Lipid nanoparticle formulations

Abstract

The present invention relates to lipid nanoparticles, formulations comprising lipid nanoparticles, and methods for treating diseases or conditions using the lipid nanoparticles and formulations thereof. The lipid nanoparticles comprise: (a) an active agent; (b) cationic lipids and / or ionized lipids comprising about 40 mol% to about 60 mol% of the total lipids present in the nanoparticles; (c) phospholipids comprising about 5 mol% to about 20 mol% of the total lipids present in the nanoparticles; (d) structured lipids comprising about 30 mol% to about 50 mol% of the total lipids present in the nanoparticles; and (e) PEGylated lipids comprising about 0.05 mol% to less than 0.5 mol% of the total lipids present in the nanoparticles.

Description

[Technical field]

[0001] TECHNICAL FIELD OF THEINVENTION The present invention relates to lipid nanoparticles, formulations comprising lipid nanoparticles and methods of treating diseases or conditions using said lipid nanoparticles and formulations thereof.

[0002] Related Applications This application claims priority to Australian Provisional Application AU 2021902567, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] 2. Background of the Invention Effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge. In particular, delivery of nucleic acids to cells is difficult due to the relative instability and low cell permeability of such species. Thus, there is a need to develop methods and compositions that facilitate the delivery of therapeutic and / or prophylactic agents, such as nucleic acids, to cells.

[0004] Lipid-containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as transporters of biologically active substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. Such compositions generally include one or more of "cationic" and / or amino (ionized) lipids, phospholipids including polyunsaturated lipids, structured lipids (e.g., sterols), and / or lipids containing polyethylene glycol (PEG lipids). Cationic and / or ionizable lipids include, for example, amine-containing lipids that can be easily protonated.

[0005] Efforts have been made to improve the delivery efficacy of lipid nanoparticle formulations. Many of these efforts have been directed to developing more suitable cationic lipids. Despite these efforts, there is still a need for improvements in terms of improved efficacy, especially for lipid nanoparticle-based drug delivery systems intended for therapeutic use.

[0006] As a result, there is a need for new and / or improved lipid nanoparticles to increase efficacy, particularly to alter organ distribution.

[0007] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0008] In one aspect, the present invention provides a lipid nanoparticle comprising: (a) an activator; (b) cationic and / or ionized lipids comprising about 40 mol % to about 60 mol % of the total lipids present in the nanoparticles; (c) phospholipids comprising about 5 mol % to about 20 mol % of the total lipids present in the nanoparticles; (d) a structured lipid comprising about 30 mol % to about 50 mol % of the total lipid present in the nanoparticle; (e) PEGylated lipids comprising about 0.05 mol % to less than 0.5 mol % of the total lipids present in the nanoparticles.

[0009] In preferred embodiments, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent or therapeutic agent in the lipid particle is resistant to enzymatic degradation, e.g., by nucleases or proteases, in aqueous solution, hi other preferred embodiments, the lipid particle is substantially non-toxic to mammals, such as humans.

[0010] In one embodiment, the lipid nanoparticles do not include a targeting ligand that specifically binds to a molecule on the surface of a target cell. Alternatively, the lipid nanoparticles do not include a targeting ligand.

[0011] In another aspect, the present invention provides a pharmaceutical composition comprising the lipid nanoparticles of the present invention and a pharma- ceutically acceptable carrier, diluent or excipient.

[0012] In another aspect, the present invention provides a method of introducing an active agent (e.g., a nucleic acid) into a cell, preferably wherein the cell is present in vivo, the method comprising contacting the cell with a lipid nanoparticle of the present invention, thereby introducing the active agent (e.g., a nucleic acid) into the cell.

[0013] In another aspect, the present invention provides a method for in vivo delivery of an active agent, said method comprising administering lipid nanoparticles of the present invention to a subject in need thereof, thereby delivering the active agent to the subject.

[0014] In another aspect, the present invention provides a method for treating or preventing a disease or condition in a subject in need thereof, said method comprising administering to the subject a lipid nanoparticle or pharmaceutical composition of the present invention, thereby treating or preventing the disease or condition in the subject in need thereof.

[0015] In another aspect, the present invention provides the lipid nanoparticles or pharmaceutical composition of the present invention in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

[0016] In another aspect, the present invention provides the lipid nanoparticles or pharmaceutical composition of the present invention for use in the treatment or prevention of a disease or condition in a subject in need thereof.

[0017] In another aspect, the present invention provides a method for producing a polypeptide of interest in a cell, preferably a mammalian cell, said method comprising contacting a cell with a lipid nanoparticle of the present invention, wherein said active agent is an mRNA encoding a polypeptide of interest, said mRNA being capable of being translated in the cell to produce the polypeptide of interest.

[0018] In another aspect, the present invention provides a method for delivering mRNA to a cell, preferably a mammalian cell, the method comprising administering to a subject lipid nanoparticles of the present invention, wherein the active agent is mRNA, thereby delivering the mRNA to the cell.

[0019] In any aspect or embodiment, the cell is a mammalian cell.

[0020] In any aspect or embodiment, the cell is a cell located in the spleen. The cell located in the spleen can be a cell of the spleen or a cell from another part of the subject that is transported to the spleen.

[0021] In any embodiment, the lipid nanoparticles preferentially target the spleen compared to the liver. In one embodiment, the lipid nanoparticles have a spleen / liver targeting ratio of more than 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, or 14.5. In one embodiment, the lipid nanoparticles have a spleen / liver targeting ratio of about 8 or more, about 14 or more, or any other value described herein, including the examples and figures. Typically, the spleen / liver targeting ratio is determined using the assays described herein, including the examples, such as the nanoluciferase assay.

[0022] In another aspect, the present invention provides a method for mRNA delivery to a target tissue, the method comprising administering to a subject lipid nanoparticles of the present invention, wherein the active agent is mRNA, thereby delivering the mRNA to the target tissue.

[0023] Preferably, the target tissue is the mammalian spleen.

[0024] In another aspect, the present invention provides a process for producing the lipid nanoparticles of the present invention, said process comprising: mixing solutions of cationic and / or ionic lipids, phospholipids, structured lipids and PEGylated lipids to obtain the desired molar ratio; Thereby producing lipid nanoparticles.

[0025] Preferably, the process further comprises adding an active agent to the lipid mixing step. In one embodiment, the lipid nanoparticles are formed such that the lipid component to active agent is between about 5:1 and about 50:1 wt:wt ratio. In one embodiment, the final lipid concentration in the solution is between about 5.5 mM and about 50 mM, preferably diluted with ethanol.

[0026] A lipid solution can be formed by mixing solutions of cationic and / or ionizable lipids, phospholipids, structured lipids, and PEGylated lipids to produce the desired molar ratio. In one embodiment, the lipid solution is rapidly injected into a solution containing an active agent using a microfluidic system, such as a Nano-Assembler microfluidic-based system, at a flow rate of between about 0.5 ml / min to about 8 ml / min, thereby producing a suspension having a water to ethanol ratio of between about 1:1 to about 4:1, preferably 3:1.

[0027] Typically, the NP ratio (nitrogen to phosphate) is maintained between 4-7.

[0028] In one embodiment, a process for producing lipid nanoparticles of the present invention comprises one or more, or all of the steps described in the Examples herein, eg, Example 1.

[0029] As used herein, unless the context requires otherwise, the term "comprise" and variations of terms such as "comprising," "comprises," and "comprised" are not intended to exclude additional additives, ingredients, integers, or steps.

[0030] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0031] [Figure 1] Figure 1. Schematic of lipid nanoparticle generation using the NanoAssembler.

[0032] [Diagram 2] Figure 2. Particle size and polydispersity index of MIPS-LNPs. (A) Initial particle size and polydispersity index (PDI) of typical MIPS-LNPs (n=3), showing that the particles fall within the pharma- ceutically acceptable ranges of both size and dispersity. (B) Particle size distribution of MIPS-LNPs versus conventional LNPs. (C) Stability of particles produced with DMG-PEG at day 0, day 7, and month 6.

[0033] [Diagram 3]Figure 3. Splenic delivery preference of MIPS-LNP using DODAP lipid. Pie charts show proportional gene expression in tissues excluding muscle after IM injection. Outside muscle, MIPS-LNP (A) delivers >75% of the dose to the spleen (pink) and actively delivers to both lymph nodes (DLN and non-DLN), whereas outside muscle tissue, most of the dose of conventional LNP (B) enters the liver after IM injection, which is somewhat similar to the particle distribution after IV injection. Key to tissue: non-DLN-non-draining lymph nodes, DLN-draining lymph nodes, SP-spleen, KD-kidney, LG-lung, LV-liver.

[0034] [Figure 4] FIG. 4. MIPS-LNP enhances gene transfer and expression in the spleen compared to conventional LNP. (A) Intramuscular injection of MIPS-LNP formulations in mice results in ~100-fold higher gene expression levels in the spleen compared to conventional LNP (highlighted box). (n=3 or 4). Tissue key: Non-DLN-non-draining lymph node, DLN-draining lymph node, SP-spleen, KD-kidney, LG-lung, LV-liver, QM-quadriceps. (B) Intravenous injection of MIPS-LNP formulations (with DODAP) in mice results in ~500-fold higher gene expression levels in the spleen compared to conventional LNP (highlighted box). (n=2 or 3). LN1 and LN2-sample lymph nodes. (C) Analysis of splenocytes sorted after intravenous injection of MIPS-LNP displays enhanced gene expression levels compared to conventional LNP. 10 μg of DODAP-formulated nanoluciferase mRNA was injected in all experiments.

[0035] [Diagram 5] Figure 5. Schematic of the mouse model protocol for ovalbumin-targeted vaccine. Analysis was performed using a flow cytometer and FloJo software.

[0036] [Figure 6] Figure 6. MIPS-LNP induces enhanced target-specific cytotoxic T cell killing (CD8+ cell response) against ovalbumin epitopes. In vivo killing of ovalbumin-pulsed target cells by cytotoxic T cells after vaccination with ovalbumin-encoding mRNA. Data presented as percentage killing compared to non-pulsed cells. A minimum of n=3 was used for each treatment. Data show that at a dose of 10 μg, the MIPS formulation induces full cell killing activity, whereas standard (conventional) LNP does not.

[0037] [Figure 7] Figure 7. Particle size of LNPs before and after freeze-thaw cycles in various buffers with and without sucrose (Suc). Different Tris buffer compositions were used. Sucrose was also used as a protectant. Although there were no major differences between the buffers, sucrose was important for the freeze-thaw stability of the MIPS formulation.

[0038] [Figure 8]Figure 8. Nanoluciferase levels expressed as relative light units per unit mass of tissue following intravenous (A) or intramuscular (quadriceps) (B) delivery of nanoluciferase mRNA in LNP formulations containing either DSPE-PEG or DMG-PEG at either 0.15 mol% or 1.5 mol%. LNP formulations contained DLin-MC3-DMA (50 mol%), distearoylphosphatidylcholine (DSPC) (10 mol%), PEGylated lipids (0.15 mol% or 1.5 mol%), cholesterol (remainder of lipid content, i.e., 39.85 mol% or 38.5 mol%, respectively). (A) ANOVA with post hoc pairwise compassion. *p<0.05, **p<0.01; (B) ANOVA with post hoc pairwise compassion. *p<0.05, **p<0.01, dotted lines indicate data analyzed by t-test *p<0.05. Tissue key: LN1 and LN2 - sample lymph nodes, DLN - draining lymph nodes after IM injection, nDLA - non-draining lymph nodes (alternate limb), QM - quadriceps muscle.

[0039] [Figure 9] Figure 9. Statistical comparison of intramuscular data (also shown in Figure 8) for LNP formulations containing DMG-PEG. In this experiment, DLinMC3 DMA was used in the MIPS formulation.

[0040] [Figure 10] Figure 10. Tissue targeting to the spleen after intramuscular injection is shown by comparing nanoluciferase activity in the spleen and liver as a ratio. (A) Both DMG-PEG and DSPE-PEG formulations containing 0.15 mol% PEGylated lipid targeted to the spleen after IM injection. (B) Splenic targeting relative to the liver is more pronounced at lower concentrations of DMG-PEG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Detailed Description of the Embodiments Specific embodiments of the present invention will now be described in detail. While the present invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims.

[0042] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the present invention.

[0043] All patents and publications mentioned herein are incorporated by reference in their entirety.

[0044] For the purposes of this specification, terms used in the singular will also include the plural and vice versa.

[0045] Unless otherwise indicated, 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. Furthermore, any methods or materials similar or equivalent to those described herein can be used in the practice of the present invention. For purposes of the present invention, the following terms are defined:

[0046] As used herein, the terms "a," "an," or "the" include not only embodiments having one member, but also embodiments having two or more members. For example, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "the agent" includes reference to one or more agents known to those of skill in the art, and so forth. For purposes of this specification, terms used in the singular include the plural, and vice versa.

[0047] The present invention is based on the surprising development of lipid nanoparticles that result in increased transfection of cells in the spleen. This is surprising because the lipid nanoparticles do not contain targeting ligands that specifically bind to molecules on the surface of cells in the spleen (e.g., splenocytes). Furthermore, compared to conventional lipid nanoparticles, the lipid nanoparticles of the present invention do not significantly increase transfection in one or more other sites in the body (e.g., heart, liver, kidney, lung, blood, and / or lymph nodes), so the increased infection of cells is specific to the spleen. This enhanced transfection of the spleen is independent of the route of administration, as observed when the lipid nanoparticles of the present invention are administered intravenously or intramuscularly. Moreover, the advantages also provided by the lipid nanoparticles of the present invention only occur when the PEG-lipid content of the lipid nanoparticles is within a narrow mol% range of the total lipid of the nanoparticle.

[0048] This low and narrow mol% range of PEG-lipid results in nanoparticles with diameters equal to or greater than 100 nm, which is larger than the current lipid nanoparticle size range of 70-100 nm in diameter. Typically, lipid nanoparticles of the present invention are greater than 125 nm in diameter, such as 140-160 nm.

[0049] Another characteristic of the lipid nanoparticles of the present invention is their unusually high negative zeta potential.Nanoparticles with relatively low positive or negative charge are generally desirable, because more highly charged species may unnecessarily interact with cells, tissues, and other elements in the body.However, the lipid nanoparticles of the present invention have high negative zeta potential and surprisingly show increased passive targeting in the spleen.

[0050] Finally, the lipid nanoparticles of the present invention are stable for extended periods of time, for example, at least 5 months at 4°C.

[0051] Without being bound by any theory or mechanism of action, it is believed that the increased particle size enhances interaction and renewal by phagocytes and, in conjunction with the highly negative zeta potential, facilitates splenic delivery and transfection of antigen presenting cells.

[0052] While lipid nanoparticles have many uses, as described herein, enhanced transfection of cells in the spleen provides a particularly beneficial delivery vehicle for antigenic or immunogenic molecules (or molecules that induce cells to produce antigenic or immunogenic molecules) such as those found in vaccines. Splenocyte targeting is attractive for many applications, such as expression of proteins for immune checkpoint inhibition, as well as other applications in inducing antigen-specific tolerance, induction of general tolerance via targeted mRNA delivery with MIPS formulations, and in combating autoimmune diseases, reducing inflammation caused by splenocytes, or reducing allergic and anaphylactic reactions via mRNA delivery, siRNA delivery, DNA or any other means of nucleic acid or mRNA delivery using other molecules (such as small drugs incorporated into MIPS LNPs).

[0053] definition As used herein, the terms "approximately" and "about" as applied to one or more values ​​of interest refer to values ​​similar to the stated reference value. In certain embodiments, the terms "approximately" or "about" refer to a range of values ​​that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value in either direction (greater or smaller), unless otherwise stated or otherwise evident from the context (except where such value exceeds 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean ±10% of the stated value. For example, a nanoparticle composition that includes a lipid component having about 40% of a given compound may contain 30-50% of the compound.

[0054] Nanoparticles The average size of the nanoparticles of the present invention may be greater than about 100 nm, for example, as measured by dynamic light scattering (DLS). For example, the average particle size may be about 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, 150 nm, 155 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 295 nm, nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, or 500 nm.

