WO2020219941A1

WO2020219941A1 - Lipid nanoparticles

Info

Publication number: WO2020219941A1
Application number: PCT/US2020/029907
Authority: WO
Inventors: James Heyes; Adam Judge; Kieu Mong LAM; Alan D. MARTIN
Original assignee: Genevant Sciences Gmbh
Priority date: 2019-04-26
Filing date: 2020-04-24
Publication date: 2020-10-29

Abstract

The invention provides lipid nanoparticles and formulations containing lipid nanoparticles.

Description

LIPID NANOPARTICLES

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of priority of U.S. application serial No. 62/839,452, filed April 26, 2019, and of U.S. application serial No. 62/867,098, filed June 26,

2019, which applications are herein incorporated by reference.

BACKGROUND

Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds, such as therapeutic nucleic acids, proteins, and peptides, which are otherwise cell impermeable. Drugs based on nucleic acids, which include large nucleic acid molecules such as, e.g., in vitro transcribed messenger RNA (mRNA) as well as smaller polynucleotides that interact with a messenger RNA or a gene, have to be delivered to the proper cellular compartment in order to be effective. For example, double-stranded nucleic acids such as double-stranded RNA molecules (dsRNA), including, e.g., siRNAs, suffer from their physico-chemical properties that render them impermeable to cells. Upon delivery into the proper compartment, siRNAs block gene expression through a highly conserved regulatory mechanism known as RNA

interference (RNAi). Typically, siRNAs are large in size with a molecular weight ranging from 12-17 kDa and are highly anionic due to their phosphate backbone with up to 50 negative charges. In addition, the two complementary RNA strands result in a rigid helix. These features contribute to the siRNA's poor "drug-like" properties. When administered intravenously, the siRNA is rapidly excreted from the body with a typical half-life in the range of only 10 minutes. Additionally, siRNAs are rapidly degraded by nucleases present in blood and other fluids or in tissues and have been shown to stimulate strong immune responses in vitro and in vivo. See, e.g., Robbins et al, Oligonucleotides 19:89- 102, 2009. mRNA molecules suffer from similar issues of impermeability, fragility, and immunogenicity. See International Patent Application Publication Number

WO2016/1 18697.

Lipid nanoparticle formulations have improved nucleic acid delivery in vivo. For example, such formulations have significantly reduced siRNA doses necessary to achieve target knockdown in vivo. See Zimmermann et al., Nature 441 : 111-114, 2006. Typically, such lipid nanoparticle drug delivery systems are multi-component

formulations comprising cationic lipids, helper lipids, and lipids containing polyethylene glycol. The positively charged cationic lipids bind to the anionic nucleic acid, while the other components support a stable self-assembly of the lipid nanoparticles.

Efforts have been directed toward improving delivery efficacy of lipid nanoparticle formulations. Many such efforts have been aimed toward developing more appropriate cationic lipids. See, e.g., Akinc et al, Nature Biotechnology 26:561-569, 2008; Love et al., Proc. Natl. Acad. Sci. USA 107:1864-1869, 2010; Baigude et al., Journal of Controlled Release 107:276-287, 2005; Semple et al., Nature Biotechnology 28:172-176, 2010.

Despites these efforts, there remains a need for lipid nanoparticle containing formulations that provide high potency following administration and that allow for the administration of lower doses of nucleic acids.

SUMMARY

Provided herewith are lipid nanoparticles and pharmaceutical compositions comprising the lipid nanoparticles. The lipid nanoparticles and pharmaceutical

compositions are particularly useful for delivering a nucleic acid to a patient (e.g., a human) or to a cell.

Lipid nanoparticle formulations useful for the delivery of nucleic acids frequently employ a PEG-lipid conjugate, which serves to help control particle size during LNP manufacture and prevent unwanted aggregation in the vial and in the blood after administration. The PEG-lipid conjugates also help to prevent unwanted opsonization in the blood. It is very typical for these PEG-lipid conjugates to use a PEG polymer component with a MW of about 2000. It is also typical for the conjugates to use a lipid moiety comprising 2 CM chains and for these PEG-lipid conjugates to be employed in molar ratios (relative to other lipids in the composition) of 0.5 to 2%. In contrast, lipid nanoparticle formulations described herein can contain PEG lipids that have a PEG MW of 500-1000 with mol ratios of 2% to 5%, as well as PEG polymer size of 5000-20000 with mol ratios of 0.2% to 0.5%. Accordingly, and as described more fully herein, new formulations have been developed that use PEG-lipid conjugate structures that are different from the ones typically used, used in differing amounts, to provide beneficial properties for the lipid nanoparticles.

Accordingly, in one embodiment, provided herewith is a lipid nanoparticle comprising: (a) a nucleic acid; (b) a cationic lipid in an amount from about 30 to about 70 mol % of the total lipid present in the particle;

(c) a non-cationic lipid in an amount from about 30 to about 70 mol % of the total lipid present in the particle, wherein the non-cationic lipid comprises a mixture of a phospholipid and a cholesterol or derivative thereof; and

(d) a polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles in an amount from about 2 to about 5 mol % of the total lipid in the particle, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety,

wherein the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 500 to about 1,000 daltons,

provided that when the lipid anchor moiety is a dialkyl moiety, at least one of the two alkyl chains is less than C₁₄.

