, Nanomaterials (Basel) 11) and the heartdiver ratio of tannic acid-linked protein complexes was 0.014, compared to 0.007 for a control formulation (Shin et al., 2018, Nat Biomed Eng 2, 304-317; Vandergriff et al., 2018, Theranostics 8, 1869-1878; Wright et al., 2021, Nat Rev Endocrinol 17, 389-399). Despite the promising results the small absolute values for cardiac uptake limit the translational utility of these strategies. Attempts to use “heart-specific” tropisms or ligands has yielded no DDS with clear clinically relevant improvement in delivery to the heart.

[0005] Thus, there is a need in the art for methods for delivery of therapeutics to the heart. The present invention addresses this need.

SUMMARY OF THE INVENTION

[0006] In some embodiments, the invention relates to a lipid nanoparticle (LNP) delivery vehicle for delivery of an agent to a cardiomyocyte comprising: a) an ionizable lipid, b) phosphatidylcholine, c) cholesterol, and d) PEG-lipid. In some embodiments, a) comprises ALC0307. In some embodiments, a), b), c) and d) are present in a molar ratio of about 50: 10:38.5:1.5, respectively.

[0007] In some embodiments, the LNP further comprises an agent for delivery to a cardiomyocyte. In some embodiments, the agent is a therapeutic agent, a diagnostic agent, a gene editing agent, an imaging agent, a contrast agent, a labeling agent, or a detection agent. In some embodiments, the therapeutic agent comprises at least one isolated nucleoside-modified RNA molecule. In some embodiments, the isolated nucleoside-modified RNA is a purified nucleoside- modified RNA.

[0008] In some embodiments, at least one nucleoside-modified RNA is encapsulated within the LNP or incorporated into the LNP.

[0009] In some embodiments, the invention relates to a combination therapy for delivering an agent to a cardiomyocyte of a subject in need thereof, the combination therapy comprising: a) a composition comprising a delivery vehicle comprising a cardiotropic lipid nanoparticle (cLNP), and b) a composition comprising an inhibitor of ApoE. In some embodiments, the cLNP is LNP-B or LNP-E. In some embodiments, the cLNP comprises: a) an ionizable lipid, b) phosphatidylcholine, c) cholesterol, and d) PEG-lipid. In some embodiments, a) comprises ALC0307. In some embodiments, a), b), c) and d) are present in a molar ratio of about 50:10:38.5: 1.5, respectively.

[0010] In some embodiments, the delivery vehicle further comprises a targeting moiety specific for binding to a cardiomyocyte.

[0011] In some embodiments, the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated as a single composition for co-administration. In some embodiments, the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated as separate compositions for co-administration. In some embodiments, the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated for sequential administration.

[0012] In some embodiments, at least one of the composition comprising the cLNP and the composition comprising the ApoE inhibitor further comprises an adjuvant.

[0013] In some embodiments, the invention relates to a method of diagnosing, preventing, evaluating the progression of or treating a disease or disorder in a subject in need thereof, the method comprising administering a cLNP or a combination therapy comprising a) a composition comprising a delivery vehicle comprising a cardiotropic lipid nanoparticle (cLNP), and b) a composition comprising an inhibitor of ApoE.

[0014] In some embodiments, the subject has or is at risk of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder. [0015] In some embodiments, the method comprises delivering an agent for the diagnosis, prevention, evaluation or treatment of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder to a cardiomyocyte of the subject.

[0016] In some embodiments, the LNP or combination therapy is administered by an intravenous, intra-myocardial, intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery route. In some embodiments, the LNP or combination therapy is administered by intra- myocardial injection. In some embodiments, the LNP or combination therapy is administered by intracoronary infusion.

[0017] In some embodiments, the composition comprising the cLNP and the composition comprising the ApoE inhibitor are co-administered. In some embodiments, the composition comprising the cLNP and the composition comprising the ApoE inhibitor are administered sequentially. In some embodiments, the composition comprising the ApoE inhibitor is administered prior to the composition comprising the cLNP.

[0018] In some embodiments, the invention relates to a method of diagnosing, preventing, evaluating or treating a disease or disorder in a subject in need thereof comprising delivering a therapeutic or diagnostic agent to a cardiomyocyte of the subject, the method comprising administering a composition comprising a cLNP to the subject, wherein the subject has an ApoE deficiency.

[0019] In some embodiments, the cLNP is LNP-B and LNP-E. In some embodiments, the cLNP comprises: a) an ionizable lipid, b) phosphatidylcholine, c) cholesterol, and d) PEG- lipid. In some embodiments, a) comprises ALC0307. In some embodiments, a), b), c) and d) are present in a molar ratio of about 50: 10:38.5:1.5, respectively.

[0020] In some embodiments, the subject has or is at risk of developing a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

[0021] In some embodiments, the therapeutic or diagnostic agent is an agent for the diagnosis, prevention, evaluation or treatment of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

[0022] In some embodiments, the composition is administered by intravenous, intra- myocardial, intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery. In some embodiments, the cLNP is administered by intra-myocardial injection. In some embodiments, the cLNP is administered by intracoronary infusion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0024] Figure 1A through Figure II depict exemplary experimental data demonstrating LNP cardiotropism. (Figure 1A) ¹²’I-cLNP (1 pg of RNA/mouse) tissue uptake in WT vs. Apoe'' mice 30 minutes after IV injection. Inset: heart-to-liver ratio, n = 28 (WT) and 47 (Apoe^). (Figure IB) Autoradiography images of 1 mm heart slices (scheme shows the approximate position of cuts) obtained from mice injected with ¹²⁵I-cLNPs. (Figure 1C) Confocal microscopy of organs (liver, lung, and heart) obtained from Apoe ^ mice 30 minutes after IV injection with PKH26-labeled cLNPs (bar = 25 pm). Anti-PECAM/CD31 antibody labeled with Alexa Fluor 647 was injected to denote vascular endothelium 10 minutes prior to sacrifice. Inset: expanded part of heart micrograph shows extravascular localization of cLNPs. (Figure ID) cLNP uptake in the diaphragm (diaph), limb skeletal muscle (limb), intercostal skeletal muscle (interc), fat tissue (fat), and heart of WT vs Apoe~'~ mice. (Figure IE) Effects of fasting (24 hours) on cLNP hepatic and cardiac uptake in Apoe ~ mice. (Figure IF) Tissue distribution of cLNP in mutant mice lacking ApoE, LDL receptor or CD36. (Figure 1G) ¹²⁵I-cLNP (10 pg of RNA/rat) tissue uptake in WT vs. Apoe'' rats 30 minutes after IV injection. (Figure 1H-I): ¹²⁵I-cLNP uptake in liver, lungs, and hearts 30 minutes post IV injection: (Figure 1G) in WT mice rendered ApoE-depleted by IV injecting 30 pg of Apoe siRNA three days prior to ¹²⁵I-LNP injection; (Figure 1H) in Apoe' ^A mice of different age. Unless stated otherwise, blue and red denote WT vs Apoe '' mice, respectively, n = 3-^5 biologically independent animals (Figure 1D-I). Data are shown as mean ± SEM, *P<0.05; **P<0.01; ***P<0.001 (t-test or two-way ANO V A with Tukey’s post-hoc test). [0025] Figure 2A through 2D depict exemplary experimental data demonstrating LNP binding to cells and GFP expression in vitro. LNP labeled with PKH-26 fluorescent dye were added to cells, incubated for 60 min, washed from unbound particles and imaged using epifluorescence microscope. (Figure 2A). Binding of PKH-26/LNP to RAW 264.7 murine macrophage cells, H9c2(2-1) rat heart myoblasts, human umbilical vein endothelial cells (HUVEC), and human pluripotent stem cell derived cardiomyocytes (hu-iPS-CM). (Figure 2B). Effect of apoE on LNP binding to hiPSC CMC. Radiolabeled LNP were preincubated with 40 pg/ml murine apoE for 16 h and added to cells for 60 min. Unbound LNP were washed out and bound fraction was measured. (Figure 2C). Dose-dependent binding of PKH-26/LNP to human pluripotent stem cell derived cardiomyocytes (hu-iPS-CM) and human fibroblasts. Right insets indicate fluorescence quantification. (Figure 2D). Dose-dependent expression of GFP by hiPSC CMC, rat adult cardiomyocytes, and human fibroblasts after incubation with eGFP mRNA-LNP. Right insets indicate fluorescence quantification.

[0026] Figure 3A through Figure 3E depict exemplary experimental data demonstrating cardiac delivery and activity of cLNP/mRNA-Cre. (Figure 3A) Scheme of Cre recombinase activity in mTmG reporter mice. Transformed cells change color from red to green. (Figure 3B) ApoE plasma level after LNP poe siRNA LNP treatment in WT vs. mTmG reporter mice. (Figure 3C) Settings of cLNP/mRNA-Cre experiments. LNP/d/wc siRNA treatment (60 pg of RNA/mouse) was followed by three consecutive cLNP/mRNA-Cre IV injections (10 pg of RNA/mouse). (Figure 3D-E) Confocal microscopy of liver (Figure 3D) and heart (Figure 3E) tissue sections from naive mTmG reporter mice and mice pre-treated with Apoe siRNA prior to injection of cLNP/mRNA-Cre. Arrows: transformed GFP-expressing cardiomyocytes outlined by dystrophin staining.

[0027] Figure 4 depicts exemplary experimental data demonstrating cardiac delivery and activity of cLNP/mRNA-Cre. Transformed cells change color from red to green. Dystrophin indicates as white, blue indicates DAPI.

[0028] Figure 5A through Figure 5C depict exemplary experimental data demonstrating parameters of LNP design modulating cardiotropism. (Figure 5 A) Biodistributions of empty cLNPs vs. poly-C RNA-cLNP. (Figure 5B) Biodistributions of IV-injected 80 nm vs. ~35O nm cLNPs. (Figure 5C) Screening for cardiotropism in different LNPs (80-100 nm) containing indicated ionizable lipids. WT vs. Apoe mice:. Five LNPs (top row) were provided by Arbutus Biopharma (Warminster, PA). Nine LNPs (middle row, first four on bottom row) were prepared with indicated ionizable lipids. cLNPs are as in Figures 1 and 3. All data indicate ¹²⁵I level in organs 30 min post IV injection of ¹²⁵I-LNP in WT (blue) vs. Apoe'¹' (red) mice, n = 3-^5 biologically independent animals. Data are shown as mean ± SEM, *P<0.05; **P<0.01; ***p<0.001 (two-way ANOVA with Tukey’s post-hoc test).

[0029] Figure 6A through Figure 61 depict exemplary experimental data demonstrating pharmacological characterization of LNP cardiotropism in mouse. (Figure 6A) Dose dependence of ¹²⁵I-CLNP uptake in organs 30 minutes post injection. (Figure 6B-C) Effect of unlabeled “cold” cLNPs (50 pg of RNA/mouse) and liposomes (1 mg/mouse) injected in WT vs. Apoe' ' mice IV 30 min prior to injection of ¹²³I-cLNPs (1 pg/mouse). (Figure 6D) Uptake of non-PEGylated vs. PEGylated ⁱⁿIn-liposomes in mice 30 min post IV injection in WT vs. Apoe '' mice. (Figure 6E) Blood, lung, and heart pharmacokinetics of ¹²⁵I-cLNPs in Apoe^' mice. Radiolabeled cLNPs were injected at specified times prior to tissue harvesting. (Figure 6F) Comparative biodistributions of ³H-cholesterol- and ¹²³I-labeled cLNPs in WT vs. Apoe'' mice. (Figure 6G) Blood clearance kinetics of cLNPs in WT vs. Apoe '~ mice as analyzed by ^12?I radiolabeling vs. tracing of fluorescent cLNPs in nanoparticle tracking analysis (NTA). (Figure 6H) Kinetics of cLNP size changes during circulation in WT vs. Apoe '' mice. Fluorescent cLNPs were IV injected, blood was drawn at indicated times, and size distributions of fluorescent species were analyzed by NTA. n = 3-^5 biologically independent animals (Figure 6D-I). Data are shown as mean ± SEM, *P<0.05; **P<0.01; ***P<0.001 (two-way ANOVA with Tukey’s post-hoc test).

[0030] Figure 7A and Figure 7B depict exemplary experimental data demonstrating dose dependence of heart delivery of ALC0307-LNP in ApoE KO mice. (Figure 7A), Heart uptake of ¹²⁵I-labeled LNP 30 min post injection. Dashed lane indicates heart delivery in WT mice. (Figure 7B), Analysis of specific LNP accumulation in heart. Specific cardiac uptake was calculated by subtracting out uptake of LNP in WT mice. Simple binding curve was fit to the observed data to calculate capacity. Capacity: 0.478 ± 0.057 pg/g tissue. ECso: 0.916 ± 0.546 pg LNP.

[0031] Figure 8 depicts exemplary experimental data demonstrating an LNP analysis by Nanoparticle tracking analysis (NTA). DiO-labeled cLNPs were diluted in PBS (1 : 1 e5 dilution, fluorescent signal from LNPs imaged).

[0032] Figure 9A and Figure 9B depict exemplary experimental data demonstrating LNP analysis by Nanoparticle tracking analysis (NTA). DiO-labeled cLNPs were injected in WT mouse. Blood was drawn after 30 min circulation. (Figure 9 A). 1 :50 dilution of plasma, scattering signal from all particles in plasma imaged. (Figure 9B) 1 :5 dilution of plasma, fluorescent signal from DiO-cLNPs imaged.

[0033] Figure 10A and Figure 10B depict exemplary experimental data demonstrating LNP analysis by Nanoparticle tracking analysis (NTA). DiO-labeled cLNPs were injected in WT vs. Apoe~'~ mice. Blood was drawn after 30 min circulation. 1 :25 dilution of plasma, fluorescent signal from DiO-cLNPs imaged in plasma of WT (Figure 10A) and Apoe (Figure 10B) mouse.

[0034] Figure 11 A through Figure 11H depict exemplary experimental data demonstrating LNP size and distribution Nanoparticle tracking analysis (NTA). DiO-labeled cLNPs were injected in mouse and drawn plasma was analyzed using fluorescent detector. (Figure 11 A) Comparison of cLNPs in PBS and plasma 30 min post-injection as detected by fluorescence with nanoparticles in plasma using light scattering. (Figure 11B-E) Kinetics of cLNP size changes during circulation in WT vs. Apoe-/- mice. (Figure 11F-H) Analysis of PK.

[0035] Figure 12 depict exemplary experimental data demonstrating protein profile of plasma fraction bound to LNP in WT vs. ApoE KO mice. Biotin-LNP were allowed to circulate in vivo for 30 min in WT and ApoE KO animals, isolated on Streptavidin-Dynabeads, and subjected to SDS-PAGE. Silver staining. Bands of apoA-IV, apoA-I, and apoE are indicated.

[0036] Figure 13A through Figure 131 depict exemplary experimental data demonstrating proteomic profiles of proteins adsorbed on cLNPs in WT vs. Apoe ' mice. (Figure 13A,C) Heat maps for proteomics profiles of proteins adsorbed on cLNPs after 30 minutes circulation in WT and Apoe ^ mice. From a mass spectrometry screen for 342 proteins, quantities of the ten most abundant proteins found on the cLNPs in WT (Figure 13A) and 4/wc ^/_ (Figure 13C) mice are depicted in heat maps. Protein quantities were determined as peptide abundance, with values for plasma samples without cLNPs subtracted to reflect magnitude of protein enrichment by presence of cLNPs. (Figure 13B, D) Breakdowns of the compositions of the protein coronae on cLNPs after circulation in WT (Figure 13B) and 4/wc ^ (Figure 13D) mice. Data in (Figure 13B, D) are divided into lipid metabolism proteins, common serum proteins, and other proteins. (Figure 13E) Abundance of all 342 tested proteins on cLNP in Apoe" (y-axis value) vs. WT (x- axis value) mice. Proteins abundant on cLNPs in WT mice, but not Apoe ^/_ mice are depicted with blue points, proteins abundant on cLNPs in Apoe mice, but not WT mice are depicted with red points, and proteins equally abundant on the cLNPs in WT and Apoe '~ mice are depicted with purple points. (Figure 13F-G) Absolute quantification of protein abundance on cLNPs, depicting quantities for the most abundant proteins on the LNPs after circulation in WT (Figure 13F) or Apoe ^ (Figure 13G) mice. Blue bars indicate quantities of protein on cLNPs in WT mice and red bars indicate quantities of protein on cLNPs in Apoe ^ mice. Blue, red, and purple boxes highlight values depicted with blue, red, and purple points, respectively, in (Figure 13E), and blue and red boxes denote p<0.05 significance in one way ANOVA. (Figure 13H-I) Illustrations of the distinct protein corona profiles for cLNPs in WT (Figure 13H) and Apoe ^ (Figure 131) mice. N=3 mice for all proteomics data in (Figure 13A-I).

[0037] Figure 14 depicts exemplary experimental data demonstrating protein profile of plasma fraction bound to LNP in WT vs. ApoE KO mice. Quantification of the most abundant proteins on the cLNPs circulated in WT vs. Apoe '~ mice depicted in heat maps.

[0038] Figure 15 depicts exemplary experimental data demonstrating protein profile of plasma fraction bound to LNP in WT vs. ApoE KO mice. Quantification of the most abundant proteins on the cLNPs circulated in Apoe~'~ mice. Breakdown of the compositions of the protein coronae.

[0039] Figure 16 depicts exemplary experimental data demonstrating protein profile of plasma fraction bound to LNP in WT vs. ApoE KO mice. Quantification of the most abundant proteins on the cLNPs circulated in WT mice. Breakdown of the compositions of the protein coronae.

[0040] Figure 17 depicts exemplary experimental data demonstrating protein profile of plasma fraction bound to LNP in WT vs. ApoE KO mice. Absolute quantification of protein abundance on cardiotropic LNPs, depicting quantities for the most abundant proteins on the LNPs after circulation in WT (Figure 17A) or Apoe" ‘ (Figure 17B) mice.

