# Patent

## Title
Lipids and compositions for intracellular delivery of biologically active compounds

## Abstract

The present invention provides novel amino-lipids, compositions comprising such amino-lipids and methods of producing them. In addition, lipid nanoparticles comprising the novel amino-lipids and a biologically active compound are provided, as well as methods of production and their use for intracellular drug delivery.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation of U.S. Ser. No. 13/466,640, filed May 8, 2012, which claims benefit of EP 11166353, filed May 17, 2011, the entire disclosures of which are incorporated by reference herein.


FIELD OF THE INVENTION
The present invention provides novel amino lipids, compositions comprising such amino-lipids and methods of producing them. In addition, lipid nanoparticles (LNPs) comprising the novel amino-lipids and a biologically active compound are provided, as well as methods of production and their use for intracellular drug delivery.
BACKGROUND OF THE INVENTION
Lipid nanoparticles (LNPs), liposomes or lipoplexes are effective drug delivery systems for biologically active compounds such as therapeutic proteins, peptides or nucleic acid based therapeutics, which are otherwise cell impermeable. Liposomal formulations have also been developed 15 for small molecule drugs with the aim to enrich the drug in certain tissues.
Drugs based on nucleic acids interact with a messenger RNA or a gene and have to be delivered to the proper cellular compartment in order to be effective. In particular double stranded nucleic acids, for example double stranded RNA molecules (dsRNA) such as 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 25 features contribute to the siRNA's poor “drug-like” properties (Nature Reviews, Drug Discovery 2007, 6:443). When administered intravenously, the siRNA is rapidly excreted from the body with a typical half-life in the range of only 10 min. 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 (Oligonucleotides 2009, 19:89).
By introduction of appropriate chemical modifications stability towards nucleases can be increased and at the same time immune stimulation can be suppressed. Conjugation of lipophilic small molecules to the siRNAs improves the pharmacokinetic characteristics of the double stranded RNA molecule. It has been demonstrated that these small molecule siRNA conjugates are efficacious in a specific down regulation of a gene expressed in hepatocytes of rodents. However, in order to elicit the desired biologic effect a large dose was needed (Nature 2004, 432:173).
With the advent of lipid nanoparticle formulations the siRNA doses necessary to achieve target knockdown in vivo could be significantly reduced (Nature 2006, 441:111). Typically, such lipid nanoparticle drug delivery systems are multi-component formulations comprising cationic lipids, helper lipids, lipids containing polyethylene glycol and cholesterol. The positively charged cationic lipids bind to the anionic nucleic acid, while the other components support a stable self-assembly of the lipid nanoparticles.
To improve delivery efficacy of these lipid nanoparticle formulations, many efforts are directed to develop more appropriate cationic lipids. These efforts include high throughput generation of cationic lipid libraries based on solvent- and protecting group free chemical reaction such as Michael additions of amines to acrylamides or acylates (Nature Biotechnology 2008, 26:561) or ring-opening reactions with amines and terminal epoxides (PNAS 2010, 107:1854). Another strategy involves structure activity studies, e.g. systematic variation of the degree of saturation in the hydrophobic part (Journal of Controlled Release 2005, 107:276) or the polar head group of the cationic lipid (Nature Biotechnology 2010, 28:172), resulting in an improved efficacy of the so-called stable nucleic acid-lipid particles (SNALP) technology (Current Opinion in Molecular Therapeutics 1999, 1:252).
Despite these efforts, improvements in terms of increased efficacy and decreased toxicity are still needed, especially for lipid nanoparticle based drug delivery systems intended for therapeutic uses. LNPs naturally accumulate in the liver after intravenous injection into an animal (Hepatology 1998, 28:1402). It has been demonstrated that gene silencing can be achieved in vivo in hepatocytes which account for the majority of the cells in the liver. Even the simultaneous down-modulation of several target genes expressed in hepatocytes could be successfully achieved (PNAS 2010, 107:1854). However, evidence of successful gene regulation in other liver cell types is lacking.
SUMMARY OF THE INVENTION
The present invention provides novel amino-lipids, compositions comprising the inventive amino-lipids, as well as methods of producing them. In particular, compositions comprising the amino-lipids of the invention that form lipid nanoparticles (LNPs) are provided, as well as methods of producing and their use for the intracellular delivery of biologically active compounds, for example nucleic acids.
The methods of producing the amino-lipids provided herein are advantageous compared to those known in prior art as the amino-lipids can be produced with a higher yield and increased purity.
The lipid nanoparticles (LNPs) comprising the inventive amino-lipids significantly enhance the intracellular delivery of nucleic acids into hepatocytes compared to LNPs comprising lipids known in prior art. In addition, the lipid nanoparticles (LNPs) comprising the inventive amino-lipids enable inhibition of gene expression in additional liver cell types apart from hepatocytes, such as Kupffer cells, Stellate cells and endothelial cells. Moreover, the lipid nanoparticles (LNPs) comprising the inventive amino-lipids are suitable for cell-type specific delivery of nucleic acids into various organs in vivo, including jujunum, liver, kidney, lung and spleen. Importantly, these lipid nanoparticles can also be administered via the air ways enabling gene silencing in the lung.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Structures representing amino-lipids of the invention.
FIG. 2. Bar graph illustrating improved LNP composition of the invention to reduce FVII mRNA levels in mice.
FIG. 3. Graph illustrating GFP mRNA levels after orotracheal instillation of LNPs of the present invention into GFP transgenic mice (n=4). Individual siRNAs directed against the targets indicated below the bars were formulated into LNPs consisting of 50% KL25, 10% DSPC, 38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administration animals were killed, bronchoalveolar lavage fluid (BALf) prepared and the GFP mRNA level determined using bDNA assay. Hatched bans represent GFP mRNA levels of animals treated with the unspecific siRNAs.
FIG. 4. Graph illustrating CD45 mRNA levels after orotracheal instillation of LNPs of the present invention into GFP transgenic mice (n=4). Individual siRNAs directed against the targets indicated below the bars were formulated into LNPs consisting of 50% KL25, 10% DSPC, 38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administration animals were killed, bronchoalveolar lavage fluid (BALf) prepared and the CD45 mRNA level determined using bDNA assay. Hatched ban represent CD45 mRNA levels of animals treated with the unspecific siRNAs.
FIG. 5. Graph illustrating CD68 mRNA levels after orotracheal instillation of LNPs of the present invention into GFP transgenic mice (n=4). Individual siRNAs directed against the targets indicated below the bars were formulated into LNPs consisting of 50% KL25, 10% DSPC, 38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administration animals were killed, bronchoalveolar lavage fluid (BALf) prepared and the CD68 mRNA level determined using bDNA assay. Hatched bars represent CD68 mRNA levels of animals treated with the unspecific siRNAs.
FIG. 6. Graph illustrating Clec7a mRNA levels after orotracheal instillation of LNPs of the present invention into GFP transgenic mice (n=4). Individual siRNAs directed against the targets indicated below the bars were formulated into LNPs consisting of 50% KL25, 10% DSPC, 38.5% cholesterol and 1.5% PEG2000-c-DOMG. 48 h post administration animals were killed, bronchoalveolar lavage fluid (BALf) prepared and the Clec7a mRNA level determined using bDNA assay. Hatched bars represent Clec7a mRNA levels of animals treated with the unspecific siRNAs.
FIG. 7. Graph illustrating Rein mRNA levels after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 8. Graph illustrating Clec4f mRNA levels after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 9. Graph illustrating Tek mRNA levels in liver after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 10. Graph illustrating Tek mRNA levels in liver after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. 5 and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 11. Graph illustrating Tek mRNA levels in lung after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 12. Graph illustrating Tek mRNA levels in jejunum after siRNA treatment employing different LNPs of the present invention. The LNPs contained a pool of five different siRNAs directed against five different targets (FVII, Rein, Clec4f, Tek, GFP). The siRNA dose was 0.5 mg/kg per siRNA (total siRNA dose 2.5 mg/kg). LNPs were dosed i.v. and 48 h post dosing mRNA levels were measured using bDNA assay. For comparison and shown has hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 13. Graph illustrating Rein mRNA expression levels of 2 individual animals per LNP relative to saline treated animals 48 h post iv dosing. LNPs contained a pool of five different siRNAs directed against FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 14. Graph illustrating Clec4f mRNA expression levels of 2 individual animals per LNP relative to saline treated animals 48 h post iv dosing. LNPs contained a pool of five different siRNAs directed against FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 15. Graph illustrating Tek mRNA expression levels of 2 individual animals per LNP relative to saline treated animals 48 h post iv dosing. LNPs contained a pool of five different siRNAs directed against FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 ml/kg (total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 16. Graph illustrating Tek mRNA expression levels of 2 individual animals per LNP relative to saline treated animals 48 h post iv dosing. LNPs contained a pool of five different siRNAs directed against FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.
FIG. 17. Graph illustrating Tek mRNA expression levels of 2 individual animals per LNP relative to saline treated animals 48 h post iv dosing. LNPs contained a pool of five different siRNAs directed against FVII, GFP, Rein, Clec4f and Tek each at a dose of 0.5 mg/kg (total siRNA dose 2.5 mg/kg). LNPs were composed of 50 mol % amino-lipid designated by the KL numbers in the graphs below, 10 mol % DSPC, 38.5 mol % cholesterol and 1.5 mol % PEG-c-OMOG. For comparison and shown as hashed bar, a benchmark LNP in which XTC2 substituted the amino-lipid was included.



DETAILED DESCRIPTION OF THE INVENTION
A. Amino-lipids and Methods of Producing them.
The amino-lipids provided herein are produced by reductive amination of a (poly)amine and an aliphatic carbonyl compound according to the general reaction scheme:

R′—NH2+R—CHO→R′—N(CHR)2 

R′—NH2+R—CO—R→R′—N(CR2)2 

The amino-lipids may be prepared by reacting the aliphatic carbonyl compound and the (poly)amine in the presence of a reducing agent.
In certain embodiments the aliphatic carbonyl compound is a ketone. In certain embodiments the aliphatic carbonyl compound is an aldehyde. Typically, the (poly)amine has two to five nitrogen atoms in its structure. In certain embodiments the (poly)amine contains primary and/or secondary and/or tertiary nitrogen atoms. Depending on the structure of the (poly)amine, and the aliphatic carbonyl compound employed regioselective alkylations can be achieved. Particularly, when ketones are reacted with polyamines displaying primary and/or secondary and/or tertiary nitrogens under reductive amination conditions selective alkylations of primary nitrogens can be achieved. The present invention covers procedures of making amino-lipids of the following structures:

    
    
        RNH(CH2)xNH2,
        R2N(CH2)xNH2,
        R2N(CH2)xNHR,
        R2N(CH2)xNR2,
        RNH[(CH2)x(CwH2wNH)y(CH2)z]NH2,
        RNH[(CH2)x(CwH2wNR)y(CH2)z]NH2,
        RNH[(CH2)x(CwH2wNR)y(CvH2vNH)u(CH2)z]NH2,
        R2N[(CH2)x(CwH2wNH)y(CH2)z]NH2,
        R2N[(CH2)x(CwH2wNR)y(CH2)z]NH2,
        R2N[(CH2)x(CwH2wNR)y(CvH2vNH)u(CH2)z]NH2,
        RNH[(CH2)x(CwH2wNH)y(CH2)z]NHR,
        RNH[(CH2)x(CwH2wNR)y(CH2)z]NHR,
        RNH[(CH2)x(CwH2wNR)y(CvH2vNH)u(CH2)z]NHR,
        R2N[(CH2)x(CwH2wNH)y(CH2)z]NHR,
        R2N[(CH2)x(CwH2wNR)y(CH2)z]NHR,
        R2N[(CH2)x(CwH2wNR)y(CvH2vNH)u(CH2)z]NHR,
        R2N[(CH2)x(CwH2wNH)y(CH2)z]NR2,
        R2N[(CH2)x(CwH2wNR)y(CH2)z]NR2.
        R2N[(CH2)x(CwH2wNR)y(CvH2vNH)u(CH2)z]NR2,
        N{[(CH2)x(CwH2wNH)y(CH2)z]NHR}3,
        N{[(CH2)x(CwH2wNH)y(CH2)z]NR2}3,
        HN{[(CH2)x(CwH2wNH)y(CH2)z]NHR}2, and
        HN{[(CH2)x(CwH2wNH)y(CH2)z]NR2}2,

wherein R is selected from alkyl, alkenyl or alkynyl carbon chains ranging from C6 to C20. In certain embodiments these chains comprise at least one, at least two or at least three sites of unsaturation, for example one or more double bonds or triple bonds. In one embodiment, R comprises at least one aromatic cycle, including for example a heterocycle. In yet another embodiment, R may comprise at least one heteroatom in the carbon chain, for example O, NH, NR′, S, SS, wherein R′ is an acyl, alkyl, alkenyl or alkynyl group consisting of two to 20 carbon atoms. In still another embodiment, at least one hydrogen in the hydrocarbon chain R may be replaced by F, Cl, Br, I. In one embodiment, w and v are independently 2, 3 or 4. In one embodiment, y and u are independently 0, 1, 2, 3 or 4. In one embodiment, x and z are independently 2, 3 or 4.

    
    


In one aspect, the present invention provides cyclic amino-lipids of the formula (I):




wherein

    R1 is independently selected from
    
        —(CH2)2—N(R)2,
        —(CH2)2—N(R)—(CH2)2—N(R)2, wherein R is independently selected from —H, C6-40 alkyl, C6-40 alkenyl and C6-40 alkynyl, provided that —N(R)2 is not NH2, and
        C6-40 alkyl, and C6-40 alkenyl;
    
    
    R2 is C6-40 alkyl, C6-40 alkenyl, or C6-40 alkynyl;
    m is 0 or 1; and
    pharmaceutically acceptable salts thereof.


