Particles, compositions and methods

Abstract

The present disclosure relates generally to nucleic acid-lipid particles, in particular functionalized nucleic acid-lipid particles, methods for producing them, to aqueous dispersions suitable for forming the nucleic acid-lipid particles and methods for producing them, and to pharmaceutical compositions containing them and their uses in medicine.

Description

[0001] PARTICLES, COMPOSITIONS AND METHODS

[0002] Technical Field

[0003] The present disclosure relates generally to nucleic acid-lipid particles, in particular functionalized nucleic acid-lipid particles, methods for producing them, to aqueous dispersions suitable for forming the nucleic acid-lipid particles and methods for producing them, and to pharmaceutical compositions containing them and their uses in medicine.

[0004] Background to the Invention

[0005] The traditional manufacturing route for the preparation of nucleic acid-containing lipid nanoparticles proceeds by a one-step process of mixing in one-part nucleic acid (such as RNA) in an aqueous buffer with three-part lipid mixture dissolved in an organic solvent. Although much success has been demonstrated with this route, the established process typically suffers from the complexity of the manufacturing process, such as requiring subsequent tangential flow filtration (TFF) steps to remove the organic solvents used in the dissolution of the lipids. This further processing increases the process’s complexity and eventually increases the manufacturing lead time and the associated costs. Furthermore, this approach is typically unfavourable for individualized patient therapy since a large number of batches needs to be manufactured with a very short turn-around time. The use of organic solvents in manufacturing RNA lipid nanoparticle formulations, where longer processing times are required, could also be a demerit when working with compounds that have limited chemical stability.

[0006] WO 2020 / 070040 relates to RNA particles for delivery of RNA to target tissues after administration, the particles comprising a lipid-polysarcosine conjugate. However, this document does not disclose a targeting compound of formula (A) as defined herein wherein the binding moiety B is a peptide or protein.

[0007] WO 2022 / 101486 describes pharmaceutical compositions comprising particles, in particular lipid nanoparticles (LNPs), and mRNA, and methods for preparing and storing them. In particular, the lipid-polymer conjugate C16-PEG2000-ceramide and formulations containing it are described. However, this document also does not disclose compositions containing a targeting compound of formula (A) as defined herein wherein the binding moiety B is a peptide or protein.

[0008] WO 2012 / 170952 describes compounds having the structure (targeting rnolecule)m- linker-(targeting moleculejn, wherein the targeting molecule is a retinoid or a fat soluble vitamin having a specific receptor on the target cell. However, this document also does not disclose compositions containing a targeting compound of formula (A) as defined herein wherein the binding moiety B is a peptide or protein.

[0009] WO 2022 / 058298 describes agents and methods for targeted delivery of payloads, such as therapeutic or diagnostic agents to target cells. The agents comprise RNA encoding a peptide or polypeptide (docking compound) comprising a binding moiety (primary targeting moiety) binding to target cells and a further binding moiety (secondary target) binding to an agent that comprises a payload (effector probe). Following expression of the RNA, the primary targeting moiety may bind to a target antigen such as a cancer antigen on cancer cells and then a secondary targeting moiety comprised in the effector probe may target the secondary target to thereby precisely deliver the payload to the target cells such as cancer cells. However, this document also does not disclose compositions containing a targeting compound of formula (A) as defined herein wherein the moiety L is hydrophobic moiety, such as a lipid, and the binding moiety B is a peptide or protein.

[0010] K. Naoya et al., Eur. J. Pharm. Sci., 2022, 176, 106239, describes a lipid-polymer conjugate of formula Cyanine 5.5-PEG2000-DSPE and formulations containing it. However, this document also does not disclose a targeting compound of formula (A) as defined herein wherein the binding moiety B is a peptide or protein.

[0011] WO 2023 / 148276 and WO 2023 / 148277 both describe functionalized nucleic acid- lipid particles and methods for their formation. In both publications, the nucleic acid- lipid particles are formed by the one-step process generally described above. Neither of these publications discloses that the aqueous dispersion formed as the first step of the two-step procedure defined herein is substantially free of organic solvents as defined herein.

[0012] WO 2018 / 089801, WO 2021 / 155274 and WO 2022 / 032087 all describe methods of producing empty lipid nanoparticle (LNP) formulations and loaded LNP formulations including nucleic acids. However, while all three documents indicate in very general terms that the lipids in the nucleic acid-lipid particle may be functionalized, neither document describes either that the aqueous dispersion containing the dispersed phase comprising the lipid mixture may contain a compound of formula (A), described below, such that the nucleic acid-lipid particle formed by the methods described herein may comprise a compound of formula (A), or that such a nucleic acid-lipid particle may be subsequently functionalized by interacting a compound of formula (I), defined below, with the compound of formula (A), defined below, to provide, for example, cell-specific targeting functionalization of the nucleic acid-lipid particle.

[0013] Moreover, whilst the methods to form the aqueous dispersion as described in all of the above three documents allows for ethanol, citrate buffer, and other destabilizing agents to be absent during the addition of mRNA, the formation of the empty lipid nanoparticles in all of these documents uses citrate buffer. The inorganic ions present in citrate buffers are thought to destabilize the colloidal properties of the lipid nanoparticle formulation, and are therefore detrimental to formulation stability.

[0014] Summary of the Invention

[0015] In a first aspect of the invention, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0016] P is absent or comprises a polymer;

[0017] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0018] B comprises a binding moiety attached to L when P is absent or to a second end of the polymer P when present;

[0019] XI is absent or a first linking moiety; and

[0020] X2 is absent or a second linking moiety, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0021] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0022] P is absent or comprises a polymer;

[0023] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0024] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0025] XI is absent or a first linking moiety; and

[0026] X2 is absent or a second linking moiety; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0027] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0028] P is absent or comprises a polymer;

[0029] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0030] B comprises a moiety capable of binding to a cell surface antigen, said moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0031] XI is absent or a first linking moiety; and

[0032] X2 is absent or a second linking moiety; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0033] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0034] L-X1-P-X2-B (A) wherein:

[0035] P is absent or comprises a polymer;

[0036] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0037] B comprises a moiety capable of binding to a peptide tag, said moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0038] XI is absent or a first linking moiety; and

[0039] X2 is absent or a second linking moiety; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0040] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0041] L-X1-P-X2-B (A) wherein:

[0042] P is absent or comprises a polymer;

[0043] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0044] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0045] XI is absent or a first linking moiety; and

[0046] X2 is absent or a second linking moiety, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion; wherein the aqueous dispersion is substantially free of organic solvents.

[0047] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0048] L-X1-P-X2-B (A) wherein:

[0049] P is absent or comprises a polymer;

[0050] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0051] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0052] XI is absent or a first linking moiety; and

[0053] X2 is absent or a second linking moiety, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion; wherein (i) the aqueous dispersion is substantially free of nucleic acids, (ii) the aqueous dispersion is substantially free of inorganic cations, and / or (iii) the aqueous dispersion contains a cryoprotectant.

[0054] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase comprising an anion of an aqueous acid; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0055] L-X1-P-X2-B (A) wherein:

[0056] P is absent or comprises a polymer; L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0057] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0058] XI is absent or a first linking moiety; and X2 is absent or a second linking moiety, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0059] The compound of formula (A) is also referred to herein as a “targeting compound”. Typically, the hydrophobic moiety (L) of the compound of formula (A) is incorporated into the particle, such that the binding moiety (B) of the compound of formula (A) is then oriented on the particle surface. The particle may be already functionalized where B is a moiety binding to a cell surface antigen on target cells. Otherwise, functionalization of the particle is possible, for example by interacting a compound of formula (I), as defined herein, (also referred to herein as a “docking compound”) with the binding moiety (B) (e.g., a peptide tag or a moiety binding to a peptide tag) of the compound of formula (A), wherein the compound of formula (I) comprises a moiety B” binding to a cell surface antigen on target cells.

[0060] Therefore, in one embodiment of the above aspects, the binding moiety B is a moiety capable of binding to a cell surface antigen.

[0061] In another embodiment of the above aspects, the binding moiety B is a peptide tag.

[0062] In another embodiment of the above aspects, the binding moiety B is a moiety capable of binding to a peptide tag.

[0063] In a second aspect of the invention, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0064] P is absent or comprises a polymer;

[0065] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0066] B comprises a binding moiety attached to L when P is absent or to a second end of the polymer P when present;

[0067] XI is absent or a first linking moiety; and

[0068] X2 is absent or a second linking moiety.

[0069] In one embodiment of this aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0070] L-X1-P-X2-B (A) wherein:

[0071] P is absent or comprises a polymer;

[0072] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0073] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0074] XI is absent or a first linking moiety; and

[0075] X2 is absent or a second linking moiety.

[0076] In another embodiment of the above aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A):

[0077] L-X1-P-X2-B (A) wherein:

[0078] P is absent or comprises a polymer;

[0079] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0080] B comprises a moiety capable of binding to a cell surface antigen, said moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0081] XI is absent or a first linking moiety; and X2 is absent or a second linking moiety.

[0082] In another embodiment of the above aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0083] P is absent or comprises a polymer;

[0084] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0085] B comprises a moiety capable of binding to a peptide tag, said moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0086] XI is absent or a first linking moiety; and

[0087] X2 is absent or a second linking moiety.

[0088] In another embodiment of the above aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0089] P is absent or comprises a polymer;

[0090] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0091] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0092] XI is absent or a first linking moiety; and

[0093] X2 is absent or a second linking moiety; wherein the aqueous dispersion is substantially free of organic solvents.

[0094] In another embodiment of the above aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0095] P is absent or comprises a polymer;

[0096] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0097] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0098] XI is absent or a first linking moiety; and

[0099] X2 is absent or a second linking moiety; wherein (i) the aqueous dispersion is substantially free of nucleic acids, (ii) the aqueous dispersion is substantially free of inorganic cations, and / or (iii) the aqueous dispersion contains a cryoprotectant.