Also provided is a lipid nanoparticle comprising:

(a) a nucleic acid;

(b) a cationic lipid in an amount from about 30 to about 70 mol % of the total lipid present in the particle;

(d) a polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles in an amount from about 0.2 to about 0.5 mol % of the total lipid in the particle, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety,

wherein the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 5,000 to about 20,000 daltons.

Also provided is a lipid nanoparticle comprising:

(a) a nucleic acid, wherein the nucleic acid is mRNA;

(b) a cationic lipid;

(c) a non-cationic lipid, wherein the non-cationic lipid comprises a mixture of a phospholipid and a cholesterol or derivative thereof; and

(d) a polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single chain C₁₈ alkyl moiety.

Also provided is a lipid nanoparticle comprising:

(a) a nucleic acid, wherein the nucleic acid is mRNA;

(d) a polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles in an amount from about 1 to about 2.5 mol % of the total lipid in the particle (e.g., about 1.6 mol %),

wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single chain C14-C22 (e.g., C ₁₄, C₁₆, C₁₈, C₂₀ or C₂₂) alkyl moiety,

wherein the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 500 to about 3,000 daltons (e.g., about 2000 daltons).

Also provided is a method for reducing the immune response of administration of a lipid nanoparticle (LNP) to a human, comprising selecting a polyethylene glycol (PEG)-lipid conjugate for use in LNP, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single alkyl chain, wherein the LNP comprises an mRNA payload.

The invention also provides a pharmaceutical composition comprising a lipid

nanoparticle of the invention, and a pharmaceutically acceptable carrier.

The invention also provides a method for delivering a nucleic acid to a cell comprising contacting the cell with a lipid nanoparticle of the invention. More generally, the invention provides methods of administering nucleic acids to a living cell, in vivo or in vitro.

The invention also provides a method for treating a disease characterized by a genetic defect that results in a deficiency of a functional protein, the method comprising: administering to a subject having the disease, a lipid nanoparticle of the invention, wherein the nucleic acid molecule is an mRNA that encodes the functional protein or a protein having the same biological activity as the functional protein.

The invention also provides a method for treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject having the disease a lipid nanoparticle of the invention, wherein the nucleic acid molecule is an siRNA that targets expression of the overexpressed polypeptide.

The invention also provides a lipid nanoparticle of the invention, for the therapeutic or prophylactic treatment of a disease characterized by a genetic defect that results in a deficiency of a functional protein.

The invention also provides a lipid nanoparticle of the invention, for the therapeutic or prophylactic treatment of a disease characterized by overexpression of a polypeptide.

The invention also provides a method for treating a disease or disorder in an animal, comprising administering a therapeutically effective amount of a lipid nanoparticle of the invention to the animal.

The invention also provides processes and intermediates disclosed herein that are useful for making lipid nanoparticles of the invention.

DETAILED DESCRIPTION

Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

The term“about” means ±5%, ±4%, ±3%, ±2%, or ±1%.

The term“interfering RNA” or“RNAi” or“interfering RNA sequence” refers to single- stranded RNA (e.g., mature miRNA) or double-stranded RNA (i.e., duplex RNA such as siRNA, aiRNA, or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity to the target gene or sequence, or may comprise a region of mismatch (i.e., a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.

Interfering RNA includes“small-interfering RNA” or“siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and is preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15- 60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferably about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may comprise 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini. Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having selfcomplementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

Preferably, siRNA are chemically synthesized. siRNA can also be generated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25 nucleotides in length) with the E. coli RNase III or Dicer. These enzymes process the dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA , 99: 14236 (2002); Byrom et al., Ambion TechNotes, 10(l):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31 :981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and

Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA are at least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotides in length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotides in length, or longer. The dsRNA can encode for an entire gene transcript or a partial gene transcript. In certain instances, siRNA may be encoded by a plasmid (e.g., transcribed as sequences that automatically fold into duplexes with hairpin loops).

As used herein, the term“mismatch motif’ or“mismatch region” refers to a portion of an interfering RNA (e.g., siRNA, aiRNA, miRNA) sequence that does not have 100%

complementarity to its target sequence. An interfering RNA may have at least one, two, three, four, five, six, or more mismatch regions. The mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides. An“effective amount” or“therapeutically effective amount” of an nucleic acid such as a nucleic acid (e.g., an interfering RNA or mRNA) is an amount sufficient to produce the desired effect, e.g., an inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of an interfering RNA; or mRNA-directed expression of an amount of a protein that causes a desirable biological effect in the organism within which the protein is expressed. Inhibition of expression of a target gene or target sequence is achieved when the value obtained with an interfering RNA relative to the control is about 90%, 85%,

80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other embodiments, the expressed protein is an active form of a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the protein that is normally expressed in the cell type of a healthy individual. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

By“decrease,”“decreasing,”“reduce,” or“reducing” of an immune response by an interfering RNA is intended to mean a detectable decrease of an immune response to a given interfering RNA (e.g., a modified interfering RNA). The amount of decrease of an immune response by a modified interfering RNA may be determined relative to the level of an immune response in the presence of an unmodified interfering RNA. A detectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,

85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of the unmodified interfering RNA. A decrease in the immune response to interfering RNA is typically measured by a decrease in cytokine production (e.g., IENg, IFNa, TNFa, IL-6, or IL-12) by a responder cell in vitro or a decrease in cytokine production in the sera of a mammalian subject after administration of the interfering RNA.