The term “C6-40 alkyl” as used herein means a linear or branched, saturated hydrocarbon consisting of 6 to 40 carbon atoms, preferably of 6 to 30 carbon atoms, most preferably of 6 to 20 carbon atoms. Especially preferred are alkyl groups containing 10, 14 or 15 carbon atoms.
The term “C6-40 alkenyl” as used herein means a linear or branched, unsaturated hydrocarbon consisting of 6 to 40 carbon atoms, preferably of 6 to 30 carbon atoms, most preferably of 6 to 15 carbon atoms. In one embodiment the C6-40 alkenyl groups comprise 1 to 4 double bonds, preferably between 1 to 3 double bonds, most preferably 1 or 2 double bonds.
The term “C6-40 alkynyl” as used herein means a linear or branched, unsaturated hydrocarbon consisting of 6 to 40 carbon atoms, preferably of 6 to 30 carbon atoms, most preferably of 6 to 20 carbon atoms. In one embodiment the C6-40 alkynyl groups comprise 1 to 4 triple bonds, preferably 1 to 3 triple bonds, most preferably 1 or 2 triple bonds.
In another embodiment there are provided the cyclic amino-lipids selected from










Preferred therein are the cyclic amino-lipids selected from




In one aspect, the present invention provides linear amino-lipids of the formula (II):




wherein

    
    
        R3 is independently selected from C1-40 alkyl or C6-40 alkenyl, wherein up to 4 carbon atoms may be replaced by a heteroatom selected from oxygen or nitrogen;
        R4 is selected from C12-40 alkyl or C6-40 alkenyl, wherein up to 4 carbon atoms may be replaced by a heteroatom selected from oxygen or nitrogen;
        R5 is selected from hydrogen, C12-30 alkyl and C6-40 alkenyl;
        R6 is selected from hydrogen and C1-12 alkyl;
        n is 1, 2, or 3; and
        k is 1, 2, 3 or 4.
    
    


In a preferred embodiment, a linear amino-lipids of the formula (II) is provided wherein

    
    
        R3 is independently selected from C1-, C12-, C14-, C27-, C30- and C37-alkyl, wherein 1 or 2 carbon atoms can be optionally replaced by an oxygen or a nitrogen atom;
        R4 is selected from C12-, C14-, C27-, C30- and C37-alkyl, wherein 1 or 2 carbon atoms can be optionally replaced by an oxygen or a nitrogen atom;
        R5 is selected from hydrogen, C12-, C14- and C30-alkyl, in which one carbon atom can be optionally replaced by a nitrogen atom;
        R6 is selected from hydrogen, C1- and C12-alkyl; and
        n and k have the meanings given in claim 1.
    
    


In one embodiment there are provided the linear amino-lipids selected from













Preferred therein is the linear amino-lipid




In yet another embodiment an amino-lipid of formula




is provided.

In yet another embodiment an amino-lipid of formula




is provided.

In yet another embodiment an amino-lipid of formula




is provided.

In particular embodiments the amino-lipids of the present invention comprise Nitrogen atoms that are protonated depending on the pH of the environment, preferably at least one Nitrogen atom is positively charged at physiological pH or below. The extent of pH dependent protonation is effected by an equilibrium reaction and hence not the entire, but only the predominant lipid species is positively charged. At physiological pH at least one of the nitrogen atoms in the lipid structure is protonated.
As used herein, the term “(poly)amine” refers to a saturated hydrocarbon linear or branched wherein 2 to 5 Carbon atoms are replaced by Nitrogen. Preferably said (poly)amine comprises two to five nitrogen atoms. Preferred therein are (poly)amines that comprise amine Nitrogens that are separated by 2 and/or 3 and/or 4 carbon atoms. Non-limiting examples of suitable (poly)amines are ethylenediamine, diethylenetriamine, triethylenetetramine, tetraethylenepentamine, tris-(2-aminoethyl)-amine, 3-dimethylamino-1-propylamine, spermine, spermidine, 2,2′-(ethylenedioxy)-bis(ethylamine). The term “aliphatic carbonyl compound” as used herein refers to a compound R—CO—R′, wherein R is a ketone or an aldehyde comprising of alkyl and/or alkenyl and/or alkynyl groups and R′ is H or a ketone or an aldehyde comprising of alkyl and/or alkenyl and/or alkynyl groups.
The term “reducing agent” as used herein refers to a reagent that enables the reduction of the iminium ion intermediate in reductive amination reactions. Examples of such reagents include, but are not limited to hydride reducing reagents such as sodium cyanoborohydride, sodium triacetoxyborohydride and sodium borotetrahydride. Catalytic hydrogenation with metal catalyst such as nickel, palladium or platinum can also be used for this purpose. As used herein, the term “lipid” refers to amphiphilic molecules comprising a polar, water soluble “headgroup” and a hydrophobic “tail”. The headgroup preferably consists of a pH dependent charged group such as an amine. The tail preferably comprises aliphatic residues. Lipids can be of natural origin or of synthetic origin. Examples include, but are not limited to, fatty acids (e.g. oleic acid, lineolic acid, stearic acid), glycerolipids (e.g. mono-, di-, triglycerols such as triglycerides), phospholipids (e.g., phosphatidylethanolamine, phosphatidylcholine), and sphingolipids (e.g. sphingomyelin)
As used herein, the term “amino-lipid” refers to lipids having at least one of the Nitrogen atoms incorporated in at least one fatty acid chain. This fatty acid chain may be an alkyl, alkenyl or alkynyl carbon chain. Lipids containing carbon chain lengths in the range from C10 to C20 are preferred. It is understood that the fatty acid portion of the amino-lipid of the present invention is incorporated through the use of suitable carbonyl compounds such as aldehydes (R—CHO) and ketones (R—CO—R). Through the use of asymmetrical ketones (R—CO—R′) corresponding unsymmetrical substituted lipids can be prepared. Likewise, through the use of carbonyl ethers, esters, carbamates and amides and suitable reducing agents the corresponding amino-lipids are accessible.
The term “cyclic amino-lipid” as used herein refers to an amino-lipid of the general formula (I).
The term “linear amino-lipid” as used herein refers to an amino-lipid of the general formula (II).
In another aspect, novel amino-lipids can be prepared by reacting a suitable (poly)amine with a carbonyl compound in the presence of a reducing agent to form a cyclic or linear amino-lipid.
In certain embodiments the alkyl, alkenyl and alkynyl groups as covered in the present invention contain 2 to 20 carbon atoms. In certain other embodiments these groups consists of 2 to 10 carbon atoms. In yet other embodiments the alkyl, alkenyl and alkynyl groups employed in this invention contain 2 to 8 carbon atoms. In still other embodiments these groups contain two to six carbon atoms. In yet other embodiments the alkyl, alkenyl and alkynyl groups of the invention contain 2 to four carbon atoms.
The term “alkyl” as used herein means a chain of saturated hydrocarbons that is aliphatic, branched or cyclo-aliphatic. Saturated aliphatic hydrocarbons include methyl, ethyl, n-propyl, n-butyl and the like. Saturated branched alkyls include isopropyl, isobutyl, tert-butyl and the like. Representative cyclo-aliphatic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
The term “alkenyl” denotes a chain of hydrocarbons that has at least one carbon-carbon double bad. For example alkenyl groups include ethenyl, propenyl, butenyl, isopropylidene and the like. The term also covers cyclic alkenyls such as cyclopropenyl, cyclobutenyl, cyclopentenyl and the like.
The term “alkynyl” denotes a chain of hydrocarbons that has at least one carbon-carbon triple bond. Exemplary alkynyl groups include ethynyl, propynyl, butynyl and the like. The term also covers cyclic alkynyls such as cyclopentynyl, cyclohexynyl, and the like.
The term “acyl” refers to any alkyl, alkenyl or alkynyl group that is linked through a carbonyl group. For example, acyl groups are —(CO)-alkyl, —(CO)-alkenyl and —(CO)-alkynyl.
B. Lipid Nanoparticles (LNPs) Comprising the Inventive Amino-lipids
Also provided herein are compositions comprising the amino-lipids of the invention that form lipid nanoparticles (LNPs). As used herein, the term “lipid nanoparticles” includes liposomes irrespective of their lamellarity, shape or structure and lipoplexes as described for the introduction of pDNA into cells (PNAS, 1987, 84, 7413). These lipid nanoparticles can be complexed with biologically active compounds such as nucleic acids and are useful as in vivo delivery vehicles. Preferably said in vivo delivery is cell-type specific.
In one embodiment, said lipid nanoparticles comprise one or more amino-lipids of the invention described above and may furthermore comprise additional lipids and other hydrophobic compounds such as sterol derivatives, e.g. cholesterol. Those additional components of a lipid nanoparticle of the present invention serve various purposes such as siding manufacturing and storage stability as well as modulation of the biodistribution. Biodistribution may also be modulated by incorporation of targeting ligands conjugated to the lipids part of the lipid nanoparticle. Specific examples of additional components of the lipid nanoparticles are given below.
In one embodiment, lipid nanoparticles are provided that comprise the amino-lipids described above and one or more additional lipids. Additional lipids suitable to be incorporated into the lipid nanoparticles of the invention comprise cationic lipids, helper lipids and PEG lipids. Hence in one embodiment lipid nanoparticles are provided that comprise the amino-lipids described above and one or more additional lipids selected from the group of cationic lipid, helper lipid and PEG lipid. “Cationic lipids” as used herein refers to any lipid comprising a quaternary amine and are consequently permanently positively charged. The term “quaternary amine” as used herein refers to a nitrogen atom having four organic substituents. For example, the nitrogen atom in Tetramethylammonium chloride is a quaternary amine.
Examples of cationic lipids comprising a quaternary amine include, but are not limited to, N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”), N,N,-Distearyl-N,N-dimethylammonium bromide (“DDBA”), 1-methyl-4-(cis-9-dioleyl)-methylpyridinium-chloride (“SAINT-solid”) N-(2,3-dioleyloxy)propyl)-N,N,N-triethylammonium chloride (“DOTMA”), N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”), (1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DIMRIE”) and the like.
“Helper lipids”, as used herein are preferably neutral zwitterionic lipids. Examples of preferred helper lipids used in this invention are 1,2-distearoyl-sa-glycero-3-phosphocholine (“DSPC”), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (“DPPC”), or any related phosphatidylcholine such as natural sphingomyelin (“SM”) and synthetic derivatives thereof such as 1-oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine (“OChemsPC”). Other preferred helper lipids include 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”), 1,2-diphytanoyl-sn-glycero-3-phosphoethanol-amine (“ME 16:0 PE”).
In one embodiment, LNPs contain uncharged lipids modified with hydrophilic polymers, e.g. polyethylene glycol (herein also referred to as “PEG-lipids”) to stabilize the lipid nanoparticle and to avoid aggregation. The polyethylene glycol (PEG) size can vary from approximately 1 to 5 approximately kDa. Depending on the relative amounts of these molecules in the formulation and the length of the hydrocarbon chain, the PEG-lipid can influence the pharmacokinetic characteristics, biodistribution, and efficacy of a formulation. PEG lipids having relatively short lipid hydrocarbon chains of about 14 carbons dissociate from the LNP in vivo in plasma with a half-life of less than 1 h. In contrast, a PEG lipid with a relatively long lipid hydrocarbon chain length of about 18 carbons circulates fully associated with the formulation for several days. Hence, in one preferred embodiment, said PEG lipid comprises a lipid hydrocarbon chain of 12 to 20 carbon atoms, 14 to 18 carbon atoms, preferably of 14 carbon atoms.
Typically, the concentration of the PEG-lipid is about 0.5 to 10 mol %. Examples of suitable PEG modified lipids include pegylated ceremide conjugates, pegylated distearoylphosphatidyl-ethanolamine (PEG-DSPE). Other compounds that can be used to stabilize lipid nanoparticles include gangliosides (GMt, GM3, etc.). Preferred PEG lipids have a PEG size ranging from about 1 to about 2 KDa. Specific examples are methoxy-polyethyleneglycol-carbamoyl-dimyristyloxy-propylamine (PEG2000-c-DMA), and (α-(3′-(1,2-dimyristoyl-3-propanoxy)carboxamide-propyl]-ω-methoxy-polyoxyethylene (PEG2000-c-DOMG).
In one embodiment lipid nanoparticles are provided that comprise the amino-lipids described above and one or more hydrophobic small molecule. The term “hydrophobic small molecule” as used herein refers to a compound with a molecular weight of about 300 to about 700 Da comprising 2 or more carbon- or heterocycles providing a rigid core structure. Preferably said hydrophobic small molecule is selected from the group of sterols such as cholesterol or stigmasterol or a hydrophobic vitamin such as tocopherol. In a preferred embodiment said hydrophobic small molecule is cholesterol.
In one embodiment the lipid nanoparticle comprises an amino-lipid of the present invention, one or more additional lipids selected from the group of cationic lipid, helper lipid and PEG lipid. and a hydrophobic small molecule selected from the group of a sterol or a hydrophobic vitamin. In one embodiment said lipid nanoparticle comprises an amino-lipid of the present invention, a helper lipid selected from DSPC or DPPC, PEG-DOMG and a hydrophobic small molecule selected from the group of a sterol or a hydrophobic vitamin.
In one embodiment the lipid nanoparticle comprises an amino-lipid of the present invention, a helper lipid, a PEG modified lipid and cholesterol. In preferred embodiments the molar ratios of these components are 30-70% amino-lipid, 0-60% helper lipid, 0.1-10% PEG lipid and 0-50% cholesterol. More preferred lipid nanoparticle compositions comprise the above mentioned components in a molar ratio of about 40% to 60% amino-lipid, 0 to 20% helper lipid, 0.1% to 5% PEG lipid and 30 to 50% cholesterol. In certain other embodiments lipid nanoparticles are provided that do not comprise cholesterol. These formulations contain up to about 60 mol % of at least one helper lipid. Preferred helper lipids in these lipid nanoparticles are DSPC, SM, DOPE, 4ME16:0PE.
In one embodiment the lipid nanoparticle comprises a cyclic amino-lipid of the present invention, DSPC or SM, PEG-c-DOMG and cholesterol. Preferably, said cyclic amino-lipid has the structure of




