WO2011056682A1 - Reverse head group lipids, lipid particle compositions comprising reverse headgroup lipids, and methods for the delivery of nucleic acids - Google Patents

Abstract

The present invention related to a novel class of zwitterionic lipids that are useful in the preparation of lipid particles suitable for the delivery of encapsulated nucleic acids to cells.

Description

REVERSE HEAD GROUP LIPIDS, LIPID PARTICLE COMPOSITIONS COMPRISING REVERSE HEADGROUP LIPIDS, AND METHODS FOR THE

DELIVERY OF NUCLEIC ACIDS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 1 19(e) of

U.S. Provisional Patent Application No. 61/255,401 filed October 27, 2009; and U.S. Provisional Patent Application No. 61/400,764 filed July 30, 2010, where these (two) provisional applications are incorporated herein by reference in their entireties. STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under University-Industry Grant 59836 awarded by Canadian Institutes for Health Research (CIHR). The government may have certain rights in this invention.

BACKGROUND Technical Field

The present invention relates to lipids and lipid particles useful in the delivery of agents, including nucleic acids, to cells, as well as related methods of use in the treatment or prevention of diseases and disorders.

Description of the Related Art

Lipid particle delivery systems are currently the leading systems for the delivery of oligonucleotides (OGN) such as small interfering RNA (siRNA) in vivo. The cationic lipids contained in current lipid particle formulations designed for nucleic acid delivery perform two functions. First, they are required to encapsulate the negatively charged OGN in the lipid nanoparticles. Second, cationic lipids enhance intracellular delivery by combining with anionic lipids in target cell membranes to form non-lamellar structures, thus allowing macromolecules such as siRNA to penetrate to intracellular sites of action.

Unfortunately, however, the permanently charged cationic lipids commonly used for transfection in vitro are not suitable for systemic use, since they are rapidly cleared from the circulation and also give rise to serious toxic effects, lonizable cationic lipids that are positively charged at low pH values and uncharged at physiological pH values have been used to at least partially overcome this problem. However, the maximum amount of these lipids that can currently be present in lipid particles is in the range of 50 mol% due to stability issues, and at least 10% of a bilayer stabilizing lipid such as DSPC must be incorporated to maintain lipid particle stability.

Clearly, there is a need in the art for new lipids that are useful in the delivery of nucleic acids, but which do not cause toxicity problems.

BRIEF SUMMARY

The present invention provides novel lipids, and lipid particles comprising the same, as well as methods of using such particles for the delivery of agents to cells, including but not limited to the delivery of therapeutic nucleic acids to cells.

In one embodiment, the present invention provides a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup.

In one embodiment the lipid has the following structure (A):

(A)

or is a pharmaceutically acceptable salt thereof, wherein: R¹ and R² are either the same or different and are each, independently, C6-C32alkyl;

R³ is either hydrogen or d-C₆alkyl; and

n and m are independently either 1 , 2, 3, or 4.

In particular embodiments, the headgroup of the lipid comprises

4-amino butyric acid (FAB).

In certain embodiments, the lipid has the following structure (I):

(I)

or is a pharmaceutically acceptable salt thereof, wherein:

Ri and R2 are each, independently, C6-C32alkyl.

In certain embodiments, the lipid has the following structure (I):

(I)

wherein:

Ri and R2 are each, independently, C6-C32alkyl.

In particular embodiments, the lipid has one of the following structures:

In a related embodiment, the present invention provides a lipid particle comprising one or more zwitterionic lipids comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup. In particular embodiments, the headgroup comprises 4-amino butyric acid (FAB). In certain embodiments, the one or more zwitterionic lipids have a structure shown above. In certain embodiments, the lipids particle further comprises cholesterol. In certain embodiments, the lipid particle further comprises a PEG-lipid. In one embodiment, the lipid particle comprises one or more zwitterionic lipids comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup, cholesterol, and a PEG-lipid. In one embodiment, the lipid particle consists of one or more zwitterionic lipids comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup, cholesterol, and a PEG-lipid. In various embodiments, the one or more zwitterionic lipids have a headgroup comprising 4-amino butyric acid (FAB) or is selected from the structures shown above.

In related embodiments, the lipid particles of the present invention comprise one or more encapsulated agent. In certain embodiments, the one or more agent is a therapeutic agent. In one embodiment, the therapeutic agent is a nucleic acid, e.g., an interfering RNA. In particular embodiments, the interfering RNA is a siRNA.

In a further related embodiment, the present invention includes a method for delivering an agent to a cell comprising contacting a cell with a lipid particle of the present invention. In various embodiments, said contacting occurs in vitro or in vivo. In particular embodiments, said cell is a mammalian cell, e.g., a human cell.

In another related embodiment, the present invention includes a method of treating or preventing a disease or disorder in a subject, comprising providing to the subject a lipid particle of the present invention comprising a therapeutic agent. In particular embodiments, the subject is a mammal, e.g., a human. In certain embodiments, the therapeutic agent is an interfering RNA. In particular embodiments, the interfering RNA is an antisense RNA or a siRNA.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figure 1 provides results from a TNS assay to determine the apparent pKa values of FABs incorporated in equimolar DLinFAB/DPPC/Cholesterol (-o-) and DOFAB/DPPC/Cholesterol (-□-) multilamellar vesicles. An increase in TNS fluorescence correlates with an increase of positive charge. pKa is defined as the point at which 50% of the molecules are charged.

Figure 2 provides results from a TNS assay to determine the apparent pKa values of DOFAB incorporated in DOFAB/PEG-c-DOMG (90:10 mol%) multilamellar vesicles. An increase in fluorescence correlates with an increase of positive surface charge. Data points plotted represents replicate trials.

Figure 3 demonstrates the influence of FAB lipids on the polymorphic phase properties of DOPC. The ³¹ P NMR spectra shown were obtained for pure DOPC and equimolar mixtures of DPFAB/DOPC, DOFAB/DOPC and DLinFAB/DOPC in HEPES buffered saline, pH 7.0, at 25°C, 37°C and 50°C. The axis shows PPM from 80 to -80. The axis indicates PPM from 80 to -80.

Figure 4 demonstrates the influence of FAB lipids on the polymorphic phase properties of POPC. ³¹ P NMR spectra corresponding to equimolar mixtures of DPFAB/POPC, DOFAB/POPC and DLinFAB/POPC in HEPES buffered saline at 25°C, 37°C and 50°C are shown. The axis indicates PPM from 80 to -80.

Figure 5 shows the influence of DOFAB on the polymorphic phase properties of DOPC at 37°C. ³¹ P NMR spectra were obtained for pure DOPC (top) and equimolar mixture of DOFAB/DOPC (bottom) in HEPES buffered saline, pH 7.0. The axis indicates PPM from 80 to -80. Figure 6 shows the influence of DOFAB on the polymorphic phase properties of various phospholipids in equimolar mixtures. ³¹ P NMR spectra were obtained for (A) DOFAB/milk sphingomyelin, (B) DOFAB/diPoPE, (C) DOFAB/DOPS, (D) DOFAB/DOPG and (E) DOFAB/DOPA. The axis indicates PPM from 80 to -80.

Figure 7 demonstrates the effect of DOPE on the phase properties of DOPC and POPC. The ³¹ P NMR spectra shown were obtained for equimolar mixtures of DOPE/DOPC and DOPE/POPC in HEPES buffered saline at 25°C, 37°C and 50°C. The axis indicates PPM from 80 to -80.

Figure 8 demonstrates the effect of cholesterol on the polymorphic phase properties of DOPC. The ³¹ P NMR spectra shown were obtained for equimolar mixtures of DPFAB/DOPC/cholesterol, DOFAB/DOPC/cholesterol and DLinFAB/DOPC/cholesterol in HEPES buffered saline at 25°C, 37°C and 50°C. The axis indicates PPM from 80 to -80.

Figure 9 demonstrates the effect of cholesterol on the polymorphic phase properties of POPC. ³¹ P NMR spectra for equimolar mixtures of DPFAB/POPC/cholesterol, DOFAB/POPC/cholesterol and DLinFAB/POPC/cholesterol in HEPES buffered saline at 25°C, 37°C and 50°C are shown. The axis indicates PPM from 80 to -80.

Figure 10 provides ³¹ P NMR spectra for equimolar mixtures of

DPFAB/diPoPE and DLinFAB/diPoPE in HEPES buffered saline at 25°C, 37°C and 50°C. Figure 10A shows ³¹ P NMR spectra of diPoPE, indicating lamellar-to- hexagonal (HII) phase transition. Figure 10B demonstrates that FAB lipids caused non-lamella phase transition in diPoPE LNs. The axis indicates PPM from 80 to -80.

Figure 1 1 provides immunoblots from cells treated for 24 or 48 hours with the indicated amounts of siRNA against the androgen receptor (siAR) or low-GC content control siRNA (loGC) encapsulated with DOFAB/cholesterol/PEG-s-DMG/SPDiO (90:4.8:5:0.2 mol%).

Figure 12 provides immunofluorescence images of LNCap cells treated with 10 g/mL of AR siRNA encapsulated with DOFAB/cholesterol/PEG-s-DMG/SPDiO (90:4.8:5:0.2 mol%) for 24 and 48 hours. Nuclei were stained blue with Hoescht's, and SPDiO fluorescence was observed in green. SPDiO fluorescence appears as light spots in the panels on the right, but was not observed in the panels to the left.

Figure 13 provides the chemical structures of DOFAB (top) and

DOPC (bottom).

Figure 14 depicts the proposed mechanisms of interaction between "reverse headgroup" and cellular phospholipids in generating membrane disruptove hexagonal phase structures. DETAILED DESCRIPTION

Lipid nanoparticle (LN or LNP) delivery systems are currently the leading systems for the delivery of oligonucleotides (OGN) such as small interfering RNA (siRNA) in vivo. The cationic lipids contained in current LN formulations perform two functions. First, they are required to encapsulate the negatively charged OGN in the lipid nanoparticles. Second, cationic lipids enhance intracellular delivery by combining with anionic lipids in target cell membranes to form non-lamellar structures, thus allowing macromolecules such as siRNA to penetrate into intracellular sites of action.

The present invention provides a new class of zwitterionic lipids that combine with naturally occurring lipids, including zwitterionic lipids such as phosphatidylcholine (PC), to result in non-lamellar structures (such as the hexagonal (Η_Ν) phase), and which also have the ability to exhibit a positive charge for the encapsulation of nucleic acids, including oligonucleotides, such as siRNA. The zwitterionic lipids of the present invention contain a "reversed" headgroup in which the positive charge is located near the acyl chain region, and the negative charge is located at the distal end of the headgroup. These "reverse headgroup" lipids, which contain 4-amino butyric acid (FAB) headgroups in particular embodiments (FAB lipids), exhibit strong bilayer destabilizing characteristics in mixture with naturally occurring zwitterionic lipids such as PC. In particular, these FAB lipids are stronger bilayer destabilizing agents than dioleoylphosphatidylethanolamine (DOPE), which is commonly used as a "helper" lipid in nucleic acid-lipid particle formulations to assist with intracellular delivery properties.

The reverse headgroup lipids of the present invention, such as dipalmitoyl-4-amino butyric acid (DPFAB), dioleoyl-4-amino butyric acid (DOFAB) and dilinoleoyl-4-amino butyric acid (DLinFAB), provide considerable advantages over previously used cationic lipids in lipid particles for the delivery of nucleic acids, such as siRNA. First, it is believed that these lipids may combine with phospholipids such as PC and PE in such a way that the positive and negative charges are apposed, leading to local charge neutralization (see Figure 14). This, in turn, may lead to dehydration at the aqueous interface and possible induction of non-lamellar structures such as the hexagonal (Hn) phase. For example, the addition of cationic lipids to anionic lipids results in formation of non-lamellar phase structure for all anionic and cationic lipids investigated (Lewis and McElhaney 2000; Hafez et al. 2001 ; Hafez and Cullis, 2001 ; Heyes et al. 2005; Koynova et al. 2006). It is believed that successful induction of non-lamellar structures by these reverse headgroup or FAB lipids may lead to efficient fusion of LN with the plasma membrane of target cells, resulting in delivery of encapsulated cargo to the cell interior.