By“decrease,”“decreasing,”“reduce,” or“reducing” of an immune response by an mRNA is intended to mean a detectable decrease of an immune response to a given mRNA (e.g., a modified mRNA). The amount of decrease of an immune response by a modified mRNA may be determined relative to the level of an immune response in the presence of an unmodified mRNA. A detectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more lower than the immune response detected in the presence of the unmodified tnRNA. A decrease in the immune response to mRNA is typically measured by a decrease in cytokine production (e.g., IENg, IFNa, TNFa, IL-6, or IL-12) by a responder cell in vitro or a decrease in cytokine production in the sera of a mammalian subject after administration of the mRNA.

As used herein, the term“responder cell” refers to a cell, preferably a mammalian cell, which produces a detectable immune response when contacted with an immunostimulatory interfering RNA such as an unmodified siRNA. Exemplary responder cells include, e.g., dendritic cells, macrophages, peripheral blood mononuclear cells (PBMCs), splenocytes, and the like. Detectable immune responses include, e.g., production of cytokines or growth factors such as TNF-a, IFN-a, IFN-b, IFN-g, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, TGF, and combinations thereof,

“Substantial identity” refers to a sequence that hybridizes to a reference sequence under stringent conditions, or to a sequence that has a specified percent identity over a specified region of a reference sequence.

The phrase“stringent hybridization conditions” refers to conditions under which a nucleic acid will hybridize to its target sequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50% formamide,

5xSSC, and 1% SDS, incubating at 42° C., or, 5xSSC, 1% SDS, incubating at 65° C., with wash in 0.2xSSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C. depending on primer length. For high stringency PCR amplification, a temperature of about 62° C. is typical, although high stringency annealing temperatures can range from about 50° C. to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec. -2 min., an annealing phase lasting 30 sec. -2 min., and an extension phase of about 72° C. for 1-2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g. , in innis et al,, PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary“moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in lxSSC at 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous references, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms“substantially identical” or“substantial identity,” in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition, when the context indicates, also refers analogously to the complement of a sequence. Preferably, the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

A“comparison window,” as used herein, includes reference to a segment of any one of a number of contiguous positions selected from the group consisting of from about 5 to about 60, usually about 10 to about 45, more usually about 15 to about 30, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g. , by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)).

A preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215 :403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids. Software for performing BLAST analyses is publicly available through the National Center for

Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)).

One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

The term“nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double- stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, precondensed DNA, a PCR product, vectors (PI, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-0-methyl ribonucleotides, and peptide- nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et ah, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et ah, Mol. Cell. Probes, 8:91- 98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups.“Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

The term“gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as an RNA transcript or a polypeptide.

The term“lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1)“simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, which can be saturated or unsaturated, having the number of carbon atoms designated (i.e., C_1-8 means one to eight carbons).

Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The term "alkenyl" refers to an unsaturated alkyl radical having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl radical having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers.

As used herein, the term lipid nanoparticle“LNP” refers to a lipid-nucleic acid particle or a nucleic acid-lipid particle (e.g., a stable nucleic acid-lipid particle). A LNP represents a particle made from lipids (e.g., a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle), and a nucleic acid, wherein the nucleic acid (e.g., siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, mRNA, self- amplifying RNA, or a plasmid, including plasmids from which an interfering RNA or mRNA is transcribed) is encapsulated within the lipid. In one embodiment, the nucleic acid is at least 50% encapsulated in the lipid; in one embodiment, the nucleic acid is at least 75% encapsulated in the lipid; in one embodiment, the nucleic acid is at least 90% encapsulated in the lipid; and in one embodiment, the nucleic acid is completely encapsulated in the lipid. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid conjugate (e.g., a PEG-lipid conjugate). LNP are extremely useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site), and they can mediate expression of the transfected gene or silencing of target gene expression at these distal sites.

The lipid particles of the invention (e.g., LNPs) typically have a mean diameter of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm, and are substantially non-toxic. In addition, nucleic acids, when present in the lipid particles of the invention, are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

As used herein,“lipid encapsulated” can refer to a lipid particle that provides a nucleic acid (e.g. , an interfering RNA or mRNA), with full encapsulation, partial encapsulation, or both. In one embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an LNP, or other nucleic acid-lipid particle).