[0100] In another embodiment of the above aspect, there is provided an aqueous dispersion comprising: an aqueous mobile phase comprising an anion of an aqueous acid; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0101] P is absent or comprises a polymer;

[0102] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0103] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0104] XI is absent or a first linking moiety; and

[0105] X2 is absent or a second linking moiety. In a third aspect of the invention, there is provided a method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion according to the method of the first aspect; and ii) mixing the aqueous dispersion with an aqueous solution comprising a nucleic acid, to produce the nucleic acid-lipid particle.

[0106] In a fourth aspect of the invention, there is provided a method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid, by mixing the lipid mixture and the aqueous phase; and ii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid, to produce an intermediate nucleic acid-lipid particle; and iii) mixing the intermediate nucleic acid-lipid particle with an aqueous solution comprising a compound of Formula (A) as defined in the first or second aspect, to produce the nucleic acid-lipid particle.

[0107] In a fifth aspect of the invention, there is provided a lipid-nucleic acid particle, obtained or obtainable by the method of the third or fourth aspect.

[0108] In a sixth aspect of the invention, there is provided a method of forming a functionalized nucleic acid-lipid particle comprising a compound of formula (I): B’-X3-B” (I) wherein:

[0109] B’ comprises a moiety binding to B, wherein B comprises a peptide tag or a moiety binding to a peptide tag;

[0110] X3 is absent or a linking moiety; and

[0111] B” comprises a moiety binding to a cell surface antigen, the method comprising:

[0112] (a) forming a nucleic acid-lipid particle according to the method of the third or fourth aspect, and (b) mixing the nucleic acid-lipid particle with the compound of formula (I), such that the compound of formula (I) interacts with the nucleic acid-lipid particle.

[0113] In one embodiment of this aspect, there is provided a method of forming a functionalized nucleic acid-lipid particle comprising: a compound of Formula (A) as defined herein, wherein B comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I):

[0114] B’-X3-B” (I) wherein B’ comprises a moiety binding to B;

[0115] X3 is absent or a linking moiety; and

[0116] B” comprises a moiety binding to a cell surface antigen, the method comprising:

[0117] (a) forming a nucleic acid-lipid particle according to the method of the third or fourth aspect; and

[0118] (b) mixing the nucleic acid-lipid particle with the compound of formula (I), such that the compound of formula (I) interacts with the nucleic acid-lipid particle.

[0119] Typically, mixing the nucleic acid-lipid particle with the compound of formula (I) results in a functionalized nucleic acid-lipid particle, where B’ of the compound of formula (I) interacts with B of the compound of formula (A), such that the compound of formula (I) is then bound on the particle surface. Typically, B” of the compound of formula (I) can then provide targeting, e.g., to specific cell types expressing the cell surface antigen to which B” binds.

[0120] In a seventh aspect of the invention, there is provided a functionalized nucleic acid- lipid particle comprising a compound of formula (I):

[0121] B’-X3-B” (I) wherein:

[0122] B’ comprises a moiety binding to B, wherein B comprises a peptide tag or a moiety binding to a peptide tag;

[0123] X3 is absent or a linking moiety; and

[0124] B” comprises a moiety binding to a cell surface antigen. In one embodiment of this aspect, there is provided a functionalized nucleic acid-lipid particle comprising: a compound of Formula (A) as defined herein, wherein B comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I):

[0125] B’-X3-B” (I) wherein B’ comprises a moiety binding to B;

[0126] X3 is absent or a linking moiety; and

[0127] B” comprises a moiety binding to a cell surface antigen.

[0128] In an eighth aspect of the invention, there is provided a pharmaceutical composition comprising a functionalized nucleic acid-lipid particle according to the seventh aspect and a pharmaceutically acceptable carrier.

[0129] In a ninth aspect of the invention, there is provided a functionalized nucleic acid-lipid particle according to the seventh aspect for use as a medicament.

[0130] In a tenth aspect of the invention, there is provided a functionalized nucleic acid-lipid particle according to the seventh aspect for use in treating a disease involving an antigen.

[0131] In an eleventh aspect of the invention, there is provided a functionalized nucleic acid- lipid particle according to the seventh aspect for use in treating a disease characterized by the presence of diseased cells expressing an antigen.

[0132] In a twelfth aspect of the invention, there is provided a functionalized nucleic acid- lipid particle according to the seventh aspect for use in treating cancer.

[0133] In a thirteenth aspect of the invention, there is provided a lyophilized (freeze-dried) functionalized nucleic acid-lipid particle according to the seventh aspect. Advantages and Surprising Findings

[0134] The present inventors have surprisingly identified methods for preparing stable preformed (i.e., empty) lipid nanoparticles comprising a compound of formula (A) (i.e., a “targeting compound”), and that such particles can be subsequently loaded with nucleic acid to form stable nucleic acid-lipid particles comprising a targeting compound. These nucleic acid-lipid particles comprising a targeting compound can then also be functionalized with a compound of formula (I) (i.e., a “docking compound”) to form functionalized nucleic acid-lipid particles.

[0135] Surprisingly, the resultant functionalized nucleic acid-lipid particles are found to be more stable than those produced by the one-step method of the prior art. The functionalized particles are able to remain intact after multiple freeze-thaw cycles. In particular, the functionalized particles produced by the methods of the invention show markedly improved stability at 2-8°C and 25°C, as compared to particles produced by the one-step methods described in the prior art. This confers the potential for the functionalized nucleic acid-lipid particles being suitable for incorporation into more stable pharmaceutical compositions, which can advantageously be stored at higher temperatures. Furthermore, the present inventors identified that the functionalized nucleic acid-lipid particles produced by the two-step methods described herein (i.e., via a pre-formed lipid particle intermediate) provide improved therapeutic efficacy, for example for targeted delivery of both RNA and DNA, as compared to corresponding functionalized nucleic acid-lipid particles produced by the one-step LNP manufacturing methods of the prior art. Thus, the present inventors have surprisingly identified particular advantages of the two-step nucleic acid-lipid particle manufacturing methods, which confer advantages specifically for functionalized particles.

[0136] It has also surprisingly been found by the present inventors that the method described herein enables preparation of the aqueous dispersion composition that does not contain organic solvents (lacking the nucleic acid) and hence, the nucleic acid-lipid particle can advantageously be prepared without using organic solvents. This avoids both the risk of the organic solvents degrading chemically unstable lipids and the need for complex purification processes to remove the organic solvents. One large batch of pre-formed aqueous dispersion can be used for manufacturing several batches of nucleic acid-lipid particle (e.g., patient-specific mRNA lipid nanoparticle) formulations and could be particularly interesting in areas such as individualized immunotherapy platforms where small-scale batches of the final products are required.

[0137] Furthermore, the methods disclosed herein provide superior manufacturing advantages with compatibility in a Class D manufacturing environment. In addition, the nucleic acid-lipid particles formed with this new process have been demonstrated to have improved colloidal stability, lipid stability, and RNA integrity in both frozen and liquid conditions when compared to other classical lipid nanoparticle manufacturing routes while maintaining biological efficacy. Furthermore, the methods disclosed herein allow much flexibility to alter the properties of the formulations such as particle size, surface charge and functionalization, without affecting the process’s robustness.

[0138] In addition, in further contrast to the methods described in WO 2018 / 089801, WO 2021 / 155274 and WO 2022 / 032087, the methods described herein, in preferred instances, avoid the use of the inorganic ions present in citrate and acetate buffers and therefore avoiding the detrimental effects of the inorganic ions on the nucleic acid- lipid particle formulation. Specifically, in some aspects, the use of malic acid is thought to further improve the maintenance of colloidal stability and / or RNA integrity, for example during RNA-lipid particle formation and subsequent storage. The addition of a cryoprotectant such as sucrose to the aqueous dispersion enhances long-term stability of the lipid particles and facilitates storage in frozen conditions.

[0139] Brief Description of the Figures

[0140] In the descriptions which follow, the term “LNP1” denotes nucleic acid-lipid particles prepared by the one-step method of the prior art, and the term “LNP2” denotes nucleic acid-lipid particles prepared by the two-step method of the present invention.

[0141] Figure 1: Characterization of functionalized RNA-lipid particles prepared by aqueous-aqueous protocol (LNP2). (A) shows the stability of the aqueous dispersion pre-formed lipid (DODMA / PSar-Ac / Alfa-Tag lipid) nanoparticles according to the invention, containing Alfa-tag lipid, when stored in liquid (4°C and 25°C) conditions, in terms of particle size (a) and polydispersity index (b); (B) shows the particle size (a) and polydispersity index (b) analysis of functionalized RNA-lipid particles according to the invention subjected to two freeze thaw (FT) cycles from -20°C to room temperature and from -80°C to room temperature; (C) shows the particle size and polydispersity index (PDI) of raw RNA-lipid (HY-501 / DSPC) particles according to the invention, alfa-tagged RNA-lipid particles with a post-insertion approach and functionalized RNA-lipid particles.

[0142] Figure 2: Particle size and PDI of functionalized RNA-lipid (DODMA / pSar- Ac / DSPE-PEG2k-Alfa) particles, prepared by aqueous-aqueous protocol) and subjected to at least two freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0143] Figure 3: Particle size and PDI of pre-formed LNPs with BNT51 and Ac-AEEA14- DMA subjected to at least three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0144] Figure 4: Characterization of BNT51 functionalized lipid nanoparticles prepared with Ac-AEEA14-DMA as stealth moiety and DSPE-PEG2k-Alfa lipid. (A) Particle size and PDI of functionalized LNP1 and LNP2. (B) Agarose Gel Electrophoresis of controls, untreated functionalized LNP1 and LNP2 (upper row) and functionalized LNP1 and LNP2 treated with release solution (lower row). (C) Particle size and PDI of functionalized LNP1 and LNP2 subjected to at least two freeze thaw cycles from - 20°C to room temperature and from -80°C to room temperature, and after 2 weeks at 2-8°C and 25°C.

