Lipid nanoparticle therapeutics to evade immune responses

Abstract

The present invention relates to compositions and methods for the effective delivery of a therapeutic agent to a subject using a delivery vehicle that comprises a domain for evading an immune response in a subject, in some embodiments, the present invention relates to compositions and methods for the targeted delivery of a therapeutic agent to a subject using a delivery vehicle that comprises a domain for evading an immune response in a subject and a domain for targeting a specific cell type. The present invention also relates to methods of using the compositions of the present invention for the treatment of diseases and disorders, including the treatment of diseases and disorders in a subject suffering from an inflammatory or autoimmune disease or disorder.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 182,605, filed April 30, 2021, which is incorporated by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under AI045008 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] 2. Background of the Invention RNA-based agents are emerging as a potential therapeutic option distinct from DNA-based gene therapy approaches. For example, mRNA (without integration into the host genome and without the need for nuclear delivery) provides transient translation of the required sequence in cells (Weissman & Kariko Mol. Ther. 2015, 23, 1416-1417). While RNA-based therapeutics are still in their early stages, there are currently over 30 registered clinical trials for mRNA-based cancer treatments and vaccines (Pardi, et al. J. Control. Release 2015, 217, 345-351). As with all drugs, and especially biologic therapies, delivery of mRNA is a major challenge for most organs other than the liver (Shuvaev, et al., J. Control. Release 2015, 219, 576-595). Drug delivery systems (DDS) that include lipid nanoparticles (LNPs) have been used to package RNA and protect the contents en route to the site of action (Kauffman, et al., J. Control. Release 2016, 240, 227-234). However, targeted delivery of RNA in organs and tissues of interest and off-target effects remain formidable barriers to the biomedical translation and utility of this class of agents.

[0004] Thus, there is a need in the art for improved targeted therapeutics that can circumvent the immune response and minimize off-target effects. The present invention addresses this need. Summary of the Invention

[0005] In one embodiment, the invention relates to a composition for delivery of a therapeutic agent to a subject in need thereof, the composition comprising a therapeutic agent and a delivery vehicle, wherein the delivery vehicle comprises a moiety for inhibiting uptake of the composition by macrophages.

[0006] In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage is a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, or an activator of LILRB1 activity. In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage comprises a CD47 polypeptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or a fragment or variant thereof.

[0007] In one embodiment, the delivery vehicle further comprises a targeting moiety specific for binding to a target cell, hi one embodiment, the target cell is an endothelial cell, an immune cell, or a stem cell. In one embodiment, the therapeutic agent comprises at least one isolated nucleoside modified RNA molecule. In one embodiment, the at least one isolated nucleoside modified RNA comprises at least one pseudouridine or 1-methyl-pseudouridine. In one embodiment, the at least one isolated nucleoside modified RNA is a purified nucleoside modified RNA.

[0008] In one embodiment, the composition further comprises an adjuvant. In one embodiment, the delivery vehicle comprises a lipid nanoparticle (LNP). In one embodiment, at least one nucleoside modified RNA is encapsulated in the LNP. In one embodiment, the present invention relates to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering to a subject in need thereof a composition for delivering a therapeutic agent, the composition comprising a therapeutic agent and a delivery vehicle, wherein the delivery vehicle comprises a moiety for inhibiting uptake of the composition by macrophages in the subject.

[0009] In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage is a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, or an activator of LILRB1 activity. In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage comprises a CD47 polypeptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or a fragment or variant thereof.

[0010] In one embodiment, the subject suffers from an inflammatory or autoimmune disease or disorder, hi one embodiment, the therapeutic agent is an agent for the treatment of an inflammatory or autoimmune disease or disorder. In one embodiment, the composition is administered by a delivery route selected from intradermal, subcutaneous, inhalation, intranasal, and intramuscular. In one embodiment, the invention relates to a method of delivering a therapeutic agent to a target cell, comprising administering to a subject in need thereof a composition for delivering a therapeutic agent, the composition comprising a therapeutic agent and a delivery vehicle, wherein the delivery vehicle comprises a moiety for inhibiting uptake of the composition by macrophages in the subject and further comprises a targeting moiety specific for binding to the target cell.

[0011] In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage is a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, or an activator of LILRB1 activity. In one embodiment, the moiety for inhibiting uptake of a composition by a macrophage comprises a CD47 polypeptide comprising the sequence of SEQ ID NO:1, SEQ ID NO:2, or a fragment or variant thereof.

[0012] In one embodiment, the target cell is an endothelial cell, an immune cell, or a stem cell. In one embodiment, the therapeutic agent is an agent for the treatment of an inflammatory or autoimmune disease or disorder. In one embodiment, the composition is administered by intradermal, subcutaneous, inhalation, intranasal, or intramuscular delivery routes.

[0013] In one embodiment, the present invention relates to a pharmaceutical composition for in vivo delivery of lipid nanoparticles (LNPs) to non-hepatic cells of a subject, while avoiding delivery to hepatic cells, wherein the LNPs comprise a pegylated lipid conjugated to an active CD47 polypeptide and a therapeutic agent.

[0014] In one embodiment, the LNPs further comprise a pegylated lipid attached to the binding moiety. In one embodiment, the binding moiety is a whole antibody or an antigen-binding fragment thereof. In one embodiment, the LNPs further comprise a non-attached pegylated lipid. In one embodiment, the active CD47 polypeptide comprises SEQ ID NO:1 or SEQ ID NO:2.

[0015] In one embodiment, the present invention relates to a method of delivering lipid nanoparticles (LNPs) to non-hepatic cells of a subject in vivo while avoiding delivery to hepatic cells, comprising administering a pharmaceutical composition for in vivo delivery of LNPs to non-hepatic cells of a subject while avoiding delivery to hepatic cells, wherein the LNPs comprise a pegylated lipid conjugated to an active CD47 polypeptide and a therapeutic agent. In one embodiment, the LNPs further comprise a pegylated lipid conjugated to a binding moiety. In one embodiment, the binding moiety is a whole antibody or an antigen-binding fragment thereof. In one embodiment, the LNPs further comprise a non-bound pegylated lipid. In one embodiment, the active CD47 polypeptide comprises SEQ ID NO:1 or SEQ ID NO:2. In one embodiment, the administration comprises intravenous administration.

[0016] In one embodiment, the inflammatory response is reduced or not exacerbated compared to LNP lacking an active CD47 polypeptide, PEG shielding, or a combination thereof, hi some embodiments, toxicity is reduced or not exacerbated compared to LNP lacking an active CD47 polypeptide, PEG shielding, or a combination thereof.

[0017] In one embodiment, the percentage of an administered therapeutic agent that reaches a target cell or tissue is enhanced compared to LNPs lacking an active CD47 polypeptide, PEG shielding, or a combination thereof, hi one embodiment, the physiologically effective dosage is reduced compared to LNPs lacking an active CD47 polypeptide, PEG shielding, or a combination thereof.

[0018] The details of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, it being understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0019] [Figure 1]Figure 1 shows a schematic diagram of the preparation method of CD47-modified targeted LNP. Schematic diagram of targeted CD47-modified mRNA-loaded LNP. A novel double post-insertion technique allows the functionalization of LNP with minimal "self" peptides, providing avoidance of macrophage uptake in conjunction with antibody targeting moieties. Targeted CD47-modified LNP provides an mRNA delivery platform with high tissue / cell targeting characteristics, minimal off-target uptake, and high safety. [Diagram 2] Figures 2A-D show data demonstrating that CD47 modification on LNPs significantly reduces non-specific uptake / mRNA translation. Figure 2A shows data demonstrating that RAW264.7 macrophage cells were incubated with mRNA-LNPs and CD47 / mRNA-LNPs (mRNA-LNPs decorated with CD47) and microscopy was performed 48 hours later. When compared to mRNA-LNPs, CD47 / mRNA-LNPs result in significantly lower mRNA translation in RAW264.7 macrophages in vitro. Figure 2B shows data demonstrating that radiolabeled CD47 / mRNA-LNPs and mRNA-LNPs were injected iv into mice and tissues were harvested 1 hour after injection. CD47 / mRNA-LNPs resulted in significantly lower tissue uptake in all organs, especially the liver, while increasing in the blood. Figure 2C shows the localization ratios showing the radioactivity in each organ normalized to the blood concentration and reaffirms the results of Figure 2B. Figure 2D shows data demonstrating that CD47 / mRNA-LNP and mRNA-LNP-encapsulated nucleoside-modified luciferase mRNA were injected iv into mice at 0.3 mg mRNA / kg and tissues were harvested 4 hours after injection. CD47 / mRNA-LNP gives signals very close to zero in all organs, especially the liver, when compared to mRNA-LNP. [Diagram 3]Figure 3 shows data demonstrating that CD47 / mRNA-LNPs recruit significantly fewer PBMCs compared to mRNA-LNPs. Total PBMC counts were measured 30 minutes after iv injection of CD47 / mRNA-LNPs and mRNA-LNPs. Significantly lower PBMC recruitment was observed at the time of iv injection of CD47-modified LNPs compared to LNPs lacking CD47. [Figure 4] Figures 4A-C show data demonstrating increased targeting efficiency when combined with CD47. Figure 4A shows data demonstrating that mice were iv injected with radiolabeled CD31-targeted mRNA-LNP and CD47 / CD31-targeted mRNA-LNP (also CD47-modified CD31-targeted mRNA-LNP) and tissues were harvested 1 hour after injection. CD47 / CD31-targeted mRNA-LNP results in higher lung uptake when compared to CD31-targeted mRNA-LNP. Figure 4B shows the localization ratio showing radioactivity in the lung normalized to blood concentration and reaffirms the results of Figure 4A. Figure 4C shows data demonstrating that mice were iv injected with CD47 / CD31-targeted mRNA-LNP and CD31-targeted mRNA-LNP encapsulated nucleoside-modified luciferase mRNA at 0.3 mg mRNA / kg and tissues were harvested 4 hours after injection. CD47 / CD31 targeted mRNA-LNPs yielded very close to zero signal in the liver but a significantly higher signal in the lung when compared to CD31 targeted mRNA-LNPs. [Diagram 5]Figures 5A-C show data demonstrating that CD47 modification enhances T cell targeting efficiency. Figure 5A shows data demonstrating that CD4-targeted mRNA-LNP and CD47 / CD4-targeted mRNA-LNP encapsulated nucleoside-modified luciferase mRNA were injected iv into mice at 0.3 mg mRNA / kg and tissues were harvested 4 hours after injection. CD47 / CD4-targeted mRNA-LNP produced a significantly lower signal in the liver but a significantly higher signal in the spleen (target organ) when compared to CD4-targeted mRNA-LNP. CD47 / CD4-targeted mRNA-LNP and CD4-targeted mRNA-LNP encapsulated nucleoside-modified Cre mRNA were injected iv into Ai6 mice at 0.3 mg mRNA / kg and lymph nodes (Figure 5B) and spleens (Figure 5C) were harvested 24 hours after injection. Ai6 as a mouse reporter model is engineered with a Cre reporter allele designed with a loxP-flanked STOP cassette that prevents transcription of a CAG promoter-driven green fluorescent reporter gene (ZsGreen1) inserted into the Gt(ROSA)26Sor locus. CD47 / CD4-targeted mRNA-LNPs result in higher ZsGreen expression in target cell populations (CD3+CD8 cells) in both lymph nodes (Figure 5B) and spleen (Figure 5C) when compared to CD4-targeted mRNA-LNPs. [Figure 6] Figures 6A and 6B show data demonstrating that proinflammatory cytokines are elevated after IV treatment of LNP-mRNA (IV-LPS) in a systemic mouse model of inflammation. The proinflammatory cytokines IL-6 in plasma (Figure 6A) and MIP-2 in liver homogenates (Figure 6B) were significantly elevated after administration of LNP-mRNA to iv LPS-treated mice. This phenomenon was termed exacerbation of inflammation. [Figure 7]Figures 7A and 7B show data demonstrating enhanced LNP uptake by monocytes / macrophages in LPS-treated mice. Figure 7A shows data demonstrating cell type distribution in untreated versus LPS-treated mice. The monocyte / macrophage population that takes up LNP-mRNA is approximately three-fold greater in the presence of LPS. Figure 7B shows data demonstrating positive cell type distribution for LNP. [Figure 8] Figures 8A and 8B show data demonstrating that depletion of macrophages using clodronate reduces systemic proinflammatory markers. Depletion of macrophages by clodronate administration significantly reduced the levels of proinflammatory cytokines (Figure 8A) IL-6 in the blood and (Figure 8B) MIP-2 in liver homogenates in LPS-treated mice receiving LNP-mRNA. [Figure 9] Figure 9 shows data demonstrating that reduced macrophage uptake by CD47-modified LNPs alleviates systemic pro-inflammatory markers in an LPS model of systemic inflammation. In the context of inflammation, iv treated LPS mice, CD47 / mRNA-LNPs showed significantly lower pro-inflammatory cytokine IL-6 when compared to mRNA-LNPs. The data demonstrated that with CD47 / mRNA-LNPs, we can improve the safety profile and use mRNA-LNPs in inflammatory conditions since there is a reduced pro-inflammatory response using CD47-optimized LNPs when compared to LNPs that do not contain CD47. [Figure 10]Figure 10 shows data demonstrating that optimized mRNA-D47 / LNP minimizes acute phase response in liver by RNA-Seq analysis. Figure 10A provides data demonstrating that there is significant downregulation of 30 genes ranging from -18.40 to -2.11 fold in CD47 / mRNA-LNP treated and untreated mice compared to unmodified control NP0. Of these genes, 8 are directly involved in the APR proinflammatory protein family. In the same RNA-seq dataset, bioinformatics principal component analysis (PCA) (Figure 10B) was performed with significantly differentially expressed genes. As expected, the results show that plot RNA-seq data from CD47 / mRNA-LNP clusters are closer to untreated mice with similar behavior, whereas unmodified mRNA-LNPs are located on the opposite side of the matrix. This data validates the transcriptional similarity of untreated control to CD47 / mRNA-LNP treated liver tissue. [Figure 11] Figure 11 shows data demonstrating protein corona analysis of optimized CD47 / mRNA-LNPs in mouse serum. The protein corona formed around CD47-modified LNPs is completely different from that of unmodified LNPs. ApoE, an acute phase protein, a protein involved in inflammatory responses and apoptosis, was found in the protein corona of unmodified LNPs but not those modified with CD47. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention The present invention relates to a composition for effective delivery of a therapeutic agent comprising a delivery vehicle, wherein the delivery vehicle comprises at least one moiety or domain for evading an immune response. In one embodiment, the delivery vehicle further comprises a targeting domain or moiety for delivery of the therapeutic agent to a target cell.

[0021] In one embodiment, the domain for evading the immune system comprises a moiety for preventing macrophage uptake. In one embodiment, the moiety for preventing macrophage uptake comprises a moiety for inhibiting phagocytosis. In one embodiment, the moiety for preventing macrophage uptake comprises a CD47 moiety, PD-L1, CD24, a polyglutamic acid peptide, or the beta-2-microglobulin subunit of the major histocompatibility class 1 complex.

[0022] In one embodiment, the targeting domain specifically binds to a marker of a cell type of interest. For example, in one embodiment, the targeting domain directs the vehicle to endothelial cells, T cells, stem cells, or another specific cell type of interest. In certain embodiments, the delivery vehicle is a lipid nanoparticle comprising at least one lipid bound to a domain for immune system evasion. In one embodiment, the delivery vehicle is a lipid nanoparticle comprising at least one lipid bound to a domain for immune system evasion and further comprising at least one lipid bound to a targeting domain.

[0023] The present invention also relates to methods of using the compositions described herein for targeted delivery of therapeutic agents, as well as methods of treating a disease or disorder in a subject suffering from an inflammatory condition using the compositions described herein that circumvent an immune response in the subject.

[0024] definition Unless otherwise defined, 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. As used herein, each of the following terms has the meaning associated with it in this section.

[0025] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0026] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1 from the stated value, as is suitable for carrying out the disclosed methods within that range.

[0027] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen or epitope. An antibody may be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is usually a tetramer of an immunoglobulin molecule. The antibodies of the present invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0028] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-specific determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0029] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation.

[0030] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa and lambda light chains refer to the two major antibody light chain isotypes.

[0031] The term "synthetic antibody" as used herein refers to an antibody generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be taken to mean an antibody generated by synthesis of a DNA molecule encoding the antibody, which DNA molecule expresses the antibody protein or amino acid sequence that specifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence technology available and well known in the art. The term should also be taken to mean an antibody generated by synthesis of an RNA molecule encoding the antibody, which RNA molecule expresses the antibody protein or amino acid sequence that specifies the antibody, where the RNA is obtained by transcription of (synthetic or cloned) DNA or other techniques available and well known in the art.

[0032] A "disease" refers to a state of health in an animal in which the animal is unable to maintain homeostasis, and if the disease is not corrected, the animal's health will continue to deteriorate. In contrast, an animal "disorder" refers to a state of health in which the animal is able to maintain homeostasis, but the animal's health is not better than it would be in the absence of the disorder. Ignoring a disorder does not necessarily result in a further decline in the animal's health.

[0033] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0034] The term "physiologically effective dosage" refers to an amount of an agent that produces a measurable biological or physiological effect in a recipient subject that is related to the activity of the agent(s). A physiologically effective dosage will vary depending on the compound, the age, weight, etc. of the subject receiving the agent, and the biological or physiological effect being measured.

[0035] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of polymers and macromolecules in biological processes and the biological properties resulting therefrom, either with a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or with a defined sequence of amino acids. Thus, a gene codes for a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually listed in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, are said to code for the protein or other product of that gene or cDNA.

[0036] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors that contain sufficient cis-acting elements for expression, as well as other elements for expression, can be supplied by a host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0037] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both of the two sequences being compared has the same base or amino acid monomer subunit, for example, if two DNA molecules have an adenine at each position, then the molecules are homologous at that position. The percentage of homology between two sequences is the number of positions where the two sequences match or are homologous divided by the total number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences match or are different, then the two sequences are 60% homologous. As an example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, the comparison is performed by aligning the two sequences to obtain the maximum homology.

[0038] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or it may exist in a non-native environment, such as, for example, a host cell.

[0039] In the context of the present invention, the following abbreviations are used for commonly occurring nucleosides (nucleobases linked to a ribose or deoxyribose sugar via an N-glycosidic bond): "A" refers to adenosine, "C" refers to cytidine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0040] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that encode the same amino acid sequence, degenerate versions of each other. Also, the term nucleotide sequence encoding a protein or RNA may include introns to the extent that the nucleotide sequence encoding the protein may contain introns in some versions.

[0041] The term "modulate" as used herein means to detectably increase or decrease the level of a response in a subject compared to the level of the response in the subject in the absence of a treatment or compound and / or compared to the level of the response in an otherwise identical but untreated subject. The term encompasses disrupting and / or affecting an innate signal or response to result in a beneficial therapeutic response in a subject, preferably a human.

[0042] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are modified versions of each other that encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may include introns. Additionally, the nucleotide sequence may include modified nucleosides that are translatable by the translational machinery in a cell. Examples include mRNAs in which some or all of the uridines are replaced with pseudouridine, 1-methylpseudouridine, or other modified nucleosides.

[0043] The term "operably linked" refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary, join two protein coding regions in the same reading frame.

[0044] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal, or cells thereof, in vitro or in situ, suitable for the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0045] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Thus, nucleic acid and polynucleotide as used herein are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide and can be hydrolyzed into monomeric "nucleotides". The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, including, but not limited to, recombinant means, i.e., cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR™, etc., or synthetic means.