Preferred molar ratios of these components are about 50% of said cyclic amino-lipid, about 10% helper lipid, about 38% cholesterol and about 2% of the PEG lipid. Preferred N/P ratios range from approximately 6.9 to approximately 8.4.
In another embodiment cholesterol free lipid nanoparticles are provided. These comprise a cyclic amino-lipid, the helper lipids DSPC and DOPE, as well as the PEG-lipid PEG-c-DMOG. LNPs comprising these components are not taken up by Kupffer cells in the liver, but mediate functional drug delivery to hepatocytes, stellate cells and endothelial cells. Preferably said cyclic amino-lipid has the structure of




In yet another embodiment cholesterol free lipid nanoparticles comprise just one helper lipid. In this case a preferred lipid nanoparticle contains a cyclic amino-lipid, the helper lipid 4ME 16:0PE and PEG-c-DMOG. LNPs comprising these components are barely taken up by hepatocytes, but mediate functional drug delivery to Kupffer cells, stellate cells and endothelial cells.
Preferably said cyclic amino-lipid has the structure of




In one embodiment the lipid nanoparticle comprises an amino-lipid of the present invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol. Preferably, said amino-lipid has the structure of




Preferred molar ratios of these components are 40% to 60% of said amino-lipid, about 0% to 20% helper lipid, about 38% cholesterol and approximately 2% of a PEG 2000 lipid. The N/P ratio preferably is at least about 15:1, more preferably at least about 10:1, even more preferably at least about 7:1, and most preferably at least about 5:1. LNPs comprising these compositions are particularly well suited to functional deliver nucleic acids into endothelial cells of various organs.
In another embodiment the lipid nanoparticle comprises an amino-lipid of the present invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol. Said amino-lipid has the structure of




Preferred molar ratios of these components are 40% to 60% of said amino-lipid, about 0% to 20% helper lipid, about 30% to 40% cholesterol and about 0.5% to about 2% of a PEG 2000 lipid. The N/P ratio preferably is at least about 8:1, more preferably at least about 15:1 and most preferably at least about 10:1.
In another preferred embodiment the lipid nanoparticle comprises an amino-lipid of the present invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol. Said amino-lipid has the structure of




In another preferred embodiment the lipid nanoparticle comprises a cyclic amino-lipid of the present invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol. Said amino-lipid has the structure of




In another preferred embodiment the lipid nanoparticle comprises a cyclic amino-lipid of the present invention, DSPC, a PEG lipid such as PEG-c-DOMG and cholesterol. Said amino-lipid has the structure of




In one embodiment, the lipid nanoparticles described above are complexed with a biologically active compound. The term “complexed” as used herein relates to the non-covalent interaction of the biologically active compound with specific components of the lipid nanoparticle. In case of a nucleic acid as the biologically active compound the negatively charged phosphate backbone of the nucleic acid interacts with the positively charged amino-lipid. This interaction supports the stable entrapment of the nucleic acid into the LNP.
The term “biologically active compound” as used herein refers to an inorganic or organic molecule including a small molecule, peptide (e.g. cell penetrating peptides), carbohydrate (including monosaccharides, oligosaccharides, and polysaccharides), protein (including nucleoprotein, mucoprotein, lipoprotein, synthetic polypeptide, or a small molecule linked to a protein, glycoprotein), steroid, nucleic acid, lipid, hormone, or combination thereof, that causes a biological effect when administered in vivo to an animal, including but not limited to birds and mammals, including humans. Preferably said biologically active compound is negatively charged.
In one embodiment the lipid nanoparticles described above are complexed with a biologically active compound selected from the group of small molecule, peptide, protein, carbohydrate, nucleic acid, or lipid. Preferably said biologically effect is a therapeutic effect.
The term “nucleic acid” as used herein means an oligomer or polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically (e.g., PNA as described in U.S. Pat. No. 5,948,902 and the references cited therein) which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Non-naturally occurring nucleic acids are oligomers or polymers which contain nucleobase sequences which do not occur in nature, or species which contain functional equivalents of naturally occurring nucleobases, sugars, or inter-sugar linkages, like peptide nucleic acids (PNA), threose nucleic acids (TNA), locked nucleic acids (LNA), or glycerol nucleic acids (GNA). This term includes oligomers that contain the naturally occurring nucleic acid nucleobases adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well as oligomers that contain base analogs or modified nucleobases. Nucleic acids can derive from a variety of natural sources such as viral, bacterial and eukaryotic DNAs and RNAs. Other nucleic acids can be derived from synthetic sources, and include any of the multiple oligonucleotides that are being manufactured for use as research reagents, diagnostic agents or potential and definite therapeutic agents. The term includes oligomers comprising a single strand nucleic acid or a double strand nucleic acid. Examples of nucleic acids useful therein are miRNA, antisense oligonucleotides, siRNA, immune-stimulatory oligonucleotides, aptamers, ribozymes, or plasmids encoding a specific gene or siRNA.
As used herein, the term “peptide” is used to refer to a natural or synthetic molecule comprising two or more amino acids linked by an amide bond consisting of the carboxylic acid group of one amino acid and the amino group by the other amino acid. A peptide is not limited by the number of amino acids and thus it can include polypeptides and proteins.
Below embodiments are exemplified for the complexation and delivery of nucleic acids. It is understood that these embodiments are also applicable for any other biologically active compound.
The complex comprising lipid nanoparticles and one or more nucleic acid are characterized by the following parameters: (1) nucleic acid to total lipid ratio; (2) nucleic acid to amino-lipid ratio; (3) encapsulation efficacy; (4) particle size; (5) particle size distribution and (6) zeta potential.
The nucleic acid to total lipid ratio is the amount of the nucleic acid in a defined volume divided by the amount of total lipid in the same volume. Total lipid refers to all components in the particle formulations except for the nucleic acid. The ratio may be expressed on a mole per mole or weight by weight basis.
The nucleic acid to amino-lipid ratio is the amount of the nucleic acid in a defined volume divided by the amount of amino-lipid in the same volume. The ratio may be expressed on a mole per mole or weight by weight basis; but is usually expressed as nitrogen to phosphorus (N/P) ratio. The (N/P) ratio is characterized by the number of positively charged nitrogen atoms present in the amino-lipid divided by the number of negatively charged phosphorus atoms present in the nucleic acid. Encapsulation efficacy is defined as the percentage of nucleic acid that is encapsulated or otherwise associated with the lipid nanoparticle. The encapsulation efficiency is usually determined by quantifying the amount of the nucleic acid in solution before and after breaking up the lipid nanoparticle by suitable organic solvents or detergents. A high encapsulation efficiency is a desirable feature of nucleic acid lipid nanoparticles particularly because of considerations regarding cost of goods.
The particle size of lipid nanoparticles is typically measured by dynamic light scattering. Sizes of lipid nanoparticles in a given formulation are typically distributed over a certain range. The size of lipid nanoparticles is typically expressed as the mean particle size or the Zaverage value. The particle size distribution of lipid nanoparticles is expressed as the polydispersity index (PI). Particles in the size range of 30 to 300 nm are considered advantageous for in vivo applications. Lipid nanoparticles with a mean particle size less than approximately 150 nm are advantageous, in particular to assess tissues characterized by a leaky vasculature as is the case for tumor tissue or liver. Lipid nanoparticles with a mean particle size greater than approximately 150 nm are advantageous, in particular to assess macrophages.
Nucleic acid lipid nanoparticles are also characterized by their surface charge as measured by the zeta (0-potential. The basis of the measurement is the movement of particles in an electrical field as measured by dynamic light scattering. Particles with a near to neutral surface charge are advantageous for in vivo applications and are thus preferred herein.
Particularly because of cost of goods and manufacturing reasons a high encapsulation efficiency of the nucleic acids complexed with the lipid nanoparticles is desirable. Particles in the size range of 30 to 300 nm with near to neutral surface charge are known to be advantageous for in vivo applications.
C. Methods of Producing Lipid Nanoparticles (LNPs) Comprising the Inventive Amino-lipids and Complexes Thereof with Biologically Active Compounds.
In general, any method known in the art can be applied to prepare the lipid nanoparticles comprising one or more amino-lipids of the present invention and to prepare complexes of biologically active compounds and said lipid nanoparticles. Examples of such methods are widely disclosed, e.g. in Biochimica et Biophysica Acta 1979, 557:9; Biochimica et Biophysica Acta 1980, 601:559; Liposomes: A practical approach (Oxford University Press, 1990); Pharmaceutica Acta Helvetiae 1995, 70:95; Current Science 1995, 68:715; Pakistan Journal of Pharmaceutical Sciences 1996, 19:65; Methods in Enzymology 2009, 464:343.
Below embodiments are exemplified for the complexation and delivery of nucleic acids. It is understood that these embodiments are also applicable for any other biologically active compound.
In one embodiment, the components of the lipid nanoparticles as outlined above are mixed in a solvent that is miscible with water, such as methanol, ethanol isopropanol or acetone. Preferred solvents are alcohols, most preferable ethanol. In most preferred embodiments the solvent is commercially available ethanol. In certain embodiments the lipid mixture consists of the above components in a molar ratio of about 30 to 70% amino-lipid:0 to 60% helper lipid:0.1 to 1 0% PEG-lipid and 0 to 50% cholesterol. More preferred lipid nanoparticle compositions comprise the above mentioned components in a molar ratio of about 40% to 60% amino-lipid, 0 to 20% helper lipid, 0.1% to 5% PEG lipid and 30 to 50% cholesterol. In certain other embodiments lipid nanoparticles lack cholesterol. These formulations contain up to about 60 mol % of at least one helper lipid. Preferred helper lipids in these lipid nanoparticles are DSPC, SM, DOPE, 4ME16:0PE.
In one embodiment, the nucleic acid is dissolved in an aqueous buffer. The pH of the buffer is such that at least one of the nitrogen atoms of the amino-lipids of the present invention will become protonated upon mixing the aqueous nucleic acid solution with the solution comprising the components of the lipid nanoparticles. Examples of appropriate buffers include, but are not limited to acetate, phosphate, citrate, EDTA and MES. Preferred concentration of the buffers are in the range of about 1 to about 500 mM. Typically the concentration of the nucleic acid in the aqueous buffer is in the range of about 0.1 to about 250 mg/mL, more preferably from about 0.5 to about 150 mg/mL.
The solution comprising the components of the lipid nanoparticles is then combined with the buffered aqueous solution of the nucleic acid. Acidic pH is preferred, particularly a pH below 6.8, more preferably a pH below 5.4 and most preferably about 4.0. Optionally, the entire mixture may be sized according to known methods, e.g. by extrusion. Particles with a mean diameter of preferably 40 to 170 nm, most preferably of about 50 to 120 nm are generated. Subsequently, the pH is neutralized yielding an at least partially surface-neutralized nucleic acid lipid nanoparticle complex. Due to the fact that amino-lipids of the present invention have at least two nitrogen atoms, pKa values can differ substantially. Formation of complexes with nucleic acids is most supported at low pH in the range of about 3 to about 5. At a pH of about 7, at least partial surface neutralization is achieved.
In one embodiment, the ratio of lipid:nucleic acid is at least about 2:1, at least about 3:1, at least about 4:1, at least about 5:1, at least about 6:1, at least about 7:1, at least about 8:1, at least about 10:1, at least about 15:1.
Techniques to combine the solutions comprising the components of the lipid nanoparticles and the buffered aqueous solution of the nucleic acid can vary widely and may be dependent on the scale of production. Preparations in the range of a few mL may be made by pipetting one solution into the other followed by mixing with e.g. a vortex mixture. Larger volumes can be preferentially prepared by a continuous mixing method using pumps, e.g. a piston pump such as Pump 33 (Harvard Apparatus) or most preferably HPLC pumps such as AKTA pumps (GE Healthcare). With the aid of such pumps the two solutions are pumped out of their individual reservoirs and combined by delivering the fluids through a suitable connector piece or mixing chamber. By varying the concentrations of the two solutions and their flow rates, the mean size of the resulting lipid nanoparticles can be controlled within a certain range. Preferably, the compositions provided herein are sized to a mean diameter from about 50 nm to about 200 nm, more preferably about 50 nm to about 150 nm and most preferably about 50 nm to 120 nm.
In certain embodiments, methods of the present invention further comprise a processing step that ensures the substantial removal of the solvent that was used to dissolve the lipid mixture and to exchange the buffer used to dissolve the therapeutically active agent. Suitable techniques to carry out this processing step include, but are not limited to diafiltration or tangential flow filtration. For buffer exchange a physiologically compatible buffer such as phosphate or HEPES buffered saline with a pH of about 7.4 or 5% dextrose solution (DSW) is used.
Optionally, nucleic acid lipid nanoparticles can be produced using the lipid film hydration method followed by extrusion. In this case, the components of the lipid nanoparticle, i.e. one of the amino-lipids as described in the present invention, a helper lipid, a PEG-lipid (e.g. PEG-c-DOMG) and a sterol, e.g. cholesterol, are dissolved in an organic solvent such as chloroform. The solvent is evaporated yielding a thin lipid film, which is subsequently hydrated with an aqueous buffer containing the therapeutically active agent to form the desired lipid nanoparticle. Alternatively, the lipid film is hydrated with buffer and the nucleic acid is added in a subsequent incubation step.
D. Pharmaceutical Compositions and Medical Uses.
In another object the present invention relates to a pharmaceutical composition comprising the amino-lipids of the invention. Preferably said pharmaceutical composition comprises the lipid nanoparticles of the present invention and a biologically active compound. In one embodiment said biologically active compound is selected from the group of a small molecule, a peptide, a protein or a nucleic acid. In a preferred embodiment, said biologically active compound is a nucleic acid. Examples of nucleic acids useful therein are miRNA, antisense oligonucleotides, siRNA, immune-stimulatory oligonucleotides, aptamers, ribozymes, or plasmids encoding a specific gene or siRNA.
The pharmaceutical compositions provided herein may additionally contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Regardless of the route of administration selected, the lipid nanoparticles of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
The phrases “administration” and “administered” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the mute of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
The pharmaceutical composition must be sterile and fluid to the extent that the composition is deliverable through syringe or infusion techniques. In addition to water, the carrier is preferably an isotonic sugar solution and most preferably an isotonic buffered saline solution.
Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
In another object the present invention relates to the use of the lipid nanoparticles of the invention complexed to a biologically active compound as a medicament for treatment of a disease. In one embodiment the biologically active compound is a nucleic acid that may comprise a single strand or a double strand DNA or RNA which may or may not be chemically modified. Examples of nucleic acids useful therein are miRNA, short interfering RNA (siRNA), antisense oligonucleotides, immune-stimulatory oligonucleotides, aptamers, ribozymes, or plasmids encoding a specific gene or a siRNA. In particular embodiments the biologically active compound is a short interfering RNA (siRNA) and is complexed with the lipid nanoparticles of the present invention, thus enabling intracellular delivery of said biologically active compound.
In one embodiment the present invention provides a method of treating a disease that is caused by the over-expression of one or several proteins in a subject, said method comprising administration of the a pharmaceutical composition of the present invention to said subject. The pharmaceutical composition comprises the LNP of the invention and a biologically active compound selected from the group of siRNA, miRNA, antisense oligonucleotides, ribozyme or a plasmid encoding for an siRNA, all being able to interfere with the expression of the disease causing protein(s).
In another embodiment, the present invention provides a method of treating a disease that is caused by a reduced expression or a suppressed expression of one or several proteins in a subject, said method comprising administration the pharmaceutical composition of the present invention to the subject. The pharmaceutical composition comprises the LNP of the invention and a biologically active compound selected from the group of a plasmid encoding for the corresponding protein(s) or a nucleic acid that interferes with the suppressor molecule.
In yet another embodiment, the present invention provides for a method of generating an immune response in a subject upon administration of a pharmaceutical composition of the present invention to said subject. The pharmaceutical composition comprises the LNP of the invention and a biologically active compound, wherein the biologically active compound is an immune-stimulatory nucleic acid such as a CpG oligonucleotide. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.
EXAMPLES
The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided above.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated in their entirety by reference.
Example 1
Amino-lipids
Examples of amino-lipids of the present invention synthesized by reaction of an amine and a carbonyl compound under reductive amination conditions are listed in Table 1. Solvents and reagents were purchased from Sigma Aldrich (Taufkirchen, Germany) or TCI Europe (Eschborn, Germany) and were used as received (FIG. 1).
A) General Synthesis Procedure for Amino-lipids Generated by Reaction of Amines and Aldehydes. The aldehydes needed for the preparation of KL5, KL6 KL7, KL8, KL12, KL15, KL16, KL34, KL35, KL37 were prepared by oxidation of the corresponding alcohols using 2-Iodoxybenzoic acid (IBX) according to the following general procedure:
The alcohol was dissolved in anhydrous ethyl acetate (EtOAc, 10 mL/1.0 mmol). IBX (1.1 eq) was added to form a suspension. The mixture was stirred briskly and refluxed at 80° C. under Argon. DMSO (2.2 eq.) was added via a syringe and the suspension was refluxed for 1.5 h. The insoluble o-iodobenzoic acid by-product was filtered off. The solvent was removed under reduced pressure and the crude product purified by flash-chromatography (Hexan−Hexcan/EtOAc=10:1, Rf=035 in Hexan/EtOAc=5:1). The final products were characterized by HPLC and mass spectrometry.
Ether containing alcohols were prepared following a published procedure (Bioconjugate Chemistry 2006, 19:1283). The subsequent oxidation to the corresponding aldehyde was again accomplished with IBX according to the procedure given above.