The term“cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7,0), It has been surprisingly found that cationic lipids comprising alkyl chains with multiple sites of

unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present invention, have been described in U.S, Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618;

5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Nonlimiting examples of cationic lipids are described in detail herein. In some cases, the cationic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, Cl 8 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

In the lipid nanoparticles described herein, the cationic lipid may comprise, e.g., one or more of the following: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3- dimethylaminopropyl)-[ 1 ,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4- dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5- dimethylaminomethyl-[ 1 ,3] -dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [l,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin- K-DMA), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1 ,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3 -(N,N -dilinoleylamino)- 1 ,2- propanediol (DLinAP), 3 -(N,N-dioleylamino)-l ,2-propanedio (DOAP), 1 ,2-dilinoleyloxo-3-(2- N,N -dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy- N,N-dimethylaminopropane (DSDMA), N-( 1 -(2,3 -dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N -distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3- (N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-( 1 ,2- dimyristyloxyprop-3 -yl)-N,N -dimethyl-N -hydroxyethyl ammonium bromide (DMRIE), 2,3- dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3 -dimethylamino-2-(cholest-5 -en-3 -beta- oxybutan-4-oxy)- 1 -(cis,cis-9, 12-octadecadienoxy)propane (CLinDMA), 2- [5 '-(cholest-5 -en-3- beta-oxy)-3'-oxapentoxy)-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N -dimethyl-3 ,4-dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), l,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof.

The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U.S. Provisional Application No. 61/104,212, filed Oct. 9, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC, DLin-MA, DLinDAP, DLin- S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT Application No. PCT/US08/88676, filed Dec. 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

Any of a variety of cationic lipids may be used in the lipid particles of the invention (e.g., LNP), either alone or in combination with one or more other cationic lipid species or non- cationic lipid species.

Cationic lipids which are useful in the present invention can be any of a number of lipid species which carry a net positive charge at physiological pH. Such lipids include, but are not limited to, N,N -dioleyl-N,N -dimethylammonium chloride (DODAC), 1 ,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), l,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N- (l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), 3-(N— (N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC -Choi), N-( 1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N- hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA),

dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan- 4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)- 3 '-oxapentoxy)-3 -dimethyl- 1 -(cis,cis-9', 1 -2'-octadecadienoxy)propane (CpLinDMA), N,N- dimethyl-3,4-dioleyloxyben2ylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 1 ,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), and mixtures thereof. A number of these lipids and related analogs have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618;

5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which are each herein incorporated by reference in their entirety for all purposes. Additionally, a number of commercial preparations of cationic lipids are available and can be used in the present invention. These include, e.g., LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE® (commercially available cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic liposomes comprising DOGS from Promega Corp., Madison, Wis., USA).

Additionally, cationic lipids of Formula I having the following structures are useful in the present invention.

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation. In certain instances, R³ and R⁴ are both the same, i.e,, R³ and R⁴ are both linoleyl (C18), etc. In certain other instances, R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C ₁₄) and R⁴ is linoleyl (Cis). In one embodiment, the cationic lipid of Formula I is symmetrical, i.e., R³ and R⁴ are both the same. In another embodiment, both R³ and R⁴ comprise at least two sites of unsaturation. In some embodiments, R³ and R⁴ are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In one embodiment, R³ and R⁴ are both linoleyl. In some embodiments, R³ and R⁴ comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl. In particular embodiments, the cationic lipid of Formula I is 1 ,2 -dilinoleyloxy-N,N - dimethylaminopropane (DLinDMA) or l,2-dilinolenyloxy-N,N-dimethylaminopropane

(DLenDMA).

Furthermore, cationic lipids of Formula II having the following structures are useful in the present invention.

wherein R¹ and R² are independently selected and are H or C1-C3 alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation. In certain instances, R³ and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (Cig), etc. In certain other instances, R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C14) and R⁴ is linoleyl (Cig). In one embodiment, the cationic lipids of the present invention are symmetrical, i.e., R³ and R⁴ are both the same. In another embodiment, both R³ and R⁴ comprise at least two sites of unsaturation. In some embodiments, R³ and R⁴ are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In one embodiment, R³ and R⁴ are both linoleyl. In some embodiments, R³ and R⁴ comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

Moreover, cationic lipids of Formula III having the following structures (or salts thereof) are useful in the present invention. wherein R¹ and R² are either the same or different and independently optionally substituted C12- C24 alkyl, optionally substituted C12-C24 alkenyl, optionally substituted C12-C24 alkynyl, or optionally substituted Ci2-C24acyl; R³ and R⁴are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted Ci-Ce alkenyl, or optionally substituted Ci-Ce alkynyl or R³ and R⁴ may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or hydrogen or Ci-Ce alkyl to provide a quaternary amine; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and independently O, S, or

NH.

In some embodiments, the cationic lipid of Formula III is 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[ 1,3] -dioxolane (DLin-K-C2 -DMA;“XTC2”), 2 ,2-dilinoleyl-4-(3 - dimethylaminopropyl) - [ 1 ,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4- dimethylaminobutyl)-[ 1 ,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5- dimethylaminomethyl- [ 1 ,3] -dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [1,3] -dioxolane (DLin-K-MPZ), 2, 2-dilinoleyl-4-dimethylaminomethyl-[ 1,3] -dioxolane (DLin- K-DMA), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), 1 ,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. Cl), 1 ,2 -dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl),

1 ,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3 - (N,N -dilinoleylamino)- 1 ,2- propanediol (DLinAP), 3 -(N,N-dioleylamino)- 1 ,2-propanedio (DOAP), 1 ,2-dilinoleyloxo-3-(2- N,N -dimethylaminojethoxypropane (DLin-EG-DM A) , or mixtures thereof. In some

embodiments, the cationic lipid of Formula III is DLin-K-C2 -DMA (XTC2).