[0041] Figure 18A through Figure 18E depict exemplary experimental data demonstrating a validation of siATP2A2 activity. Human induced pluripotent stem cell derived cardiomyocytes were treated with lipofectamine and siATP2A2 (gray) versus scrambled siRNA (blue) for 48 hours. Figure 18A: Quantitative PCR of ATP2A2 transcript relative to GAPDH normalized to scrambled-treated cells (n=4, *p<0.05). Figure 18: Immunoblot and densitometric quantification of protein isolates for SERCA2 relative to [Lactin (n=3, *p<0.05). Figure 18C: Representative fluorescent micrographs of actinin (red), SERCA2 (green), and DAPI (blue), scale: 100 pm. Figure 18D: Representative relative contraction profile (mean +/- s.e.m.) of iPS- CMs using image correlation software, and quantification of contraction and relaxation times (Paced at 0.3 Hz, N=3, n=3-5 measurements per replicate, *p<0.05). Figure 18E: Fura-2 calcium indicator-treated iPS-cardiomyocytes, and transient times (Paced at 0.3 Hz, N=3, n=3 measurements per replicate, *p<0.05).

[0042] Figure 19A through Figure 19G depict exemplary experimental data demonstrating cLNP delivery of Atp2a2 siRNA modulates CMC function. (Figure 19A) Proposed action c Atp2a2 siRNA in CMCs. (Figure 19B) Atp2a2 siRNA-loaded LNPs were IV injected intoApoe ^J~ mice and SERCA2 level was detected in a 5-day time course. (Figure 19C) Immunoblot of heart protein isolates following LNP injection, for SERCA2 relative to GAPDH, with associated densitometry (n=3, *p<0.05). (Figure 19D) Heart weight (HW) relative to tibia length (TL) (n=3, dashed line represents average HW/TL in uninjected controls, boxes show the median and twenty -fifth and seventy-fifth percentiles, *p<0.05). (Figure 19E) Representative cardiac sections stained with hematoxylin and eosin. Scale bars: 1 mm (top) and 50 pm (bottom). (Figure 19F) Representative cardiomyocyte cross sections stained for cell membranes with wheat-germ agglutinin (green), and nuclei with DAPI (blue), and associated quantitation of cardiomyocyte cross sectional area (N=3 animals per group, n=100 cardiomyocytes per animal, boxes show the median and twenty -fifth and seventy-fifth percentiles, *p<0.01). (Figure 19G) Average +/- s.e.m. traces of sarcomere shortening, and associated contraction and relaxation parameters averaged over 6-10 transients during pacing at 1 Hz (N=3 animals per group, n=22- 49 cardiomyocytes per animal). Boxes show the median and twenty-fifth and seventy-fifth percentiles, *p<0.05.

[0043] Figure 20 depicts a comparison of Arbutus LNP B&F injection in apoE ^/_ vs. WT mice.

[0044] Figure 21 depicts a comparison of Arbutus LNP C&E injection in apoE'^/_vs. WT mice.

DETAILED DESCRIPTION

[0045] The present invention relates to compositions and systems for efficient delivery of a therapeutic agent to cardiomyocytes. In one embodiment, the composition or system further comprises an ApoE inhibitor. Definitions

[0046] 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.

[0047] As used herein, each of the following terms has the meaning associated with it in this section.

[0048] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0049] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0050] The term “antibody,” as used herein, refers to an immunoglobulin molecule, which specifically binds with an antigen or epitope. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0051] The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0052] An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. [0053] An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations, k and 1 light chains refer to the two major antibody light chain isotypes.

[0054] By the term “synthetic antibody” as used herein, is meant an antibody, which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The term should also be construed to mean an antibody, which has been generated by the synthesis of an RNA molecule encoding the antibody. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.

[0055] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0056] An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

[0057] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0058] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0059] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0060] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0061] In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N- glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0062] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0063] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject. In some embodiments, the subject is a human.

[0064] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1 -methyl pseudouridine, or another modified nucleoside.

[0065] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0066] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0067] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0068] In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside-modified nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196- 197).

[0069] In certain embodiments, “pseudouridine” refers, in another embodiment, to m¹acp³Y (l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine. In another embodiment, the term refers to m'Y (1 -methylpseudouridine). In another embodiment, the term refers to Ym (2'- O-methylpseudouridine. In another embodiment, the term refers to m⁵D (5- methyldihydrouridine). In another embodiment, the term refers to m³Y (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

[0070] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0071] The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

[0072] By the term “specifically binds,” as used herein with respect to an affinity ligand, in particular, an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0073] The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

[0074] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0075] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0076] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0077] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0078] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0079] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and/or triple bonds), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1-methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-l-enyl, pent-l-enyl, penta- 1,4- dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless specifically stated otherwise, an alkyl group is optionally substituted. [0080] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated or unsaturated (z.e., contains one or more double (alkenylene) and/or triple bonds (alkynylene)), and having, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, //-butylene, ethenylene, propenylene, n-butenylene, propynylene, w-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0081] “Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. In some embodiments the ring system has from three to ten carbon atoms. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like. Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0082] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0083] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered non-aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted.

[0084] The term “substituted” used herein means any of the above groups (e.g., alkyl, cycloalkyl or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a nonhydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=0); hydroxyl groups (-OH); alkoxy groups (-OR^a, where R^a is C1-C12 alkyl or cycloalkyl); carboxyl groups (-OC(=O)R^a or -C(=O)OR^a, where R^a is H, C1-C12 alkyl or cycloalkyl); amine groups (-NR^aR^b, where R^a and R^b are each independently H, C1-C12 alkyl or cycloalkyl); C1-C12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0085] “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0086] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

[0087] The present invention relates in part to cardiomyocyte targeted delivery vehicles and methods for cardiomyocyte targeted delivery of therapeutic agents. The invention is based, in part, on the demonstration that LNP with high levels of ionizable lipid show cardiotropism (cLNP). Therefore, in one embodiment, the invention provides compositions comprising a cLNP comprising a high level of ionizable lipid for delivery to a cardiomyocyte.

[0088] The present invention relates in part to cardiomyocyte targeted delivery vehicles and methods for cardiomyocyte targeted delivery of therapeutic agents. The invention is based, in part, on the demonstration that ApoE interferes with LNP delivery to cardiomyocytes. Therefore, in one embodiment, the invention provides compositions comprising a cLNP for delivery to a cardiomyocyte in a subject having an ApoE deficiency or reduced level of ApoE. In some embodiments, the invention provides compositions comprising a combination of a cLNP and an ApoE inhibitor for delivery to a cardiomyocyte. In one embodiment, the ApoE inhibitor increases the level of cardiomyocyte uptake of the cLNP.

[0089] Inhibition of ApoE, can be assessed using a wide variety of methods, including those disclosed herein, as well as methods known in the art or to be developed in the future. That is, the person of skill in the art would appreciate, based upon the disclosure provided herein, that inhibiting the level or activity of a gene, or gene product, can be readily assessed using methods that assess the level of a nucleic acid encoding a gene product (e.g., mRNA), the level of polypeptide gene product present in a biological sample, the activity of polypeptide gene product present in a biological sample, or combinations thereof.

Small molecule Inhibitors

[0090] One of skill in the art would readily appreciate, based on the disclosure provided herein, that an inhibitor of ApoE encompasses a chemical compound that modulates the level or activity of a gene, or gene product. Additionally, an inhibitor of ApoE encompasses a chemically modified compound, and derivatives, as is well known to one of skill in the chemical arts.

[0091] When the inhibitor of the invention is a small molecule or a derivative thereof, a small molecule inhibitor may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

[0092] Methods of identifying, or generating derivatives of, small molecule inhibitors are known in the art. Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

[0093] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores.

Nucleic Acid Inhibitors

[0094] In one embodiment, the composition of the invention comprises one or more antisense nucleic acid molecules. For example, in one embodiment, the one or more antisense nucleic acid molecules are specific for targeting ApoE. Antisense oligonucleotides are DNA or RNA molecules that are complementary to some portion of ApoE mRNA. When present in a cell, antisense oligonucleotides hybridize to an existing mRNA molecule and inhibit translation into a gene product or promote degradation of the RNA molecule. Inhibiting the expression of a gene using an antisense oligonucleotide is well known in the art, as are methods of expressing an antisense oligonucleotide in a cell. The methods of the invention include the use of antisense oligonucleotide to diminish the amount of ApoE activity or ApoE mRNA. Contemplated in the present invention are antisense oligonucleotides that are synthesized and provided to the cell by way of methods well known to those of ordinary skill in the art. As an example, an antisense oligonucleotide can be synthesized to be between about 10 and about 100, more preferably between about 15 and about 50 nucleotides long. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of modified antisense oligonucleotides to improve biological activity in comparison to unmodified antisense oligonucleotides.

[0095] Similarly, the expression of a gene may be inhibited by the hybridization of an antisense molecule to a promoter or other regulatory element of a gene, thereby affecting the transcription of the gene. Methods for the identification of a promoter or other regulatory element that interacts with a gene of interest are well known in the art.

[0096] Alternatively, inhibition of ApoE can be accomplished through the use of an siRNA, shRNA, antisense oligonucleotide or ribozyme. Given the nucleotide sequence of the molecule, one of ordinary skill in the art could synthesize an antisense oligonucleotide or ribozyme without undue experimentation, provided with the disclosure and references incorporated herein.

[0097] In one embodiment, siRNA is used to decrease the level of ApoE is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216.

Therefore, the present invention also includes methods of decreasing levels of ApoE at the mRNA or protein level using RNAi technology.

[0098] In certain embodiments, the modulators described herein comprise short hairpin RNA (shRNA) molecules. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleaves the shRNA to form siRNA.

[0099] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA, shRNA, gapmer or antisense molecule, inhibits ApoE, a regulator thereof, or an activator thereof.

Peptide inhibitors

[0100] In one embodiment, the composition of the present invention comprises an isolated peptide inhibitor of ApoE.

[0101] The peptide of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0102] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction. [0103] The peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation. A variety of approaches are available for introducing unnatural amino acids during protein translation.

[0104] A peptide or protein of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins provided that the resulting fusion protein retains the functionality of inhibiting ApoE.

[0105] A peptide or protein of the invention may be phosphorylated using conventional methods such as the method described in Reedijk et al. (The EMBO Journal 11(4): 1365, 1992).

[0106] Cyclic derivatives of the peptides of the invention are also part of the present invention. Cyclization may allow the peptide to assume a more favorable conformation for association with other molecules. Cyclization may be achieved using techniques known in the art. For example, disulfide bonds may be formed between two appropriately spaced components having free sulfhydryl groups, or an amide bond may be formed between an amino group of one component and a carboxyl group of another component. Cyclization may also be achieved using an azobenzene-containing amino acid as described by Ulysse, L , et al., J. Am. Chem. Soc. 1995, 117, 8466-8467. The components that form the bonds may be side chains of amino acids, nonamino acid components or a combination of the two. In an embodiment of the invention, cyclic peptides may comprise a beta-turn in the right position. Beta-turns may be introduced into the peptides of the invention by adding the amino acids Pro-Gly at the right position.

[0107] It may be desirable to produce a cyclic peptide which is more flexible than the cyclic peptides containing peptide bond linkages as described above. A more flexible peptide may be prepared by introducing cysteines at the right and left position of the peptide and forming a disulphide bridge between the two cysteines. The two cysteines are arranged so as not to deform the beta-sheet and turn. The peptide is more flexible as a result of the length of the disulfide linkage and the smaller number of hydrogen bonds in the beta-sheet portion. The relative flexibility of a cyclic peptide can be determined by molecular dynamics simulations.

[0108] The peptides and fusion proteins of the invention may be converted into pharmaceutical salts by reacting with inorganic acids such as hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid, etc., or organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benezenesulfonic acid, and toluenesulfonic acids.

Antibody Inhibitors

[0109] In some embodiments, the invention relates to compositions comprising at least one inhibitory antibody, or fragment thereof, specific for binding to ApoE. In one embodiment, the anti-ApoE antibody is a neutralizing antibody.

[0110] As used herein, the term “antibody” or “immunoglobulin” refers to proteins (including glycoproteins) of the immunoglobulin (Ig) superfamily of proteins. An antibody or immunoglobulin (Ig) molecule may be tetrameric, comprising two identical light chain polypeptides and two identical heavy chain polypeptides. The two heavy chains are linked together by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. Each full-length Ig molecule contains at least two binding sites for a specific target or antigen.

[0111] An anti-ApoE neutralizing antibody, or antigen-binding fragment thereof, includes, but is not limited to a polyclonal antibody, a monoclonal fusion proteins, antibodies or fragments thereof , chimerized or chimeric fusion proteins, antibodies or fragments thereof , humanized fusion proteins, antibodies or fragments thereof , deimmunized humfusion proteins, antibodies or fragments thereof , fully humfusion proteins, antibodies or fragments thereof , single chain antibody, single chain Fv fragment (scFv), Fv, Fd fragment, Fab fragment, Fab' fragment, F(ab')2 fragment, diabody or antigen- binding fragment thereof, minibody or antigenbinding fragment thereof, triabody or antigen- binding fragment thereof, domain fusion proteins, antibodies or fragments thereof , camelid fusion proteins, antibodies or fragments thereof , dromedary fusion proteins, antibodies or fragments thereof , phage-displayed fusion proteins, antibodies or fragments thereof , or antibody, or antigen- binding fragment thereof, identified with a repetitive backbone array (e.g. repetitive antigen display).

[0112] As used throughout the present disclosure, the term “antibody” further refers to a whole or intact antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that is generated by any one of a variety of methods that are known in the art and described herein. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a deimmunized human antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non- human primates (e.g., monkeys, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.

Genetic modification

[0113] In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor of ApoE, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0114] The desired polynucleotide can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora of vectors which are readily available and/or well-known in the art. For example, a desired polynucleotide of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal viruse, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0115] In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0116] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.

[0117] For expression of the desired polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation.

[0118] Additional promoter elements, i.e., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0119] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0120] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0121] A promoter sequence exemplified in the experimental examples presented herein is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue specific promoter, which promoter is active only in a desired tissue. [0122] In order to assess the expression of the vector, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neo and the like.

[0123] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0124] Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of siRNA polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0125] In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. It is readily understood that the introduction of the expression vector comprising the polynucleotide of the invention yields a silenced cell with respect to a regulator. [0126] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0127] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0128] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid nanoparticles. A preferred colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0129] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0130] Any DNA vector or delivery vehicle can be utilized to transfer the desired polynucleotide to a cell in vitro or in vivo. In the case where a non-viral delivery system is utilized, exemplary delivery vehicles include, but are not limited to, a liposome or lipid nanoparticle. The above-mentioned delivery systems and protocols therefore can be found in Gene Targeting Protocols, 2ed., pp 1 -35 (2002) and Gene Transfer and Expression Protocols, Vol. 7, Murray ed., pp 81-89 (1991).

[0131] “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine nucleic acid complexes.

Targeting Molecule

[0132] In various embodiments, the delivery vehicle of the invention comprises a targeting domain that binds to a cell surface molecule of a cardiomyocyte. In certain embodiments, the targeting domain binds to a cell surface molecule of a cardiomyocyte, thereby directing the composition to the cardiomyocyte. In one embodiment, the composition comprises a delivery vehicle conjugated to a targeting domain that binds a cell surface molecule of a cardiomyocyte, thereby directing the composition to the cardiomyocyte.

[0133] In some embodiments, the invention provides a method for treating a disease or disorder in subjects in need thereof, the method comprising the administration of a composition including a delivery vehicle conjugated to a cardiomyocyte targeting domain to a subject having an ApoE deficiency or in combination with an ApoE inhibitor.

Delivery Vehicle

[0134] In some embodiments, the delivery vehicle is a colloidal dispersion system, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e g., an artificial membrane vesicle). In one embodiment, the delivery vehicle is a cardiomyocyte targeted lipid nanoparticle (cLNP).

[0135] The use of lipid formulations is contemplated for the delivery vehicle for delivery of an agent to a cardiomyocyte (in vitro, ex vivo or in vivo). In another aspect, the at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/nucleic acid or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0136] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-agent complexes.

[0137] In one embodiment, delivery of at least one agent comprises any suitable delivery method, including exemplary delivery methods described elsewhere herein. In certain embodiments, delivery of at least one agent to a subject comprises mixing the at least one agent with a transfection reagent prior to the step of contacting. In another embodiment, a method of the present invention further comprises administering at least one agent together with the transfection reagent. In another embodiment, the transfection reagent is a cationic lipid reagent.

[0138] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethyleneimine based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0139] In another embodiment, the transfection reagent forms a liposome. Liposomes, in another embodiment, increase intracellular stability, increase uptake efficiency and improve biological activity. In another embodiment, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. In some embodiments, the liposomes comprise an internal aqueous space for entrapping water-soluble compounds. In another embodiment, liposomes can deliver the at least one agent to cells in an active form.

[0140] In one embodiment, the composition comprises a lipid nanoparticle (LNP) and at least one agent.

[0141] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids. In various embodiments, the particle includes a lipid of Formula (I), (II) or (III). In some embodiments, lipid nanoparticles are included in a formulation comprising at least one agent as described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound IVa). In some embodiments, the at least one agent is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0142] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 83 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 102 nm. In one embodiment, the lipid nanoparticles have a mean diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the at least one agent, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation by intra- or intercellular enzymes

[0143] The LNP may comprise any lipid capable of forming a particle to which the at least one agent is attached, or in which the at least one agent is encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0144] In one embodiment, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

[0145] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or 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 nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0146] In certain embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, 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), N- (l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), N,N-dimethyl-2,3-bis(((9Z,12Z,15Z)-octadeca-9,12,15- trien- 1 -yl)oxy)propan- 1 -amine (DLenDMA).

[0147] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-l,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA).

[0148] Suitable amino lipids include those having the formula:

wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted Cio-C24acyl; 3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R? and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;

Rs is either absent or present and when present is hydrogen or Ci-Ce alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.

[0149] In one embodiment, Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0150] A representative useful dilinoleyl amino lipid has the formula:

JL -K-DMA wherein n is 0, 1, 2, 3, or 4.

[0151] In one embodiment, the cationic lipid is a DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA above, wherein n is 2).

[01 2] In one embodiment, the cationic lipid component of the LNPs has the structure of Formula (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹ and L² are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;

R^la and R^lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or

(b) R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^2a and R^2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or

(b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^3a and R^3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^4a and R^4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R³ and R⁶ are each independently methyl or cycloalkyl;

R⁷ is, at each occurrence, independently H or C1-C12 alkyl;

R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.

[0153] In certain embodiments of Formula (I), at least one of R^la, R^2a, R^3a or R^4a is Cl- C12 alkyl, or at least one of L¹ or L² is -O(C=O)- or -(C=O)O-. In other embodiments, R^1a and R^lb are not isopropyl when a is 6 or n-butyl when a is 8.