The aldehydes needed for the preparation of KL52 and KL56 were synthesized by oxidation of the corresponding alcohols employing pyridinium chlorochromate (PCC) in Dichloromethane according to standard procedures (Synthesis, 1982, 245). Other aldehydes are commercially available. The amine in KL12, KL22, KL33, KL34 and KL35 was made pursuing a published synthesis route (PNAS 2010, 5:1864).
B) Preparation of N-peralkylated Amino-lipids. KL4, KL9, KL22, KL33, KL34, KL35, KL36, KL37, KL51, KL52, KL53, and KL56 were prepared by combining the corresponding aldehyde and corresponding amine in dichloroethane (DCE) in a ratio of 1.75 equivalents per amino function of the amine. To this solution was added at room temperature sodium triacetoxyborohydride (NaBH(OAc)3) (1.3 equivalents per aldehyde) and acetic acid (HOAc, 1.3 equivalents per aldehyde). The reaction mixture was stirred at ambient temperature until thin layer chromatography indicated completion of reaction. After hydrolysis with 2N NaOH, the reaction mixture was extracted twice with dichloromethane (DCM). The combined organic layers were washed with saturated NaCl solution and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography or reversed phase (RP) HPLC.
Amino-lipids were analyzed for purity using analytical reversed phase HPLC. For this purpose, an XBridge C4 column (2.1×50 mm, 3.5 μm) from Waters (Eschborn, Germany) was used. Eluent was A 0.1% TFA in water and eluent B was 0.1% TFA in 90% Acetonitrile (ACN). Elution was achieved at a column temperature of 60° C. running a gradient from 50% to 100% B in 20 min at a flow rate of 0.5 mL/min. Amino-lipids were detected using an evaporative light scattering detector (PL-ELS 2100, Agilent, Waldbronn, Germany) with evaporation temperature set to 90° C., nebulizer temperature set to 40° C. and a nitrogen flow of 1 mL/sec. Identity was established by electrospray ionization (ESI) mass spectrometry (MS) and direct infusion technique.
1) Synthesis of KL22.




To a solution of Dodecanal (11.6 g, 62.8 mmol, 9.0 eq, 1.73 eq/amine function) and 2-[4-(2-Amino-ethyl)-piperazin-1-yl]-ethyl}-ethane-1,2-diamine (1.5 g, 7.0 mmol. 1.0 eq) in 100 mL dichloroethane (DCE) was added sodium triacetoxyborohydride (NaBH(OAc)3) (17.3 g, 81.6 mmol, 11.7 eq) and acetic acid (HOAc) (81.6 mmol, 5.0 mL) at room temperature. The reaction mixture was stirred for 16 h at ambient temperature. After hydrolysis with 2N NaOH the reaction mixture was extracted twice with dichloromethane (DCM). The combined organic layers were washed with saturated NaCl-solution and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography (DCM-DCM/CH3OH=100:6, Rf=0.05 in DCM/CH3OH=100:1 (0.5% NEt3)) to afford the title compound as a pale yellow oil.
ESI-MS (direct infusion): [M+H]+: 1058.5
A comparison of crude synthesis products obtained by an alternate peralkylation procedure (ring opening reaction of terminal epoxides by amines) detailed in WO2010/053572A2 and PNAS 2010, 5:1864 underscores the high efficiency of the chemistry disclosed herein allowing for high isolated yields (FIG. 1).
2) Synthesis of KL10.




To a solution of Dodecanal (15.1 g, 82.1 mmol, 6.0 eq) and Tris-(2-aminoethyl)-amine (2.0 g, 13.7 mmol. 1.0 eq) in 100 mL DCE was added NaBH(OAc)3 (26.1 g, 123.1 mmol, 9.0 eq) and HOAc (123.1 mmol, 7.0 mL) at room temperature. The reaction mixture was stirred for 16 h at ambient temperature. After hydrolysis with 2N NaOH the reaction mixture was extracted twice with DCM. The combined organic layers were washed with saturated NaCl-solution and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography (DCM-DCM/CH3OH=10:1-DCM/CH3OH=5:1, Rf=0.01 in DCM (0.5% NEt3)) to concentrate the title compound as a pale yellow oil. KL 0 was further purified to homogeneity using RP HPLC. ESI-MS (direct infusion): [M+H]+: 986.1
3) Synthesis of KL36.




To a solution of Dodecanal (3.22 g, 17.5 mmol, 4.5 eq) and Diethylentriamine (0.40 g, 3.88 mmol. 1.0 eq) in 50 mL DCE was added NaBH(OAc)3 (5.56 g, 26.3 mmol, 6.75 eq) and HOAc (263 mmol, 1.6 mL) at room temperature. The reaction mixture was stirred for 16 h at ambient temperature. After hydrolysis with 2N NaOH the reaction mixture was extracted twice with DCM. The combined organic layers were washed with saturated NaCl-solution and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography (DCM-DCM/CH3OH=10:1-DCM/CH3OH=5:1, Rf=0.01 in DCM (0.5% NEt3)) to concentrate the title compound as a pale yellow oil and to remove the excess aldehyde. The crude product was finally purified by HPLC employing a C4 reversed phase column (YMC-Pack C4, 150×20 mm, 0 μm. Dienslaken, Germany). Eluent A was H2O containing 0.1% Trifluoroacetic acid (TF A) and eluent B was 90% ACN containing 0.1% TFA. For elution at room temperature, a gradient from 70-100% Eluent B and a flow rate of 45 mL/min was used. 5 ESI-MS (direct infusion): [M+H]+: 776.7
4) Synthesis of KL37.




To a solution of octadecanal (1.20 g, 4.51 mmol, 5.5 eq) and Tris-(2-aminoethyl)-amine (0.12 g, 0.82 mmol. 1.0 eq) in 50 mL DCE was added NaBH(OAc)3 (1.43 g, 6.77 mmol, 6.75 eq) and HOAc (6.77 mmol, 0.4 mL) at room temperature. The reaction mixture was stirred for 16 h at ambient temperature. After hydrolysis with 2N NaOH, the reaction mixture was extracted twice with DCM. The combined organic layers were washed with saturated NaCl-solution and dried over Na2SO4. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography (DCM−DCM/CH3OH=10:1-DCM/CH3OH=5:1, Rf=0.01 in DCM (0.5% NEt3)) to concentrate the title compound as a pale yellow oil and to remove the excess aldehyde. The crude product was finally purified by HPLC on a C4 reversed phase column (YMC-Pack C4, 150×20 mm, 10 μm). Eluent A was H2O containing 0.1% Trifluoroacetic acid (TFA) and eluent B was 90% ACN containing 0.1% TFA. For elution at room temperature, a gradient from 70-100% Eluent Band a flow rate of 45 mL/min was used.
ESI-MS (direct infusion): [M+H]+: 1386.2
C) Preparation of Selectively Alkylated Amino-lipids. Amino-lipids KL5, KL6, KL7, KL8, KL2, KL15, KL16, were generated by a stepwise synthetic protocol published in J Org. Chem. 1996, 61:3849-3862:




The corresponding aldehyde (2.0 eq) and amine (1.0 eq) were mixed in MeOH (20 mL/1.0 mmol) at room temperature under an Argon atmosphere. The mixture was stirred at ambient temperature for 3 h, until the aldimine formation was completed. The solvent was removed under reduced pressure and the crude product was dissolved in DCE (20 mL/1.0 mmol) and treated with NaBH(OAc)3 (3.0 eq) and AcOH (3.0 eq) and stirred under Argon for 3 h. The reaction was quenched with 2M NaOH and the product was extracted with EtOAc. The combined organic layers were dried over Mg2SO4 and the solvent was evaporated under reduced pressure. The crude product was subject to flash chromatography (DCM-DCM/MeOH=19:1, Rf 0.25 in DCM/MeOH=0:1 (0.5% NEt3)) to afford the desired compound as a pale yellow oil. The final product was characterized by HPLC and muss spectrometry.
D. General Synthesis Procedure for Amino-lipids Derived from Amines and Ketones. The ketone needed for the preparation of KL32 and KL39 was prepared in 4 steps according to a published synthesis route (Nature Biotechnology 2010, 28:172). Other ketones are commercially available. The amine in KL23, KL30 and KL39 was made pursuing a published synthesis route (PNAS 2010, 5:1864).
The amino-lipids KL23, KL24, KL25, KL26, KL27, KL28, KL30, KL32, KL39, KL43, KL49 and KL58 were prepared by combining the corresponding ketone and the corresponding amine in DCE in a ratio of one equivalent of ketone per amine group. Subsequently, NaBH(OAc)3 (3.0 eq) and HOAc was added and stirred at room temperature until thin layer chromatography indicated completion of reaction. The reaction mixture was worked up by addition of 2N NaOH and extraction with DCM. The organic phase was dried and the solvent removed under reduced pressure. The amino-lipids were purified by flash column chromatography and analyzed by analytical reversed phase HPLC and direct infusion ESI-MS.
1) Preparation of KL25.