[0046] In some cases, the polynucleotide or nucleic acid of the present invention is a "nucleoside-modified nucleic acid", which refers to a nucleic acid that contains at least one modified nucleoside. A "modified nucleoside" refers to a nucleoside that has been modified. For example, more than 100 different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0047] In some embodiments, "pseudouridine" is, in another embodiment, m 1 acp 3 (1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine). In another embodiment, the term refers to 1 In another embodiment, the term refers to Ψm (2'-O-methylpseudouridine). In another embodiment, the term refers to m 5 D (5-methyldihydrouridine). In another embodiment, the term refers to m 3Ψ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to the monophosphate, diphosphate, or triphosphate of any of the pseudouridines listed above. In another embodiment, the term refers to other pseudouridines known in the art. Each possibility represents a separate embodiment of the present invention.

[0048] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up the sequence of a protein or peptide. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, also commonly referred to in the art as peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0049] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence, such as the promoters recognized by bacteriophage RNA polymerase and used to generate mRNA by in vitro transcription.

[0050] The term "specifically binds" as used herein with respect to affinity ligands, particularly antibodies, refers to an antibody that recognizes a particular antigen in a sample but does not substantially recognize or bind other molecules. For example, an antibody that specifically binds to an antigen of one species may also bind to one or more other antigens. However, such cross-species reactivity does not in itself change the classification of the antibody's specificity. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of that antigen. However, such cross-reactivity does not in itself change the classification of the antibody's specificity. In some examples, the terms "specific binding" or "specific binding" may be used to mean that the interaction of an antibody, protein, or peptide with a second chemical species is due to the presence of a particular structure (e.g., an antigenic determinant or epitope) of the chemical species. For example, antibodies generally recognize and bind to specific protein structures, but not proteins. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free unlabeled A) in a reaction involving labeled "A" and an antibody will reduce the amount of labeled A that binds to the antibody.

[0051] As used herein, the term "therapeutic" refers to treatment and / or prophylaxis. A therapeutic effect is obtained by suppressing, reducing, ameliorating, or eradicating at least one sign or symptom of a disease or disorder.

[0052] The term "therapeutically effective amount" refers to an amount of a compound of interest that elicits the biological or medical response in a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of a compound that is sufficient when administered to prevent or alleviate to some extent the onset of one or more signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease being treated and its severity, age, weight, etc.

[0053] As used herein, the term "treating" a disease means reducing the frequency or severity of at least one sign or symptom of the disease or disorder experienced by a subject.

[0054] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0055] As used herein, the term "under transcriptional control" or "operably linked" means that a promoter is in the correct position and orientation relative to a polynucleotide to control initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0056] A "vector" is a complex of substances that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the inside of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides bound to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

[0057] "Alkyl" means an alkyl group containing 1 to 24 carbon atoms (C1-C 24 Alkyl), 1 to 12 carbon atoms (C1-C 12"C-C alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, saturated or unsaturated (i.e., containing one or more double and / or triple bonds), having from 1 to 8 carbon atoms (C1-C8 alkyl) or from 1 to 6 carbon atoms (C1-C6 alkyl), and attached to the remainder of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise specified, alkyl groups are optionally substituted.

[0058] An "alkylene" or "alkylene chain" is saturated or unsaturated (i.e., contains one or more double bonds (alkenylene) and / or triple bonds (alkynylene)) and can have, for example, 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C15 alkylene), 1 to 12 carbon atoms (C1-C 12 (C1-C8 alkylene), 1-8 carbon atoms (C1-C6 alkylene), 1-6 carbon atoms (C2-C4 alkylene), 2-4 carbon atoms (C2-C4 alkylene), 1-2 carbon atoms (C1-C2 alkylene), a straight or branched divalent hydrocarbon chain consisting solely of carbon and hydrogen atoms that attaches the remainder of the molecule to a radical group, e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, etc. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless otherwise specified in this specification, alkylene chains may be optionally substituted.

[0059] "Cycloalkyl" or "carbocyclic ring" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having from 3 to 15 carbon atoms, preferably from 3 to 10 carbon atoms, and which is saturated or unsaturated and attached to the remainder of the molecule by a single bond. Examples of monocyclic radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic radicals include adamantyl, norbornyl, decalinyl, 7,7-dimethylbicyclo[2.2.1]heptanyl, and the like. Unless otherwise specified, cycloalkyl groups are optionally substituted.

[0060] "Cycloalkylene" is a divalent cycloalkyl group. Unless stated otherwise in the specification, a cycloalkylene group may be optionally substituted.

[0061] "Heterocyclyl" or "heterocyclic ring" refers to a stable 3- to 18-membered non-aromatic ring radical which consists of 2 to 12 carbon atoms and 1 to 6 heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, the heterocyclyl radical can be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which can include fused or bridged ring systems, the nitrogen, carbon, or sulfur atoms in the heterocyclyl radical can be optionally oxidized, the nitrogen atoms can be optionally quaternized, and the heterocyclyl radical can be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Unless otherwise specified, heterocyclyl groups may be optionally substituted.

[0062] As used herein, "substituted" means that at least one hydrogen atom has been replaced with a halogen atom, such as F, Cl, Br, and I; an oxo group (=O); a hydroxyl group (-OH); an alkoxy group (-ORa, where Ra is C1-C 12 alkyl or cycloalkyl), carboxyl group (-OC(=O)Ra or -C(=O)ORa, where Ra is H, C1-C 12 alkyl or cycloalkyl; an amine group (-NRaRb, where Ra and Rb are each independently H, C1-C 12 alkyl or cycloalkyl); C1-C 12alkyl groups; and cycloalkyl groups, meaning any of the above groups (e.g., alkyl, cycloalkyl, or heterocyclyl) substituted by a bond to a non-hydrogen atom. In some embodiments, the above substituents are C1-C 12 In other embodiments, the substituent is an alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0063] "Optionally" or "optionally" (e.g., optionally substituted) means that the subsequently described circumstance event may or may not occur, and that the description encompasses cases where the described event or circumstance occurs as well as cases where they do not occur. For example, "optionally substituted alkyl" means that the alkyl radical may or may not be substituted, and that the description encompasses both substituted alkyl radicals and alkyl radicals that have no substituents.

[0064] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0065] explanation The present invention relates in part to compositions and methods for targeted delivery of delivery vehicles with enhanced efficacy and reduced off-target effects. In one aspect, the present invention relates to compositions comprising a delivery vehicle linked to a domain for evasion of the host immune system. In one aspect, the present invention relates to compositions comprising a delivery vehicle linked to a domain for evasion of the host immune system and further linked to a targeting domain.

[0066] In one embodiment, the domain for immune system evasion comprises a moiety for reducing or preventing macrophage uptake. In some embodiments, the domain for immune system evasion can be a peptide, protein, or peptidomimetic. Exemplary moieties that can be incorporated into the delivery vehicles of the present invention to prevent macrophage uptake include, but are not limited to, a CD47 moiety, PD-L1, CD24, a polyglutamic acid peptide, or the beta-2-microglobulin subunit of the major histocompatibility class 1 complex (β2M) or a functional fragment thereof, or a combination thereof. In one embodiment, the domain for immune response evasion comprises a CD47 polypeptide comprising a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment or variant thereof.

[0067] In some embodiments, the domain for immune system evasion binds to the macrophage inhibitory receptor SIRPα and activates SIRPα signaling, thereby reducing or preventing macrophage uptake of the therapeutic agent. Thus, in one embodiment, the present invention relates to a composition comprising a moiety for activating SIRPα signaling. In some embodiments, the moiety for activating SIRPα signaling is a nucleic acid molecule, a small molecule, a protein, a peptide, or a peptidomimetic.

[0068] In some embodiments, the domain for immune system evasion binds to PD-1 on macrophages and activates PD-1 signaling, thereby reducing or preventing macrophage uptake of the therapeutic. Thus, in one embodiment, the invention relates to a composition comprising a moiety for activating PD-1 signaling. In some embodiments, the moiety for activating PD-1 signaling is a nucleic acid molecule, a small molecule, a protein, a peptide, or a peptidomimetic.

[0069] In some embodiments, the domain for immune system evasion binds to the Siglec-10 receptor on macrophages and activates Siglec-10 receptor signaling, thereby reducing or preventing macrophage uptake of the therapeutic agent. Thus, in one embodiment, the present invention relates to a composition comprising a moiety for activating Siglec-10 signaling. In some embodiments, the moiety for activating Siglec-10 signaling is a nucleic acid molecule, a small molecule, a protein, a peptide, or a peptidomimetic.

[0070] In some embodiments, the domain for immune system evasion binds to the inhibitory receptor LILRB1 on macrophages and activates LILRB1 receptor signaling, thereby reducing or preventing macrophage uptake of therapeutic agents. Therefore, in one embodiment, the present invention relates to a composition comprising a moiety for activating LILRB1 signaling. In some embodiments, the moiety for activating LILRB1 signaling is a nucleic acid molecule, a small molecule, a protein, a peptide, or a peptidomimetic.

[0071] Phagocytosis inhibitors The present invention relates to the prevention and treatment of diseases or disorders by administration of a therapeutic agent for the treatment of the disease or disorder formulated with a delivery vehicle expressing a moiety for evasion of an immune response. In one embodiment, the moiety for evasion of an immune response comprises an inhibitor of phagocytosis. In one embodiment, the composition comprises a delivery vehicle conjugated to a phagocytosis inhibitor that binds to an inhibitory cell surface molecule of macrophages and provides an inhibitory signal, thereby preventing phagocytosis of the delivery vehicle and associated therapeutic molecule.

[0072] In some embodiments, the moiety for evading an immune response comprises a CD47 polypeptide, an active CD47 polypeptide fragment, or an activator of SIRPα activity. In some embodiments, the moiety for evading an immune response comprises a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, or an activator of PD-1 activity. In some embodiments, the moiety for evading an immune response comprises a CD24 polypeptide, an active CD24 polypeptide fragment, or an activator of Siglec-10 activity. In some embodiments, the moiety for evading an immune response comprises a polyglutamic acid peptide. In some embodiments, the moiety for evading an immune response comprises a β2M polypeptide, an active β2M polypeptide fragment, or an activator of LILRB1 activity.

[0073] In one embodiment, the moiety for evading an immune response comprises a CD47 polypeptide comprising a sequence set forth in SEQ ID NO:1 or SEQ ID NO:2, or a fragment or variant thereof.

[0074] There are two main mechanisms by which LNPs are taken up by the liver. Hepatocytes can take up LNPs by receptor-mediated processes, for example, based on interaction with ApoE. This can be avoided by shielding the LNP surface with pegylated lipids. Lipids can bind targeting moieties and / or active CD47 polypeptides can contribute to shielding. Kupffer cells (which are macrophages) in the liver can take up LNPs by phagocytosis. This is avoided by SIRPα signaling activation (e.g., by binding active CD47 polypeptides). Thus, LNPs with sufficient surface shielding in combination with phagocytosis inhibitors can substantially reduce or even completely avoid uptake by the liver. This offers several advantages. The liver is the first destination of systemically administered LNPs. By reducing or avoiding liver uptake, more administered LNPs are available to be taken up by target cells, regardless of whether targeting is achieved by lipid composition or encapsulation of targeting moieties in the LNP, enhancing the efficiency of transfection of target cells or reducing the required dosage. This also reduces lipid exposure to and accumulation in the liver, and reduces or avoids the toxic and / or proinflammatory effects that lipid components may have on the liver. Finally, in some cases, therapeutic agents may have deleterious or undesirable effects in the liver. By avoiding delivery to the liver, these effects are reduced or eliminated.

[0075] Thus, in some embodiments, the invention relates to compositions and methods for delivering LNPs to non-hepatic cells of a subject in vivo while avoiding delivery to hepatic cells, where the LNPs comprise a pegylated lipid conjugated to an activator of SIRPα activity and a therapeutic agent. In some embodiments, the activator of SIRPα activity is a CD47 polypeptide, or an active CD47 polypeptide fragment. In some embodiments, the LNPs further comprise a pegylated lipid conjugated to a targeting moiety. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. In some embodiments, the LNPs further comprise an unconjugated pegylated lipid.

[0076] Protein activation can be assessed using a variety of methods, including those disclosed herein as well as methods known in the art or that will be developed in the future. That is, based on the disclosure provided herein, one of skill in the art will appreciate that enhanced activity of SIRPα, PD-1, Siglec-10 or LILRB1 activity can be readily assessed using methods to assess the level of phagocytosis of a composition comprising an activator of the present invention.

[0077] Activators of the present invention include, but are not limited to, chemicals, proteins, peptidomimetics, antibodies, and nucleic acid molecules. Based on the disclosure provided herein, one skilled in the art will readily understand that SIRPα, PD-1, Siglec-10, or LILRB1 activators encompass chemicals that enhance SIRPα, PD-1, Siglec-10, or LILRB1 signaling, activity, or the like. In some embodiments, the activity is to reduce or inhibit phagocytosis. Furthermore, SIRPα, PD-1, Siglec-10, or LILRB1 activators encompass chemically modified compounds and derivatives that are well known to those skilled in the chemical arts.

[0078] Moreover, one of skill in the art, when armed with this disclosure and the methods exemplified herein, will appreciate that the present invention encompasses future discovered phagocytosis inhibitors (which have the physiological consequence of preventing or reducing the level of phagocytosis) that may be identified by criteria well known in the art of pharmacology. As such, the present invention is not limited in any way to any particular activators or inhibitors exemplified or disclosed herein; rather, the present invention encompasses those activators or inhibitors known in the art and discovered in the future that will be appreciated by those of skill in the art to be useful.

[0079] Methods for identifying and producing CD47, PD-L1, CD24, polyglutamic acid peptide, β2M polypeptides or functional fragments thereof are well known to those of skill in the art and include, but are not limited to, obtaining the polypeptide from a naturally occurring source. Alternatively, CD47, PD-L1, CD24, polyglutamic acid peptide, β2M polypeptides or functional fragments thereof may be chemically synthesized. Furthermore, one of skill in the art will be well aware, based on the teachings provided herein, that CD47, PD-L1, CD24, polyglutamic acid peptide, β2M polypeptides or functional fragments thereof may be obtained from genetically modified organisms. Compositions and methods for chemically synthesizing polypeptide molecules and obtaining them from natural sources are well known and described in the art.

[0080] targeting molecule In various embodiments, the delivery vehicle of the present invention comprises a targeting domain that binds to a cell surface molecule of a target cell of interest, including, but not limited to, an endothelial cell, a T cell, or a stem cell. In certain embodiments, the targeting domain binds to a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell. In one embodiment, the composition comprises a delivery vehicle that binds to a targeting domain that binds a cell surface molecule of a target cell of interest, thereby directing the composition to the target cell.

[0081] For example, in various embodiments, with respect to compositions targeting endothelial cells, the targeting domain binds to a molecule selected from the group including, but not limited to, interferon-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, angiotensin-converting enzyme (ACE), aminopeptidase P (APP), plasma membrane vesicle protein 1 (PV1), P-selectin, VE-cadherin, receptors for cytokines, plasma proteins, and microorganisms.

[0082] In some embodiments, the targeted delivery vehicle of the present invention comprises a targeting moiety that binds to a surface molecule of an immune cell, including, but not limited to, a T cell (including killer T cell, helper T cell, regulatory T cell, and gamma delta T cell), a natural killer (NK) cell, an antigen-presenting cell, a dendritic cell, a B cell, or a Langerhans cell. In some embodiments, the targeted delivery vehicle comprises a targeting moiety that binds to a surface molecule of a T cell. Exemplary targeting moieties that can be used to target the compositions of the present invention to T cells include, but are not limited to, anti-CD4, anti-CD8, anti-CD5, anti-CD3, or anti-CD25 targeting ligands. Exemplary compositions and methods for targeting T cells in vivo are described in WO2022 / 081694, WO2022 / 081699, and WO2022 / 081702, which are incorporated by reference in their entirety.

[0083] In some embodiments, the targeted delivery vehicle of the present invention comprising a targeting moiety that binds to a surface molecule of a stem cell includes, but is not limited to, a somatic stem cell, a mesenchymal stem cell, or a hematopoietic stem cell. Exemplary surface molecules of stem cells include, but are not limited to, CD34, CD117, CD90, CD133, CD105, ABCG2, bone morphogenetic protein receptor (BMPR), CD44, Sca-1, Thy-1, CD133, alkaline phosphatase, alpha-fetoprotein, CD70, CD90, CD105, CD73, Stro-1, SSEA-4, CD271, CD146, GD2, SSEA-3, SUSD2, Stro-4, MSCA-1, CD56, CD200, PODXL, CD13, CD29, CD44, and CD10. Exemplary compositions and methods for targeting stem cells in vivo are described in International Patent Application Nos. PCT / US22 / 26933 and PCT / US22 / 26981, which are incorporated by reference in their entireties.

[0084] However, the present invention is not limited to vehicles that are directed to endothelial cells, T cells, or stem cells. Rather, the present invention encompasses delivery vehicles that include a targeting domain that directs the vehicle to any particular target cell, as this is mediated by the binding of the targeting domain to a specific marker. In some embodiments, the vehicle is targeted to the specific treatment site required. For example, the targeting domain can be specifically directed to the site of inflammation.

[0085] The invention also relates in part to a method of treating a disease or disorder in a subject in need thereof, the method comprising administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response, hi some embodiments, the method comprises administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response and further linked to a targeting domain.

[0086] In some embodiments, the invention provides a method for treating an inflammatory disease or disorder in a subject in need thereof, the method comprising administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response, hi some embodiments, the method comprises administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response and further linked to a targeting domain.

[0087] In some embodiments, the invention provides a method for treating an inflammatory or non-inflammatory disease or disorder in a subject with an ongoing or prior diagnosis of an inflammatory disease or disorder, the method comprising administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response, hi some embodiments, the method comprises administering a composition comprising a delivery vehicle linked to a domain for evasion of an immune response and further linked to a targeting domain.

[0088] Delivery Vehicle In some embodiments, delivery vehicles include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use in vitro and in vivo as a delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0089] The use of lipid formulations is contemplated for introducing at least one agent into host cells (in vitro, ex vivo or in vivo). In another embodiment, at least one agent may be associated with a lipid. The at least one agent associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule attached to both the liposome and the oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included as a lipid suspension, included in or complexed with micelles, or otherwise associated with lipids. The lipid, lipid / nucleic acid or lipid / expression vector associated compositions are not limited to a particular structure in solution. For example, they may exist in a bilayer structure such as micelles, or in a "disintegrated" structure. They may also simply be interspersed in a solution and form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be natural or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons, such as fatty acids, alcohols, amines, aminoalcohols, aldehydes, and their derivatives.

[0090] Lipids suitable for use are available from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") is available from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") is available from K & K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components first undergo self-reorganization and then form a closed structure, trapping water and solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions whose structure in solution differs from the normal vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as a heterogeneous collection of lipid molecules. Lipofectamine-agent complexes are also contemplated.

[0091] In one embodiment, the delivery of at least one active substance comprises any suitable delivery method, including the exemplary delivery method described herein.In certain embodiments, the delivery of at least one active substance to a subject comprises mixing at least one active substance with a transfection reagent before the contact step.In another embodiment, the method of the present invention further comprises administering at least one active substance together with a transfection reagent.In another embodiment, the transfection reagent is a cationic lipid reagent.

[0092] In another embodiment, the transfection reagent is a lipid-based transfection reagent. In another embodiment, the transfection reagent is a protein-based transfection reagent. In another embodiment, the transfection reagent is a polyethylenimine-based transfection reagent. In another embodiment, the transfection reagent is calcium phosphate. In another embodiment, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In another embodiment, the transfection reagent is any other transfection reagent known in the art.

[0093] In another embodiment, the transfection reagent forms liposomes. In another embodiment, the liposomes enhance intracellular stability, enhance uptake efficiency, and improve biological activity. In another embodiment, the liposomes are hollow spherical vesicles composed of lipids arranged in a manner similar to the lipids that compose the cell membrane. In some embodiments, the liposomes contain an internal aqueous space for entrapment of water-soluble compounds. In another embodiment, the liposomes can deliver at least one agent to cells in an active form.