[0145] Figure 5: Percentages of transfected cells (CD 14+ Monocytes, CD 19+ B cells, CD4+ T cells or CD8+ T cells) within all transfected PBMCs for both RNA (Thy 1.1) (A) and DNA (Venus) (B), delivered using LNP1 or LNP2.

[0146] Figure 6: Particle size and PDI of preformed LNPs with BNT51 and Ac-AEEA14-a- tocopherol subjected to at least three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0147] Figure 7: Characterization of BNT51 functionalized lipid nanoparticles prepared with Ac-AEEA14-a-tocopherol as stealth moiety and DSPE-PEG2k-Alfa lipid. (A) Particle size and PDI of functionalized LNP1 and LNP2. (B) Agarose Gel Electrophoresis of controls, untreated functionalized LNP1 and LNP2 (upper row) and functionalized LNP1 and LNP2 treated with release solution (lower row). (C) Particle size and PDI of functionalized LNP1 and LNP2, subjected to at least two freeze thaw cycles from - 20°C to room temperature and from -80°C to room temperature, and after 2 weeks at 2-8°C and 25°C.

[0148] Figure 8: Percentages of transfected cell (CD 14+ Monocytes, CD 19+ B cells, CD4+ T cells or CD8+ T cells) within all transfected PBMCs for both RNA (Thy 1.1) (A) and DNA (Venus) (B), delivered using LNP1 or LNP2.

[0149] Figure 9: Characterization of BNT51 functionalized lipid nanoparticles prepared with Ac-AEEA14-DMA as stealth moiety and DSPE-pAEEA14-Alfa lipid. (A) Particle size and PDI of preformed LNPs subjected to at least two freeze thaw cycles from - 20°C to room temperature and from -80°C to room temperature. (B) Particle size and PDI of functionalized RNA / DNA-LNP2 at tO and subjected to at least two freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0150] Figure 10: Characterization of BNT52 functionalized lipid nanoparticles prepared with Ac-AEEA14-DMA as stealth moiety and DSPE-pAEEA14-Alfa lipid. (A) Particle size and PDI of preformed LNPs subjected to at least two freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature. (B) Particle size and PDI of functionalized RNA / DNA-LNP2 at tO and subjected to at least two freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature.

[0151] Figure 11: Particle size and PDI of preformed LNPs prepared with HY501 / PEG2k- Cer and subjected to at least three freeze thaw cycles from -20°C to room temperature or from -80°C to room temperature.

[0152] Figure 12: (A) Particle size and PDI of functionalized RNA-lipid particles, prepared with HY501 and C16-PEG2k-Cer, formulated with addition of alfa-tag lipid in the RNA phase (post-insertion). (B) Particle size and PDI of functionalized RNA-lipid particles, prepared with HY501 and C16-PEG2k-Cer, with alfa-tag lipid added in the RNA phase (post-insertion), subjected to at least three freeze thaw cycles from -20°C to room temperature and from -80°C to room temperature. (C) Agarose Gel Electrophoresis of controls, functionalized RNA-lipid particles and functionalized RNA-lipid particles treated with release solution.

[0153] Figure 13: (A) Particle size and PDI of preformed LNPs prepared with HY501 / pSar(23)-Ac after 1 month at -20°C and 5 days at 4°C. (B) Particles size and PDI of RNA-lipid particles, prepared with HY501 / pSar(23)-Ac before and after the addition of alfa-tag lipid (post insertion). (C) Western blot of RNA-lipid particles and controls.

[0154] Detailed Description

[0155] In the following, the elements of the present disclosure will be described in more detail. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present disclosure to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0156] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995). The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques which are 25 explained in the literature in the field (cf., e.g., Organikum, Deutscher Verlag der Wissenschaften, Berlin 1990; Streitwieser / Heathcook, "Organische Chemie", VCH, 1990; Bey er / W alter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey / Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rbmpp Chemie Lexikon, Falbe / Regitz (Hrsg.), Georg Thieme Verlag Stuttgart, New York, 1989; Molecular Cloning: A 30 Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989.

[0157] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "such as"), provided herein is intended merely to better illustrate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0158] Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

[0159] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0160] Definitions

[0161] In the following, definitions will be provided which apply to all aspects of the present disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0162] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps. The term "consisting essentially of' means excluding other members, integers or steps of any essential significance. The term "comprising" encompasses the term "consisting essentially of' which, in turn, encompasses the term "consisting of'. Thus, at each occurrence in the present application, the term "comprising" may be replaced with the term "consisting essentially of' or "consisting of'. Likewise, at each occurrence in the present application, the term "consisting essentially of' may be replaced with the term "consisting of'.

[0163] The terms "a", "an" and "the" and similar references used in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context.

[0164] Where used herein, "and / or" is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "X and / or Y" is to be taken as specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, just as if each is set out individually herein.

[0165] In the context of the present disclosure, the term "about" denotes an interval of accuracy that the person of ordinary skill will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value by ±5%, such as ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example ±0.01%. For example, with respect to a pH value, the term “about” may in preferred instances indicate deviation from the indicated numerical value by up to 0.3. As will be appreciated by the person of ordinary skill, the specific such deviation for a numerical value for a given technical effect will depend on the nature of the technical effect. For example, a natural or biological technical effect may generally have a larger such deviation than one for a man-made or engineering technical effect.

[0166] The expression "substantially free of X", as used herein, means that the composition described herein is free of X in such manner as it is practically and realistically feasible. For example, if the mixture is substantially free of X, the amount of X in the mixture may be less than 1% by weight (e.g., less than 0.5% by weight, less than 0.4% by weight, less than 0.3% by weight, less than 0.2% by weight, less than 0.1% by weight, less than 0.09% by weight, less than 0.08% by weight, less than 0.07% by weight, less than 0.06% by weight, less than 0.05% by weight, less than 0.04% by weight, less than 0.03% by weight, less than 0.02% by weight, less than 0.01% by weight, less than 0.005% by weight, or less than 0.001% by weight), based on the total weight of the mixture. Specific meanings of the term “substantially free” in relation to certain components of the composition are defined herein.

[0167] "Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0168] "Physiological conditions" as used herein refer to the conditions (in particular pH and temperature) in a living subject, in particular a human. Preferably, physiological conditions mean a physiological pH and / or a temperature of about 37°C.

[0169] As used in the present disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components, multiplied by 100.

[0170] As used in the present disclosure, "mol % of the lipid mixture" is defined as the ratio of the number of moles of that particular lipid component to the total number of moles of all lipids in the lipid mixture, multiplied by 100. In this context, in some embodiments, the term "total lipid" and / or “total lipid mixture” includes lipids and lipid-like material.

[0171] The term "hydrocarbyl" as used herein relates to a monovalent organic group obtained by removing one H atom from a hydrocarbon molecule. In some embodiments, hydrocarbyl groups are non-cyclic, e.g., linear (straight) or branched. Typical examples of hydrocarbyl groups include alkyl, alkenyl, alkynyl, cycloalkyl, aryl groups, and combinations thereof (such as arylalkyl (aralkyl), etc.). Particular examples of hydrocarbyl groups are Ci-40 alkyl (such as Ce-40 alkyl, Ce-30 alkyl, C6-20 alkyl, or C10-20 alkyl), C2-40 alkenyl (such as Ce-40 alkenyl, Ce-30 alkenyl, or C6-20 alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(Ci-6 alkyl). In some embodiments, the hydrocarbyl group is optionally substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. The term "heterohydrocarbyl" means a hydrocarbyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the hydrocarbyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. In one embodiment, the heterohydrocarbyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0172] The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “cycloaliphatic”), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., Ci-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.

[0173] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2-propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1,2- dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2-ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n- tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n- nonadecyl, n-icosyl, n-triacontyl, n-tetracontyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. Examples of a substituted alkyl include chloromethyl, dichloromethyl, fluoromethyl, and difluoromethyl.