To a solution of Heptacosan-14-one (11.0 g, 27.9 mmol, 2.0 eq, 1.0 eq/amine function) and Triethylenetetramine (2.03 g, 13.9 mmol. 1.0 eq) in 200 mL DCE was added NaBH(OAc)3 (8.90 g, 41.9 mmol, 3.0 eq) and HOAc (41.9 mmol, 2.5 mL) at room temperature. The reaction mixture was stirred for 72 h at ambient temperature. After hydrolysis with 2N NaOH the reaction mixture was extracted twice with DCM. The combined organic layers were washed with saturated NaCl solution and dried over Na2S04. The solvent was removed under reduced pressure and the crude product was subject to flash chromatography (DCM-DCM/CH3OH=10:1-DCM/CH3OH 5:1, Rf=0.3 in DCM/CH3OH=4:1 (0.1% aq. NH3)) to afford the title compound as a pale yellow wax.
ESI-MS (direct infusion): [M+H]+: 904.0
Example 2
siRNA Synthesis
siRNAs were synthesized by standard solid phase RNA oligomerization using the phosphoramidite technology. Depending on the scale either an ABI 394 synthesizer (Applied Biosystems) or an Äkta oligopilot 100 (GE Healthcare, Freiburg, Germany) was used. In order to increase siRNA stability and abrogate immune responses, 2′-O-methyl modified nucleotides were placed within certain positions in the siRNA duplex. Ancillary synthesis reagents, RNA and 2′-O-Methyl RNA phosphoramidites were obtained from SAFC Proligo (Hamburg, Germany). Specifically, 5′-O-(4,4′-dimethoxytrityl)-3′-O-(2-cyanoethyl-N,N-disopropyl)phosphoramidite monomers of uridine (U), 4-N-acetylcytidine (CAc), 6-N-benzoyladenosine (Abz) and 2-N-isobutyriguanosine (GiBu) with 2′-O-t-butyldimethylsilyl were used to build the oligomers sequence. 2′-O-Methyl modifications were introduced employing the corresponding phosphoramidites carrying the same nucleobase protecting groups as the regular RNA building blocks. Coupling time for all phosphoramidites (100 mM in Acetonitrile) was 6 min employing 5-Ethylthio-1-tetrazole (ETT) as activator (0.5 M in Acetonitrile). Phosphorothioate linkages were introduced using 50 mM 3-((Dimethylamino-methylidene)amino)-3H-1,2,4-dithiazole-3-thione (DDTT, AM Chemicals, Oceanside, Calif., USA) in a 1:1 (v/v) mixture of pyridine and Acetonitrile. Upon completion of the solid phase synthesis oligoribonucleotides were cleaved from the solid support and deprotected using slight modification of published methods (Wincott F. et al. “Synthesis, deprotection, analysis and purification of RNA and ribozymes.” Nucleic Acids Res 1995, 23:2677-2684).
Crude oligomers were purified by anionic exchange HPLC using a column packed with Source Q15 (GE Healthcare) and an Äkta Explorer system (GE Healthcare). Buffer A was 10 mM sodium perchlorate, 20 mM Tris, 1 mM EDTA, pH 7.4 (Fluka, Buchs, Switzerland) and contained 20% Acetonitrile. Buffer B was the same as buffer A with the exception of 500 mM sodium perchlorate. A gradient of 22% B to 42% B within 32 column volumes (CV) was employed. UV traces at 280 nm were recorded. Appropriate fractions were pooled and precipitated with 3M NaOAc, pH 5.2 and 70% ethanol. Finally, the pellet was washed with 70% ethanol.
Isolated RNAs were shown to be at least 85% pure by analytical strong anion exchange chromatography. Identity of the RNA single strands was confirmed by LC-ESI-MS.
siRNAs were prepared by combining equimolar amounts of the complementary RNA strands in sodium citrate buffer (10 mM Na-Citrate, 30 mM NaCl, pH 6), hating to 70° C. for 5 min and slow cooling to room temperature over a time period of 2 h. siRNAs were further characterized by capillary gel electrophoresis and were stored frozen until use.
siRNA sequences are listed in Table 2. As indicated in the table, these siRNAs are directed against gene targets that are exclusively expressed in certain cells in the liver. In certain embodiments the individual siRNAs were employed in the inventive formulations. In certain other embodiments a mixture of all the indicated siRNAs were incorporated into the inventive formulations to address siRNA delivery to other cell types than hepatocytes. Those additional cell types are listed in Table 2 as well.







TABLE 2







siRNAs employed in LNPs of the present invention.












Duplex-ID
ssRNA ID
Sequence 5′-3′
type
Target
Cell type





1/2
 1
GcAAAGGcGuGccAAcucAdTsdT
s
Factor VII
 hepatocytes


1/2
 2
UGAGUUGGcACGCCUUUGCdTsdT
as




 


3/4
 3
AcGucuAuAucAuGGccGAdTsdT
s
EGFP
ubiquitous


3/4
 4
UCGGCcAUGAuAuAGACGUdTsdT
as




 


7/8
 7
cuuuucucGuGAcAAGAAGdTsdT
s
CLEC4F
Kupffer 


7/8
 8
CUUCUUGUcACGAGAAAAGdTsdT
as

cells


 


 9/10
 9
GGucucAAGccAcucGuuudTsdT
s
RELN
Stellate 


 9/10
10
AAACGAGUGGCUUGAGACCdTsdT
as

cells


 


11/12
11
GAAGAuGcAGuGAuuuAcAdTsdT
s
TEK
endothelial


11/12
12
UGuAAAUcACUGcAUCUUCdTsdT
as

cells


 


13/14
13
GcGcAGAAuucAucucuucdTsdT
s
CD68
Macrophages


13/14
14
GAAGAGAUGAAUUCUGCGCdTsdT
as




 


15/16
15
cuGGcuGAAuuucAGAGcAdTsdT
s
CD45
Leukocytes


15/16
16
UGCUCUGAAAUUcAGCcAGdTsdT
as




 


5/6
 5
AAcGAGAAGcGcGAucAcAdTsdT
s
EGFP
Ubiquitous


5/6
 6
UGUGAUCGCGCUUCUCGUUdTsdT
as




 


17/18
17
agAuGGAuAuAcucAAuuAdTsdT
s
Clec7a
Macrophages


17/18
18
uAAUUGAGuAuAUCcAUCUdTsdT
as





Key:


Upper case letters A, C, G, U represent RNA nucleotides;


lower case letters a, c, g, u, are 2′-O-Methyl nucleotides.


A phosphorothioate linkage is symbolized with a lower case “s”.


dT is deoxythimidine.






Example 3
Lipid Nanoparticles 
A. siRNA Lipid Nanoparticle Preparation. Helper lipids were purchased from Avanti Polar Lipids (Alabaster, Ala., USA). PEGylated lipids were obtained from NOF (Bouwelven, Belgium). Small molecules such as cholesterol were purchased from Sigma-Aldrich (Taufkirchen, Germany).
Lipid nanoparticles containing siRNAs as described in the section below were compared against a standard formulation containing the lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC2) discovered by Tekmira Pharmaceuticals. XTC2 was synthesized according to published procedures (Nature Biotechnology, 2010, 28:172). The corresponding standard formulation was prepared, unless otherwise stated, according to a published composition (PNAS, 2010, 107:1854). For this purpose, stock solutions of 1,2-distearoyl-3-phosphatidylcholine (DSPC, 10%), XTC2 (50%), cholesterol (38.5%), and α-[3′-(1,2-dimyristoyl-3-propanoxy)carboxamide-propyl]-ω-methoxy-polyoxyethylene (PEG-c-DOMG 1.5%) were prepared at concentrations of 50 mM in ethanol.
The inventive lipid nanoparticle formulations of the present invention contain the amino-lipids disclosed herein instead of XTC2. Initially, the other components were kept unchanged.
siRNA stock solutions at a concentration of 10-20 mg/mL in 10 mM sodium citrate buffer, 30 mM NaCl, pH 6 were diluted in 50 mM citrate buffer, pH 4 to the desired total siRNA concentration ( ̃1 mg/mL).
siRNA lipid nanoparticles were manufactured at a total lipid to siRNA mass ratio of 7 by combining the lipid solution in ethanol with the buffered siRNA solution in a mixing tee (e.g. CMIXPK, VICI AG International, Schenkon, Switzerland) by using either a Harvard Pump 33 Syringe Pump (Harvard Apparatus Holliston, Mass.) or for larger batches (>15 mL) an Äkta 900 HPLC Pump (GE Healthcare Bio-Sciences Corp., Piscataway, N.J.). Flow rates ranged from (17 mL/min to 67 mL/min for the siRNA solution and from 8 mL/min to 33 mL/min for the lipid solution.
Subsequent to the initial testing in mice efficacious siRNA lipid nanoparticle formulations were further optimized. Formulation variations were generated by variation of the compositions. Differences between formulations reflect differences in the lipid species or differences in the molar percentages of the lipid components, or differences in the ratio between positively and negatively charged components of the formulation.
The primary product, i.e. the product resulting by combining the two input solutions, was dialyzed 2× against phosphate buffered saline (PBS), pH 7.4 at volumes 100× of that of the primary product using Spectra/Por dialysis tubing (Spectrum Europe B.V, Breda, The Netherlands) with a MWCO of 100 or 250 kDa (CE, or PVDF membrane) or using Slide-A-Lyzer cassettes (Thermo Fisher Scientific inc. Rockford, Ill.) with a MWCO of 10 kD (RC membrane).
If desired siRNA lipid nanoparticles were concentrated at a 4° C. by using Vivaspin 20 centrifugation tubes (Sartorius AG, Gottingen, Germany) with a MWCO of 50 kD at 700 g using a table-top centrifuge.
The lipid nanoparticle suspension was filtered through a Filtropur S 0.2 filter with a pore size of 0.2 μm (Sarstedt, Numbrecht, Germany) and filled into glass vials with a crimp closure.
B. siRNA Lipid Nanoparticle Characterization. To determine the siRNA concentration, formulations were diluted to a theoretical siRNA concentration of approximately 0.02 mg/mL in phosphate buffered saline (PBS). A volume of 100 μL of the diluted formulation was added to 900 μL of a 4:1 (vol/vol) mixture of methanol and chloroform. After vigorous mixing for 1 min, the absorbance spectrum of the solution was recorded at wavelengths between 230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). The siRNA concentration in the liposomal formulation was determined based on the extinction coefficient of the siRNA used in the formulation. If extinction coefficient was not known, an average value of 22 OD/mg was used. The siRNA concentration was calculated based on the difference between the absorbance maximum at a wavelength of  ̃260 nm and the baseline value at a wavelength of 330 nm.
To determine the mean size of siRNA lipid nanoparticles, formulations were diluted in PBS to a concentration of approximately 0.05 mg/mL siRNA in a disposable polystyrene cuvette. The mean particle size was determined by using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).
To determine the zeta potential of siRNA lipid nanoparticles, formulations were diluted in PBS, pH 7.4 and in citrate buffer, pH 4 to a concentration of approximately 0.01 mg/mL siRNA in a disposable zeta cell (DTS1060C, Malvern Instruments Ltd, Malvern, Worcestershire, UK). The zeta potential was determined by using a Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK).
To determine the percentage of siRNA entrapped in lipid nanoparticles the Quant-iTTM RiboGreen® RNA assay (invitrogen Corporation Carlsbad, Calif.) was used according to the manufacture's instructions. In brief, samples were diluted to a concentration of approximately 5 μg/mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). Volumes of 50 μL of the diluted samples were transferred to a polystyrene 96 well plate. To the samples, either 50 μL of TE buffer or 50 μL of a 2% Triton X-100 solution was added. The plate was incubated as 35° C. for 15 min. The RiboGreen reagent was diluted 1:100 in TE buffer and a volume of 100 μL of was added to each well. The fluorescence intensity in each well was determined using a fluorescence plate reader (Wallac Victor 1420 Multilabel Counter; Perkin Elmer, Waltham, Mass.) at an excitation wavelength of  ̃480 nm and an emission wavelength of  ̃520 nm. The fluorescence values of the reagent blank were subtracted from that of each of the samples and siRNA concentrations were determined based on a standard curve of fluorescence intensities versus RNA concentrations. The percentage of free siRNA was determined by dividing the fluorescence intensity of the intact sample (without addition of Triton X-1 00) by the fluorescence value of the disrupted sample (with addition of Triton X-100).
Example 4
Animal Experiments
Mice (strain C57BL/6) were obtained from Charles River (Sulzfeld, Germany) or were bred in house (EGFP transgenic mice in CS7Bl/6 background) and were between 6 and 8 weeks 30 old at the time of the experiments. Intravenously administered LNPs were injected by infusion of 200 μL into the tail vein. LNPs administered to the lung were orotracheally instilled by applying 50 μL into the pharynx of isoflurane anaesthetized mice and making mice breathe in the LNP solution while blocking their obligate nose breathing. 48 h post administration, mice were anaesthetized by CO2 inhalation and sacrificed by cervical dislocation. Blood was collected during the experiment by submandibular vein bleed or—after sacrificing the animals—by cardiac puncture and serum isolated with serum separation tubes (Greiner Bio-One, Frickenhausen, Germany). Factor VII protein levels were analyzed by a chromogenic assay (see below). For preparation of bronchoalveolar lavage (BAL) fluid, the lungs were flushed 3× with 1 ml PBS via an intratracheally inserted canula and cells were pelleted by centrifugation. For quantitation of mRNA levels, organs were harvested and organ homogenates were prepared. Tissues were snap frozen in liquid nitrogen and powdered with mortar and pestle on dry ice. 30-50 mg of tissue was transferred to a chilled 1.5 mL reaction tube. 1 mL Lysis Mixture (Epicenter Biotechnologies, Madison, USA) and 33 μL Proteinase K (50 μg/μL) (Epicenter Biotechnologies, Madison, USA) was added and tissues were lysed by sonication for several seconds using a sonicator (HD2070, Bandelin, Berlin, Germany) and digested with Proteinase K for 15 min at 65° C. in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany). BAL lysates were obtained by resuspending BAL cells in 200 μL Lysis Mixture, followed by incubation at 53° C. for 30 min. Lysates were stored at −80° C. until analysis. For mRNA analyses, lysates were thawed and mRNA levels were measured using either QuantiGene 1.0 or Quantigene 2.0 branched DNA (bDNA) Assay Kit (Panomics, Fremont, Calif., USA, Cat-No: QG00004) according to the manufacturer's recommendations. In order to assess the FVII, EGFP, Clec4f, RELN, TEK, CD45, CD68, Clec7a and GAPDH mRNA content, the following probe sets were employed:







TABLE 3







Quantigene 1.0 probe sets.