The cationic lipid typically comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about

65 mol %, or from about 55 mol % to about 65 mol % of the total lipid present in the particle. It will be readily apparent to one of skill in the art that depending on the intended use of the particles, the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay.

In certain embodiments, the term“cationic lipid” refers to a compound of formula CLi or a salt thereof:

In certain embodiments, the term“cationic lipid” refers to a compound of formula CL₂ or a salt thereof:

In certain embodiments, the term“cationic lipid” refers to a compound of formula CL3 or a salt thereof:

CL₃.

The non-cationic lipids used in the lipid particles of the invention (e.g., LNP) can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC),

dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG),

dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N -maleimidomethyl)-cyclohexane- 1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE),

monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are typically acyl groups derived from fatty acids having C10-C24 carbon chains, e.g. , lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl,

Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'- hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

In some embodiments, the non-cationic lipid present in the lipid particles (e.g., LNP) comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid-free lipid particle formulation. In other embodiments, the non-cationic lipid present in the lipid particles (e.g., LNP) comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation. In further embodiments, the non-cationic lipid present in the lipid particles (e.g., LNP) comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof.

Other examples of non-cationic lipids suitable for use in the present invention include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.

The term“hydrophobic lipid” refers to compounds having apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N— N- dialkylamino, l,2-diacyloxy-3-aminopropane, and l,2-dialkyl-3-aminopropane.

The term“fusogenic” refers to the ability of a lipid particle, such as a LNP, to fuse with the membranes of a cell. The membranes can be either the plasma membrane or membranes surrounding organelles, e.g., endosome, nucleus, etc.

As used herein, the term“aqueous solution” refers to a composition comprising in whole, or in part, water. As used herein, the term“organic lipid solution” refers to a composition comprising in whole, or in part, an organic solvent having a lipid.

“Distal site,” as used herein, refers to a physically separated site, which is not limited to an adjacent capillary bed, but includes sites broadly distributed throughout an organism.

“Serum-stable” in relation to lipid nanoparticles such as LNP means that the particle is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA. Suitable assays include, for example, a standard serum assay, a DNAse assay, or an RNAse assay,

“Systemic delivery,” as used herein, refers to delivery of lipid particles that leads to a broad biodistribution of an nucleic acid, such as an interfering RNA or mRNA, within an organism. Some techniques of administration can lead to the systemic delivery of certain agents, but not others. Systemic delivery means that a useful, preferably therapeutic, amount of an agent is exposed to most parts of the body. To obtain broad biodistribution generally requires a blood lifetime such that the agent is not rapidly degraded or cleared (such as by first pass organs (liver, lung, etc.) or by rapid, nonspecific cell binding) before reaching a disease site distal to the site of administration. Systemic delivery of lipid particles can be by any means known in the art including, for example, intravenous, subcutaneous, and intraperitoneal. In a preferred embodiment, systemic delivery of lipid particles is by intravenous delivery.

“Local delivery,” as used herein, refers to delivery of an nucleic acid, such as an interfering RNA or mRNA, directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like.

The term“mammal” refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like.

The term“cancer” refers to any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, lung cancer, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, stomach (gastric) cancer, esophageal cancer; gallbladder cancer, liver cancer, pancreatic cancer, appendix cancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer, renal cancer (e.g., renal cell carcinoma), cancer of the central nervous system, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head and neck cancers, osteogenic sarcomas, and blood cancers. Non-limiting examples of specific types of liver cancer include hepatocellular carcinoma (HCC), secondary liver cancer (e.g., caused by metastasis of some other non-liver cancer cell type), and hepatoblastoma. As used herein, a“tumor” comprises one or more cancerous cells.

Description of the Embodiments

In certain embodiments, provided here are lipid nanoparticles comprising:

(a) a nucleic acid;

In certain embodiments, the lipid anchor moiety is a single C₁₀-C₂₄ alkyl chain (e.g., C ₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, or C₂₄).

In certain embodiments, the lipid anchor moiety is a single C₁₀, C₁₂, C ₁₄, C ₁₆or C₁₈ chain.

In certain embodiments, the lipid anchor moiety is a single C₁₈ chain.

In certain embodiments, the lipid anchor moiety is a single C₁₆-C₂₄ alkyl chain.

In certain embodiments, the lipid anchor moiety is a single C₁₈-C₂₂ alkyl chain.

In certain embodiments, the lipid anchor moiety is, or comprises, a sterol or sterol derivative.

In certain embodiments, the lipid anchor moiety is, or comprises, cholesterol or a cholesterol derivative.

In certain embodiments, the lipid anchor moiety is, or comprises, a polycyclic structure. In certain embodiments, the lipid anchor moiety is a dialkyl moiety.

In certain embodiments, the lipid anchor moiety is a symmetric dialkyl moiety.

In certain embodiments, the lipid anchor moiety is an asymmetric dialkyl moiety.