[0094] In one embodiment, a composition comprises a lipid nanoparticle (LNP) and at least one agent.

[0095] The term "lipid nanoparticle" refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) that includes one or more lipids. In various embodiments, the particle includes a lipid of formula (I), (II), or (III). In some embodiments, the lipid nanoparticle is included in a formulation that includes at least one agent described herein. In some embodiments, such lipid nanoparticles include one or more excipients selected from cationic lipids (e.g., lipids of formula (I), (II), or (III)) and neutral lipids, charged lipids, steroids, and polymer-bound lipids (e.g., pegylated lipids, such as pegylated lipids of structure (IV), such as compound Iva). In some embodiments, the at least one agent is encapsulated within the lipid portion of the lipid nanoparticle or within the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects, such as, for example, a deleterious immune response, elicited by mechanisms of the host organism or cells.

[0096] In various embodiments, the lipid nanoparticles may be about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or have an average diameter of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm and are substantially non-toxic. In one embodiment, the lipid nanoparticles have an average diameter of about 83 nm. In one embodiment, the lipid nanoparticles have an average diameter of about 102 nm. In one embodiment, the lipid nanoparticles have an average diameter of about 103 nm. In some embodiments, the lipid nanoparticles are substantially non-toxic. In certain embodiments, the at least one agent, when present in the lipid nanoparticles, is resistant to degradation by intracellular or intercellular enzymes in aqueous solution.

[0097] LNPs may include any lipid capable of forming a particle having at least one agent attached thereto or in which at least one agent is encapsulated. The term "lipid" refers to a group of organic compounds that are derivatives (e.g., esters) of fatty acids and are generally characterized as being insoluble in water but soluble in many organic solvents. Lipids are generally divided into at least three classes: (1) "simple lipids," including fats, oils, and waxes; (2) "complex lipids," including phospholipids and glycolipids; and (3) "derivative lipids," such as steroids. In one embodiment, the LNPs comprise one or more cationic lipids and one or more stabilizing lipids, including neutral lipids and pegylated lipids.

[0098] In one embodiment, the LNP comprises a cationic lipid. As used herein, the term "cationic lipid" refers to a lipid that is cationic or cationic (protonated) when the pH is lower than the pK of the ionizable group of the lipid, but becomes gradually more neutral at higher pH values. At pH values ​​below the pK, the lipid can bind to negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that becomes positively charged when the pH is decreased.

[0099] In certain embodiments, cationic lipids include any of a number of lipid species that carry an overall positive charge at a selected pH, such as physiological pH. Such lipids include, but are not limited to, the following: N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol ), N-(1-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamidoglycylcarboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercial formulations of cationic lipids that can be used in the present invention are available. Examples include: LIPOFECTIN® (a commercially available cationic liposome containing DOTMA and 1,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), GIBCO / BRL, Grand Island, NY); LIPOFECTAMINE® (a commercially available cationic liposome containing N-(1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), GIBCO / BRL); and TRANSFECTAM® (a commercially available cationic lipid containing dioctadecylamidoglycylcarboxyspermine (DOGS) in ethanol, Promega Corp., Madison, Wis.).The following lipids are cationic and have a positive charge at sub-physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

[0100] In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids useful in the present invention include those described in WO2012 / 016184, the entirety of which is incorporated herein by reference. Representative amino lipids include, but are not limited to, 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-T). DLin-MA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0101] Suitable amino lipids include those having the following formula: [ka]

[0102] {In the formula, R1 and R2 are the same or different and independently represent optionally substituted C 10 -C 24 Alkyl, optionally substituted C 10 -C 24 Alkenyl, optionally substituted C 10 -C 24 Alkynyl or optionally substituted C 10 -C 24 is acyl; R3 and R4 are the same or different and independently an optionally substituted C1-C6 alkyl, an optionally substituted C2-C6 alkenyl, or an optionally substituted C2-C6 alkynyl, or R3 and R4 may join together to form an optionally substituted heterocycle having 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R5 is absent or present, and if present is hydrogen or C1-C6 alkyl; m, n, and p are the same or different and independently 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 are O, S, or NH.

[0103] In one embodiment, R1 and R2 are each linoleyl and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

[0104] Representative useful dilinoleyl amino lipids have the following formula: [ka] wherein n is 0, 1, 2, 3, or 4.

[0105] In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (n is 2 in the formula of DLin-K-DMA above).

[0106] In one embodiment, the cationic lipid portion of the LNP has the structure of formula (I): [ka]

[0107] {In the formula, L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a carbon-carbon double bond; R 1a and R 1b is, for each occurrence, independently either (a) or (b) of the following: (a) H or C1-C12 alkyl; or (b) R 1a is H or C1-C 12 alkyl, and R 1b forms a carbon-carbon double bond with adjacent carbon atoms; R 2a and R 2b is, for each occurrence, independently either (a) or (b) of the following: (a) H or C1-C12 alkyl; or (b) R 2a or C1-C 12 alkyl, and R 2b forms a carbon-carbon double bond with adjacent carbon atoms; R 3a and R 3b is, for each occurrence, independently either (a) or (b) of the following: (a) H or C1-C12 alkyl; or (b) R 3a or C1-C 12 alkyl, and R 3b forms a carbon-carbon double bond with adjacent carbon atoms; R 4a and R 4bis, for each occurrence, independently either (a) or (b) of the following: (a) H or C1-C12 alkyl; or (b) R 4a or C1-C 12 alkyl, and R 4b forms a carbon-carbon double bond with adjacent carbon atoms; R 5 and R 6 are each independently methyl or cycloalkyl; R 7 is independently H or C1-C 12 is alkyl; R 8 and R 9 are each independently C1-C 12 alkyl or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring containing one nitrogen atom; a and d each independently represent an integer of 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2} or a pharma- ceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.

[0108] In certain embodiments of formula (I), R 1a , R 2a , R 3a or R 4a At least one of the following is C1-C 12 alkyl or L 1 Or L 2 At least one of R is -O(C=O)- or -(C=O)O-. 1a and R 1b is not isopropyl when a is 6, and is n-butyl when a is 8.

[0109] In a further embodiment of formula (I), R 1a , R 2a , R 3a or R 4aAt least one of the following is C1-C 12 alkyl or L 1 Or L 2 is -O(C=O)- or -(C=O)O-; and R 1a and R 1b is not isopropyl when a is 6, and is n-butyl when a is 8.

[0110] In another embodiment of formula (I), R 8 and R 9 are each independently an unsubstituted C-C 12 alkyl; or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocyclic ring containing one nitrogen atom.

[0111] In certain embodiments of formula (I), L 1 Or L 2 Either one of L may be -O(C=O)- or a carbon-carbon double bond. 1 and L 2 may each be -O(C=O)- or each be a carbon-carbon double bond.

[0112] In some embodiments of Formula (I), L 1 Or L 2 is -O(C=O)-. 1 and L 2 All of the above are -O(C=O)-.

[0113] In some embodiments of Formula (I), L 1 Or L 2 In another embodiment, L 1 and L 2 All of these are -(C=O)O-.

[0114] In some other embodiments of Formula (I), L 1 Or L 2is a carbon-carbon double bond. 1 and L 2 All of these are carbon-carbon double bonds.

[0115] In yet another embodiment of formula (I), L 1 Or L 2 One of the groups is -O(C=O)-, and L 1 Or L 2 and the other is -(C=O)O-. 1 Or L 2 One of the groups is -O(C=O)-, and L 1 Or L 2 The other of L is a carbon-carbon double bond. 1 Or L 2 One of the groups is -(C=O)O-, and L 1 Or L 2 The other is a carbon-carbon double bond.

[0116] As used throughout this specification, a "carbon-carbon" double bond has the following structure: [ka] {where, R a and R b is, for each occurrence, independently H or a substituent. For example, in some embodiments, R a and R b are each independently H, C 12 Alkyl or cycloalkyl, e.g., H or C1-C 12 It is understood to refer to either

[0117] In another embodiment, the lipid compound of formula (I) has the following structure (Ia): [ka] has.

[0118] In another embodiment, the lipid compound of formula (I) has the following structure (Ib): [ka] has.

[0119] In yet another embodiment, the lipid compound of formula (I) has the following structure (Ic): [ka] has.

[0120] In certain embodiments of the lipid compound of formula (I), a, b, c, and d are each independently an integer from 2 to 12, or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12, or an integer from 5 to 9. In some embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In a further embodiment, a is 3. In yet another embodiment, a is 4. In some embodiments, a is 5. In another embodiment, a is 6. In a further embodiment, a is 7. In a further embodiment, a is 8. In some embodiments, a is 9. In another embodiment, a is 10. In a further embodiment, a is 11. In a further embodiment, a is 12. In some embodiments, a is 13. In another embodiment, a is 14. In a further embodiment, a is 15. In a further embodiment, a is 16.

[0121] In some further embodiments of Formula (I), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In yet other embodiments, b is 16.

[0122] In some further embodiments of Formula (I), c is 1. In another embodiment, c is 2. In a further embodiment, c is 3. In a further embodiment, c is 4. In some embodiments, c is 5. In another embodiment, c is 6. In a further embodiment, c is 7. In a further embodiment, c is 8. In some embodiments, c is 9. In another embodiment, c is 10. In a further embodiment, c is 11. In a further embodiment, c is 12. In some embodiments, c is 13. In another embodiment, c is 14. In a further embodiment, c is 15. In a further embodiment, c is 16.

[0123] In some particular embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In yet other embodiments, d is 16.

[0124] In some other various embodiments of formula (I), a and d are the same. In some other embodiments, b and c are the same. In some other particular embodiments, a and d are the same, and b and c are the same.

[0125] In formula (I), the sum of a and b and the sum of c and d are factors that can be varied to obtain lipids of formula (I) with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In yet another embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer in the range of 14 to 24. In further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or more. In some embodiments of formula (I), e is 1. In some other embodiments, e is 2.

[0126] R in formula (I) 1a , R 2a , R 3a and R 4aThe substituents of R are not particularly limited. In certain embodiments, 1a , R 2a , R 3a and R 4a is H in each occurrence. In certain other embodiments, at least one R 1a , R 2a , R 3a and R 4a is C1-C 12 In certain other embodiments, at least one R 1a , R 2a , R 3a and R 4a is C1-C8 alkyl. In certain other embodiments, at least one R 1a , R 2a , R 3a and R 4a is C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0127] In certain embodiments of formula (I), R 1a , R 1b , R 4a and R 4b is C1-C 12 In a further embodiment of formula (I), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H at each occurrence. In certain embodiments of formula (I), R 1b forms a carbon-carbon double bond together with the adjacent carbon atom. 4b forms a carbon-carbon double bond with the adjacent carbon atom.

[0128] R in formula (I) 5 and R6 The substituents in R are not particularly limited in the above embodiment. 5 or R 6 One or both of R 5 or R 6 One or both of is cycloalkyl, e.g., cyclohexyl. In these embodiments, the cycloalkyl may be substituted or unsubstituted. In certain other embodiments, the cycloalkyl is C-C 12 It is substituted with alkyl, for example tert-butyl.

[0129] R 7 The substituents in are not particularly limited in the above embodiments of formula (I). In certain embodiments, at least one R 7 is H. In certain other embodiments, R 7 is H at each occurrence. In certain other embodiments, R 7 is C1-C 12 Certain other of the foregoing embodiments of formula (I) are 8 or R 9 is methyl. In another embodiment, R 8 and R 9 and R are both methyl. In some different embodiments of Formula (I), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, such as a pyrrolidinyl ring.

[0130] In various different embodiments, exemplary lipids of formula (I) include:

[0131] [ka]

[0132] [ka]

[0133] [ka]

[0134] [ka]

[0135] [ka]

[0136] [ka]

[0137] [ka]

[0138] In some embodiments, the LNP comprises a lipid of formula (I), at least one agent, and one or more excipients selected from a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (I) is compound I-5. In some embodiments, the lipid of formula (I) is compound I-6.

[0139] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (II): [ka]

[0140] {In the formula, L 1 and L 2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O- or a direct bond; G 1 is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NR a C(=O)- or a direct bond; G 2 -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NR a or a direct bond; G 3 is C1-C6 alkylene; R a or C1-C 12 is alkyl; R 1a and R 1b is, independently at each occurrence, either (a) or (b) below: (a) H or C1-C 12 or (b) R 1a or C1-C 12 alkyl, and R 1b forms a carbon-carbon double bond with the adjacent carbon atom; R 2a and R 2b is, independently at each occurrence, either (a) or (b) below: (a) H or C1-C 12 or (b) R 2a or C1-C 12 alkyl, and R 2b forms a carbon-carbon double bond with the adjacent carbon atom; R 3a and R 3b is, independently at each occurrence, either (a) or (b) below: (a) H or C1-C 12or (b) R 3a or C1-C 12 alkyl, and R 3b forms a carbon-carbon double bond with the adjacent carbon atom; R 4a and R 4b is, independently at each occurrence, either (a) or (b) below: (a) H or C1-C 12 or (b) R 4a or C1-C 12 alkyl, and R 4b forms a carbon-carbon double bond with the adjacent carbon atom; R 5 and R 6 are each independently H or methyl; R 7 is C4-C 20 is alkyl; R 8 and R 9 are each independently C1-C 12 Alkyl, or R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6- or 7-membered heterocyclic ring; a, b, c, and d are each independently an integer from 1 to 24; and and x is 0, 1, or 2} or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0141] In some embodiments of Formula (II), L 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond. 1 and G 2 are each independently -(C=O)- or a direct bond. 1 and L 2 are each independently -O(C=O)-, -(C=O)O-, or a direct bond; and G 1 and G 2are each independently -(C=O)- or a direct bond.

[0142] In some different embodiments of Formula (II), L 1 and L 2 are each independently -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a -, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a , -OC(=O)NR a -, -NR a C(=O)O-, -NR a S(O) x NR a -, -NR a S(O) x -or-S(O) x NR a -It is.

[0143] In another example of the above embodiment of formula (II), the lipid compound has the following structure (IIA) or (IIB): [ka] The present invention has either

[0144] In some embodiments of formula (II), the lipid compound has the structure (IIA). In other embodiments, the lipid compound has the structure (IIB).

[0145] In any of the foregoing embodiments of formula (II), L 1 Or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is -O(C=O)-. In some different embodiments of Formula (II), L 1 Or L 2 One of L is -(C=O)O-. For example, in some embodiments, L1 and L 2 Each of is -(C=O)O-. In another embodiment of formula (II), L 1 Or L 2 As used herein, a "direct bond" refers to a bond that is bonded to a group (e.g., L 1 Or L 2 For example, in some embodiments, L 1 and L 2 Each of is a direct bond.

[0146] In yet another embodiment of formula (II), R 1a and R 1b At least one occurrence of R 1a is H or C1-C12 alkyl, and R 1b forms a carbon-carbon double bond with the adjacent carbon atom. In yet another embodiment of formula (II), R 4a and R 4b At least one occurrence of R 4a is H or C1-C12 alkyl, and R 4b forms a carbon-carbon double bond with the adjacent carbon atom. In a further embodiment of formula (II), R 2a and R 2b At least one occurrence of R 2a is H or C1-C12 alkyl, and R 2b forms a carbon-carbon double bond with the adjacent carbon atom. In yet another embodiment of formula (II), R 3a and R 3b At least one occurrence of R 3a is H or C1-C12 alkyl, and R 3b forms a carbon-carbon double bond with the adjacent carbon atom.

[0147] In other various embodiments of formula (II), the lipid compound has the following structure (IIC) or (IID): [ka] {wherein e, f, g and h each independently represent an integer of 1 to 12}.

[0148] In some embodiments of formula (II), the lipid compound has the structure (IIC). In other embodiments, the lipid compound has the structure (IID).

[0149] In various embodiments of structure (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

[0150] In certain embodiments of formula (II), a, b, c, and d are each independently an integer from 2 to 12, or an integer from 4 to 12. In other embodiments, a, b, c, and d are each independently an integer from 8 to 12, or an integer from 5 to 9. In some particular embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In further embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In further embodiments, a is 7. In further other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In further embodiments, a is 11. In further other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In further embodiments, a is 15. In still other embodiments, a is 16.

[0151] In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In further embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In further embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In further embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In further embodiments, b is 15. In yet other embodiments, b is 16.

[0152] In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In further embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In further embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In further embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In further embodiments, c is 15. In yet other embodiments, c is 16.

[0153] In some particular embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In further embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6. In further embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In further embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In further embodiments, d is 15. In yet other embodiments, d is 16.

[0154] In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In further embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In further embodiments, e is 7. In further embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In further embodiments, e is 11. In yet other embodiments, e is 12.

[0155] In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In further embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In further embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In further embodiments, f is 11. In yet other embodiments, f is 12.

[0156] In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In further embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In further embodiments, g is 7. In further embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In further embodiments, g is 11. In yet other embodiments, g is 12.

[0157] In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In further embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In further embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In further embodiments, h is 11. In yet other embodiments, h is 12.

[0158] In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other particular embodiments, a and d are the same, and b and c are the same.

[0159] In formula (II), the sum of a and b and the sum of c and d are factors that can be varied to obtain lipids with desired properties. In one embodiment, a and b are selected such that their sum is an integer in the range of 14 to 24. In another embodiment, c and d are selected such that their sum is an integer in the range of 14 to 24. In yet another embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments, the sum of a and b and the sum of c and d are both the same integer in the range of 14 to 24. In further embodiments, a, b, c, and d are selected such that the sum of a and b and the sum of c and d are 12 or more.

[0160] R in formula (II) 1a , R 2a , R 3a and R 4a The substituents in are not particularly limited. In some embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is H. In certain embodiments, 1a , R 2a , R 3a and R 4a is H at each occurrence. In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of the following is C1-C 12 In certain other embodiments, R 1a , R 2a , R 3a and R 4a At least one of R is C-C alkyl. 1a , R 2a , R 3a and R 4a At least one of is C1-C6 alkyl. In some of the foregoing embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0161] In certain embodiments of formula (II), R 1a , R 1b , R 4a and R 4b is C1-C 12 It is an alkyl.

[0162] In a further embodiment of formula (II), R 1b , R 2b , R 3b and R 4b At least one of is H or R 1b , R 2b , R 3b and R 4b is H in each occurrence.

[0163] In certain embodiments of formula (II), R 1b forms a carbon-carbon double bond together with the adjacent carbon atom. 4b forms a carbon-carbon double bond with the adjacent carbon atom.

[0164] R in formula (II) 5 and R 6 In the above embodiment, the substituents in are not particularly limited. 5 or R 6 is methyl. In another embodiment, R 5 or R 6 Each of is methyl.

[0165] R in formula (II) 7 In the above embodiment, the substituents in are not particularly limited. 7 is C6-C 16 In some other embodiments, R 7 is C6-C9 alkyl. In some of these embodiments, R 7 -(C=O)OR b , -O(C=O)R b , -C(=O)R b , -OR b , -S(O)x R b , -S-SR b , -C(=O)SR b , -SC(=O)R b , -NR a R b , -NR a C(=O)R b , -C(=O)NR a R b , -NR a C(=O)NR a R b , -OC(=O)NR a R b , -NR a C(=O)OR b , -NR a S(O) x NR a R b , -NR a S(O) x R b Or -S(O) x NR a R b wherein R a is H or C1-C 12 R is alkyl; b is C1-C 15 alkyl; and x is 0, 1, or 2. For example, in some embodiments, R 7 -(C=O)OR b or -O(C=O)R b is replaced by.

[0166] In the various embodiments of formula (II) above, R b is branch C1-C 15 For example, in some embodiments, R b has the following structure: [ka] The present invention has either

[0167] Other particular embodiments of the foregoing of formula (II) are 8 or R 9 is methyl. In another embodiment, R8 and R 9 Both are methyl.