[0154] In still further embodiments of Formula (I), at least one of R^la, R^2a, R^3a or R^4a is C1-C12 alkyl, or at least one of L¹ or L² is -O(C=O)- or -(C=O)O-; and

R^la and R^lb are not isopropyl when a is 6 or n-butyl when a is 8.

[0155] In other embodiments of Formula (I), R⁸ and R⁹ are each independently unsubstituted C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom.

[0156] In certain embodiments of Formula (I), any one of L¹ or L² may be -O(C=O)- or a carbon-carbon double bond. L¹ and L² may each be -O(C=O)- or may each be a carbon-carbon double bond.

[0157] In some embodiments of Formula (I), one of L¹ or L² is -O(C=O)-. In other embodiments, both L¹ and L² are -O(C=O)-.

[0158] In some embodiments of Formula (I), one of L¹ or L² is -(C=O)O-. In other embodiments, both L¹ and L² are -(C=O)O-. [0159] In some other embodiments of Formula (I), one of L¹ or L² is a carbon-carbon double bond. In other embodiments, both L¹ and L² are a carbon-carbon double bond.

[0160] In still other embodiments of Formula (I), one of L¹ or L² is -O(C=O)- and the other of L¹ or L² is -(C=O)O- In more embodiments, one of L¹ or L² is -O(C=O)- and the other of L¹ or L² is a carbon-carbon double bond. In yet more embodiments, one of L¹ or L² is -(C=O)O- and the other of L¹ or L² is a carbon-carbon double bond.

[0161] It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures:

wherein R^a and R^b are, at each occurrence, independently H or a substituent. For example, in some embodiments R^a and R^b are, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

[0162] In other embodiments, the lipid compounds of Formula (I) have the following structure (la):

[0163] In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):

[0164] In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic):

[0165] In certain embodiments of the lipid compound of Formula (I), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0166] In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0167] In some more embodiments of Formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0168] In some certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 1 1 . In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0169] In some other various embodiments of Formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

[0170] The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

[0171] In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

[0172] The substituents at R^la, R^2a, R^3a and R^4a of Formula (I) are not particularly limited. In certain embodiments R^la, R^2a, R^3a and R^4a are H at each occurrence. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is C1-C12 alkyl. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is Ci-Cs alkyl. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0173] In certain embodiments of Formula (I), R^la, R^lb, R^4a and R^4b are C1-C12 alkyl at each occurrence.

[0174] In further embodiments of Formula (I), at least one of R^lb, R^2b, R^3b and R^4b is H or R^lb, R^2b, R^3b and R^4b are H at each occurrence.

[0175] In certain embodiments of Formula (I), R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0176] The substituents at R⁵ and R⁶ of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R⁵ or R⁶ is methyl. In certain other embodiments one or both of R⁵ or R⁶ is cycloalkyl for example cyclohexyl. In these embodiments the cycloalkyl may be substituted or not substituted. In certain other embodiments the cycloalkyl is substituted with C1-C12 alkyl, for example tert-butyl.

[0177] The substituents at R⁷ are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments at least one R⁷ is H. In some other embodiments, R⁷ is H at each occurrence. In certain other embodiments R⁷ is C1-C12 alkyl.

[0178] In certain other of the foregoing embodiments of Formula (I), one of R⁸ or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

[0179] In some different embodiments of Formula (I), R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

[0180] In various different embodiments, exemplary lipid of Formula (I) can include

[0181] In some embodiments, the LNPs comprise a lipid of Formula (I), at least one agent, and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.

[0182] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹ and L² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-,

-S(O)_X-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, -NR^aC(=O)NR^a,

-OC(=O)NR^a-, -NR^aC(=O)O-, or a direct bond; G¹ is C1-C2 alkylene, -(C=0)- , -0(C=0)-, -SC(=O)-, -NR^aC(=O)- or a direct bond;

G² is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NR^a or a direct bond;

G³ is C1-C6 alkylene;

R^a is H or C1-C12 alkyl;

R^la and R^lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^2a and R^2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^3a and R^3b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^4a and R^4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R³ and R⁶ are each independently H or methyl;

R⁷ is C4-C20 alkyl;

R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0183] In some embodiments of Formula (II), L¹ and L² are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G¹ and G² are each independently -(C=O)- or a direct bond. In some different embodiments, L¹ and L² are each independently -0(C=0)-, -(C=0)0- or a direct bond; and G¹ and G² are each independently - (C=O)- or a direct bond.

[0184] In some different embodiments of Formula (II), L¹ and L² are each independently -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^a-, -NR^aC(=O)-, -C(=O)NR^a-, -NR^aC(=O)NR^a, -OC(=O)NR^a-, -NR^aC(=O)O-, -NR^aS(O)_xNR^a-, -NR^aS(O)_x- or -S(O)xNR^a-.

[0185] In other of the foregoing embodiments of Formula (II), the lipid compound has one of the fo

(IIA) (IIB)

[0186] In some embodiments of Formula (II), the lipid compound has structure (1IA). In other embodiments, the lipid compound has structure (IIB).

[0187] In any of the foregoing embodiments of Formula (II), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-.

[0188] In some different embodiments of Formula (II), one of L¹ or L² is -(C=O)O-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

[0189] In different embodiments of Formula (II), one of L¹ or L² is a direct bond. As used herein, a “direct bond” means the group (e.g., L¹ or L²) is absent. For example, in some embodiments each of L¹ and L² is a direct bond.

[0190] In other different embodiments of Formula (II), for at least one occurrence of R^la and R^lb, R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carboncarbon double bond.

[0191] In still other different embodiments of Formula (II), for at least one occurrence of R^4a and R^4b, R^4a is H or Ci-C 12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carboncarbon double bond.

[0192] In more embodiments of Formula (II), for at least one occurrence of R^2a and R^2b, R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[0193] In other different embodiments of Formula (II), for at least one occurrence of R^3a and R^3b, R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carboncarbon double bond.

[0194] In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID):

wherein e, f, g and h are each independently an integer from 1 to 12.

[0195] In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID).

[0196] In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10. [0197] In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

[0198] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

[0199] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

[0200] In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

[0201] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11 . In yet other embodiments, e is 12.

[0202] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

[0203] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

[0204] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

[0205] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

[0206] The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In one embodiment, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

[0207] The substituents at R^la, R^2a, R^3a and R^4a of Formula (II) are not particularly limited. In some embodiments, at least one of R^la, R^2a, R^3a and R^4a is H. In certain embodiments R^la, R^2a, R^3a and R^4a are H at each occurrence. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is C1-C12 alkyl. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is Ci-Cs alkyl. In certain other embodiments at least one of R^la, R^2a, R^3a and R^4a is Ci-Ce alkyl.

In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

[0208] In certain embodiments of Formula (II), R^la, R^lb, R^4a and R^4b are C1-C12 alkyl at each occurrence.

[0209] In further embodiments of Formula (II), at least one of R^lb, R^2b, R^3b and R^4b is H or R^lb, R^2b, R^3b and R^4b are H at each occurrence.

[0210] In certain embodiments of Formula (II), R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

[021 1] The substituents at R⁵ and R⁶ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R⁵ or R⁶ is methyl. In other embodiments each of R⁵ or R⁶ is methyl.

[0212] The substituents at R⁷ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R⁷ is Ce-Ci6 alkyl. In some other embodiments, R⁷ is Ce- C9 alkyl. In some of these embodiments, R⁷ is substituted with: -NR^a

-NR^aS(O)_xNR^aR^b, -NR^aS(O)_xR^b or -S(O)_xNR^aR^b, wherein: R^a is H or C1-C12 alkyl; R^b is C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R⁷ is substituted with -(C=O)OR^b or -O(C=O)R^b.

[0213] In various of the foregoing embodiments of Formula (II), R^b is branched C1-C15 alkyl. For example, in some embodiments R^b has one of the following structures:

[0214] In certain other of the foregoing embodiments of Formula (II), one of R⁸ or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

[0215] In some different embodiments of Formula (II), R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

[0216] In still other embodiments of the foregoing lipids of Formula (II), G³ is C2-C4 alkylene, for example C3 alkylene.

[0217] In various different embodiments, the lipid compound has one of the following structures:

[0218] In some embodiments, the LNPs comprise a lipid of Formula (II), at least one agent, and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II- 10. In some embodiments, the lipid of Formula (II) is compound II- 11. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32.

[0219] In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-,

-C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-,

-S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, ,NR^aC(=O)NR^a-, -OC(=O)NR^a- or - NR^aC(=O)O- or a direct bond;

G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G³ is C1-C24 alkylene, C1-C24 alkenylene, Ca-Cs cycloalkylene, Ca-Cs cycloalkenylene;

R^a is H or C1-C12 alkyl;

R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R³ is H or C1-C6 alkyl; and x is 0, 1 or 2.

[0220] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB) :

wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R⁶ is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15. In some of the foregoing embodiments of Formula (III), the lipid has structure

(IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of Formula (III), the lipid has one of the following structures

wherein y and z are each independently integers ranging from 1 to 12.

[0221] In any of the foregoing embodiments of Formula (III), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-. In some different embodiments of any of the foregoing, L¹ and L² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

[0222] In some different embodiments of Formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

(HIE) (IIIF)

[0223] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ) :

[0224] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3,

4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is

5. In some embodiments, n is 6.

[0225] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0226] In some of the foregoing embodiments of Formula (III), R⁶ is H. In other of the foregoing embodiments, R⁶ is C1-C24 alkyl. In other embodiments, R⁶ is OH.

[0227] In some embodiments of Formula (III), G³ is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G³ is linear C1-C24 alkylene or linear C1-C24 alkenylene.

[0228] In some other foregoing embodiments of Formula (III), R¹ or R², or both, is Ce- C24 alkenyl. For example, in some embodiments, R¹ and R² each, independently have the following structure:

wherein:

R^7a and R^7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R^7a, R^7b and a are each selected such that R¹ and R² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

[0229] In some of the foregoing embodiments of Formula (III), at least one occurrence of R^7a is H. For example, in some embodiments, R^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R^7b is Ci-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

[0230] In different embodiments of Formula (III), R¹ or R², or both, has one of the

CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NHC(=0)R⁴. In some embodiments, R⁴ is methyl or ethyl.

[0232] In various different embodiments, the cationic lipid of Formula (III) has one of the following structures:

[0233] In some embodiments, the LNPs comprise a lipid of Formula (III), at least one agent, and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound 111-7.

[0234] In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In one embodiment, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In one embodiment, the LNP comprises only cationic lipids.

[0235] In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

[0236] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0237] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0238] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1 ,2-di stearoyl -sn- glycero-3 -phosphocholine (DSPC).

[0239] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0240] In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

[0241] In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2:1 to 1 : 1.

[0242] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidyl ethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0243] In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GM1). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

[0244] In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[0245] In certain embodiments, the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate di acylglycerol (PEG-S-DAG) such as 4- O-(2’, 3 ’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(poly ethoxy )ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-( co-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

[0246] In some embodiments, the LNPs comprise a pegylated lipid having the following structure (IV):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.

[0247] In some of the foregoing embodiments of the pegylated lipid (IV), R¹⁰ and R¹¹ are not both n-octadecyl when z is 42. In some other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R¹⁰ is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R¹¹ is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0248] In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average z is about 45.

[0249] In other embodiments, the pegylated lipid has one of the following structures:

wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g/mol.

[0250] In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in about 1 mole percent or about 1 .5 mole percent.

[0251] In some embodiments, the LNPs comprise a lipid of Formula (I), a nucleoside- modified RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments the lipid of Formula (I) is compound 1-6. In different embodiments, the neutral lipid is DSPC. In other embodiments, the steroid is cholesterol. In still different embodiments, the pegylated lipid is compound IVa.

[0252] In certain embodiments, the LNP comprises one or more targeting moieties that targets the LNP to a cell or cell population. For example, in one embodiment, the targeting domain is a ligand which directs the LNP to a receptor found on a cardiomyocyte.

[0253] Exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172- 176; Akinc et al., 2010, Mol Then, 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440- 18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, el39; Maier et al., 2013, Mol Then, 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

[0254] The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III).

GENERAL REACTION SCHEME 1

[0255] Embodiments of the lipid of Formula (I) (e.g., compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-1, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A-5 after any necessarily workup and or purification step. GENERAL REACTION SCHEME 2

[0256] Other embodiments of the compound of Formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-l (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and/or purification.

[0257] It should be noted that although starting materials A-l and B-l are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds. GENERAL REACTION SCHEME 3

[0258] Different embodiments of the lipid of Formula (I) (e.g., compound C-7 or C9) can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-1 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.

GENERAL REACTION SCHEME 4

[0259] Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R^la, R^lb, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R⁵, R⁶, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as defined herein, and R⁷ represents R⁷ or a C3-C19 alkyl. Referring to General Reaction Scheme 1, compounds of structure D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and/or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary work up and/or purification.

GENERAL REACTION SCHEME 5

[0260] Embodiments of the lipid of Formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein R^la, R^lb, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², G³, a, b, c and d are as defined herein. Referring to General Reaction Scheme 2, compounds of structure E-l and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-l (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and/or purification.

GENERAL REACTION SCHEME 6

[0261] General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III). G¹, G³, R¹ and R³ in General Reaction Scheme 6 are as defined herein for Formula (III), and Gl’ refers to a one-carbon shorter homologue of Gl. Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

[0262] It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L¹ and L² are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G¹ and G² are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G¹ and G² are different.

[0263] It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkyl silyl (for example, Lbutyldimethylsilyl, Lbutyldiphenyl silyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include /-butoxy carbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), /?-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

Lipid Nanoparticle Formulations

[0264] The present invention relates, in part, to a lipid nanoparticle (LNP) comprising at least one lipid compound of the present invention. In various embodiments, the LNP comprises one or more ionizable lipid in a concentration range of about 0.1 mol% to about 100 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration range of about 0.1 mol% to about 99.99 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration range of about 10 mol% to about 70 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration range of about 10 mol% to about 50 mol%.

[0265] For example, in some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 1 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 2 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 5 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 5.5 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 10 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 12 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 15 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 16 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 20 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 25 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 30 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 35 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 37 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 40 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 45 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 50 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 60 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 70 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 80 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 90 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 95 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 95.5 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 99 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 99.9 mol%. In some embodiments, the LNP comprises one or more ionizable lipids in a concentration of about 100 mol%.

[0266] In various embodiments, the LNP further comprises at least one helper compound. In various embodiments, the LNP comprises one or more helper compound in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.01 mol% to about 99.99 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.1 mol% to about 99.9 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0. 1 mol% to about 90 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.1 mol% to about 70 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 5 mol% to about 95 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.5 mol% to about 50 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.5 mol% to about 47 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 2.5 mol% to about 47 mol%.

[0267] For example, in some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.01 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.1 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 1 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 1.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 2 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 2.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 10 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 12 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 15 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 16 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 20 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 25 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 30 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 35 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 37 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 40 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 45 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 46.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 47 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 50 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 60 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 63 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 70 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 80 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 90 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 95 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 95.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 99 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 100 mol%.

[0268] In some embodiments, the helper compound is a helper lipid, helper polymer, or any combination thereof. In some embodiments, the helper lipid is phospholipid, cholesterol lipid, polymer, cationic lipid, neutral lipid, charged lipid, steroid, steroid analogue, polymer conjugated lipid, stabilizing lipid, or any combination thereof.

[0269] In some embodiments, the phospholipid is dioleoyl-phosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoyl-phosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, l-stearioyl-2-oleoyl- phosphatidy ethanol amine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof. For example, in some embodiments, the LNP comprises a phospholipid in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 15 mol% to about 50 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 10 mol% to about 40 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 16 mol% to about 40 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 16 mol%. In some embodiments, the LNP comprises DOPE in a concentration of about 16 mol%.

[0270] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof, such as a substituted cholesterol molecule. In some embodiments, the LNP comprises a mixture of cholesterol and a substituted cholesterol molecule. For example, in some embodiments, the LNP comprises total cholesterol lipid including cholesterol and one or more substituted cholesterol in a concentration range of about 0 mol% to about 100 mol%. Tn some embodiments, the LNP comprises a total cholesterol lipid in a concentration range of about 20 mol% to about 50 mol%. In some embodiments, the LNP comprises total cholesterol lipid in a concentration range of about 20 mol% to about 47 mol%. In some embodiments, the LNP comprises total cholesterol lipid in a concentration of about 46.5 mol%.

[0271] In some embodiments, the polymer is polyethylene glycol (PEG) or a derivative thereof. For example, in some embodiments, the LNP comprises a polymer in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 10 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 2.5 mol%. For example, in some embodiments, the LNP comprises a polymer in a concentration of about 2.5 mol%.

[0272] As used herein, the term “cationic lipid” refers to a lipid that is cationic or 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 nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0273] In some embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, 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), N-(l- (2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0274] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-3 - morpholinopropane (DLin-MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3 -dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-l,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA).

[0275] In one embodiment, the lipid is a PEGylated lipid, including, but not limited to, DSPE-PEG-DBCO, DOPE-PEG-Azide, DSPE-PEG- Azide, DPPE-PEG- Azide, DSPE-PEG- Carboxy-NHS, DOPE-PEG-Carboxylic Acid, DSPE-PEG-Carboxylic acid.

[0276] The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include phosphatidylcholine, diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0277] Exemplary neutral lipids include, for example, phosphatidylcholine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG, distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearioyl-2-oleoyl- phosphatidy ethanol amine (SOPE), stearoyloleoylphosphatidylcholine (SOPC), and 1,2- dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0278] In some embodiments, the composition comprises a neutral lipid selected from phosphatidylcholine, DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0279] A “steroid” is a compound comprising the following carbon skeleton:

[0280] In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid.

[0281] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0282] The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include polyethylene glycol (PEG), maleimide PEG (mPEG), DSPE-PEG-DBCO, l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG), DOPE-PEG- Azide, DSPE-PEG- Azide, DPPE-PEG-Azide, DSPE-PEG-Carboxy-NHS, DOPE-PEG- Carboxylic Acid, DSPE-PEG-Carboxylic acid and the like.

[0283] In certain embodiments, the cLNP comprises an additional, stabilizing-lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2ooo)carbamyl]-l,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(m on om ethoxy-poly ethyl eneglycol)-2, 3 -dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- O-(2’ ,3 ’-di(tetradecanoyloxy)propyl- 1 -0-(co -methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

[0284] In various embodiments, the cLNP have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In one embodiment, the cLNP of the invention comprise a mean diameter of about 80 nm.