A second point is that reverse headgroup lipids, such as FAB lipids, may exhibit unusual pH-dependent properties. At pH values below the pKa of the carboxyl function, the FAB lipid is expected to exhibit a net positive charge, whereas a net negative charge is expected at pH values above the amino pKa of the FAB. At intermediate pH values, a net neutral lipid is expected. These physical properties, particularly the phase behaviour of the FAB lipids, should therefore be sensitive to the pH of the aqueous medium. For example, unsaturated phosphatidyserines preferentially adopt the H_N phase at pH values below the pKa of the carboxyl function (Hope and Cullis, 1980), where the headgroup changes from being negatively charged to being net neutral. Another important point is that reverse headgroup lipids have potential for encapsulation and delivery of anionic macromolecules such as siRNA. The reverse headgroup lipids, such as the FAB lipids, are expected to exhibit a positive charge at pH values below the pKa of the carboxyl function and should therefore be able to be used to load negatively charged nucleic acid polymers such as siRNA into lipid-based nanoparticles (Maurer et al. 2001 , 2007). In addition, at higher or neutral pH, LNs comprising reverse headgroup lipids have a low surface charge, which is required for long circulating LN to access target tissues other than the fixed and free macrophages of the reticuloendothelial system.

Thus, the present prevention further include a novel LN system that may induce fusion with the plasma membrane and, therefore, deliver nucleic acids, such as siRNA, directly into the cytoplasm of cells. This may bypass the endocytic pathway in certain embodiments. Electrostatic interactions between the cationic lipids of the LN carrier and the anionic lipids of the endosome can result in membrane-disruptive lipid structures. This disrupts the endosomal membrane, promoting the release of the siRNA payload.

Without wishing to be bound to any particular theory or mechanism, it is believed that this new class of lipids may form similar electrostatic interactions with zwitterionic lipids (such as phosphatidylcholine, PC) of the plasma membrane. These novel lipids are specifically designed to induce charge interaction with zwitterionic lipids such as PC by having a reversed charge orientation in the headgroup, hence the name reverse headgroup (RH) lipids. Reversal of charge orientation in the headgroup allows RH lipids to form electrostatic interactions with PC in a similar fashion as the interaction between cationic lipids and anionic endosomal lipids (Figure 1 1 ). Thus, LN formulated with RH lipids are anticipated to form membrane-disruptive structures with PC, resulting in fusion with the plasma membrane and delivery of their nucleic acid, e.g., siRNA, cargo directly into the cytoplasm. Reverse Headgroup Lipids

The present invention includes a zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup. Such lipids are referred to herein as reverse headgroup lipids, which are defined as zwitterionic lipids comprising a headgroup and acyl chains {e.g., two acyl chains), wherein the headgroup comprises a positive charge and a negative charge under certain pHs, and where the positive charge is located in a region of the headgroup near or proximal to the acyl chains as compared to the negative charge, which is located in a region of the headgroup distal to the acyl chains as compared to the positive charge.

In certain embodiments, a reverse headgroup lipid of the present invention has the followin structure (A):

(A)

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are either the same or different and are each, independently, C6-C32alkyl;

R³ is either hydrogen or Ci-C6alkyl; and

n and m are independently either 1 , 2, 3, or 4.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e. , contains one or more double and/or triple bonds), having from one to thirty-two carbon atoms, preferably one to six carbon atoms (Ci-C6alkyl), six to 32 carbon atoms (C6-C32alkyl), eight to twenty- four carbon atoms (C₈-C₂₄alkyl), or eight to twenty carbon atoms (C₈-C₂oalkyl), and which is attached to the rest of the molecule by a single bond. In particular embodiments of lipids of structure (A), Ri and F¾ are each, independently, a saturated C6-C32alkyl, a saturated Ci2-C2₄ alkyl, a C12- C₂₄ alkenyl, or a Ci₂-C₂₄ alkynyl.

"Pharmaceutically acceptable salt" includes both acid and base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4- acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, 2- hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene- 1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, 1 -hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p- toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

"Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, /V-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

In particular embodiments, the headgroup of a reverse headgroup lipids of the present invention comprises 4-amino butyric acid (FAB). The structure of FAB is provided below:

Reverse headgroup lipids comprising FAB headgroups are also referred to herein as FAB lipids.

In certain embodiments wherein the headgroup comprises a FAB the lipid has the following structure I):

(I)

or is a pharmaceutically acceptable salt thereof,

wherein Ri and R₂ are each, independently, C6-C₃₂alkyl. In certain embodiments, the lipid has the following structure (I):

(I)

wherein Ri and F¾ are each, independently, C6-C32alkyl.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e. , contains one or more double and/or triple bonds), having from one to thirty-two carbon atoms, preferably one to six carbon atoms (Ci-C6alkyl), six to 32 carbon atoms (C6-C32alkyl), eight to twenty- four carbon atoms (C₈-C₂₄alkyl), or eight to twenty carbon atoms (C₈-C₂oalkyl), and which is attached to the rest of the molecule by a single bond.

In particular embodiments lipids of structure (I), Ri and R₂ are each, independently, a saturated C6-C32alkyl, a saturated Ci2-C2₄ alkyl, a C12- C2₄ alkenyl, or a Ci2-C2₄ alkynyl.

In particular embodiments, a RH lipid of the present invention has one of the following structures:

The lipids of the present invention may be synthesized as described in the accompanying Synthetic Examples. In addition, it is understood that one skilled in the art may be able to make these lipids by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other lipids of structures (A) and (I) not specifically illustrated below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described in herein.

Lipid Particles and Pharmaceutical Compositions

The present invention further includes lipid particles, e.g., LNs or liposomes, comprising one or more reverse headgroup lipids of the present invention.

In certain embodiments, lipid particles comprise one or more reverse headgroup li ids having the following structure (A):

(A)

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are either the same or different and are each, independently, C₆-C₃₂alkyl;

either hydrogen or d-C₆alkyl; and

n and m are independently either 1 , 2, 3, or 4.

In particular embodiments, lipid particles of the present invention comprise a reverse head roup lipid having the following structure (I):

(I) or a pharmaceutically acceptable salt thereof, wherein

Ri and F¾ are each, independently, C6-C32alkyl.

In particular embodiments, lipid particles of the present invention comprise a reverse headgroup lipid having the following structure (I):

(I)

wherein

Ri and F¾ are each, independently, C6-C32alkyl.

In particular embodiments, the reverse headgroup lipid is DLinFAB, DOFAB, or DPFAB, or a combination thereof.

In particular embodiments, the one or more reverse headgroup lipids of the present invention comprise at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the total lipid present in the lipid particle (mol%). In one embodiment, the one or more reverse headgroup lipids of the present invention comprise between 50% and 90% of the total lipid (mol%).

In certain embodiments, lipid particles of the present invention, which comprise one of more reverse headgroup lipids of the present invention, further comprise one or more phosphatidylcholine or phosphatidylethanolamine lipids. In particular embodiments, the phosphatidylcholine lipid is selected from DOPC and POPC. In certain embodiments, the phosphatidylethanolamine lipid is 1 ,2-dipalmitoleoyl-sn-glycero-3-phophoethanolamin (diPoPE).

In additional embodiments, lipid particles of the present invention, which comprise one or more one of more reverse headgroup lipids of the present invention, further comprise cholesterol . In particular embodiments, lipid particles of the present invention comprise: one of more reverse headgroup lipids of the present invention, one or more phosphatidylcholine or phosphatidylethanolamine lipids, and cholesterol. In one embodiment, the molar amount of reverse headgroup lipid: PE or PC lipid: cholesterol is about equimolar or about 1 :1 :1 . In other embodiments, lipid particles of the present invention comprise or consist of one of more reverse headgroup lipids of the present invention, cholesterol, and a PEG-lipid. In certain embodiments, the reverse headgroup lipid comprises greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the total lipid (mol%). In one embodiment, the one or more reverse headgroup lipids of the present invention comprise between 50% and 90% or between 50% and 95% of the total lipid (mol%).

PEG-lipid conjugates include, but are not limited to, PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols (DAG), PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Patent No. 5,885,613); cationic PEG lipids; cationic- polymer-lipid conjugates (CPLs). Examples of PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates {e.g., PEG-CerC14 or PEG-CerC20) which are described in copending USSN 08/486,214, incorporated herein by reference, PEG-modified dialkylamines and PEG-modified 1 ,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modified diacylglycerols and dialkylglycerols. In one embodiment, the PEG-lipid conjugate is 3-N-[( -methoxypoly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxy-propylamine (PEG-DMG). In particular embodiments, a PEG-lipid is selected from:

P

PEG-lipid conjugates are also described, e.g., in U.S. Patent Nos. 5,820,873, 5,534,499 and 5,885,613. In particular embodiments, a lipid particle of the present invention comprises between 1 to 20 mol% PEG-lipid of total lipid, or between 1 to 15 mol% PEG-lipid of total lipid. The term "between" in this context is inclusive of the two values defining the range.

In certain embodiments, lipid particles of the present invention comprises between 50 to 95 mol% reverse headgroup lipid of the present invention, between 0 and 20 mol% cholesterol, and between 0 and 20 mol% PEG-lipid, where these values indicate mol% of total lipid. In particular embodiments, lipid particles of the present invention comprises between 75 to 95 mol% reverse headgroup lipid of the present invention, between 1 and 10 mol% cholesterol, and between 1 and 10 mol% PEG-lipid, where these values indicate mol% of total lipid. In particular embodiments, lipid particles of the present invention comprises about 90 mol% reverse headgroup lipid of the present invention, about 5 mol% cholesterol, and about 5 mol% PEG-lipid, where these values indicate mol% of total lipid. In particular embodiments, the reverse headgroup lipid is DOFAB. In particular embodiments, lipid particles of the present invention comprises about 90 mol% DOFAB, about 5 mol% cholesterol, and about 5 mol% PEG-S-DMG.

In particular embodiments, lipid particles of the present invention further comprise or encapsulate a nucleic acid, such as, e.g., an siRNA, which may target a therapeutic target, such as the androgen receptor, and may be used e.g., for the treatment or prevention of a disease or disorder in a subject, such as, e.g., prostate cancer.

Other lipids may be present in a lipid particle of the present invention, including, e.g., one or more cationic lipids or non-cationic lipids. The term "cationic lipid" refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH. Other cationic lipids that may be used in the lipid particles of the present invention include, but are not limited to, DLinDMA, DLin-K-DMA, N,N-dioleyl- Ν,Ν-dimethylammonium chloride (DODAC), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N-(1 -(2,3-dioleyloxy)propyl)- Ν,Ν,Ν-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N- dimethylammonium bromide (DDAB), N-(1 -(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)- carbamoyl)cholesterol (DC-Choi), N-(1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl- N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-1 -propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3- beta-oxybutan-4-oxy)-1 -(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2- [5'-(cholest-5-en-3 -oxy)-3'-oxapentoxy)-3-dimethy-1 -(cis,cis-9',1 -2'- octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1 ,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), 1 ,2-N,N'-Dilinoleylcarbamyl-3- dimethylaminopropane (DLincarbDAP), 1 ,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), and mixtures thereof. A number of these lipids and related analogs have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390. Other cationic lipids that may be used include those described in International Patent Application No. PCT/US2008/088676.

The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid. Non-cationic lipids used in the lipid particles, e.g., SNALP, of the present invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex. The term "neutral lipid" refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol- phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl- phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl- phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having Ci₀-C₂₄ carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Examples of non-cationic lipids suitable for use in the present invention include, but are not limited to, nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolncinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.