SEQ ID



Oligo Name
Sequence 5′ → 3′
No.
mRNA













mGAP 2001
gagagcaatgccagccccTTTTTctcttggaaagaaagt
19
mouse GAPDH


 


mGAP 2002
ggtccagggtttcttactccttgTTTTTctcttggaaagaaagt
20
mouse GAPDH


 


mGAP 2003
ccctaggcccctcctgttattTTTTTctcttggaaagaaagt
21
mouse GAPDH


 


mGAP 2004
tgcagcgaactttattgatggTTTTTctcttggaaagaaagt
22
mouse GAPDH


 


mGAP 2005
gcacgtcagatccacgacgTTTTTaggcataggacccgtgtct
23
mouse GAPDH


 


mGAP 2006
ggcaggtttctccaggcgTTTTTaggcataggacccgtgtct
24
mouse GAPDH


 


mGAP 2007
gccctcagatgcctgcttcaTTTTTaggcataggacccgtgtct
25
mouse GAPDH


 


mGAP 2008
gccgtattcattgtcataccaggTTTTTaggcataggacccgtgtct
26
mouse GAPDH


 


mGAP 2009
gtccaccaccctgttgctgtaTTTTTaggcataggacccgtgtct
27
mouse GAPDH


 


mGAP 2010
aattgtgagggagatgctcagtTTTTTaggcataggacccgtgtct
28
mouse GAPDH


 


mGAP 2011
ccaccttcttgatgtcatcatactt
29
mouse GAPDH


 


mGAP 2012
cccaagatgcccttcagtgg
30
mouse GAPDH


 


mGAP 2013
gagacaacctggtcctcagtgtag
31
mouse GAPDH


 


mGAP 2014
ggagttgctgttgaagtcgcag
32
mouse GAPDH


 


mGAP 2015
ggcatcgaaggtggaagagtg
33
mouse GAPDH


 


mGAP 2016
aaatgagcttgacaaagttgtcatt
34
mouse GAPDH


 


mGAP 2017
gaggccatgtaggccatgag
35
mouse GAPDH


 


mGAP 2018
gtccttgctggggtgggt
36
mouse GAPDH


 


mGAP 2019
tagggcctctcttgctcagt
37
mouse GAPDH


 


mGAP 2020
gttgggggccgagttggga
39
mouse GAPDH


 


mGAP 2021
atgggggtctgggatgga
39
mouse GAPDH


 


mGAP 2022
tattcaagagagtagggagggct
40
mouse GAPDH


 


mmFak7 001
gagaagcagcagcccatgcTTITTctcttggaaagaaagt
41
mouse Factor VII


 


mmFak7 002
tggagctggagcagaaagcaTTTTTctcttggaaagaaagt
42
mouse Factor VII


 


mmFak7 003
tgcttcctcctgggttatgaaaTTTTTctcttggaaagaaagt
43
mouse Factor VII


 


mmFak7 004
ccgggccaaagctcctccTTTTTctcttggaaagaaagt
44
mouse Factor VII


 


mmFak7 005
cttgaagatctcccgggccTTTTTctcttggaaagaaagt
45
mouse Factor VII


 


mmFak7 006
ctgcttggtcctctcagggctTTTTTctcttggaaagaaagt
46
mouse Factor VII


 


mmFak7 007
actgcagtccctagaggtcccTTTTTaggcataggacccgtgtct
47
mouse Factor VII


 


mmFak7 008
tttgcctgtgtaggacaccatgTTTTTaggcataggacccgtgtct
48
mouse Factor VII


 


mmFak7 009
tcctcaaaggagcactgttccTTTTTaggcataggacccgtgtct
49
mouse Factor VII


 


mmFak7 010
ccccatcactgtaaacaatccagaaTTTTTaggcataggacccgtgtct
50
mouse Factor VII


 


mmFak7 011
tggattcgaggcacactggtTTTTTaggcataggacccgtgtct
51
mouse Factor VII


 


mmFak7 012
tggcaggtacctacgttctgacaTTTTTaggcataggacccgtgtct
52
mouse Factor VII


 


mmFak7 013
cttgcttttctcacagttccgaTTTTTaggcataggacccgtgtct
53
mouse Factor VII


 


mmFak7 014
aggagtgagttggcacgcc
54
mouse Factor VII


 


mmFak7 015
tcattgcactctctctccagagag
55
mouse Factor VII


 


mmFak7 016
gcagacgtaagacttgagatgatcc
56
mouse Factor VII


 


mmFak7 017
ccctcaaagtctaggaggcagaa
57
mouse Factor VII


 


mmFak7 018
ttgcacagatcagctgctcatt
58
mouse Factor VII


 


EGFP 001
ggcacgggcagcttgcTTTTTctcttggaaagaaagt
59
EGFP


 


EGFP 002
ggtagcggctgaagcactgTTTTTctcttggaaagaaagt
60
EGFP


 


EGFP 003
cctggacgtagccttcgggTTTTTctcttggaaagaaagt
61
EGFP


 


EGFP 004
ccttgaagaagatggtgcgctTTTTTctcttggaaagaaagt
62
EGFP


 


EGFP 005
cgaacttcacctcggcgcTTTTTctcttggaaagaaagt
63
EGFP


 


EGFP 006
ccttcagctcgatgcggtTTTTTctcttggaaagaaagt
64
EGFP


 


EGFP 007
gtcacgagggtgggccagTTTTTaggcataggacccgtgtct
65
EGFP


 


EGFP 008
cacgccgtaggtcagggtgTTTTTaggcataggacccgtgtct
66
EGFP


 


EGFP 009
gtgctgcttcatgtggtcggTTTTTaggcataggacccgtgtct
67
EGFP


 


EGFP 010
tcaccagggtgtcgccctTTTTTaggcataggacccgtgtct
68
EGFP


 


EGFP 011
cggtggtgcagatgaacttca
69
EGFP


 


EGFP 012
catggcggacttgaagaagtc
70
EGFP


 


EGFP 013
cgtcctccttgaagtcgatgc
71
EGFP


 


mmClec4f 001
ggtcccttctcagggtctgtaaTTTTTctcttggaaagaaagt
72
mouse Clec4f


 


mmClec4f 002
tctgtcttggccctctgaagatTTTTTctcttggaaagaaagt
73
mouse Clec4f


 


mmClec4f 003
ccccaggcgattctgctcTTTTTctcttggaaagaaagt
74
mouse Clec4f


 


mmClec4f 004
tctgctcctgcttctgtgcagTTTTTctcttggaaaeaaagt
75
mouse Clec4f


 


mmClec4f 005
cagaacttctcagcctcccgTTTTTctcttggaaagaaagt
76
mouse Clec4f


 


mmClec4f 006
tgcgctccctgggacgtaTTTTTctcttggaaagaaagt
77
mouse Clec4f


 


mmClec4f 007
gacttcaaagctgagacatcactcaTTTTTaggcataggacccgtgtct
78
mouse Clec4f


 


mmClec4f 008
gatctgccttcaaactctgcatcTTTTTaggcataggacccgtgtct
79
mouse Clec4f


 


mmClec4f 009
ggctttggtcgcctgcaTTTTTaggcataggacccgtgtct
80
mouse Clec4f


 


mmClec4f 010
ttccagttctgcgcgatcaTTTTTaggcataggacccgtgtct
81
mouse Clec4f


 


mmClec4f 011
agaggtcaccgaagccaggTTTTTaggcataggacccgtgtct
82
mouse Clec4f


 


mmClec4f 012
tgctctgtagcatctggacattg
83
mouse Clec4f


 


mmClec4f 013
cccctgaatcttggcagtgag
84
mouse Clec4f


 


mmClec4f 014
ccacagcttcctgcagggc
85
mouse Clec4f


 


mmClec4f 015
gctggagaacctgattctgagtct
86
mouse Clec4f


 


mmClec4f 016
aaaagtaataaaagtttccattgaagtac
87
mouse Clec4f


 


mmClec4f 017
ccacggcttcttgtcacgag
88
mouse Clec4f


 


mmReln 001
cagagatcttgaactgcatgatccTTTTTctcttggaaagaaagt
89
mouse Reln


 


mmReln 002
tcggcgggtaagcactgaTTTTTctcttggaaagaaagt
90
mouse Reln


 


mmReln 003
cgcttccagaacactttgggTTTTTctcttggaaagaaagt
91
mouse Reln


 


mmReln 004
ttcaggaagcgggtaggtgaTTTTTctcttggaaagaaagt
92
mouse Reln


 


mmReln 005
gtccatcatggctgccacaTTTTTaggcataggacccgtgtct
93
mouse Reln


 


mmReln 006
tcatgagtcactgcatacacctctcTTTTTaggcataggacccgtgtct
94
mouse Reln


 


mmReln 007
ttcaggcactttgcatccaaTTTTTaggcataggacccgtgtct
95
mouse Reln


 


mmReln 008
tgaatttgattctgggcaattttTTTTTaggcataggacccgtgtct
96
mouse Reln


 


mmReln 009
gggactaaataactccagctcacgTTTTTaggcataggacccgtgtct
97
mouse Reln


 


mmReln 010
tccttttccacccttcagttgTTTTTaggcataggacccgtgtct
98
mouse Reln


 


mmReln 011
ttacaggattccccgttaagctTTTTTaggcataggacccgtgtct
99
mouse Reln


 


mmReln 012
gggtcacagatacactgttcctttTTTTTaggcataggacccgtgtct
100
mouse Reln


 


mmReln 013
agtcacagaatctttccactgtacag
101
mouse Reln


 


mmReln 014
gagcatgacaccatctggcg
102
mouse Reln


 


mmReln 015
agttctcagtgggcgtcagg
103
mouse Reln


 


mmReln 016
ccaaagtcagtagaaaactgcacg
104
mouse Reln


 


mmReln 017
ggaagaacacagacggttgagaaa
105
mouse Reln


 


mmReln 018
tctgagtacttttggtagaacctaaatc
106
mouse Reln


 


mmTek 001
tgagtccctgggaagctttcaTTTTTctcttggaaagaaagt
107
mouse Tek


 


mmTek 002
acttccccagatctccccatTTTTTctcttggaaagaaagt
108
mouse Tek


 


mmTek 003
taagccggctaaagagtccatTTTTTctcttggaaagaaagt
109
mouse Tek


 


mmTek 004
aatgcaggtgagggatgtttTTTTTctcttggaaagaaagt
110
mouse Tek


 


mmTek 005
tcctatggtgatgggctcatggTTTTTctcttggaaagaaagt
111
mouse Tek


 


mmTek 006
ccgctcgcatggtccacgTTTTTaggcataggacccgtgtct
112
mouse Tek


 


mmTek 007
acaactcacaactttgcgacttcTTTTTaggcataggacccgtgtct
113
mouse Tek


 


mmTek 008
ccagcgtccacagatgagcaTTTTTaggcataggacccgtgtct
114
mouse Tek


 


mmTek 009
agcaagctgactccacagagaacTTTTTaggcataggacccgtgtct
115
mouse Tek


 


mmTek 010
gcgccttctactactccataaaggTTTTTaggcataggacccgtgtct
116
mouse Tek


 


mmTek 011
cggcatcagacacaagaggtaggTTTTTaggcataggacccgtgtct
117
mouse Tek


 


mmTek 012
gggtgccacccagaggcTTTTTaggcataggacccgtgtct
118
mouse Tek


 


mmTek 013
gcaaggagaaacaccacagaag
119
mouse Tek


 


mmTek 014
cgctcttgtttacaagttggcg
120
mouse Tek


 


mmTek 015
gaattgatcaagatcaggtccatg
121
mouse Tek
















TABLE 4







Quantigene 2.0 probe sets.