[0168] In some different embodiments of Formula (II), R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-, 6-, or 7-membered heterocycle. In some of the foregoing embodiments, R 8 and R 9 together with the nitrogen atom to which they are attached form a 5-membered heterocyclic ring, e.g., a pyrrolidinyl ring. In some other embodiments of the foregoing, R 8 and R 9 together with the nitrogen atom to which they are attached form a six-membered heterocyclic ring, such as a piperazinyl ring.

[0169] In yet another embodiment of the aforementioned lipid of formula (II), G 3 is C2-C4 alkylene, for example C3 alkylene.

[0170] In various different embodiments, the lipid compound has any of the following structures:

[0171] [ka]

[0172] [ka]

[0173] [ka]

[0174] [ka]

[0175] [ka]

[0176] [ka]

[0177] In some embodiments, the LNP comprises a lipid of formula (II), at least one agent, and one or more excipients selected from a neutral lipid, a steroid, and a pegylated lipid. In some embodiments, the lipid of formula (II) is compound II-9. In some embodiments, the lipid of formula (II) is compound II-10. In some embodiments, the lipid of formula (II) is compound II-11. In some embodiments, the lipid of formula (II) is compound II-12. In some embodiments, the lipid of formula (II) is compound II-32.

[0178] In some other embodiments, the cationic lipid component of the LNP has the structure of formula (III): [ka]

[0179] {In the formula, L 1 Or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O-, L 1 Or L 2 The other is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR aC(=O)NR a -, -OC(=O)NR a -OR-NR a C(=O)O- or a direct bond; G 1 and G 2 are each independently an unsubstituted C-C 12 Alkylene or C1-C 12 alkenylene; G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; R a is H or C1-C 12 is alkyl; R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and; R 4 is C1-C 12 is alkyl; R 5 is H or C1-C6 alkyl; and and x is 0, 1, or 2} or a pharma- ceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof.

[0180] In some of the foregoing embodiments of formula (III), the lipid has the following structure (IIIA) or (IIIB): [ka]

[0181] {In the formula, A is a 3- to 8-membered cycloalkyl or cycloalkylene ring; R 6 is independently, for each occurrence, H, OH, or C1-C 24 is alkyl; and n is an integer ranging from 1 to 15.

[0182] In some of the foregoing embodiments of formula (III), the lipid has the structure (IIIA), and in other embodiments, the lipid has the structure (IIIB).

[0183] In another embodiment of formula (III), the lipid has the following structure (IIIC) or (IIID): [ka] {wherein y and z are each independently an integer in the range of 1 to 12}.

[0184] In any of the foregoing embodiments of formula (III), L 1 Or L 2 One of L is -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of L is -O(C=O)-. In some different embodiments of any of the foregoing, L 1 and L 2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L 1 and L 2 Each of is -(C=O)O-.

[0185] In some different embodiments of formula (III), the lipid has the following structure (IIIE) or (IIIF): [ka] The present invention has one of the following:

[0186] In some different embodiments of formula (III), the lipid has the following structure (IIIG), (IIIH), (IIII), or (IIIJ): [ka] The present invention has one of the following:

[0187] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, such as from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[0188] In some other such embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

[0189] In some of the foregoing embodiments of formula (III), R 6 is H. In other embodiments of the above, R 6 is C1-C 24 In another embodiment, R 6 is OH.

[0190] In some embodiments of Formula (III), G 3 is unsubstituted. In another embodiment, G is substituted. In various different embodiments, G 3 is a straight chain C1-C 24 Alkylene or linear C1-C 24 It is alkenylene.

[0191] In some other aforementioned embodiments of formula (III), R 1 or R 2 Either or both are C6-C 24 For example, in some embodiments, R 1 and R 2 each independently have the structure: [ka]

[0192] {In the formula, R 7a and R 7b is, for each occurrence, independently, H or C 12 is alkyl; and a is an integer from 2 to 12; Here, R 7a , R 7b and a are each R 1 and R 2 are each independently selected to contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.

[0193] In some of the foregoing embodiments of formula (III), R 7a is H at least one occurrence. For example, in some embodiments, R 7a is H in each occurrence. In other different embodiments of the above, R 7b is C1-C8 alkyl at least one occurrence. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, or n-octyl.

[0194] In another embodiment of formula (III), R 1 or R 2 , or both, have the following structure: [ka] The present invention has either

[0195] In some of the foregoing embodiments of formula (III), R 3 OH, CN, -C(=O)OR 4 , -OC(=O)R 4 or -NHC(=O)R 4 In some embodiments, R4 is methyl or ethyl.

[0196] In various different embodiments, the cationic lipid of formula (III) has any of the structures shown in Table 3 below:

[0197] [ka]

[0198] [ka]

[0199] [ka]

[0200] [ka]

[0201] [ka]

[0202] [ka]

[0203] In some embodiments, the LNP comprises a lipid of formula (III), at least one agent, and optionally one or more excipients selected from neutral lipids, steroids, and pegylated lipids. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the lipid of formula (III) is compound III-7.

[0204] In certain embodiments, the cationic lipids are present in the LNP in an amount of about 30 to about 95 molar percent. In one embodiment, the cationic lipids are present in the LNP in an amount of about 30 to about 70 molar percent. In one embodiment, the cationic lipids are present in the LNP in an amount of about 40 to about 60 molar percent. In one embodiment, the cationic lipids are present in the LNP in an amount of about 50 molar percent. In one embodiment, the LNP is composed solely of cationic lipids.

[0205] In some embodiments, the LNPs include one or more additional lipids that stabilize the particle during formation.

[0206] Suitable stabilizing lipids include neutral lipids and anionic lipids.

[0207] The term "neutral lipid" refers to any of a number of lipid species that exist in uncharged or neutral zwitterionic form at physiological pH. Exemplary neutral lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides.

[0208] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethylPE, 16-O-dimethylPE, 18-1-trans. PE, 1-stearioyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielideyl-sn-glycero-3-phosphoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0209] In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of cationic lipid (e.g., a lipid of formula (I)) to neutral lipid ranges from about 2:1 to about 8:1.

[0210] In various embodiments, the LNP further comprises a steroid or steroid analog. A "steroid" is a compound that contains the following carbon skeleton: [ka]

[0211] In certain embodiments, the steroid or steroid analog is cholesterol. In some of these embodiments, the molar ratio of cationic lipid (e.g., a lipid of Formula (I)) to cholesterol ranges from about 2:1 to about 1:1.

[0212] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. Such lipids include the following: phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other neutral lipids with anionic modifying groups attached.

[0213] In certain embodiments, the LNPs comprise a glycolipid (eg, monosialoganglioside GM1). In certain embodiments, the LNPs comprise a sterol, such as cholesterol.

[0214] In some embodiments, the LNP comprises a lipid-linked polymer. The term "lipid-linked polymer" refers to a molecule that includes both a lipid portion and a polymer portion. One example of a lipid-linked polymer is a pegylated lipid. The term "pegylated lipid" refers to a molecule that includes both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoyl glycerol (PEG-DMG), etc.

[0215] In certain embodiments, the LNPs comprise an additional stabilizing lipid that is a polyethylene glycol-lipid (a PEGylated lipid). Suitable polyethylene glycol-lipids include: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Exemplary polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxypoly(ethylene glycol) 2000 )carbamyl]-1,2-dimyristoylpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In another embodiment, the LNP includes PEGylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), PEGylated phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy) PEG-S-ethyl)butanedioate (PEG-S-DMG), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of cationic lipid to PEGylated lipid ranges from about 100:1 to about 25:1.

[0216] In some embodiments, the LNP has the following structure (IV): [ka]

[0217] {In the formula, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, which alkyl chain is optionally interrupted by one or more ester linkages; and The average value of z is 30 to 60}, or a pharma- ceutically acceptable salt, tautomer, or stereoisomer thereof.

[0218] In some of the foregoing embodiments of the PEGylated lipid (IV), when z is 42, R 10 and R 11 In some other embodiments, R 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 18 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. 10 and R 11 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. 10 is a linear or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms, and R 11 is a linear or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

[0219] In various embodiments, z is in a range selected so that the average molecular weight of the PEG portion of (II) is from about 400 to about 6000 g / mol. In some embodiments, z has an average of about 45.

[0220] In another embodiment, the pegylated lipid has the following structure: [ka] where n is an integer selected such that the average molecular weight of the PEGylated lipid is about 2500 g / mol.

[0221] In certain embodiments, the additional lipid is present in the LNP in an amount of about 1 to about 10 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 to about 5 mole percent. In one embodiment, the additional lipid is present in the LNP in an amount of about 1 mole percent or about 1.5 mole percent.

[0222] In some embodiments, the LNP comprises a lipid of formula (I), a nucleoside-modified RNA, a neutral lipid, a steroid, and a PEGylated lipid. In some embodiments, the lipid of formula (I) is compound I-6. In a different embodiment, the neutral lipid is DSPC. In another embodiment, the steroid is cholesterol. In yet a different embodiment, the PEGylated lipid is compound IVa.

[0223] In certain embodiments, the LNPs comprise one or more immune evasion moieties that reduce or prevent phagocytosis of the LNPs by macrophages, for example, in one embodiment, the immune evasion moiety binds to an inhibitory receptor on macrophages and inhibits phagocytosis of the LNPs.

[0224] In certain embodiments, the LNPs comprise one or more targeting moieties that target the LNP to a cell or cell population. For example, in one embodiment, the targeting domain is a ligand that directs the LNP to a receptor on the cell surface.

[0225] In one embodiment, the LNP is coated with a polypeptide comprising a domain for immune system evasion. For example, in some embodiments, the LNP is contacted with a peptide micelle comprising a polypeptide comprising a domain for immune system evasion. Following contact with the micelle, the polypeptide comprising a domain for immune system evasion coats the LNP. In some embodiments, the LNP comprises at least one lipid bound to a targeting domain and is further coated with a polypeptide comprising a domain for immune system evasion.

[0226] In one embodiment, the LNP comprises at least one lipid bound to a domain for evasion of the immune system and at least one lipid bound to a targeting domain. In various embodiments, the LNP that comprises a combination of at least one lipid bound to a domain for evasion of the immune system and at least one lipid bound to a targeting domain may comprise them in any ratio. For example, in some embodiments, at least one lipid bound to a domain for evasion of the immune system and at least one lipid bound to a targeting domain are present in the LNP of the present invention in a ratio of 1:1. However, the combination is not limited to any particular ratio. Rather, any ratio shown to be effective is included.

[0227] In certain embodiments, LNPs can bind biomolecules in vivo, where the LNP-bound biomolecules can then be recognized by cell surface receptors to a) inhibit phagocytosis by macrophages, b) induce internalization by target cells, or a combination thereof.

[0228] Exemplary LNPs and their manufacture are described in the art, e.g., U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic acid, 131(5): E781-90; Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74.

[0229] The following reaction schemes illustrate methods for preparing lipids of formula (I), (II) or (III).

[0230] General reaction scheme 1 [ka]

[0231]

[0033] Embodiments of lipids of formula (I) (e.g., compound A-5) can be prepared according to general reaction scheme 1 ("Method A"), where R is a saturated or unsaturated C1-C 24alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. With reference to General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. A mixture of A-1, A-2, and DMAP is treated with DCC to produce bromide A-3. A mixture of bromide A-3, a base (e.g., N,N-diisopropylethylamine) and N,N-dimethyldiamine A-4 is heated at a sufficient temperature and time to produce A-5 after any necessary work-up and / or purification steps.

[0232] General reaction scheme 2 [ka]

[0233] Another embodiment of the compound of formula (I) (e.g., compound B-5) can be prepared according to general reaction scheme 2 ("Method B"), where R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. As shown in general reaction scheme 2, compounds of structure B-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. A solution of B-1 (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinium chlorochromate) to recover intermediate product B-3. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to give B-5 after any necessary workup and / or purification.

[0234] It should be noted that although starting materials A-1 and B-1 are shown above as containing only saturated methylene carbons, starting materials containing carbon-carbon double bonds can also be used to prepare compounds containing carbon-carbon double bonds.

[0235] General Reaction Scheme 3 [ka]

[0236] Different embodiments of lipids of formula (I) (e.g., compounds C-7 or C9) can be prepared according to general reaction scheme 3 ("Method C"), where R is a saturated or unsaturated C1-C 24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1, and n is an integer from 1 to 24. With reference to General Reaction Scheme 3, compounds of structure C-1 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art.

[0237] General Reaction Scheme 4 [ka]

[0238] Embodiments of compounds of formula (II) (e.g., compounds D-5 and D-7) can be prepared according to general reaction scheme 4 ("Method D"), where R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 8 , R 9 , L 1 , L 2 , G 1 , G 2 , G 3 , a, b, c and d are as defined herein; R 7’ is R 7 or C3-C 19Represents alkyl. With reference to General Reaction Scheme 1, compounds of structure D-1 and D-2 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. A solution of D-1 and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to give D-3 after any necessary workup. A solution of D-3 and a base (e.g., trimethylamine, DMAP) is treated with an acyl chloride D-4 (or a carboxylic acid and DCC) to give D-5 after any necessary workup and / or purification. D-5 can be reduced with LiAlH4 D-6 to give D-7 after any necessary workup and / or purification.

[0239] General Reaction Scheme 5 [ka]

[0240] Embodiments of lipids of formula (II) (e.g., compound E-5) can be prepared according to general reaction scheme 5 ("Method E"), where R 1a , R 1b , R 2a , R 2b , R 3a , R 3b , R 4a , R 4b , R 5 , R 6 , R 7 , R 8 , R 9 , L 1 , L 2 , G 3 , a, b, c and d are as described herein. With reference to General Reaction Scheme 2, compounds of structure E-1 and E-2 can be purchased from commercial sources or prepared according to methods well known to those skilled in the art. Heating a mixture of E-1 (in excess), E-2 and a base (e.g., potassium carbonate) affords E-3 after any necessary workup. Treating a solution of E-3 and a base (e.g., trimethylamine, DMAP) with an acyl chloride E-4 (or a carboxylic acid and DCC) affords E-5 after any necessary workup and / or purification.

[0241] General Reaction Scheme 6 [ka]

[0242] General Reaction Scheme 6 provides an exemplary method (Method F) for the preparation of lipids of formula (III). 1 , G 3 , R 1 and R 3 is as described herein for formula (III); G 1’ is G 1 F-1 refers to a one-carbon short homologue of. Compounds of structure F-1 are either purchased or prepared according to methods known in the art. Reaction of F-1 with a diol F-2 under suitable condensation conditions (e.g., DCC) produces an ester / alcohol F-3, which can be oxidized (e.g., PCC) to an aldehyde F-4. Reaction of F-4 with an amine F-5 under reductive amination conditions gives lipids of formula (III).

[0243] It should be noted that various alternative strategies for the preparation of lipids of formula (III) are available to the skilled artisan. For example, L 1 and L 2 Other lipids of formula (III) other than G esters can be prepared according to similar methods using appropriate starting materials. Further, General Reaction Scheme 6 shows the G 1 and G 2 The preparation of lipids of formula (III) in which G is identical is shown, however, this is not an essential aspect of the invention. 1 and G 2 The above reaction scheme can be modified to produce different compounds.

[0244] It will be appreciated by those skilled in the art that in the processes described herein, functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto, and carboxylic acid. Suitable protecting groups include the following: trialkylsilyl or diarylalkylsilyl (e.g., t-butyldimethylsilyl, t-butyldiphenylsilyl, or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino, and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (wherein R" is alkyl, aryl, or arylalkyl), p-methoxybenzyl, trityl, and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl, or arylalkyl esters. Protecting groups can be added or removed according to standard techniques known to those skilled in the art and described herein. The use of protecting groups is described in detail in Green, TW and PGM Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art will appreciate, the protecting group may be a polymeric resin such as a Wang resin, a Rink resin, or a 2-chlorotrityl chloride resin.

[0245] PEG Shielding In one embodiment, the LNPs comprise one or more pegylated lipids that serve to protect the LNPs from hepatic uptake by ApoE (also referred to herein as "PEG shielding"). In one embodiment, the pegylated lipid is conjugated to a targeting domain or therapeutic agent. In some embodiments, the LNPs further comprise a pegylated lipid conjugated to a targeting moiety. In some embodiments, the targeting moiety is an antibody or antigen-binding fragment thereof. In one embodiment, the pegylated lipid is conjugated to a phagocytosis inhibitor. In one embodiment, the pegylated lipid is conjugated to an active CD47 polypeptide fragment. In one embodiment, the pegylated lipid is conjugated to SEQ ID NO:1 or SEQ ID NO:2. In some embodiments, the LNPs further comprise one or more unconjugated pegylated lipids.

[0246] agent In one embodiment, the delivery vehicle comprises at least one active agent. In some embodiments, the active agent is a therapeutic agent, an imaging agent, a diagnostic agent, a contrast agent, a labeling agent, a detection agent or a disinfectant. The active agent also includes substances with biological activity that are not typically considered to be active ingredients, such as, for example, flavors, sweeteners, flavorings and flavor enhancers, pH adjusters, foaming agents, emollients, swelling agents, soluble organic salts, permeabilizing agents, antioxidants, colorants or dyes.

[0247] In one embodiment, the delivery vehicle comprises at least one therapeutic agent. The present invention is not limited to any particular therapeutic agent, but rather encompasses any suitable therapeutic agent that may be included in the delivery vehicle. Exemplary therapeutic agents include, but are not limited to, antiviral agents, antibacterial agents, antioxidants, thrombolytic agents, chemotherapeutic agents, anti-inflammatory agents, immunogenic agents, antiseptics, anesthetics, analgesics, pharmaceutical agents, small molecules, peptides, nucleic acids, and the like.

[0248] In some embodiments, the LNP or nanoparticle composition of the invention further comprises a nucleic acid. In various embodiments, the nucleic acid is an mRNA, a self-replicating RNA, an siRNA, an miRNA, an antisense oligonucleotide, a DNA, a DNA-RNA hybrid, a gene editing component (e.g., a guide RNA, a tracr RNA, an sgRNA, an RNA-guided nuclease encoding an mRNA, a gene or base editing protein, a zinc finger nuclease, a CRISPR nuclease such as Talen, Cas9, a DNA molecule that is inserted or serves as a template for repair), or the like, or a combination thereof. In some embodiments, the mRNA encodes a gene editing or base editing protein. In some embodiments, the nucleic acid is a guide RNA. In even further embodiments, the mRNA encodes a biological response modifier, a chemokine, a cytokine, a gamma chain receptor cytokine, such as IL-2, IL-7, IL-15, and IL-21, or an immune checkpoint agonist or antagonist. In some embodiments, the LNP or tLNP comprises both an mRNA encoding a gene or base editing protein and one or more guide RNAs. The CRISPR nuclease may have altered activity, for example, modifying the nuclease so that it is a nickase instead of making a double-stranded break, or so that it binds the sequence specified by the guide RNA but has no enzymatic activity. Base editing proteins are often fusion proteins that contain a deaminase domain and a sequence-specific DNA binding domain (such as an inactive CRISPR nuclease). In alternative embodiments, rather than containing an RNA-guided nuclease and an mRNA encoding the guide RNA, the LNP or nanoparticle contains a ribonucleoprotein, i.e., a complex containing a guide RNA bound to an RNA-guided nuclease. In other embodiments, the nanoparticle contains RNA and a reverse transcriptase. In yet other embodiments, the LNP or nanoparticle contains a virus particle, a virus-like particle, or a nucleocapsid.