[0285] In various embodiments, the lipids or the cLNP of the present invention are substantially non-toxic.

[0286] In various embodiments, the cLNPs described herein show uptake in cardiomyocytes. In some embodiments, the cLNP of the present invention comprise an ionizable lipid in a concentration range of about 0.1 mol% to about 99.99 mol%. In some embodiments, the compound of the present invention is present in concentration range of about 1 mol% to about 65 mol%. In some embodiments, the compound of the present invention is present in a molar ratio of about 50 or a concentration of about 50 mol%.

[0287] In some embodiments, the LNP formulated for stability for in vivo cell targeting comprises a phospholipid in a concentration range of about 5 mol% to about 45 mol%. In one embodiment, the phospholipid is phosphatidylcholine, and the phosphatidylcholine is present in a molar ratio of about 10 or at a molar percentage of about 10%.

[0288] In some embodiments, the cLNP comprises total cholesterol lipid in a concentration range of about 5 mol% to about 50 mol%. In one embodiment, the total cholesterol is present in a molar ratio of about 38.5, or at a molar percentage of about 38.5%.

[0289] In some embodiments, the cLNP comprises total PEG in a concentration range of about 0.5 mol% to about 12.5 mol%. In one embodiment, the total PEG is present in a molar ratio of about 1.5, or at a molar percentage of about 1.5%.

[0290] In some embodiments, the cardiomyocyte targeted LNP (cLNP) of the invention comprises an ionizable cationic lipid, phosphatidylcholine, cholesterol, and PEG-lipid at molar ratio or 50: 10:38.5: 1.5, respectively. In one embodiment, the ionizable cationic lipid is ALC0307 (Acuitas, Vancouver, BC, Canada) Therefore in some embodiments, the invention comprises ALC0307, phosphatidylcholine, cholesterol, and PEG-lipid at molar ratio 50:10:38.5: 1.5, respectively.

[0291] In some embodiments, the cardiomyocyte targeted LNP (cLNP) of the invention comprises LNP-B or LNP-E from Arbutus.

LNP Compositions

[0292] In one aspect, the present invention relates to a composition comprising at least one cLNP of the present invention. In one aspect, the present invention relates to a composition comprising at least one cLNP of the present invention for delivery of an cargo molecule or agent to a cardiomyocyte.

[0293] In one aspect, the invention is not limited to any particular cargo or otherwise agent for which the cLNP is able to carry or transport. Rather, the invention includes any agent that can be carried by the cLNP. For example, agents that can be carried by the LNP of the invention include, but are not limited to, diagnostic agents, detectable agents, and therapeutic agents. For example, in some embodiments, the LNPs are effective for delivery of prosurvival RNA drugs.

[0294] In various embodiments, the composition comprises an in vitro transcribed (IVT) RNA molecule. For example, in certain embodiments, the composition of the invention comprises an IVT RNA molecule, which encodes an agent. In certain embodiments, the IVT RNA molecule of the present composition is a nucleoside-modified mRNA molecule.

[0295] In one embodiment, the composition comprises at least one RNA molecule encoding a combination of at least two agents. In one embodiment, the composition comprises a combination of two or more RNA molecules encoding a combination of two or more agents.

[0296] In one embodiment, the present invention provides a method for gene editing of a cell of interest of a subject. For example, the method can be used to provide one or more component of a gene editing system (e.g., a component of a CRISPR system) to a cell of interest of a subject. In some embodiments, the method comprises administering to the subject a composition comprising one or more cLNP molecule formulated for targeted delivery comprising one or more nucleoside-modified RNA molecule for gene editing.

[0297] In one embodiment, the delivery vehicle comprises at least one agent. In some embodiments, the agent is a therapeutic agent, a diagnostic agent, a gene editing agent, an imaging agent, a contrast agent, a labeling agent, a detection agent, or a disinfectant. In some embodiments the agent is a prosurvival RNA (e.g., an mRNA, a IncRNA, a circRNA, or a miRNA).

[0298] The agent may also include substances with biological activities which are not typically considered to be active ingredients, such as fragrances, sweeteners, flavorings and flavor enhancer agents, pH adjusting agents, effervescent agents, emollients, bulking agents, soluble organic salts, permeabilizing agents, anti-oxidants, colorants or coloring agents, and the like.

[0299] In one embodiment, the delivery vehicle comprises at least one therapeutic agent. The present invention is not limited to any particular therapeutic agent, but rather encompasses any suitable therapeutic agent that can be included within the delivery vehicle. Exemplary therapeutic agents include, but are not limited to, anti-viral agents, anti-bacterial agents, antioxidant agents, thrombolytic agents, chemotherapeutic agents, anti-inflammatory agents, immunogenic agents, antiseptics, anesthetics, analgesics, pharmaceutical agents, small molecules, peptides, nucleic acids, inhibitors and activators of cellular receptors, ion channels, cholesterol quenchers, enzymes and inhibitors thereof and the like. Exemplary enzymes include, but are not limited to hydrolases, ligases, proteases, and peptidases of diverse specificities.

Imaging Agents

[0300] In one embodiment, the delivery vehicle comprises an imaging agent. Imaging agents are materials that allow the delivery vehicle to be visualized after exposure to a cell or tissue. Visualization includes imaging for the naked eye, as well as imaging that requires detecting with instruments or detecting information not normally visible to the eye, and includes imaging that requires detecting of photons, sound or other energy quanta. Examples include stains, vital dyes, fluorescent markers, radioactive markers, enzymes or plasmid constructs encoding markers or enzymes. Many materials and methods for imaging and targeting that may be used in the delivery vehicle are provided in the Handbook of Targeted delivery of Imaging Agents, Torchilin, ed. (1995) CRC Press, Boca Raton, Fla.

[0301] Visualization based on molecular imaging typically involves detecting biological processes or biological molecules at a tissue, cell, or molecular level. Molecular imaging can be used to assess specific targets for gene therapies, cell-based therapies, and to visualize pathological conditions as a diagnostic or research tool. Imaging agents that are able to be delivered intracellularly are particularly useful because such agents can be used to assess intracellular activities or conditions. Imaging agents must reach their targets to be effective; thus, in some embodiments, an efficient uptake by cells is desirable. A rapid uptake may also be desirable to avoid the RES, see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001).

[0302] Further, imaging agents should provide high signal to noise ratios so that they may be detected in small quantities, whether directly, or by effective amplification techniques that increase the signal associated with a particular target. Amplification strategies are reviewed in Allport and Weissleder, Experimental Hematology 1237-1246 (2001), and include, for example, avidin-biotin binding systems, trapping of converted ligands, probes that change physical behavior after being bound by a target, and taking advantage of relaxation rates. Examples of imaging technologies include magnetic resonance imaging, radionuclide imaging, computed tomography, ultrasound, and optical imaging. [0303] Delivery vehicles as set forth herein may advantageously be used in various imaging technologies or strategies, for example by incorporating imaging agents into delivery vehicles. Many imaging techniques and strategies are known, e.g., see review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001); such strategies may be adapted to use with delivery vehicles. Suitable imaging agents include, for example, fluorescent molecules, labeled antibodies, labeled avidimbiotin binding agents, colloidal metals (e.g., gold, silver), reporter enzymes (e.g., horseradish peroxidase), superparamagnetic transferrin, second reporter systems (e.g., tyrosinase), and paramagnetic chelates.

[0304] In some embodiments, the imaging agent is a magnetic resonance imaging contrast agent. Examples of magnetic resonance imaging contrast agents include, but are not limited to, 1,4,7, 10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA), diethylenetriaminepentaacetic (DTP A), 1,4,7, 10-tetraazacyclododecane-N,N', N'',N'"- tetraethylphosphorus (DOTEP), 1,4,7, 10-tetraazacyclododecane-N,N',N"-triacetic acid (DOTA) and derivatives thereof (see U.S. Pat. Nos. 5,188,816, 5,219,553, and 5,358,704). In some embodiments, the imaging agent is an X-Ray contrast agent. X-ray contrast agents already known in the art include a number of halogenated derivatives, especially iodinated derivatives, of 5-amino-isophthalic acid.

Small molecule therapeutic agents

[0305] In various embodiments, the delivery vehicle comprises a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, a small molecule may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means. Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art. In one embodiment, a small molecule therapeutic agent comprises an organic molecule, inorganic molecule, biomolecule, synthetic molecule, and the like.

[0306] Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art, as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development. In some embodiments of the invention, the therapeutic agent is synthesized and/or identified using combinatorial techniques.

[0307] In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (“focused libraries”) or synthesized with less structural bias using flexible cores. In some embodiments of the invention, the therapeutic agent is synthesized via small library synthesis.

[0308] The small molecule and small molecule compounds described herein may be present as salts even if salts are not depicted, and it is understood that the invention embraces all salts and solvates of the therapeutic agents depicted here, as well as the non-salt and non-solvate form of the therapeutic agents, as is well understood by the skilled artisan. In some embodiments, the salts of the therapeutic agents of the invention are pharmaceutically acceptable salts.

[0309] Where tautomeric forms may be present for any of the therapeutic agents described herein, each and every tautomeric form is intended to be included in the present invention, even though only one or some of the tautomeric forms may be explicitly depicted. For example, when a 2-hydroxypyridyl moiety is depicted, the corresponding 2-pyridone tautomer is also intended.

[0310] The invention also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms of the therapeutic agents described. The recitation of the structure or name herein is intended to embrace all possible stereoisomers of therapeutic agents depicted. All forms of the therapeutic agents are also embraced by the invention, such as crystalline or non-crystalline forms of the therapeutic agent. Compositions comprising a therapeutic agent of the invention are also intended, such as a composition of substantially pure therapeutic agent, including a specific stereochemical form thereof, or a composition comprising mixtures of therapeutic agents of the invention in any ratio, including two or more stereochemical forms, such as in a racemic or non-racemic mixture. [0311] The invention also includes any or all active analog or derivative, such as a prodrug, of any therapeutic agent described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. As such, in certain instances, the analogs of the small molecules described herein that have modulated potency, selectivity, and solubility are included herein and provide useful leads for drug discovery and drug development. Thus, in certain instances, during optimization new analogs are designed considering issues of drug delivery, metabolism, novelty, and safety.

[0312] In some instances, small molecule therapeutic agents described herein are derivatives or analogs of known therapeutic agents, as is well known in the art of combinatorial and medicinal chemistry. The analogs or derivatives can be prepared by adding and/or substituting functional groups at various locations. As such, the small molecules described herein can be converted into derivatives/analogs using well known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. Also, the linking atoms or groups can be modified into longer or shorter linkers with carbon backbones or hetero atoms. Also, the ring groups can be changed so as to have a different number of atoms in the ring and/or to include hetero atoms. Moreover, aromatics can be converted to cyclic rings, and vice versa. For example, the rings may be from 5-7 atoms, and may be carbocyclic or heterocyclic.

[0313] As used herein, the term “analog,” “analogue,” or “derivative” is meant to refer to a chemical compound or molecule made from a parent compound or molecule by one or more chemical reactions. As such, an analog can be a structure having a structure similar to that of the small molecule therapeutic agents described herein or can be based on a scaffold of a small molecule therapeutic agents described herein, but differing from it in respect to certain components or structural makeup, which may have a similar or opposite action metabolically. An analog or derivative of any of a small molecule inhibitor in accordance with the present invention can be used to treat a disease or disorder.

[0314] In one embodiment, the small molecule therapeutic agents described herein can independently be derivatized, or analogs prepared therefrom, by modifying hydrogen groups independently from each other into other substituents. That is, each atom on each molecule can be independently modified with respect to the other atoms on the same molecule. Any traditional modification for producing a derivative/analog can be used. For example, the atoms and substituents can be independently comprised of hydrogen, an alkyl, aliphatic, straight chain aliphatic, aliphatic having a chain hetero atom, branched aliphatic, substituted aliphatic, cyclic aliphatic, heterocyclic aliphatic having one or more hetero atoms, aromatic, heteroaromatic, polyaromatic, polyamino acids, peptides, polypeptides, combinations thereof, halogens, halo- substituted aliphatics, and the like. Additionally, any ring group on a compound can be derivatized to increase and/or decrease ring size as well as change the backbone atoms to carbon atoms or hetero atoms.

Nucleic acid therapeutic agents

[0315] In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is one of a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, IncRNA, circRNA, siRNA, shRNA or miRNA molecule. In some embodiments, the therapeutic agent is an antisense molecule (e.g., an siRNA, miRNA, or shRNA) which inhibits a targeted nucleic acid including those encoding proteins that are involved in aggravation of the pathological processes.

[0316] In one embodiment, the nucleic acid comprises a promoter/regulatory sequence such that the nucleic acid is capable of directing expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous nucleic acid into cells with concomitant expression of the exogenous nucleic acid in the cells such as those described, for example, in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein.

[0317] In one aspect of the invention, a targeted gene or protein, can be inhibited by way of inactivating and/or sequestering the targeted gene or protein. As such, inhibiting the activity of the targeted gene or protein can be accomplished by using a nucleic acid molecule encoding a transdominant negative mutant.

[0318] In one embodiment, siRNA is used to decrease the level of a targeted protein. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA-induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3’ overhang. See, for instance, Schwartz et al., 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 115:209-216. Therefore, the present invention also includes methods of decreasing levels of PTPN22 using RNAi technology.

[0319] In one aspect, the invention includes a vector comprising an siRNA or an antisense polynucleotide. In some embodiments, the siRNA, or antisense polynucleotide, is capable of inhibiting the expression of a target polypeptide. The incorporation of a desired polynucleotide into a vector and the choice of vectors are well-known in the art as described in, for example, Sambrook et al. (2012), and in Ausubel et al. (1997), and elsewhere herein.

[0320] In certain embodiments, the expression vectors described herein encode a short hairpin RNA (shRNA) therapeutic agent. shRNA molecules are well known in the art and are directed against the mRNA of a target, thereby decreasing the expression of the target. In certain embodiments, the encoded shRNA is expressed by a cell, and is then processed into siRNA. For example, in certain instances, the cell possesses native enzymes (e.g., dicer) that cleave the shRNA to form siRNA.

[0321] In order to assess the expression of the siRNA, shRNA, or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification of expressing cells from the population of cells sought to be transfected or infected using the delivery vehicle of the invention. In other embodiments, the selectable marker may be carried on a separate piece of DNA and also be contained within the delivery vehicle. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibioticresistance genes, such as neomycin resistance and the like.

[0322] Therefore, in one aspect, the delivery vehicle may contain a vector, comprising the nucleotide sequence or the construct to be delivered. The choice of the vector will depend on the host cell in which it is to be subsequently introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0323] By way of illustration, the vector in which the nucleic acid sequence is introduced can be a plasmid, which is or is not integrated in the genome of a host cell when it is introduced in the cell. Illustrative, non-limiting examples of vectors in which the nucleotide sequence of the invention or the gene construct of the invention can be inserted include a tet-on inducible vector for expression in eukaryote cells.

[0324] The vector may be obtained by conventional methods known by persons skilled in the art (Sambrook et al., 2012). In a particular embodiment, the vector is a vector useful for transforming animal cells.

[0325] In one embodiment, the recombinant expression vectors may also contain nucleic acid molecules, which encode a peptide or peptidomimetic.

[0326] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0327] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression. The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0328] The recombinant expression vectors may also contain a selectable marker gene, which facilitates the selection of host cells. Suitable selectable marker genes are genes encoding proteins such as G418 and hygromycin, which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin (e.g., an IgG.) The selectable markers may be introduced on a separate vector from the nucleic acid of interest.

[0329] Following the generation of the siRNA polynucleotide, a skilled artisan will understand that the siRNA polynucleotide will have certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Therefore, the siRNA polynucleotide may be further designed to resist degradation by modifying it to include phosphorothioate, or other linkages, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0330] Any polynucleotide may be further modified to increase its stability in vivo. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends; the use of phosphorothioate or 2' O-methyl rather than phosphodiester linkages in the backbone; and/or the inclusion of nontraditional bases such as inosine, queuosine, and wybutosine and the like, as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0331] In one embodiment of the invention, an antisense nucleic acid sequence, which is expressed by a plasmid vector is used as a therapeutic agent to inhibit the expression of a target protein. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of the target protein.

[0332] Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

[0333] The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5,190,931.

[0334] Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. In some embodiments, antisense oligomers of between about 10 to about 30 nucleotides are used since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0335] In one embodiment of the invention, a ribozyme is used as a therapeutic agent to inhibit expression of a target protein. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure, which are complementary, for example, to the mRNA sequence encoding the target molecule. Ribozymes targeting the target molecule, may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

[0336] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, where a guide RNA (gRNA) targeted to a gene encoding a target molecule, and a CRISPR-associated (Cas) peptide form a complex to induce mutations within the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0337] In one embodiment, the agent comprises a miRNA or a mimic of a miRNA. In one embodiment, the agent comprises a nucleic acid molecule that encodes a miRNA or mimic of a miRNA.

[0338] MiRNAs are small non-coding RNA molecules that are capable of causing post- transcriptional silencing of specific genes in cells by the inhibition of translation or through degradation of the targeted mRNA. A miRNA can be completely complementary or can have a region of noncomplementarity with a target nucleic acid, consequently resulting in a "bulge" at the region of non-complementarity. A miRNA can inhibit gene expression by repressing translation, such as when the miRNA is not completely complementary to the target nucleic acid, or by causing target RNA degradation, which is believed to occur only when the miRNA binds its target with perfect complementarity. The disclosure also can include double-stranded precursors of miRNA. A miRNA or pri-miRNA can be 18- 100 nucleotides in length, or from 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. MiRNA precursors typically have a length of about 70-100 nucleotides and have a hairpin conformation. miRNAs are generated in vivo from pre- miRNAs by the enzymes Dicer and Drosha, which specifically process long pre- miRNA into functional miRNA. The hairpin or mature microRNAs, or pri-microRNA agents featured in the disclosure can be synthesized in vivo by a cell-based system or in vitro by chemical synthesis.

[0339] In various embodiments, the agent comprises an oligonucleotide that comprises the nucleotide sequence of a disease-associated miRNA. In certain embodiments, the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in a pre - microRNA, mature or hairpin form. In other embodiments, a combination of oligonucleotides comprising a sequence of one or more disease-associated miRNAs, any pre -miRNA, any fragment, or any combination thereof is envisioned.

[0340] MiRNAs can be synthesized to include a modification that imparts a desired characteristic. For example, the modification can improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell -type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism.