SEQ ID



Oligo Name
Sequence 5′ → 3′
No.
mRNA





Q2 mCD45 1
tgacgagttttacaccgcgatTTTTTgaagttaccgtttt
122
mouse CD45


 


Q2 mCD45 2
aatctgtctgcacatttataacattttTTTTTctgagtcaaagcat
123
mouse CD45


 


Q2 mCD45 3
ggcgtttctggaaatccccaTTTTTctcttggaaagaaagt
124
mouse CD45


 


Q2 mCD45 4
tggatccccacaactaggcttaTTTTTgaagttaccgtttt
125
mouse CD45


 


Q2 mCD45 5
agagactaacgtttttcttgcagcTTTTTctgagtcaaagcat
126
mouse CD45


 


Q2 mCD45 6
gtttagatacaggctcaggccaTTTTTctcttggaaagaaagt
127
mouse CD45


 


Q2 mCD45 7
tggggtttagatgcagactcagTTTTTgaagttaccgtttt
128
mouse CD45


 


Q2 mCD45 8
attgttcttatagcataaaacatatccaTTTTTctgagtcaaagcat
129
mouse CD45


 


Q2 mCD45 9
taggcaaacttttacatttttctgaTTTTTctcttggaaagaaagt
130
mouse CD45


 


Q2 mCD45 10
ccacctcaaaactggtcacattatTTTTTgaagttaccgtttt
131
mouse CD45


 


Q2 mCD45 11
tcatagtatttataaggtttcaagctttTTTTTctgagtcaaagcat
132
mouse CD45


 


Q2 mCD45 12
ttgacataggcaagtagggacact
133
mouse CD45


 


Q2 mCD45 13
cccatttctttgaatcttcccaTTTTTctcttggaaagaaagt
134
mouse CD45


 


Q2 mCD45 14
tgaaaattgcacttctcagcagtTTTTTgaagttaccgtttt
135
mouse CD45


 


Q2 mCD45 15
ccggacgatctgcttttgtgTTTTTctgagtcaaagcat
136
mouse CD45


 


Q2 mCD45 16
ggttttcattccattgaccttgtTTTTTctcttggaaagaaagt
137
mouse CD45


 


Q2 mCD45 17
ttgtctgtcggccgggaTTTTTgaagttaccgtttt
138
mouse CD45


 


Q2 mCD45 18
ggaggaccacatgtaacatttatactaTTTTTctgagtcaaagcat
139
mouse CD45


 


Q2 mCD45 19
ggttttagggccattagtttcataaTTTTTctcttggaaagaaagt
140
mouse CD45


 


mGAPDH QG2 1
cgaggctggcactgcacaaTTTTTctcttggaaagaaagt
141
mouse GAPDH


 


mGAPDH QG2 2
cttcaccattttgtctacgggaTTTTTgaagttaccgtttt
142
mouse GAPDH


 


mGAPDH QG2 3
ccaaatccgttcacaccgacTTTTTctgagtcaaagcat
143
mouse GAPDH


 


mGAPDH QG2 4
ccaggcgcccaatacggTTTTTctcttggaaagaaagt
144
mouse GAPDH


 


mGAPDH QG2 5
caatggcagccctggtgaTTTTTgaagttaccgtttt
145
mouse GAPDH


 


mGAPDH QG2 6
aacaatctccactttgccactgTTTTTctgagtcaaagcat
146
mouse GAPDH


 


mGAPDH QG2 7
tgaaggggtcgttgatggc
147
mouse GAPDH


 


mGAPDH QG2 8
catgtagaccatgtagttgaggtcaa
148
mouse GAPDH


 


mGAPDH QG2 9
ccgtgagtggagtcatactggaaTTTTTctcttggaaagaaagt
149
mouse GAPDH


 


mGAPDH QG2 10
ttgactgtgccgttgaatttgTTTTTctcttggaaagaaagt
150
mouse GAPDH


 


mGAPDH QG2 11
agcttcccattctcggccTTTTTgaagttaccgtttt
151
mouse GAPDH


 


mGAPDH QG2 12
gggcttcccgttgatgacaTTTTTctgagtcaaagcat
152
mouse GAPDH


 


mGAPDH QG2 13
cgctcctggaagatggtgatTTTTTctcttggaaagaaagt
153
mouse GAPDH


 


mGAPDH QG2 14
cccatttgatagttagtggggtct
154
mouse GAPDH


 


mGAPDH QG2 15
atactcagcaccggcctcacTTTTTctcttggaaagaaagt
155
mouse GAPDH


 


QG2 mCD68 1
ctgggagccgttggccTTTTTctcttggaaagaaagt
156
mouse CD68


 


QG2 mCD68 2
ggcttggagctgaacacaaggTTTTTgaagttaccgtttt
157
mouse CD68


 


QG2 mCD68 3
ggtataggattcggatttgaatttgTTTTTctgagtcaaagcat
158
mouse CD68


 


QG2 mCD68 4
acctttcttccaccctgaattgTTTTTctcttggaaagaaagt
159
mouse CD68


 


QG2 mCD68 5
tctttaagccccactttagctttTTTTTgaagttaccgtttt
160
mouse CD68


 


QG2 mCD68 6
acagatatgccccaagcccTTTTTctgagtcaaaagcat
161
mouse CD68


 


QG2 mCD68 7
cttggttttgttgggattcaaaTTTTTgaagttaccgtttt
162
mouse CD68


 


QG2 mCD68 8
ccgtcacaacctccctggacTTTTTctgagtcaaagcat
163
mouse CD68


 


QG2 mCD68 9
agagacaggtggggatgggtaTTTTTctcttggaaagaaagt
164
mouse CD68


 


QG2 mCD68 10
ggtaagctgtccataaggaaatgagTTTTTgaagttaccgtttt
165
mouse CD68


 


QG2 mCD68 11
tgtaggtcctgtttgaatccaaaTTTTTctgagtcaaagcat
166
mouse CD68


 


QG2 mCD68 12
ggtagactgtactcgggctctgaTTTTTctcttggaaagaaagt
167
mouse CD68


 


QG2 mCD68 13
tccaccgccatgtagtccaTTTTTgaagttaccgtttt
168
mouse CD68


 


QG2 mCD68 14
cctgtgggaaggacacattgtatTTTTTctgagtcaaagcat
169
mouse CD68


 


QG2 mCD68 15
ccatgaatgtccactgtgctgTTTTTgaagttaccgtttt
170
mouse CD68


 


QG2 mCD68 16
tctcgaagagatgaattctgcgTTTTTctgagtcaaagcat
171
mouse CD68


 


QG2 mCD68 17
cccaagggagcttggagcTTTTTctcttggaaagaaagt
172
mouse CD68


 


QG2 mCD68 18
tttccacagcagaagctttgg
173
mouse CD68


 


QG2 mCD68 19
ctggagaaagaactatgcttgca
174
mouse CD68


 


QG2 mCD68 20
agagagcaggtcaaggtgaacagTTTTTctcttggaaagaaagt
175
mouse CD68


 


QG2 eGFP 1
ggctgaagcactgcacgcTTTTgaagttaccgtttt
176
EGFP


 


QG2 eGFP 2
gaagtcgatgcccttcagctTTTTTctgagtcaaa.e;cat
177
EGFP


 


QG2 eGFP 3
ggatgttgccgtcctccttTTTTTgaagttaccgtttt
178
EGFP


 


QG2 eGFP 4
tactccagcttgtgccccaTTTTTctgagtcaaagcat
179
EGFP


 


QG2 eGFP 5
agacgttgtggctgttgtagttgTTTTTgaagttaccgtttt
180
EGFP


 


QG2 eGFP 6
tctgcttgtcggccatgatatTTTTTctgagtcaaagcat
181
EGFP


 


QG2 eGFP 7
aagttcaccttgatgccgttctTTTTTgaagttaccgtttt
182
EGFP


 


QG2 eGFP 8
cgatgttgtggcggatcttgTTTTTctgagtcaaagcat
183
EGFP


 


QG2 eGFP 9
ctgcacgctgccgtcctTTTTctcttggaaagaaagt
184
EGFP


 


QG2 eGFP 10
gctggtagtggtcggcgag
185
EGFP


 


QG2 eGFP 11
cgccgatgggggtgttct
186
EGFP


 


QG2 eGFP 12
cttcatgtggtcggggtagcTTTTTctgagtcaaagcat
187
EGFP


 


QG2 eGFP 13
gcagcacggggccgt
188
EGFP


 


QG2 eGFP 14
caggtagtggttgtcgggca
189
EGFP


 


QG2 eGFP 15
agggcggactgggtgctTTTTctcttggaaagaaagt
190
EGFP


 


QG2 eGFP 16
tctcgttggggtctttgctc
191
EGFP


 


QG2 eGFP 17
aggaccatgtgatcgcgctTTTTTctcttggaaagaaagt
192
EGFP


 


QG2 eGFP 18
gcggtcacgaactccagcTTTTTctcttggaaagaagt
193
EGFP


 


QG2 eGFP 19
cggacttgaagaagtcgtgctg
194
EGFP


 


QG2 eGFP 20
cgtagccttcgggcatggTTTTTctcttggaaagaaagt
195
EGFP


 


QG2 eGFP 21
aagatggtgcgctcctggaTTTTTgaagttaccgtttt
196
EGFP


 


QG2 eGFP 22
agttgccgtcgtccttgaagTTTTTctgagtcaaagcat
197
EGFP


 


QG2 eGFP 23
tcggcgcgggtcttgt
198
EGFP


 


QG2 eGFP 24
gtcgccctcgaacttcaccTTTTTctcttggaaagaaagt
199
EGFP


 


QG2 eGFP 25
cgatgcggttcaccagggtTTTTTgaagttaccgtttt
200
EGFP


 


QG2 mClec7a 1
tttctctgatcccctgggcTTTTTgaagttaccgtttt
201
mouse Clec7a


 


QG2 mClec7a 2
gaagatggagcctggcttccTTTTTctgagtcaaagcat
202
mouse Clec7a


 


QG2 mClec7a 3
gcaatgggcctccaaggtTTTTTctcttggaaagaaagt
203
mouse Clec7a


 


QG2 mClec7a 4
gcacaggattcctaaacccactTTTTTgaagttaccgtttt
204
mouse Clec7a


 


QG2 mClec7a 5
gcagcaaccactactaccacaaaTTTTTctgagtcaaagcat
205
mouse Clec7a


 


QG2 mClec7a 6
tgctagggcacccagcactTTTTTctcttggaaagaaagt
206
mouse Clec7a


 


QG2 mClec7a 7
cctgaattgtgtcgccaaaaTTTTgaagttaccgtttt
207
mouse Clec7a


 


QG2 mClec7a 8
ttgtctttctcctctggatttctcTTTTTctgagtcaaagcat
208
mouse Clec7a


 


QG2 mClec7a 9
tggttctctttatttcttgataggaagTTTTTctcttggaaagaaagt
209
mouse Clec7a


 


QG2 mClec7a 10
ctaaagatgattctgtgggcttgTTTTTgaagttaccgtttt
210
mouse Clec7a


 


QG2 mClec7a 11
ggagggagccaccttctcatTTTTTctgagtcaaagcat
211
mouse Clec7a


 


QG2 mClec7a 12
ctcctgtagtttgggatgccttTTTTTctcttggaaagaaagt
212
mouse Clec7a


 


QG2 mClec7a 13
ggaaggcaaggctgagaaaaacTTTTTgaagttaccgtttt
213
mouse Clec7a


 


QG2 mClec7a 14
cttccatgcatgatccaattaTTTTTctgagtcaaagcat
214
mouse Clec7a


 


QG2 mClec7a 15
cctgagaagctaaataggtaacagctTTTTTctcttggaaagaaagt
215
mouse Clec7a


 


QG2 mClec7a 16
tctcttacttccataccaggaatttTTTTTgaagttaccgtttt
216
mouse Clec7a


 


QG2 mClec7a 17
gcacctagctgggagcagtgTTTTTctgagtcaaagcat
217
mouse Clec7a


 


QG2 mClec7a 18
ttttgagttgtctatcttcagtagatga
218
mouse Clec7a


 


QG2 mClec7a 19
tggctttcaatgaactcaaattcTTTTTctcttggaaagaaagt
219
mouse Clec7a


 


QG2 mClec7a 20
gcattaatacggtgagacgatgttTTTTTgaagttaccgtttt
220
mouse Clec7a


 


QG2 mClec7a 21
cgggaaaggcctatccaaaatTTTTTctgagtcaaagcat
221
mouse Clec7a


 


QG2 mClec7a 22
catggcccttcactctgattgTTTTTctcttggaaagaagt
222
mouse Clec7a


 


QG2 mClec7a 23
gctgatccatcctcccagaacTTTTTctcttggaaagaaagt
223
mouse Clec7a


 


QG2 mClec7a 24
ttgaaacgattggggaagaatTTTTTctcttggaaagaaagt
224
mouse Clec7a


 


QG2 mClec7a 25
cctggggagctgtatttctgacTTTTTgaagttaccgtttt
225
mouse Clec7a


 


QG2 mClec7a 26
catacacaattgtgcagtaagctttTTTTTctgagtcaaagcat
226
mouse Clec7a









The bDNA assay was performed using 20 μL lysate and the corresponding gene specific probe sets. For normalization purposes GAPDH mRNA expression was analyzed using 40 μL lysate and Rattus norvegicus probe sets shown to be cross-react with mice (sequences of probe sets see above). As assay readout the chemiluminescence signal was measured in a Victor 2 Light luminescence counter (Perkin Elmer, Wiesbaden, Germany) as relative light units (RLU). The signal for the corresponding mRNA was divided by the signal for GAPDH mRNA from the same lysate. Values are reported as mRNA expression normalized to GAPDH.
For measurement of FVII activity, plasma samples from mice were prepared by collecting blood (9 volumes) by submandibular bleeding into microcentrifuge tubes containing 0.109 mol/L sodium citrate anticoagulant (1 volume) following standard procedures. FVII activity in plasma was measured with a chromogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason, Ohio) following manufacturer's recommendations. Absorbance of colorimetric development was measured using a Tecan Safire2 microplate reader (Tecan, Crailsheim, Germany) at 405 nm. Results are shown in FIGS. 2-17.







TABLE 5







Composition and physico-chemical parameters of the benchmark formulation


RLX-165 and the LNP RLK-044 containing lipid KL22 of the present invention. The


benchmark formulation was prepared according to a published protocol (Nature


Biotechnology 2010, 28: 172) with the exception of a slightly different PEG-lipid.


Instead of PEG2000-c-DMA (methoxypolyethyleneglycol-carbamoyl-dimyristyloxy-


propylamine), PEG2000-c-DMOG (α-[3′-(1,2-dimyristoyl-3-propanoxy)-


carboxamide-propyl]-ω-methoxy-polyoxyethylene) was used.