In certain embodiments, the lipid anchor moiety is an asymmetric dialkyl moiety having Cio and Cu alkyl chains.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two Cs alkyl chains.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two Cio alkyl chains.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two C12 alkyl chains.

In certain embodiments, the lipid anchor moiety is a trialkyl moiety.

In certain embodiments, the lipid anchor moiety is a trialkyl moiety having three alkyl chains of Cio or less.

In certain embodiments, the lipid anchor moiety is a symmetric trialkyl moiety.

In certain embodiments, the lipid anchor moiety is an asymmetric trialkyl moiety.

In certain embodiments, the lipid anchor moiety is a tetraalkyl moiety.

In certain embodiments, the lipid anchor moiety is a tetraalkyl moiety having three alkyl chains of Cs or less.

In certain embodiments, the lipid anchor moiety is a symmetric tetraalkyl moiety.

In certain embodiments, the lipid anchor moiety is an asymmetric tetraalkyl moiety.

In certain embodiments, the PEG moiety of the PEG-lipid conjugate has an average molecular weight of about 750 daltons.

In certain embodiments, the polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles is present in an amount of about 2 mol % of the total lipid in the particle.

In certain embodiments, the polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles is present in an amount of about 3 mol % of the total lipid in the particle.

In certain embodiments, the polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles is present in an amount of about 4 mol % of the total lipid in the particle.

In certain embodiments, the polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles is present in an amount of about 5 mol % of the total lipid in the particle.

In certain embodiments, provided here are lipid nanoparticles comprising:

(a) a nucleic acid;

In certain embodiments, the lipid anchor moiety is a single alkyl chain.

In certain embodiments, the lipid anchor moiety is a symmetric dialkyl moiety.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having alkyl chains longer than C ₁₄.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having C14-C22 alkyl chains (e.g., C ₁₄, C15, Ci6, C n, Cis, C19, C20, C21, or C22).

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two CM alkyl chains.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two Cie alkyl chains.

In certain embodiments, the lipid anchor moiety is a dialkyl moiety having two Cis alkyl chains.

In certain embodiments, the lipid anchor moiety is a trialkyl moiety.

In certain embodiments, the lipid anchor moiety is a trialkyl moiety having three alkyl chains of Cs or greater.

In certain embodiments, the lipid anchor moiety is a symmetric trialkyl moiety.

In certain embodiments, the lipid anchor moiety is a tetraalkyl moiety.

In certain embodiments, the lipid anchor moiety is a tetraalkyl moiety having three alkyl chains of Ce or greater.

In certain embodiments, the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 5,000 to about 10,000 daltons.

In certain embodiments, the PEG moiety of the PEG-lipid conjugate has an average molecular weight of from about 8,000 to about 10,000 daltons.

In certain embodiments, the PEG moiety of the PEG-lipid conjugate has an average molecular weight of about 5,000 daltons.

In certain embodiments, the PEG moiety of the PEG-lipid conjugate has an average molecular weight of about 10,000 daltons.

In certain embodiments, the nucleic acid is at least 80 bases in length.

In certain embodiments, the nucleic acid is at least 100 bases in length.

In certain embodiments, the nucleic acid is at least 500 bases in length.

In certain embodiments, the nucleic acid is DNA, plasmid DNA, minicircle DNA, ceDNA (closed ended DNA), mRNA, self-replicating RNA, CRISPR RNA, a gene editing construct, an RNA editing construct, or a base editing construct.

In certain embodiments, the nucleic acid is mRNA.

In certain embodiments, the nucleic acid is siRNA.

In certain embodiments, the nucleic acid is not siRNA.

In certain embodiments, the cationic lipid is a compound of formula CLi or a salt thereof:

In certain embodiments, the cationic lipid is a compound of formula CL2 or a salt thereof:

In certain embodiments, the cationic lipid is a compound of formula CL3 or a salt thereof:

CL₃.

In certain embodiments, the phospholipid is DSPC.

In certain embodiments, the lipid anchor moiety comprises at least one saturated alkyl chain.

In certain embodiments, the lipid anchor moiety comprises at least one unsaturated alkyl chain.

In certain embodiments, the lipid anchor moiety comprises at least one alkyl chain having at least one double bond.

Certain embodiments provide a method for reducing the immune response of administration of a lipid nanoparticle (LNP) to a human, comprising selecting a polyethylene glycol (PEG)-lipid conjugate for use in LNP, wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single alkyl chain, wherein the LNP comprises an mRNA payload.

In certain embodiments, the lipid anchor moiety is a single C₁₀-C₂₄ alkyl chain (e.g., Cm, C₁₁, C_12, C₁₃, C ₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, or C₂₄).

In certain embodiments, the lipid anchor moiety is a single C₁₀, C₁₂, C ₁₄, C ₁₆, C₁₈ C₂₀,

C₂₂ or C₂₄, chain.

In certain embodiments, the lipid anchor moiety is a single C₁₈ chain.

In certain embodiments, the method further comprises treating a human in need thereof with an initial administration of the LNP and at least one subsequent administration of the LNP.