[0249] Imaging Agents In one embodiment, the delivery vehicle comprises an imaging agent. An imaging agent is a substance that allows the delivery vehicle to be visualized after exposure to a cell or tissue. Visualization includes imaging, including imaging for the naked eye, as well as imaging that is not usually visible to the eye, must be detected using an instrument or detector, and requires the detection of photons, sound waves, or other energy quanta. Examples include stains, vital dyes, fluorescent markers, radioactive markers, enzymes, or plasmid constructs that code for markers or enzymes. Many materials and methods for imaging and targeting that can be used in the delivery vehicle are provided in Handbook of Targeted delivery of Imaging Agents, Torchilin, ed. (1995) CRC Press, Boca Raton, Fla.

[0250] Molecular imaging-based visualization typically involves detecting biological processes or biological molecules at the tissue, cell, or molecular level. Molecular imaging can be used to evaluate specific targets for gene therapy, cell-based therapeutics, and to visualize pathological conditions as diagnostic or research tools. Imaging agents that can be delivered intracellularly are particularly useful because such agents can be used to evaluate intracellular activities or conditions. Imaging agents must reach their targets to be effective; thus, in some embodiments, efficient uptake by cells is desirable. Rapid uptake is also desirable to avoid RES, see review by Allport and Weissleder, Experimental Hematology 1237-1246 (2001).

[0251] Furthermore, imaging agents should preferably provide a high signal-to-noise ratio so that they can be detected in small amounts, whether directly or through efficient amplification techniques that enhance the signal associated with a particular target. Amplification strategies are reviewed in Allport and Weissleder, Experimental Hematology 1237-1246 (2001), and include, for example, avidin-biotin binding systems, capture of probes that are converted ligands and change their physical behavior after being bound by the target, and exploitation of relaxation rates. Examples of imaging techniques include magnetic resonance imaging, radionuclide imaging, computed tomography imaging, ultrasound, and optical imaging.

[0252] The delivery vehicles provided herein can be advantageously used in a variety of imaging techniques or strategies, for example by incorporating an imaging agent into the delivery vehicle. Many imaging techniques and strategies are known. See, for example, the review in Allport and Weissleder, Experimental Hematology 1237-1246 (2001); such strategies can be adapted for use with the delivery vehicle. Suitable imaging agents include, for example, fluorescent molecules, labeled antibodies, labeled avidin:biotin binding agents, colloidal metals (e.g., gold, silver), reporter enzymes (e.g., horseradish peroxidase), superparamagnetic transferrin, second reporter systems (e.g., tyrosinase), and paramagnetic chelates.

[0253] In some embodiments, the imaging agent is a magnetic resonance imaging contrast agent. Non-limiting examples of magnetic resonance imaging agents include 1,4,7,10-tetraazacyclododecane-N,N',N"N'"-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraethylphosphorus (DOTEP), 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (DOTA), and derivatives thereof (see U.S. Pat. Nos. 5,188,816, 5,219,553, and 5,358,704). In some embodiments, the imaging agent is an X-ray contrast agent. X-ray contrast agents already known in the art include a number of halogenated derivatives, particularly iodinated derivatives, of 5-amino-isophthalic acid.

[0254] Small molecule therapeutics In various embodiments, the agent is a therapeutic agent. In various embodiments, the therapeutic agent is a small molecule. When the therapeutic agent is a small molecule, the small molecule can be obtained using standard methods known to those of skill in the art. Such methods include chemical organic synthesis or biological means. Biological means include purification from biological sources, recombinant synthesis, and in vitro translation systems using methods well known in the art. In one embodiment, small molecule therapeutic agents include organic molecules, inorganic molecules, biomolecules, synthetic molecules, and the like.

[0255] Combinatorial libraries of molecularly diverse compounds that may be useful in treating a variety of diseases and conditions, and methods for generating such libraries, are well known in the art. The methods may utilize a variety of techniques well known to those of skill in the art. Techniques include solid-phase synthesis, solution methods, parallel synthesis of multiple single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear arrays, deconvolution strategies, tagging techniques, and the generation of unbiased molecular landscapes for lead discovery versus biased structures for lead development. In some embodiments of the invention, therapeutic agents are synthesized and / or identified using combinatorial techniques.

[0256] In one common method for small library synthesis, a single activated core molecule is condensed with multiple building blocks resulting in a combinatorial library of a collection of covalently linked core building blocks. The shape and rigidity of the core determines the orientation of the building blocks in shape space. Libraries can be biased by varying the core, linkages, or building blocks to target characterized biological structures ("focused libraries") or synthesized with less structural bias using flexible cores. In some embodiments of the invention, therapeutic agents are synthesized through the synthesis of small libraries.

[0257] Because the small molecules and small molecule compounds described herein can exist as salts even when no salt is shown, the invention is understood to encompass all salts and solvates of the therapeutic agents shown herein, as well as non-salt and non-solvated forms of the therapeutic agents, as would be well understood by one of skill in the art. In some embodiments, the salts of the therapeutic agents of the invention are pharma- ceutically acceptable salts.

[0258] Where tautomeric forms are possible for any of the therapeutic agents described herein, all tautomeric forms are contemplated as being included in the invention, even if only one or some of the tautomeric forms are explicitly shown, For example, when a 2-hydroxypyridyl moiety is shown, the corresponding 2-pyridone tautomer is also contemplated.

[0259] The present invention also includes any or all stereochemical forms, including any enantiomeric or diastereomeric forms of the therapeutic agents described. Reference to a structure or name herein is intended to encompass all possible stereoisomers of the indicated therapeutic agent. All forms of the therapeutic agent, such as crystalline or non-crystalline forms of the therapeutic agent, are also encompassed by the present invention. Compositions comprising the therapeutic agents of the present invention are also contemplated, including compositions of a substantially pure therapeutic agent (including its particular stereochemical form) or compositions comprising mixtures of the therapeutic agents of the present invention in any ratio, such as in racemic or non-racemic mixtures, including two or more stereochemical forms thereof.

[0260] The present invention also includes any or all active analogs or derivatives (such as prodrugs) of any of the therapeutic agents described herein. In one embodiment, the therapeutic agent is a prodrug. In one embodiment, the small molecules described herein are candidates for derivatization. Thus, in some cases, analogs of the small molecules described herein with altered potency, selectivity, and solubility are included herein, which provide useful leads for drug discovery and development. Thus, in some cases, new analogs are provided that take into account issues of drug delivery, metabolism, novelty, and safety during optimization.

[0261] In some cases, the small molecule therapeutics described herein are derivatives or analogs of known therapeutics well known in the art of combinatorial chemistry and medicinal chemistry. These analogs or derivatives can be prepared by adding and / or substituting functional groups at various positions. Thus, the small molecules described herein can be converted into derivatives / analogs using well-known chemical synthesis procedures. For example, all of the hydrogen atoms or substituents can be selectively modified to generate new analogs. The linking atoms or groups can also be modified to longer or shorter linkers with carbon backbones or heteroatoms. Ring groups can also be altered to have different numbers of atoms in the ring and / or to include heteroatoms. Additionally, aromatics can be converted into ring groups and vice versa. For example, the ring can be 5-7 atoms and can be carbocyclic or heterocyclic.

[0262] As used herein, the terms "analog," "analogue," or "derivative" refer to a compound or molecule made from a parent compound or molecule by one or more chemical reactions. Thus, an analog can be structurally similar to the structure of a small molecule therapeutic described herein, or based on a scaffold of a small molecule therapeutic described herein, but differs in some component or structural makeup such that it has a similar or opposite metabolic effect. Analogs or derivatives of any small molecule inhibitor according to the present invention can be used to treat a disease or disorder.

[0263] In one embodiment, the small molecule therapeutics described herein can be independently derivatized by changing hydrogen groups to other substituents independently of each other, or analogs can be prepared from the small molecule therapeutics. That is, each atom on each molecule can be independently modified relative to other atoms on the same molecule. Any traditional modification for generating derivatives / analogs can be utilized. For example, the atoms and substituents can independently consist of hydrogen, alkyl, aliphatic, straight chain aliphatic with chain heteroatoms, branched aliphatic, substituted aliphatic, cycloaliphatic, heterocyclic aliphatic with one or more heteroatoms, aromatic, heteroaromatic, polyaromatic, polyamino acid, peptide, polypeptide, combinations thereof, halogen, halo-substituted aliphatic, etc. Additionally, any ring group on the compound can be derivatized to increase and / or decrease the size of the ring, as well as to change backbone atoms to carbon atoms or heteroatoms.

[0264] Nucleic Acid Therapeutics In other related aspects, the therapeutic agent is an isolated nucleic acid. In certain embodiments, the isolated nucleic acid molecule is a DNA molecule or an RNA molecule. In certain embodiments, the isolated nucleic acid molecule is a cDNA, mRNA, siRNA, shRNA, or miRNA molecule. In some embodiments, the therapeutic agent is an siRNA, miRNA, shRNA, or antisense molecule, which inhibits the targeted nucleic acid, including those encoding proteins involved in the deterioration of physiological processes.

[0265] In one embodiment, the nucleic acid comprises a promoter / regulatory sequence such that the nucleic acid can direct expression of the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of an exogenous nucleic acid into a cell with co-expression of the exogenous nucleic acid in the cell, e.g., as described in Sambrook et al. (2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York), and elsewhere herein.

[0266] In one embodiment of the invention, the activity of a targeted gene or protein may be inhibited by inactivating and / or sequestering the targeted gene or protein. Thus, inhibition of the activity of a targeted gene or protein may be achieved by using a nucleic acid molecule that encodes a transdominant negative mutant.

[0267] In one embodiment, siRNAs are used to reduce the levels of targeted proteins. RNA interference (RNAi) is a phenomenon in which the introduction of double-stranded RNA (dsRNA) into a variety of organisms and cell types results in the degradation of complementary mRNAs. In cells, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs are then assembled into RNA-induced silencing complexes (RISCs) that combine with protein components and unwind during the process. The activated RISC then binds to the complementary transcripts through base-pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved, resulting in sequence-specific degradation of the mRNA, resulting in gene silencing. See, e.g., U.S. Patent No. 6,506,559; Fire et al., 1998, Nature 391(19):306-311; Timmons et al., 1998, Nature 395:854; Montgomery et al., 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432:173-178). Soutschek et al. (2004, Nature 432:173-178) describe chemical modifications that can be made to siRNAs to aid in intravenous systemic delivery. Optimization of siRNAs includes consideration of overall G / C content, C / T content at the termini, Tm, and nucleotide content of the 3' overhangs.See, e.g., Schwartz et al., 2003, Cell, 115:199-208 and Khvorova et al., 2003, Cell 115:209-216. Thus, the present invention also includes methods of reducing levels of PTPN22 using RNAi technology.

[0268] In one aspect, the present invention includes a viral vector comprising siRNA or antisense polynucleotide.Preferably, siRNA or antisense polynucleotide can inhibit the expression of target polypeptide.The incorporation of desired polynucleotide into viral vector and the selection of viral vector are well known in the art, for example, as described in Sambrook et al. (2012) and Ausubel et al. (1997) and elsewhere herein.

[0269] In some embodiments, the expression vector described herein encodes a short hairpin RNA (shRNA) therapeutic. shRNA molecules are well known in the art and are directed to target mRNA, thereby reducing the expression of the target. In some embodiments, the encoded shRNA is expressed by cells and then processed into siRNA. For example, in some cases, cells have a natural enzyme (e.g., dicer) that cleaves shRNA to form siRNA.

[0270] To assess the expression of siRNA, shRNA, or antisense polynucleotides, the expression vector introduced into the cells can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification of expressing cells from a population of cells to be transfected or infected with the delivery vehicle of the present invention. In other embodiments, the selectable marker can be carried on separate DNA and can also be included in the delivery vehicle. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers are known in the art and include, for example, antibiotic resistance genes (such as neomycin resistance).

[0271] Thus, in one aspect, the delivery vehicle can contain a viral vector that includes the nucleotide sequence or construct to be delivered. The choice of viral vector will depend on the host cell into which it is to be introduced. In a particular embodiment, the vector of the invention is an expression vector. Suitable host cells include a variety of prokaryotic and eukaryotic host cells. In a particular embodiment, the expression vector is selected from the group consisting of viral vectors, bacterial vectors, and mammalian cell vectors. Systems based on prokaryotic and / or eukaryotic cell vectors can be utilized in the present invention to produce polynucleotides, or their cognate polypeptides. Many such systems are commercially available and widely available.

[0272] By way of example, the viral vector into which the nucleic acid sequence is introduced can be a plasmid. When the plasmid is introduced into the genome of a host cell, it may or may not be integrated into the cell. Non-limiting illustrative examples of viral vectors into which the nucleotide sequence of the invention or the genetic construct of the invention can be inserted include tet-on inducible viral vectors for expression in eukaryotic cells.

[0273] The vectors can be obtained by conventional methods known to those skilled in the art (Sambrook et al., 2012). In a particular embodiment, the viral vector is a viral vector useful for transforming animal cells.

[0274] In one embodiment, the recombinant expression vector can also contain a nucleic acid molecule encoding a peptide or peptidomimetic.

[0275] A promoter can be one that is naturally associated with a gene or polynucleotide sequence, obtained by isolating 5' non-coding sequences located upstream of the coding segment and / or exons. Such a promoter can be called "endogenous". Similarly, an enhancer can be one that is naturally associated with a polynucleotide sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages may be obtained by placing the coding polynucleotide segment under the control of a recombinant or heterologous promoter, a promoter that is not originally associated with the polynucleotide sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not originally associated with the polynucleotide sequence in its natural environment. Such promoters or enhancers can include promoters or enhancers of other genes, promoters or enhancers isolated from any other prokaryotic, viral or eukaryotic cell, and promoters or enhancers that are not "natural", i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can be produced in conjunction with the compositions disclosed herein using recombinant cloning and / or nucleic acid amplification techniques, including PCR™ (U.S. Patent Nos. 4,683,202 and 5,928,906).Furthermore, it is contemplated that control sequences that direct transcription and / or expression of sequences in non-nuclear organelles (e.g., mitochondria, chloroplasts, etc.) can also be used.

[0276] Naturally, it will be important to use promoters and / or enhancers that efficiently direct the expression of the DNA compartment in the cell type, organelle, and organism selected for expression. Those skilled in the art of molecular biology generally know how to use combinations of promoters, enhancers, and cell types to express proteins. See, for example, Sambrook et al. (2012). The promoters used can be constitutive, tissue-specific, inducible, and / or promoters that are useful for directing high levels of expression of the introduced DNA compartment under appropriate conditions (e.g., advantageous for large-scale production of recombinant proteins and / or peptides). The promoters can be heterologous or endogenous promoters.

[0277] The recombinant expression vector may also contain a selection marker gene that facilitates the selection of host cells. Suitable selection marker genes are genes that code for proteins (such as G418 or hygromycin) that confer resistance to certain drugs, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or immunoglobulins or portions thereof (such as the Fc portion of immunoglobulins, preferably IgG). Selection markers can be introduced into a viral vector separate from the nucleic acid of interest.

[0278] One of skill in the art will appreciate that after generation of a siRNA polynucleotide, the siRNA polynucleotide has certain characteristics that can be modified to improve the siRNA as a therapeutic compound. Thus, siRNA polynucleotides can be further designed to resist degradation by modification to include phosphorothioate or other linkages (methyl phosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate, etc.) (see, e.g., Agrawal et al., 1987, Tetrahedron Lett. 28:3539-3542; Stec et al., 1985 Tetrahedron Lett. 26:2191-2194; Moody et al., 1989 Nucleic Acids Res. 12:4769-4782; Eckstein, 1989 Trends Biol. Sci. 14:97-100; Stein, In: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, ed., Macmillan Press, London, pp. 97-117 (1989)).

[0279] Any polynucleotide can be further modified to increase its stability in vivo. Non-limiting examples of possible modifications include the addition of flanking sequences at the 5' and / or 3' ends, the use of phosphorothioate or 2'O-methyl rather than phosphodiester linkages in the backbone, and / or the inclusion of non-standard bases (such as inosine, queosine, and wt.) as well as acetyl, methyl, thio, and other modified forms of adenine, cytidine, guanine, thymine, and uridine.

[0280] In one embodiment of the invention, antisense nucleic acid sequences expressed by a plasmid viral vector are used as therapeutic agents to inhibit expression of a target protein. Antisense expressing viral vectors are used to transfect mammalian cells or whole mammals, thereby reducing endogenous expression of the target protein.

[0281] Antisense molecules and their use to inhibit gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary (as that term is defined elsewhere herein) to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA to form a double-stranded molecule, thereby inhibiting the translation of the gene.

[0282] The use of antisense methods to inhibit gene translation is known in the art and is described, for example, in Marcus-Sakura (1988, Anal. Biochem. 172:289). Such antisense molecules can be provided to cells through gene expression using DNA encoding the antisense molecule, as taught in Inoue, 1993, U.S. Patent No. 5,190,931.

[0283] Alternatively, the antisense molecules of the present invention can be made synthetically and then provided to cells.Antisense oligomers consisting of between about 10 and about 30 nucleotides, more preferably about 15 nucleotides, are preferred because they are easy to synthesize and introduce into target cells.Synthetic antisense molecules contemplated by the present invention include oligonucleotide derivatives known in the art that have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

[0284] In one embodiment of the present invention, ribozymes are used as therapeutic agents to inhibit the expression of target proteins. Ribozymes useful for inhibiting the expression of target molecules can be designed to incorporate target sequences into the basic ribozyme structure that is complementary to, for example, the mRNA sequence that encodes the target molecule. Ribozymes that target target molecules can be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or genetically expressed from DNA that encodes them.

[0285] In one embodiment, the therapeutic agent may comprise one or more components of a CRISPR-Cas system, in which a guide RNA (gRNA) targets a gene encoding a target molecule and a CRISPR-associated (Cas) peptide forms a complex to induce a mutation in the targeted gene. In one embodiment, the therapeutic agent comprises a gRNA or a nucleic acid molecule encoding a gRNA. In one embodiment, the therapeutic agent comprises a Cas peptide or a nucleic acid molecule encoding a Cas peptide.

[0286] In one embodiment, the agent comprises an miRNA or a miRNA mimic.In one embodiment, the agent comprises a nucleic acid molecule encoding an miRNA or a miRNA mimic.

[0287] miRNAs are small non-coding RNA molecules that can cause post-transcriptional silencing of specific genes in cells by inhibiting translation or through degradation of targeted mRNAs. miRNAs can be fully complementary or have regions that are not complementary to the target nucleic acid (resulting in "protruding" non-complementary regions). miRNAs can inhibit gene expression by suppressing translation, such as when the miRNA is not fully complementary to the target nucleic acid, or by causing degradation of the target RNA, which is believed to occur only when the miRNA binds to its corresponding target with full complementarity. The present disclosure can also include double-stranded precursors of miRNAs. miRNAs or pri-miRNAs can be 18-100 nucleotides in length, or 18-80 nucleotides in length. Mature miRNAs can have a length of 19-30 nucleotides, or 21-25 nucleotides, particularly 21, 22, 23, 24, or 25 nucleotides. miRNA precursors are typically about 70-100 nucleotides in length and have a hairpin structure. miRNAs are generated in vivo from pre-miRNAs by the enzymes Dicer and Drosha, which specifically process the long pre-miRNA into functional miRNA. Hairpin or mature microRNA, or pri-microRNA agents appearing in this disclosure can be synthesized in vivo by cell-based systems or in vitro by chemical synthesis.

[0288] In various embodiments, the agent comprises an oligonucleotide comprising the nucleotide sequence of a disease-associated miRNA. In certain embodiments, the oligonucleotide comprises the nucleotide sequence of a disease-associated miRNA in pre-microRNA form, mature form, or hairpin form. In other embodiments, combinations of oligonucleotides comprising the sequence of one or more disease-associated miRNAs, any pre-miRNA, any fragment, or any combination thereof are contemplated.

[0289] miRNAs can be synthesized to contain modifications that confer desired characteristics, e.g., modifications can improve stability, thermodynamics of hybridization with target nucleic acids, targeting to particular tissues or cell types, or cell permeability, e.g., by endocytosis-dependent or -independent mechanisms.