[0341] Modifications can also increase sequence specificity, and consequently decrease off-site targeting. Methods of synthesis and chemical modifications are described in greater detail below. If desired, miRNA molecules may be modified to stabilize the miRNAs against degradation, to enhance half-life, or to otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Publication Nos. 20070213292, 20060287260, 20060035254. 20060008822. and 2005028824, each of which is hereby incorporated by reference in its entirety. For increased nuclease resistance and/or binding affinity to the target, the single- stranded oligonucleotide agents featured in the disclosure can include 2'-O-methyl, 2'- fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and/or phosphorothioate linkages. Inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA), e.g., 2'-4'-ethylene- bridged nucleic acids, and certain nucleotide modifications can also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone can also decrease endonucleolytic cleavage. An oligonucleotide can be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3 '-terminus with a 3 -3' linkage. In another alternative, the 3 '-terminus can be blocked with an aminoalkyl group. Other 3' conjugates can inhibit 3'-5' exonucleolytic cleavage. While not being bound by theory, a 3' may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 3' end of the oligonucleotide. Even small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose etc.) can block 3'-5'-exonucleases.

[0342] In one embodiment, the miRNA includes a 2'-modified oligonucleotide containing oligodeoxynucleotide gaps with some or all intemucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure may be used in conjunction with any technologies that may be developed to enhance the stability or efficacy of an inhibitory nucleic acid molecule.

[0343] miRNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleotide oligomers. Nucleotide oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. Various salts, mixed salts and free acid forms are also included.

[0344] A miRNA described herein, which may be in the mature or hairpin form, may be provided as a naked oligonucleotide. In some cases, it may be desirable to utilize a formulation that aids in the delivery of a miRNA or other nucleotide oligomer to cells (see, e.g., U.S. Pat. Nos. 5,656,61 1, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

[0345] In some examples, the miRNA composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the miRNA composition is in an aqueous phase, e.g., in a solution that includes water. The aqueous phase or the crystalline compositions can be incorporated into a delivery vehicle, e g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the miRNA composition is formulated in a manner that is compatible with the intended method of administration. A miRNA composition can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Still other agents include chelators, e.g., EDTA (e g., to remove divalent cations such as Mg), salts, and RNAse inhibitors (e.g., a broad specificity RNAse inhibitor). In one embodiment, the miRNA composition includes another miRNA, e.g., a second miRNA composition (e.g., a microRNA that is distinct from the first). Still other preparations can include at least three, five, ten, twenty, fifty, or a hundred or more different oligonucleotide species.

[0346] In certain embodiments, the composition comprises an oligonucleotide composition that mimics the activity of a miRNA. In certain embodiments, the composition comprises oligonucleotides having nucleobase identity to the nucleobase sequence of a miRNA, and are thus designed to mimic the activity of the miRNA. In certain embodiments, the oligonucleotide composition that mimics miRNA activity comprises a double-stranded RNA molecule which mimics the mature miRNA hairpins or processed miRNA duplexes.

[0347] In one embodiment, the oligonucleotide shares identity with endogenous miRNA or miRNA precursor nucleobase sequences. An oligonucleotide selected for inclusion in a composition of the present invention may be one of a number of lengths. Such an oligonucleotide can be from 7 to 100 linked nucleosides in length. For example, an oligonucleotide sharing nucleobase identity with a miRNA may be from 7 to 30 linked nucleosides in length. An oligonucleotide sharing identity with a miRNA precursor may be up to 100 linked nucleosides in length. In certain embodiments, an oligonucleotide comprises 7 to 30 linked nucleosides. In certain embodiments, an oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In certain embodiments, an oligonucleotide comprises 19 to 23 linked nucleosides. In certain embodiments, an oligonucleotide is from 40 up to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0348] In certain embodiments, an oligonucleotide has a sequence that has a certain identity to a miRNA or a precursor thereof. Nucleobase sequences of mature miRNAs and their corresponding stem-loop sequences described herein are the sequences found in miRBase, an online searchable database of miRNA sequences and annotation. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence. The compositions of the present invention encompass oligomeric compound comprising oligonucleotides having a certain identity to any nucleobase sequence version of a miRNAs described herein.

[0349] In certain embodiments, an oligonucleotide has a nucleobase sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. Accordingly, in certain embodiments the nucleobase sequence of an oligonucleotide may have one or more non-identical nucleobases with respect to the miRNA.

[0350] In certain embodiments, the composition comprises a nucleic acid molecule encoding a miRNA, precursor, mimic, or fragment thereof. For example, the composition may comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, precursor, mimic, or fragment thereof in a desired mammalian cell or tissue.

In vitro transcribed RNA

[0351] In one embodiment, the therapeutic agent of the invention comprises in vitro transcribed (IVT) RNA. In one embodiment, the composition of the invention comprises in vitro transcribed (IVT) RNA encoding a therapeutic protein. In one embodiment, the composition of the invention comprises IVT RNA encoding one or more therapeutic protein.

[0352] In one embodiment, an IVT RNA can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In one embodiment, the desired template for in vitro transcription is a therapeutic protein, as described elsewhere herein.

[0353] In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest of a portion of a gene. The gene can include some or all of the 5' and/or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0354] Genes that can be used as sources of DNA for PCR include genes that encode polypeptides that induce or enhance an adaptive immune response in an organism. In some embodiments, the genes are genes which are useful for a short-term treatment, or where there are safety concerns regarding dosage or the expressed gene.

[0355] In various embodiments, a plasmid is used to generate a template for in vitro transcription of RNA which is used for transfection.

[0356] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. In some embodiments, the RNA has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0357] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0358] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

[0359] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0360] In one embodiment, the RNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized RNA which is effective in eukaryotic transfection when it is polyadenylated after transcription. [0361] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0362] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

[0363] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase RNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0364] 5' caps can also provide stability to RNA molecules. In some embodiments,

RNAs produced by the methods to include a 5' cap-1 structure, comprising a methylated 2'- hydroxy group on the first ribose sugar. The cap-1 structure can be generated using Vaccinia capping enzyme and 2’-O-methyltransferase enzymes (CellScript, Madison, WI). Alternatively, 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0365] In one embodiment, the nucleoside modified RNA encodes a polypeptide, protein or therapeutic agent for the treatment of a cardiac disease or disorder. As a non-limiting example, in some embodiments, the IVT RNA encodes SERCA2.

Nucleoside-modified RNA

[0366] In one embodiment, the therapeutic agent comprises a nucleoside-modified nucleic acid. In one embodiment, the composition of the invention comprises a nucleoside- modified RNA encoding a therapeutic protein. [0367] For example, in one embodiment, the composition comprises a nucleoside- modified RNA. In one embodiment, the composition comprises a nucleoside-modified mRNA. Nucleoside-modified mRNA have particular advantages over non-modified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, which is incorporated by reference herein in its entirety.

[0368] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16: 1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy.

[0369] In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0370] In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16: 1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23: 165-175).

[0371] It has been demonstrated that the presence of modified nucleosides, including pseudouridines in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). A preparative HPLC purification procedure has been established that was critical to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:el42). Administering HPLC-purified, pseudourine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948- 953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0372] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises an isolated nucleic acid, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises a vector, comprising an isolated nucleic acid, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.

[0373] In one embodiment, the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside- modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside-modified RNA is synthesized by T3 phage RNA polymerase.

[0374] In one embodiment, the modified nucleoside is mlacpST (l-methyl-3-(3-amino- 3 -carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is ml (1- methylpseudouridine). In another embodiment, the modified nucleoside is I'm (2'-O- methylpseudouridine. In another embodiment, the modified nucleoside is m5D (5- methyldihydrouridine). In another embodiment, the modified nucleoside is m3'P (3- methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0375] In another embodiment, the nucleoside that is modified in the nucleoside- modified RNA the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0376] In another embodiment, the modified nucleoside of the present invention is m⁵C (5-methylcytidine). In another embodiment, the modified nucleoside is nr U (5-methyluridine). In another embodiment, the modified nucleoside is m⁶A (N⁶-methyladenosine). In another embodiment, the modified nucleoside is s²U (2-thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0377] In other embodiments, the modified nucleoside is rrriA (1 -methyladenosine); m²A (2 -methyladenosine); Am (2'-O-methyladenosine); ms²m⁶A (2-methylthio-N⁶-methyladenosine); i⁶A (N⁶-isopentenyladenosine); ms²i6A (2-methylthio-N⁶isopentenyladenosine); io⁶A (N⁶-(cis- hydroxyisopentenyl)adenosine); ms²io⁶A (2-methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine); g⁶A (N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶ -threonylcarbamoyladenosine); ms²t⁶A (2- methylthio-N⁶ -threonyl carbamoyladenosine); m⁶t⁶A (N⁶-methyl-N⁶- threonylcarbamoyladenosine); hn⁶A(N⁶ -hydroxynorvalylcarbamoyladenosine); ms²hn⁶A (2- methylthio-N⁶ -hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m¹! (1 -methylinosine); nflm (l,2'-O-dimethylinosine); m³C (3- methylcytidine); Cm (2'-O-methylcytidine); s²C (2 -thiocytidine); ac⁴C (N⁴-acetylcytidine); f^C (5-formylcytidine); m⁵Cm (5,2'-O-dimethylcytidine); ac⁴Cm (N⁴-acetyl-2'-O-methylcytidine); k²C (lysidine); nriG (1 -methylguanosine); m²G (N²-methylguanosine); m⁷G (7- methylguanosine); Gm (2'-O-methylguanosine); m²?G (N²,N²-dimethylguanosine); m²Gm (N²,2'- O-dimethylguanosine); m²2Gm (N²,N²,2'-O-trimethylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); yW (wybutosine); O2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7-deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m’Um (5,2'-O-dimethyluridine); s⁴U (4-thiouridine); m⁵s²U (5-methyl-2- thiouridine); s²Um (2-thio-2'-O-methyluridine); acp³U (3-(3-amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U (5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo’U (uridine 5-oxyacetic acid methyl ester); chm⁵U (5-(carboxyhydroxymethyl)uridine)); mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U (5-methoxycarbonylmethyluridine); mcm’Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm⁵s²U (5- methoxycarbonylmethyl-2-thiouridine); nm⁵s²U (5-aminomethyl-2-thiouridine); mnm’U (5- methylaminomethyluridine); mnm U (5-methylaminomethyl-2-thiouridine); mmrfse²U (5- methylaminomethyl-2-selenouridine); ncm⁵U (5-carbamoylmethyluridine); ncm⁵Um (5- carbamoylmethyl-2'-O-methyluridine); cmnm⁵U (5-carboxymethylaminomethyluridine); cmnm⁵Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm U (5- carboxymethylaminomethyl-2-thiouridine); m⁶2A (N⁶,N⁶-dimethyladenosine); Im (2'-O- methylinosine); m⁴C (N⁴-methylcytidine); m⁴Cm (N⁴,2'-O-dimethylcytidine); hm⁵C (5- hydroxymethylcytidine); m³U (3 -methyluridine); crn’U (5-carboxymethyluridine); m⁶Am (N⁶,2'- O-dimethyladenosine); nfbAm (N⁶,N⁶,O-2'-trimethyladenosine); m^2,7G (N²,7- dimethylguanosine); m²’^2,7G (N²,N²,7-trimethylguanosine); m³Um (3,2'-O-dimethyluridine); m⁵D (5-methyldihydrouridine); f⁵Cm(5-formyl-2'-O-methylcytidine); m'Gm (l,2'-O- dimethylguanosine); m'Am (l,2'-O-dimethyladenosine); rm⁵U (5-taurinomethyluridine); xm⁵s²U (5-taurinomethyl-2 -thiouridine)); imG- 14 (4-demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶-acetyladenosine).

[0378] In another embodiment, a nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

[0379] In another embodiment, between 0.1% and 100% of the residues in the nucleoside-modified of the present invention are modified (e.g. either by the presence of pseudouridine or a modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0380] In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0381] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of the given nucleotide that is modified is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 100%.

[0382] In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

[0383] In another embodiment, a nucleoside-modified RNA of the present invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3- fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10-100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000-fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50-1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200-1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0384] In one embodiment, the nucleoside modified RNA encodes a polypeptide, protein or therapeutic agent for the treatment of a cardiac disease or disorder. As a non-limiting example, in some embodiments, the nucleoside modified RNA encodes SERCA2.

Polypeptide therapeutic agents

[0385] In other related aspects, the therapeutic agent includes an isolated peptide that modulates a target. For example, in one embodiment, the peptide of the invention inhibits or activates a target directly by binding to the target thereby modulating the normal functional activity of the target. In one embodiment, the peptide of the invention modulates the target by competing with endogenous proteins. In one embodiment, the peptide of the invention modulates the activity of the target by acting as a transdominant negative mutant.

[0386] The variants of the polypeptide therapeutic agents may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the polypeptide is an alternative splice variant of the polypeptide of the present invention, (iv) fragments of the polypeptides and/or (v) one in which the polypeptide is fused with another polypeptide, such as a

I l l leader or secretory sequence or a sequence which is employed for purification (for example, His- tag) or for detection (for example, Sv5 epitope tag). The fragments include polypeptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0387] In one embodiment, the polypeptide, protein comprises a therapeutic agent for the treatment of a cardiac disease or disorder. As a non-limiting example, in some embodiments, the polypeptide is SERCA2.

Antibody therapeutic agents

[0388] The invention also contemplates a delivery vehicle comprising an antibody, or antibody fragment, specific for a target. That is, the antibody can inhibit a target to provide a beneficial effect.

[0389] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain FV molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0390] Antibodies can be prepared using intact polypeptides or fragments containing an immunizing antigen of interest. The polypeptide or oligopeptide used to immunize an animal may be obtained from the translation of RNA or synthesized chemically and can be conjugated to a carrier protein, if desired. Suitable carriers that may be chemically coupled to peptides include bovine serum albumin and thyroglobulin, keyhole limpet hemocyanin. The coupled polypeptide may then be used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

Genetic modification

[0391] In other related aspects, the invention includes an isolated nucleic acid encoding a target therapeutic agent, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0392] The desired polynucleotide can be cloned into a number of types of vectors. However, the present invention should not be construed to be limited to any particular vector. Instead, the present invention should be construed to encompass a wide plethora of vectors which are readily available and/or well-known in the art. For example, a desired polynucleotide of the invention can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0393] In specific embodiments, the expression vector is selected from the group consisting of a viral vector, a bacterial vector and a mammalian cell vector. Numerous expression vector systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and/or eukaryote-vector based systems can be employed for use with the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially and widely available.

[0394] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001), and in Ausubel et al. (1997), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. (See, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193.

[0395] For expression of the desired polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 genes, a discrete element overlying the start site itself helps to fix the place of initiation. [0396] Additional promoter elements, i.e., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

[0397] A promoter may be one naturally associated with a gene or polynucleotide sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding polynucleotide segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a polynucleotide sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other prokaryotic, viral, or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.

[0398] Naturally, it will be important to employ a promoter and/or enhancer that effectively directs the expression of the DNA segment in the cell type, organelle, and organism chosen for expression. Those of skill in the art of molecular biology generally know how to use promoters, enhancers, and cell type combinations for protein expression, for example, see Sambrook et al. (2001). The promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides. The promoter may be heterologous or endogenous.

[0399] A promoter sequence exemplified in the experimental examples presented herein is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, Moloney virus promoter, the avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the muscle creatine promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter in the invention provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Further, the invention includes the use of a tissue specific promoter, which promoter is active only in a desired tissue.

[0400] In order to assess the expression of the vector, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic-resistance genes, such as neo and the like. [0401 ] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. Reporter genes that encode for easily assayable proteins are well known in the art. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a protein whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.

[0402] Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (see, e.g., Ui-Tei et al., 2000 FEBS Lett. 479:79-82). Suitable expression systems are well known and may be prepared using well known techniques or obtained commercially. Internal deletion constructs may be generated using unique internal restriction sites or by partial digestion of non-unique restriction sites. Constructs may then be transfected into cells that display high levels of siRNA polynucleotide and/or polypeptide expression. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0403] In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical or biological means. It is readily understood that the introduction of the expression vector comprising the polynucleotide of the invention yields a silenced cell with respect to a regulator.

[0404] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0405] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0406] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid nanoparticles. An example of a colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art.

[0407] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

[0408] Any DNA vector or delivery vehicle can be utilized to transfer the desired polynucleotide to a cell in vitro or in vivo. In the case where a non-viral delivery system is utilized, exemplary delivery vehicles include, but are not limited to, a liposome or lipid nanoparticle. The above-mentioned delivery systems and protocols therefore can be found in Gene Targeting Protocols, 2ed., pp 1-35 (2002) and Gene Transfer and Expression Protocols, Vol. 7, Murray ed., pp 81-89 (1991).

[0409] “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes may be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). However, the present invention also encompasses compositions that have different structures in solution than the normal vesicular structure. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine nucleic acid complexes.

Combinations

[0410] In one embodiment, the composition of the present invention comprises a combination of agents described herein. In certain embodiments, a composition comprising a combination of agents described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual agent. In other embodiments, a composition comprising a combination of agents described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual agent.

[0411] A composition comprising a combination of agents comprises individual agents in any suitable ratio. For example, in one embodiment, the composition comprises a 1 : 1 ratio of two individual agents. However, the combination is not limited to any particular ratio. Rather, any ratio that is shown to be effective is encompassed.

Conjugation

[0412] In various embodiments of the invention, the delivery vehicle is conjugated to a targeting domain or therapeutic agent. Exemplary methods of conjugation can include, but are not limited to, covalent bonds, electrostatic interactions, hydrophobic interactions and “van der Waals” interactions. In one embodiment, the conjugation is a reversible conjugation, such that the delivery vehicle can be disassociated from the targeting domain or therapeutic agent upon exposure to certain conditions or chemical agents. In another embodiment, the conjugation is an irreversible conjugation, such that under normal conditions the delivery vehicle does not dissociate from the targeting domain or therapeutic agent.

[0413] In some embodiments, the conjugation comprises a covalent bond between an activated polymer conjugated lipid and at least one of the targeting domain or therapeutic agent. The term “activated polymer conjugated lipid” refers to a molecule comprising a lipid portion and a polymer portion that has been activated via functionalization of a polymer conjugated lipid with a first coupling group. In one embodiment, the activated polymer conjugated lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer conjugated lipid is an activated pegylated lipid. In one embodiment, the first coupling group is bound to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is bound to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to at least one of the targeting domain or therapeutic agent.