Therapeutic Agents

In certain embodiments, lipid particles of the present invention comprise a therapeutic agent. While any agent, e.g., antibodies, polypeptides, toxins, or small molecules, may be delivered to a cell or tissue using the lipid particles of the present invention, in particular embodiments, the lipid particles of the present invention comprise a nucleic acid. The nucleic acid may comprise DNA, RNA, or both, including modified forms of DNA and/or RNA. In certain embodiments, the nucleic acid is single-stranded or double-stranded.

In particular embodiments, the lipid particle of the present invention comprises an interfering RNA capable of mediating knockdown (i.e., reduced expression) of a target gene {e.g., a siRNA, microRNA (miRNA), short hairpin RNA (shRNA), including plasmids from which an interfering RNA is transcribed may be encapsulated within the lipid particle. As used herein, "lipid encapsulated" refers to a lipid formulation that provides a compound, such as a nucleic acid {e.g., a siRNA), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle. In both instances, the nucleic acid is protected from nuclease degradation.

RNA interference methods using interfering RNAi molecules may be used to disrupt the expression of a gene or polynucleotide of interest, such as a gene associated with an inflammatory or immune disease or disorder, e.g., a gene overexpressed in such as disease or disorder. In particular embodiments, the interfering RNA is a small interfering RNA (siRNA). SiRNAs are RNA duplexes typically 19-30 nucleotides long that can associate with a cytoplasmic multi-protein complex known as RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts. Therefore, siRNA can be designed to knock down protein expression with high specificity. While the first described RNAi molecules were RNA:RNA hybrids comprising both an RNA sense and an RNA antisense strand, it has now been demonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNA antisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi (Lamberton, J.S. and Christian, AT., (2003) Molecular Biotechnology 24:1 1 1 -1 19). Thus, the invention includes the use of RNAi molecules comprising any of these different types of double-stranded molecules. In addition, it is understood that RNAi molecules may be used and introduced to cells in a variety of forms. Accordingly, as used herein, RNAi molecules encompasses any and all molecules capable of inducing an RNAi response in cells, including, but not limited to, double-stranded polynucleotides comprising two separate strands, i.e. a sense strand and an antisense strand, e.g., small interfering RNA (siRNA); polynucleotides comprising a hairpin loop of complementary sequences, which forms a double-stranded region, e.g., small hairpin RNA (shRNA) molecules, and expression vectors that express one or more polynucleotides capable of forming a double-stranded polynucleotide alone or in combination with another polynucleotide.

RNAi molecules targeting specific polynucleotides can be readily prepared according to procedures known in the art. Structural characteristics of effective siRNA molecules have been identified. Elshabir, S.M. et al. (2001 ) Nature 41 1 :494-498 and Elshabir, S.M. et al. (2001 ), EMBO 20:6877-6888. Accordingly, one of skill in the art would understand that a wide variety of different siRNA molecules may be used to target a specific gene or transcript. In certain embodiments, siRNA molecules according to the invention are typically double-stranded and 16-30 or 18 - 25 nucleotides in length, including each integer in between. In one embodiment, a siRNA is about 21 nucleotides in length. In certain embodiments, siRNAs have 0-7 nucleotide 3' overhangs or 0-4 nucleotide 5' overhangs. In one embodiment, a siRNA molecule has a two nucleotide 3' overhang. In one embodiment, a siRNA is 21 nucleotides in length with two nucleotide 3' overhangs (i.e. they contain a 19 nucleotide complementary region between the sense and antisense strands). In certain embodiments, the overhangs are UU or dTdT 3' overhangs.

In one embodiment, siRNA target sites are selected by scanning the target mRNA transcript sequence for the occurrence of AA dinucleotide sequences. Each AA dinucleotide sequence in combination with the 3' adjacent approximately 19 nucleotides are potential siRNA target sites. In one embodiment, siRNA target sites are preferentially not located within the 5' and 3' untranslated regions (UTRs) or regions near the start codon (within approximately 75 bases), since proteins that bind regulatory regions may interfere with the binding of the siRNP endonudease complex (Elshabir, S. et al. Nature 41 1 :494-498 (2001 ); Elshabir, S. et al. EMBO J. 20:6877-6888 (2001 )). In addition, potential target sites may be compared to an appropriate genome database, such as BLASTN 2.0.5, available on the NCBI server at www.ncbi.nlm, and potential target sequences with significant homology to other coding sequences eliminated.

In particular embodiments, short hairpin RNAs constitute the nucleic acid component of nucleic acid-lipid particles of the present invention. ShRNAs contain a stem loop structure. In certain embodiments, they may contain variable stem lengths, typically from 19 to 29 nucleotides in length, or any number in between. In certain embodiments, hairpins contain 19 to 21 nucleotide stems, while in other embodiments, hairpins contain 27 to 29 nucleotide stems. In certain embodiments, loop size is between 4 to 23 nucleotides in length, although the loop size may be larger than 23 nucleotides without significantly affecting silencing activity. ShRNA molecules may contain mismatches, for example G-U mismatches between the two strands of the shRNA stem without decreasing potency. In fact, in certain embodiments, shRNAs are designed to include one or several G-U pairings in the hairpin stem to stabilize hairpins during propagation in bacteria, for example. However, complementarity between the portion of the stem that binds to the target mRNA (antisense strand) and the mRNA is typically required, and even a single base pair mismatch is this region may abolish silencing. 5' and 3' overhangs are not required, although they may be present.

In certain aspects, an interfering RNA or siRNA comprises one or more modification, such as a modified nucleoside or a modified phosphate linkage. In one embodiment, a siRNA comprises at least one modified nucleotide in the double-stranded region. In some embodiments, the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide such as a 2'OMe-guanosine, 2'OMe-uhdine, 2'OMe-adenosine, and/or 2'OMe-cytosine nucleotide. Examples of modified nucleotides suitable for use in the present invention include, but are not limited to, ribonucleotides having a 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group.

Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) that may be present in interfering RNA of the present invention include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331 -417 (1995); Mesmaeker et ai, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the 5'-end and/or 3'-end of the sense strand, antisense strand, or both strands of the siRNA. Interfering RNA of the present invention may comprise a morpholino backbone.

In particular embodiments, cells contacted with lipid particle of the present invention comprising an interfering RNA under conditions and for a time sufficient for RNA interference to occur express less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% the amount of the targeted gene as expressed by the same cell type not contacted with the lipid particle. Expression may be measured as either protein expression or mRNA expression, or as microRNA expression. Levels of the protein expressed by the target gene may be readily determined using routine procedures, e.g., such as Western blotting or FACS. Levels of RNA expressed by a targeted gene may be readily determined using routine procedures such as RT-PCR.

An interfering RNA used in the present invention comprises a region corresponding to or complementary to a region of a target gene. In preferred embodiments, this complementary region is completely complementary, while in other embodiments, it may comprise one or more mismatches. In certain embodiments, the complementary region is between 19 and 25 bases in length.

In one embodiment, the target gene is the androgen receptor, and lipid particles comprise an siRNA directed against the androgen receptor mRNA. According, the interfering RNA comprises a region that is complementary to an mRNA expressed by an androgen gene. In preferred embodiments, this complementary region is completely complementary, while in other embodiments, it may comprise one or more mismatches. In certain embodiments, the complementary region is between 19 and 25 bases in length. In one particular embodiment, the interfering RNA is a siRNA comprising the following two strands: 5^'-AGCACUGCUACUCUUCAGCAUdTdT-3^' (AR sense) and 5^'-AUGCUGAAGAGUAGCAGUGCdTdT-3^' (AR anti-sense). This 23-mer AR siRNA may be purchased from Thermo Scientific (Dharmacon). The coding sequences and mRNA sequences of various mammalian androgen receptor genes are known in the art. Methods of Producing Lipid Particles

Lipid particles of the present invention may be prepared by procedures described in the art, including those described in the accompanying Example and those described in WO 96/40964, WO 01/05374, U.S. Patent No. 5,981 ,501 , U.S. Patent No. 6,1 10,745, WO 1999/18933, and WO 1998/51278. In the exemplary methods described herein, a mixture of lipids is combined with a buffered aqueous solution of nucleic acid to produce an intermediate mixture containing nucleic acid encapsulated in lipid particles, e.g., wherein the encapsulated nucleic acids are present in a nucleic acid/lipid ratio of about 10 wt% to about 20 wt%. The intermediate mixture may optionally be sized to obtain lipid-encapsulated nucleic acid particles wherein the lipid portions are unilamellar vesicles, preferably having a diameter of 30 to 150 nm, more preferably about 40 to 90 nm. The pH may then be raised to neutralize at least a portion of the surface charges on the lipid particles, thus providing an at least partially surface-neutralized lipid particle composition.

In preparing the lipid particles of the invention, the mixture of lipids may be a solution of lipids in an organic solvent. This mixture of lipids can then be dried to form a thin film or lyophilized to form a powder before being hydrated with an aqueous buffer to form liposomes. Alternatively, in a preferred method, the lipid mixture can be solubilized in a water miscible alcohol, such as ethanol, and this ethanolic solution added to an aqueous buffer resulting in spontaneous liposome formation. In most embodiments, the alcohol is used in the form in which it is commercially available. For example, ethanol can be used as absolute ethanol (100%), or as 95% ethanol, the remainder being water. This method is described in more detail in US Patent 5,976,567.

In one exemplary embodiment, the mixture of lipids is a mixture of a reverse headgroup lipid of the present invention, cholesterol, and a PEG-lipid in an alcohol solvent. In preferred embodiments, the lipid mixture consists essentially of a cationic lipid, a non-cationic lipid, cholesterol and a PEG- modified lipid in alcohol, more preferably ethanol. In certain embodiments, the lipid mixture is combined with a buffered aqueous solution that may contain the nucleic acids. The buffered aqueous solution is typically a solution in which the buffer has a pH of less than the pK_a of the protonatable lipid in the lipid mixture. Examples of suitable buffers include citrate, phosphate, acetate, and MES. A particularly preferred buffer is citrate buffer. Preferred buffers will be in the range of 1 -1000 mM of the anion, depending on the chemistry of the nucleic acid being encapsulated, and optimization of buffer concentration may be significant to achieving high loading levels (see, e.g., US Patent 6,287,591 and US Patent 6,858,225). Alternatively, pure water acidified to pH 5-6 with chloride, sulfate or the like may be useful. In this case, it may be suitable to add 5% glucose, or another non- ionic solute which will balance the osmotic potential across the particle membrane when the particles are dialyzed to remove ethanol, increase the pH, or mixed with a pharmaceutically acceptable carrier such as normal saline. The amount of nucleic acid in buffer can vary, but will typically be from about 0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL to about 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of nucleic acids is combined to provide an intermediate mixture. The intermediate mixture is typically a mixture of lipid particles having encapsulated nucleic acids. Additionally, the intermediate mixture may also contain some portion of nucleic acids which are attached to the surface of the lipid particles (liposomes or lipid vesicles) due to the ionic attraction of the negatively-charged nucleic acids and positively-charged lipids on the lipid particle surface (the cationic lipids or other lipid making up the protonatable first lipid component are positively charged in a buffer having a pH of less than the pK_a of the protonatable group on the lipid). In one group of preferred embodiments, the mixture of lipids is an alcohol solution of lipids and the volumes of each of the solutions is adjusted so that upon combination, the resulting alcohol content is from about 20% by volume to about 45% by volume. The method of combining the mixtures can include any of a variety of processes, often depending upon the scale of formulation produced. For example, when the total volume is about 10-20 ml_ or less, the solutions can be combined in a test tube and stirred together using a vortex mixer. Large-scale processes can be carried out in suitable production scale glassware.