Amino-lipid
Helper lipid 
Chol
PEG2000-lipid

Size
Encap


LNP
mol %
mol %
mol %
mol %
N/P
(nm)
%

















RLX-165
57.1 XTC2
7.1 DPPC
34.4
1.4 DMOG
2.2
92
92


RLX-044
  50 KL22
 10 DSPC
38.5
1.5 DMOG
7.6
92
80
















TABLE 6







LNPs based on the amino-lipid KL10. Compositions, physico-chemical properties


and serum FVII activity upon treatment with 0.1 mg/kg siRNA directed against FVII.



















PEG2000-







KL10
DSPC
Chol
c-DOMG

Size
Encap
FVII


No
mol %
mol %
mol %
mol %
N/P
(nm)
(%)
(%)


















1
50
10
38.5
1.5
4.9
106
74
81


2
50
10
38.5
1.5
6.9
108
78
55


3
60
0
38.5
1.5
5.9
79
36
54


4
50
10% C16
38.5
1.5
5.9
110
84
69




Diether PC








5
50
10
38.5
1.5
8.4
80
83
23


6
65
3.5
30
1.5
6.9
83
56
107


7
60
8.5
30
1.5
6.9
93
71
52


8
60
9.5
30
0.5
6.9
121
75
60


9
60
0
30
10
6.9
49
36
84


10
65
0
30
5
6.9
64
29
129


11
98.5
0
0
1.5
6.9
122
53
110


12
50
10% DMPC
38.5
1.5
6.9
99
79
78


13
50
10% DPPC
38.5
1.5
6.9
100
81
43


14
50
10% DOPC
38.5
1.5
6.9
90
75
122


15
50
10% DLIPC
38.5
1.5
6.9
89
74
128


16
50
10% POPC
38.5
1.5
6.9
86
76
128


17
50
10% C18
38.5
1.5
6.9
96
86
46




Diether PC








18
50
10% C16
38.5
1.5
6.9
91
75
77




Lyso-PC








19
50
10% DOPE
38.5
1.5
6.9
88
69
124


20
50
10% DOPG
38.5
1.5
6.9
92
39
77


21
50
10% SM
38.5
1.5
6.9
101
90
27


22
50
10
38.5%
1.5
6.9
75
89
140





OChemsPC







23
50
10
38.5% DOPE
1.5
6.9
99
89
153


24
50
10
38.5
1.5% mPEG-
6.9
129
88
97






1000 DM






25
50
10
38.5
1.5% mPEG-
6.9
120
88
108






2000 DS






26
50
10
38.5
1.5% PEG-
6.9
109
88
91






2000 Chol






29
50
0
48.5%
1.5
6.9
111
86
81





4ME16:0PE







XTC2
57.1
7.1% DPPC
34.4
1.4
2.2
95
92
65
















TABLE 7







LNPs based on the amino-lipid KL22. Compositions, physico-chemical


properties and serum FVII activity upon treatment with 0.1 mg/kg siRNA directed


against FVII.



















PEG2000-c-







KL22
DSPC
Chol
DOMG

Size
Encap
FVII


No
mol %
mol %
mol %
mol %
N/P
(nm)
%
%


















1
50
10
38.5
1.5
5.5
78
46
68


2
50
10
38.5
1.5
10
76
63
45


3
50
10
38.5
1.5
15
81
69
61


4
50
10
39.5
0.5
7.6
126
84
64


5
50
10
40
0
7.6
736
40
78


6
50
20
30
0
7.6
414
9
nd


7
60
0
38.5
1.5
7.6
84
28
nd


8
60
8.5
30
1.5
7.6
92
36
80


9
40
20
38.5
1.5
7.6
93
82
79


10
65
0
30
5
7.6
59
5
nd


11
98.5
0
0
1.5
7.6
95
0
nd


12
50
10% DMPC
38.5
1.5
7.6
78
57
92


13
50
10% DPPC
38.5
1.5
7.6
90
47
73


14
50
10% DOPC
38.5
1.5
7.5
71
62
57


15
50
10% DLiPC
38.5
1.5
7.6
84
59
63


16
50
10% POPC
38.5
1.5
7.6
74
55
81


17
50
10% C18
38.5
1.5
7.6
96
61
103




Diether PC








18
50
10% C16
38.5
1.5
7.6
75
38
75




Lyso-PC








19
50
10% DOPE
38.5
1.5
7.6
75
49
59


20
50
10% DOPG
38.5
1.5
7.6
80
48
131


21
50
10% SM
38.5
1.5
7.6
92
64
68


22
50
10
38.5%
1.5
7.6
76
84
107





OChemsPC







23
50
10
38.5% DOPE
1.5
7.6
93
75
127


24
50
10
38.5% DSPC
1.5
7.6
246
1
nd


25
50
10
38.5
1.5% mPEG-
7.6
122
86
112






1000 DM






26
50
10
38.5
1.5% mPEG-
7.6
91
36
nd






5000 DM






27
50
10
38.5
1.5% mPEG-
7.6
119
86
106






1000 DS






28
50
10
38.5
1.5% mPEG-
7.6
106
68
107






2000 DS






29
50
10
38.5
1.5% PEG-1000
7.6
147
90
110






Chol






30
50
10
38.5
1.5% PEG-2000
7.6
101
64
134






Chol






XTC2
57.1
7.1% OPPC
34.4
1.4
2.2
95
92
44
















TABLE 8







LNPs based on amino-lipid KL25. Compositions, physico-chemical


properties and FVII mRNA levels upon treatment with 0.1 mg/kg 


siRNA directed against FVII.
















KL25
DSPC
Cholesterol
PEG2000c-DOMG

Size
Encap
FVII


No
mol %
mol %
mol %
mol %
N/P
(nm)
%
%


















Std
50
10
38.5
1.5
6.7
142
90
81


1
50
10
38.5% DOPE
1.5
6.7
nd
—
nd


2
50
10
40
0
6.7
nd
—
nd


3
50
10
38.5
1.5
5
109
31
nd


4
50
10
38.5
1.5
10
128
68
35


5
50
10
38.5
1.5
15
120
72
34


6
40
20
38.5
1.5
6.7
111
47
39


7
60
0
38.5
1.5
6.7
113
34
40


8
50
10% C16
38.5
1.5
6.7
132
65
67




Diether PC








9
50
10% SM
38.5
1.5
6.7
123
58
51


10
50
10
38.5
1.5% mPEG-1000 
6.7
177
59
47






DM






11
50
10
38.5
1.5% mPEG-1000 
6.7
122
80
50






DS






12
50
10
38.5
1.5% mPEG-2000 
6.7
123
49
74






DS






13
50
10
38.5
1.5% PEG-1000 
6.7
207
9
nd






Chol






14
50
10
38.5
1.5% PEG-2000 
6.7
141
46
68






Chol
















TABLE 9







LNPs based on the amino-lipid KL25. Compositions 


and physico-chemical properties.


















PEG2000-






KL25
DSPC
Chol
c-DOMG

Size
Encap


No
mol %
mol %
mol %
mol %
N/P
(nm)
%

















std
50
10
38.5
1.5
6.7
142
90


1
50
10
38.5%
1.5
6.7
nd
—





DOPE






2
50
10
40
0
6.7
nd
—


3
50
10
38.5
1.5
5
109
31


4
50
10
38.5
1.5
10
128
68


5
50
10
38.5
1.5
15
120
72


6
40
20
38.5
1.5
6.7
111
47


7
60
0
38.5
1.5
6.7
113
34


8
50
10% C16
38.5
1.5
6.7
132
65




Diether PC







9
50
10% SM
38.5
1.5
6.7
123
58


10
50
10
38.5
1.5% mPEG-
6.7
177
59






1000 DM





11
50
10
38.5
1.5% mPEG-
6.7
122
80






1000 DS





12
50
10
38.5
1.5% mPEG-
6.7
123
49






2000 DS





13
50
10
38.5
1.5% PEG-1000
6.7
207
9






Chol





14
50
10
38.5
1.5% PEG-2000
6.7
141
46






Chol



















TABLE 10







Tek mRNA levels in various organs upon treatment with LNPs based


on amino-lipid KL25. LNPs contained a pool of 5 siRNA of which


one was directed against Tek (0.2 mg/kg of each siRNA,


total siRNA dose 1 mg/kg). Values are given as the mean of


two treated animals relative to saline treated animals.















Kidney
Lung
Jejunum
Spleen
Muscle



No
% Tek
% Tek
% Tek
% Tek
% Tek


















std
41
43
72
47
77



3
20
21
25
31
34



4
18
17
27
25
38



5
35
35
58
32
40



6
22
18
41
28
25



7
38
33
57
31
48



8
42
43
47
40
65



9
51
48
39
32
43



10 
40
42
26
27
47



11 
45
37
67
60
88



12 
54
49
60
62
56



13 
54
64
47
48
73



14 
53
52
54
51
68



saline
100
100
100
100
100

















TABLE 11







mRNA levels in liver after treatment with LNPs based on


amino-lipid KL25. LNPs contained a pool of 5 siRNAs directed against


the targets listed in the table (0.2 mg/kg of each siRNA, total siRNA


dose 1 mg/kg). Values are given as the mean of two treated animals


relative to saline treated animals.












No
% FVII
% Clec4f
% Rein
% Tek
% GFP















std
81
51
32
51
80


3
35
29
28
57
61


4
34
29
23
60
58


5
39
31
51
91
75


6
40
27
58
66
72


7
67
44
41
64
80


8
51
36
40
59
84


9
47
36
57
58
74


10 
50
33
30
61
80


11 
74
46
43
65
92


12 
68
59
55
66
89


13 
65
63
55
59
72


14 
55
42
17
67
70


saline
100
100
100
100
100
















TABLE 12







Composition and physico-chemical properties of selected 


LNPs based on amino-lipid KL10.


















PEG2000-c-






KL10
DSPC
Chol
DOMG

Size
Encap


No
mol %
mol %
mol %
mol %
N/P
(nm)
%

















21
50
10% SM
38.5
1.5
6.9
92
64


23
50
10
38.5 DOPE
1.5
6.9
93
75





48.5%






29
50
0
4ME16:0PE
1.5
6.9
111
86
















TABLE 13







mRNA levels of targets expressed in the liver upon treatment with LNPs


based on amino-lipid KL10. LNPs contained a pool of 5 siRNA directed 


against the targets in the table below (0.5 mg/kg of each siRNA).













LNP
Animal
Rein 
GFP
Tek
Clec4f
FVII





KL 10-21
K1
 13%
 82%
 25%
 19%
102%



K2
 15%
104%
 30%
 13%
106%


KL 10-23
L1
 17%
 10%
 46%
136% 
 28%



L2
 14%
 8%
 35%
113%
 22%


KL 10-29 
H1
 12%
 77%
 33%
 5%
 88%



H2
 13%
 65%
 33%
 12%
 86%


Saline
S1
 95%
101%
100%
 97%
101%



S2
105%
 99%
100%
103%
 99%

## Claims

1. A linear amino-lipid having the structure:

2. A lipid nanoparticle comprising the linear amino lipid of claim 1.

3. The lipid nanoparticle of claim 2, further comprising an additional lipid selected from the group consisting of: a cationic lipid, a helper lipid, and a PEG-lipid.

4. The lipid nanoparticle of claim 3, further comprising a hydrophobic small molecule.

5. The lipid nanoparticle of claim 3, further comprising a biologically active compound.

6. The lipid nanoparticle of claim 5, wherein the biologically active compound is selected from the group consisting of: a small molecule, a peptide, a protein, a carbohydrate, a nucleic acid, and a lipid.

7. The lipid nanoparticle of claim 3, wherein the cationic lipid is a lipid comprising a quaternary amine with a nitrogen atom having four organic substituents.

8. The lipid nanoparticle of claim 3, wherein the helper lipid is a neutral zwitterionic lipid.

9. The lipid nanoparticle of claim 4, wherein the hydrophobic small molecule is selected from the group consisting of a sterol and a hydrophobic vitamin.

10. The lipid nanoparticle of claim 9, wherein the hydrophobic small molecule is cholesterol.

11. The lipid nanoparticle of claim 6, wherein the protein is selected from the group consisting of a nucleoprotein, a mucoprotein, a lipoprotein, a synthetic polypeptide, a small molecule linked to a protein and a glycoprotein.

12. The lipid nanoparticle of claim 6, wherein the nucleic acid is in the form of a single stranded or partially double stranded oligomer or a polymer composed of ribonucleotides.

13. The lipid nanoparticle of claim 6, wherein the nucleic acid is selected from the group consisting of miRNA, antisense oligonucleotides, siRNA, immune-stimulatory oligonucleotides, aptamers, ribozymes, and plasmids encoding a specific gene or siRNA.

14. A pharmaceutical composition comprising a linear amino-lipid having the structure:

15. The pharmaceutical composition of claim 14, further comprising a biologically active compound.

16. The pharmaceutical composition of claim 15, wherein the biologically active compound is a nucleic acid.

17. The pharmaceutical composition of claim 16, wherein the nucleic acid is in the form of a single stranded or partially double stranded oligomer or a polymer composed of ribonucleotides.

18. A method of treating a disease that is caused by the over-expression of one or several proteins in a subject, said method comprising the administration of a lipid nanoparticle according to claim 5 to the subject.

19. A method of treating a disease that is caused by a reduced, suppressed or missing expression of one or several proteins in a subject, said method comprising the administration of a lipid nanoparticle according to claim 5 to the subject.

20. A method for generating an immune response in a subject, said method comprising the administration of a lipid nanoparticle according to claim 5 to the subject, wherein the biologically active compound is an immune-stimulatory nucleic acid.