[0174] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1,1 -ethylene, 1,2-ethylene), propylene i.e., 1,1- propylene, 1,2-propylene (-CH(CH3)CH2-), 2,2-propylene (-C(CH3)2-), and 1,3- propylene), the butylene isomers (e.g., 1,1-butylene, 1,2-butylene, 2,2-butylene, 1,3- butylene, 2,3-butylene (cis or trans or a mixture thereof), 1,4-butylene, 1 , 1 -isobutylene, 1,2-iso-butylene, and 1,3 -iso-butylene), the pentylene isomers (e.g., 1,1- pentylene, 1,2-pentylene, 1,3-pentylene, 1,4-pentylene, 1,5-pentylene, 1,1-iso- pentylene, 1,1 -sec-pentyl, 1,1-neo-pentyl), the hexylene isomers (e.g., 1,1-hexylene, 1,2-hexylene, 1,3-hexylene, 1,4-hexylene, 1,5-hexylene, 1,6-hexylene, and 1,1- isohexylene), the heptylene isomers (e.g., 1,1 -heptylene, 1,2-heptylene, 1,3 -heptylene, 1,4-heptylene, 1,5 -heptylene, 1,6-heptylene, 1,7-heptylene, and 1,1 -isoheptylene), the octylene isomers (e.g., 1,1-octylene, 1,2-octylene, 1,3-octylene, 1,4-octylene, 1,5- octylene, 1,6-octylene, 1,7-octylene, 1,8-octylene, and 1,1 -isooctylene), and the like. The straight alkylene moieties having at least 3 carbon atoms and a free valence at each end can also be designated as a multiple of methylene (e.g., 1,4-butylene can also be called tetramethylene). Generally, instead of using the ending "ylene" for alkylene moieties as specified above, one can also use the ending "diyl" (e.g., 1,2- butylene can also be called butan-l,2-diyl). A "substituted alkylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituent may be the same or different). In one embodiment, the alkylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0175] The term "alkenyl" refers to a monoradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkenyl group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenyl group has 1 to 6 (such as 1 to 4), z.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenyl group comprises from 2 to 40 carbon atoms, such as 2 to 30 carbon atoms, such as 2 to 20 carbon atoms, such as 2 to 12 carbon atoms, such as 2 to 10 carbon atoms, such as 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenyl group comprises from 2 to 40, such as 2 to 30, such as 2 to 20, such as 2 to 12, such as 2 to 10 carbon atoms and 1, 2, 3, 4, 5, or 6 (e.g., 1, 2, 3, 4, or 5) carboncarbon double bonds, such as comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carboncarbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carboncarbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenyl groups include vinyl, 1 -propenyl, 2-propenyl (z.e., allyl), 1-butenyl, 2-butenyl, 3-butenyl, 1 -pentenyl, 2-pentenyl, 3 -pentenyl, 4-pentenyl, 1 -hexenyl, 2-hexenyl, 3- hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5- heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7- nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6- decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4- undecenyl, 5 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10- undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6- dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, and the like. A "substituted alkenyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0176] The term "alkenylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Generally, the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer. For example, for an alkenylene group having 9 carbon atoms, the maximum number of carbon-carbon double bonds is 4. Preferably, the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as 1, 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 5 it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1, 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-l,2-diyl, vinylidene (also called ethenylidene), 1- propen-l,2-diyl, 1 -propen- 1,3 -diyl, l-propen-2,3-diyl, allylidene, l-buten-l,2-diyl, 1- buten- 1,3 -diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, l-buten-3,4-diyl, 2-buten-l,2-diyl, 2-buten- 1,3 -diyl, 2-buten-l,4-diyl, 2-buten-2,3-diyl, 2-buten-2,4- diyl, 2-buten-3,4-diyl, and the like. A "substituted alkenylene" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkenylene group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 15 up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkenylene group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced, the substituents may be the same or different). In one embodiment, the alkenylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0177] The term "alkynyl" refers to a linear or branched monovalent hydrocarbon moiety having at least one carbon-carbon triple bond in which the total carbon atoms may be six to forty, such as six to thirty, typically six to twenty, such as six to eighteen. Alkynyl groups can optionally have one or more carbon-carbon triple bonds. Generally, the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer. For example, for an alkynyl group having 9 carbon atoms, the maximum number of carbon-carbon triple bonds is 4. Preferably, the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds. A "substituted alkynyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkynyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkynyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkynyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0178] The term "alkynylene" refers to a diradical of an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Preferably, the alkynylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon triple bonds. Preferably, the alkynylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkynylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1, 2, 3, 4, 5, or 6 (such as

[0179] 1, 2, 3, 4, or 5) carbon-carbon triple bonds, more preferably it comprises 2 to 8 carbon atoms and 1, 2, 3, or 4 carbon-carbon triple bonds, such as 2 to 6 carbon atoms and 1,

[0180] 2, or 3 carbon-carbon triple bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon triple bonds. Exemplary alkenylene groups include ethyn-l,2-diyl, l-propyn-l,2-diyl, 1-propyn- 1,3 -diyl.. In one embodiment, the alkenylene is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0181] The terms "cycloalkyl" and “cycloalkenyl” represents cyclic non-aromatic versions of "alkyl" and "alkenyl" with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, and adamantyl. Exemplary cycloalkenyl groups include cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, cyclononenyl, and cyclodecenyl. The cycloalkyl or cycloalkenyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic). A "substituted cycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the cycloalkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the cycloalkyl or cycloalkenyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0182] The terms "cycloalkylene" and “cycloalkenylene” represents cyclic non-aromatic versions of "alkylene" and "alkenylene" with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Exemplary cycloalkylenene groups include cyclopentenylene and cy cl ohexeny 1 ene .

[0183] The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon.

[0184] Preferably, the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl). Exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl. Preferably, "aryl" refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes. A "substituted aryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an aryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 5 or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the aryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the aryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. Examples of a substituted aryl include biphenyl, 2-fluorophenyl, 2-chloro-6- methylphenyl, anilinyl, 4-hydroxyphenyl, and methoxyphenyl (z.e., 2-, 3-, or 4- methoxyphenyl).

[0185] The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O- CH2-CH2-O-CH3, and the like.

[0186] The term "heteroaryl" or "heteroaromatic ring" means an aryl group as defined above in which one or more carbon atoms in the aryl group are replaced by heteroatoms of O, S, or N. Preferably, heteroaryl refers to a five or six-membered aromatic monocyclic ring wherein 1, 2, or 3 carbon atoms are replaced by the same or different heteroatoms of O, N, or S. Alternatively, it means an aromatic bicyclic or tricyclic ring system wherein 1, 2, 3, 4, or 5 carbon atoms are replaced with the same or different heteroatoms of O, N, or S. Preferably, in each ring of the heteroaryl group the maximum number of O atoms is 1, the maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. Exemplary heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, IH-indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotri azolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrol othi azolyl, phenothiazinyl, isobenzofuranyl, chromenyl, xanthenyl, pyrrolizinyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phthalazinyl, naphthyridinyl, cinnolinyl, pteridinyl, carbazolyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, and phenazinyl. Exemplary 5- or 6-memered heteroaryl groups include furanyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl, pyrrolyl, imidazolyl (e.g., 2-imidazolyl), pyrazolyl, triazolyl, tetrazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl (e.g., 4-pyridyl), pyrimidinyl, pyrazinyl, triazinyl, and pyridazinyl. A "substituted heteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heteroaryl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1, 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1, the 5 maximum number of S atoms is 1, and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heterocyclyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0187] The term “alkylcycloalkyl” means a cycloalkyl group, as defined above, which is substituted with an alkyl group, as defined above, the cycloalkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylcycloalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. The term “cycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted cycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the cycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0188] The term “alkylcycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule and the cycloalkyl portion in turn being substituted with a further alkyl group. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylcycloalkylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0189] The term “alkylaryl” means an aryl group, as defined above, which is substituted with an alkyl group, as defined above, the aryl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0190] The term “arylalkyl” means an alkyl group, as defined above, which is substituted with an aryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted arylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a arylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the arylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0191] The term “alkylheteroaryl” means a heteroaryl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heteroaryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheteroaryl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylheteroaryl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0192] The term “heteroarylalkyl” means an alkyl group, as defined above, which is substituted with a heteroaryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heteroarylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heteroarylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0193] The term “alkylheterocyclyl” means a heterocyclyl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheterocyclyl group, e.g., 1, 2, 3, 4, 5, 6,

[0194] 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the alkylheterocyclyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0195] The term “heterocyclylalkyl” means an alkyl group, as defined above, which is substituted with a heterocyclyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heterocyclylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclylalkyl group, e.g., 1, 2, 3, 4, 5, 6, 7,

[0196] 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heterocyclyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In one embodiment, the heterocyclylalkyl is substituted with one or more, such as 1, 2 or 3, such as 1 or 2, such as 1 substituents selected from List A. The term “organosulfuric acid” or “sulfate” means a compound of formula R-OSO2- OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfate” is used when the group is deprotonated. Depending on the pH, the sulfate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0197] The term “sulfonic acid” or “sulfonate” means a compound of formula R-SO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfonate” is used when the group is deprotonated. Depending on the pH, the sulfonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonate group is typically deprotonated at physiological pH).

[0198] The term “carboxylic acid” or “carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “carboxylate” is used when the group is deprotonated. Depending on the pH, the carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0199] The term “dicarboxylic acid” or “di carb oxy late” means a compound of formula HChC-R’-CChH, wherein R’ is alkylene or alkenylene group (all as defined above, either in a broadest aspect or a preferred aspect). The term “di carb oxy late” is used when the group is deprotonated. Depending on the pH, the dicarboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the dicarboxylic acid group is typically protonated at acidic or neutral pH and deprotonated at alkaline pH).

[0200] The term “hydroxy carboxylic acid” or “hydroxy carboxylate” means a compound of formula R-CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), which is substituted by one or more (preferably 1 to 5, such as 1, 2 or 3) hydroxy groups. The term “hydroxy carboxylate” is used when the group is deprotonated. Depending on the pH, the hydroxy carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0201] The term "ester" as used herein means, depending on context, a bivalent linkage where both ends are to the rest of a molecule, of the structure -C(=O)O- or -OC(=O)- where each end is attached to the rest of a molecule, or to a compound having the structure R-C(O)O-R’ (including its isomerically arranged structure R-OC(O)-R’, unless it is specified to the contrary) wherein R and R’ are each independently hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). When the term denotes a substituent connected to the rest of a molecule, the ester moiety may have the structure R-C(O)O- or R-OC(O)-, where R is as defined above. In one embodiment, each of both ends of the ester structure is covalently linked to a C atom of the same organic group or of two separate organic groups (e.g., an alkylene group as further component of the linker). The term "thioester" as used herein means a bivalent linkage of the structure -C(=S)O- , -C(=O)S-, -SC(=O)- or -OC(=S)- where one end is attached to the carbon atom and the other the oxygen o sulfur atom.

[0202] The term “phosphate” means a compound of formula R0-P(=0)(0H)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphate group is typically deprotonated at physiological pH).

[0203] The term “phosphonate” means a compound of formula R-P(=0)(0H)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0204] “Halo” means fluoro (-F), chloro (-C1), bromo (-Br) or iodo (-1).

[0205] “Amine” means the group -NR2, wherein each R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. When both groups R are hydrogen, the amine group is a primary amine group. When one R is hydrogen and the other R is other than hydrogen, the amine group is a secondary amine group. When both groups R are other than hydrogen, the amine group is a tertiary amine group. A “quaternary ammonium” salt is a compound containing a group -N R3, wherein each R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. In contrast to some amines as defined above which are protonated only at certain pH, a quaternary ammonium salt carries a constitutive positive charge (as defined herein) at all pH.