[0055] In some embodiments, the average particle size of the nanoparticles of the present invention is about 75 nm to about 500 nm, about 80 nm to about 500 nm, about 90 nm to about 500 nm, about 100 nm to about 500 nm, about 110 nm to about 500 nm, about 120 nm to about 500 nm, about 130 nm to about 500 nm, about 140 nm to about 500 nm, about 150 nm to about 500 nm, about 160 nm to about 500 nm, about 170 nm to about 500 nm, about 180 nm to about 500 nm, about 190 nm to about 500 nm, about 200 nm to about 500 nm, about 210 nm to about 500 nm, about 220 nm to about 500 nm, about 230 nm to about 500 nm, about 240 nm to about 500 nm, about 250 nm to about 500 nm, about 260 nm to about 500 nm, about 270 nm to about 500 nm, about 280 nm to about 500 nm, about 290 nm to about 500 nm, about 300 nm to about 500 nm, about 310 nm to about 500 nm, about 320 nm to about 500 nm, about 330 nm to about 500 nm, about 340 nm to about 500 nm, about 350 nm to about 500 nm, about 360 nm to about 500 nm, about 370 nm to about 500 nm, about 380 nm to about 500 nm, about 390 nm to about 500 nm, about 400 nm to about 500 nm, about 410 nm to about 500 nm, about 420 0nm to about 500nm, about 170nm to about 500nm, about 180nm to about 500nm, about 190nm to about 500nm, about 200nm to about 500nm, about 210nm to about 500 nm, about 220nm to about 500nm, about 230nm to about 500nm, about 240nm to about 500nm, about 250nm to about 500nm, about 260nm to about 500nm, about 270n m ~ about 500nm, about 280nm - about 500nm, about 300nm - about 500nm, about 310nm - about 500nm, about 320nm - about 500nm, about 330nm - about 500nm , about 340nm to about 500nm, about 350nm to about 500nm, about 360nm to about 500nm, about 370nm to about 500nm, about 380nm to about 500nm, about 390nm to about It may be about 500 nm, about 400 nm to about 500 nm, about 410 nm to about 500 nm, about 420 nm to about 500 nm, about 430 nm to about 500 nm, about 440 nm to about 500 nm, about 450 nm to about 500 nm, about 460 nm to about 500 nm, about 470 nm to about 500 nm, about 480 nm to about 500 nm, or about 490 nm to about 500 nm.

[0056] In some embodiments, the average particle size of the nanoparticles of the present invention is about 100 nm to about 490 nm, about 100 nm to about 480 nm, about 100 nm to about 470 nm, about 100 nm to about 460 nm, about 100 nm to about 450 nm, about 100 nm to about 440 nm, about 100 nm to about 430 nm, about 100 nm to about 420 nm, about 100 nm to about 430 nm, about 100 nm to about 440 nm, about 100 nm to about 450 nm, about 100 nm to about 460 nm, about 100 nm to about 470 nm, about 100 nm to about 480 nm, about 100 nm to about 49 ... m, about 100nm to about 420nm, about 100nm to about 410nm, about 100nm to about 400nm, about 100nm to about 390nm, about 100nm to about 380nm, about 100 nm~about 370nm, about 100nm~about 360nm, about 100nm~about 350nm, about 1000nm~about 340nm, about 100nm~about 330nm, about 100nm~about 32 0nm, about 100nm to about 310nm, about 100nm to about 300nm, about 100nm to about 290nm, about 100nm to about 280nm, about 100nm to about 270nm, about 1 00nm to about 260nm, about 100nm to about 250nm, about 100nm to about 240nm, about 100nm to about 230nm, about 100nm to about 220nm, about 100nm to about 2 It may be 10 nm, about 100 nm to about 200 nm, about 100 nm to about 190 nm, about 100 nm to about 180 nm, about 100 nm to about 170 nm, about 100 nm to about 160 nm, about 100 nm to about 150 nm, about 100 nm to about 140 nm, about 100 nm to about 130 nm, about 100 nm to about 120 nm, or about 100 nm to about 110 nm.

[0057] The nanoparticles may be relatively homogeneous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, for example, the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition may have a polydispersity index of about 0 to about 0.25, such as, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition may be about 0.10 to about 0.20.

[0058] As used herein, "zeta potential" is the electrokinetic potential of, for example, lipids in a nanoparticle.

[0059] The zeta potential of a nanoparticle can be used to indicate the interfacial kinetic potential of the particle. For example, the zeta potential can represent the surface charge of the nanoparticle. Nanoparticles with a relatively low charge, positive or negative, are generally desirable because more highly charged species may have undesirable interactions with cells, tissues, and other elements in the body. However, current lipid nanoparticles have an unusually high negative zeta potential. In some embodiments, the zeta potential of the nanoparticle can be about -50 mV to about +10 mV, preferably about -10 mV to about +5 mV. In some embodiments, the zeta potential of the nanoparticle can be -50 mV to +10 mV, preferably -10 mV to +5 mV. Furthermore, in some embodiments, the zeta potential of the nanoparticle can be about -20 mV to about -5 mV, about -15 mV to about -5 mV, about -10 mV to about -5 mV, about -20 mV to about -10 mV. Further, in some embodiments, the zeta potential of the nanoparticles may be -20mV to -5mV, -15mV to -5mV, -10mV to -5mV, -20mV to -10mV. Further, in some embodiments, the zeta potential of the nanoparticles may be about -5mV, about -10MV, about -15mV, or about -20mV. Furthermore, in some embodiments, the zeta potential of the nanoparticles may be -5mV, -10mV, -15mV, or -20mV.

[0060] The lipid nanoparticles may be any one of those described herein, including those listed in the examples, such as Example 1.

[0061] Cationic and / or Ionizable Lipids Any of a variety of cationic lipids can be used in the lipid nanoparticles of the present invention.

[0062] Cationic lipids useful in the present invention can be any of a number of lipid species that have a net positive charge at physiological pH. Such lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N' ,N'-Dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-Dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-Dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-Dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3.β.N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLin ... , 2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), and mixtures thereof. Many of these lipids and related analogs are described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96 / 10390, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.In addition, numerous commercial formulations of cationic lipids are available and can be used in the present invention. These include, for example, LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and DOPE, available from GIBCO / BRL, Grand Island, NY, USA); LIPOFECTAMINE® (a commercially available cationic liposome containing DOSPA and DOPE, available from GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic liposome containing DOGS, available from Promega Corp., Madison, Wis., USA).

[0063] Additionally, cationic lipids of Formula I, having the structure: [ka] Here, R 1 and R 2 are independently selected from H or C 1 -C 3 is alkyl, R 3 and R 4 is an independently selected alkyl group having from about 10 to about 20 carbon atoms; R 3 and R 4 At least one of R contains at least two unsaturated sites. 3 and R 4 are the same for both, i.e., R 3 and R 4 Both are Linoleil (C 18 ) etc. In certain other examples, R 3 and R 4 are different, i.e., R 3 is tetradectryenyl (C 14 ) and R 4 Linoleil (C 18 In a preferred embodiment, the cationic lipid of formula I is symmetrical, i.e., R 3 and R 4 In another preferred embodiment, R 3 and R 4 Both of R contain at least two sites of unsaturation. 3 and R 4 are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In preferred embodiments, R and R are both linoleyl. In some embodiments, R 3 and R 4contains at least three unsaturated sites, for example, independently selected from dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl. In a particularly preferred embodiment, the cationic lipid of formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA).

[0064] Additionally, cationic lipids of Formula II, having the structure: [ka] Here, R 1 and R 2 are independently selected from H or C 1 -C 3 is alkyl, R 3 and R 4 is an independently selected alkyl group having from about 10 to about 20 carbon atoms; R 3 and R 4 At least one of R contains at least two unsaturated sites. 3 and R 4 are the same for both, i.e., R 3 and R 4 Both are Linoleil (C 18 ) etc. In certain other examples, R 3 and R 4 are different, i.e., R 3 is tetradectryenyl (C 14 ) and R 4 Linoleil (C 18 In a preferred embodiment, the cationic lipids of the present invention are symmetrical, i.e., R 3 and R 4 In another preferred embodiment, R 3 and R 4 Both of R contain at least two sites of unsaturation. 3 and R 4is independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. 3 and R 4 and R are both linoleyl. 3 and R 4 contains at least three sites of unsaturation and is independently selected from, for example, dodecatrienyl, tetradecatrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0065] Additionally, cationic lipids of formula III having the following structure (or a salt thereof) are useful in the present invention: [ka] Here, R 1 and R 2 are the same or different and are independently optionally substituted C 12 -C 24 Alkyl, optionally substituted C 12 -C 24 Alkenyl, optionally substituted C 12 -C 24 Alkynyl, or optionally substituted C 12 -C 24 Acyl; R 3 and R 4 are the same or different and are independently optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 alkenyl, or optionally substituted C 1 -C 5 alkynyl or R 3 and R 4 may combine to form an optionally substituted heterocycle of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is absent or hydrogen or C to provide a quaternary amine. 1 -C 6m, n and p are 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 the same or different and independently O, S or NH.

[0066] In some embodiments, the cationic lipid of formula III is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C5-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C6-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C7-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C8-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C9-DMA), 2,2-dilinoleyl-5-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-C1 ... 2,2-Dilinoleyl-4-N-methyl-[1,3]-dioxolane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methyl-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin- DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-Linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleoyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMAP), In a preferred embodiment, the cationic lipid of formula III is DLin-K-C2-DMA (XTC2).

[0067] Preferably, the cationic lipid is DODAP, DLin-DMA, DLin-K-DMA, DLin-K2-DMA or DLin-MC3-DMA.

[0068] The cationic lipid typically comprises about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 45 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipid present in the particle.

[0069] The cationic lipid typically comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol % of the total lipid present in the particle.

[0070] Phospholipids As used herein, a "phospholipid" is a lipid that includes one or more carbon chains, such as a phosphate moiety and an unsaturated fatty acid chain. A phospholipid can include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Certain phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cell membrane or an intracellular membrane). The fusion of a phospholipid to a membrane allows one or more elements of a lipid-containing composition to pass through the membrane, for example, allowing one or more elements to be delivered to a cell.

[0071] The lipid component of the lipid nanoparticle or composition may comprise one or more phospholipids, such as one or more (poly)unsaturated lipids. The phospholipids may be assembled into one or more lipid bilayers. In general, the phospholipids may comprise a phospholipid moiety and one or more fatty acid moieties. For example, the phospholipid may be a lipid according to formula (IV): [ka] This represents a phospholipid moiety, where R and R' may be the same or different and represent fatty acid moieties with or without unsaturation. The phospholipid moiety can be selected from the non-limiting group consisting of: Phosphatidylcholine, Phosphatidylethanolamine, Phosphatidylglycerol, Phosphatidylserine, Phosphatidic acid, 2-Lysophosphatidylcholine, and -Sphingomyelin.

[0072] The fatty acid moiety may be selected from the non-limiting group consisting of: Lauric acid, Myristic acid, Myristoleic acid, Palmitic acid, Palmitoleic acid, Stearic acid, Oleic acid, Linoleic acid, α-linolenic acid, Erucic acid, Phytanic acid, Arachidic acid, Arachidonic acid, Eicosapentaenoic acid, Behenic acid, Docosapentaenoic acid, Docosahexaenoic acid.

[0073] Non-natural species are also contemplated, including natural species with modifications and substitutions, including branching, oxidation, cyclization, and alkynes. For example, phospholipids may be functionalized or crosslinked with one or more alkynes (e.g., alkenyl groups with one or more double bonds replaced with triple bonds). Under appropriate reaction conditions, the alkyne groups undergo copper-catalyzed cycloaddition reactions when exposed to azides. Such reactions are useful for functionalizing the lipid bilayer of nanoparticle compositions to facilitate membrane permeation or cell recognition, or for conjugating nanoparticle compositions with useful components, such as targeting or imaging moieties (e.g., dyes).

[0074] Contemplated are, for example, lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine ... Phospholipids such as palmitoyloleoyl-phosphatidylglycerol (POPG), dioleylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably C 10 -C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0075] In some embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0076] The phospholipids typically comprise from about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 10 mol% to about 20 mol%, or about 15 mol% to about 20 mol% of the total lipids present in the particle.

[0077] The phospholipids typically comprise 5 mol% to 20 mol%, 5 mol% to 15 mol%, 5 mol% to 10 mol%, 10 mol% to 20 mol%, or 15 mol% to 20 mol% of the total lipids present in the particle.

[0078] structural lipids The lipid component of the nanoparticle composition may include one or more structured lipids. The structured lipid may be selected from the group consisting of, but is not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structured lipid is cholesterol. In some embodiments, the structured lipid includes cholesterol and a corticosteroid (e.g., prednisolone, dexamethasone, prednisone, and hydrocortisone, etc.), or a combination thereof. Additionally, the structured lipid may be squalene, squalene, or a combination thereof.

[0079] Structured lipids may include lipids containing geranyl acetate, farnesyl acetate or geranyl-geranyl, or ether, ester, or other derivatives.

[0080] The structured lipid typically comprises about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% of the total lipid present in the particle.

[0081] The structured lipid typically comprises 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 30 mol% to 35 mol%, 35 mol% to 50 mol%, 40 mol% to 50 mol%, or 45 mol% to 50 mol% of the total lipid present in the particle.

[0082] PEGylated lipids The lipid component of the lipid nanoparticle or composition can include one or more PEG or PEG-modified lipids. Such species may alternatively be referred to as PEGylated lipids. As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid that includes a polyethylene glycol moiety. The PEG lipid can be selected from the non-limiting group consisting of: ·PEG-modified phosphatidylethanolamine, ·PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamines, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and A mixture of them.

[0083] For example, the PEG lipid can be a PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipid.

[0084] In another embodiment, the PEGylated lipid may be 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, also known as DMG-PEG.

[0085] In another embodiment, the PEGylated lipid may be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0086] The PEGylated lipid may have a PEG moiety having a molecular weight of about 100 Daltons (Da) to 10,000 Da or more as desired (including but not limited to, optionally 0.1 to 10 kDa) and any molecular mass that is practically desirable. The molecular weight of the PEG may be in a wide range, including but not limited to, from about 100 Da to about 10,000 Da or more. The PEG may be from about 100 Da to about 100,000 Da, including but not limited to 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the PEG is about 100 Da to 10,000 Da, about 1000 Da to 9,000 Da, about 1000 Da to 8,000 Da, about 1000 Da to 7,000 Da, about 1000 Da to 6,000 Da, about 1000 Da to 5,000 Da, about 1000 Da to 4,000 Da, about 1000 Da to 3,000 Da, or about 1000 Da to 2,000 Da. In some embodiments, the PEG is about 1000 Da to 5000 Da. In some embodiments, the PEG is about 2,000 Da to 5,000 Da.

[0087] The PEGylated lipid typically comprises from about 0.05 mol% to about 0.5 mol%, from about 0.06 mol% to about 0.5 mol%, from about 0.07 mol% to about 0.5 mol%, from about 0.08 mol% to about 0.5 mol%, from about 0.09 mol% to about 0.5 mol%, from about 0.1 mol% to about 0.5 mol%, from about 0.15 mol% to about 0.5 mol%, from about 0.2 mol% to about 0.5 mol%, from about 0.25 mol% to about 0.5 mol%, from about 0.3 mol% to about 0.5 mol%, from about 0.3 mol% to about 0.5 mol%, from about 0.35 mol% to about 0.5 mol%, from about 0.4 mol% to about 0.5 mol%, from about 0.45 mol% to about 0.5 mol%, from about 0.05 mol% to about 0.45 mol%, from about 0.05 mol% to about 0.4 mol%, from about 0.05 mol% to about 0.35 mol%, from about 0.05 mol% to about 0.3 mol%, from about 0.05 mol% to about 0.25 mol%, from about 0.05 mol% to about 0.2 mol%, from about 0.05 mol% to about 0.15 mol%, from about 0.05 mol% to about 0.1 mol%, from about 0.05 mol% to about 0.09 mol%, from about 0.05 mol% to about 0.08 mol%, from about 0.05 mol% to about 0.07 mol%, or from about 0.05 mol% to about 0.06 mol% of the total lipids present in the particles.