Certain embodiments provide a lipid nanoparticle comprising: (a) a nucleic acid, wherein the nucleic acid is mRNA;

(b) a cationic lipid;

(d) a polyethylene glycol (PEG)-lipid conjugate that inhibits aggregation of lipid nanoparticles,

wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single chain Cis alkyl moiety.

Certain embodiments provide a lipid nanoparticle comprising:

(a) a nucleic acid, wherein the nucleic acid is mRNA;

wherein the PEG-lipid conjugate comprises a PEG moiety linked to a lipid anchor moiety, wherein the lipid anchor moiety is a single chain C14-C22 (e.g., C ₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, or C₂₂, e.g., C ₁₄, C ₁₄, C₁₈, C₂₀ or C22) alkyl moiety,

Certain embodiments provide a pharmaceutical composition comprising a lipid nanoparticle as described herein, and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.

Certain embodiments provide a method for delivering a nucleic acid to a cell comprising contacting the cell with a lipid nanoparticle as described herein.

Certain embodiments provide a method for treating a disease characterized by a genetic defect that results in a deficiency of a functional protein, the method comprising: administering to a subject having the disease, a lipid nanoparticle as described herein, wherein the lipid nanoparticle comprises mRNA that encodes the functional protein or a protein having the same biological activity as the functional protein.

Certain embodiments provide a method for treating a disease characterized by overexpression of a polypeptide, comprising administering to a subject having the disease a lipid nanoparticle as described herein, wherein the lipid nanoparticle comprises siRNA that targets expression of the overexpressed polypeptide.

Certain embodiments provide a lipid nanoparticle as described herein for the therapeutic or prophylactic treatment of a disease characterized by a genetic defect that results in a deficiency of a functional protein.

Certain embodiments provide a lipid nanoparticle as described herein for the therapeutic or prophylactic treatment of a disease characterized by overexpression of a polypeptide.

In certain embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid particle such that the nucleic acid in the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease or protease. In certain other embodiments, the lipid particles are substantially non-toxic to mammals such as humans.

In certain instances, the nucleic acid comprises an interfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, or mixtures thereof. In certain other instances, the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid such as, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, an immunostimulatory oligonucleotide, or mixtures thereof. In certain other instances, the nucleic acid comprises one or more mRNA molecules (e.g., a cocktail).

In one embodiment, the nucleic acid comprises an siRNA. In one embodiment, the siRNA molecule comprises a double-stranded region of about 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). The siRNA molecules of the invention are capable of silencing the expression of a target sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule comprises at least one modified nucleotide. In certain preferred embodiments, the siRNA molecule comprises one, two, three, four, five, six, seven, eight, nine, ten, or more modified nucleotides in the double-stranded region. In certain instances, the siRNA comprises from about 1% to about 100% (e.g., about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or

100%) modified nucleotides in the double-stranded region. In preferred embodiments, less than about 25% (e.g. , less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% to about 25% (e.g., from about l%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%, or 10%-20%) of the nucleotides in the double-stranded region comprise modified nucleotides.

In other embodiments, the siRNA molecule comprises modified nucleotides including, but not limited to, 2'-0-methyl (2'OMe) nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'- deoxy nucleotides, 2'-0-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA) nucleotides, and mixtures thereof. In preferred embodiments, the siRNA comprises 2'OMe nucleotides (e.g., 2'OMe purine and/or pyrimidine nucleotides) such as, for example, 2'OMe- guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe- cytosine nucleotides, and mixtures thereof. In certain instances, the siRNA does not comprise 2'OMe-cytosine nucleotides. In other embodiments, the siRNA comprises a hairpin loop structure.

The siRNA may comprise modified nucleotides in one strand (i.e., sense or antisense) or both strands of the double-stranded region of the siRNA molecule. Preferably, uridine and/or guanosine nucleotides are modified at selective positions in the double-stranded region of the siRNA duplex. With regard to uridine nucleotide modifications, at least one, two, three, four, five, six, or more of the uridine nucleotides in the sense and/or antisense strand can be a modified uridine nucleotide such as a 2'OMe-uridine nucleotide. In some embodiments, every uridine nucleotide in the sense and/or antisense strand is a 2'OMe-uridine nucleotide. With regard to guanosine nucleotide modifications, at least one, two, three, four, five, six, or more of the guanosine nucleotides in the sense and/or antisense strand can be a modified guanosine nucleotide such as a 2'OMe-guanosine nucleotide. In some embodiments, every guanosine nucleotide in the sense and/or antisense strand is a 2'OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six, seven, or more 5'-GU-3' motifs in an siRNA sequence may be modified, e.g. , by introducing mismatches to eliminate the 5'-GU-3' motifs and/or by introducing modified nucleotides such as 2'OMe nucleotides. The 5'-

GU-3' motif can be in the sense strand, the antisense strand, or both strands of the siRNA sequence. The 5'-GU-3' motifs may be adjacent to each other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.