Optionally, the lipid particles that are produced by combining the lipid mixture and the buffered aqueous solution of nucleic acids can be sized to achieve a desired size range and relatively narrow distribution of lipid particle sizes. Preferably, the compositions provided herein will be sized to a mean diameter of from about 70 to about 200 nm, more preferably about 90 to about 130 nm. Several techniques are available for sizing liposomes to a desired size. One sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) less than about 0.05 microns in size. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure, multilamellar vesicles are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size determination. For certain methods herein, extrusion is used to obtain a uniform vesicle size.

Extrusion of lipid particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane results in a relatively well- defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired liposome complex size distribution is achieved. The liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in lipid particle siz. In some instances, the lipid particles which are formed can be used without any sizing.

In particular embodiments, methods of the present invention further comprise a step of neutralizing at least some of the surface charges on the lipid portions of the lipid-nucleic acid compositions. By at least partially neutralizing the surface charges, unencapsulated nucleic acid is freed from the lipid particle surface and can be removed from the composition using conventional techniques. Preferably, unencapsulated and surface adsorbed nucleic acids are removed from the resulting compositions through exchange of buffer solutions. For example, replacement of a citrate buffer (pH about 4.0, used for forming the compositions) with a HEPES-buffered saline (HBS pH about 7.5) solution, results in the neutralization of liposome surface and nucleic acid release from the surface. The released nucleic acid can then be removed via chromatography using standard methods, and then switched into a buffer with a pH above the pKa of the lipid used.

Optionally, the lipid vesicles (i.e., lipid particles) can be formed by hydration in an aqueous buffer and sized using any of the methods described above prior to addition of the nucleic acid. As described above, the aqueous buffer may be of a pH below the pKa of the reverse headgroup lipid. A solution of the nucleic acids can then be added to these sized, preformed vesicles. To allow encapsulation of nucleic acids into such "pre-formed" vesicles, the mixture should contain an alcohol, such as ethanol. In the case of ethanol, it should be present at a concentration of about 20% (w/w) to about 45% (w/w). In addition, it may be necessary to warm the mixture of pre-formed vesicles and nucleic acid in the aqueous buffer-ethanol mixture to a temperature of about 25° C to about 50° C depending on the composition of the lipid vesicles and the nature of the nucleic acid. It will be apparent to one of ordinary skill in the art that optimization of the encapsulation process to achieve a desired level of therapeutic agent, e.g., nucleic acid, in the lipid vesicles will require manipulation of variable such as ethanol concentration and temperature. Once the therapeutic agents, e.g., nucleic acids, are encapsulated within the preformed vesicles, the external pH can be increased to at least partially neutralize the surface charge. Unencapsulated and surface adsorbed therapeutic agent, e.g., nucleic acids, can then be removed as described above. Pharmaceutical Compositions

The lipid particles of present invention may be formulated as a pharmaceutical composition, e.g., which further comprises a pharmaceutically acceptable diluent, excipient, or carrier, such as physiological saline or phosphate buffer, selected in accordance with the route of administration and standard pharmaceutical practice. In a related embodiment, the present invention includes a kit comprising a lipid particle of the present invention.

Suitable carriers include, e.g., physiological saline, water, buffered water, 0.9% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. In compositions comprising saline or other salt containing carriers, the carrier is preferably added following lipid particle formation. Thus, after the lipid particle formulations are formed, the compositions can be diluted into pharmaceutically acceptable carriers, such as normal saline.

The resulting pharmaceutical preparations may be sterilized by conventional, well known sterilization techniques. The aqueous solutions can then be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Additionally, the lipidic suspension may include lipid- protective agents which protect lipids against free-radical and lipid-peroxidative damages on storage. Lipophilic free-radical quenchers, such as a-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are suitable.

The concentration of lipid particle in the pharmaceutical formulations can vary widely, i.e., from less than about 0.01 %, usually at or at least about 0.05-5% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Methods for Delivering a Nucleic Acid to a Cell and for Treating or Preventing Diseases and Disorders

The present invention further includes methods of delivering an agent, e.g., a therapeutic agent, to a cell, comprising contacting a cell with a lipid particle of the present invention comprising the agent. In particular embodiments said contacting occurs in vitro or in vivo. In certain embodiments, the cell is a mammalian cell, e.g., a human cell.

In a related embodiment, the present invention provides a method of treating or preventing a disease or disorder in a subject, comprising providing to the subject a lipid particle of the present invention comprising a therapeutic agent. In particular embodiments, the subject is provided with an effective amount or a therapeutically effective amount of the lipid particle. In particular embodiments, the subject is a mammal, e.g., a human. In particular embodiments, the disease or disorder is a tumor {e.g., prostate cancer), an inflammatory disease or disorder, a metabolic disease or disorder, a neurological disease or disorder, or a cardiac disease or disorder. The present methods may be used to deliver an encapsulated agent to a variety of different cells and subcellular locations. Accordingly, the methods of the invention may be used to modulate the expression of a variety of different genes, modulate an immune response, and treat or prevent various related diseases and disorders, including tumors, inflammatory or immune-related diseases and disorders.

As used herein, unless the context makes clear otherwise, "treatment," and similar words such as "treated," "treating" etc., indicates an approach for obtaining beneficial or desired results, including and preferably clinical results. Treatment can involve optionally either the amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition.

As used herein, unless the context makes clear otherwise, "prevention," and similar words such as "prevented," "preventing" etc., indicates an approach for preventing, inhibiting, or reducing the likelihood of, the onset or recurrence of a disease or condition. It also refers to preventing, inhibiting, or reducing the likelihood of, the occurrence or recurrence of the symptoms of a disease or condition, or optionally an approach for delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.

As used herein, an "effective amount" or a "therapeutically effective amount" of a substance is that amount sufficient to affect a desired biological effect, such as beneficial results, including clinical results.

In particular embodiments of the methods of the present invention, the lipid particle comprises a therapeutic agent {e.g., a nucleic acid such as an siRNA) and one or more reverse headgroup lipids {e.g., a reverse headgroup lipid of structure (I), DLinFAB, DOFAB, or DPFAB). In particular embodiments, it further comprises cholesterol and a PEG-lipid.

Various methods of the present invention may be carried out in vitro or in vivo.

Contact between the cells and the lipid particles, when carried out in vitro, may take place in a biologically compatible medium. The concentration of lipid particles in the medium can vary widely depending on the particular application, but is generally between about 1 μιτιοΙ and about 10 mmol. In certain embodiments, treatment of the cells with the lipid particles will generally be carried out at physiological temperatures (about 37°C) for periods of time from about 1 to 24 hours, preferably from about 2 to 8 hours. For in vitro applications, the cell may be grown or maintained in culture, whether of plant or animal origin, vertebrate or invertebrate, and of any tissue or type. In preferred embodiments, the cells will be animal cells, more preferably mammalian cells, and most preferably human cells.

For in vivo administration, pharmaceutical compositions comprising lipid particles of the present invention may be administered by any means available in the art. For example, they may be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In particular embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. For one example, see Stadler, et al., U.S. Patent No. 5,286,634, which is incorporated herein by reference. Intracellular nucleic acid delivery has also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY, Academic Press, New York. 101 :512-527 (1983); Mannino, et ai, Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Still other methods of administering lipid-based therapeutics are described in, for example, Rahman et al., U.S. Patent No. 3,993,754; Sears, U.S. Patent No. 4,145,410; Papahadjopoulos et al., U.S. Patent No. 4,235,871 ; Schneider, U.S. Patent No. 4,224,179; Lenk et al., U.S. Patent No. 4,522,803; and Fountain et al., U.S. Patent No. 4,588,578. In other methods, the pharmaceutical preparations may be contacted with a desired tissue by direct application of the preparation to the tissue. The lipid particles can also be administered in an aerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci. 298(4):278-281 (1989)) or by direct injection at the site of disease (Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)).

The methods of the present invention may be practiced in a variety of subjects or hosts. Preferred subjects or hosts include mammalian species, such as humans, non-human primates, dogs, cats, cattle, horses, sheep, and the like. In particular embodiments, the subject is a mammal, such as a human, in need of treatment or prevention of a disease or disorder, e.g., a subject diagnosed with or considered at risk for a disease or disorder.

Dosages for the lipid particles of the present invention will depend on the ratio of therapeutic agent, e.g., nucleic acid, to lipid and the administrating physician's opinion based on age, weight, and condition of the patient. SYNTHETIC EXAMPLES

The reverse headgroup lipids described herein were prepared by O-alkylation of 1 -(2,3-dihydroxypropyl)pyrrolidin-2-one (2), obtained as depicted and described below, with the commercially available mesylates of linoleyl (3), oleyl (4), or palmitoyl (5) alcohol, followed by base hydrolysis of the lactam ring.

\^ \^ \^/- 0-S0₂Me 4

Preparation of 1 -Allylpyrrolidin-2-one (1 ). A solution of 2- pyrrolidinone (20.0 g, 0.23 moles) and allyl bromide (33.8 g, 0.28 moles) in acetonitrile (100 mL) containing suspended K2CO3 (39.0 g, 0.28 moles) was refluxed under an argon atmosphere for 12 h with good stirring. The mixture was cooled to room temperature, filtered, and evaporated under reduced pressure. The residue was purified by column chromatography (25-50% ethyl acetate/hexanes), to afford 12.8 g (64%) of 1 -allylpyrrolidin-2-one (1 ), a known compound (Vepkhishvili et al., 1991 ), as yellow viscous liquid. ¹ H-NMR (300 MHz, CDCI3): 5.62-5.85 (1 H, m), 5.10-5.30 (2H, m), 3.90 (2H, dd), 3.35 (2H, t), 2.40 (2H, t), 2.00 (2H, m). ESI-MS: [M + H]⁺ 126.2.

Preparation of 1 -(2,3-dihvdroxypropyl)pyrrolidin-2-one (2). A 4% (w/w) aqueous solution of OsO (2 mL) was added to a solution of 1 - allylpyrrolidin-2-one (1 , 12.8 g, 0.08 moles) and N-methyl morpholine N-oxide (1 1 .8 g, 0.10 moles) in 80% acetone:water (100 mL) and the mixture was stirred at room temperature for 12 h. The reaction was quenched with saturated sodium sulfite solution (5 mL) and solvent was removed under reduced pressure. The residue was purified by column chromatography (5% methanol/CH₂CI₂) to afford 10.2 g (79%) of 1 -(2,3-dihydroxypropyl)pyrrolidin-2- one (2) as yellow viscous liquid. ¹H-NMR (300 MHz, CDCI₃) δ: 3.80-4.00 (3H, m), 3.35-3.52 (4H, m), 2.40 (2H, m), 2.00 (2H, m). ¹³C: 177.03, 70.13, 63.72, 49.47, 45.81 , 30.84, 18.17. HRMS: calc. for C₇Hi₄NO₃ [M + H]⁺ = 160.0974, found 160.0969.

O-alkylation of 1 -(2,3-dihvdroxypropyl)pyrrolidin-2-one. A solution of diol 1 -(2,3-dihydroxypropyl)pyrrolidin-2-one (2, 500 mg, 3.16 mmol) in benzene (5 mL) was carefully added to a suspension of NaH (250 mg, 10.48 mmol, 60% oil suspension) in dry benzene (10 mL), at 0 °C under an argon atmosphere, and the resulting mixture was stirred for 12 h. A solution of linoleyl, oleyl or palmitoyl methanesulfonate in benzene (10 mL) was then added, and the mixture was refluxed under argon for 12 h, with good stirring. The solution was cooled to room temperature and ethanol (10 mL) was carefully added, with vigorous stirring under argon, to destroy excess sodium hydride. Water (25 mL) was then added, the organic phase was separated and retained, and the aqueous layer was extracted with ethyl acetate (3 x 100 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Purification of the residue by column chromatography (5% Methanol/ CH2CI2) afforded a yellow viscous liquid.