[0206] “Hydroxyl” - means the group -OH. “Sulfhydryl” - means the group -SH. “Nitro” means the group -NO2.

[0207] “Ether” means an oxygen atom to which two hydrocarbyl or heterohydrocarbyl groups, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl groups (all as defined above, either in a broadest aspect or a preferred aspect) are attached. The ether may be a cyclic ether, wherein the two hydrocarbyl groups together form a ring, and may include dioxolane groups.

[0208] “Thioether” means or a bivalent linkage of formula -S- where both connected moieties are via the sulfur atom, or a group of formula -SR wherein R is a C1-10 alkyl group.

[0209] “Disulfide” means or a bivalent linkage of formula - S-S- where one moiety is connected to the first sulfur atom and another to the second sulfur atom.

[0210] “Amide” means the group -C(=O)NR(R’), wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect) and is preferably an alkyl group, such as a Ci-6 alkyl group, or a bivalent linkage of the formula -C(=O)NR-, wherein R is hydrogen or hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect) group, wherein one moiety is connected to the rest of the molecule via the carbon atom and the other via the nitrogen atom.

[0211] “Thioamide” means a bivalent linkage of the formula -C(=S)NR-, wherein R is hydrogen or a Ci-6 alkyl group, wherein one moiety is connected to the rest of the molecule via the carbon atom and the other via the nitrogen atom.

[0212] “Hydroxylamide” means the group -C(=O)O-NR(R’), wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect).

[0213] “Sulfonyl” means the group -S(=O)2R, wherein R is a C1-13 alkyl or C2-3 alkenyl group (all as defined above, either in a broadest aspect or a preferred aspect), or a bivalent linkage of formula -S(=O)2- where each moiety is connected to the rest of the molecule via the sulfur atom.

[0214] “Sulfonamide” means the group -S(=0)2NRR’, wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-30 alkyl group. “Carbamate” means the group -O-C(=O)NRR’ wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0215] “Amidine” means the group -C(=NR)NR’R” wherein R, R’ and R” are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0216] “Guanidine” means the group -NR-C(=NR’)NR”R”’ or =N-C(NRR’)(NR”R”’) wherein R, R’, R” and R’” are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a Ci-6 alkyl group.

[0217] The above definitions, when relating to any basic nitrogen atom which is protonated, may be modified by the substitution of the suffix “-ium” in accordance with normal chemical nomenclature. For example, a guanidinium group is a protonated guanidine, an ammonium group is a protonated ammonia or a protonated primary, secondary tertiary amine, an imidazolium group is a protonated imidazole, a pyridinium group is a protonated pyridine, an amidinium group is a protonated amidine, and a piperazinium group is a protonated piperazine.

[0218] “Carbohydrate” means a compound having the empirical formula Cm(H20)n where m may or may not be different from n. The term “carbohydrate residue” or “carbohydrate moiety” defines a residue attached to another atom, where one hydrogen atom of the carbohydrate is replaced by a bond attached to the rest of the molecule. The carbohydrate moiety may be a monosaccharide moiety. The monosaccharide moiety may have the D- or L-configuration. Furthermore, the monosaccharide moiety may be an aldose or ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6, carbon atoms. In one embodiment, the monosaccharide moiety is a hexose moiety (i.e. it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose, and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0219] In another embodiment, the monosaccharide moiety is a pentose moiety (i.e. it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0220] In another embodiment, the carbohydrate may be a higher saccharide (i.e. a di-, or oligosaccharide) comprising more than one monosaccharide moiety joined together by glycoside bonds. When the monosaccharide moieties are hexose moieties, the glycoside bonds may be l-a,l'-a glycoside bonds, l,2'-gly coside bonds (which maybe l-a2’ or 1 '-P-2' glycoside bonds), l,3'-glycoside bonds (which may be l-a-3' or 1-P- 3 '-glycoside bonds), 1 ,4'-gly coside bonds (which may be l-a-4' or l-P-4'-gly coside bonds), l,6'-gly coside bonds (which may be l-a-6' or l-P-6'-gly coside bonds), or any combination thereof. In one embodiment, the higher saccharide comprises 2 monosaccharide units (i.e. is a di saccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose and trehalulose. In another embodiment, the higher saccharide comprises 3 to 10 monosaccharide units (i.e. is an oligosaccharide) in a chain, which may be branched or unbranched. Preferably, the oligosaccharide comprises 3 to 8, more preferably 3 to 6, monosaccharide units. Examples of suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose.

[0221] “List A” substituents are selected from the group consisting of Ci-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, 6- to 14-membered (such as 6- to 10-membered) aryl, 3- to 14- membered (such as 5- or 6- membered) heteroaryl, 3- to 14-membered (such as 3- to 7-membered) cycloalkyl, 3- to 14-membered (such as 3- to 7-membered) heterocyclyl, halogen, -CN, azido, -NO2, -OR’, -N(R’)2, -S(0)o-2R’, -S(O)I-2OR’, wherein X1is independently selected from O, S, NH and N(CHa); and each R’ is independently selected from the group consisting of H, Ci-4 alkyl, C2-4 alkenyl, C2-4 alkynyl, 5- or 6-membered cycloalkyl, 5- or 6-membered aryl, 5- or 6-membered heteroaryl, and 5- or 6-membered heterocyclyl, wherein each of the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, and heterocyclyl groups is optionally substituted with one, two or three substituents independently selected from the group consisting of C1-3 alkyl, halogen, -CF3, -CN, azido, -NO2, -OH, -O(Ci-3 alkyl), -S(Ci-3 alkyl), - NH2, -NH(CI-3alkyl), -N(CI-3 alkyl)2, -NHS(O)2(CI-3 alkyl), -S(O)2NH2-Z(CI-3 alkyl)z, -C(=O)OH, -C(=O)O(Cl-3alkyl), -C(=O)NH2-Z(CI-3 alkyl)z, -NHC(=0)(Cl-3 alkyl), - NHC(=NH)NHZ-2(C1-3 alkyl)z, and -N(CI-3 alkyl)C(=NH)NH2-z(Ci-3 alkyl)z, wherein each z is independently 0, 1, or 2 and each C1-3 alkyl is independently methyl, ethyl, Al, consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2- z(CH3)z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1, or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, List A substituents are selected from List A2, consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3.

[0222] Nucleic Acid

[0223] The lipid particle compositions of the present application (both when functionalized and prior to functionalization) contain an active ingredient. The active ingredient is a nucleic acid. Preferably the lipid particle compositions of the present application contain RNA, such as mRNA, and DNA. Typically, the lipid particle compositions described herein comprise lipid particles that encapsulate the nucleic acid. The term "nucleic acid" comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. The term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the active ingredient (e.g., which is to be delivered to target cells to genetically modify the target cells and enable the target cells to express a biomolecule (such as a peptide or protein, encoded by the nucleic acid)) comprises DNA, RNA, or a mixture thereof.

[0224] A nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule. A nucleic acid can be isolated. The term "isolated nucleic acid" means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.

[0225] The term "nucleoside" relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nucleobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine. Nucleic acids may include one or more modified nucleosides or nucleotides. Examples of modified nucleosides or nucleotides which may be incorporated into nucleic acids include N7-alkylguanine, N6-alkyl-adenine, 5- alkyl-cytosine, 5-alkyl-uracil, and N(l)-alkyl-uracil, such as N7-Cl-4 alkylguanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(l)-Cl-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y), and Nl-methyl-pseudouri dine (ml'P).

[0226] RNA

[0227] In some embodiments of all aspects of the disclosure, the nucleic acid is RNA. According to the present disclosure, the term "RNA" means a nucleic acid molecule which includes ribonucleotide residues. RNA typically comprises the naturally occurring nucleic acids adenosine (A), uridine (U), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'- position of a P-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides (z.e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (z.e., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The active ingredient may be mRNA, saRNA, taRNA, or mixtures thereof. The active ingredient is preferably mRNA. In some instances, the active ingredient is not siRNA.

[0228] In a preferred embodiment, the RNA comprises an open reading frame (ORF) encoding a peptide, polypeptide or protein. Said RNA may capable of or configured to express the encoded peptide, polypeptide, or protein. For example, said RNA may be RNA encoding and capable of or configured for expressing a pharmaceutically active peptide or protein. In some embodiments, RNA is able to interact with the cellular translation machinery allowing translation of the peptide or protein. A cell may produce the encoded peptide or protein intracellularly (e.g. in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface.

[0229] Alternatively, the RNA can be non-coding RNA such as antisense-RNA, micro RNA (miRNA) or siRNA. mRNA

[0230] In preferred embodiments of all aspects of the disclosure, the nucleic acid is mRNA. According to the present disclosure, the term "mRNA" means "messenger-RNA" and includes a "transcript" which may be generated by using a DNA template. Generally, mRNA encodes a peptide, polypeptide or protein. As established in the art, the RNA (such as mRNA) generally contains a 5' untranslated region (5'-UTR), a peptide / polypeptide / protein coding region and a 3' untranslated region (3'-UTR). mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.

[0231] According to the present disclosure, "dsRNA" means double-stranded RNA and is RNA with two partially or completely complementary strands.

[0232] In preferred embodiments of the present disclosure, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

[0233] In some embodiments, the RNA which preferably encodes a peptide, polypeptide or protein has a length of at least 45 nucleotides (such as at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, such as up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides or up to 10,000 nucleotides.

[0234] In some embodiments, the RNA (such as mRNA) is produced by in vitro transcription or chemical synthesis. Preferably, the RNA (such as mRNA) is produced by in vitro transcription using a DNA template. The term "in vitro transcription" or "IVT" as used herein means that the transcription (z.e., the generation of RNA) is conducted in a cell-free manner. I.e., IVT does not use living / cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)). The in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989. Furthermore, a variety of in vitro transcription kits is commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAid™ T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (such as RiboMAX™, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribe™).