[0088] The PEGylated lipid typically comprises 0.05 mol%-0.5 mol%, 0.06 mol%-0.5 mol%, 0.07 mol%-0.5 mol%, 0.08 mol%-0.5 mol%, 0.09 mol%-0.5 mol%, 0.1 mol%-0.5 mol%, 0.15 mol%-0.5 mol%, 0.2 mol%-0.5 mol%, 0.25 mol%-0.5 mol%, 0.3 mol%-0.5 mol%, 0.3 mol%-0.5 mol%, 0.35 mol%-0.5 mol%, 0.4 ... ol%, 0.45mol%~0.5mol%, 0.05mol%~0.45mol%, 0.05mol%~0.4mol%, 0.05mol%~0.35mol%, 0.05mol%~0.3mol%, 0.05mol%~0.25mol%, 0.05mol%~0 .2mol%, 0.05mol%~0.15mol%, 0.05mol%~0.1mol%, 0.05mol%~0.09mol%, 0.05mol%~0.08mol%, 0.05mol%~0.07mol%, or 0.05mol%~0.06mol%.

[0089] The PEGylated lipid typically comprises 0.05 mol%, 0.06 mol%, 0.07 mol%, 0.08 mol%, 0.09 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, or 0.45 mol% of the total lipid present in the particle.

[0090] As used herein, mole % and mol% are used interchangeably.

[0091] Activator The lipid nanoparticles can include one or more therapeutic and / or prophylactic agents. The disclosure features methods of delivering a therapeutic and / or prophylactic agent to a mammalian cell or organ, optionally producing a polypeptide of interest in a mammalian cell, and methods of treating a disease or disorder in a mammal in need thereof comprising administering to the mammal and / or contacting a mammalian cell with lipid nanoparticles that include a therapeutic and / or prophylactic agent.

[0092] Therapeutic and / or prophylactic agents include biologically active substances and are alternatively referred to as "active agents." Therapeutic and / or prophylactic agents can be substances that, once delivered to a cell or organ, effect a desired change in a cell, organ, or other body tissue or system. Such agents can be useful in the treatment of one or more diseases, disorders, or conditions. In some embodiments, the therapeutic and / or prophylactic agents are small molecule drugs useful in the treatment of a particular disease, disorder, or condition. Examples of agents useful in nanoparticle compositions include antineoplastic agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), antitumor agents, anticancer agents, anticancer drugs ... agents (e.g., actinomycin D, vincristine, vinblastine, cystine arabinoside, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs such as methotrexate and purine and pyrimidine analogs), anti-infectives, local anesthetics (e.g., dibucaine and chlorpromazine), beta-adrenergic blockers (e.g., propranolol, timolol, and labetolol), antihypertensives (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepim), anti-convulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorphenirimine, and promethazine), antibiotics / antibacterials (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungals (e.g., miconazole, terconazole, econazole, isoconazole, butaconazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitics, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma medications, vitamins, anesthetics, and contrast media.

[0093] The present invention provides novel lipid nanoparticles comprising one or more active agents, methods of making the lipid nanoparticles, and methods of delivering and / or administering the lipid nanoparticles (e.g., for the treatment of a disease or disorder).

[0094] In one aspect, the present invention provides a lipid nanoparticle comprising: (a) Activator (b) cationic and / or ionized lipids comprising about 40 mol % to about 60 mol % of the total lipids present in the nanoparticles (c) phospholipids comprising about 5 mol % to about 20 mol % of the total lipids present in the nanoparticles; (d) a structured lipid comprising about 30 mol % to about 50 mol % of the total lipid present in the nanoparticle; (e) PEGylated lipids comprising about 0.05 mol % to less than 0.5 mol % of the total lipids present in the nanoparticles.

[0095] As used herein, (b), (c), (d) and (e) may be referred to as the "lipid components."

[0096] In certain embodiments, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent in the lipid nanoparticle is resistant in aqueous solution to enzymatic degradation, e.g., by nucleases or proteases, hi certain other embodiments, the lipid nanoparticles are substantially non-toxic to mammals, such as humans.

[0097] In some embodiments, the active or therapeutic agent comprises a nucleic acid. In certain embodiments, the nucleic acid comprises an interfering RNA molecule, such as, for example, siRNA, aiRNA, miRNA, or a mixture thereof. In certain other embodiments, the nucleic acid comprises a single-stranded or double-stranded DNA, RNA, or a DNA / RNA hybrid, such as, for example, an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory oligonucleotide, or a mixture thereof.

[0098] The amount of mRNA in the lipid nanoparticles may depend on the size, sequence, and other properties of the mRNA. The amount of mRNA in the lipid nanoparticles may also depend on the size, composition, desired target, and other properties of the lipid nanoparticles. The relative amounts of mRNA and other elements (e.g., lipids) may also vary. In some embodiments, the wt / wt ratio of lipid components to mRNA in the nanoparticle composition can be about 5:1 to about 50:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the wt / wt ratio of lipid components to mRNA may be about 10:1 to about 40:1. The amount of mRNA in the nanoparticle composition can be measured, for example, using absorbance spectroscopy (eg, UV-Visible spectroscopy).

[0099] In some embodiments, the wt / wt ratio of lipid component to mRNA in the nanoparticle composition is from about 5:1 to about 50:1. In certain embodiments, the wt / wt ratio is from about 10:1 to about 40:1.

[0100] In some embodiments, the mRNA(s), lipids, and amounts thereof can be selected to provide a particular N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the lipid(s) to the number of phosphate groups in the mRNA. In general, a lower N:P ratio is preferred. The mRNA(s), lipids, and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In certain embodiments, the N:P ratio can be about 2:1 to about 5:1. In preferred embodiments, the N:P ratio can be about 4:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0101] In some embodiments, the N:P ratio of the nanoparticle composition is about 2:1 to about 8:1. In certain embodiments, the N:P ratio is about 2:1 to about 5:1. In preferred embodiments, the N:P ratio is about 4:1. In certain embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0102] In other embodiments, the active or therapeutic agent comprises a peptide or polypeptide. In certain instances, the peptide or polypeptide comprises an antibody, such as, for example, a polyclonal antibody, a monoclonal antibody, an antibody fragment; a humanized antibody, a recombinant antibody, a recombinant human antibody, a Primatized™ antibody, or a mixture thereof. In certain other embodiments, the peptide or polypeptide comprises a cytokine, a growth factor, an apoptotic factor, a differentiation inducer, a cell surface receptor, a ligand, a hormone, a small molecule (e.g., a small organic molecule or compound), or a mixture thereof.

[0103] In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. Therapeutic drug derivatives may be therapeutically active in their own right or may be prodrugs that become active upon further modification. Thus, in one embodiment, a therapeutic drug derivative retains some or all of the therapeutic activity compared to the unmodified drug, while in another embodiment, a therapeutic drug derivative is a prodrug that lacks therapeutic activity but becomes active upon further modification.

[0104] A. Nucleic acid In certain embodiments, the lipid nanoparticles of the present invention are associated with nucleic acids, resulting in nucleic acid-lipid nanoparticles (e.g., NALPs). In some embodiments, the nucleic acid is fully encapsulated in the lipid particle. As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally referred to as oligonucleotides and longer fragments referred to as polynucleotides.

[0105] In certain embodiments, the oligonucleotides of the present invention are about 15 to about 60 nucleotides in length. Nucleic acids can be administered alone in the lipid particles of the present invention or in combination (e.g., co-administration) with lipid nanoparticles of the present invention that include small molecules such as peptides, polypeptides, or conventional drugs.

[0106] In the context of the present invention, the terms "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers containing non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over natural forms due to properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0107] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose, covalently linked to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0108] The nucleic acid present in the nucleic acid-lipid nanoparticle according to the present invention includes any form of nucleic acid known. The nucleic acid used herein can be single-stranded DNA or RNA (e.g., pre-mRNA, mature mRNA, mRNA), or double-stranded DNA or RNA, or DNA-RNA hybrid. Examples of double-stranded DNA are described herein, and include, for example, structural genes, genes including control and termination regions, and self-replicating systems such as viral DNA or plasmid DNA. Examples of double-stranded RNA are described herein, and include, for example, siRNA, and other RNAi agents such as aiRNA and pre-miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.

[0109] The mRNA contained in or encapsulated by the lipid nanoparticles of the present invention can encode a polypeptide of interest. Preferably, the mRNA can be translated in the cell to produce the polypeptide of interest. The polypeptide of interest can be an antigenic or immunogenic polypeptide, such as those used to stimulate the humoral (e.g., B-cell or T-cell) immune system. The polypeptide of interest can be useful for therapeutic or prophylactic immunization of a mammal, preferably a human. The polypeptide of interest can be useful for providing a therapeutic or prophylactic effect against a disease or condition, preferably, the condition being an infectious disease. The infectious disease can be due to any microorganism, such as a bacterium, a virus, a fungus, or a protozoan. The virus can be any virus, including, but not limited to, a coronavirus, preferably, SARS-CoV or SARS-CoV-2.

[0110] The nucleic acids of the invention may be of various lengths, generally depending on the particular form of the nucleic acid. For example, in certain embodiments, a plasmid or gene may range in length from about 100 to about 100,000 nucleotide residues. In certain embodiments, an oligonucleotide may be from about 10 to about 100 nucleotides in length. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides may range in length from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides.

[0111] In certain embodiments, the oligonucleotide (or a strand thereof) of the present invention specifically hybridizes or is complementary to a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. It is understood that an oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In a preferred embodiment, an oligonucleotide is specifically hybridizable and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., under physiological conditions for in vivo assays or therapeutic treatments, or in vitro assays, under conditions where the assay is performed, if the binding of the oligonucleotide to the target sequence would disrupt the normal function of the target sequence, thereby causing loss of utility or expression. Thus, an oligonucleotide can contain one, two, three, or more base substitutions compared to the region of the gene or mRNA sequence to which it is targeted or to which it specifically hybridizes.

[0112] 1. siRNA The siRNA component of the nucleic acid-lipid particles of the present invention is capable of silencing the expression of a target gene of interest. Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. Modified siRNAs are generally less immunostimulatory than the corresponding unmodified siRNA sequences and retain RNAi activity against a target gene of interest. In some embodiments, the modified siRNA comprises at least one 2'OMe purine or pyrimidine nucleotide, such as a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and / or 2'OMe-cytosine nucleotide. In preferred embodiments, one or more uridine and / or guanosine nucleotides are modified. The modified nucleotides may be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequence may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nyilnen et al., Cell, 107:309 (2001)) or may lack overhangs (i.e., have blunt ends).

[0113] Modified siRNAs generally comprise about 1% to about 100% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in the double-stranded region of the siRNA duplex. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleotides in the double-stranded region of the siRNA comprise modified nucleotides.

[0114] In some embodiments, less than about 25% (e.g., less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the nucleotides in the double-stranded region of the siRNA comprise modified nucleotides.

[0115] In other embodiments, the double-stranded region of the siRNA is about 1% to about 25% (e.g., about 1% to 25%, 2% to 25%, 3% to 25%, 4% to 25%, 5% to 25%, 6% to 25%, 7% to 25%, 8% to 25%, 9% to 25%, 10% to 25%, 11% to 25%, 12% to 25%, 13% to 25%, 14% to 25%, 15% to 25%, 16% to 25%, 17% to 25%, 18% to 25%, 19% to 25%, 20% to 25%, 21% to 25%, 22% to 25%, 23% to 25%, 24% to 25%, etc.), or about 1% to about 20% (e.g., about 1% to 20%, 2% to 20%, 3%~20%, 4%~20%, 5%~20%, 6%~20%, 7%~20%, 8%~20%, 9%~20%, 10%~20%, 11%~20%, 12%~20%, 13%~20%, 14%~20%, 15%~20%, 16%~20%, 17%~20%, 18%~20%, 19%~20%, 1%~19%, 2%~19%, 3%~19%, 4%~19%, 5%~19%, 6%~19%, 7%~19%, 8%~19%, 9%~19%, 10%~19%, 11%~19%, 12%~19%, 13%~19%, 14%~19%, 15%~19%, 16%~19 %, 17%~19%, 18%~19%, 1%~18%, 2%~18%, 3%~18%, 4%~18%, 5%~18%, 6%~18%, 7%~18%, 8%~18%, 9%~18%, 10%~18%, 11%~18%, 12%~18%, 13%~18%, 14%~18%, 15%~ 18%, 16%~18%, 17%~18%, 1%~17%, 2%~17%, 3%~17%, 4%~17%, 5%~17%, 6%~17%, 7%~17%, 8%~17%, 9%~17%, 10%~17%, 11%~17%, 12%~17%, 13%~17%, 14%~17%, 15 %-17%, 16%-17%, 1%-16%, 2%-16%, 3%-16%, 4%-16%, 5%-16%, 6%-16%, 7%-16%, 8%-16%, 9%-16%, 10%-16%, 11%-16%, 12%-16%, 13%-16%, 14%-16%, 15%-16%, 1%-15%, 2%-15%, 3%-15%, 4%-15%, 5%-15%, 6%-15%, 7%-15%, 8%-15%, 9%-15%, 10%-15%, 11%-15%, 12%-15%, 13%-15%, 14%-15%, etc.) of the nucleotides include modified nucleotides.

[0116] In further embodiments, for example, when one or both strands of the siRNA are selectively modified with uridine and / or guanosine nucleotides, the resulting modified siRNA contains less than about 30% modified nucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% modified nucleotides), or between about 1% and about 30% modified nucleotides. modified nucleotides) (e.g., about 1% to 30%, 2% to 30%, 3% to 30%, 4% to 30%, 5% to 30%, 6% to 30%, 7% to 30%, 8% to 30%, 9% to 30%, 10% to 30%, 11% to 30%, 12% to 30%, 13% to 30%, 14% to 30%, 15% to 30%, 16% to 30%, 17% to 30%, 18% to 30%, 19% to 30%, 20% to 30%, 21% to 30%, 22% to 30%, 23% to 30%, 24% to 30%, 25% to 30%, 26% to 30%, 27% to 30%, 28% to 30%, or 29% to 30% modified nucleotides).

[0117] a. Selection of siRNA sequences Suitable siRNA sequences can be identified by any means known in the art.Typically, the method described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) is used in conjunction with the rational design rules described in Reynolds et al., Nature Biotech., 22(3):326-330 (2004).

[0118] Generally, the nucleotide sequence of the AUG start codon 3' of the transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, where N=C, G, or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). The nucleotides immediately 3' of the dinucleotide sequence are identified as potential siRNA sequences (i.e., target or sense strand sequences). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35, or more nucleotides immediately 3' of the dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and the 19 nucleotides immediately 3' of the AA or NA dinucleotide are identified as potential siRNA sequences. The siRNA sequences are usually spaced at different positions along the length of the target gene. To further enhance the silencing efficiency of the siRNA sequence, the potential siRNA sequence can be analyzed to identify sites that do not contain regions of homology with other coding sequences in, for example, a target cell or organism. For example, a suitable siRNA sequence of about 21 base pairs typically does not have more than 16-17 contiguous base pairs of homology with coding sequences in the target cell or organism. If the siRNA sequence is expressed from an RNA Pol III promoter, an siRNA sequence that lacks more than 4 contiguous A's or T's is selected.

[0119] Once potential siRNA sequences are identified, complementary sequences (i.e., antisense strand sequences) can be designed. Potential siRNA sequences can also be analyzed using various criteria known in the art. For example, siRNA sequences can be analyzed by rational design algorithms to enhance their silencing efficiency to identify sequences with one or more of the following features: (1) about 25% to about 60% G / C content, (2) at least three A / Us at positions 15-19 of the sense strand, (3) no internal repeats, (4) A at position 19 of the sense strand, (5) A at position 3 of the sense strand, (6) U at position 10 of the sense strand, (7) no G / C at position 19 of the sense strand, (8) no G at position 13 of the sense strand. siRNA design tools that incorporate algorithms that assign appropriate values ​​for each of these features and are useful for selecting siRNAs can be found, for example, at http: / / boz094.ust.hk / RNAi / siRNA. One of skill in the art will appreciate that sequences that possess one or more of the aforementioned properties may be selected for further analysis and testing as potential siRNA sequences.