[0414] The first coupling group and second coupling group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or second coupling group are selected from the group consisting of maleimides, N- hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first coupling group or second coupling group is selected from the group consisiting of free amines (-NH2), free sulfhydryl groups (-SH), free hydroxide groups (-OH), carboxylates, hydrazides, and alkoxyamines. In some embodiments, the first coupling group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first coupling group is a maleimide.

[0415] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain or therapeutic agent using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain or therapeutic agent, such as through reaction with 2- mercaptoethylamine. In one embodiment, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S- acetylthioacetate (SATA).

[0416] In some embodiments, the polymer conjugated lipid and the targeting domain or therapeutic agent, are functionalized with groups used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group with a 1,3-dipole, such as an azide, a nitrile oxide, a nitrone, an isocyanide, and the link, with an alkene or an alkyne dipolarophiles. Exemplary dipolarophiles include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes, difluorinated cyclooctynes, and biarylazacyclooctynone

Targeting Domain

[0417] In one embodiment, the cardiomyocyte targeted LNP of the invention comprises a targeting domain that increases the targeting of the delivery vehicle to a cardiomyocyte. The targeting domain may comprise a nucleic acid, peptide, antibody, small molecule, organic molecule, inorganic molecule, glycan, sugar, hormone, and the like that targets the particle to a site in particular need of the therapeutic agent. In certain embodiments, the particle comprises multivalent targeting, wherein the particle comprises multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with a site in need of an agent comprised within the delivery vehicle. For example, the targeting domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In some embodiments, the targeting domain is an affinity ligand which specifically binds to a target. In certain embodiments, the target (e.g. antigen) associated with a site in need of a treatment with an agent. In some embodiments, the targeting domain may be co-polymerized with the composition comprising the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to the composition comprising the delivery vehicle, such as through a chemical reaction between the targeting domain and the composition comprising the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. Targeting domains of the instant invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

Peptides [0418] In one embodiment, the targeting domain of the invention comprises a peptide. In certain embodiments, the peptide targeting domain specifically binds to a target of interest.

[0419] The peptide of the present invention may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0420] The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0421] The variants of the peptides according to the present invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present invention, (iv) fragments of the peptides and/or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which is employed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0422] As known in the art the “similarity” between two peptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide to a sequence of a second peptide. Variants are defined to include peptide sequences different from the original sequence. In some embodiments, the variant is different from the original sequence in less than 40% of residues per segment of interest. In some embodiments, the variant is different from the original sequence in less than 25% of residues per segment of interest. In some embodiments, the variant is different from the original sequence by less than 10% of residues per segment of interest. In some embodiments, the variant is different from the original sequence in just a few residues per segment of interest and at the same time retains sufficient homology or sequence identity to the original sequence to preserve the functionality of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences can be determined by using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0423] The peptides of the invention can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristoylation, protein folding and proteolytic processing, etc. Some modifications or processing events require introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes or Xenopus egg extracts (U.S. Pat. No. 6,103,489) to a standard translation reaction.

[0424] The peptides of the invention may include unnatural amino acids formed by post- translational modification or by introducing unnatural amino acids during translation.

Nucleic acids

[0425] In one embodiment, the targeting domain of the invention comprises an isolated nucleic acid, including for example a DNA oligonucleotide and a RNA oligonucleotide. In certain embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0426] The nucleotide sequences of a nucleic acid targeting domain can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting nucleic acid functions as the original and specifically binds to the target of interest.

[0427] In the sense used in this description, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences describe herein when its nucleotide sequence has a degree of identity with respect to the original or parental nucleotide sequence of at least 60%, at least 70%, at least 85%, at least 95% or greater than 95%. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two nucleotide sequences can be determined by using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

Antibodies

[0428] In one embodiment, the targeting domain of the invention comprises an antibody, or antibody fragment. In certain embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0429] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0430] Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species. Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity.

Methods

[0431] In some embodiments, the invention provides methods and systems for administering a cardiomyocyte targeted LNP (cLNP) to a subject having a reduced level of ApoE, or an ApoE insufficiency. In some embodiments, the invention provides methods and systems for administering a combination of a cardiomyocyte targeted LNP and an ApoE inhibitory composition to increase the level of uptake of the LNP by cardiomyocytes.

[0432] In some embodiments, the invention provides methods and systems for delivery of an agent to a cardiomyocyte of a subject having a reduced level of ApoE, or an ApoE insufficiency. In some embodiments, the invention provides methods and systems for delivery of an agent to a cardiomyocyte of a subject, wherein the method comprises delivery of an cLNP comprising the agent in combination with an ApoE inhibitor.

[0433] In some embodiments, the invention provides methods of diagnosing a disease or disorder in a subject comprising delivering a diagnostic agent for the disease or disorder to a cardiomyocyte by administering a cLNP comprising a diagnostic agent to a subject having an ApoE deficiency. In some embodiments, the invention provides methods of diagnosing a disease or disorder in a subject comprising delivering a diagnostic agent for the disease or disorder to a cardiomyocyte by administering a combination of a cLNP comprising a diagnostic agent and an ApoE inhibitor.

[0434] In some embodiments, the invention provides methods of preventing a disease or disorder in a subject comprising delivering a preventative agent for the disease or disorder to a cardiomyocyte by administering a cLNP comprising a preventative agent to a subject having an ApoE deficiency. In some embodiments, the invention provides methods of preventing a disease or disorder in a subject comprising delivering a preventative agent for the disease or disorder to a cardiomyocyte by administering a combination of a cLNP comprising a preventative agent and an ApoE inhibitor.

[0435] In some embodiments, the invention provides methods of evaluating the stage, progression or the efficacy of a treatment for a disease or disorder in a subject comprising delivering an agent for evaluating the stage, progression or the efficacy of a treatment for the disease or disorder to a cardiomyocyte by administering a cLNP comprising an agent for evaluating the stage, progression or the efficacy of a treatment for the disease or disorder to a subject having an ApoE deficiency. In some embodiments, the invention provides methods of evaluating the stage, progression or the efficacy of a treatment for a disease or disorder in a subject comprising delivering an agent for evaluating the stage, progression or the efficacy of a treatment for the disease or disorder to a cardiomyocyte by administering a combination of a cLNP comprising an agent for evaluating the stage, progression or the efficacy of a treatment for the disease or disorder and an ApoE inhibitor to the subject.

[0436] In some embodiments, the invention provides methods of treating a disease or disorder in a subject comprising delivering a therapeutic agent for the disease or disorder to a cardiomyocyte by administering a cLNP comprising a therapeutic agent to a subject having an ApoE deficiency. In some embodiments, the invention provides methods of treating a disease or disorder in a subject comprising delivering a therapeutic agent for the disease or disorder to a cardiomyocyte by administering a combination of a cLNP comprising a therapeutic agent and an ApoE inhibitor.

[0437] The present invention also provides methods of delivering at least one agent to a cardiomyocyte of a subject in need thereof. In certain embodiments, the method is used to diagnose, evaluate, treat or prevent a disease or disorder in a subject, wherein the subject has a disease or disorder for which delivery of a therapeutic agent to the cardiomyocyte would be beneficial. In some embodiments, the agent is a therapeutic agent for the treatment of at least one of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

[0438] Exemplary cardiac diseases or disorders include, but are not limited cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, diabetic cardiomyopathy, genetic cardiomyopathy, atherosclerosis, coronary artery disease, contractility and cardiac rhythm disorders, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, acute myocardial infarction, congestive heart failure, arrhythmias, diseases resulting in remodeling of the heart, heart failure, systolic and diastolic heart failure, ischemic disease, transplantation, restenosis, angina pectoris, rheumatic heart disease, injuries-physical or otherwise, or congenital cardiovascular defects.

[0439] In some embodiments, the LNPs are effective for delivery of RNA based therapeutics to cardiomyocytes for the treatment of acute conditions. Exemplary acute conditions that can be treated include, but are not limited to, acute myocardial infarction (AMI), heart failure (HF), cardiac edema; acute myocarditis, arrhythmias, cardiogenic shock, pericarditis, and endocarditis.

[0440] In an embodiment, the term “administering” means that the compounds of the present invention are introduced into a subject using one or more known routes of administration. In some embodiments, the compositions of the invention are administrated by way of injection.

[0441] In one embodiment, one or more ApoE inhibitor of the invention is coadministered with one or more LNP delivery molecule comprising at least one therapeutic agent or adjuvant. “Co-administration” as used herein is understood as administration of one or more agents to a subject such that the agents are present and active in the subject at the same time. Coadministration does not require a preparation of an admixture of the agents or simultaneous administration of the agents.

[0442] One of skill in the art will appreciate that one or more ApoE inhibitor of the invention is co-administered with one or more LNP delivery molecule comprising at least one therapeutic agent can be administered singly or in any combination thereof. Further, one or more ApoE inhibitor of the invention is co-administered with one or more LNP delivery molecule comprising at least one therapeutic agent can be administered singly or in any combination thereof in a temporal sense, in that they may be administered simultaneously, before, and/or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that one or more ApoE inhibitor of the invention is co-administered with one or more LNP delivery molecule comprising at least one therapeutic agent can be used to prevent or treat diseases or disorders for which administration of a therapeutic agent to a cardiomyocyte would be beneficial for the prevention or treatment of the disease or disorder.

[0443] One of skill in the art, when armed with the disclosure herein, would appreciate that the prevention of a disease or disorder, encompasses administering to a subject one or more ApoE inhibitor of the invention is co-administered with one or more LNP delivery molecule comprising at least one therapeutic agent as a preventative measure against the development of, or progression of, the disease or disorder.

[0444] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the invention is not limited to treatment of diseases or disorders that are already established. Particularly, the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant signs or symptoms of diseases or disorders do not have to occur before the present invention may provide benefit. Therefore, the present invention includes a method for preventing diseases or disorders, in that a composition, as discussed previously elsewhere herein, can be administered to a subject prior to the onset of diseases or disorders, thereby preventing diseases or disorders.

[0445] In various embodiments, the invention encompasses delivery of a LNP comprising at least one agent to a subject having an ApoE deficiency or in combination with an ApoE inhibitor. In one embodiment, the delivery vehicle further comprises at least one targeting domain. To practice the methods of the invention; the skilled artisan would understand, based on the disclosure provided herein, how to formulate and administer the appropriate composition(s) to a subject. The present invention is not limited to any particular method of administration or treatment regimen.

[0446] One of skill in the art will appreciate that compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the compositions of the invention can be used to prevent or to treat a disease or disorder, and that a composition can be used alone or in any combination with another composition to affect a therapeutic result. In various embodiments, any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with diseases or disorders.

[0447] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In certain embodiments, the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.

[0448] The method comprises administering a combination of compositions in any suitable ratio. For example, in one embodiment, the method comprises administering two individual compositions at a 1 : 1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.

Pharmaceutical Compositions

[0449] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0450] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0451] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, intra-myocardial, intracoronary, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations. [0452] In some embodiments, the cLNPs of the invention are delivered by intra- myocardial injection. In some embodiments, the cLNPs of the invention are delivered through intracoronary infusion, for example, via a catheter placed in the coronary artery.

[0453] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0454] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0455] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[0456] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[0457] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0458] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0459] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0460] A pharmaceutical composition of the invention may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers. In some embodiments, the diameter is in the range from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent/powder-dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. In some embodiments, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. In some embodiments, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. In some embodiments, dry powder compositions include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0461] Low boiling propellants generally include liquid propellants having a boiling point of below 65°F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w/w) of the composition, and the active ingredient may constitute 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent. In some embodiments, the additional ingredient(s) have a particle size of the same order as particles comprising the active ingredient.

[0462] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0463] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenteral ly-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0464] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

EXPERIMENTAL EXAMPLES

[0465] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0466] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : Cardiotropic solid lipid nanoparticles

[0467] The investigations in this paper demonstrate and characterize the phenomenon of cLNP cardiotropism. The discovery of cLNPs provides momentum for design of biotherapeutics. The field of nucleoside-modified RNA/LNP therapeutics has made major advances with the production of highly effective vaccines (Baden et al., 2021, N Engl J Med 384, 403-416; Hogan et al., 2022, Annu Rev Med 73, 17-39) and other in vivo gene delivery therapeutics (Gillmore et al., 2021, N Engl J Med 385, 493-502). Without being bound by theory, it is believed that future RNA/LNP therapeutics will be enhanced by the ability to target specific cells and organs. In this report, a novel cardiac drug delivery system is presented. The findings that cLNPs/RNA gene edit CMCs in the myocardium, including knockdown of one of the most abundant proteins in CMCs, indicate potential utility of cLNPs for treatment of acquired and hereditary car di omy opathi es .

[0468] Cardiotropic LNPs may provide a novel platform for cardiac delivery of nucleic acid cargoes to achieve effective therapeutic, diagnostic, and prophylactic interventions in the heart. The envisioned utility of cLNPs is not limited to any sole cardiac pathology. Gene editing CMCs with an IV agent could help systolic and diastolic heart failure, acute myocardial infarction, ischemic disease, genetic cardiomyopathy, and myocarditis. These studies will be important for both basic and translational science.

[0469] RNA therapeutics may enable effective and precise interventions in heart diseases. Delivery of RNA requires: A) Optimization of pharmacokinetics, providing sufficient circulation time for the RNA to find and enter the target; B) Protection of the RNA from degradation; C) Effective cytosolic delivery of the RNA. Diverse DDSs have been tested for RNA delivery. Successful delivery of nucleic acids to the liver has provided proof of principle for RNA delivery and clinical application with solid lipid nanoparticles (LNPs) (Hou et al., 2021, Nat Rev Mater 6, 1078-1094). LNPs typically consist of lipid membrane components (e.g., phospholipids and cholesterol), excipients (e.g., lipid-modified polyethylene glycol, PEG), ionizable lipids enabling endosomal escape, and nucleic acid cargo (Paunovska et al., 2022, Nat Rev Genet 23, 265-280). LNPs have enabled delivery of nucleoside-modified mRNA in COVID- 19 vaccines (Baden et al., 2021, N Engl J Med 384, 403-416; Hogan et al., 2022, Annu Rev Med 73, 17-39) and siRNA in Alnylam’s Onpattro, among others (Evers et al., 2022, J Control Release 343, 207-216; Shin et al., 2018, Nat Biomed Eng 2, 304-317; Hou et al., 2021, Nat Rev Mater 6, 1078-1094; Hogan et al., 2022, Annu Rev Med 73, 17-39; Gillmore et al., 2021, N Engl J Med 385, 493-502; Mullard 2021, Nat Rev Drug Discov 20, 728; Cheraghi et al., 2017, Biomed Pharmacother 86, 316-323; Li et al., 2022, Biomaterials 284, 121529; Sullivan et al.,

2021, Biomater Sci 9, 1204-1216). Therefore, LNPs have proven to be a viable DDS in billions of patients (Hou et al., 2021, Nat Rev Mater 6, 1078-1094; Hogan et al., 2022, Annu Rev Med 73, 17-39; Mullard 2021, Nat Rev Drug Discov 20, 728).

[0470] In the blood, LNPs containing neutral ionizable lipids interact with apolipoprotein E (apoE) (Chen et al., 2019, Nanoscale 11, 18806-18824; Francia et al., 2020, Bioconjug Chem 31, 2046-2059), changing the LNP structure (Sebastiani et al., 2021, ACS Nano 15, 6709-6722) and promoting binding to lipoprotein receptors in the liver (Niemietz et al., 2020, Amyloid 27, 45-51). Intravenous LNPs thus predominantly achieve nucleic acid delivery to the liver. Study of LNP tropism to organs other than the liver has been limited, with some success in delivery to the spleen (Kranz et al., 2016, Nature 534, 396-401; Krienke et al., 2021, Science 371, 145-153) and lungs (Cheng et al., 2020, Nat Nanotechnol 15, 313-320; Liu et al., 2021, Nat Mater 20, 701- 710) based on inclusion of cationic lipids in LNP formulations. Since apoE mediates hepatic uptake of LNPs, apoE may interfere with extrahepatic delivery of LNPs (Da Silva Sanchez et al.,

2022, Nano Lett). Radiolabeled LNP/RNA formulations in WT vs Apoe-/- mice were traced to probe for enhanced extrahepatic delivery. It was found that an LNP/RNA formulation reaching cardiac uptake as high as 30-40% injected dose per gram tissue (%ID/g) in Apoe-/- mice. Studies of mRNA and siRNA delivery establish that nucleic acid delivery to CMCs is achieved with this LNP formulation. Thus investigations of the pharmacological, mechanistic, and therapeutic features and implications of cardiotropic LNPs (cLNPs) are reported. The magnitude of cardiac delivery achieved in these studies, and focal effects of delivery in CMCs, are compelling for potential translation applications. Results presented establish that cLNPs are a cardiac DDS platform providing effective and precise delivery of therapeutic RNA into the heart via systemic vascular administration.

The materials and methods used for the experiments are now described. Antibodies.

[0471] Monoclonal antibody MEC-13.3 towards murine PECAM was from BD Biosciences (San Jose, CA). Antibodies to GAPDH (clone D4C6R; product #97166), ATP2A2/SERCA2 (product #4388), P-Actin (clone 8H10D10; product #3700), anti-mouse IgG, HRP linked (product #7076), anti-rabbit IgG, HRP linked (product #7074), anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) (product #4412), anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 594 Conjugate) (product #8890) were purchased from Cell Signaling Technology. Anti-a-Actinin (Sarcomeric) antibody produced in mouse (product #A7811) was purchased from Sigma Aldrich.

Formulation of lipid nanoparticles.

[0472] mRNAs were produced as described (Pardi et al., 2013, Methods Mol Biol 969, 29-42). To make modified nucleoside-containing mRNA, ml ¥-5 '-triphosphate (TriLink) was incorporated instead of UTP. mRNAs were transcribed to contain 101 nucleotide-long poly(A) tails. They were capped using the m7G capping kit with 2'-O-methyltransferase (ScriptCap, CellScript) to obtain capl. mRNA was purified by Fast Protein Liquid Chromatography (FPLC) (Akta Purifier, GE Healthcare)(Weissman et al., 2013, Methods Mol Biol 969, 43-54). All prepared mRNAs were analyzed by electrophoresis using denaturing or native agarose gels and stored at -20°C. FPLC-purified mRNAs were encapsulated in LNPs using a self-assembly process in which an aqueous solution of mRNA at pH=4.0 is rapidly mixed with a solution of lipids dissolved in ethanol (Maier et al., 2013, Mol Ther 21, 1570-1578). Composition of LNPs used in this study contain an ionizable cationic lipid ALC0307 (Acuitas, Vancouver, BC, Canada), phosphatidylcholine, cholesterol, and PEG-lipid at molar ratio 50: 10:38.5: 1.5, respectively, and were encapsulated at an RNA to total lipid ratio of -0.05 (wt/wt). They had a diameter of -80 nm and PDI<0.1 as measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, UK) instrument. mRNA-LNP formulations were stored at -80°C at a concentration of mRNA of -1 mg/ml.