[0235] For providing modified RNA (such as mRNA), correspondingly modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be effected in and / or added to the mRNA after transcription. The RNA (such as mRNA) may be modified. The RNA (such as mRNA) may comprise modified nucleotides or nucleosides, such as 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (y) or N(l)-methyl-pseudouridine (mly). One or more uridine in the RNA described herein may be replaced by a modified nucleoside. The modified nucleoside may be a modified uridine. The RNA may comprise a modified nucleoside in place of at least one uridine. Preferably, the RNA may comprise a modified nucleoside in place of each uridine (e.g., all of the uridines in the RNA are replaced with a modified nucleoside). The modified nucleoside may be independently selected from pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (\| / ) or Nl-methyl-pseudouridine (mly).

[0236] In some embodiments, RNA (such as mRNA) is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Particular examples of RNA polymerases are the T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription is controlled by a T7 or SP6 promoter. A DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription. The cDNA may be obtained by reverse transcription of RNA.

[0237] In some embodiments of the present disclosure, the RNA (such as mRNA) is "replicon RNA" (such as "replicon mRNA") or simply a "replicon", in particular "self-replicating RNA" (such as "self-replicating mRNA") or "self-amplifying RNA" (or "self-amplifying mRNA"). The lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. The lipid particles containing RNA as described herein may contain an mRNA encoding a replicase protein, and one or more RNA molecules capable of being replicated or amplified by the replicase.

[0238] Inhibitory RNA

[0239] In some embodiments of all aspects of the disclosure, the nucleic acid is an inhibitory RNA.

[0240] The term "inhibitory RNA" as used herein means RNA which selectively hybridizes to and / or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and / or translation thereof. Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.

[0241] The term "antisense RNA" as used herein refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and / or translation of said mRNA. The size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0242] By "small interfering RNA" or "siRNA" as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs. Typically siRNAs comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.

[0243] As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer.

[0244] According to the present disclosure, siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide", revised Oct. 11, 2002, the entire disclosure of which is herein incorporated by reference. Further guidance with respect to the selection of target sequences and / or the design of siRNA can be found on the webpages of Protocol Online (www.protocol-online.com) using the keyword "siRNA". Thus, in some embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA. siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Selection of other suitable promoters is within the skill in the art. Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.

[0245] The term "miRNA" (microRNA) as used herein relates to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and / or prevent translation of target mRNAs. miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter.

[0246] DNA

[0247] In some embodiments of all aspects of the disclosure, the nucleic acid is DNA. Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" represents "deoxy"). In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a P-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxy-ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (z.e., naturally occurring) nucleotide residues or analogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA. The DNA may comprise a plasmid, a nanoplasmid, a minicircle, a transposon, or linear DNA such as doggybone DNA.

[0248] Pharmaceutically active peptides or polypeptides

[0249] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (preferably mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (z.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of RNA (preferably mRNA) corresponding to that gene produces the protein in a cell or other biological system. Similarly, an RNA (such as mRNA) encodes a protein if translation of that RNA (e.g., in a cell) produces that protein. In some embodiments, the active ingredient is an RNA (preferably mRNA) or a DNA, as described in the present disclosure, which comprises a nucleic acid sequence (e.g., an ORF) encoding one or more polypeptides, e.g., a peptide or protein, preferably a pharmaceutically active peptide or protein. In some embodiments, the RNA (preferably mRNA) or DNA described in the present disclosure is capable of expressing said peptide or protein, in particular if transferred into a cell or subject. Thus, in some embodiments, the RNA (preferably mRNA) or DNA described in the present disclosure contains a coding region (ORF) encoding a peptide or protein, preferably encoding a pharmaceutically active peptide or protein. In this respect, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon. Such RNA (preferably mRNA) or DNA encoding a pharmaceutically active peptide or protein is also referred to herein as "pharmaceutically active RNA" (or "pharmaceutically active mRNA") or "pharmaceutically active DNA". In some embodiments, RNA (preferably mRNA) or DNA described in the present disclosure comprises a nucleic acid sequence encoding more than one peptide or polypeptide, e.g., two, three, four or more peptides or polypeptides. In some embodiments, RNA (preferably mRNA) or DNA described in the present disclosure comprises a nucleic acid sequence encoding one or more (e.g., 1, 2, 3, 4, 5, or more) patient-specific antigens suitable for personalized cancer therapy. In some embodiments, the lipid particle compositions comprising RNA or DNA may comprise one or more species of RNA or DNA, wherein each RNA or DNA encodes a different peptide or protein.

[0250] Preferably, the RNA (i) contains structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, 3' UTR, poly(A) sequence); (ii) is modified for optimized efficacy of the RNA (e.g, increased translation efficacy, decreased immunogenicity, and / or decreased cytotoxicity) (e.g., by replacing (partially or completely, preferably completely) naturally occurring nucleosides (in particular cytidine) with synthetic nucleosides (e.g., modified nucleosides selected from the group consisting of pseudouridine (y), Nl-methyl-pseudouridine (mly), and 5-methyl-uridine); and / or codon-optimization), or (iii) both (i) and (ii). The term "pharmaceutically active peptide or protein" may be understood to mean a peptide or protein that can be used in the treatment of an individual where the expression of the peptide or protein would be of benefit, e.g., in ameliorating the symptoms of a disease or disorder. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or disorder or to lessen the severity of such disease or disorder.

[0251] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, interferons, such as interferon-alpha (IFN-a), interferon beta (IFNP) or interferon-gamma (IFN-y), interleukins, such as interleukin 2 (IL2), IL-4, IL-7, IL-10, IL-11, IL-12, IL-15, IL-21 and IL23, colony stimulating factors, such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic protein (BMP); immunoglobulin superfamily members including antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD 19), antigen receptor accessory molecules (e.g., CD-3y, CD3-6, CD-3s, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds such as tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0252] In some embodiments, the nucleic acid encodes an antigen receptor such as a T cell receptor (TCR) or chimeric antigen receptor (CAR). The pharmaceutically active peptide or protein may be or comprise a TCR or a CAR. Delivering a nucleic acid encoding an antigen receptor such as a TCR or CAR to cells may be useful for generating immune effector cells genetically modified to express an antigen receptor. The functionalized nucleic acid-lipid particles described herein may be used for targeted delivery of a nucleic acid encoding an antigen receptor e.g., for generating in vitro / ex vivo or in vivo immune effector cells genetically modified to express an antigen receptor. The term "genetically modified", "genetic modification" or simply "modification" includes the transfection of cells with nucleic acid. The term "transfection" relates to the introduction of nucleic acids, e.g., DNA and / or RNA, into a cell. The cell may be present in a subject (e.g., a patient) or the cell may be in vitro, (e.g., outside of a patient). Transfection can be transient or stable. For example, RNA or DNA can be transfected into cells to transiently express its coded protein. Typically, the nucleic acid is not integrated into the nuclear genome, and will be diluted through mitosis or degraded. Alternatively, a stable transfection is usually required for the transfected nucleic acid to enter the genome of the cell and remain in its daughter cells. Such stable transfection can be achieved by using virus-based systems or transposon-based systems for transfection, for example. Thus, at least a portion of transfected DNA can be inserted into the genome for stable transfection. Generally, cells that are genetically modified to express an antigen receptor are stably transfected with nucleic acid encoding the antigen receptor. RNA can be transfected into cells to transiently express its coded protein.

[0253] Targeting compounds

[0254] The aqueous dispersions, nucleic acid-lipid particles and functionalized nucleic acid- lipid particles comprise a hydrophobic moiety, such as a lipid, conjugated to a binding moiety. The lipid L having a binding moiety B covalently attached thereto, optionally via polymer P (when present) and / or linking moieties XI and X2, comprises a compound of formula (A), as described further herein. This is also referred to in the present specification as a “targeting compound”.

[0255] The hydrophobic moiety of the targeting compound relates to the part of the targeting compound that integrates into the particle comprising a nucleic acid payload. The binding moiety of the targeting compound relates to the part of the targeting compound that binds to target cells or forms the binding partner for a docking compound, as defined herein, which binds to target cells. Generally, the targeting compound is non-covalently incorporated into the particle comprising the active ingredient, i.e., it forms an integral part of the particle, and the binding moiety of the targeting compound is covalently attached to a hydrophobic moiety in a manner such that it is available for binding to target cells or a docking compound. In some embodiments, the binding moiety B of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag).

[0256] In some embodiments, the binding moiety B of the targeting compound comprises a peptide or protein (e.g., an antibody or antibody fragment or a peptide tag) and is chemically linked, e.g., through a linker, to the hydrophobic moiety (e.g., lipid). In some embodiments, the binding moiety B of the targeting compound comprises an antibody or antibody fragment.

[0257] The targeting compound described herein comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle. In some embodiments, the hydrophobic component comprises a moiety selected from a vitamin E compound (which may be a-tocopherol, P-tocopherol, y-tocopherol, 6- tocopherol, a-tocotrienol, P-tocotrienol, y-tocotrienol, 6-tocotrienol, preferably a- tocopherol), a dialkylamine, e.g., dimyristylamine (DMA), diacylglyceride, e.g., 1,2- dimyristoyl-sn-glycerol (DMG) and ceramide. In some embodiments, the hydrophobic moiety comprises two C8-C24 hydrocarbyl chains. In some embodiments, the hydrophobic moiety comprises two C10-C18 hydrocarbyl chains.

[0258] In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a phospholipid, e.g., a biodegradable phospholipid such as phosphatidylethanolamine. In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a glycerophospholipid. In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In some embodiments, as a phospholipid, DSPE will be used for its qualities of stability in the particles described herein. Moreover, as hydrophobic group (e.g., lipid), a compound having at least one alkyl chain providing hydrophobic anchoring to a particle as described herein may be used.

[0259] In some embodiments, the targeting compound comprises a polymer, defined as P herein when present. In some embodiments, the hydrophobic moiety (e.g., lipid) of the targeting compound and the binding moiety of the targeting compound are connected (typically covalently) through the polymer.