[0120] Furthermore, potential siRNA sequences having one or more of the following criteria can often be excluded as siRNAs: (1) sequences containing a stretch of four or more identical bases in a row; (2) sequences containing homopolymers of G (i.e., to reduce non-specific effects that may result from the structural features of these polymers); (3) sequences containing triple base motifs (e.g., GGG, CCC, AAA, or ITT); (4) sequences containing a stretch of seven or more G / C in a row; and (5) sequences containing direct repeats of four or more bases within the candidate resulting in internal fold-back structures. However, those skilled in the art will appreciate that sequences having one or more of the aforementioned characteristics may still be selected for further analysis and testing as potential siRNA sequences.

[0121] In some embodiments, potential siRNA sequences can be further analyzed based on siRNA duplex asymmetry, e.g., as described in Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003). In other embodiments, potential siRNA sequences can be further analyzed based on secondary structure at the target site, e.g., as described in Luo et al., Biophys.Res.Commun., 318:303-310 (2004). For example, secondary structure at the target site can be modeled using the Mfold algorithm (available at http: / / www.bioinfo.rpi.edu / applications / mfold / rna / forml.cgi) to select siRNA sequences that favor accessibility at the target site with less base-pairing or stem-loop shaped secondary structure.

[0122] Once a potential siRNA sequence is identified, the sequence can be analyzed for any immunostimulatory properties, for example, using in vitro cytokine assays or in vivo animal models. Motifs in the sense and / or antisense strands of the siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.), can also provide an indication of whether the sequence is immunostimulatory. Once an siRNA molecule is found to be immunostimulatory, it can be modified to reduce its immunostimulatory properties as described herein. As a non-limiting example, the siRNA sequence can be contacted with a mammalian responder cell under conditions such that the cell produces a detectable immune response to determine whether the siRNA is an immunostimulatory siRNA or a non-immunostimulatory siRNA. The mammalian responder cell can be from a naive mammal (i.e., a mammal that has not previously been contacted with the gene product of the siRNA sequence). The mammalian responder cells may be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. The detectable immune response may include, for example, the production of cytokines or growth factors, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or combinations thereof. The siRNA molecule identified as being immunostimulatory may then be modified to reduce its immunostimulatory properties by replacing nucleotides on at least one sense and / or antisense strand with modified nucleotides. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded region of the siRNA duplex may be replaced with modified nucleotides, such as 2'OMe nucleotides. The modified siRNA may then be contacted with a mammalian responder cell as described above to confirm that its immunostimulatory properties have been reduced or abrogated.

[0123] Suitable in vitro assays for detecting immune responses include the double monoclonal antibody sandwich immunoassay of David et al. (U.S. Pat. No. 4,376,110); the monoclonal-polyclonal antibody sandwich assay (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the "Western blot" method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligands (Brown et al., J. Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays (ELISAs), e.g., as described by Raines et al., J. Biol. Chem., 257:5154-5160 (1982); immunocytochemical techniques involving the use of fluorescent dyes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980)); and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad. Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassays mentioned above, numerous other immunoassays are available, including those described in U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0124] Non-limiting examples of in vivo models for detecting immune responses include assays such as those described in Judge et al., Mol. Ther., 13:494-505 (2006). In certain embodiments, the assays can be performed as follows: (1) siRNA can be administered by standard intravenous injection into the lateral tail vein; (2) blood can be collected by cardiac puncture approximately 6 hours after administration and processed as plasma for cytokine analysis; and (3) cytokines can be quantified using sandwich ELISA kits according to the manufacturer's instructions (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-α (eBioscience; San Diego, Calif.); and mouse IL-6, TNF-α and IFN-γ (BD Biosciences; San Diego, Calif.).

[0125] Monoclonal antibodies that specifically bind cytokines and growth factors are commercially available from several sources and can be made using methods known in the art (see, e.g., Kohler et al., Nature, 256:495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). The production of monoclonal antibodies has been previously described and can be accomplished by any means known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, the monoclonal antibodies are labeled for ease of detection (e.g., with any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means).

[0126] b. Creation of siRNA molecules siRNAs can be provided in several forms, including, for example, as one or more isolated small-interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid, etc. The siRNA sequence may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or may lack overhangs (i.e., have blunt ends).

[0127] A population of RNA can be used to provide long precursor RNAs, or long precursor RNAs with substantial or complete identity to a selected target sequence can be used to make siRNA. RNAs can be isolated from cells or tissues, synthesized, and / or cloned according to methods well known to those of skill in the art. RNA can be a mixed population (obtained from cells or tissues, transcribed from cDNA, subtracted, selected, etc.) or can represent a single target sequence. RNA can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or cloned cDNA), or chemically synthesized.

[0128] For synthetic RNA, to form long dsRNA, the complement is also transcribed in vitro and hybridized to form dsRNA. When using naturally occurring RNA population, the complement of RNA is also provided by transcribing cDNA corresponding to the RNA population or by using RNA polymerase (e.g., to form dsRNA for digestion by E.coli RNAse III or Dicer). Then, the RNA precursor is hybridized to form double-stranded RNA for digestion. dsRNAs can be directly administered to a subject or can be digested in vitro before administration.

[0129] Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990)). Expression libraries are also well known to those of skill in the art. Additional basic texts disclosing the general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed., 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0130] Preferably, siRNA is chemically synthesized. Oligonucleotides, including siRNA molecules of the present invention, can be synthesized using any of a variety of techniques known in the art, such as, for example, Usman et al., J.Am.Chem.Soc., 109:7845 (1987); Scaringe et al., Nucl.Acids Res., 18:5433 (1990); Wincott et al., Nucl.Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol.Bio., 74:59 (1997). Synthesis of oligonucleotides utilizes common nucleic acid protecting and coupling groups, such as, for example, dimethoxytrityl at the 5' end and phosphoramidite at the 3' end. As a non-limiting example, small-scale synthesis can be carried out on an Applied Biosystems synthesizer using a 0.2 μmol scale protocol. Alternatively, synthesis on a 0.2 μmol scale can be performed on a Protogene (Palo Alto, Calif.) 96-well plate synthesizer. However, larger or smaller scale syntheses are also within the scope of the present invention. Reagents suitable for synthesis of oligonucleotides, methods of RNA deprotection, and methods of RNA purification are also known to those of skill in the art.

[0131] siRNA molecules can also be synthesized by a tandem synthesis technique, where both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker, which is then cleaved to provide separate fragments or strands that hybridize to form the siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA can be easily adapted to both multi-well / multi-plate synthesis platforms and large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, siRNA molecules can be assembled from two, different oligonucleotides, one oligonucleotide containing the sense strand of the siRNA and the other containing the antisense strand. For example, each strand can be synthesized separately and joined by hybridization or ligation after synthesis and / or deprotection. In certain other examples, siRNA molecules can be synthesized as a single continuous oligonucleotide fragment, where the self-complementary sense and antisense regions hybridize to form an siRNA duplex with a hairpin secondary structure.

[0132] c. Modification of siRNA sequence In certain aspects, the siRNA molecule comprises a duplex having two strands and at least one modified nucleotide in the duplex region, each strand being about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, but retains the ability to silence the expression of the target sequence. In a preferred embodiment, the degree of chemical modification introduced into the siRNA molecule strikes a balance between reducing or eliminating the immunostimulatory properties of the siRNA and retaining RNAi activity. As a non-limiting example, an siRNA molecule targeting a gene of interest can be minimally modified (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5% modified) at selective uridine and / or guanosine nucleotides within the siRNA duplex to eliminate the immune response generated by the siRNA while retaining the ability to silence the expression of its target gene.

[0133] Examples of modified nucleotides suitable for use in the present invention include, but are not limited to, ribonucleotides with 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl groups.Modified nucleotides with northern conformation, such as those described in Saenger, Principles of Nucleic Acid Structure, edited by Springer-Verlag, (1984), are also suitable for use in siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thioethyl nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides. In certain embodiments, the siRNA molecules described herein contain one or more G-clamp nucleotides. G-clamp nucleotide refers to a modified cytosine analogue, where the modification confers the ability to hydrogen bond with both the Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J Am. Chem. Soc., 120:8531-8532 (1998)). Additionally, nucleotides having nucleotide base analogs such as, for example, C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives, e.g., 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)), can be incorporated into siRNA molecules.

[0134] In certain embodiments, the siRNA molecule can further comprise one or more chemical modifications, such as terminal cap moieties, phosphate backbone modifications, etc. Examples of terminal cap moieties include, but are not limited to, inverted deoxy abasic residues, glyceryl modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic ... moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 5'-5'-inverted nucleotide moieties, 5'-5'-inverted abasic moieties, 3'-5'-inverted deoxy abasic moieties moieties), 5'-aminoalkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and bridged or unbridged methylphosphonate or 5'-mercapto moieties (see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron 49:1925 (1993)).Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the 5'-end and / or 3'-end of the sense strand, the antisense strand, or both strands of the siRNA. The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0135] In some embodiments, the sense and / or antisense strands of the siRNA molecule may further comprise a 3'-terminal overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Additional examples of the types of modified nucleotides and chemical modifications that can be introduced into the siRNA molecule are described, for example, in British Patent No. GB ​​2,397,818 B and U.S. Patent Publication Nos. 20040192626, 20050282188, and 20070135372, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0136] The siRNA molecules described herein may optionally contain one or more non-nucleotides in one or both strands of siRNA.As used herein, the term "non-nucleotide" refers to any group or compound that can be incorporated into nucleic acid strand in place of one or more nucleotide units, including sugar and / or phosphate substitution, and allows the remaining bases to exhibit their activity.The group or compound is non-basic in that it does not contain a commonly recognized nucleotide base, such as adenosine, guanine, cytosine, uracil, or thymine, and therefore lacks a base at 1' position.

[0137] In other embodiments, chemical modification of siRNA comprises attaching a conjugate to siRNA molecule. The conjugate can be attached to the 5' and / or 3' end of the sense strand and / or antisense strand of siRNA via a covalent bond, such as a biodegradable linker. The conjugate can also be attached to siRNA via, for example, a carbamate group or other linking group (see, for example, US Patent Publication Nos. 25000074771, 20050043219, and 20050158727). In certain embodiments, the conjugate is a molecule that promotes the delivery of siRNA into cells. Examples of conjugate molecules suitable for attachment to siRNA include, but are not limited to, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Publication Nos. 20030130186, 20040110296, and 20040249178; U.S. Patent No. 6,753,429). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleaving agents, and crosslinker conjugates described in U.S. Patent Publication Nos. 20050119470 and 20050107325. Still other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationically modified pyrimidines, cationic peptides, guanidinium groups, amidinium groups, cationic amino acid conjugates described in U.S. Patent Publication No. 20050153337.Additional examples include hydrophobic groups, membrane active compounds, cell permeable compounds, cell targeting signals, interaction modifiers, and steric stabilizer conjugate molecules described in US Patent Publication No. 20040167090. Further examples include conjugate molecules described in US Patent Publication No. 20050239739. The type of conjugate used and the degree of conjugation to the siRNA molecule can be evaluated for improving the pharmacokinetic profile, bioavailability, and / or stability of the siRNA while retaining RNAi activity. Thus, one skilled in the art can screen siRNA molecules with various conjugates attached to identify those with improved properties and full RNAi activity using any of a variety of well-known in vitro cell culture or in vivo animal models. The disclosures of the above-mentioned patent documents are incorporated herein by reference in their entirety for all purposes.

[0138] D. Target gene The siRNA component of the nucleic acid-lipid particles described herein can be used to downregulate or silence the translation (i.e., expression) of a gene of interest, including, but not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenic genes, immunomodulatory genes, such as those associated with inflammation and autoimmune responses, and the like.

[0139] Genes associated with metabolic diseases and disorders (e.g., disorders in which the liver is a target and liver diseases and disorders) include, for example, Genes expressed in dyslipidemia (e.g., liver X receptors such as LXRα and LXRβ (Genbank Accession No. NM_007121), farnesoid X receptor (FXR) (Genbank Accession No. NM_005123), sterol regulatory element binding protein (SREBP), site 1 protease (SIP), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB) (Genbank Accession No. NM_000384), apolipoprotein CIII (ApoC3) (Genbank Accession Nos. NM_000040 and NG_008949 REGION:5001.8164), and apolipoprotein E (ApoE) (Genbank Accession Nos. NM_000041 and NG_007084) were identified. REGION:5001.8612); as well as genes expressed in diabetes (e.g., glucose 6-phosphatase) (e.g., Forman et al., Cell, 81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995); Zavacki et al., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J.Biol.Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731 (1996); and Peet et al., Cell, 93:693-704 (1998). Those skilled in the art will appreciate that genes associated with metabolic diseases and disorders (e.g., diseases and disorders in which the liver is a target, and liver diseases and disorders) include genes expressed in the liver itself and genes expressed in other organs and tissues. Silencing of sequences encoding genes associated with metabolic diseases and disorders can be advantageously used in combination with the administration of conventional drugs used to treat diseases or disorders.Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent Publication No. 20060134189, the disclosure of which is incorporated herein by reference in its entirety for all purposes.Non-limiting examples of siRNA molecules targeting the ApoC3 gene include those described in U.S. Provisional Application No. 61 / 147,235, filed January 26, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0140] Examples of gene sequences associated with oncogenesis and cell transformation (e.g., cancer or other neoplasia) include mitotic kinesins such as Eg5 (KSP, KIF11; Genbank Accession No. NM_004523); serine / threonine kinases such as polo-like kinase 1 (PLK-1) (Genbank Accession No. NM_005030; Barr et al., Nat. Cell Biol. 5:429-440 (2004)); tyrosine kinases such as WEE1 (Genbank Accession Nos. NM_003390 and NM_001143976); apoptosis inhibitors such as XIAP (Genbank Accession No. NM_001167); COP9 signalsome subunits such as COP1 (RFWD2; Genbank Accession Nos. NM_022457 and NM_001001740); ubiquitin ligases such as COP1, HDAC2 (Genbank Accession Nos. NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, FIDAC8, HDAC9, and the like. Non-limiting examples of siRNA molecules targeting Eg5 and XIAP genes include those described in U.S. Patent Application Serial No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Publication Nos. 20050107316 and 20070265438; and U.S. Patent Application Serial No. 12 / 343,342, filed December 23, 2008, the disclosures of which are incorporated by reference in their entireties for all purposes.Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the disclosure of which is incorporated by reference in its entirety herein for all purposes.

[0141] Further examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences such as MLL fusion genes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al., Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003)); multidrug resistance genes (Nieth et al., FEBS Lett, 545:144 (2003); Wu et al., Cancer Res., 63:1515 (2003)), cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes, 2003, 10:1011 (2003); Dev., 16:2923 (2002)), β-catenin (Verma et al., Clin Cancer Res., 9:1291 (2003)), telomerase gene (Kosciolek et al., Mol Cancer Ther., Mol Cancer Ther., 2:209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank Accession Nos. NM_005228, NM_201282, NM_201283, and NM_201284; see also Nagy et al., Exp. Cell Res., 285:39-49 (2003)), ErbB2 / HER-2 (Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (Genbank Accession Nos. NM_00200571 and NM_00100571), and the HER2 / HER-2 gene (Genbank Accession Nos. NM_00100571 and NM_00100571). and overexpressed sequences such as EGFR (Genbank Accession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank Accession Nos. NM_005235 and NM_001042599); and mutated sequences such as RAS (reviewed in Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Non-limiting examples of siRNA molecules targeting the EGFR gene include those described in U.S. Patent Application Serial No. 11 / 807,872, filed May 29, 2007, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0142] Silencing of sequences encoding DNA repair enzymes is used in combination with the administration of chemotherapeutic agents (Collis et al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences of interest, such as integrins, selectins, and metalloproteases. The foregoing examples are not exclusive. Those skilled in the art will appreciate that the whole or part of any gene sequence that facilitates or promotes tumor formation or cell transformation, tumor growth, or tumor migration may be included as a template sequence.