[0290] Modifications can also increase sequence specificity and consequently reduce off-target targeting. Methods of synthesis and chemical modification are described in more detail below. If desired, miRNA molecules can be modified to stabilize the miRNA against degradation, increase half-life, or otherwise improve efficacy. Desirable modifications are described, for example, in U.S. Patent Publication Nos. 20070213292, 20060287260, 20060035254, 20060008822, and 2005028824, each of which is incorporated herein by reference in its entirety. To increase nuclease resistance and / or binding affinity to a target, single-stranded oligonucleotide agents appearing in this disclosure can include 2'-O-methyl linkages, 2'-fluorine linkages, 2'-O-methoxyethyl linkages, 2'-O-aminopropyl linkages, 2'-amino linkages, and / or phosphorothioate linkages. The inclusion of locked nucleic acids (LNA), ethylene nucleic acids (ENA) (e.g., 2'-4'-ethylene bridged nucleic acids), and certain nucleotide modifications can also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone can also reduce endonucleolytic cleavage. Oligonucleotides can be further modified by including a 3' cationic group or by inverting the nucleoside at the 3' end with a 3-3' linkage. In another alternative, the 3' end can be blocked with an aminoalkyl group. Other 3' complexes can inhibit 3'-5' exonucleolytic cleavage. Without being bound by theory, the 3' may inhibit exonucleolytic cleavage by sterically blocking exonucleases from binding to the 3' end of the oligonucleotide. Small alkyl chains, aryl groups, or even heterocyclic complexes or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

[0291] In one embodiment, the miRNA comprises a 2' modified oligonucleotide containing an oligodeoxynucleotide gap in which some or all internucleotide linkages are modified to phosphorothioate for nuclease resistance. The presence of phosphonate methyl modifications increases the affinity of the oligonucleotide for the corresponding target RNA and lowers the IC5Q. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present disclosure can be used in combination with any technology that may be developed to enhance the stability or efficacy of inhibitory nucleic acid molecules.

[0292] miRNA molecules include nucleotide oligomers containing modified backbones or non-natural internucleoside linkages. Oligomers with modified backbones include oligomers that retain a phosphorus atom in the backbone and oligomers that do not have a phosphorus atom in the backbone. For the purposes of this disclosure, modified oligonucleotides that do not have a phosphorus atom in the internucleoside backbone are also considered to be nucleotide oligomers. Nucleotide oligomers with modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl phosphonates and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates. Various salts, mixed salts, and free acid forms are also included.

[0293] The miRNAs described herein may be in mature or hairpin form and may be provided as naked oligonucleotides. In some cases, it may be desirable to use formulations that aid in the delivery of miRNAs or other nucleotide oligomers to cells (see, e.g., U.S. Patent Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055; each of which is incorporated herein by reference).

[0294] In some examples, the miRNA composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the miRNA composition is in an aqueous phase (e.g., a solution containing water). The aqueous phase or crystalline composition can be incorporated into a delivery vehicle (e.g., a liposome (particularly for the aqueous phase), or a particle (e.g., a microparticle, which may be appropriate for a crystalline composition)). In general, the miRNA composition is formulated to be compatible with the intended method of administration. The miRNA compositions can be formulated in combination with another agent, e.g., another therapeutic agent, or an agent that stabilizes the oligonucleotide agent, e.g., a protein that complexes with the oligonucleotide agent. Further agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg), salts, and RNAse inhibitors, e.g., broad specificity RNAse inhibitors. In one embodiment, the miRNA composition includes another miRNA, e.g., a second miRNA composition, e.g., a microRNA different from the first. Further preparations can include at least 3, 5, 10, 20, 50, or 100 or more different oligonucleotide species.

[0295] In some embodiments, the composition comprises an oligonucleotide composition that mimics the activity of a miRNA. In some embodiments, the composition comprises an oligonucleotide having a nucleobase sequence that matches the nucleobase sequence of a miRNA, and is therefore designed to mimic the activity of a miRNA. In some embodiments, the oligonucleotide composition that mimics the activity of a miRNA comprises a double-stranded RNA molecule that mimics a mature miRNA hairpin, or a processed miRNA duplex.

[0296] In one embodiment, the oligonucleotide shares a nucleobase sequence identity with an endogenous miRNA or miRNA precursor. The oligonucleotides selected for inclusion in the compositions of the invention can be one of many lengths. Such oligonucleotides can be 7-100 linked nucleosides in length. For example, oligonucleotides that share a nucleobase identity with an miRNA can be 7-30 linked nucleosides in length. Oligonucleotides that share a nucleobase identity with an miRNA precursor can be up to 100 linked nucleosides in length. In some embodiments, the oligonucleotide comprises 7-30 linked nucleosides. In some embodiments, the oligonucleotide comprises 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28, 29, or 30 linked nucleotides. In some embodiments, the oligonucleotide comprises 19-23 linked nucleosides. In certain embodiments, the oligonucleotide is from 40 to 50, 60, 70, 80, 90, or 100 linked nucleosides in length.

[0297] In some embodiments, the oligonucleotide has a sequence that has a portion that matches a miRNA or its precursor. The nucleobase sequences of mature miRNAs and their corresponding stem-loop sequences described herein are sequences found in miRBase, an online searchable database of miRNA sequences and annotations. Entries in the miRBase sequence database include information about the predicted hairpin portion (stem-loop) of a miRNA transcript and the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and in some cases may include pre-miRNAs and some flanking sequences from the predicted primary transcript. The miRNA nucleobase sequences described herein encompass all versions of miRNAs, including those described in release 10.0 of the miRBase sequence database and in any previous release of the miRBase sequence database. The name of a miRNA may change as a result of a release of the sequence database. Variations in a mature miRNA sequence may occur as a result of a release of the sequence database. The compositions of the invention include oligomeric compounds that include oligonucleotides having a portion of correspondence to any of the nucleobase sequence versions of the miRNAs described herein.

[0298] In certain embodiments, an oligonucleotide has a nucleobase sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to an miRNA over a region of 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases. Thus, in certain embodiments, the nucleobase sequence of an oligonucleotide may have one or more nucleobases that do not match an miRNA.

[0299] In certain embodiments, the composition comprises a nucleic acid molecule encoding the miRNA, precursor, mimetic, or fragment thereof, e.g., the composition can comprise a viral vector, plasmid, cosmid, or other expression vector suitable for expressing the miRNA, precursor, mimetic, or fragment thereof in a desired mammalian cell or tissue.

[0300] In vitro transcribed RNA In one embodiment, a therapeutic agent of the invention comprises an in vitro transcribed (IVT) RNA. In one embodiment, a therapeutic agent of the invention comprises an in vitro transcribed (IVT) RNA encoding a therapeutic protein. In one embodiment, a therapeutic agent of the invention comprises an IVT RNA encoding multiple therapeutic proteins.

[0301] In one embodiment, IVT RNA can be introduced into cells in the form of transient transfection. RNA is produced by in vitro transcription using synthetically produced plasmid DNA templates. DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. In one embodiment, the desired template for in vitro transcription is a therapeutic protein as described elsewhere herein.

[0302] In one embodiment, the DNA used in the PCR contains an open reading frame. The DNA can be derived from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full-length gene of interest that is a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA used in the PCR is a human gene. In another embodiment, the DNA used in the PCR is a human gene that includes a 5'UTR and a 3'UTR. In another embodiment, the DNA used in the PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA used in the PCR is derived from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, and includes a 5'UTR and a 3'UTR. Alternatively, the DNA can be an artificial DNA sequence that is not normally expressed in a natural organism. One representative artificial DNA sequence is a sequence that contains portions of genes that are linked together to form an open reading frame that encodes a fusion protein. The linked portions of DNA can be derived from a single organism, or from two or more organisms.

[0303] Genes that can be used as DNA sources for PCR include genes that encode polypeptides that induce or enhance adaptive immune responses in an organism. Preferred genes are those that are useful for short-term therapy or where there are safety concerns regarding dosage or the gene being expressed.

[0304] In various embodiments, a plasmid is used to generate a template for in vitro transcription of RNA used for transfection.

[0305] Chemical structures capable of improving stability and / or translation efficiency may also be used. Preferably, the RNA has a 5'UTR and a 3'UTR. In one embodiment, the 5'UTR is 0-3000 nucleotides in length. The length of the 5'UTR and 3'UTR sequences added to the coding region can be varied by various methods. Non-limiting examples of methods include designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can vary the length of the 5'UTR and 3'UTR required to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0306] The 5'UTR and 3'UTR can be the natural endogenous 5'UTR and 3'UTR for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating UTR sequences into the forward and reverse primers or by any other modification of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for altering RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3'UTR sequence can reduce RNA stability. Thus, the 3'UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs that are well known in the art.

[0307] In one embodiment, the 5'UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5'UTR that is not endogenous to the gene of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5'UTR sequence. Although the Kozak sequence can improve the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to allow efficient translation. It is known in the art that the Kozak sequence is necessary for many RNAs. In another embodiment, the 5'UTR can be derived from an RNA virus whose RNA genome is stable in the cell. In another embodiment, various nucleotide analogs are used in the 3'UTR or 5'UTR to prevent exonuclease degradation of the RNA.

[0308] To allow synthesis of RNA from a DNA template without the need for gene cloning, a transcription promoter must be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In a preferred embodiment, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Non-limiting examples of other useful promoters include the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for the T7, T3, and SP6 promoters are known in the art.

[0309] In a preferred embodiment, the RNA has both a 5'-cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On a circular DNA template (e.g., plasmid DNA), RNA polymerase produces long chain products that are not suitable for expression in eukaryotic cells. Plasmid DNA linearized at the end of the 3'UTR is transcribed into normal-sized RNA, which is effective when posttranscriptionally polyadenylated for transfection into eukaryotic cells.

[0310] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of a transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0311] The traditional method of incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination-deficient bacterial cells for plasmid propagation.

[0312] The poly(A) tail of the RNA can be further extended using poly(A) polymerase (such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase) after in vitro transcription. In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides increases the translation efficiency of the RNA by about 2-fold. In addition, attachment of different chemical groups to the 3' end can increase the stability of the RNA. Such attachments can include modified / artificial nucleotides, aptamers, and other compounds. For example, poly(A) polymerase can be used to introduce ATP analogs into the poly(A) tail. The ATP analogs can further increase the stability of the RNA.

[0313] The 5' cap also provides stability to the RNA molecule. In a preferred embodiment, the RNA produced by the present method includes a 5' Cap 1 structure. Such Cap 1 structures can be generated using Vaccinia capping enzyme and 2'-O-methyltransferase enzymes (CellScript, Madison, Wis.). Alternatively, the 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0314] Nucleoside-modified RNA In one embodiment, the composition of the invention comprises a nucleoside modified nucleic acid. In one embodiment, the composition of the invention comprises a nucleoside modified RNA encoding a therapeutic protein.

[0315] For example, in one embodiment, the composition comprises nucleoside-modified RNA. In one embodiment, the composition comprises nucleoside-modified mRNA. Nucleoside-modified mRNA has specific advantages over unmodified mRNA, including, for example, increased stability, low or no natural immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent No. 8,278,036, each of which is incorporated herein by reference in its entirety.

[0316] In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiological pathways, is translated very efficiently and almost immediately after delivery, and serves as a template for continuous protein production in vivo, continuing for several days (Kariko et al., 2008, Mol Ther 16:1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small, making it applicable to human treatment.

[0317] In certain instances, expression of proteins by delivering coding mRNA has many advantages over methods using proteins, plasmid DNA, or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only material delivered to the cell, thus avoiding all side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA and viral-based vectors, mRNA does not carry the risk of integration into the genome, and protein production begins immediately after mRNA delivery. For example, high concentrations of circulating proteins have been measured within 15-30 minutes of in vivo injection of coding mRNA. In certain embodiments, using mRNA rather than protein also has many advantages. Proteins often have a short half-life in the circulation, thus requiring frequent dosing for protein treatments, whereas mRNA provides a template for continuous protein production over several days. Protein purification is problematic and proteins can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

[0318] In certain embodiments, the nucleoside modified RNA comprises pseudouridine, a naturally occurring modified nucleoside. In certain embodiments, the inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et al., 2008, Mol Ther 16:1833-1840; Anderson et al., 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:e142; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23:165-175).

[0319] It has been demonstrated that the presence of modified nucleosides, including pseudouridine, in RNA suppresses its natural immunogenicity (Kariko et al., 2005, Immunity 23:165-175). Furthermore, in vitro transcribed protein-coding RNAs containing pseudouridine can be translated more efficiently than RNAs that do not contain modified nucleosides or contain other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). It has subsequently been shown that the presence of pseudouridine improves RNA stability (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and reduces both PKR activation and translation inhibition (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892). We established that a preparative HPLC purification procedure is important to obtain pseudouridine-containing RNA with good translational capacity and no natural immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:e142). Administration of HPLC-purified pseudouridine-containing RNA encoding erythropoietin to mice and rhesus monkeys resulted in a significant increase in serum EPO concentrations (Kariko et al., 2012, Mol Ther 20:948-953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy.

[0320] The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules that contain pseudouridine or modified nucleosides. In certain embodiments, the compositions include isolated nucleic acids, where the nucleic acids contain pseudouridine or modified nucleosides. In certain embodiments, the compositions include vectors that contain isolated nucleic acids, where the nucleic acids contain pseudouridine or modified nucleosides.

[0321] In one embodiment, the nucleoside modified RNA of the present invention is the IVT RNA described elsewhere herein.For example, in certain embodiments, the nucleoside modified RNA is synthesized by T7 phage RNA polymerase.In another embodiment, the nucleoside modified mRNA is synthesized by SP6 phage RNA polymerase.In another embodiment, the nucleoside modified RNA is synthesized by T3 phage RNA polymerase.

[0322] In one embodiment, the modified nucleoside is 1 acp 3 In another embodiment, the modified nucleoside is m 1 (1-methylpseudouridine). In another embodiment, the modified nucleoside is Ψm (2'-O-methylpseudouridine). In another embodiment, the modified nucleoside is m 5 D (5-methyldihydrouridine). In another embodiment, the modified nucleoside is 3 Ψ (3-methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified. In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the pseudouridines described above. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

[0323] In another embodiment, the modified nucleoside in the nucleoside-modified RNA of the present invention is uridine (U). In another embodiment, the modified nucleoside is cytidine (C). In another embodiment, the modified nucleoside is adenosine (A). In another embodiment, the modified nucleoside is guanosine (G).

[0324] In another embodiment, the modified nucleoside of the present invention is 5In another embodiment, the modified nucleoside is m 5 In another embodiment, the modified nucleoside is m 6 A(N 6 In another embodiment, the modified nucleoside is s 2 In another embodiment, the modified nucleoside is U (2-thiouridine). In another embodiment, the modified nucleoside is Ψ (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

[0325] In other embodiments, the modified nucleoside is 1 A(1-methyladenosine);m 2 A(2-methyladenosine); Am(2'-O-methyladenosine); ms 2 m 6 A(2-methylthio-N 6 -Methyladenosine);i 6 A(N 6 -Isopentenyladenosine);ms 2 i6A(2-methylthio-N 6 Isopentenyladenosine;io 6 A(N 6 -(cis-hydroxyisopentenyl)adenosine);ms 2 io 6 A(2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine);g 6 A(N 6 -glycinylcarbamoyl adenosine);t 6 A(N 6 -Threonylcarbamoyl adenosine);ms 2 t 6 A(2-methylthio-N 6 -Threonylcarbamoyl adenosine);m 6 t 6 A(N 6 -Methyl-N 6 -Threonylcarbamoyl adenosine);hn 6 A(N 6-Hydroxynorvalylcarbamoyl adenosine);ms 2 hn 6 A(2-methylthio-N 6 -Hydroxynorvalylcarbamoyl adenosine; Ar(p)(2'-O-ribosyladenosine (phosphate)); I(inosine); m 1 I(1-methylinosine);m 1 Im(1,2'-O-dimethylinosine);m 3 C(3-methylcytidine); Cm(2'-O-methylcytidine); s 2 C(2-thiocytidine);ac 4 C(N 4 -Acetylcytidine);f 5 C(5-formylcytidine);m 5 Cm(5,2'-O-dimethylcytidine);ac 4 Cm(N 4 -acetyl-2'-O-methylcytidine);k 2 C(lysidine);m 1 G(1-methylguanosine);m 2 G(N 2 -Methylguanosine);m 7 G(7-methylguanosine);Gm(2'-O-methylguanosine);m 2 2G(N 2 ,N 2 -Dimethylguanosine);m 2 Gm(N 2 ,2'-O-dimethylguanosine);m 2 2Gm(N 2 ,N 2 ,2'-O-trimethylguanosine;Gr(p)(2'-O-ribosylguanosine (phosphate));yW(wybutosine);o2yW(peroxywybutosine);OHyW(hydroxywybutosine);OHyW*(undermodified hydroxywybutosine);imG(wybutosine);mimG(methylwybutosine);Q(queuosine);oQ(epoxyqueuosine);galQ(galactosyl-queuosine);manQ(mannosyl-queuosine);preQ0(7-cyano-7-deazaguanosine);preQ1(7-aminomethyl-7-deazaguanosine);G +(Archaeosine);D(Dihydrouridine);m 5 Um(5,2'-O-dimethyluridine);s 4 U(4-thiouridine);m 5 s 2 U(5-methyl-2-thiouridine);s 2 Um(2-thio-2'-O-methyluridine); acp 3 U(3-(3-amino-3-carboxypropyl)uridine);ho 5 U(5-hydroxyuridine); 5 U(5-methoxyuridine); cmo 5 U(uridine 5-oxyacetic acid);mcmo 5 U(uridine 5-oxyacetic acid methyl ester);chm 5 U(5-(carboxyhydroxymethyl)uridine));mchm 5 U(5-(carboxyhydroxymethyl)uridine methyl ester);mcm 5 U(5-Methoxycarbonylmethyluridine);mcm 5 Um(5-methoxycarbonylmethyl-2'-O-methyluridine); mcm 5 s 2 U(5-methoxycarbonylmethyl-2-thiouridine); nm 5 s 2 U(5-aminomethyl-2-thiouridine);mnm 5 U(5-methylaminomethyluridine); mnm 5 s 2 U(5-methylaminomethyl-2-thiouridine);mnm 5 se 2 U(5-methylaminomethyl-2-selenouridine);ncm 5 U(5-carbamoylmethyluridine);ncm 5 Um(5-carbamoylmethyl-2'-O-methyluridine); cmnm 5 U(5-carboxymethylaminomethyluridine);cmnm 5 Um(5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm 5 s 2 U(5-carboxymethylaminomethyl-2-thiouridine);m6 2A(N 6 ,N 6 -Dimethyladenosine; Im (2'-O-methylinosine); m 4 C(N 4 -Methylcytidine);m 4 Cm(N 4 ,2'-O-dimethylcytidine);hm 5 C(5-hydroxymethylcytidine);m 3 U(3-methyluridine); cm 5 U(5-carboxymethyluridine);m 6 Am(N 6 ,2'-O-dimethyladenosine);m 6 2Am(N 6 ,N 6 ,O-2'-trimethyladenosine);m 2,7 G(N 2 ,7-Dimethylguanosine);m 2,2,7 G(N 2 ,N 2 ,7-trimethylguanosine);m 3 Um(3,2'-O-dimethyluridine);m 5 D(5-methyldihydrouridine);f 5 Cm(5-formyl-2'-O-methylcytidine); 1 Gm(1,2'-O-dimethylguanosine);m 1 Am(1,2'-O-dimethyladenosine); τm 5 U(5-taurinomethyluridine); τm 5 s 2 U(5-taurinomethyl-2-thiouridine);imG-14(4-demethylwyosine);imG2(isowyosine);orac 6 A(N 6 -acetyladenosine).