Liposome synthesis.

[0473] Liposomes were prepared as described (Ferguson et al., 2022, ACS Nano 16, 4666-4683; Hood et al., 2018, Bioconjug Chem 29, 3626-3637). For bare liposome formulation DPPC (l,2-dipalmitoyl-sn-glycero-3-phosphocholine) and cholesterol (all lipids used were purchased from Avanti Polar Lipids, Alabaster, AL) were combined at a phospholipid to cholesterol molar ratio of 3: 1 in HPLC-grade chloroform. Organic phase was evaporated; lipid fdms were rehydrated with sterile PBS and underwent three cycles of freeze/thaw between liquid N2 and a 50 °C water bath, followed by 10 extrusion cycles through 200 nm polycarbonate fdters using an Avanti Mini Extruder (Avanti Polar Lipids). PEGylated liposomes were supplemented with 6% methyl PEG k DSPE, relative to total phospholipid. Liposomes requiring ^U1ln radiolabeling contained additional 0.2 mol % DTPA-PE (l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-diethylenetriaminepentaacetic acid) and further labeled with '" In by surface chelation (Hood et al., 2018, Bioconjug Chem 29, 3626-3637). Particles were characterized using a Zetasizer Nano ZSP (Malvern Panalytical, Malvern UK). Bare liposomes were ca. 120 nm (PDI < 0.1); PEGylated liposome were 130 nm (PDI < 0.1).

Cell lines.

[0474] Human umbilical vein endothelial cells (HUVEC) were purchased at passage 1 from Lonza (Walkersville, MD) and subcultured to passage 4 in endothelial basal medium (EBM) supplemented with EGM-bulletkit (Lonza). Macrophage cell line RAW 264.7 was cultured in DMEM supplemented with 10% FCS. Rat BDIX heart myoblasts H9c2(2-1) (ATCC; product CRL-1446) were grown in DMEM supplemented with 10% FCS. HUVEC, RAW 264.7 and H9c2(2-1) cells were grown in 24-well plate on glass inserts (012 mm) coated with 1% gelatin for 5 min at 37°C.

[0475] Human induced pluripotent stem cell derived ventricular cardiomyocytes (hiPSC CMC; Ncardia; product NC-038) were proliferated and maintained on BR-CM culture medium (Ncardia) in accordance to manufacturer’s recommendations. Glass inserts (012 mm) were placed into 24-well plate and coated with 10 pg/ml fibronectin in PBS (Ca²⁺/Mg²⁺) for 3 hours at 37°C. Half a million hiPSC CMC cells were plated on 24-well plate in 0.5 ml of BR-CM culture medium. Human ventricular cardiac fibroblasts (Lonza) were maintained in fibroblast growth medium-3 (FGM-3, Lonza). All cells were cultured at 37°C and 5% CO2.

Adult Cardiomyocyte Isolation and Cell Culture. [0476] Adult rodent cardiomyocyte isolations were performed as previously described (Chen et al., 2020, Circ Res 127, el4-e27). Briefly, rats or mice were anesthetized using isoflurane. Once the heart was excised, the aorta was cannulated and coronary arteries were flushed with cell isolation buffer (CIB, 130 mM NaCl, 1 mM Sodium lactate, 5.4 mM KC1, 25 mM HEPES, 0.5 mM MgCh*6H2O, 0.33 mM NaH2PO4, 22 mM dextrose, 20 mM creatine, 10 U/L insulin) supplemented with 100 mM EGTA. The cannulated heart was connected to a Langendorff retrograde perfusion apparatus. The heart was rinsed with CIB without EGTA and then digested in a CIB containing type II collagenase (180 units/mL) and 50 pM CaCh until the heart was pale in appearance (8-12 minutes). The digested heart was minced, sheared using a transfer pipette, and centrifuged at 300 rpm for 2 minutes. The pellet was then serially resuspended in CIB containing 0.5 w/v% bovine serum albumin (BSA) and increasing concentrations of calcium (100 pM, 400 pM, 9 mM), with gravity settling between each solution. The final cardiomyocyte pellet was resuspended in adult cardiomyocyte media (Media 199 (ThermoFisher) supplemented with Primocin (InvivoGen), 25 mM HEPES, and Insulin- Transferrin-Selenium-Ethanolamide (Gibco)). The media was further supplemented with 25 pM cytochalasin D if culture for greater than 12 hours was required.

Animals.

[0477] All mice strains were purchased from The Jackson Laboratory (Farmington, CT). Black mice C57BL/6J are indicated as WT throughout the manuscript, ApoE knock-out mice B6. 129P2-Apoe^tmlUn7J on C57BL/6J background are indicated as ApoE KO or ApoE' '. In gene editing experiments mTmG reporter mice B6. 129(Cg)-Gt(ROSA)26Sor^{tm4(ACTB tdTomato EGFP)Lu}7J on C57BL/6J background with two-color fluorescent Cre-reporter allele were used. Wistar ratsRattus norvegicus) were obtained from Charles River Laboratories, Inc. (Boston, MA). ApoE KO rats were received from Envigo (Indianapolis, IN).

Tissue biodistribution and pharmacokinetics of nanoparticles

[0478] Radiolabeled with ¹²⁵I or ³H-Cholesterol LNP were injected in mice IV (1 pg of RNA or equivalent) retro-orbitally. After 30 min, blood was drawn and the internal organs (liver, lung, kidney, heart, and spleen) were harvested, rinsed with saline, blotted dry, and weighed. Tissue radioactivity in organs and 100-pl samples of blood was determined in a Wallac 1470 Wizard™ gamma counter. Alternately, tissues were homogenated and lipids were extracted with hexane :2-propanol 3:2 mixture (v/v). Hexane phase was collected, dried, extracted lipids were reconstituted with scintillation liquid and radioactivity was measured in a LS 6500 Beckman beta-counter (Wang et al., 2007, Arterioscler Thromb Vase Biol 27, 1837-1842). Radioactivity values and weight of the samples were then used to calculate tissue uptake as percent of injected dose per gram of tissue (%ZD/g) (Scherpereel et al., 2002, J Pharmacol Exp Ther 300, 777-786). In competition experiments unlabeled LNP or liposomes were pre-injected 30 minutes prior the injection of ¹²⁵I-RNA-LNP. In experiments with empty LNP lipid equivalent to 1 pg of RNA- LNP was injected. In experiments with rats 10 pg of ¹²³I-RNA-LNP were injected via tail vein. In experiments with liposomes ^luIn-Liposomes or ¹¹¹In-PEG-Liposomes were injected. In pharmacokinetics experiments animals were sacrificed at 5, 15, 30, 60, and 120 minutes postinjection. In experiments with rats anesthesia was induced with 5% isoflurane, animals were intubated and ventilated, and sedation was maintained with 3% isoflurane. Injection of ¹²⁵I-LNP was performed via tail vein and allowed to circulate for 30 minutes. Blood was collected from the inferior vena cava, and the heart, lung, liver, kidney and spleen were harvested.

Autoradiography.

[0479] Twenty pg of ¹²⁵I-LNP was injected retroorbitally in WT and ApoE KO mice. Hearts were harvested in 30 minutes, washed and fixed overnight in 4% paraformaldehyde. Fixed tissues were cut on four 2-mm slices and exposed on X-ray Film for 72 hours.

Apolipoprotein E depletion.

[0480] For transiently depletion of apoE in mice siRNA to murine ApoE was used (Dharmacon, A Horizon Discovery Group Company; ON-TARGETplus siRNA; product J- 040885-06). siRNA was complexed in LNP using Invivofectamine 3.0 Reagent (Invitrogen, #IVF3001) in accordance to manufacturer’s recommendation. Sixty pg of ApoE siRNA-LNP was injected. Level of plasma apoE was measured by Mouse Apolipoprotein E ELISA (Abeam, product ab215086).

Confocal microscopy studies of LNP accumulation in heart. [0481] I.NP were labeled with PKH26 red fluorescent dye (Sigma) and injected retroorbitally in mice. In some experiments 10 min before organ harvesting 40 pg of Alexa Fluor 647-labeled anti-PECAM monoclonal antibody (MEC13.3; BD Pharmingen) was injected for in vivo staining of blood vessels. Organs were harvested, frozen in O.C.T. Compound (Fisher Healthcare) and cryosectioned using cryostat Leica CM 1950 (Leica BioSystems, Nussloch, Germany). Cardiomyocytes were visualized with rabbit antibodies to mouse dystrophin (abeam, product #15277) and Alexa-647-labeled anti-rabbit secondary antibodies (Invitrogen, ThermoFisher). Finally, samples were mounted using ProLong Gold Antifade Reagent with DAPI (Molecular Probes, ThermoFisher). Microscopy studies were performed on a confocal laser scanning microscope Leica TCS-SP8 (Leica, Germany) using HC PL APO CS2 63x/1.40 Oil objective and 488/552/638 lasers. Images were processed using Volocity 6.3 Cellular Imaging & Analysis.

In vitro Uptake of Lipid Nanoparticles.

[0482] Adult rat cardiomyocytes, human iPS cardiomyocytes, or human cardiac fibroblasts were incubated with varying concentrations of lipid nanoparticles loaded with mRNA to GFP or labeled with PKH26 red fluorescent dye. After 12 hours, cells were washed with phosphate buffered saline (PBS) and counterstained with NucBlue (Hoechst 33342, Invitrogen). Cells were imaged on Nikon Ti-U inverted fluorescence microscope using Photometries Cool SNAP HQ². Images were analyzed for fluorescence intensity per cell relative to background using ImageJ.

SDS-PAGE and Silver Staining.

[0483] Samples were mixed with sample buffer for SDS-PAGE and then were subjected to 4-15 % gradient SDS-electrophoresis. In some experiments, gels were stained using Silver Xpress (Invitrogen, Thermo Fisher Sci., product LC6100) silver staining in accordance with manufacturer’s recommendation.

Western Blotting.

[0484] Protein, either from cells or pulverized tissues, were lysed using RIPA Buffer (CellSignal Technology) with protease inhibitor cocktail (CellSignal Technology). Protein quantity was verified using BCA Protein Assay Kit (Pierce). Protein was combined with 4x Laemmli protein sample buffer (Bio-Rad) and 2-Mercaptoethanol, and boiled for 6 minutes. Samples were run on a 4-20% Criterion TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked using the Pierce Protein Free Blocking Buffer (ThermoFisher) for one hour. Primary antibodies were shaken over the membrane overnight at 4°C. Membranes were washed three times with TBST (CellSignal Technology) and secondary antibodies were added for 1 hour prior to another 3 washes with TBST. SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher) was added to the membranes and chemiluminescence was immediately imaged using ImageQuant LAS (GE Healthcare).

Dynamic light scattering (DLS).

[0485] The effective diameter of the prepared particles was measured by DLS using Zetasizer Nano ZSP (Malvern Instruments Ltd., Malvern, UK).

Nanoparticle Tracking Analysis.

[0486] DiO-labeled LNP (Acuitas) were injected in WT or ApoE KO mice. Blood was drawn on heparin at specific times, centrifuged at 2,000 g for 15 min and plasma was used for analysis. Nanoparticle Tracking Analysis was done with NanoSight NS300 (Malvern Pananalitical, Malvern, UK). DiO-LNP size distribution and concentration were measured in NanoSight fluorescence mode to study pharmacokinetics of LNP as well as nanoparticle size enlargement in vivo.

LNP isolation from blood and proteomics analysis.

[0487] Biotin-LNP were prepared using post-insertion techniques. DSPE-PEG(2000) and DSPE-PEG(2000)-biotin (Avanti Polar Lipids, Alabaster, AL; products 880126C and 880129C) were mixed at a molar ratio 4: 1, solvent was evaporated and lipids were rehydrated in PBS at 65°C with intense vortexing to final concentration 4 mM of biotin. Size of DSPE micelles was monitored to be 14-20 nm by DLS. 1 mg/ml RNA-LNP were mixed with DSPE micelles (final concentration 0.2 mM of biotin) and insertion reaction was performed at 37°C for 3 hours. Size of LNP did not significantly changed and remained about 80 nm. Twenty pg of biotin-RNA-LNP were injected retroorbitally in mice. After the specified time 1 ml of blood was collected on heparin and centrifuged at 10,000 g for 10 minutes. Two mg of SA-Dynabeads (Dynabeads M- 280 Streptavidin, Invitrogen by ThermoFisher Sci., product 112050) were washed at least three times with 1 ml PBS, 400 pl of plasma were added to SA-Dynabeads and rotated for 30 min at room temperature. SA-Dynabeads were pooled with magnets and unbound material was washed out five times with 1 ml PBS. Bound material was lysed with 100 pl of RIP A Lysis Buffer (Upstate Cell Signaling Solutions, Lake Placid, NY), separated from dynabeads. Plasma proteins bound to LNP were analyzed by SDS-PAGE with following silver staining or by mass spectrometry analysis (Proteomics analysis).

Validation of siATP2A2.

[0488] Two siRNAs were used, a scrambled pool of non-targeting siRNAs (ON- Targetplus, Horizon Discovery Biosciences) or siRNA targeting ATP2A2. Human iPS cardiomyocytes were incubated with 25 pmol siRNA in Lipofectamine RNAiMAX (Invitrogen) for up to 48 hours. Cells were washed and used for downstream experiments. For gene expression analysis, RNA was isolated using Qiazol (Qiagen) and RNeasy Kit (Qiagen). Reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and qPCR performed using Luna Universal qPCR Master Mix (New England BioLabs) and Applied Biosystems QuantStudio 6 flex. Data were analyzed using the AACt method. For immunofluorescence, iPS cardiomyocytes were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton-X, and non-specific binding blocked with 5% BSA. Primary and secondary antibodies were incubated for 1 hour each with 3 PBS washes in between. Cells were counterstained with DAPI and imaged using Nikon Ti-U inverted fluorescence microscope. For relative contraction and calcium analysis, iPS-CMs were initially seeded on MatTek 35 mm glass bottom dishes incubated with siRNA as above. For calcium studies, cells were incubated with 1 pM Fura-2 AM (Invitrogen) for 30 minutes and then allowed to de-esterify for 30 minutes. Data were acquired using the MultiCell High Throughput System with CytoMotion software and analyzed using CytoSolver Transient Analysis Tool (lonOptix).

Delivery of LNP-si ATP2A2

[0489] Mice received retro-orbital injections of 10 pg LNP-scrambled or LNP- si ATP2A2. At 1, 3, or 5 days following injection, mice were anesthetized using isoflurane and hearts excised and weighed. Tibia lengths were measured. Hearts were halved along the short axis. Half was fixed with paraformaldehyde and subsequently embedded in paraffin and stained with hematoxylin and eosin. To determine cardiomyocyte cross-sectional area, heart sections were stained with wheat germ agglutin, Texas Red conjugate (Invitrogen) and 4’, 6- diamidino-2- phenylindole (DAPI) to visualize the surface of cell membranes and nuclei, respectively. The other half was flash frozen, and mechanically pulverized for downstream protein isolation (see below). Image analyses were performed on ImageJ. For contractility analysis, whole hearts were excised, and cardiomyocytes isolated as above. Contractility data were acquired using the MultiCell High Throughput System and analyzed using CytoSolver Transient Analysis Tool (lonOptix).

The experimental results are now described.

Demonstration of LNP cardiotropism

[0490] Intravenous (IV)-injected ¹²⁵I-labeled LNPs/RNA containing components used and described in previous studies (Parhiz et al., 2022, J Control Release 344, 50-61; Tombacz et al., 2021, Mol Ther 29, 3293-3304; Parhiz et al., 2018, J Control Release 291, 106-115) were traced. The size of these cLNPs is 78 to 80 nm, with polydispersity index (PDI) 0.06 to 0.1, and zeta potential -5 to -6 mV. Synthesis, stability, storage, PK and metabolism of this LNP formulation in normal organisms have been described (Maier et al., 2013, Mol Ther 21, 1570- 1578; Pardi et al., 2013, Methods Mol Biol 969, 29-42). Here Apoe'~ vs. wild-type (WT) mice were used. Thirty minutes after injection, the cLNPs predominantly distribute to the liver and spleen, but uptake in these organs is 3-4 fold lower in Apoe ' vs. WT mice. In other splanchnic organs, LNP uptake is minimal across both strains, with one exception: cardiac uptake of these cLNPs is an order of magnitude higher in Apoe ^J~ vs. WT mice (Fig. 1 A). Due to concomitant decrease of hepatic uptake, the heart/liver ratio (HLR) is 25-fold higher in Apoe '' mice.

[0491] Autoradiography (Fig. IB) and confocal microscopy (Fig. 1C) confirmed cardiac uptake of cLNPs in Apoe " mice. Consistent with ¹²⁵I isotope tracing data, fluorescently labeled cLNP uptake was high in the liver, marginal in the lungs, and abundant in the heart (Fig. 1 C). Co-staining for endothelial cells revealed that cLNPs accumulate in extravascular myocardial tissue more than blood vessels (Fig.lC, right panel) and 3D images confirm this finding. [0492] cLNP uptake in the Apoe ^/_ heart is significantly higher than in other tissues with high metabolic demand for nutrients: the diaphragm, skeletal muscle and brown fat (Fig. ID). However, it is noteworthy that Apoe ^/_ skeletal muscles, particularly the diaphragm, have higher cLNP uptake than their WT counterparts. Fasting has no effect on cLNP uptake in the heart (Fig. IE). Genetic ablation of other proteins involved in metabolism of lipoproteins, i.e., long-chain lipid transporter CD36 and receptor for low density lipoprotein (LDL-R), does not induce cardiotropism (Fig. IF). On the other hand, cLNPs accumulate in the heart of Apoe'¹' rats (Fig. 1G), as well as WT mice made transiently ApoE-deficient by ApoE-specific siRNA (Fig.1H). Age of Apoe' ‘ mice has no effect on LNP cardiotropism (Fig. II). These data indicate that LNP cardiotropism is not due to mild lipid pathologies that may develop in Apoe " animals, or due to factors in lipid metabolism, but rather cardiotropism is a heart-specific phenomenon requiring only the absence of a single inhibitory factor, ApoE. In vitro, cLNPs bind to several cell types, including macrophages, endothelial cells, myoblasts, and cardiomyocytes, with similar extent (Fig. 2A). The binding of LNP to cardiomyocytes in vitro is apoE-independent (Fig. 2B). Binding and GFP expression were found to be dose-dependent in vitro (Fig. 2C). These results confirm published findings showing in vitro LNP data has low correlation with in vivo results (Paunovska et al., 2018, Nano Lett 18, 2148-2157).