Base hydrolysis of the lactam ring. A solution of alkylated 1 -(2,3- dihydroxypropyl)pyrrolidin-2-one and NaOH (1 .5 g, 37.0 mmol) in 1 -butanol (75 mL) was refluxed under argon for 6 days. The progress of the slow hydrolysis of the pyrrolidinone ring was monitored by TLC and by mass spectrometry. When starting material was no longer apparent, the mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was redissolved in CH2CI2 (75 mL) Aqueous 4N HCI solution was added until an apparent pH of 6 was attained. The organic layer was separated and the aqueous phase was extracted with more CH2CI2 (3 x 150 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (10-15% methanol/CH₂Cl2) afforded a yellow viscous liquid. SYNTHETIC EXAMPLE 1

SYNTHESIS OF 4-[1 -(2,3-BIS[(9Z, 1 2Z)-9, 1 2-OCTADECADIEN-1 - YLOXY])PROPYL]AMINOBUTANOIC ACID (DLINFAB, 7)

DLinFAB was prepared as described above and depicted in the synthetic scheme shown below, as described in further detail below.

Preparation of Compound 6. A solution of diol 2 (780 mg, 4.9 mmol) in benzene (5 mL) was carefully added to a suspension of NaH (431 mg, 18 mmol, 60% oil suspension) in dry benzene (15 mL), at 0 °C under an argon atmosphere (CAUTION: evolution of flammable H₂) and the resulting mixture was stirred for 30 minutes. A solution of linoleyl methanesulfonate (3, 3.44 g, 10 mmol) in benzene (10 mL) was then added, and the mixture was refluxed under argon for 12 h, with good stirring. The solution was cooled to room temperature and ethanol (10 mL) was carefully added, with vigorous stirring under argon, to destroy excess sodium hydride. Water (25 mL) was then added, the organic phase was separated and retained, and the aqueous layer was extracted with ethyl acetate (3 x 150 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Purification of the residue by column chromatography (5% Methanol/ CH2CI2) afforded 2.10 g (65%) of 6 as yellow viscous liquid. ¹ H-NMR (300 MHz, CDCI₃) δ: 5.30-5.48 (8H, m), 3.30-3.70 (9H, m), 2.70 (4H, t), 2.35 (t, 2H) 2.12 (8H, m), 1 .65 (8H, m), 1 .25-1 .42 (32H, m), 0.82-0.92 (6H, t). ESI-MS: [M + H]⁺ 656.8, [M + Na]⁺ 678.7.

Preparation of DLinFAB (7). A solution of compound 6 (2.10 g, 3.2 mmol) and NaOH (3.0 g, 76 mmol) in 1 -butanol (75 mL) was refluxed under argon for 6 days. The progress of the slow hydrolysis of the pyrrolidinone ring was monitored by TLC and by mass spectrometry. When starting material was no longer apparent, the mixture was cooled to room temperature and the solvent was removed under reduced pressure and the residue was redissolved in CH2CI2 (75 mL). Aqueous 4N HCI solution was added until an apparent pH of 6 was attained. The organic layer was separated and the aqueous phase was extracted with more CH2CI2 (3 x 150 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (10-15% methanol/CH₂Cl2) to afford 1 .62 g (63%) of 7 as a yellow viscous liquid. ¹ H-NMR (300 MHz, CDCI₃) δ: 6.24- 6.33 (2H, m), 5.90-6.01 (2H, m), 5.60-5.70 (2H, m), 5.24-5.34 (2H, m), 3.53- 3.61 (5H, m), 3.41 -3.46 (2H, t), 3.06-3.27 (4H, m), 2.54 (2H, t), 2.05-2.18 (8H, m), 1 .54-1 .58(41-1, m), 1 .28-1 .37 (40H, m), 0.82-0.92 (6H, t). ¹³C: 175.51 , 134.67, 134.56, 130.05, 129.91 , 128.60, 128.55, 125.59, 125.55, 74.06, 71 .96, 70.30, 69.63, 49.12, 47.99, 32.88, 31 .71 , 31 .45, 29.86, 29.73, 29.61 , 29.56, 29.49, 29.43, 29.38, 29.28, 29.25, 28.89, 27.68, 27.63, 26.03, 22.59, 22.53, 14.08, 14.04. IR: 1400, 1710, 2950, 3050, HRMS: calc. for C₄₃H₈₀NO₄ [M + H]⁺ = 674.6087, found 674.6100.

SYNTHETIC EXAMPLE 2

SYNTHESIS OF 4-[1 -(2,3-BIS[(9Z)-9-OCTADECEN-1 -YLOXY])PROPYL]AMINOBUTANOIC

ACID (DOFAB, 9)

DOFAB was prepared as described above and depicted in the synthetic scheme shown below, as described in further detail below.

Preparation of Compound 8. A solution of diol 2 (500 mg, 3.16 mmol) in benzene (5 ml_) was carefully added to a suspension of NaH (250 mg, 10.48 mmol, 60% oil suspension) in dry benzene (10 ml_), at 0 °C under an argon atmosphere, and the resulting mixture was stirred for 12 h. A solution of oleyl methanesulfonate (4, 2.10 g, 6.06 mmol) in benzene (10 ml_) was then added, and the mixture was refluxed under argon for 12 h, with good stirring. The solution was cooled to room temperature and ethanol (10 ml_) was carefully added, with vigorous stirring under argon, to destroy excess sodium hydride. Water (25 ml_) was then added, the organic phase was separated and retained, and the aqueous layer was extracted with ethyl acetate (3 x 100 ml_). The combined extracts were dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure. Purification of the residue by column chromatography (5% Methanol/ CH₂CI₂) afforded 1 .23 g (60%) of 8 as a yellow viscous liquid. ¹ H-NMR (300 MHz, CDCI₃) δ: 5.30-5.48 (4H, m), 3.30-3.70 (9H, m), 2.40 (2H, t), 2.12 (8H, m), 1 .65 (4H, m), 1 .25-1 .42 (48H, m), 0.82-0.92 (6H, t). ESI-MS: [M + H]⁺ 660.8, [M + Na]⁺ 683.7.

Preparation of DOFAB (9). A solution of compound 8 (1 .3 g, 1 .9 mmol) and NaOH (1 .5 g, 37.0 mmol) in 1 -butanol (75 ml_) was refluxed under argon for 6 days. The progress of the slow hydrolysis of the pyrrolidinone ring was monitored by TLC and by mass spectrometry. When starting material was no longer apparent, the mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was redissolved in CH2CI2 (75 ml_) Aqueous 4N HCI solution was added until an apparent pH of 6 was attained. The organic layer was separated and the aqueous phase was extracted with more CH2CI2 (3 x 150 ml_). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (10-15% methanol/CH₂Cl2) to afford 775 mg (63%) of 9 as a yellow viscous liquid. ¹H-NMR (300 MHz, CDCI₃) δ: 6.25-6.33 (1 H, m), 5.90-5.97 (1 H, m), 5.60-5.70 (1 H, m), 5.25-5.40 (1 H, m), 3.26-3.66 (7H, m), 2.31 -2.40 (2H, t), 1 .94-2.19 (6H, m), 1 .54-1 .55 (4H, m), 1 .25- 1 .42 (52H, m), 0.82-0.92 (6H, t). ESI-MS: [M + H]⁺ 678.8.

SYNTHETIC EXAMPLE 3

SYNTHESIS OF 4-[1 -(2,3-BIS(OCTADECYLOXY)-PROPYL]AMINOBUTANOIC ACID

(DPFAB, 1 1 ) DPFAB was prepared as described above and depicted in the synthetic scheme shown below, as described in further detail below.

11

Preparation of Compound 10. A solution of diol 2 (500 mg, 3.16 mmol) in benzene (5 mL) was carefully added to a suspension of NaH (251 mg, 10.5 mmol, 60% oil suspension) in dry benzene (10 mL), at 0 °C under an argon atmosphere, and the resulting mixture was stirred for 12 h. A solution of palmitoyl methanesulfonate (5, 2.0 g, 6.32 mmol) in benzene (10 mL) was then added, and the mixture was refluxed under argon for 12 h, with good stirring. The solution was cooled to room temperature and ethanol (10 mL) was carefully added, with vigorous stirring under argon, to destroy unreacted sodium hydride. Water (25 mL) was then added, the organic phase was separated and retained, and the aqueous layer was extracted with ethyl acetate (3 x 100 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo. Purification of the residue by column chromatography (5% Methanol/ CH₂CI₂) afforded 1 .25 g (65%) of 9 as a pale yellow solid, m.p. 87-90 °C. ¹H-NMR (300 MHz, CDCI₃) δ: 3.25-3.70 (9H, m), 2.70 (2H, t), 2.12 (4H, m), 1 .65 (4H, m), 1 .25-1 .42 (52H, m), 0.82-0.92 (6H, t). ESI-MS: [M + H]⁺ 608.8, [M + Na]⁺ 630.7.

Preparation of DPFAB (1 1 ). A solution of compound 10 (1 .25 g,

2.05 mmol) and NaOH (1 .9 g, 49.0 mmol) in 1 -butanol (75 mL) was refluxed under argon for 6 days. The progress of the slow hydrolysis of the pyrrolidinone ring was monitored by TLC and by mass spectrometry. When starting material was no longer apparent, the mixture was cooled to room temperature, the solvent was removed under reduced pressure, and the residue was redissolved in CH2CI2 (60 mL) Aqueous 4N HCI solution was added until an apparent pH of 6 was attained. The organic layer was separated and the aqueous phase was extracted with more CH2CI2 (3 x 100 mL). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by column chromatography (10-15% methanol/CH₂CI₂) to afford 955 mg (63%) of 1 1 as a yellow solid, m.p. 136-140 °C. ¹ H-NMR (300 MHz, CDCI3) δ: 3.39-3.76 (7H, m), 3.02-3.26 (2H, m), 2.32-2.53 (4H, m), 2.12 (2H, m), 1 .65 (4H, m), 1 .25-1 .42 (52H, m), 0.82-0.92 (6H, t). HRMS: calc. for C₃9H₈₀NO₄ [M + H]⁺ = 626.6087, found 626.6071 . SYNTHETIC EXAMPLE 4

SYNTHESIS OF MPEG2000-1 ,2-DI-0-ALKYL-SA/3-CARBOMOYLGLYCERIDE

(PEG-C-DOMG)

PEG-lipids, such as mPEG2000-1 ,2-Di-O-Alkyl-sn3- Carbomoylglyceride (PEG-C-DOMG) are synthesized as shown in the schematic diagram and described below. "OH

,o

R^'

la R C-14H29

lb R = C16H33

lie R = C₁₈H₃₇ 1δΗ₃7

Synthesis of IVa

1 ,2-Di-O-tetradecyl-sn-glyceride la (30 g, 61 .80 mmol) and Ν,Ν'- succinimidylcarboante (DSC, 23.76 g, 1 .5eq) were taken in dichloromethane (DCM, 500 mL) and stirred over an ice water mixture. Triethylamine (TEA, 25.30 mL, 3 eq) was added to the stirring solution and subsequently the reaction mixture was allowed to stir overnight at ambient temperature. Progress of the reaction was monitored by TLC. The reaction mixture was diluted with DCM (400 mL) and the organic layer was washed with water (2X500 mL), aqueous NaHCO3 solution (500 mL) followed by standard work-up. The residue obtained was dried at ambient temperature under high vacuum overnight. After drying, the crude carbonate Ma thus obtained was dissolved in dichloromethane (500 mL) and stirred over an ice bath. To the stirring solution, mPEG₂ooo-NH₂ (III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) and anhydrous pyridine (Py, 80 mL, excess) were added under argon. In some embodiments, the x in compound III has a value of 45-49, preferably 47-49, and more preferably 49. The reaction mixture was then allowed to stir at ambient temperature overnight. Solvents and volatiles were removed under vacuum and the residue was dissolved in DCM (200 mL) and charged on a column of silica gel packed in ethyl acetate. The column was initially eluted with ethyl acetate and subsequently with gradient of 5-10 % methanol in dichloromethane to afford the desired PEG-Lipid IVa as a white solid (105.30g, 83%). ¹ H NMR (CDCI3, 400 MHz) δ = 5.20-5.12(m, 1 H), 4.18-4.01 (m, 2H), 3.80- 3.70(m, 2H), 3.70-3.20(m, -O-CH₂-CH₂-O-, PEG-CH₂), 2.10-2.01 (m, 2H), 1 .70- 1 .60 (m, 2H), 1 .56-1 .45(m, 4H), 1 .31 -1 .15(m, 48H), 0.84(t, J= 6.5Hz, 6H). MS range found: 2660-2836.