[0143] Angiogenic genes can promote the formation of new blood vessels. Of particular interest is vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis., 9:210 (2003)) or VEGFR. siRNA sequences targeting VEGFR are described, for example, in GB 2396864; U.S. Patent Publication No. 20040142895; and CA 2456444, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0144] Antiangiogenic genes can inhibit neovascularization. These genes are particularly useful for treating cancers in which angiogenesis plays a role in the pathological progression of the disease. Examples of antiangiogenic genes include, but are not limited to, endostatin (see, e.g., U.S. Pat. No. 6,174,861), angiostatin (see, e.g., U.S. Pat. No. 5,639,725), and VEGFR2 (see, e.g., Decaussin et al., J. Pathol., 188:369-377 (1999)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0145] Immunomodulatory genes are genes that modulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to, growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fas ligand genes are also immunomodulatory target sequences of interest (Song et al., Nat. Med., 9:347 (2003)). Genes encoding secondary signaling molecules in hematopoietic and lymphoid cells are also included in the present invention, for example, Tec family kinases such as Bruton's tyrosine kinase (Btk) (Heinonen et al., FESS Lett., 527:274 (2002)).

[0146] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and modulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., regulation of glucose levels, blood cell development, mitosis, etc.) Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, and the like. Templates encoding expansions of trinucleotide repeats (e.g., CAG repeats) are used to silence pathogenic sequences in neurodegenerative disorders caused by trinucleotide repeat expansions, such as spinobulbular muscular atrophy or Huntington's disease (Caplen et al., Hum. Mol. Genet., 11:175 (2002)).

[0147] In addition to being useful in silencing the expression of any of the above-mentioned genes for therapeutic purposes, the siRNA described herein is also useful for research and development applications, as well as diagnostic, preventive, prognostic, clinical and other health care applications.As a non-limiting example, siRNA can be used in target validation studies, which are aimed at testing whether a gene of interest has the potential to be a therapeutic target.siRNA can also be used in target identification studies, which are aimed at discovering genes as potential therapeutic targets.

[0148] 2.aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can result in effective silencing of various genes in mammalian cells by employing the RNA-induced silencing complex (RISC) to mediate sequence-specific cleavage of the target sequence between nucleotides 10 and 11 relative to the 5' end of the antisense strand (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNA duplex having a sense strand and an antisense strand, the duplex comprising overhangs at the 3' and 5' ends of the antisense strand. Because the sense strand is shorter at both ends compared to the complementary antisense strand, aiRNA is generally asymmetric. In some embodiments, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences may be selected and generated using the methods described above for selecting siRNA sequences.

[0149] In another embodiment, aiRNA duplexes of various lengths (e.g., about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) can be designed with overhangs at the 3' and 5' ends of the antisense strand to target the mRNA of interest. In certain examples, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain other instances, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length.

[0150] In some embodiments, the 5' antisense overhang comprises one, two, three, four, or more non-target nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3' antisense overhang comprises one, two, three, four, or more non-target nucleotides (e.g., "AA", "UU", "dTdT", etc.). In certain aspects, the aiRNA molecules described herein can comprise one or more modified nucleotides, for example, in the double-stranded (duplex) region and / or the antisense overhang. As non-limiting examples, the aiRNA sequence may comprise one or more modified nucleotides as described above with respect to the siRNA sequence. In preferred embodiments, the aiRNA molecule comprises 2'OMe nucleotides, such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0151] In certain embodiments, the aiRNA molecule can comprise an antisense strand of an siRNA molecule, e.g., an antisense strand that corresponds to the antisense strand of one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule can be used to silence the expression of any of the target genes defined above, such as, for example, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immune regulatory genes such as genes associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0152] 3.miRNA In general, microRNAs (miRNAs) are single-stranded RNA molecules of about 21-23 nucleotides in length that control gene expression. Although miRNAs are encoded by genes that are transcribed from DNA, miRNAs are not translated into proteins (non-coding RNA); instead, each primary transcript (a pri-miRNA) is processed into a short stem-loop structure called a pre-miRNA, and finally into a functional mature miRNA. Mature miRNA molecules are partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression. Identification of miRNA molecules is described, for example, in Lagos-Quintana et al., Science, 294:853-858; Lau et al., Science, 294:858-862; and Lee et al., Science, 294:862-864.

[0153] Genes encoding miRNAs are much longer than the processed mature miRNA molecules. miRNAs are initially transcribed as primary transcripts or pri-miRNAs with a cap and polyA tail, and are expressed in the cell nucleus as short, ~70 nucleotide (~100 nucleotide) miRNAs known as pre-miRNAs. ~Pre-miRNAs are then processed into a 70-nucleotide (70-nucleotide) stem-loop structure. In animals, this processing is carried out by a protein complex known as the microprocessor complex, which consists of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These pre-miRNAs are then processed into mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001)). Either the sense or antisense strand of DNA can serve as a template to generate miRNAs.

[0154] When Dicer cleaves the stem loop of the pre-miRNA, two complementary short RNA molecules are formed, but only one is incorporated into the RISC complex. This strand, known as the guide strand, is selected by the Argonaute protein, the catalytically active RNase of the RISC complex, based on the stability of its 5' end (Preall et al., Curr. Biol., 16:530-535 (2006)). The remaining strand, known as the anti-guide strand or passenger strand, is degraded as a substrate for the RISC complex (Gregory et al., Cell, 123:631-640 (2005)). After being incorporated into the active RISC complex, miRNAs base-pair with their complementary mRNA molecules and induce degradation and / or translational repression of the target mRNA.

[0155] Mammalian miRNA molecules are typically complementary to a site in the 3'UTR of a target mRNA sequence. In certain instances, annealing of the miRNA to the target mRNA inhibits protein translation by interfering with the protein translation machinery. In certain other instances, annealing of the miRNA to the target mRNA promotes cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs may also target methylation of genomic sites corresponding to the target mRNA. Generally, miRNAs function in association with a complement of proteins collectively referred to as miRNPs.

[0156] In certain embodiments, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In certain other embodiments, the miRNA molecules may include one or more modified nucleotides. As a non-limiting example, the miRNA sequence may include one or more modified nucleotides as described above with respect to the siRNA sequence. In a preferred embodiment, the miRNA molecule includes 2'OMe nucleotides, such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, or mixtures thereof.

[0157] In some embodiments, miRNA molecules can be used to silence the expression of any of the target genes defined above, such as, for example, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immune regulatory genes such as genes associated with inflammation and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0158] In other embodiments, one or more agents that interfere with the activity of miRNA targeting an mRNA of interest are administered with the lipid particles (e.g., nucleic acid-lipid particles) of the present invention. Examples of blocking agents include, but are not limited to, steric blocking oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents may bind directly to the miRNA or may bind to the miRNA binding site on the target mRNA.

[0159] 4. Antisense Oligonucleotides In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The term "antisense oligonucleotide" or "antisense" includes oligonucleotides that are complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence. An antisense RNA oligonucleotide inhibits translation of a complementary RNA strand by binding to the RNA. An antisense DNA oligonucleotide can be used to target a specific complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In certain embodiments, the antisense oligonucleotide comprises about 10 to about 60 nucleotides, more preferably about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, the present invention may be utilized when the antisense finds non-target specific activity or when an antisense sequence that contains one or more mismatches with the target sequence is most preferred for a particular application.

[0160] Antisense oligonucleotides have been proven to be effective and targeted inhibitors of protein synthesis, and can therefore be used to specifically inhibit the synthesis of proteins by target genes.The effectiveness of antisense oligonucleotides in inhibiting protein synthesis has been well established.For example, the synthesis of polygalactauronase and muscarinic type 2 acetylcholine receptors is inhibited by antisense oligonucleotides directed to their respective mRNA sequences (see U.S. Patent Nos. 5,739,119 and 5,759,829). Additionally, examples of antisense inhibition have been demonstrated for the nuclear protein cyclin, the multidrug resistance gene (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF (see Jaskulski et al., Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun., 1:225-32 (1989); Penis et al., Brain Res Mal Brain Res., 15;57:310-20 (1998); and U.S. Patent Nos. 5,801,154; 5,789,573; 5,718,709, and 5,610,288). Moreover, antisense constructs that can be used to inhibit and treat various abnormal cell proliferations, such as cancer, have also been described (see U.S. Patent Nos. 5,747,470; 5,591,317; and 5,783,683), the disclosures of which are incorporated herein by reference in their entireties for all purposes.

[0161] Methods for producing antisense oligonucleotides are known in the art and can be easily adapted to produce antisense oligonucleotides targeting any polynucleotide sequence. Selection of antisense oligonucleotide sequences specific to a given target sequence is based on analysis of the selected target sequence and determination of secondary structure, Tm, binding energy, and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to the target mRNA in the host cell. Highly preferred target regions of mRNA include the region at or near the AUG translation initiation codon, and sequences that are substantially complementary to the 5' region of the mRNA. Analysis of these secondary structures and target site selection considerations can be performed, for example, using OLIGO primer analysis software (Molecular Biology Insights) v.4 and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0162] 5. Ribozymes In another embodiment of the present invention, the nucleic acid-lipid particle is associated with a ribozyme. Ribozymes are RNA-protein complexes that have a specific catalytic domain with endonuclease activity (see Kim et al., Proc. Natl. Acad Sci. USA., 84:8788-92 (1987); and Forster et al., Cell, 49:211-20 (1987)). For example, many ribozymes catalyze phosphoester transfer reactions with high specificity, often cleaving only one of several phosphates in an oligonucleotide substrate (see Cech et al., Cell, 27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990); Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity results from the requirement that the substrate bind, via specific base-pairing interactions, to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.

[0163] Currently, at least six basic types of naturally occurring enzymatic RNA molecules are known. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through a target binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, an enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base pairing, and once bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of a target RNA would impair its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to seek another target, and can repeatedly bind and cleave new targets.

[0164] The enzymatic nucleic acid molecule may be formed, for example, with a hammerhead, hairpin, hepatitis delta virus, group I intron, or RNaseP RNA (in conjunction with an RNA guide sequence), or Neurospora VS RNA motif. Specific examples of hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP 0360257, Hampel et al., Biochemistry, 28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990); and U.S. Patent No. 5,631,359. Examples of hepatitis delta virus motifs are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of RNaseP motifs are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of Neurospora VS RNA ribozyme motifs are described, for example, in Saville et al., Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Examples of Group I introns are described, for example, in U.S. Pat. No. 4,987,071. An important feature of the enzymatic nucleic acid molecules used in accordance with the present invention is that they have a specific substrate binding site that is complementary to one or more target gene DNA or RNA regions, and that they have nucleotide sequences within or surrounding that substrate binding site that confer RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the particular motifs mentioned herein. The disclosures of these references are incorporated herein by reference in their entireties for all purposes.

[0165] Methods for producing ribozymes that target any polynucleotide sequence are known in the art. Ribozymes can be designed, for example, as described in PCT Publication Nos. WO 93 / 23569 and WO 94 / 02595, and synthesized to be tested in vitro and / or in vivo as described therein. The disclosures of these PCT Publications are incorporated herein by reference in their entirety for all purposes.

[0166] Ribozyme activity can be optimized by varying the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent degradation by serum ribonucleases (see, e.g., PCT Publication Nos. WO 92 / 07065, WO 93 / 15187, WO 91 / 03162, and WO 94 / 13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, which describe various chemical modifications that can be made to the sugar moiety of enzymatic RNA molecules, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes), modifications that enhance efficacy within cells, and removal of stem II bases to shorten RNA synthesis time and reduce chemical requirements.

[0167] 6. Immunostimulatory Oligonucleotides The nucleic acid associated with the lipid particles of the present invention may be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that can induce an immune response when administered to a subject, such as a mammalian subject, such as a human. ISS includes, for example, specific palindromes that lead to hairpin secondary structures (see Yamamoto et al., J.Immunol., 148:4072-6 (1992)), or CpG motifs, and other known ISS features (such as multi-G domains; see PCT Publication No. WO 96 / 11266; the disclosure of which is incorporated herein by reference in its entirety for all purposes).

[0168] An immunostimulatory nucleic acid is considered to be non-sequence specific if it is not required to specifically bind to and reduce the expression of a target sequence in order to elicit an immune response. Thus, a particular immunostimulatory nucleic acid may contain a sequence that corresponds to a region of a naturally occurring gene or mRNA and still be considered a non-sequence specific immunostimulatory nucleic acid.

[0169] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in said CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one cytosine in said CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of said CpG dinucleotides comprises a methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for use in the compositions and methods of the invention are described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, PCT Publication Nos. WO 02 / 069369 and WO 01 / 15726, U.S. Patent No. 6,406,705, and Raney et al., J. Pharm. Exper. Ther., 298:1185-92 (2001), the disclosures of which are incorporated herein by reference in their entireties for all purposes. In certain embodiments, the oligonucleotides used in the compositions and methods of the invention have a phosphodiester ("PO") or phosphorothioate ("PS") backbone, and / or at least one methylated cytosine residue in a CpG motif.

[0170] B. Other Active Agents In certain embodiments, the active agent associated with the lipid nanoparticles of the present invention can include one or more therapeutic proteins, polypeptides, or small organic molecules or compounds. Non-limiting examples of such therapeutically active agents or drugs include oncology drugs (e.g., chemotherapeutic drugs, hormonal therapy drugs, immunotherapy drugs, radiotherapy drugs, etc.), lipid-lowering drugs, antiviral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenics, cytovascular agents, signal transduction inhibitors, cardiovascular drugs such as antiarrhythmic drugs, hormones, vasoconstrictors, and steroids. These active agents can be administered alone in the lipid particles of the present invention or in combination (e.g., co-administration) with the lipid particles of the present invention that contain a nucleic acid such as an interfering RNA.

[0171] Non-limiting examples of chemotherapeutic agents include platinum-based agents (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin, satraplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine, uramustine, thiotepa, nitrosoureas, etc.), antimetabolites (e.g., 5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, etc.), and the like. , paclitaxel (Taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan (CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g., gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®; OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib (SU5416), vatalanib (PTK787 / ZK222584), sorafenib (BAY43-9006), imatinib (Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and combinations thereof.

[0172] Examples of conventional hormone therapy drugs include, but are not limited to, steroids (eg, dexamethasone), finasteride, aromatase inhibitors, tamoxifen, and goserelin and other gonadotropin-releasing hormone agonists (GnRH).

[0173] Examples of conventional immunotherapeutic agents include immune stimulants (e.g., Bacillus Calmette-Guerin (BCG), levamisole, interleukin-2, α-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas aeruginosa exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111 In, 90 Y, 131 Antibody compositions that can be used to treat or prevent the development of a pulmonary artery disease include, but are not limited to, anti-CD20 monoclonal antibodies conjugated to I, and the like.

[0174] Examples of conventional radiation treatment drugs include: 47 Sc, 64 Cu, 67 Cu, 37 Sr, 86 Y, 37 Y, 90 Y, 105 Rh, 111 Ag, 111 In, 117m Sn, 149 Pm, 153 Sm, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 At, and 212 These include, but are not limited to, radionuclides such as Bi, optionally conjugated to an antibody against a tumor antigen.

[0175] Additional anti-cancer drugs that can be used in accordance with the present invention include alkeran, allopurinol, altretamine, amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU, carmustine, CCNU, celecoxib, cladribine, cyclosporine A, cytosine arabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane, FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin, interferon, letrozole, roysta Examples of anti-cancer drugs that can be used in accordance with the present invention include, but are not limited to, ellipticin, leuprolide, litertinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, pegademase, pentostatin, porfimer sodium, prednisone, rituximab, streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene, tretinoin, ATRA, valrubicin, and velban. Other examples of anti-cancer drugs that can be used in accordance with the present invention are ellipticin and ellipticine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecin.