In some preferred embodiments, a modified siRNA molecule is less immunostimulatory than a corresponding unmodified siRNA sequence. In such embodiments, the modified siRNA molecule with reduced immunostimulatory properties advantageously retains RNAi activity against the target sequence. In another embodiment, the immunostimulatory properties of the modified siRNA molecule and its ability to silence target gene expression can be balanced or optimized by the introduction of minimal and selective 2'OMe modifications within the siRNA sequence such as, e.g., within the double-stranded region of the siRNA duplex. In certain instances, the modified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than the corresponding unmodified siRNA. It will be readily apparent to those of skill in the art that the immunostimulatory properties of the modified siRNA molecule and the corresponding unmodified siRNA molecule can be determined by, for example, measuring INF-a and/or IL-6 levels from about two to about twelve hours after systemic administration in a mammal or transfection of a mammalian responder cell using an appropriate lipid-based delivery system (such as the LNP delivery system disclosed herein).

In certain embodiments, a modified siRNA molecule has an IC₅₀ (i.e., half-maximal inhibitory concentration) less than or equal to ten-fold that of the corresponding unmodified siRNA (i.e., the modified siRNA has an IC₅₀ that is less than or equal to ten-times the IC₅₀ of the corresponding unmodified siRNA). In other embodiments, the modified siRNA has an IC₅₀ less than or equal to three-fold that of the corresponding unmodified siRNA sequence. In yet other embodiments, the modified siRNA has an IC₅₀ less than or equal to two-fold that of the corresponding unmodified siRNA. It will be readily apparent to those of skill in the art that a dose-response curve can be generated and the IC₅₀ values for the modified siRNA and the corresponding unmodified siRNA can be readily determined using methods known to those of skill in the art.

In yet another embodiment, a modified siRNA molecule is capable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the expression of the target sequence relative to the corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double -stranded region. In other embodiments, the siRNA comprises one, two, three, four, or more phosphate backbone modifications, e.g., in the sense and/or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not comprise phosphate backbone modifications.

In further embodiments, the siRNA does not comprise 2'-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In yet further embodiments, the siRNA comprises one, two, three, four, or more 2'-deoxy nucleotides, e.g., in the sense and/or antisense strand of the double-stranded region. In preferred embodiments, the siRNA does not comprise 2'-deoxy nucleotides.

In certain instances, the nucleotide at the 3'-end of the double-stranded region in the sense and/or antisense strand is not a modified nucleotide. In certain other instances, the nucleotides near the 3 '-end (e.g., within one, two, three, or four nucleotides of the 3 '-end) of the double-stranded region in the sense and/or antisense strand are not modified nucleotides.

The siRNA molecules described herein may have 3' overhangs of one, two, three, four, or more nucleotides on one or both sides of the double-stranded region, or may lack overhangs (i.e., have blunt ends) on one or both sides of the double-stranded region. Preferably, the siRNA has 3' overhangs of two nucleotides on each side of the double-stranded region. In certain instances, the 3' overhang on the antisense strand has complementarity to the target sequence and the 3' overhang on the sense strand has complementarity to a complementary strand of the target sequence. Alternatively, the 3' overhangs do not have complementarity to the target sequence or the complementary strand thereof. In some embodiments, the 3' overhangs comprise one, two, three, four, or more nucleotides such as 2'-deoxy (2Ή) nucleotides. In certain preferred embodiments, the 3' overhangs comprise deoxythymidine (dT) and/or uridine nucleotides. In other embodiments, one or more of the nucleotides in the 3' overhangs on one or both sides of the double-stranded region comprise modified nucleotides. Non-limiting examples of modified nucleotides are described above and include 2'OMe nucleotides, 2'-deoxy-2'F nucleotides, 2'-deoxy nucleotides, 2'-0-2-M0E nucleotides, LNA nucleotides, and mixtures thereof. In preferred embodiments, one, two, three, four, or more nucleotides in the 3' overhangs present on the sense and/or antisense strand of the siRNA comprise 2'OMe nucleotides (e.g., 2'OMe purine and/or pyrimidine nucleotides) such as, for example, 2'OMe-guanosine nucleotides, 2'OMe-uridine nucleotides, 2'OMe-adenosine nucleotides, 2'OMe-cytosine nucleotides, and mixtures thereof.

The siRNA may comprise at least one or a cocktail (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) of unmodified and/or modified siRNA sequences that silence target gene expression. The cocktail of siRNA may comprise sequences which are directed to the same region or domain (e.g., a“hot spot”) and/or to different regions or domains of one or more target genes. In certain instances, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) modified siRNA that silence target gene expression are present in a cocktail. In certain other instances, one or more (e.g., at least two, three, four, five, six, seven, eight, nine, ten, or more) unmodified siRNA sequences that silence target gene expression are present in a cocktail.

In some embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%

complementary to the target sequence or a portion thereof. In other embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that is 100% complementary to the target sequence or a portion thereof. In further embodiments, the antisense strand of the siRNA molecule comprises or consists of a sequence that specifically hybridizes to the target sequence or a portion thereof.

In further embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the target sequence or a portion thereof. In additional embodiments, the sense strand of the siRNA molecule comprises or consists of a sequence that is 100% identical to the target sequence or a portion thereof.

Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl -2 '-hydroxyethyl ether is described herein.

As used herein, DSPC means distearoylphosphatidylcholine.

In the lipid particles of the invention, the nucleic acid may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from enzymatic degradation. In pr