Synthesis of Ivb. 1 ,2-Di-O-hexadecyl-sn-glyceride lb (1 .00 g, 1 .848 mmol) and DSC (0.710 g, 1 .5eq) were taken together in dichloromethane (20 ml_) and cooled down to 0°C in an ice water mixture. Triethylamine (1 .00 ml_, 3eq) was added and the reaction was stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solution and dried over sodium sulfate. Solvents were removed under reduced pressure and the resulting residue of Mb was maintained under high vacuum overnight. This compound was directly used for the next reaction without further purification. MPEG2000-NH2 Ml (1 .50g, 0.687 mmol, purchased from NOF Corporation, Japan) and Mb (0.702g, 1 .5eq) were dissolved in dichloromethane (20 ml_) under argon. In some embodiments, the x in compound Ml has a value of 45-49, preferably 47-49, and more preferably 49. The reaction was cooled to 0°C. Pyridine (1 ml_, excess) was added and the reaction stirred overnight. The reaction was monitored by TLC. Solvents and volatiles were removed under vacuum and the residue was purified by chromatography (first ethyl acetate followed by 5-10% MeOH/DCM as a gradient elution) to obtain the required compound IVb as a white solid (1 .46 g, 76 %). ¹ H NMR (CDCI₃, 400 MHz) δ = 5.17(t, J= 5.5Hz, 1 H), 4.13(dd, J= 4.00Hz, 1 1 .00 Hz, 1 H), 4.05(dd, J= 5.00Hz, 1 1 .00 Hz, 1 H), 3.82-3.75(m, 2H), 3.70-3.20(m, -O-CH₂-CH₂-O-, PEG- CH₂), 2.05-1 .90(m, 2H), 1 .80-1 .70 (m, 2H), 1 .61 -1 .45(m, 6H), 1 .35-1 .17(m, 56H), 0.85(t, J= 6.5Hz, 6H). MS range found: 2716-2892.

Synthesis of IVc. 1 ,2-Di-O-octadecyl-sn-glyceride lc (4.00 g, 6.70 mmol) and DSC (2.58 g, 1 .5eq) were taken together in dichloromethane (60 ml_) and cooled down to 0°C in an ice water mixture. Triethylamine (2.75 ml_, 3eq) was added and the reaction was stirred overnight. The reaction was followed by TLC, diluted with DCM, washed with water (2 times), NaHCO3 solution, and dried over sodium sulfate. Solvents were removed under reduced pressure and the residue was maintained under high vacuum overnight. This compound was directly used for the next reaction without further purification. MPEG2000-NH2 III (1 .50g, 0.687 mmol, purchased from NOF Corporation, Japan) and lie (0.760g, 1 .5eq) were dissolved in dichloromethane (20 mL) under argon. In some embodiments, the x in compound III has a value of 45- 49, preferably 47-49, and more preferably 49. The reaction was cooled to 0°C. Pyridine (1 mL, excess) was added and the reaction was stirred overnight. The reaction was monitored by TLC. Solvents and volatiles were removed under vacuum and the residue was purified by chromatography (ethyl acetate followed by 5-10% MeOH/DCM as a gradient elution) to obtain the desired compound IVc as a white solid (0.92 g, 48 %). ¹ H NMR (CDCI3, 400 MHz) δ = 5.22-5.15(m, 1 H), 4.16(dd, J= 4.00Hz, 1 1 .00 Hz, 1 H), 4.06(dd, J= 5.00Hz, 1 1 .00 Hz, 1 H), 3.81 -3.75(m, 2H), 3.70-3.20(m, -O-CH₂-CH₂-O-, PEG-CH₂), 1 .80-1 .70 (m, 2H), 1 .60-1 .48(m, 4H), 1 .31 -1 .15(m, 64H), 0.85(t, J= 6.5Hz, 6H). MS range found: 2774-2948.

BIOLOGICAL EXAMPLES

The physical properties of various FAB lipids, as well as their effect on bilayer stability and their ability to deliver nucleic acids to cells is described in the following biological examples. BIOLOGICAL EXAMPLE 1

CHARACTERIZATION OF FAB LIPIDS AND FAB LIPID PARTICLES

Reverse headgroup lipids comprisig FAB headgroups were characterized with respect to the pKa values exhibited by the FAB headgroups and the abilities of these FAB lipids to combine with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) to induce non-lamellar structures.

MATERIALS AND METHODS Materials:

Bovine milk sphingomyelin (SM),1 ,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), cholesterol, 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-dioleoyl-sn-glycero-3-phosphate sodium salt (DOPA), 1 ,2-dioleoyl-sn-glycero-3-phosphoserim sodium salt (DOPS), 1 ,2- dioleoyl-sn-glycero-3-phospho-(1 '-rac-glycerol) sodium salt (DOPG), 1 ,2- dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (diPoPE), 1 ,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC) and 1 -Palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) were obtained from Avanti Polar Lipids (Alabaster, AL). 2-(Cyclohexylamino)ethanesulfonic acid (CHES), 3-(Cyclohexylamino)-1 - propanesulfonic acid (CAPS), 4-(2-Hydroxyethyl)piperazine-1 -ethanesulfonic acid (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES) and 2-(p-toluidino)- 6-naphthalenesulfonic acid potassium salt (TNS) were obtained from Sigma- Aldrich (St. Louis, MO). 3-N-[(ro-methoxypoly(ethyleneglycol)2000)carbamoyl]- 1 ,2-dimyristyloxy-propylamine (PEG-s-DMG) and R-3-[(co-methoxy- poly(ethyleneglycol)2000)carbamoyl)]-1 ,2-dimyristyloxypropyl-3-amine (PEG-c- DOMG) were obtained from Tekmira Pharmaceuticals (Vancouver, BC). Glacial acetic acid and sodium chloride were obtained from Fisher Scientific (Fair Lawn, NJ).

TNS Assays:

The pKa's of various FAB lipids were determined using the fluorescence probe, TNS, which increases in fluorescence quantum yield upon binding to lipid membrane.

xln certain experiments, FAB lipid, DPPC, and cholesterol stocks were dissolved in chloroform. Equimolar components were mixed by vortexing. Solvent was removed by drying under a stream of nitrogen gas and subsequently by vacuum overnight. Multilamellar vesicles (MLVs) were formed by rehydration of lipid film in HEPES buffered saline (100 mM HEPES, 50 mM NaCI, pH 7.0) followed by vigorous vortexing. MLVs composed of equimolar FAB/DPPC/cholesterol were diluted to 200 μΜ in buffered solution at various pH containing 10 mM acetate, 10 mM MES, 10 mM HEPES, 10 mM CAPS, 10 mM CHES and 100 mM of NaCI. Ammonium acetate was added to a final concentration of 10 mM to abolish any pH gradients in the liposomes.

Alternatively, DOFAB and PEG-c-DOMG lipid stocks were dissolved in chloroform. DOFAB and PEG-c-DOMG stock solutions were mixed at appropriate molar ratio, and solvent was removed by drying under a stream of nitrogen gas and subsequently by vacuum overnight. Multilamellar vesicles were formed by rehydration of lipid film in a poly-buffer solution containing 10mM ammonium acetate, 10mM HEPES, 10mM MES, and 130mM NaCI at pH 7.0 followed by vigorous vortexing. MLVs were diluted to 100 μΜ in buffered solution at various pH containing 10 mM ammonium acetate, 10 mM MES, 10 mM HEPES and 130 mM of NaCI.

Fluorescence was monitored using a Perkin Elmer LS-55 spectrofluorometer at excitation wavelength of 321 nm and emission wavelength at 445 nm. TNS was added into the cuvette at a final concentration of 2 μΜ (FAB/DPPC/cholesterol) or 1 μΜ (DOFAB/PEG-c-DOMG) just prior to the commencement of measurement. Fluorescence was measured for 1 minute (FAB/DPPC/cholesterol) or 30 seconds (DOFAB/PEG-c-DOMG), and the fluorescence intensity at 1 minute or 30 seconds, respectively, was used for calculation. Readings were subtracted by the reading obtained at the highest pH, which represents the baseline fluorescence. All readings were normalized to the reading obtained at the lowest pH. The final pH of the solutions was determined after each measurement. All measurements were done in replicates.

³¹ P NMR spectroscopy:

Lipid films were hydrated in the NMR tube with 100 mM HEPES,

50 mM NaCI (pH 7.0) to achieve a final concentration of 25 mM phospholipids. The samples were freeze-thawed 5 times with liquid nitrogen and vigorous vortexing. Samples were stored at -20 ^°C until data acquisition. Proton decoupled ³¹P NMR spectra were obtained using a Bruker AVII 400 spectrometer operating at 162MHz. Free induction decay (FID) corresponding to 1000 scans were obtained with a 15 s, 55° pulse with a 1 s interpulse delay and a spectral width of 64 kHz. An exponential multiplication corresponding to 50 Hz of line broadening was applied to the FID prior to Fourier transformation. The sample temperature was regulated using a Bruker BVT 3200 temperature unit.

RESULTS

The FAB headqroup exhibited two pKa values

The TNS assay was used to determine the pKa values of FAB lipids. In a first set of experiments, the TNS assay was conducted on MLVs consisting of equimolar mixture of a FAB lipid, DPPC and cholesterol. The FAB lipids tested were DLinFAB, DOFAB, and DPFAB. DPPC was used because it is a saturated lipid that does not readily adopt non-lamellar structures such as the H ii phase and is expected to maintain lamellar structure despite the presence of equimolar quantities of FAB lipids. Furthermore, the phosphate group in DPPC exhibits a pKa of approximately 2 (Boggs, 1987), which is considerably lower than that expected for the carboxyl group of FAB. Therefore, interference by the phosphate moiety in the TNS assay was expected to be minimal.

The titration curve of equimolar DLinFAB/DPPC/cholesterol liposomes against pH clearly showed 2 pKa values, one at 4.5 and the other at 7.9, presumably representing the carboxylic acid and amine moieties of FAB respectively (Figure 1 ). Similarly, the pKa values of DOFAB were observed to be 4.6 and 7.9, also presumably representing the carboxylic acid and amine moieties of FAB respectively (Figure 1 ). The TNS assay did not reveal any pKa values for DPFAB (data not shown).

The TNS fluorescence of different FAB liposomes was determined at different pH values. Since the quantum yield of TNS increases when it binds to membranes, comparing the TNS fluorescence of different formulations can provide an estimate of the relative affinity of TNS for each type of liposome. Table 1 shows the fluorescence intensity of 2 μΜ TNS with various FAB/DPPC/cholesterol (1 :1 :1 ) liposomes at different pH. It was expected that increasing the pH decreases the fluorescence of TNS, as it loses affinity to the liposomes when FAB is deprotonated. The TNS fluorescence of DOFAB and DLinFAB liposomes decreased from approximately 871 .5 to 27.8 units and 861 .1 to 50.3 units, respectively, with increasing pH (Table 1 ). However, the fluorescence intensity of DPFAB-containing liposomes was approximately 7 regardless of the pH and was much lower than that of the other two formulations. This suggests that the affinity of TNS was significantly higher for both DOFAB and DLinFAB-containing systems than for DPFAB-ontaining systems. The lower affinity of TNS for DPFAB may result from phase separation of the DPFAB in mixtures with DPPC or could arise if the lipid mixture is in a gel phase, which would limit the ability of TNS to partition into the lipid bilayer. TABLE 1

Fluorescence intensity of TNS in the presence of FAB/DPPC/cholesterol (1 :1 :1 ) at different pH. TNS was added to lipid mixture at various pH, and fluorescence was measured for a 1 -minute duration. F_max indicates the maximum fluorescence intensity, and F₆o represents the fluorescence intensity at the end of the measurement.