[0493] Therefore, cLNP cardiotropism; A) does not involve or depend on abnormalities known to develop in old Apoe '' animals; B) resists tested transient and genetic metabolic changes; C) can be studied in diverse animal models; D) is highly reproducible. These findings are favorable for translation to human patients.

Cardiotropic LNPs deliver active mRNA to cardiomyocytes

[0494] To define activity and cellular localization of cLNP cargo, testing was devised based on altering Cre recombinase activity in established mTmG reporter mice (Muzumdar et al. 2007, Genesis 45, 593-605), which contain a constitutively active Cre-LoxP -flanked tdTomato (tdTm) reporter introduced into the genome, followed by a constitutively silent GFP reporter (Fig. 3A).

[0495] To induce LNP cardiotropism in mTmG reporter mice, Apoe siRNA was injected prior to cLNPs, inducing an 85-90% reduction of ApoE blood level for at least 5 days (Fig. 3B). IV-injected cLNPs loaded with Cre recombinase-encoding mRNA (cLNP/mRNA-Cre) (Fig. 3C) blocked tdTomato production and induced GFP expression in the liver (Fig. 3D). In the heart, cLNP/mRNA-Cre induced cardiac expression of GFP in several cell types (Fig. 4), including cardiomyocytes (CMCs), as identified by dystrophin co-staining in confocal microscopy (Fig. 3E).

[0496] Confocal imaging data; A) shows that cLNPs/mRNA achieve nucleic acid delivery to CMCs; B) shows a spatially non-uniform pattern of gene modification. The latter may be due to low levels of reporter protein expression, which may or may not be sufficient for therapeutic effect. Nonetheless, the data establish that mRNA delivered by cLNPs causes protein expression in CMCs, justifying studies of therapeutic RNA delivery presented later in this article.

Cardiotropic and non-cardiotropic LNPs

[0497] Variation of lipid composition, synthesis methods, and nucleic acid cargo yield LNPs with different structures and functions (Cheng et al., 2020, Nat Nanotechnol 15, 313-320; Liu et al., 2021, Nat Mater 20, 701-710; Dillard et al., 2021, Proc Natl Acad Sci U S A 118; Johnson et al., 2022, Mol Pharm; Yu et al., 2021, Pharmaceutics 13). Some of these features may confer, modulate or inhibit cardiotropism. Poly-C RNA-loaded cLNPs and empty cLNPs have similar cardiotropism, indicating that cargo does not modulate cardiotropism (Fig. 5A). But enlarging cLNPs from ~70 to ~350nm obliterates cardiotropism (Fig. 5B).

[0498] Screening of different LNP formulations in Apoe^ vs. WT mice shows that most LNPs have no cardiotropism (Fig. 5C). Out of fifteen formulations, only two formulations other than cLNPs, namely LNP-B and LNP-E have exerted significant cardiotropism (Fig. 5, Fig. 20 and Fig. 21), achieving approximately twofold increase in cardiac uptake and ~8-fold increase in heartdiver ratio in Apoe ~ vs. WT mice. While cLNPs achieving 10-fold increase in cardiac uptake and 25-fold increase in heart diver ratio, dwarfed the values for LNP-B and LNP-E, it is plausible to suggest that fine-tuning the design of LNP-B and LNP-E could yield alternative cLNPs.

Pharmacological characteristics of cardiotropic LNPs

[0499] Dose escalation of ¹²⁵I-LNP injection leads to saturation of specific cardiac uptake in Apoe ' mice, while uptake in the liver and lungs increases linearly (Fig. 6A). LNP-specific capacity of heart tissue was estimated as 0.48 ± 0.06 pg LNP per gram (Fig. 7). Pre-injection of 50-fold excess of unlabeled (“cold”) cLNPs, but not liposomes, inhibits cardiac uptake of ^12?I- cLNPs in Apoe mice (Fig. 6B-C). ¹¹¹In-liposomes (size —130 nm) do not go to the heart, regardless of PEGylation (Fig. 6D, 7). PEGylation limits hepatic uptake and prolongs circulation of liposomes, just as ApoE depletion limits hepatic uptake and prolongs circulation of cLNPs. Thus, prolonged circulation and limited hepatic uptake alone do not yield cardiotropism.

[0500] Isotope tracing shows ¹²⁵I-cLNP cardiac uptake at a dose of 1 pg/mouse (40 pg/kg) gradually increases to a peak of -30% ID/g at 30 min (10-fold increase from level at 5 min), while cLNPs gradually clear from blood (Fig. 6E). Minimal uptake in the lungs, a representative well- perfused organ, was noted in PK studies. Comparison of tissue biodistribution using ¹²⁵I-LNP and ³H-Cholesterol-LNP show that cardiotropism is not unique to ¹²⁵I labeling, even as tissue uptake measurements can vary with tracer characteristics (Fig. 6F).

[0501] Nanoparticle Tracking Analysis (NTA; Fig. 8-10) was3 used to determine cLNP concentration and size in blood by measuring; A) Baseline pre-injection size of fluorescent cLNPs in PBS; B) The size distribution of endogenous light scattering of species in plasma; C) The size distribution and concentration of fluorescent species in plasma after fluorescent cLNP injection. The size distributions of cLNPs in PBS and in plasma closely resembled one another, and were each distinct from the size distribution of endogenous species in plasma, showing that NTA data reflect plasma concentration and size of cLNPs (Fig. 11). NTA measurements of cLNP concentration in plasma matched radiotracing data in both WT and Apoe '' mice, confirming slower clearance in Apoe~'~ mice vs. WT mice (Fig. 6G). This is consistent with the role of ApoE in hepatic uptake of LNPs, leading to fast depletion of the circulating pool (Sebastiani et al., 2021, ACS Nano 15, 6709-6722; Akinc et al., 2010, Mol Ther 18, 1357-1364; Mahley et al., 2000, Annu Rev Genomics Hum Genet 1, 507-537).

[0502] NTA assessment of LNP size in circulation showed that cLNPs undergo in vivo size transformations that differ between WT and Apoe~'~ mice. Over one hour in circulation, the modal diameter of cLNPs increases from 67 nm to 101 nm in WT mice and from 67 nm to 77 nm in d/w ⁴ mice (Fig. 6H). This result indicates differences in LNP interactions with blood components in Apoe ^ vs. WT mice.

[0503] Accumulation of cLNPs in the heart over 30 minutes and cLNP enlargement in blood over 1 hour in circulation imply that cardiotropism occurs via relatively slow processes as compared with 5-min culmination of the vascular targeting provided by ligands of endothelial surface determinants (Marcos-Contreras et al., 2020, Proc Natl Acad Sci U S A 117, 3405-3414; Chrastina et al., 2010, J Vase Res 47, 531-543; Kowalski et al., 2014, J Control Release 176, 64- 75). Biological mechanisms for cLNP cardiac uptake may involve direct interactions with a receptor or more complex processes involving cLNP interactions with blood component(s) that help transport the cLNPs to targets, possibly including poorly accessible extravascular sites following particle extravasation. ApoE may inhibit cardiotropism via; A) Boosting hepatic uptake depleting cLNPs in blood; B) Inhibiting interaction of cLNPs with hypothetical receptor or blood component(s), which re-target cLNPs to the heart in ApoE-deficient animals; C) A combination of these mechanisms. The following section addresses cLNP association with blood components in WT vs. Apoe ' mice.

Profdes of protein coronae on cLNPs in WT vs. Apoe^' animals.

[0504] Deposition of blood protein components on cLNPs in WT and Apoe^' mice was studied. First, silver staining in SDS-PAGE was used to assess cLNP -bound plasma proteins after circulation in vivo, showing differences between the proteins found on cLNPs after circulation in WT vs. Apoe~ ~ mice. Bands at ~90 kDa and ~36 kDa are more prominent on cLNPs extracted from WT mice, compared to Apoe ^ mice. A band at ~46 kDa is more prominent for cLNPs extracted from Apoe mice, compared to WT mice. A prominent band is found for both types of sample at ~28 kDa (Fig. 12).

[0505] Proteomics analysis of cLNPs after circulation in WT vs. Apoe ^ mice decodes preliminary silver staining data. Samples of circulating cLNPs were isolated from specimens of drawn blood, as above for silver staining experiments, and subjected to proteomics screening (Fig. 19). Direct quantification of the most abundant proteins on the cLNPs shows that ApoE, albumin, vitronectin, ApoAl, and serotransferrin form the bulk of the protein corona in WT mice (Fig. 13A-B, 13E; Fig. 14 and 15), while ApoB, ApoAl, and ApoA4 form the bulk of the protein corona in Apoe^ mice (Fig. l3B-C, 13E; Fig. 14 and 16). These findings fit silver staining data, where the 46 kDa band corresponds to ApoA4 found primarily on cLNPs in Apoe~ ~ mice, the 36 kDa band corresponds to ApoE found only on cLNPs in WT mice, and the 28 kDa band corresponds to ApoAl found on LNPs in both WT and Apoe ' mice. In addition to ApoE, the common serum proteins albumin, vitronectin, and serotransferrin are abundant on cLNPs in WT mice, but almost absent on cLNPs in Apoe ' mice (Fig. 13F). While the absence of ApoE is expected, it is noteworthy that cLNPs in Apoe ' mice are instead coated with ApoB and ApoA4, which are diminished on cLNPs in WT mice. On cLNPs in^oe^7- mice, ApoB, ApoA4, and ApoAl are present in equal quantities and far exceed the quantities of any other proteins (Fig. 13G, Fig. 16). Only ApoAl is found in significant quantities on cLNP in both WT and Apoe ' mice.

[0506] NTA and proteomics show that blood proteins coat cLNPs differently in WT vs. Apoe'¹' mice. The surface of opsonized cLNPs is completely redefined in Apoe vs. WT mice, with only ApoAl being shared as a surface protein between the two conditions (Fig. 13H-I, Fig. 17). Although this should be clarified by further studies, the different coronae may in theory modulate LDLR-mediated hepatic delivery vs. heart delivery (Fig. 13 J), with the ApoA4/ApoB- enriched corona in ApoE-depleted conditions, for example, allowing cLNPs to enter the myocardium (Fig. 13K). The causative relationship between protein coronae on cLNPs and cardiac uptake animals deserves future mechanistic studies.

Functional suppression of the Ca²⁺ pump SERCA2A in the heart by cLNPs/ Atp2a2-siRNA

[0507] To determine the potential utility of cLNPs as agents that alter cardiac function, cLNPs were loaded with siRNA for the gene Atp2a2, which encodes the cardiac sarcoplasmic reticulum calcium ATPase (SERCA2A), a central modulator of contractility and relaxation of cardiomyocytes (Fig. 18A-B). Atp2a2 siRNA suppressed contractility of CMCs in culture, whereas scrambled siRNA did not (Fig. 19). Based on this in vitro study, it was established that; K) Atp2a2 siRNA and scrambled siRNA as positive and negative controls, respectively; B) irrelevant nucleic acid cargo are clearly not toxic to CMCs.

[0508] A single IV injection of 10 pg Atp2a2 siRNA-cLNPs in Apoe ^/_ mice dramatically reduced SERCA2 protein levels in the heart at day 3 after dosing (Fig. 18C), while similar treatment of WT mice did not reduce SERCA2 protein levels. This was correlated with compensatory heart enlargement and a transient increase in cardiomyocyte cross-sectional area without overt myocardial damage (Fig. 18D-F). Cardiomyocytes isolated from 3 day Atp2a2 siRNA-cLNP-treated mice, but not from mice treated with scrambled siRNA-cLNPs, have reduced and slowed contractility ex vivo, as anticipated with reduction of SERCA2 expression (Fig. 18G). [0509] These data show that IV injected, RNA-cLNPs; A) enable specific and potent modulation of cardiac gene expression; B) induce functional effects in the heart consistent with targeted uptake in CMCs. Scrambled siRNA-cLNPs; A) did not change CMC contractility or cause other abnormalities in vitro, B) did not cause impairment of cardiac morphology and contractile function in vivo.

[0510] cLNPs that change gene expression in CMCs have vast potential as therapeutics. SERCA2a is a potential therapeutic target because diminished SERCA2a function leads to defects in CMC calcium cycling associated with advanced heart failure. Notably, after a positive phase I/II clinical trial of SERCA2a gene therapy using an AAV vector (Jaski et al., 2009, J Card Fail 15, 171-181), the neutral phase III study yielded a failure to increase SERCA expression in the active treatment group (Yla-Herttuala, 2015, Mol Ther 23, 1551-1552). This result was due in part to lower than expected viral uptake in the heart (Hulot et al., 2016, Eur Heart J, 37(21): 1651-8), a delivery challenge potentially addressable with cLNPs. More broadly, preclinical target-engagement studies suggest a possible therapeutic benefit of targeting a range of defects contributing to CMC dysfunction in acquired heart disease, including abnormalities in calcium cycling, beta-adrenergic signaling, cytoskeletal architecture, and metabolism. cLNP gene editing in CMCs might mitigate pathologic cardiac remodeling in the large variety of hereditary cardiomyopathies.

[0511] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A lipid nanoparticle (LNP) delivery vehicle for delivery of an agent to a cardiomyocyte comprising: a) an ionizable lipid, b) phosphatidylcholine, c) cholesterol, and d) PEG-lipid.

2. The LNP of claim 1, wherein a) comprises ALC0307.

3. The LNP of any one of claims 1 to 2, wherein a), b), c) and d) are present in a molar ratio of about 50: 10:38.5: 1.5, respectively.

4. The LNP of any one of claims 1 to 3, wherein the LNP further comprises an agent for delivery to a cardiomyocyte.

5. The LNP of claim 4, wherein the agent is selected from the group consisting of a therapeutic agent, a diagnostic agent, a gene editing agent, an imaging agent, a contrast agent, a labeling agent, and a detection agent.

6. The LNP of claim 5, wherein the therapeutic agent comprises at least one isolated nucleoside-modified RNA molecule.

7. The LNP of claim 6, wherein the at least one isolated nucleoside-modified RNA is a purified nucleoside-modified RNA.

8. The LNP of claim 5, wherein the at least one nucleoside-modified RNA is encapsulated within the LNP or incorporated into the LNP.

9. A combination therapy for delivering an agent to a cardiomyocyte of a subject in need thereof, the combination therapy comprising: a) a composition comprising a delivery vehicle comprising a cardiotropic lipid nanoparticle (cLNP), and b) a composition comprising an inhibitor of ApoE.

10. The combination therapy of claim 9, wherein the cLNP is selected from the group consisting of an LNP of any one of claims 1 to 8, LNP-B and LNP-E.

11. The combination therapy of claim 9, wherein the delivery vehicle further comprises a targeting moiety specific for binding to a cardiomyocyte.

12. The combination therapy of claim 9, wherein the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated as a single composition for co-admini strati on.

13. The combination therapy of claim 9, wherein the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated as separate compositions for co-administration.

14. The combination therapy of claim 9, wherein the composition comprising the cLNP and the composition comprising the ApoE inhibitor are formulated for sequential administration.

15. The combination therapy of claim 9, wherein at least one of the composition comprising the cLNP and the composition comprising the ApoE inhibitor further comprises an adjuvant.

16. A method of diagnosing, preventing, evaluating the progression of or treating a disease or disorder in a subject in need thereof, the method comprising administering a cLNP or a combination therapy of any one of claims 9-15 to the subject.

17. The method of claim 16, wherein the cLNP is selected from the group consisting of an LNP of any one of claims 1 to 8, LNP-B and LNP-E.

18. The method of claim 16, wherein the subject has or is at risk of a disease or disorder selected from the group consisting of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

19. The method of claim 16, wherein the method comprises delivering an agent for the diagnosis, prevention, evaluation or treatment of a disease or disorder selected from the group consisting of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder to a cardiomyocyte of the subject.

20. The method of claim 16, wherein the cLNP or combination therapy is administered by a delivery route selected from the group consisting of intravenous, intra- myocardial, subcutaneous, inhalation, intranasal, and intramuscular.

21. The method of claim 16, wherein the cLNP or combination therapy is administered by intra-myocardial injection.

22. The method of claim 16, wherein the cLNP or combination therapy is administered by intracoronary infusion.

23. The method of claim 16, wherein the composition comprising the cLNP and the composition comprising the ApoE inhibitor are co-administered.

24. The method of claim 16, wherein the composition comprising the cLNP and the composition comprising the ApoE inhibitor are administered sequentially.

25. The method of claim 16, wherein the composition comprising the ApoE inhibitor is administered prior to the composition comprising the cLNP.

26. A method of diagnosing, preventing, evaluating or treating a disease or disorder in a subject in need thereof comprising delivering a therapeutic or diagnostic agent to a cardiomyocyte of the subject, the method comprising administering a composition comprising a cLNP to the subject, wherein the subject has an ApoE deficiency.

27. The method of claim 26, wherein the cLNP is selected from the group consisting of an LNP of any one of claims 1 to 8, LNP-B and LNP-E.

28. The method of claim 26, wherein the subject has or is at risk of developing a disease or disorder selected from the group consisting of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

29. The method of claim 26, wherein the therapeutic or diagnostic agent is an agent for the diagnosis, prevention, evaluation or treatment of a disease or disorder selected from the group consisting of a cardiac disease or disorder, hypertension, metabolic disease or disorder, pulmonary disease or disorder, neurological disease or disorder, hematological disease or disorder, vascular disease or disorder, cancer, inflammatory disease or disorder, or an autoimmune disease or disorder.

30. The method of claim 26, wherein the composition is administered by a delivery route selected from the group consisting of intravenous, intra-myocardial, intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

31. The method of claim 26, wherein the LNP is administered by intra- myocardial injection.

32. The method of claim 26, wherein the LNP is administered by intracoronary infusion.