[0260] In some embodiments, the polymer is a hydrophilic polymer and the targeting compound comprises an amphiphilic derivative of the polymer. In some embodiments, the amphiphilic derivative of a polymer comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle and a hydrophilic component of the polymer facing the outside of said particle, conferring hydrophilic properties at the surface thereof. In some embodiments, the amphiphilic derivatives of a polymer is inserted into the particle via its hydrophobic end. Consequently, the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle. In some embodiments, the polymer portion of the amphiphilic derivative contributes to conferring stealth properties on the particles. In some embodiments, the polymer portion of the amphiphilic derivative confers stealth properties on the particles. In some embodiments, the plasmatic half-life of the particles described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the particles to accumulate at the target cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

[0261] The term "stealth" is used herein to describe the ability of the particles described herein not to be detected and then sequestered and / or degraded, or to be hardly detected and then sequestered and / or degraded, and / or to be detected and then sequestered and / or degraded late, by the immune system of the host to which they are administered.

[0262] In some embodiments, the polymer for use herein is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N- methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof), as defined herein. In some embodiments, a polymer is designed to sterically stabilize a particle by forming a protective hydrophilic layer. In some embodiments, a polymer can reduce association of a particle with serum proteins and / or the resulting uptake by the reticuloendothelial system when such particles are administered in vivo.

[0263] In some embodiments, the polymer is PEG, and the PEG is an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In some embodiments, the PEG is unsubstituted. In some embodiments, the PEG is substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy or aryl groups. In some embodiments, the PEG has a molecular weight of from about 130 to about 50,000, in another embodiment about 150 to about 30,000, in another embodiment about 150 to about 20,000, in another embodiment about 150 to about 15,000, in another embodiment about 150 to about 10,000, in another embodiment about 150 to about 6000, in another embodiment about 150 to about 5000, in another embodiment about 150 to about 4000, in another embodiment about 150 to about 3000, in another embodiment about 300 to about 3000, in another embodiment about 1000 to about 3000, and in still another embodiment about 1500 to about 2500.

[0264] In some embodiments, the PEG moiety of the amphiphilic derivative of a polymer has a molecular weight of 1000 or more. In some embodiments, the PEG moiety of the amphiphilic derivative of a polymer comprises 2 units or more, such as 5 units of more, such as 10 units or more of formula (O-CH2-CH2)n(where n is the number of ethylene oxide units). In some embodiments, the PEG comprises from 20 to 200 ethylene oxide units, such as about 45 ethylene oxide units.

[0265] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.

[0266] In some embodiments, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as the amphiphilic derivative of a polymer.

[0267] In some embodiments, the polymer is a pSar and the pSar comprises between 2 and 200 sarcosine units, such as between 5 and 100 sarcosine units, between 10 and 50 sarcosine units, between 15 and 40 sarcosine units, e.g., about 23 sarcosine units. In some embodiments, a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.

[0268] In some embodiments, the polymer is POX and / or POZ, and the POX and / or POZ polymer comprises between 2 and 200, between 2 and 190, between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between

[0269] 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between

[0270] 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between

[0271] 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 10 and 70 POX and / or POZ repeating units.

[0272] In some embodiments, the POX and / or POZ polymer comprises the following general formula: wherein a is an integer between 1 and 2; Rn is alkyl, in particular C1-3 alkyl, such as methyl, ethyl, iso-propyl, or n-propyl, and is independently selected for each repeating unit; and m refers to the number of POX and / or POZ repeating units.

[0273] In some embodiments, the POX and / or POZ polymer is a polymer of POX and comprises repeating units of the following general formula: wherein Rn is as defined above.

[0274] In some embodiments, the POX and / or POZ polymer is a polymer of POZ and comprises repeating units of the following general formula: wherein Rn is as defined above.

[0275] In any of the above embodiments of formulas, m (i.e., the number of repeating units in the polymer) preferably is between 2 and 190, such as between 2 and 180, between 2 and 170, between 2 and 160, between 2 and 150, between 2 and 140, between 2 and 130, between 2 and 120, between 2 and 110, between 2 and 100, between 2 and 90, between 2 and 80, between 2 and 70, between 5 and 200, between 5 and 190, between 5 and 180, between 5 and 170, between 5 and 160, between 5 and 150, between 5 and 140, between 5 and 130, between 5 and 120, between 5 and 110, between 5 and 100, between 5 and 90, between 5 and 80, between 5 and 70, between 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between

[0276] 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between

[0277] 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between

[0278] 10 and 70. In certain embodiments, m is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0279] In some embodiments, the POX and / or POZ polymer is a copolymer comprising repeating units of the following general formulas: wherein Rn is as defined above. In some embodiments, the number of repeating units shown on the left in the copolymer is 1 to 199. In some embodiments, the number of repeating units of formula on the right in the copolymer is 1 to 199. In some embodiments, the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 200.

[0280] In some embodiments of the oxazolinylated and / or oxazinylated hydrophobic moiety (e.g., lipid), the number of repeating units of formula on the left in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; the number of repeating units of formula on the right in the copolymer is 1 to 179, such as 1 to 159, 1 to 139, 1 to 119 or 1 to 99; and the sum of the number of repeating units of formula on the left and the number of repeating units of formula on the right in the copolymer is 2 to 180, such as 4 to 160, 6 to 140, 8 to 120 or 10 to 100, e.g., 20 to 80, 30 to 70, or 40 to 50.

[0281] In some of the above embodiments, Rn at each occurrence (i.e., in each repeating unit) may be the same alkyl group (e.g., Rn may be methyl in each repeating unit). In some alternative embodiments, Rn in at least one repeating unit differs from Rn in another repeating unit (e.g., for at least one repeating unit Rn is one specific alkyl (such as ethyl), and for at least one different repeating unit Rn is a different specific alkyl (such as methyl)). For example, each Rn may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl. In any of the above embodiments, Rn preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rn is methyl or each Rn is ethyl. In some alternative embodiments, Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn is ethyl.

[0282] In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)- acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof, as defined herein.

[0283] In some embodiments, the polymer comprises the following general formula: wherein

[0284] X11and X12taken together are optionally substituted amide, optionally substituted thioamide or ester;

[0285] Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

[0286] In some embodiments,

[0287] (i) when X11is -C(O)- then X12is -NR1-;

[0288] (ii) when X11is -NR1- then X12is -C(O)-;

[0289] (iii) when X11is -C(S)- then X12is -NR1-;

[0290] (iv) when X11is -NR1- then X12is -C(S)-;

[0291] (v) when X11is -C(O)- then X12is -O-; or

[0292] (vi) when X11is -O- then X12is -C(O)-; wherein R1is hydrogen or Ci-8 alkyl.

[0293] In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen or Ci-8 alkyl. In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen or methyl. In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen.

[0294] In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0295] In some embodiments, the polymer comprises the following general formula: wherein

[0296] R1is hydrogen or C1-8 alkyl; z is 2 to 24; and n is 1 to 100. In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7. In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0297] In some embodiments, the polymer comprises the following general formula: wherein

[0298] R1is hydrogen or Ci-8 alkyl; and n is 1 to 100.

[0299] In some embodiments of the above formulas, R1is hydrogen or methyl. In some embodiments, R1is hydrogen.

[0300] In some embodiments, the polymer comprises the following general formula: wherein n is 1 to 100.

[0301] In some embodiments of the above formulas, n is 5 to 50. In some embodiments, n is 5 to 25. In some embodiments, n is 7 to 14. In some embodiments, n is 10 to 25. In some embodiments, n is 14 to 17. In some embodiments, n is 8 or 14.

[0302] In some embodiments, the molar proportion of the amphiphilic derivative of a polymer integrated into the particles is between 0.5 and 20 mol% of the lipid molecules making up the particle, preferably between 1 and 10 mol%.

[0303] In some embodiments, the targeting compound is present in an amount of 0.01 to 10 mol%, optionally 0.05 to 5 mol%, of the lipid mixture. In some embodiments, the targeting compound is present in an amount of 0.1 to 2 mol%, optionally 0.1 to 1 mol%, of the lipid mixture.

[0304] In some embodiments, the targeting compound is a compound of Formula (A):

[0305] L-X1-P-X2-B (A) wherein:

[0306] P is absent or comprises a polymer;

[0307] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0308] B comprises a binding moiety attached to L when P is absent or to a second end of the polymer P when present;

[0309] XI is absent or a first linking moiety; and

[0310] X2 is absent or a second linking moiety.

[0311] In one embodiment of formula (A), the hydrophobic moiety comprises a lipid. In one embodiment of formula (A), the hydrophobic moiety comprises a phospholipid. In one embodiment of formula (A), the hydrophobic moiety comprises a moiety selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), and mixtures thereof. In one embodiment of formula (A), the hydrophobic moiety comprises a DSPE moiety.

[0312] In one embodiment of formula (A), P is absent. In one embodiment of formula (A), P is a polymer. In one embodiment of formula (A), P is a hydrophilic polymer. In one embodiment of formula (A), P is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly (N-m ethylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.

[0313] In one embodiment of formula (A), P comprises polyethylene glycol (PEG), preferably wherein the average molecular weight of the PEG is from about 200 to about 10,000, more preferably 500 to 5000, even more preferably 1000 to 4000, most preferably 2000. In one embodiment of formula (A), P comprises the following general formula: wherein n is 1 to 100.

[0314] In one embodiment of formula (A), XI comprises a carbonyl group.

[0315] In one embodiment of formula (A), X2 comprises the reaction product of a thiol or cysteine reactive group with a thiol or cysteine group of a compound comprising the binding moiety B. In one embodiment of formula (A), the thiol or cysteine reactive group comprises a maleimide group.

[0316] In one embodiment of formula (A), the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoyl-glycero-phosphoethanolamine- polyethylene glycol-conjugate (DSPE-PEG).