Sample pH Fmax Feo

3.78 7.7 7.4

DPFAB 6.76 8.9 8.8

10.12 8.7 8.5

3.77 871 .5 867.3

DOFAB 6.74 329.4 329.4

10.13 27.8 27.8

3.76 861 .1 858.4

DLinFAB 6.73 321 .0 321 .0

10.12 50.3 50.3

DOFAB was zwitterionic at physiological pH

The TNS assay was also used to determine the pKa values of DOFAB in MLVs consisting of mixture of DOFAB with 10% PEG-c-DOMG. PEG-c-DOMG was incorporated into the particle to prevent aggregation as pure DOFAB does not hydrate readily. Furthermore, PEG-c-DOMG does not contain any ionizable groups; therefore interference of the TNS assay was expected to be minimal. The titration curve of DOFAB/PEG-c-DOMG liposomes against pH showed the isoelectric point (pi) of the particle to be at pH 6.7 (Figure 2). The apparent pKa of the amino and carboxyl groups in DOFAB can be estimated by using the half-way point between the maximal fluorescence and the pi (carboxyl moiety) and between the pi and minimal fluorescence (amino moiety). The apparent pKa for the carboxyl and amino moieties were estimated to be 4.5 and 8.0, respectively. This suggests that DOFAB is zwitterionic and does not carry a net charge at physiological pH, an important requirement for increasing circulation lifetime.

FAB lipids exhibited strong bilayer destabilizing capabilities in mixtures with DOPC, POPC and other phospholipids

Pure DOPC multilamellar vesicles at pH 7.0 exhibited the characteristic ³¹ P NMR lineshape composed of a high-field peak and a low-field shoulder that is diagnostic for phospholipids in a lamellar organization (Figure 3). The addition of equimolar DPFAB to DOPC resulted in the appearance of a narrower component indicating isotropic motional averaging at higher temperatures (Figure 3); however, the bulk of the lipid remained in the lamellar organization. DOFAB, on the other hand, exhibited a strong bilayer- destabilizing capacity as the addition of equimolar DOFAB to DOPC resulted in the presence of a significant H_N component at 25 C and isotropic components at higher temperatures (Figure 3). Interestingly, the most unsaturated version of FAB, DLinFAB, was not the most potent bilayer-destabilizer, as equimolar mixture with DOPC remained in the lamellar phase at 25 C (Figure 3). A narrow lineshape indicating isotropic motional averaging began to appear at around 37^°C and completely replaced the lamellar phase at 50 C.

These results showed that DOFAB could act as a strong bilayer destabilizing agent capable of inducing non-lamellar structures when mixed with the bilayer-preferring lipid DOPC. It is unclear why the DLinFAB/DOPC system did not form Hn phase, even though DLinFAB lipid would be expected to be the most effective bilayer destabilizing agent due to the very unsaturated nature of its acyl chains. It is possible that the DLinFAB did not mix well with DOPC, resulting in reduced bilayer-destabiling capabilities.

The effects of FABs on the more saturated POPC lipid were also investigated (Figure 4). Addition of equimolar quantities of DPFAB did not induce detectable Η_Ν structure at any temperature tested, although significant isotropic signals consistent with the presence of non-lamellar phases such as the cubic phase were observed. Some non-lamellar H_N signals were detected in the mixture containing DOFAB at 37^°C and higher (Figure 4). Again, the behavior of the DLinFAB/POPC system was anomalous, as it remained lamellar up to 37^°C and only adopted the isotropic lineshape upon further heating to 50^°C (Figure 4). These results suggest possible mixing issues, as it is expected that the most unsaturated FAB lipids would exert the maximum bilayer- destabilizing effects.

The results of these experiments indicated that FAB lipids, such as DOFAB, can act as strong bilayer destabilizing agents in mixture with phosphatidylcholines such as DOPC and phosphatidylethanolamines such as DOPE. It was of interest to extend these observations to mixtures with other phospholipids such as, sphingomyelin (SM), phosphatidylserine (PS) phosphatidylglycerol (PG) and phosphatidic acid (PA). Based on the hypothesized reverse-headgroup mechanism of DOFAB, it is expected that FAB lipids, such as DOFAB, would be effective in inducing H_M phase formation with zwitterionic lipids such SM, while they would be ineffective against anionic phospholipids such as PS, PG and PA.

Therefore, in another set of experimets, the influence of DOFAB on the polymorphic phase properties of DOPC as compared to a variety of other phospholipids at 37°C was determined. ³¹P NMR spectra were obtained for pure DOPC (Figure 5, top) and equimolar mixture of DOFAB/DOPC (Figure 5, bottom) in HEPES buffered saline, pH 7.0. The influence of DOFAB on the polymorphic phase properties of various other phospholipids in equimolar mixtures was also examined. ³¹ P NMR spectra were obtained for DOFAB/milk sphingomyelin (Figure 6A), DOFAB/diPoPE (Figure 6B), DOFAB/DOPS (Figure 6C), DOFAB/DOPG (Figure 6D) and DOFAB/DOPA (Figure 6E). These studies demonstrated that DOFAB is a potent bilayer destabilizer when mixed with unsaturated phospholipids. As expected, DOFAB was an effective bilayer destailizing agent in mixtures with diPoPE, however in mixtures with sphingomyelin, which is a relatively saturated lipid, it was ineffective. Surprisingly, DOFAB was also capable in inducing non-lamellar structures in mixtures with all anionic phospholipids tested. The ability to disrupt anionic phospholipids indicates that DOFAB is an extremely potent bilayer-destabilizer.

FAB lipids exhibited stronger bilayer destabilizing effects than DOPE

DOPE is commonly used as a "helper" lipid in lipid nanoparticle carrier systems to aid in destabilizing cellular membranes. In order to put the bilayer-destabilizing abilities of the FAB lipids in context, the effect of DOPE on phase behaviour of DOPC and POPC was examined (Figure 7). Equimolar mixtures of DOPE/DOPC and DOPE/POPC remained lamellar at all temperatures tested, indicating that the DOPE was not able to destabilize bilayer structure for DOPC in our experimental conditions. Since the FAB lipids were able to induce non-lamellar structures in mixture with DOPC and POPC (Figures 2 and 3), they were much more potent bilayer-destabilizing agents than DOPE.

The bilayer destabilizing properties of FAB lipids were enhanced by the presence of cholesterol

It is well known that the presence of cholesterol in model membrane systems encourages the mixing of components that would otherwise undergo phase separation into gel and liquid crystalline regions (Silvius et ai, 1996). Furthermore, plasma membranes of target cells have equimolar levels of cholesterol with respect to phospholipid. It is also well-established that cholesterol plays a strong bilayer-destabilizing role in mixtures of lamellar and non-lamellar lipids. It was, therefore, of considerable interest to examine the phase behavior of equimolar FAB, DOPC and cholesterol mixtures.

As shown in Figure 8, cholesterol exerted a remarkable effect in these lipid mixtures. Formation of Η_Ν phase structure was observed in DOPC mixtures with DOFAB and DLinFAB at all temperatures. Even the mixture containing DPFAB exhibited detectable Hn phase formation at 37^°C and 50 C (Figure 8). These results indicated that the incorporation of cholesterol strongly promoted the formation of Hn phase in FAB/DOPC systems.

The incorporation of equimolar cholesterol in FAB/POPC mixtures yielded similar results (Figure 9). Equimolar mixtures of DPFAB/POPC/cholesterol remained in the lamellar phase at temperatures up to 37 C with some evidence of an isotropic component at 50 C. The DOFAB and DLinFAB-containing systems exhibited evidence of non-bilayer structure at 50 C, as indicated by components indicating H_N phase structure or structure giving rise to isotropic motional averaging. These results were consistent with the expectation that POPC would be more difficult to force into the H_N and other non-lamellar structures due to its more saturated nature as compared to DOPC.

FAB lipids exhibited strong bilayer destabilizing properties in mixtures with PE

The results presented to this point indicate that the FAB lipids can act as strong bilayer destabilizing agents in mixtures with phosphatidylcholines such as DOPC and POPC. It was of interest to extend these observations to mixtures with phosphatidylethanolamines (PEs) for two reasons. First, in contrast to PCs, both the phosphate group and the amino function of PEs are titratable and may interact differently with the FAB headgroup. Second, while the PEs found in biological membranes are unsaturated and preferentially adopt H ii phase in isolation (Cullis and de Kruijff, 1978), an ability to combine with endogenous PEs to further promote non-bilayer organization would be expected to be of benefit from an intracellular delivery point of view.

In order to investigate the ability of FAB lipids to destabilize bilayer structure in mixtures with PE, a species of PE that adopts lamellar phase organization initially was used, namely 1 ,2-dipalmitoleoyl-sn-glycero-3- phosphoethanolamine (diPoPE), which underwent a bilayer-to-H_N transition between 30 C and 40 C (Figure 10A). The appearance of H_N phase in DPFAB and DLinFAB mixture at as low as 25°C was observed, suggesting a strong bilayer-destabilizing capacity of the FAB lipids for PE species (Figure 10B).

DISCUSSION

This example demonstrates that lipids with "reverse headgroup" charge distributions have potent bilayer destabilizing characteristics when mixed with phosphatidylcholines and phosphatidylethanolamines and that cholesterol further enhances this bilayer destabilizing capacity. The mechanism whereby DOFAB induces non-lamellar structures is of interest.

It is well-established that charge neutralization of anionic lipids by cationic species can promote non-lamellar phase formulation by dehydration of water interface and reduction in the effective headgroup area caused by reduced inter-headgroup electrostatic repulsion (Boggs et al. 1987). For example, the addition of Ca²⁺ to cardiolipin MLVs results in a transition from the bilayer organization to the H_N phase, an effect that has been attributed to formation of Ca²⁺-cardiolipin pairs that exhibit a "cone" shape compatible with H ii phase formation (Cullis et al. 1978). Similarly, negatively charged lipids such as phosphatidylserine (PS), phosphatidylglycerol (PG) and phosphatidylinositol (PI) all adopt bilayer structure on hydration and are all converted into the Η_Ν organization in the presence of cationic lipids such as 3β- [N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC- Chol), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1 ,2- dioleoyloxy-3-(trimethylammonio) propane (DOTAP) (Hafez et al. 2001 ). Again, these effects are attributed to the formation of net neutral ion pairs between the anionic and cationic lipids leading to complexes that exhibit a reduced headgroup area and increased cone shape, thus engendering H_N phase formation.

The mechanism whereby FAB lipids induce non-lamellar structure in mixtures with zwitterionic phospholipids such PC and PE is likely due to similar effects. It has been well-established that dehydration of the polar headgroup of PCs results in enhanced Η_Ν phase formation (Luzzati et al. 1968; Cullis et al. 1986). Such dehydration has also been associated with changes in the headgroup orientation (Hsieh et al. 1997). Dehydration and reduced inter- headgroup electrostatic repulsion effects resulting from charge neutralization between DOFAB and PC/PE leads to a reduction in the effective headgroup area of the FAB-phospholipid complex. It is, therefore, possible that FAB lipids encourage non-lamellar structure by associating directly via ionic interactions with PC or PE headgroups to reduce headgroup hydration. Alternatively, it may be that the presence of FAB lipids directly affects PC and PE headgroup orientation. Finally, it is possible that the unsaturated FAB lipids could act solely via their influence in the acyl chain region, acting to increase the effective cross-sectional area. In the case of sphingomyelin, the apparent failure to adopt the Η_Ν phase is likely to be caused by highly saturated acyl chains found in milk sphingomyelin, which effectively protects it against any disruption from the bilayer conformation despite charge neutralization in the headgroup.