[0326] In another embodiment, the nucleoside modified RNA of the present invention comprises a combination of two or more of the above modifications.In another embodiment, the nucleoside modified RNA comprises a combination of three or more of the above modifications.In another embodiment, the nucleoside modified RNA comprises a combination of more than three of the above modifications.

[0327] In another embodiment, 0.1% to 100% of the residues in the nucleoside modification of the invention are modified (e.g., by the presence of pseudouridine or another modified nucleoside base). In another embodiment, 0.1% of the residues are modified. In another embodiment, the percentage of modified residues is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0328] In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0329] In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the percentage of a given nucleotide that is modified is 0.2%. In another embodiment, the percentage is 0.3%. In another embodiment, the percentage is 0.4%. In another embodiment, the percentage is 0.5%. In another embodiment, the percentage is 0.6%. In another embodiment, the percentage is 0.8%. In another embodiment, the percentage is 1%. In another embodiment, the percentage is 1.5%. In another embodiment, the percentage is 2%. In another embodiment, the percentage is 2.5%. In another embodiment, the percentage is 3%. In another embodiment, the percentage is 4%. In another embodiment, the percentage is 5%. In another embodiment, the percentage is 6%. In another embodiment, the percentage is 8%. In another embodiment, the percentage is 10%. In another embodiment, the percentage is 12%. In another embodiment, the percentage is 14%. In another embodiment, the percentage is 16%. In another embodiment, the percentage is 18%. In another embodiment, the percentage is 20%. In another embodiment, the percentage is 25%. In another embodiment, the percentage is 30%. In another embodiment, the percentage is 35%. In another embodiment, the percentage is 40%. In another embodiment, the percentage is 45%. In another embodiment, the percentage is 50%. In another embodiment, the percentage is 60%. In another embodiment, the percentage is 70%. In another embodiment, the percentage is 80%. In another embodiment, the percentage is 90%. In another embodiment, the percentage is 100%.

[0330] In another embodiment, the percentage of a given nucleotide that is modified is less than 8%. In another embodiment, the percentage is less than 10%. In another embodiment, the percentage is less than 5%. In another embodiment, the percentage is less than 3%. In another embodiment, the percentage is less than 1%. In another embodiment, the percentage is less than 2%. In another embodiment, the percentage is less than 4%. In another embodiment, the percentage is less than 6%. In another embodiment, the percentage is less than 12%. In another embodiment, the percentage is less than 15%. In another embodiment, the percentage is less than 20%. In another embodiment, the percentage is less than 30%. In another embodiment, the percentage is less than 40%. In another embodiment, the percentage is less than 50%. In another embodiment, the percentage is less than 60%. In another embodiment, the percentage is less than 70%.

[0331] In another embodiment, the nucleoside modified RNA of the invention is translated intracellularly more efficiently than an unmodified RNA molecule having the same sequence. In another embodiment, the nucleoside modified RNA has an enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2 relative to its unmodified counterpart. In another embodiment, translation is enhanced by a factor of 3. In another embodiment, translation is enhanced by a factor of 5. In another embodiment, translation is enhanced by a factor of 7. In another embodiment, translation is enhanced by a factor of 10. In another embodiment, translation is enhanced by a factor of 15. In another embodiment, translation is enhanced by a factor of 20. In another embodiment, translation is enhanced by a factor of 50. In another embodiment, translation is enhanced by a factor of 100. In another embodiment, translation is enhanced by a factor of 200. In another embodiment, translation is enhanced by a factor of 500. In another embodiment, translation is enhanced by a factor of 1000. In another embodiment, translation is enhanced by a factor of 2000. In another embodiment, the factor is between 10 and 1000 fold. In another embodiment, the factor is 10-100 fold. In another embodiment, the factor is 10-200 fold. In another embodiment, the factor is 10-300 fold. In another embodiment, the factor is 10-500 fold. In another embodiment, the factor is 20-1000 fold. In another embodiment, the factor is 30-1000 fold. In another embodiment, the factor is 50-1000 fold. In another embodiment, the factor is 100-1000 fold. In another embodiment, the factor is 200-1000 fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

[0332] Polypeptide Therapeutics In other related aspects, the therapeutic agent comprises an isolated peptide that modulates a target. For example, in one embodiment, a peptide of the invention directly inhibits or activates a target by binding to the target, thereby altering the normal functional activity of the target. In one embodiment, a peptide of the invention modulates a target by competing with an endogenous protein. In one embodiment, a peptide of the invention modulates the activity of a target by acting as a transdominant negative mutant.

[0333] Variants of polypeptide therapeutics can be (i) those in which one or more of the amino acid residues contained therein have been replaced with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), where such replaced amino acid residues may or may not be encoded by the genetic code; (ii) those in which one or more modified amino acid residues (e.g., residues modified by attachment of a substituent group) are present; (iii) those in which the polypeptide is an alternative splice variant of a polypeptide of the invention; (iv) those fragments of the polypeptide; and / or (v) those in which the polypeptide is fused to another polypeptide, such as a leader sequence, or a secretory sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include polypeptides generated through proteolytic (including multi-site proteolytic) cleavage of the original sequence. Variants may be post-translationally modified or chemically modified. Such variants are considered to be within the scope of one of skill in the art from the teachings herein.

[0334] Antibody Therapeutics The present invention also contemplates delivery vehicles that include an antibody or antibody fragment specific for a target. That is, the antibody can bind to the target to direct the delivery vehicle to a cell expressing the target. In some embodiments, the antibody can inhibit the target to provide a beneficial effect.

[0335] As used herein, the term "antibody" refers to a protein that contains an immunoglobulin domain with hypervariable regions that determine the specificity of the antibody with respect to the antigen to which it binds; the so-called complementarity determining regions (CDRs). Thus, the term antibody refers to complete or whole antibodies, as well as antibody fragments and constructs that contain antigen-binding portions of whole antibodies. While standard natural antibodies have a set of heavy and light chains, camelids (camels, alpacas, llamas, etc.) produce antibodies with both standard structures and antibodies that contain only heavy chains. The variable regions of camelid heavy chain-only antibodies have a unique structure with an extended CDR3, called VHHs, or nanobodies when produced as fragments. Antigen-binding fragments and constructs of antibodies include F(ab)2, F(ab), minibodies, Fvs, single chain Fvs (scFvs), diabodies, and VHs. Such elements may be combined to create bi- and multispecific reagents, such as bispecific T cell engagers. The term "monoclonal antibody" arose from hybridoma technology, but is now used to refer to any single molecular species of antibody, regardless of how it originated or was produced. Antibodies can be obtained by immunization, selection (e.g., by phage display) from naive or immune libraries, modification of isolated antibody coding sequences, or any combination thereof.

[0336] Antibody variable regions may originate from the germline of a particular species, or they may be chimeric, containing segments from multiple species that may be further modified to optimize characteristics such as binding affinity or low immunogenicity. For treating humans, it is desirable for the antibody to have human sequences. If a human antibody of the desired specificity is not available, but such an antibody from a non-human species is available, the non-human antibody is humanized, for example, by CDR grafting (wherein the CDRs from the non-human antibody are placed in their respective positions within a compatible human antibody framework by genetically engineering the coding DNA). Similar considerations and procedures can be applied mutatis mutandis to antibodies for treating other species.

[0337] The antibodies can be complete monoclonal or polyclonal antibodies, immunologically active fragments (e.g., ScFv, Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, engineered single chain Fv molecules (Ladner et al., U.S. Pat. No. 4,946,778), or chimeric antibodies (e.g., antibodies that contain the binding specificity of a mouse antibody, but the remainder is of human origin). Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art.

[0338] Antibodies can be prepared using complete polypeptides or fragments containing the immunizing antigen of interest. The polypeptides or oligopeptides used to immunize animals can be obtained from the translation of RNA or can be chemically synthesized and can be conjugated to a vehicle protein if desired. Suitable substrates that can be chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. The coupled polypeptide can then be used to immunize animals (e.g., mice, rats, or rabbits).

[0339] combination In one embodiment, a composition of the invention comprises a combination of therapeutic agents as described herein. In some embodiments, a composition comprising a combination of therapeutic agents as described herein has an additive effect, where the overall effect of the combination is approximately equal to the sum of the effects of each individual therapeutic agent. In other embodiments, a composition comprising a combination of therapeutic agents as described herein has a synergistic effect, where the overall effect of the combination is greater than the sum of the effects of each individual therapeutic agent.

[0340] Compositions that include a combination of therapeutic agents include the individual therapeutic agents in any suitable ratio. For example, in one embodiment, the composition includes two individual therapeutic agents in a 1:1 ratio. However, the combination is not limited to any particular ratio. Rather, any ratio shown to be effective is encompassed.

[0341] join In various embodiments of the present invention, the delivery vehicle is bound to at least one of the domain for evading immune response and the targeting domain. Non-limiting examples of representative methods of binding include covalent bonds, electrostatic interactions, and hydrophobic ("van der Waals forces") interactions. In one embodiment, the binding is a reversible binding, so that the delivery vehicle can dissociate from at least one of the domain for evading immune response and the targeting domain upon exposure to a condition or chemical therapeutic agent. In another embodiment, the binding is an irreversible binding, so that the delivery vehicle does not separate from at least one of the domain for evading immune response and the targeting domain under normal conditions.

[0342] In some embodiments, the linkage comprises a covalent bond between the activated polymer-linked lipid and at least one of the domain for evasion of an immune response and the targeting domain. The term "activated polymer-linked lipid" refers to a molecule comprising a lipid portion and a polymer portion, where the polymer portion is activated through functionalization of the polymer-linked lipid with a first coupling group. In one embodiment, the activated polymer-linked lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated polymer-linked lipid is an activated pegylated lipid. In one embodiment, the first coupling group is attached to the lipid portion of the pegylated lipid. In another embodiment, the first coupling group is attached to the polyethylene glycol portion of the pegylated lipid. In one embodiment, the second functional group is covalently attached to at least one of the domain for evasion of an immune response and the targeting domain.

[0343] The first coupling group and the second coupling group can be any functional group known to those skilled in the art that forms a covalent bond with each other, for example, under mild reaction conditions or physiological conditions. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of maleimide, N-hydroxysuccinimide (NHS) ester, carbodiimide, hydrazide, pentafluorophenyl (PFP) ester, phosphine, hydroxymethylphosphine, psoralen, imidoester, pyridyl disulfide, isocyanate, vinyl sulfone, alpha-haloacetyl, aryl azide, acyl azide, alkyl azide, diazirine, benzophenone, epoxide, carbonate, anhydride, sulfonyl chloride, cyclooctyne, aldehyde, and sulfhydryl group. In some embodiments, the first coupling group or the second coupling group is selected from the group consisting of free amine (-NH2), free sulfhydryl group (-SH), free hydroxide group (-OH), carboxylate, hydrazide, and alkoxyamine. In some embodiments, the first coupling group is a functional group that reacts with sulfhydryl groups (such as maleimide, pyridyl disulfide, or haloacetyl). In one embodiment, the first coupling group is a maleimide.

[0344] In one embodiment, the second coupling group is a sulfhydryl group. The sulfhydryl group can be attached to the surface of the targeting domain using any method known to those skilled in the art. In one embodiment, the sulfhydryl group is present on the surface of a free cysteine ​​residue. In one embodiment, the sulfhydryl group is revealed by reduction of a disulfide on the targeting domain (e.g., through reaction with 2-mercaptoethylamine). In one embodiment, the sulfhydryl group can be attached by chemical reaction (such as reaction between a free amine and 2-iminothiolane or N-succinimidyl S-acetylthioacetate (SATA)).

[0345] In some embodiments, the polymer-linked lipid and the domain for evasion of immune responses, and in some embodiments, the targeting domain, are functionalized with groups used in "click" chemistry. Bioorthogonal "click" chemistry involves the reaction between a functional group having a 1,3-dipole (such as azide, nitrile oxide, nitrone, isocyanide, etc.) and a link having an alkene or alkyne dipolarophile. Representative dipolarophiles include any of the strained cycloalkenes and cycloalkynes known to those skilled in the art, non-limiting examples of which include cyclooctyne, dibenzocyclooctyne, monofluorinated cyclooctyne, difluorinated cyclooctyne, and biarylazacyclooctynones.

[0346] Targeting Domains In one embodiment, the composition includes a targeting domain that directs the delivery vehicle to a site. In one embodiment, the site is a site that requires the agent contained in the delivery vehicle. The targeting domain may include a nucleic acid, a peptide, an antibody, a small molecule, an organic molecule, an inorganic molecule, a glycan, a sugar, a hormone, etc., that targets the particle to a site that specifically requires a therapeutic agent. In certain embodiments, the particle includes multivalent targeting, where the particle includes multiple targeting mechanisms described herein. In certain embodiments, the targeting domain of the delivery vehicle specifically binds to a target associated with a site that requires the agent contained in the delivery vehicle. For example, the targeting domain may be selected to identify a ligand that functions as a cell surface marker on target cells associated with a particular disease state. Such targets are proteins, protein fragments, antigens, or other biomolecules associated with the targeting site. In some embodiments, the targeting domain is an affinity ligand that specifically binds to the target. In certain embodiments, the target (e.g., an antigen) is associated with a site that requires treatment with the agent. In some embodiments, the targeting domain may be copolymerized with the composition that includes the delivery vehicle. In some embodiments, the targeting domain may be covalently attached to a composition that includes a delivery vehicle, such as by chemical reaction of the targeting domain with the composition that includes the delivery vehicle. In some embodiments, the targeting domain is an additive in the delivery vehicle. Targeting domains of the present invention include, but are not limited to, antibodies, antibody fragments, proteins, peptides, and nucleic acids.

[0347] peptide In one embodiment, a targeting domain of the invention comprises a peptide. In certain embodiments, a peptide targeting domain specifically binds to a target of interest.

[0348] The peptides of the present invention can be produced using chemical methods. For example, peptides can be synthesized by solid-phase techniques (Roberge JY et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis can be achieved, for example, using an ABI 431 A peptide synthesizer (Perkin Elmer) following the instructions provided by the manufacturer.

[0349] Alternatively, the peptide may be produced by recombinant means, or by cleavage from a longer polypeptide. The composition of the peptide may be confirmed by amino acid analysis or sequencing.

[0350] Variants of the peptides according to the invention can be (i) those in which one or more of the amino acid residues are replaced by conserved or non-conserved amino acid residues (preferably conserved amino acid residues), where such replaced amino acid residues may or may not be encoded by the genetic code, (ii) those in which one or more modified amino acid residues are present (e.g., residues modified by attachment of a substituent group), (iii) those in which the peptide is a splice variant of the peptide of the invention, (iv) a fragment of the peptide, and / or (v) those in which the peptide is fused to another peptide, such as a leader or secretory sequence, or a sequence used for purification (e.g., His tag) or detection (e.g., Sv5 epitope tag). Fragments include peptides generated through proteolytic (including multi-site proteolytic) cleavage of the original sequence. Variants can be modified post-translationally or chemically. Such variants are considered to be within the scope of the skilled artisan from the teachings herein.

[0351] As known in the art, the "similarity" between two peptides is determined by comparing the amino acid sequence of one peptide and its conserved amino acid substitutions with the sequence of a second peptide. A variant is defined to include a peptide sequence that differs from the original sequence, preferably differs from the original sequence by less than 40% of the residues per segment of interest, more preferably differs from the original sequence by less than 25% of the residues per segment of interest, more preferably differs from the original sequence by less than 10% of the residues per segment of interest, and most preferably differs from the original protein sequence by only a few residues per segment of interest, while at the same time being sufficiently homologous to the original sequence to retain the function of the original sequence. The present invention includes amino acid sequences that are at least 60%, 65%, 70%, 72%, 74%, 76%, 78%, 80%, 90%, or 95% similar or identical to the original amino acid sequence. The degree of identity between two peptides is determined using computer algorithms and methods well known to those skilled in the art. Preferably, the identity between two amino acid sequences is determined using the BLASTP algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0352] The peptides of the invention can be post-translationally modified. For example, post-translational modifications within the scope of the invention include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis, myristylation, protein folding, and proteolytic processing. Some modifications or processing events require the introduction of additional biological machinery. For example, processing events (such as signal peptide cleavage and core glycosylation) can be examined by adding dog microsomal membranes or Xenopus egg extracts (US Pat. No. 6,103,489) to a standard translation reaction.

[0353] The peptides of the invention can include unnatural amino acids, formed by post-translational modification or by introducing unnatural amino acids during translation.

[0354] nucleic acid In one embodiment, the targeting domain of the present invention comprises an isolated nucleic acid, including, for example, DNA and RNA oligonucleotides. In some embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0355] The nucleotide sequence of the nucleic acid targeting domain can alternatively contain sequence variations (e.g., substitutions, insertions, and / or deletions of one or more nucleotides) relative to the original nucleotide sequence, provided that the resulting nucleic acid is functionally identical to the original and specifically binds to the target of interest.

[0356] As used herein, a nucleotide sequence is "substantially homologous" to any of the nucleotide sequences described herein when the nucleotide sequence is at least 60%, advantageously at least 70%, preferably at least 85%, more preferably at least 95% identical to the nucleotide sequence described herein. Other examples of possible modifications include the insertion of one or more nucleotides into the sequence, the addition of one or more nucleotides to either end of the sequence, or the deletion of one or more nucleotides at either end or within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods well known to those skilled in the art. The degree of identity between two amino acid sequences is preferably determined using the BLASTN algorithm [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)].

[0357] antibody In one embodiment, the targeting domain of the present invention comprises an antibody or an antibody fragment. In some embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human antibodies, and humanized antibodies.

[0358] The antibodies can be complete monoclonal or polyclonal antibodies, immunologically active fragments (e.g., Fab or (Fab)2 fragments), antibody heavy chains, antibody light chains, humanized antibodies, engineered single chain Fv molecules (Ladner et al., U.S. Pat. No. 4,946,778), or chimeric antibodies (e.g., antibodies that contain the binding specificity of a mouse antibody but the remainder is of human origin). Antibodies, including monoclonal and polyclonal antibodies, fragments, and chimeras, can be prepared using methods known to those skilled in the art.

[0359] Such antibodies can be produced in a variety of ways, including hybridoma culture, recombinant expression in bacterial or mammalian cell culture, and recombinant expression in transgenic animals. The choice of production method depends on several factors, including the desired antibody structure, the importance of carbohydrate moieties on the antibody, ease of culture and purification, and cost. Many different antibody structures can be produced using standard expression techniques, including full-length antibodies, antibody fragments (such as Fab and Fv fragments), as well as chimeric antibodies containing components from different species. Antibody fragments of small size (such as Fab and Fv fragments) that lack effector functions and have limited pharmacokinetic activity can be produced in bacterial expression systems. Single chain Fv fragments exhibit low immunogenicity.

[0360] Treatment method In some embodiments, the invention provides a method of treating a disease or disorder in a subject comprising delivering a therapeutic agent for the treatment of the disease or disorder, wherein the delivery vehicle comprises a moiety for evasion of an immune response. In some embodiments, the invention provides a method for stem cell targeted delivery of a therapeutic agent for the treatment of a disease or disorder in a subject, comprising delivering a therapeutic agent for the treatment of the disease or disorder, wherein the delivery vehicle comprises a moiety for evasion of an immune response and further comprises a moiety for targeting a cell type of interest.

[0361] The present invention also provides a method of delivering at least one agent to a subject in need thereof. In certain embodiments, the method is used to treat or prevent a disease or disorder in a subject, where the subject is suffering from an inflammatory condition, or an autoimmune disease or disorder. In some embodiments, the agent is a therapeutic agent for treating at least one of an inflammatory condition, or an autoimmune disease or disorder. In some embodiments, the agent is a therapeutic agent for treating at least one of an inflammatory condition, or an autoimmune disease or disorder in a subject diagnosed with an inflammatory condition, or an autoimmune disease or disorder. A therapeutic agent for treating an inflammatory condition or a disease or disorder that is not associated with an autoimmune disease or disorder. For example, in some embodiments, the compositions of the invention allow for the delivery of a therapeutic agent that avoids an enhanced or hypersensitive immune response in a subject.