[0176] Non-limiting examples of lipid-lowering agents for treating lipid diseases or disorders associated with elevated triglycerides, cholesterol, and / or glucose include statins, fibrates, ezetimibe, thiazolidinediones, niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, and mixtures thereof.

[0177] Examples of antiviral drugs include abacavir, aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atriptidine, cidofovir, combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, entry inhibitors, famciclovir, fixed-dose combination drugs, fomivirsen, fosane, Prenavir, foscarnet, phosphonates (fosfonet), fusion inhibitors, ganciclovir, ibatitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine, integrase inhibitors, interferon type III (e.g., IFN-λ molecules such as IFN-λ1, IFN-λ2, IFN-λ3), interferon type II (e.g., IFN-γ), interferon type I (e.g., PEGylated IFN-α, IFN- IFN-α (IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ξ), interferons, lamivudine, lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir, nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir These include, but are not limited to, valaciclovir, stavudine, synergistic enhancer, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, truvada, valacyclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and mixtures thereof.

[0178] The additional agents include zotarolimus, sirolimus, rapamycin, everolimus, biolimus, myolimus, novolimus, temsirolimus, deforolimus, melilimus, tacrolimus, pimecrolimus, ridaforolimus, pharmaceutically acceptable salts thereof, stereoisomers thereof, derivatives thereof, analogs thereof, and mixtures thereof.

[0179] Pharmaceutical Compositions The lipid nanoparticles can be formulated in whole or in part as a pharmaceutical composition. The pharmaceutical composition can include one or more nanoparticles. For example, the pharmaceutical composition can include one or more nanoparticles that include one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further include one or more pharma- ceutically acceptable excipients or accessory ingredients as described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and medicaments can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st Edition, AR Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and accessory ingredients can be used in any pharmaceutical composition, except insofar as any conventional excipient or accessory ingredient may be incompatible with one or more of the components of the lipid nanoparticles. An excipient or accessory ingredient may be incompatible with a component of the lipid nanoparticle if combination with the component of the lipid nanoparticle may result in undesirable biological effects or other adverse effects.

[0180] In some embodiments, one or more excipients or accessory ingredients can comprise more than 50% of the total mass or volume of a pharmaceutical composition comprising lipid nanoparticles. For example, one or more excipients or accessory ingredients may comprise 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention. In some embodiments, a pharma- ceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0181] The relative amounts of one or more lipid nanoparticles, one or more pharma- ceutically acceptable excipients, and / or any additional components in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the pharmaceutical composition may contain from 0.1% to 100% (wt / wt) of one or more lipid nanoparticles.

[0182] In certain embodiments, the lipid nanoparticles and / or pharmaceutical compositions of the present invention are refrigerated or frozen for storage and / or shipping (e.g., stored at a temperature of about 4°C or less, such as about -150°C to about 0°C, or about -80°C to about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)).

[0183] In certain embodiments, the pharmaceutical composition of the present disclosure comprises a lipid nanoparticle disclosed herein and a pharma- ceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical composition of the present disclosure has a pH value of about 7-8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5-8 or 7-7.8). For example, the pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, e.g., suitable for storage and / or shipping at about -20°C. For example, the pharmaceutical composition of the present disclosure comprises a lipid nanoparticle disclosed herein and PBS, and has a pH of about 7-7.8, e.g., suitable for storage and / or shipping at about 4°C or below. "Stability," "stabilization," and "stable" in the context of this disclosure refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, change in particle size, aggregation, change in encapsulation, etc.) under given conditions of manufacture, preparation, transportation, storage, and / or use, e.g., when subjected to stresses such as shear forces, freeze / thaw stress, etc.

[0184] In a preferred embodiment, the lipid nanoparticles of the present invention are formulated with one or more disaccharides or disaccharide containing molecules such as sucrose, lactose, maltose, trehalose, maltitol, or lactitol in any one or more buffers described herein, including TrisHCl, TrisAcetate, TT / AA. As shown in the examples below, lipid nanoparticles formulated with sucrose can be reconstituted after thawing without aggregation.

[0185] The lipid nanoparticles and / or pharmaceutical compositions comprising one or more lipid nanoparticles can be administered to any patient or subject, including those who may benefit from a therapeutic effect provided by the delivery of a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. The description of lipid nanoparticles and pharmaceutical compositions comprising lipid nanoparticles provided herein is directed primarily to compositions suitable for administration to humans, but it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications of compositions suitable for administration to humans are well understood, and a reasonably skilled veterinary pharmacologist can design and / or implement such modifications, if any, using no more than routine experimentation, to render the compositions suitable for administration to a variety of animals. Subjects to which the compositions may be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals, such as, for example, cows, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0186] Pharmaceutical compositions containing one or more lipid nanoparticles can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such methods of preparation include bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into the desired single or multiple dosage units.

[0187] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient (e.g., lipid nanoparticles). The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0188] Pharmaceutical compositions can be prepared in a variety of forms suitable for different routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0189] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms can contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizers, and emulsions such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1.3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, oral compositions can contain additional therapeutic and / or prophylactic agents, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or aromatic agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as Cremophor®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0190] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenterally acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Water, Ringer's solution, USP, and isotonic sodium chloride solution are acceptable vehicles and solvents that can be used. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any sterile fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0191] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0192] Compositions for rectal or vaginal administration are typically suppositories and can be prepared by mixing the composition with a suitable non-irritating excipient, such as cocoa butter, polyethylene glycol or a suppository wax; these compositions are solid at ambient temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active ingredient.

[0193] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert pharma- ceutically acceptable excipient and / or filler or extender, such as sodium citrate or dicalcium phosphate (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), binder (e.g., carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectant (e.g., glycerol), disintegrant (e.g., agar, charcoal, etc.), and / or sorbitol (e.g., sorbitol ... Examples of suitable additives include calcium stearate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarders (e.g., paraffin), absorption enhancers (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g., kaolin and bentonite clay, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may also contain buffering agents.

[0194] Solid compositions of a similar type can be used as fillers for soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulation art. These can optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers for soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

[0195] Dosage forms for topical and / or transdermal administration of the composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. In general, the active ingredient is mixed under sterile conditions with pharma- ceutically acceptable excipients and / or any required preservatives and / or buffers that may be required. In addition, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Alternatively or additionally, the rate can be controlled by either providing a rate-controlling membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0196] Suitable devices for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositions can be administered by devices that limit the effective penetration length of the needle into the skin, such as those described in PCT Publication WO 99 / 34850 and its functional equivalents. Jet injection devices are suitable that deliver liquid compositions to the dermis via a liquid jet injector and / or via a needle that creates a jet that penetrates the stratum corneum and reaches the dermis. Jet injection devices are described, for example, in U.S. Patent Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT publication WO These are described in WO 97 / 37705 and WO 97 / 13537. Ballistic powder / particle delivery devices, which use compressed gas to accelerate the powdered vaccine through the outer layer of the skin to the dermis, are suitable. Alternatively or additionally, a conventional syringe can be used for the classical Mantoux technique for intradermal administration.

[0197] Formulations suitable for topical administration include, but are not limited to, liquid and / or semi-liquid formulations, such as oil-in-water and / or water-in-oil emulsions, and / or solutions and / or suspensions, such as liniments, lotions, creams, ointments and / or pastes. The concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent, but topically administrable formulations may contain, for example, about 1% to about 10% (wt / wt) active ingredient. Formulations for topical administration may further include one or more additional ingredients described herein.

[0198] The pharmaceutical composition can be prepared, packaged, and / or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles comprising the active ingredient. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a dry powder reservoir into which a stream of propellant can be directed to disperse the powder, and / or using a self-propelling solvent / powder dispensing container, such as a device comprising the active ingredient dissolved and / or suspended in a low boiling propellant in a closed container. Dry powder compositions may comprise a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0199] Low boiling point propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant may comprise 50%-99.9% (wt / wt) of the composition and the active ingredient may comprise 0.1%-20% (wt / wt) of the composition. The propellant may further comprise additional components such as a liquid non-ionic surfactant and / or a solid anionic surfactant and / or a solid diluent (which may have a particle size on the same order as the particles containing the active ingredient).

[0200] Pharmaceutical compositions formulated for pulmonary delivery can provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations can be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions, optionally sterile, containing the active ingredient, and can be conveniently administered using any nebulization and / or atomization device. Such formulations may further comprise one or more additional ingredients, including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methylhydroxybenzoate. The droplets provided by this route of administration can have an average diameter in the range of about 1 nm to about 200 nm.

[0201] The formulations described herein that are useful for pulmonary delivery are useful for nasal delivery of pharmaceutical compositions. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle size of about 0.2 μm to about 500 μm. Such formulations are administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passages from a container of the powder held close to the nose.

[0202] Formulations suitable for nasal administration may, for example, contain as little as 0.1% (wt / wt) to as much as 100% (wt / wt) of the active ingredient, and may include one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges manufactured using conventional methods, and may contain, for example, 0.1% to 20% (wt / wt) of the active ingredient, the balance comprising an orally soluble and / or degradable composition, and, optionally, one or more of the additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, have an average particle and / or droplet size in the range of about 0.1 nm to about 200 nm and can further include one or more of any of the additional ingredients described herein.

[0203] A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for ocular administration. Such a formulation may be in the form of, for example, eye drops comprising a 0.1 / 1.0% (wt / wt) solution and / or suspension of the active ingredient in an aqueous or oily liquid vehicle. Such eye drops may further comprise buffering agents, salts, and / or one or more of any other additional ingredients described herein. Other ophthalmically administrable formulations that are useful include those which comprise the active ingredient in microcrystalline form and / or in a liposomal formulation. Ear drops and / or eye drops are contemplated as being within the scope of the present disclosure.

[0204] Methods for Treating Diseases and Disorders The lipid nanoparticles can be useful for treating diseases, disorders, or conditions. In particular, such compositions can be useful for treating diseases, disorders, or conditions characterized by missing or abnormal protein or polypeptide activity. For example, a nanoparticle composition containing an active, such as an mRNA encoding a missing or abnormal polypeptide, can be administered or delivered to a cell. Subsequent translation of the mRNA can produce the polypeptide, thereby reducing or eliminating problems caused by missing or abnormal activity due to the polypeptide. Because translation can occur rapidly, the methods and compositions can be useful for treating acute diseases, disorders, or conditions, such as sepsis, stroke, myocardial infarction, and the like. The therapeutic and / or prophylactic agents included in the nanoparticle composition can also alter the transcription rate of a given species, thereby affecting gene expression.

[0205] Diseases, disorders, and / or conditions characterized by dysfunction or abnormality of protein or polypeptide activity for which the compositions may be administered include, but are not limited to, rare diseases, infectious diseases (both as vaccines and therapeutics), cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. Several diseases, disorders, and / or conditions can be characterized by a lack of protein activity (or substantially reduced such that proper protein function does not occur). Such proteins may be absent or essentially non-functional. A specific example of a dysfunctional protein is a missense mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produces a dysfunctional protein variant of the CFTR protein that causes cystic fibrosis.

[0206] The present disclosure provides methods of treating such diseases, disorders, and / or conditions in a subject by administering lipid nanoparticles of the present invention containing RNA, which can be mRNA encoding a polypeptide that antagonizes or otherwise overcomes aberrant protein activity present in the subject's cells.

[0207] The present disclosure provides methods comprising administering lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents, and pharmaceutical compositions comprising the same. The terms therapeutic and prophylactic may be used interchangeably herein with respect to features and embodiments of the present disclosure. The therapeutic composition, or its imaging, diagnostic, or prophylactic composition, may be administered to a subject using any appropriate amount and any route of administration effective for the prevention, treatment, diagnosis, or imaging of a disease, disorder, and / or condition, and / or any other purpose. The specific amount administered to a given subject may vary depending on the species, age, and general condition of the subject; the purpose of administration; the particular composition; the mode of administration; and the like. The compositions according to the present disclosure may be formulated in dosage unit form for ease of administration and uniformity of dosage. However, it will be understood that the total daily usage of the lipid nanoparticles or pharmaceutical compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment. A particular therapeutically effective, prophylactically effective, or otherwise appropriate dose level (e.g., for imaging) for a particular patient will depend on a variety of factors, including the severity and identification of the disorder being treated, if any; the therapeutic and / or prophylactic agent(s) employed; the particular composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and rate of excretion of the particular pharmaceutical composition employed; the duration of treatment; drugs used in combination or concomitantly with the particular pharmaceutical composition employed; and similar factors well known in the medical arts.

[0208] The lipid nanoparticles comprising one or more therapeutic and / or prophylactic agents can be administered by any route. In some embodiments, the compositions comprising the prophylactic, diagnostic, or imaging compositions comprising one or more lipid nanoparticles described herein are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal or intradermal, interdermal, rectal, intravaginal, intraperitoneal, intraocular, subretinal, intravitreal, topical (e.g., by powder, ointment, cream, gel, lotion, and / or liquid drop), mucosal, nasal, buccal, enteral, vitreous, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as oral spray and / or powder, nasal spray, and / or aerosol, and / or via a portal vein catheter. In some embodiments, the compositions can be administered intravenously, intramuscularly, intradermal, intraarterial, intratumoral, subcutaneous, intraocular, subretinal, intravitreal, or by inhalation. Generally, the most appropriate route of administration will depend on a variety of factors, including the properties of the nanoparticle composition comprising one or more therapeutic and / or prophylactic agents (e.g., its stability in various body environments, such as the bloodstream and gastrointestinal tract), the condition of the patient (e.g., whether the patient can tolerate a particular route of administration), and the like.

[0209] In certain embodiments, compositions according to the present disclosure may be administered at a dose of from about 0.0001 mg / kg to about 10 mg / kg, from about 0.001 mg / kg to about 10 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg , about 0.001mg / kg to about 5mg / kg, about 0.005mg / kg to about 5mg / kg, about 0.01mg / kg to about 5mg / kg, about 0.05mg / kg to about 5mg / kg, about 0.1mg / kg to about 5mg / kg, about 1mg / kg to about 5mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05mg / kg~about 2.5mg / kg, about 0.1mg / kg~about 2.5mg / kg, about 1mg / kg~about 2.5mg / kg, about 2mg / kg~about 2.5mg / kg, about 0.0001mg / kg~about 1mg / kg, about 0.001mg / kg~ Approximately 1 mg / kg, approximately 0.005 mg / kg to approximately 1 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, approximately 0.05 mg / kg to approximately 1 mg / kg, approximately 0.1 mg / kg to approximately 1 mg / kg, approximately 0.0001 mg / kg to approximately 0.25 mg / kg, approximately The nanoparticle compositions may be administered at dosage levels sufficient to deliver from about 0.001 mg / kg to about 0.25 mg / kg, from about 0.005 mg / kg to about 0.25 mg / kg, from about 0.01 mg / kg to about 0.25 mg / kg, from about 0.05 mg / kg to about 0.25 mg / kg, or from about 0.1 mg / kg to about 0.25 mg / kg of a therapeutic and / or prophylactic agent (e.g., mRNA) at a given dose, where a dose of 1 mg / kg (mpk) provides 1 mg of therapeutic and / or prophylactic agent per kg of subject body weight. In some embodiments, a dose of from about 0.001 mg / kg to about 10 mg / kg of a therapeutic and / or prophylactic agent (e.g., mRNA) of the nanoparticle composition may be administered.In other embodiments, a dose of about 0.005 mg / kg to about 2.5 mg / kg of therapeutic and / or prophylactic agent can be administered. In certain embodiments, a dose of about 0.1 mg / kg to about 1 mg / kg can be administered. In other embodiments, a dose of about 0.05 mg / kg to about 0.25 mg / kg can be administered. Doses can be administered one or more times per day, in the same or different amounts, to obtain a desired level of mRNA expression and / or therapeutic, diagnostic, preventive, or imaging effect. The desired dose can be delivered, for example, three times per day, twice per day, once per day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dose can be delivered using multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses). In some embodiments, a single dose can be administered, for example, before or after a surgical procedure or in the case of an acute disease, disorder, or condition.