Based on the proposed model of "reverse headgroup" mechanism (Figure 14), it is interesting that DOFAB can induce non-lamellar structures in anionic phospholipids such as DOPS, DOPG and DOPA.

BIOLOGICAL EXAMPLE 2

The ability of lipid nanoparticles comprising reverse headgroup lipids to deliver siRNA to cells and mediate gene knockdown was determined. Specifically, knockdown of the androgen receptor (AR) in LNCap cells with DOFAB-encapsulated siRNA was measured.

MATERIALS AND METHODS siRNA:

The siRNA targeting AR comprised two strands: 5^'- AGCACUGCUACUCUUCAGCAUdTdT-3^' (AR sense) and 5^'- AUGCUGAAGAGUAGCAGUGCdTdT-3^' (AR anti-sense). This 23-mer AR siRNA was purchased from Thermo Scientific (Dharmacon). Negative control sequence was purchased from Invitrogen, Stealth™ RNAi Negative Control Duplexes, Lo GC duplexs (Cat. No. 12935 - 200). siRNA Encapsulation into Lipid Nanoparticles (LNPs):

All lipid stock solutions were dissolved in ethanol and were mixed at appropriate molar ratios (e.g., 85 mol% DOFAB, 4.8 mol% cholesterol, 10% PEG-s-DMG, 0.2 mol% SPDiO). The lipid/ethanol mixtures were added dropwise into a vortexing buffer solution containing 150 mM acetate at pH 4.0. The resulting multilamellar vesicles (MLVs) were extruded 5 times through two Nuclepore polycarbonate filters with a pore size of 80nm at -400 psi. siRNA was hydrated in citrate buffer (10 mM citrate, 30 mM NaCI, pH 6.0) and quantified by measuring absorbance at 260 nm.

siRNA solution was added into the lipid mixture to make a total lipid:siRNA ratio of 10:1 and incubated at room temperature for 30 minutes. The resulting vesicles were dialyzed with citrate buffer (50mM citrate, pH 4.0) for 4 hours and then overnight in PBS (pH 7.4). The mean diameter of the vesicles was determined by dynamic light scattering using a NICOMP 370 particle sizer (Nicomp Particle Sizing Inc., Santa Barbara, CA) and was measured to be 403.3 ± 267.8 nm. Lipid concentrations were determined by measuring cholesterol content using the Cholesterol E Enzymatic Assay Kit (Wako Chemicals USA, Richmond, VA). siRNA content was verified by measuring absorbance at 260 nm in 70% to lyze the vesicles.

Gene Knockdown:

LNCap cells were seeded in 12-well plates (-2.0 x 10⁵ cells/well) with RPMI media supplemented with 5% FBS overnight prior to siRNA treatment. Cells were treated continuously for either 24 or 48 hours with DOFAB/cholesterol/PEG-s-DMG/SPDiO (90:4.8:5:0.2 mol%) LNPs comprising siRNA against the androgen receptor (siAR) or low-GC content control siRNA (loGC) was encapsulated with and protein expression was analyzed with immunoblotting.

Cells were lysed in detergent buffer (1 % NP-40 and 0.5% deoxycholic acid) supplemented with protease inhibitor (Roche Diagnostics). Total protein was quantified by Bradford Assay and was analyzed by immunoblotting. Antibodies to the androgen receptor were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to β-actin were purchased from Invitrogen (Eugene, OR). Antigen-antibody complexes were detected using Millipore Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA).

Confocal Microscopy:

LNCap cells were plated in 12-well plates (-2.0 x 10⁵ cells/well) with a L-lysine coated coverslip (BD Biosciences, Mississauga, ON) and treated with 10 g/mL of encapsulated siRNA for 24 and 48 hours. Cells were fixed with 3% parformaldehyde in the prescence of Hoescht for 15 minutes. Coverslips were rinsed with PBS, followed by water and were mounted onto slides with SlowFade (Invitrogen, Eugene, OR). Slides were imaged and analyzed with confocal microscopy (Olympus FV1000). Fluorochromes were excited with lasers operating at 488 nm (DiO), and 350 nm (Hoescht). Images were collected with a 60 x oil immersion objective lens.

RESULTS

DOFAB encapsulated siRNA was able to transfect LNCap cells and mediate the knockdown of the androgen receptor.

DOFAB acts as an effective transfection reagent in lipoplexes containing siRNA

The structure of DOFAB and the pKa results described in Biological Example 1 suggested that DOFAB exhibited a positive charge at lower pH values {e.g., pH values below 7) and becomes progressively net neutral and then negatively charged at higher pH values. At lower pH values, DOFAB should therefore behave as a cationic lipid. Cationic lipids form complexes characterized as "lipoplexes" with RNA and DNA-based macromolecules, and can act as effective transfection agents to introduce these macromolecules into cells. In order to test whether DOFAB could induce formation of lipoplexes and act as a transfection reagent, lipoplexes formed of DOFAB with siRNA against the androgen receptor or control siRNA (loGC) were expressed in LNCaP prostate cancer cells. The formulation comprised DOFAB/cholesterol/PEG-s-DMG/SPDiO (90:4.8:5:0.2 mol%) and was approximately 400 nm in diameter. Cells were treated continuously with the DOFAB lipoplex for both 24 and 48 hours. Immunoblots were used to determine the expression of the androgen receptor and actin (control).

Cells without treatment or treated with loGC showed normal expression of AR. Treatment with DOFAB-encapsulated siAR led to a dose dependent reduction in AR expression, as shown in Figure 1 1 . This became apparent at a siRNA dose of 10 g/mL after 24 hours incubation (Figure 1 1 ). After 48 hours, reduction of AR expression was observed at as low as 2 g/mL of siRNA. These results illustrate DOFAB is an effective transfection agent for siRNA. DOFAB lipoplexes were internalized via endocytic pathways

The proposed mechanism of action for "reverse headgroup" lipids suggests the delivery of siRNA payload directly into the cytoplasm through direct fusion with the plasma membrane. It was of interest to examine the delivery mechanism of DOFAB lipoplex. DOFAB lipoplex was labeled with SPDiO, and distribution of the lipid was examined with confocal microscopy. It was expected that direct plasma membrane fusion would distribute the SPDiO fluorescence around the plasma membrane, while entry via the endocytic pathway would lead to punctate fluorescence caused by the entrapment of the lipoplex in endocytic vesicles. Cellular uptake of DOFAB lipoplex was examined by confocal microscopy. LNCap cells were treated with 10 g/mL of AR siRNA encapsulated with DOFAB/cholesterol/PEG-s-DMG/SPDiO (90:4.8:5:0.2 mol%) for 24 and 48 hours. Nuclei were stained blue with Hoescht's, and SPDiO fluorescence was observed in green. Figure 12 shows that the cellular uptake of DOFAB lipoplex was mainly through the endocytic pathway, as suggested by the punctate fluorescence pattern from SPDiO. However, several cells had green fluorescence around the plasma membrane at the 24 hours time-point (data not shown). Therefore, uptake via plasma membrane fusion could not be completely ruled out.

DISCUSSION

These experiments demonstrate that the "reverse headgroup" lipids, such as DOFAB, can be used to deliver siRNA to cells for mediating gene knockdown. The utility of LNP systems containing DOFAB and other "reverse headgroup" lipids for delivery of macromolecules such as siRNA warrants further discussion.

The potential utility of reverse headgroup lipids such as FAB lipids for delivery of macromolecules such as siRNA stems from three factors. First, in order for LN systems to exhibit the long circulation lifetimes required to reach tissues other than the liver, they should be small (i.e., diameter < 100 nm) and should have neutral or near-neutral surface charge. As a result, permanently charged cationic lipids commonly used for transfection in vitro are not suitable for systemic use, as they are rapidly cleared from the circulation and also give rise to serious toxic side effects. This problem was addressed in the past by using ionizable cationic lipids that are positively charged at low pH values (e.g., pH 4), thereby allowing anionic polymers such as siRNA to be loaded into the LN, but relatively uncharged at physiological pH values. The FAB lipids of the present invention would be expected to have similar properties but with one important advantage. At pH values at or below the pKa of the carboxyl function (pKa~4.5), the FAB lipids exhibit a net positive charge and should therefore be expected to act as efficient agents for loading oligonucleotides in lipid nanoparticles (LN). At physiological pH values, the LNs exhibit net neutral surface charge and, hence, should exhibit long circulation lifetimes.

Another important advantage could be that, in distinction to ionizable cationic lipids, it should be possible to construct LN containing siRNA where the only lipid components are the FAB lipid, cholesterol, and a limited amount of PEG-lipid for stability. The maximum amount of bilayer-destabilizing cationic lipid that can currently be associated with LN systems is in the range of 50 mol% due to stability issues. Furthermore, at least 10% of a bilayer- stabilizing lipid such as DSPC must be incorporated to maintain LN stability. LN systems containing FAB could contain substantially more of the bilayer- destabilizing lipid without compromising LN stability or circulation lifetimes, with the additional feature that it could act on PC and PE lipid species which are the majority lipids in target membranes.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure. REFERENCES

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Claims

1. A zwitterionic lipid comprising a headgroup wherein the positive charge is located near the acyl chain region and the negative charge is located at the distal end of the headgroup.

2. The zwitterionic lipid of claim 1 , wherein said lipid has the following structure (A :

(A)

or is a pharmaceutically acceptable salt thereof, wherein:

R¹ and R² are either the same or different and are each independently, C6-C32alkyl;

R³ is either hydrogen or Ci-C₆alkyl; and

n and m are independently either 1 , 2, 3, or 4.

3. The zwitterionic lipid of claim 2, wherein the headgroup comprises 4-amino butyric acid (FAB).

4. The zwitterionic lipid of claim 2, wherein the lipid has the following structure (I):

(I)

wherein Ri and R2 are each, independently, C₆-C32alkyl.

5. The zwitterionic lipid of claim 4, wherein the lipid has one of the followin structures:

6. A lipid particle comprising a lipid of any one of claims 1 -5.

7. The lipid particle of claim 6, further comprising cholesterol.

8. The lipid particle of claim 6 or claim 7, further comprising a

PEG-lipid.

9. The lipid particle of any one of claim 6-8, wherein said lipid particle further comprises an encapsulated agent.

10. The lipid particle of claim 9, wherein the agent is a therapeutic agent.

1 1 . The lipid particle of claim 10, wherein the therapeutic agent is an interfering RNA.

12. The lipid particle of claim 1 1 , wherein the interfering RNA is a siRNA.

13. A method for delivering an agent to a cell comprising contacting a cell with the lipid particle of any one of claims 9-12.

14. The method of claim 13, wherein said contacting occurs in vitro.

15. The method of claim 13, wherein said contacting occurs in vivo.

16. The method of any one of claims 13-15, wherein said cell is a mammalian cell.

17. The method of claim 16, wherein said cell is a human cell.

18. A method of treating or preventing a disease or disorder in a subject, comprising providing to the subject the lipid particle of any one of claims 10-12.

The method of claim 18, wherein said subject is a mammal

The method of claim 19, wherein said subject is a human.

21 . The method of any one of claims 18-20, wherein the therapeutic agent is a nucleic acid.

22. The mehod of claim 21 , wherein the nucleic acid is an interfering RNA.

23. The method of claim 22, wherein the interfering RNA is an siRNA.