[0362] Exemplary inflammatory conditions and autoimmune diseases include, but are not limited to, rheumatoid arthritis, systemic lupus erythematosus, alopecia areata, spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, pemphigoid, cardiomyopathy, celiac disease-dermatitis, chronic fatigue syndrome-immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cicatricial pemphigoid , cold agglutinin disease, CREST syndrome, Crohn's disease, Dego's disease, dermatomyositis, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura (ITP), Iga nephropathy, insulin-dependent diabetes mellitus (type I), juvenile arthritis, Meniere's disease, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome syndromes), polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff man syndrome, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, and Wegener's granulomatosis, as well as other organ-specific inflammatory diseases.

[0363] Thus, in some embodiments, the present invention provides a method of treating a non-inflammatory disease or disorder in a subject previously diagnosed with an inflammatory disease or disorder comprising delivering a therapeutic agent for the treatment of the non-inflammatory disease or disorder, wherein the delivery vehicle comprises a moiety for evasion of an immune response.

[0364] Those skilled in the art, when armed with the present disclosure, including the methods detailed herein, will recognize that the present invention is not limited to the treatment of already established diseases or disorders. In particular, the disease or disorder does not have to reach the point where it is harmful to the subject; in fact, the disease or disorder does not have to be detected in the subject before the treatment is applied. That is, significant signs or symptoms of the disease or disorder do not have to occur before the present invention provides benefits. Thus, the present invention includes methods of preventing a disease or disorder, in the sense that the compositions previously discussed elsewhere herein can be administered to a subject before the onset of the disease or disorder, thereby preventing the disease or disorder.

[0365] One of skill in the art, when armed with the present disclosure, will recognize that prevention of a disease or disorder includes administering a composition to a subject as a prophylactic measure against the development or progression of the disease or disorder.

[0366] The present invention encompasses the delivery of a delivery vehicle comprising at least one therapeutic agent bound to at least one domain for evading at least one immune response. In one embodiment, the delivery vehicle further comprises at least one targeting domain. To carry out the method of the present invention; a person skilled in the art would know how to formulate and administer a suitable composition to a subject based on the disclosure provided herein. The present invention is not limited to any particular method of administration or treatment regimen.

[0367] Those skilled in the art will understand that the compositions of the present invention can be administered alone or in any combination. Furthermore, the compositions of the present invention can be administered alone or in any combination in terms of time, i.e., simultaneously, before, and / or after each other. Those skilled in the art will recognize, based on the disclosure provided herein, that the compositions of the present invention can be used to prevent or treat a disease or disorder, and that the compositions can be used alone or in any combination with another composition to affect the outcome of the treatment. In various embodiments, any of the compositions of the present invention described herein can be administered alone or in combination with other modulators of other molecules associated with a disease or disorder.

[0368] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In some embodiments, the method has an additive effect, where the overall effect of administering the combination of compositions is approximately equal to the sum of the effects of administering each individual inhibitor. In other embodiments, the method has a synergistic effect, where the overall effect of administering the combination of compositions is greater than the sum of the effects of administering each individual composition.

[0369] The methods include administering a combination of compositions in any suitable ratio. For example, in one embodiment, the methods include administering two separate compositions in a 1:1 ratio. However, the methods are not limited to any particular ratio. Rather, any ratio shown to be effective is encompassed. In some embodiments, the invention encompasses methods of preparing a therapeutic composition for delivering at least one agent to endothelial cells lining the lumen of a blood vessel.

[0370] Compositions and methods for non-hepatic delivery One aspect relates to a pharmaceutical composition for in vivo delivery of lipid nanoparticles (LNPs) to non-hepatic cells of a subject, while avoiding delivery to hepatic cells, where the LNPs are pegylated lipids bound to an active CD47 polypeptide and a therapeutic or diagnostic agent. In some embodiments, the LNPs further comprise a pegylated lipid bound to a targeting moiety. In some embodiments, the targeting moiety is an antibody or an antigen-binding fragment thereof. In some embodiments, the LNPs further comprise a non-bound pegylated lipid.

[0371] In some embodiments, active CD47 polypeptide refers to the entire CD47 protein or a CD47 protein fragment that binds to and activates SIRPα signaling and inhibits uptake (phagocytosis) by macrophages. In some embodiments, the CD47 fragment comprises SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, a CD47 polypeptide comprising SEQ ID NO: 1 or SEQ ID NO: 2 constitutes a means of activating SIRPα signaling or a means of reducing or preventing macrophage uptake.

[0372] In some embodiments of this method, the harmful or undesirable effects of the therapeutic agent of the LNP are substantially reduced or effectively eliminated. By substantially reduced, it is meant that the reduction is merely detectable, but leads to a clinically significant physiological improvement. By effectively eliminated, it is meant that there may be some detectable presence of the therapeutic agent in the liver, but there is also no significant physiological impact. In some embodiments, the inflammatory response is reduced or not exacerbated compared to LNPs lacking active CD47 polypeptide and / or PEG shielding. In some embodiments, the toxicity is reduced or not exacerbated compared to LNPs lacking active CD47 polypeptide and / or PEG shielding. In some embodiments, the physiologically effective dosage is reduced compared to LNPs lacking active CD47 polypeptide and / or PEG shielding. In some embodiments, the percentage of the administered therapeutic agent that reaches the target cell or tissue is enhanced compared to LNPs lacking active CD47 polypeptide and / or PEG shielding.

[0373] Pharmaceutical Compositions The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparation methods include the step of bringing into association the active ingredient with the carrier or one or more other accessory ingredients and then, if necessary or desired, shaping or packaging the product into the desired single or multiple dosage units.

[0374] The description of pharmaceutical compositions provided herein is primarily of pharmaceutical compositions suitable for ethical administration to humans, but those skilled in the art will understand that such compositions are generally suitable for administration to animals of any kind. Modifications to make pharmaceutical compositions suitable for administration to humans suitable for administration to a variety of animals are well understood, and an ordinary skilled veterinary pharmacologist can design and implement such modifications, if necessary, with routine experimentation. Subjects to which the pharmaceutical compositions of the present invention are intended to be administered include, but are not limited to, humans and other primates, non-human primates, and various mammals, including commercial mammals such as cows, pigs, horses, sheep, cats, and dogs.

[0375] Pharmaceutical compositions useful in the methods of the invention can be prepared, packaged, or sold in formulations suitable for intraocular, oral, enteral, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intraventricular, intradermal, intramuscular, or another route of administration. Other possible formulations include projected nanoparticles, liposomal formulations, resealed red blood cells containing the active ingredient, and immunogen-based formulations.

[0376] The pharmaceutical composition of the present invention can be prepared, packaged or sold in bulk as one single unit dose or as multiple single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject, or a convenient fraction thereof, such as, for example, half or a third of such dosage.

[0377] The relative amounts of the active ingredient, pharma- ceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route of administration of the composition. By way of example, the composition may contain from 0.1% to 100% (w / w) active ingredient. In addition to the active ingredient, the pharmaceutical compositions of the present invention may further comprise one or more additional pharma- ceutical active agents. Controlled or sustained release formulations of the pharmaceutical compositions of the invention can be made using conventional techniques.

[0378] "Parenteral administration" of a pharmaceutical composition as used herein includes any administration route characterized by the physical destruction of the tissue of a subject and the administration of the pharmaceutical composition by the destruction of the tissue. Thus, parenteral administration includes, but is not limited to, the administration of a pharmaceutical composition by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intraventricular, and kidney dialysis infusion techniques.

[0379] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, and sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, and sold in unit dosage form, such as ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0380] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using, for example, non-toxic parenterally acceptable diluents or solvents, such as water or 1,3 butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, in liposomal preparations, or as components of biodegradable polymer systems. Sustained release or implantation compositions may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0381] The pharmaceutical compositions of the invention may be prepared, packaged, and sold in a formulation suitable for pulmonary administration via the buccal cavity. Such formulations may comprise dry particles having a diameter in the range of about 0.5 to about 7 nanometers, preferably about 1 to about 6 nanometers, and 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 capable of directing a stream of propellant to disperse the powder, or a device using a self-propelling solvent / powder dispensing container, such as a device in which the active ingredient is dissolved or suspended in a low boiling propellant in a closed container. Preferably, such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0382] Low boiling point propellants generally include liquid propellants having a boiling point below 65° F. at atmospheric pressure. Generally, the propellant will comprise 50-99.9% (w / w) of the composition and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size on the same order as the particles containing the active ingredient).

[0383] A formulation of a pharmaceutical composition suitable for parenteral administration comprises the active ingredient in combination with a pharma- ceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, and sold in a form suitable for bolus or continuous administration. Injectable formulations may be prepared, packaged, and sold in unit dosage form, such as ampoules, or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional components, including, but not limited to, suspending agents, stabilizing agents, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0384] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations may be prepared using, for example, non-toxic parenterally acceptable diluents or solvents, such as water or 1,3 butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides. Other parenterally administrable formulations that are useful include those that contain the active ingredient in microcrystalline form, in liposomal preparations, or as components of biodegradable polymer systems. Sustained release or implantation compositions may include pharma-ceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, poorly soluble polymers, or poorly soluble salts.

[0385] As used herein, "additional ingredients" include, but are not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharma-ceutical acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference. EXAMPLES

[0386] The present invention will be described in more detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but should be construed as embracing any and all variations that become evident as a result of the teachings provided herein.

[0387] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and use the present invention and practice the methods of the claims. Accordingly, the following examples are not to be construed as limiting the disclosure in any way.

[0388] Example 1: Eliminating the off-target effects of lipid-based carriers using a novel peptide-based dual post-insertion technique to enhance tissue-specific delivery and safety Lipid nanoparticles (LNPs) have been used to deliver nucleoside-modified mRNA in small and large animal models. With systemic delivery, LNPs target the liver, which must be reduced or avoided when delivering targeted mRNA to other cells or tissues. The data presented herein demonstrate the development of a method to target specific cell types and tissues by linking antibodies, antibody fragments, and ligands to LNPs. In naive mice, mRNA-LNP delivery does not result in the release of proinflammatory mediators. However, in a pre-existing inflammatory state, treatment of mice with LNP-mRNA exacerbated the pre-existing inflammation to a level where lethality could be observed. It was found that macrophage uptake is responsible for the exacerbation of inflammation by mRNA-LNPs. Therefore, a method to reduce off-target delivery of LNPs specific to macrophages was developed by both attaching targeting ligands and inhibiting LNP uptake by macrophages. CD47 was used to reduce inflammatory exacerbation, which was observed when mRNA-LNPs were administered to inflamed mice (Figure 8). LNP is not only decorated with targeting moiety, but also decorated with CD47 peptide lipid.This new custom-designed CD47 peptide lipid provides avoidance of uptake by phagocyte system, while the targeting moiety allows cell-specific targeting.Any type of lipid can be used to make CD47 peptide lipid, as long as it can be effectively fixed on the surface of LNP.In addition, for CD47 peptide, any sequence that spans the binding site of CD47 protein can be used.

[0389] The CD47 peptide-lipids are used in a micellar form and subsequently incorporated into the LNP surface or as a structural component of the LNP itself. Figure 1 shows a procedure involving the use of micelles to generate CD47-modified targeted LNP constructs.

[0390] Inflammation in mice challenged with LPS was exacerbated by mRNA-LNPs, but the use of mRNA-LNPs containing CD47 significantly alleviated the exacerbation of inflammation. Targeting efficiency was also enhanced. For example, using PECAM delivery to endothelial cells, localization in the tissue of interest was doubled by using CD47 peptides to prevent macrophage uptake. Moreover, the ratio of mRNA expression in the tissue of interest compared to the liver as an off-target can be enhanced up to 100-fold by this technique. The data also show enhanced targeted delivery to T cells when combining anti-CD4 targeting ligands with the CD47 moiety. Thus, linking the CD47 peptide to the surface of the LNP significantly enhances targeting and reduces the exacerbation of inflammation.

[0391] Experiments are performed to examine the effect of CD47 on LNP uptake into macrophages and demonstrate improved delivery efficacy to specific cell types when CD47 modification is combined with specific cell targeting.

[0392] Figure 2 shows that CD47 modification on LNPs significantly reduces non-specific uptake / mRNA translation, and Figure 3 shows that CD47 / mRNA-LNPs recruit significantly fewer PBMCs when compared to mRNA-LNPs.

[0393] Figure 4A shows that when combined with CD47, targeting efficiency is increased. Data for endothelial targeting are shown. Figure 5 shows that targeting efficiency is increased when combined with CD47. Data regarding T cell targeting are shown.

[0394] FIG. 6 shows that proinflammatory cytokines are elevated following IV treatment with LNP-mRNA (IV-LPS) in a systemic mouse model of inflammation. FIG. 7 shows enhanced LNP uptake by monocytes / macrophages in LPS-treated mice.

[0395] FIG. 8 shows that depletion of macrophages with clodronate reduces systemic pro-inflammatory markers. FIG. 9 shows that reducing macrophage uptake by CD47-modified LNPs ameliorates systemic pro-inflammatory markers in the LPS model of systemic inflammation.

[0396] FIG. 10 shows that optimized mRNA-D47 / LNP minimizes the acute phase response in the liver by RNA-Seq analysis. FIG. 11 shows that the protein corona formed around CD47-modified LNPs in mouse serum is completely different from that of unmodified LNPs.

[0397] The unique design and combination of a targeting ligand together with the CD47 moiety significantly expands the therapeutic potential of mRNA-LNPs with enhanced efficacy and reduced adverse events.

[0398] The present invention allows both improved delivery of nucleic acid therapeutics to target tissues in vivo using LNP targeting technology and reduced exacerbation of inflammation when LNPs are administered to mice suffering from basal inflammation. This technology has the potential to revolutionize both targeted delivery of carriers and minimizing their side effects by reducing the possibility of off-target delivery. With the present invention, the dose of lipid-based carriers can be reduced and LNPs are directed to the tissue of interest with minimal side effects. Many therapeutics may be developed with a wide range of targeted CD47-modified carriers, including lipids, polymers, peptides, etc. By minimizing off-target effects, targeted therapeutics have the potential to access difficult-to-reach and rare targets, including stem cells.

[0399] array: The CD47 peptide sequences used in the experiments disclosed herein are as follows: Mouse: Lys(N3)-GGGNYTCEVTELSREGKTVIELK (SEQ ID NO: 1) Human: Lys(N3)-GGGNYTCEVTELTREGETIIELK (SEQ ID NO:2)

[0400] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it will be apparent to others skilled in the art that other embodiments and variations of the present invention can be made without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A composition for delivery of a therapeutic agent to a subject in need thereof, comprising a therapeutic agent and a delivery vehicle, wherein the delivery vehicle comprises a moiety for inhibiting uptake of the composition by macrophages.

2. 2. The composition of claim 1, wherein the moiety for inhibiting uptake of the composition by macrophages is selected from the group consisting of a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, and an activator of LILRB1 activity.

3. The composition of claim 2, wherein the portion for inhibiting uptake of the composition by macrophages comprises a CD47 polypeptide comprising an sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or a fragment or variant thereof.

4. The composition of claim 1 , wherein the delivery vehicle further comprises a targeting moiety specific for binding to a target cell.

5. The composition of claim 4 , wherein the target cells are selected from the group consisting of endothelial cells, immune cells and stem cells.

6. The composition of claim 1 , wherein the therapeutic agent comprises at least one isolated nucleoside modified RNA molecule.

7. The at least one isolated nucleoside modified RNA comprises pseudouridine and The composition of claim 6, comprising at least one selected from the group including 1-methyl-pseudouridine.

8. The composition of claim 6, wherein the at least one isolated nucleoside modified RNA is a purified nucleoside modified RNA.

9. The composition of claim 1 , wherein the composition further comprises an adjuvant.

10. The composition of claim 1 , wherein the delivery vehicle comprises a lipid nanoparticle (LNP).

11. The composition of claim 10, wherein the at least one nucleoside modified RNA is encapsulated within a LNP.

12. 13. Use of the composition of claim 1 in the manufacture of a medicament for treating a disease or disorder in a subject in need thereof.

13. 13. The use of claim 12, wherein the moiety for inhibiting uptake of the composition by macrophages is selected from the group consisting of a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, and an activator of LILRB1 activity.

14. 14. The use of claim 13, wherein the moiety for inhibiting uptake of the composition by macrophages comprises a CD47 polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, or a fragment or variant thereof.

15. The use of claim 12, wherein the disease or disorder is an inflammatory or autoimmune disease or disorder.

16. 16. The use according to claim 15, wherein the therapeutic agent is an agent for the treatment of an inflammatory or autoimmune disease or disorder.

17. 13. The use of claim 12, wherein the composition is formulated for administration by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

18. 13. Use of the composition of claim 1 in the manufacture of a medicament for delivering a therapeutic agent to a target cell.

19. 19. The use of claim 18, wherein the moiety for inhibiting uptake of the composition by macrophages is selected from the group consisting of a CD47 polypeptide, an active CD47 polypeptide fragment, an activator of SIRPα activity, a PD-L1 polypeptide, an active PD-L1 polypeptide fragment, an activator of PD-1 activity, a CD24 polypeptide, an active CD24 polypeptide fragment, an activator of Siglec-10 activity, a polyglutamic acid peptide, a β2M polypeptide, an active β2M polypeptide fragment, and an activator of LILRB1 activity.

20. 20. The use of claim 19, wherein the moiety for inhibiting uptake of the composition by macrophages comprises a CD47 polypeptide comprising a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, or a fragment or variant thereof.

21. 19. The use according to claim 18, wherein the target cells are selected from the group consisting of endothelial cells, immune cells and stem cells.

22. 19. The use according to claim 18, wherein the therapeutic agent is an agent for the treatment of an inflammatory or autoimmune disease or disorder.

23. 20. The use of claim 18, wherein the composition is formulated for administration by a delivery route selected from the group consisting of intradermal, subcutaneous, inhalation, intranasal, and intramuscular.

24. A pharmaceutical composition for in vivo delivery of lipid nanoparticles (LNPs) to non-hepatic cells of a subject, while avoiding delivery to hepatic cells, wherein the LNPs comprise a pegylated lipid conjugated to an active CD47 polypeptide and a therapeutic agent.

25. 25. The pharmaceutical composition of claim 24, wherein the LNP further comprises a pegylated lipid attached to a binding moiety.

26. 25. The pharmaceutical composition of claim 24, wherein the binding moiety is a whole antibody or an antigen-binding fragment thereof.

27. 25. The pharmaceutical composition of claim 24, wherein the LNP further comprises an unbound pegylated lipid.

28. 25. The pharmaceutical composition of claim 24, wherein the active CD47 polypeptide comprises SEQ ID NO:1 or SEQ ID NO:

2.

29. 25. Use of the composition of claim 24 in the manufacture of a medicament for delivering LNPs to non-hepatic cells of a subject in vivo, while avoiding delivery to hepatic cells.

30. 30. The use of claim 29, wherein the composition is formulated for intravenous administration.

31. The use of claim 29, wherein inflammatory responses or toxicity are reduced or not exacerbated compared to LNPs lacking active CD47 polypeptide and / or PEG shielding.

32. The use of claim 29, wherein the percentage of the administered therapeutic agent that reaches the target cell or tissue is enhanced compared to LNPs lacking an active CD47 polypeptide and / or PEG shielding.

33. The use of claim 29, wherein the physiologically effective dosage is reduced compared to LNPs lacking an active CD47 polypeptide and / or PEG shielding.

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