[0210] The lipid nanoparticles of the present invention comprising one or more therapeutic and / or prophylactic agents can be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By "in combination," it is meant that these delivery methods are within the scope of this disclosure, but the agents must be administered simultaneously and / or formulated for delivery together. It is not intended to imply that the lipid nanoparticles are administered in combination with one or more lipid nanoparticles containing one or more different therapeutic and / or prophylactic agents. The lipid nanoparticles may be administered simultaneously with, prior to, or after one or more other desired therapeutic agents or medical procedures. In general, each agent is administered at a dose and / or time schedule determined for that agent. In some embodiments, the present disclosure encompasses delivery of lipid nanoparticles, compositions, or imaging, diagnostic, or prophylactic compositions thereof in combination with agents that improve bioavailability, reduce and / or modify metabolism, inhibit excretion, and / or modify biodistribution.

[0211] It will be further understood that therapeutic, prophylactic, diagnostic, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination may be lower than the levels at which they are utilized individually.

[0212] The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be understood that the therapies employed may achieve a desired effect for the same disorder (e.g., a composition useful for treating cancer is administered simultaneously with a chemotherapeutic agent) or may achieve a different effect (e.g., control of any side effects such as infusion related reactions). EXAMPLES

[0213] Example 1 - Generation of lipid nanoparticles Nanoparticles can be created by mixing processes such as microfluidics and T-junction mixing of two fluid streams, where one fluid stream contains the therapeutic and / or prophylactic agent and the other has a lipid component. The lipid composition is prepared by combining cationic lipids (such as DODAC, DODAP, or Dlim-MC3-DMA), phospholipids (such as DOPE or DSPC), PEG lipids (such as 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000, also known as DMG-PEG), and structural lipids (such as cholesterol or corticosteroids (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or combinations thereof) in ethanol at a concentration of about 50 mM. Unless otherwise specified, nanoparticles referred to herein as "MIPS-LNP" include any one of the lipid nanoparticles 1-4 referred to below. The solution should be refrigerated, for example, when stored at 4°C, or frozen, for example, when stored at -20°C. The lipids are combined to provide the desired molar ratio and diluted with ethanol to a final lipid concentration of about 5.5 mM to about 50 mM. Nanoparticle compositions containing a therapeutic and / or prophylactic agent and a lipid component are prepared by combining the lipid solution with a solution containing a therapeutic and / or prophylactic agent in a wt:wt ratio of lipid component to therapeutic and / or prophylactic agent of about 5:1 to about 50:1. The lipid solution is mixed with the nucleic acid solution, for example, using a microfluidic or T-junction-based system at a flow rate of about 0.5 ml / min to about 8 ml / min to produce a lipid nanoparticle suspension with a water to ethanol ratio of about 1:1 to about 4:1, preferably 3:1. Here, the NP ratio (nitrogen phosphate) is maintained between 4 and 7. The solution can be immediately diluted or buffer exchanged with a buffer of choice, as described below.

[0214] Examples of lipid nanoparticles produced include: [Table 1] [Table 2] [Table 3] [Table 4]

[0215] For nanoparticle compositions containing RNA, a 0.133-0.6 mg / ml concentration of RNA solution in deionized water is diluted with 25 mM sodium acetate buffer at pH 3-4.5 to form a stock solution (Figure 1). After nanoparticle formulation, the nanoparticle composition may be processed by dialysis or tangential flow filtration to remove ethanol and achieve buffer exchange. For example, the formulation is dialyzed twice against phosphate buffered saline (PBS) at pH 7.4 at 200 times the volume of the primary product using a Slide-A-Lyzer cassette with a molecular weight cut-off of 10 kD (Thermo Fisher Scientific Inc., Rockford, III.). The first dialysis is performed for 3 hours at room temperature. The resulting nanoparticle suspension is filtered through a 0.4 or 0.22 μm sterile filter in a glass vial and sealed. A nanoparticle composition solution of 0.01 mg / ml to 0.50 mg / ml is generally obtained. In some embodiments, a tangential flow filtration (TFF) from Repligen was used instead of a dialysis cassette. Additionally, a Tris-sucrose solution at pH 7.4 was used to allow portions of the formulation to be frozen at -10 to -80°C.

[0216] The method described above uses NanoAssemblr microfluidics to induce nanoprecipitation and particle formation. Alternative processes, including but not limited to T-junction and direct injection, can be used to achieve similar nanoprecipitation. Higher or lower concentrations of mRNA stock and encapsulating lipids can also be used during nanoprecipitation.

[0217] LNPs are generally formulated to be passively delivered to the liver after intravenous injection. LNPs targeting siRNA to the liver (OnPattro, Alnylam Pharmaceuticals) were approved for human use by the FDA in 2018. We refer to this type of product as conventional LNPs.

[0218] Example 2 - Structural characterization of lipid nanoparticles Lipid nanoparticle size, polydispersity index (PDI), and zeta potential of nanoparticle composition were measured using a Zetasizer (Malvern instruments). Briefly, dialyzed particles were placed in a disposable cuvette (e.g., Zetacell (Malvern) or UV cuvette). The cuvette was placed in the Zetasizer and measurements were recorded. Size calculations were recoded using Zetasizer software 12.0.

[0219] MIPS-LNPs can be produced by reducing the molar ratio of PEGylated lipid content below the 0.5 mol% concentration used in conventional LNP compositions. Conventional LNPs are typically formed with 1.5 mol% PEGylated lipid. We observed that reducing the concentration of PEGylated lipid content in LNPs results in particle sizes greater than 100 nm (Figure 2A). In contrast, conventional LNP formulations produce particles less than 100 nm in size, but both MIPs-LNPs and conventional LNPs maintain similar particle size distribution profiles (Figure 2B). Furthermore, we have shown that MIPS-LNPs with 0.15 mol% PEGylated lipid are stable at 4°C for at least 6 months at reduced PEGylated mol% concentrations (Figure 2C). The overall formulation of MIPS-LNPs provides a very strong negative zeta potential that is beneficial for increasing access to immune organs such as the spleen (data not shown).

[0220] The biophysical characterizations described herein were carried out on the lipid nanoparticles 3 or 4 mentioned above.

[0221] Example 3 - Functional characterization of lipid nanoparticles Intramuscular injection (IM) is a common method of administration used for vaccination, especially for viral and bacterial diseases. Using DODAP as an ionized lipid (e.g., lipid nanoparticles 3 or 4 mentioned above), we demonstrated that after IM injection, >75% of gene expression outside of muscle tissue induced by MIPS-LNPs is in the spleen, compared to ~10% induced by conventional LNPs (Figure 3). Delivery of mRNA by MIPS-LNPs also surprisingly results in ~100-fold higher concentrations of the reporter gene nanoluciferase in the spleen, compared to conventional small LNPs (Figure 4A). In this study, a dose of 10 μg nanoluciferase mRNA was used. Furthermore, MIPS-LNPs have been shown to gain access to lymph nodes that are not immediately downstream of the lymphatic flow from the injection site (known as non-draining lymph nodes).

[0222] The inventors have shown that a larger average size of LNPs leads to better uptake by antigen-presenting cells, i.e., immune cells that direct vaccine responses. The typical size of current LNPs is in the range of 70-100 nm. MIPS-LNPs are designed to be larger than 100 nm, specifically larger than 125 nm in diameter (typically 140-160 nm). However, larger particles can further add selective passive delivery to the spleen (data not shown).

[0223] Intravenous (IV) injection is another administration method that can be used for hospitalized patient treatments - for example, cancer vaccination. We have demonstrated that delivery of mRNA with MIPS-LNPs (ultra-large LNPs) via IV injection using a dose of 10 μg nanoluciferase mRNA results in ~500-fold higher concentration of reporter gene nanoluciferase in the spleen (highlighted by box) compared to conventional small LNPs (Figure 4B). Furthermore, MIPS-LNPs demonstrated a substantial increase in gene transfer via reporter gene expression in various sorted spleen cells compared to conventional LNPs (Figure 4C). MIPS-LNPs also have the advantage of diverting the formulation from the liver (the main use target of conventional LNP formulations designed for delivery of siRNA to the liver). For vaccines, reducing gene expression in the liver is considered beneficial - i.e., reducing side effects by reducing "off-target" regions. To analyze gene expression in these studies, tissues were harvested 16-24 hours after injection, weighed, 1 ml of GloLysis buffer was added, and homogenized using M-tubes (Miltenyl). The tubes were centrifuged (10000g x 1min) and the supernatant was collected. Analysis of nanoluciferase gene expression was performed as suggested by the NanoGlo assay (Promega). Gene expression levels were quantified with a plate reader (Perkin elmer) using the luciferase setting. Gene expression per gram of tissue was calculated and plotted.

[0224] Gene expression in splenocytes was measured approximately 16 hours after injection of nanoluciferase (10ug). Splenocytes were isolated by collagenase digestion (2mg / ml) and first labeled with CD8, CD11C, F4 / 80, mPDCA-1, and separated using FACS. Separated populations were then analyzed for luciferase expression (i.e., specific uptake and expression per cell population) using the NanoGlo assay as described above.

[0225] In this example, MIPS-LNPs have a 2.3-fold lower delivery to the liver compared to conventional LNPs (Table 1). [Table 5]

[0226] Example 4 - Cellular immune responses to lipid nanoparticle delivery of antigenic molecules To determine the level of immune response, an antigen-specific killing assay was used with ovalbumin antigen. C57BL mice (8-24 weeks old) were vaccinated intravenously.

[0227] Regarding the cellular immune response to antigen molecule delivery by MIPS-LNP, we used an in vivo mRNA vaccine model. Mice were immunized with MIPS-LNP containing an antigenic ovalbumin mRNA construct on day 0. Cell-tracking labeled SIINFEKL splenocytes were transferred from donor mice to immunized mice on day 5, and splenocytes from vaccinated mice were isolated and analyzed 24 hours later (Figure 5). The level of killing of SIINFEKL-pulsed cells (antigen-bearing cells) was compared with cell control (no SIINFEKL). MIPS-LNP induced enhanced killing of target-specific cytotoxic T cells against ovalbumin epitopes (Figure 6). MIPS-LNP induced nearly complete killing when small amounts of mRNA were administered to mice (10ug). In comparison, conventional LNP did not produce effective vaccination with the same dose of mRNA. The difference in antigen loss rate and antigen content (killing) compared to pre-injection was measured using a flow cytometer and plotted as a percentage.

[0228] Example 5 - Physical stability of MIPS-LNPs To prove that MIPS formulations can be used to manufacture pharmaceutical products, we tested whether the formulations could be "freeze-thawed," allowing storage in a freezer for transportation prior to use. The product (lipid nanoparticle 1 above) was formulated and dialyzed in various buffers as described above and frozen multiple times (Figure 7). All formulations with the addition of sucrose as a cryoprotectant demonstrated particle reconstitution after thawing without significant aggregation. Thus, MIPS-LNP formulations were shown to be stable for transportation, demonstrating their potential for use as pharmaceutical products.

[0229] Example 6 - Effect of PEGylated lipid content on LNP delivery To prove the effect of PEGylated lipid concentration on LNP delivery, we prepared two formulations containing DLin-MC3-DMA (50 mol%), distearoylphosphatidylcholine (DSPC) (10 mol%), PEGylated lipid (0.15 mol% or 1.5 mol%), and cholesterol (remainder of lipid content - i.e., 39.85 mol% or 38.5 mol%, respectively). DSPE-PEG or DMG-PEG were used as PEGylated lipid. Nanoluciferase mRNA was packaged in LNP formulations ("0.15 mol%" or "1.5 mol%) and injected intramuscularly (IM) or intravenously (IV) into BalbC mice (8-24 weeks old). After 14-18 hours, organs were isolated and weighed. Organs were homogenized using M-tubes (Miltenyi Biotec) and Glo-lysis buffer (Promega). Nanoluciferase activity as (RLU) was measured using a nanoluciferase lysis kit (Nano-Glo® Luciferase Assay System, Promega) according to the manufacturer's instructions.

[0230] After IV injection, nanoluciferase concentrations in the spleen and lymph nodes were consistently 50-100 times higher for the 0.15 mol% formulation than the 1.5 mol% formulation. The trend for lymphatic tissues after IM injection was also consistent, but the difference was 5-10 times (Figure 8). Notably, the 0.15 mol% DMG-PEG formulation injected IM produced significantly higher concentrations of nanoluciferase in the spleen and lower concentrations in the liver compared to the 1.5 mol% DMG-PEG formulation (Figure 9). Both DMG-PEG and DSPE-PEG formulations containing 0.15 mol% PEGylated lipids redirected nanoluciferase mRNA from the liver to the spleen (Figure 10A).

[0231] Example 7 - PEGylated lipid content in lipid nanoparticles Having established that a PEGylated lipid content below 1.5 mol % had beneficial effects on LNP mRNA delivery, we sought to determine the optimal PEGylated lipid range for LNP formulation.

[0232] Various DMG-PEG lipid compositions were tested ranging from 0.1 mol% to 1.5 mol%, and the cholesterol composition was adjusted appropriately. Nanoluciferase mRNA was packaged into LNP formulations, and the compositions were injected IM into BalbC mice (8-24 weeks old). [Table 6]

[0233] DMG-PEG at 0.2 mol% to 0.5 mol% resulted in higher expression of nanoluciferase in the spleen compared to the standard 1.5 mol% PEGylated lipid formulation, suggesting enhanced splenic delivery at this particular range (Table 2). However, the 0.1 mol% to 0.5 mol% DMG-PEG LNP formulations all had significantly higher spleen / liver expression ratios than the 1.5 mol% DMG-PEG LNP formulation. A similar effect was also seen in the non-draining lymph, where nanoluciferase expression was highest with 0.2 mol% DMG-PEG (data not shown). There was no significant difference in nanoluciferase expression in the draining lymph nodes, injected quadriceps and liver, although the average expression level of 0.1 to 0.2 mol% was lower than the higher range (data not shown).

[0234] To further elucidate the scope of PEGylated lipids, we tested 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol% and 0.35 mol% DMG-PEG in the LNP formulation. As described above, nanoluciferase mRNA was packaged in the LNP formulation and the composition was injected IM. Similar to the previous results, the highest expression level of nanoluciferase in the spleen, non-draining lymph nodes and lymph nodes was seen at 0.15 mol%, with a trend towards decreased expression as the DMG-PEG content increased (data not shown).

[0235] A decrease in DMG-PEG content was associated with an increase in nanoluciferase expression in the spleen and a decrease in nanoluciferase expression in the liver (data not shown). Similarly, there is a relationship between a decrease in DMG-PEG and an increase in non-draining lymphocyte delivery and expression. Furthermore, low concentrations of DMG-PEG switched nanoluciferase expression from the liver to the spleen route (Table 3). [Table 7]

[0236] Taken together, these results demonstrate that reduced PEGylation of LNPs, as formulated in MIPS-LNPs, promotes particle uptake and gene expression in the spleen. MIPS-LNP formulations (using DODAP as an example) enhance immune responses and possibly improve the efficiency of both preventive and therapeutic vaccines for cancer. Spleen cell targeting is attractive for many applications, such as expression of proteins for immune checkpoint inhibition, as well as other applications, such as inducing antigen-specific tolerance, induction of general tolerance, via targeted mRNA delivery by MIPS formulations, and in combating autoimmune diseases, reducing inflammation caused by splenocytes, or reducing allergic and anaphylactic reactions, via DNA or any other means of nucleic acid or mRNA delivery using mRNA delivery, siRNA delivery, other molecules (e.g., small drugs incorporated into MIPS LNPs, etc.). Conventional LNPs do not deliver to splenocytes and are therefore inferior when used for the applications outlined above.

[0237] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or apparent from the text or drawings, all of which different combinations constitute various alternative aspects of the invention.

Claims

1. 1. A lipid nanoparticle comprising: (a) activator (b) cationic lipids and / or ionizable lipids, comprising about 40 mol % to about 60 mol % of the total lipids present in the nanoparticles; (c) a phospholipid comprising about 5 mol % to about 20 mol % of the total lipid present in the nanoparticle; (d) structural lipids comprising about 30 mol% to about 50 mol% of the total lipids present in the nanoparticles; (e) a PEGylated lipid comprising about 0.05 mol% to less than 0.5 mol% of the total lipid present in the nanoparticle; A lipid nanoparticle comprising:

2. The PEGylated lipid comprises from about 0.06 mol% to about 0.5 mol%, from about 0.07 mol% to about 0.5 mol%, from about 0.08 mol% to about 0.5 mol%, from about 0.09 mol% to about 0.5 mol%, from about 0.1 mol% to about 0.5 mol%, from about 0.15 mol% to about 0.5 mol%, from about 0.2 mol% to about 0.5 mol%, from about 0.25 mol% to about 0.5 mol%, from about 0.3 mol% to about 0.5 mol%, from about 0.3 mol% to about 0.5 mol%, from about 0.35 mol% to about 0.5 mol%, from about 0.4 mol% to about 0.5 mol%, from about 0.45 mol% to about 0.5 mol% of the total lipid present in the particle.

2. The lipid nanoparticle of claim 1, comprising: about 0.05 mol% to about 0.45 mol%, about 0.05 mol% to about 0.4 mol%, about 0.05 mol% to about 0.35 mol%, about 0.05 mol% to about 0.3 mol%, about 0.05 mol% to about 0.25 mol%, about 0.05 mol% to about 0.2 mol%, about 0.05 mol% to about 0.15 mol%, about 0.05 mol% to about 0.1 mol%, about 0.05 mol% to about 0.09 mol%, about 0.05 mol% to about 0.08 mol%, about 0.05 mol% to about 0.07 mol%, or about 0.05 mol% to about 0.06 mol%.

3. The PEGylated lipid comprises 0.06 mol% to 0.5 mol%, 0.07 mol% to 0.5 mol%, 0.08 mol% to 0.5 mol%, 0.09 mol% to 0.5 mol%, 0.1 mol% to 0.5 mol%, 0.15 mol% to 0.5 mol%, 0.2 mol% to 0.5 mol%, 0.25 mol% to 0.5 mol%, 0.3 mol% to 0.5 mol%, 0.3 mol% to 0.5 mol%, 0.35 mol% to 0.5 mol%, 0.4 mol% to 0.5 mol%, 0.45 mol% to 0.5 mol 1 mol%, 0.05 mol% to 0.45 mol%, 0.05 mol% to 0.4 mol%, 0.05 mol% to 0.35 mol%, 0.05 mol% to 0.3 mol%, 0.05 mol% to 0.25 mol%, 0.05 mol% to 0.2 mol%, 0.05 mol% to 0.15 mol%, 0.05 mol% to 0.1 mol%, 0.05 mol% to 0.09 mol%, 0.05 mol% to 0.08 mol%, 0.05 mol% to 0.07 mol%, or 0.05 mol% to 0.06 mol%.

4. 2. The lipid nanoparticle of claim 1, wherein the PEGylated lipid comprises 0.05 mol%, 0.06 mol%, 0.07 mol%, 0.08 mol%, 0.09 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, or 0.45 mol% of the total lipid present in the particle.

5. The lipid nanoparticle of claim 1, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

6. The lipid nanoparticle of claim 1, wherein the PEGylated lipid is selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

7. The lipid nanoparticle of claim 6, wherein the PEGylated lipid is PEG-DSPE.

8. 2. The lipid nanoparticle of claim 1, wherein the PEGylated lipid has a PEG moiety with a molecular weight of about 100 Da to about 100,000 Da, about 100 Da to about 100,000 Da, about 1000 Da to about 9000 Da, about 1000 Da to about 8000 Da, about 1000 Da to about 7000 Da, about 1000 Da to about 6000 Da, about 1000 Da to about 5000 Da, about 1000 Da to about 4000 Da, about 1000 Da to about 3000 Da, or about 1000 Da to about 2000 Da.

9. The lipid nanoparticle of claim 1, wherein the PEGylated lipid has a PEG moiety with a molecular weight of about 1,000 Da to 5,000 Da, preferably about 2,000 Da to 5,000 Da.

10. 2. The lipid nanoparticle of claim 1, wherein the PEGylated lipid has a PEG moiety having a molecular weight of 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da.

11. The lipid nanoparticle of claim 1, wherein the PEGylated lipid is DSPE-PEG, and the PEG has a molecular weight of 2000 Da.

12. The cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide ( DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (D OSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3.β.oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2- N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-3015), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid,The lipid nanoparticles according to claim 1, which are any one of 1-octylnonyl ester (SM-102) and mixtures thereof.

13. The cationic lipid has formula I: 【Chemical 1】 R １ and R ２ are independently selected and are H or C １ -C ３ alkyl, and R ３ and R ４ is an independently selected alkyl group having from about 10 to about 20 carbon atoms; R ３ and R ４ The lipid nanoparticle of claim 1, wherein at least one of the cationic lipids of formula I contains at least two unsaturated sites, and preferably the cationic lipid of formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA).

14. The cationic lipid has the formula II: 【Chemistry 2】 R １ and R ２ are independently selected and are H or C １ -C ３ alkyl, and R ３ and R ４ is an independently selected alkyl group having from about 10 to about 20 carbon atoms; R ３ and R ４ The lipid nanoparticle of claim 1, wherein at least one of the groups comprises at least two unsaturated sites.

15. The cationic lipid has formula III: 【Chemistry 3】 R １ and R ２ are the same or different and independently represent optionally substituted C １２ -C ２４ Alkyl, optionally substituted C １２ -C ２４ Alkenyl, optionally substituted C １２ -C ２４ Alkynyl, or optionally substituted C １２ -C ２４ acyl; R ３ and R ４ are the same or different and independently represent optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ alkenyl, or optionally substituted C １ -C ５ alkynyl, or R ３ and R ４ may be joined to form an optionally substituted heterocycle of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R ５ is absent or is either hydrogen or C1-C6 alkyl, providing a quaternary amine; m, n, and p are the same or different and independently are either 0 or 1, provided that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are the same or different and independently are O, S, or NH.

16. The cationic lipids of formula III include 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3 ]-dioxolane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyl 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleyloxy-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 16. The lipid nanoparticle of claim 15, wherein the lipid nanoparticle is selected from the group consisting of 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)propane chloride salt (DLin-TAP.C1), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and mixtures thereof.

17. The lipid nanoparticle of claim 1, wherein the cationic lipid is DODAP, DLin-DMA, DLin-K-DMA, DLin-K2-DMA or DLin-MC3-DMA.

18. 2. The lipid nanoparticle of claim 1, wherein the cationic lipid comprises about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 45 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipid present in the particle.

19. 2. The lipid nanoparticle of claim 1, wherein the cationic lipid comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol% of the total lipid present in the particle.

20. The lipid nanoparticle of claim 1, wherein the phospholipid is a cationic phospholipid.

21. The lipid nanoparticle of claim 1 , wherein the phospholipid is an unsaturated lipid.

22. The lipid nanoparticle of claim 1, wherein the phospholipid is a polyunsaturated lipid.

23. The phospholipid is according to formula (IV): 【Chemistry 4】 2. The lipid nanoparticle of claim 1, wherein R and R' represent lipid moieties, which may be the same or different, and which may or may not have unsaturation.

24. The lipid nanoparticle of claim 1, wherein the phospholipid moiety is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

25. 2. The lipid nanoparticle of claim 1, wherein the phospholipid has a fatty acid moiety selected from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, erucic acid, phytanic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

26. The phospholipids include lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (P 2. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

27. The lipid nanoparticle of claim 1, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

28. 2. The lipid nanoparticle of claim 1, wherein the phospholipid comprises about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 10 mol% to about 20 mol%, or about 15 mol% to about 20 mol% of the total lipid present in the particle.

29. The lipid nanoparticle of claim 1, wherein the phospholipid typically comprises 5 mol% to 20 mol%, 5 mol% to 15 mol%, 5 mol% to 10 mol%, 10 mol% to 20 mol%, or 15 mol% to 20 mol% of the total lipid present in the particle.

30. The lipid nanoparticle of claim 1, wherein the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, α-tocopherol, and mixtures thereof.

31. The lipid nanoparticle of claim 1, wherein the structural lipid is cholesterol.

32. 2. The lipid nanoparticle of claim 1, wherein the structured lipid comprises about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% of the total lipid present in the particle.

33. The lipid nanoparticle of claim 1, wherein the structured lipid typically comprises 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 30 mol% to 35 mol%, 35 mol% to 50 mol%, 40 mol% to 50 mol%, or 45 mol% to 50 mol% of the total lipid present in the particle.

34. The lipid nanoparticle of claim 1 , wherein the active agent comprises or consists of a peptide or polypeptide.

35. The lipid nanoparticle of claim 34, wherein the polypeptide comprises an antibody, preferably a polyclonal antibody, a monoclonal antibody, an antibody fragment; a humanized antibody, a recombinant antibody, a recombinant human antibody, a Primatized™ antibody, or a mixture thereof.

36. The lipid nanoparticle of claim 34, wherein the peptide or polypeptide comprises a cytokine, growth factor, apoptotic factor, differentiation inducer, cell surface receptor, ligand, hormone, small molecule (e.g., small organic molecule or compound), or a mixture thereof.

37. The lipid nanoparticle of claim 1 , wherein the active agent comprises or consists of a nucleic acid.

38. The lipid nanoparticle of claim 37, wherein the nucleic acid comprises an interfering RNA molecule, preferably siRNA, aiRNA, miRNA, or a mixture thereof.

39. The lipid nanoparticle of claim 37, wherein the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or a DNA / RNA hybrid, preferably an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory oligonucleotide, or a mixture thereof.

40. The lipid nanoparticle of claim 1 , wherein the active agent is mRNA.

41. The lipid nanoparticle of claim 1, wherein the active agent is completely encapsulated within the lipid portion of the lipid particle, and the active agent or therapeutic agent within the lipid particle is resistant to enzymatic degradation, e.g., by nucleases or proteases, in aqueous solution.

42. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle does not contain a targeting ligand that specifically binds to a molecule on the surface of a target cell.

43. The lipid nanoparticles of claim 1, wherein the average size of the lipid nanoparticles is greater than about 100 nm, for example, as measured by dynamic light scattering (DLS).

44. The average size is about 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, 150 nm, 155 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, 2. The lipid nanoparticle of claim 1, wherein the average diameter of the lipid nanoparticle is greater than, or greater than, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410 nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455 nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, or 500 nm.

45. The average size of the nanoparticles of the present invention may be from about 75 nm to about 500 nm, from about 80 nm to about 500 nm, from about 90 nm to about 500 nm, from about 100 nm to about 500 nm, from about 110 nm to about 500 nm, from about 120 nm to about 500 nm, from about 130 nm to about 500 nm, from about 140 nm to about 500 nm, from about 150 nm to about 500 nm, from about 160 nm to about 500 nm, 170 nm to about 500 nm, about 180 nm to about 500 nm, about 190 nm to about 500 nm, about 200 nm to about 500 nm, about 210 nm to about 500 nm, about 220 nm to about 5 00nm, about 230nm to about 500nm, about 240nm to about 500nm, about 250nm to about 500nm, about 260nm to about 500nm, about 270nm to about 500nm, about 280nm nm to about 500 nm, about 300 nm to about 500 nm, about 310 nm to about 500 nm, about 320 nm to about 500 nm, about 330 nm to about 500 nm, about 340 nm to about 500 nm m, about 350 nm to about 500 nm, about 360 nm to about 500 nm, about 370 nm to about 500 nm, about 380 nm to about 500 nm, about 390 nm to about 500 nm, about 400 nm to The lipid nanoparticle of claim 1, which may be about 500 nm, about 410 nm to about 500 nm, about 420 nm to about 500 nm, about 430 nm to about 500 nm, about 440 nm to about 500 nm, about 450 nm to about 500 nm, about 460 nm to about 500 nm, about 470 nm to about 500 nm, about 480 nm to about 500 nm, or about 490 nm to about 500 nm.

46. The average size of the nanoparticles of the present invention may be from about 100 nm to about 490 nm, from about 100 nm to about 480 nm, from about 100 nm to about 470 nm, from about 100 nm to about 460 nm, from about 100 nm to about 450 nm, from about 100 nm to about 440 nm, from about 100 nm to about 430 nm, from about 100 nm to about 420 nm, from about 100 nm to about 430 nm, from about 100 nm to about 420nm, about 100nm to about 410nm, about 100nm to about 400nm, about 100nm to about 390nm, about 100nm to about 380nm, about 100nm to about 370nm, about 100nm to about 360nm, about 100nm to about 350nm, about 1000nm to about 340nm, about 100nm to about 330nm, about 100nm to about 320nm, about 100nm to about 310nm, about 100nm to about 300nm, about 100nm to about 290nm, about 100nm to about 280nm, about 100nm to about 270nm, about 100nm to about 260nm, about 100 nm to about 250 nm, about 100 nm to about 240 nm, about 100 nm to about 230 nm, about 100 nm to about 220 nm, about 100 nm to about 210 nm, about 100 nm to about 2 The lipid nanoparticle of claim 1, wherein the diameter of the lipid nanoparticle may be about 100 nm, about 100 nm to about 190 nm, about 100 nm to about 180 nm, about 100 nm to about 170 nm, about 100 nm to about 160 nm, about 100 nm to about 150 nm, about 100 nm to about 140 nm, about 100 nm to about 130 nm, about 100 nm to about 120 nm, or about 100 nm to about 110 nm.

47. 2. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has a polydispersity index of about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.

25.

48. The lipid nanoparticle of claim 1, wherein the lipid nanoparticle has a polydispersity index of about 0.10 to about 0.

20.

49. The lipid nanoparticle of claim 1, wherein the zeta potential of the nanoparticle composition is from about −50 mV to about +10 mV, preferably from about −20 mV to about +5 mV.

50. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient.

51. A method for introducing an active agent into a cell, preferably wherein the cell is present in vivo, comprising contacting the cell with a lipid nanoparticle described in any one of claims 1 to 49, thereby introducing the active agent (e.g., a nucleic acid) into the cell.

52. 50. A method for in vivo delivery of an active agent, comprising administering to a subject in need thereof a lipid nanoparticle according to any one of claims 1 to 49, or a pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, thereby delivering the active agent in vivo.

53. 50. A method for treating or preventing a disease or condition in a subject in need thereof, comprising administering to the subject a lipid nanoparticle according to any one of claims 1 to 49 or a pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, thereby treating or preventing the disease or condition in the subject in need thereof.

54. 50. A method for producing a polypeptide of interest in a cell, preferably a mammalian cell, said method comprising contacting the cell with a lipid nanoparticle according to any one of claims 1 to 47, or a pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, wherein the active agent is an mRNA encoding the polypeptide of interest, said mRNA being capable of being translated in the cell to produce the polypeptide of interest.

55. 50. A method for delivering mRNA to a cell, preferably a mammalian cell, the method comprising administering to a subject a lipid nanoparticle according to any one of claims 1 to 49, or a pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, wherein the active agent is mRNA, thereby delivering the mRNA to the cell.

56. 52. The method of claim 51, wherein the cell is a cell present in the spleen.

57. 57. The method of claim 56, wherein the cells present in the spleen are cells of the spleen or cells derived from another part of the subject that are transported to the spleen.

58. 50. A method for delivering mRNA to a target tissue, comprising administering to a subject a lipid nanoparticle according to any one of claims 1 to 49, or a pharmaceutical composition comprising the lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, wherein the active agent is mRNA, thereby delivering the mRNA to the target tissue.

59. 56. The method of claim 55, wherein the target tissue is the spleen of a mammal.

60. 50. The lipid nanoparticles of any one of claims 1 to 49, or a pharmaceutical composition comprising the lipid nanoparticles of any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, in the manufacture of a medicament for treating or preventing a disease or condition in a subject in need thereof.

61. 50. A lipid nanoparticle according to any one of claims 1 to 49, or a pharmaceutical composition comprising a lipid nanoparticle according to any one of claims 1 to 49 and a pharmaceutically acceptable carrier, diluent or excipient, for use in the treatment or prevention of a disease or condition in a subject in need thereof.

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