[0317] In some embodiments, the targeting compound is a compound of Formula (Al): L-X1-P-X2-B (Al) wherein

[0318] P comprises a polymer;

[0319] L comprises a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer; B comprises a binding moiety attached to a second end of the polymer;

[0320] XI is absent or a first linking moiety; and

[0321] X2 is absent or a second linking moiety.

[0322] In some embodiments of formula (Al), XI comprises a carbonyl group.

[0323] In some embodiments of formula (Al), L comprises a phosphatidylethanolamine which may be linked to P by an amide group. In some embodiments of formula (Al), X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a thiol or cysteine group of a compound comprising the binding moiety.

[0324] In some embodiments of formula (Al), L comprises a lipid as described above. In some embodiments of formula (Al), L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group.

[0325] In some embodiments of formula (Al), P comprises a polymer as described above. In some embodiments of formula (Al), P comprises a polymer which provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion.

[0326] In some embodiments of formula (Al), P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N- methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2- aminoethoxy)-ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments of formula (Al), P comprises poly(ethylene glycol) (PEG); e.g., PEG as described above.

[0327] In some embodiments of formula (Al), L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments of formula (Al), the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero- phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments of formula (Al), the amphiphilic derivative of a polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG).

[0328] In some embodiments of formula (Al), the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the binding moiety. In some embodiments of formula (Al), the thiol or cysteine reactive group comprises a maleimide group.

[0329] In some embodiments of formula (Al), the PEG reagent comprises DSPE-PEG- maleimide. In some embodiments of formula (Al), the compound comprising the binding moiety comprises the formula HS-(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments, n is 2.

[0330] In some embodiments of formula (Al), the targeting compound comprises the reaction product of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)] with a compound comprising the formula HS- (CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the binding moiety. In some embodiments of formula (Al), n is 2.

[0331] In some embodiments, the targeting compound is of the general formula (A2):

[0332] L-X1-P-X2-B (A2) wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety.

[0333] In some embodiments, the targeting compound comprises the following general formula (A2’) wherein B comprises the binding moiety, and PEG is polyethylene glycol, as defined above (either in its broadest aspect or a preferred aspect).

[0334] In some embodiments of formula (A2) or (A2’), B comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein the peptide moiety is as defined herein.

[0335] In some embodiments, the targeting compound has the following general formula (A3): wherein P, X2 and B are as described above and Ri and R2 independently comprise an alkyl moiety, as defined herein (either in its broadest aspect or a preferred aspect). In some embodiments, at least one, e.g., each alkyl moiety is straight or branched, preferably straight. In some embodiments, at least one, e.g., each alkyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol, more preferably at least one, e.g., each alkyl moiety is the alkyl moiety of a fatty acid alcohol having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of alkyl moieties include -(CH2)i7CH3 (stearyl), -(CE^isCEE (palmityl), and -(CH2)i3CH3 (myristyl).

[0336] In some embodiments of formula (A3), R1R2N- in the above formula (A3) is 1,2- dimyristylamine, wherein both alkyl groups are -(CH2)i3CH3 (myristyl).

[0337] In some embodiments of formula (A3), the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylamino- ethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments of formula (A3), the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments of formula (A3), n is 8 or 14. In some embodiments of formula (A3), n is 14. In some embodiments of formula (A3), Ri and R2 in the above formula are - (CH2)i3CH3 (myristyl) and the polymer P comprises the following general formula: wherein n is 14. In some embodiments, the targeting compound is of the general formula (A4): wherein P, X2 and B are as described above and each of Rti and Rt2 is independently H or methyl. In some embodiments of formula (A4), Rti and Rt2 are both methyl. In some embodiments of formula (A4), Rti is methyl, and Rt2 is H. In some embodiments of formula (A4), Rti is H, and Rt2 is methyl. In some embodiments of formula (A4), Rti and Rt2 are both H.

[0338] In some embodiments, the targeting compound is of the general formula (A4’): wherein P, X2 and B are as described above.

[0339] In some embodiments of formula (A4) or (A4’), the polymer P in the above formulas comprises poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2- methylaminoethoxy)-ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments of formula (A4) or (A4’), the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments of formula (A4) or (A4’), n is 8 or 14. In some embodiments of formula (A4) or (A4’), n is 8. In some embodiments of formula (A4) or (A4’), n is 14.

[0340] In some embodiments, the targeting compound is of the general formula (A5): wherein XI, P, X2 and B are as described above and Ri and R2 independently comprise an acyl moiety. In some embodiments of formula (A5), at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments of formula (A5), at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moi eties include CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5), both acyl groups are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A5), both acyl groups are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5), XI is absent or comprises -UPCh' (CH2)n-NH-, wherein n is 1 to 5, e.g., 2.

[0341] In some embodiments of formula (A5), the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)- ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments of formula (A5), the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments of formula (A5), n is 8 or 14. In some embodiments of formula (A5), n is 8. In some embodiments, n is 14.

[0342] In some embodiments of formula (A5), the polymer P comprises a pSar. In some embodiments of formula (A5), the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments of formula (A5), s is 20 or 23.

[0343] In some embodiments, the targeting compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula (A5’): wherein P, X2 and B are as described above and Ri and R2 independently comprise an acyl moiety. In some embodiments of formula (A5’), at least one, e.g., each acyl moiety is straight or branched, preferably straight. In some embodiments of formula (A5’), at least one, e.g., each acyl moiety has at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Preferably, at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid, more preferably at least one, e.g., each acyl moiety is the acyl moiety of a fatty acid having at least 8 carbon atoms, e.g., 8 to 24 such as 10 to 18 carbon atoms. Examples of acyl moi eties include CH3(CH2)ieC(O)- (stearoyl), CH3(CH2)i4C(O)- (palmitoyl), and CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5’), both acyl groups are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A5’), both acyl groups are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5’), the polymer P comprises poly-2- (2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)- ethoxy)acetic acid (pMAEEA), or a derivative thereof. In some embodiments of formula (A5’), the polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17. In some embodiments of formula (A5’), n is 8 or 14. In some embodiments of formula (A5’), n is 8. In some embodiments of formula (A5’), n is 14. In some embodiments of formula (A5’), n is 8 and R1 and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A5’), n is 14 and R1 and R2 are CH3(CH2)i6C(O)- (stearoyl). In some embodiments of formula (A5’), n is 8 and R1 and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5’), n is 14 and R1 and R2 are CH3(CH2)i2C(O)- (myristoyl).

[0344] In some embodiments of formula (A5’), the polymer P comprises a pSar. In some embodiments of formula (A5’), the polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40. In some embodiments, s is 20 or 23. In some embodiments, s is 20 and R1 and R2 are CH3(CH2)i6C(O)- (stearoyl). In some embodiments, s is 20 and R1 and R2 are CH3(CH2)i2C(O)- (myristoyl).

[0345] In some embodiments of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a compound comprising a thiol or cysteine group. In some embodiments of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the compound comprising a thiol or cysteine group comprises the formula HS(CH2)nC(O)-, wherein n ranges from 1 to 5. In some embodiments, n is 2. In some embodiments, X2 comprises a thiosuccinimide moiety. In some embodiments, X2 comprises the following general formula:

[0346] In some embodiments of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), comprises the following general formula: wherein nl and n2 are independently 1 to 5. In some embodiments, nl is 1 and n2 is 2. In some embodiments, nl is 2 and n2 is 1.

[0347] In some embodiments of the targeting compound, such as the compound of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the binding moiety B comprises a moiety binding to a cell surface antigen, e.g., a primary targeting moiety described herein. In some embodiments of the targeting compound, such as the compound of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the binding moiety B comprises a moiety binding to a docking compound as defined herein. In some embodiments of the targeting compound, such as of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the binding moiety B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0348] In one embodiment, the targeting compound is of the general formula (A10): L-X1-P-X2-B (A10) wherein:

[0349] P comprises a polymer;

[0350] L comprises a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;

[0351] B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein, attached to a second end of the polymer;

[0352] XI is absent or a first linking moiety; and

[0353] X2 is absent or a second linking moiety.

[0354] In one embodiment, the targeting compound is of the general formula (A10): L-X1-P-X2-B (A10) wherein:

[0355] P comprises a polymer;

[0356] L comprises a hydrophobic moiety (e.g., lipid) attached to a first end of the polymer;

[0357] B comprises (i) a moiety binding to a cell surface antigen, (e.g., a primary targeting moiety described herein), or (ii) a moiety binding to a peptide tag (e.g., an ALFA-tag binding moiety, such as a single-domain antibody (sdAb), NbALFA-nanobody, as defined herein), attached to a second end of the polymer;

[0358] XI is absent or a first linking moiety; and X2 is absent or a second linking moiety. In some embodiments of formula (A10), XI comprises a carbonyl group. In some embodiments of formula (A10), L comprises a phosphatidylethanolamine which may be linked to P by an amide group.

[0359] In some embodiments of formula (A10), X2 comprises the reaction product of a thiol or cysteine reactive group, e.g., a maleimide group, with a thiol or cysteine group of a compound comprising the epitope tag. In some embodiments of formula (A10), X2 comprises a thiosuccinimide moiety.

[0360] In some embodiments of formula (A10), L comprises a lipid as described above. In some embodiments of formula (A10), L comprises DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine) which may be linked to P by an amide group.

[0361] In some embodiments of formula (A10), P comprises a polymer as described above. In some embodiments of formula (A10), P comprises a polymer which provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion. In some embodiments of formula (A10), P comprises a polymer selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof). In some embodiments of formula (A10), P comprises polyethylene glycol (PEG); e.g., PEG as described above.

[0362] In some embodiments of formula (A10), L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments of formula (A10), the amphiphilic derivative of a polymer comprises a conjugate of disteroyl-glycero- phosphoethanolamine (DSPE) and a polymer, e.g., a polymer as described above. In some embodiments of formula (A10), the amphiphilic derivative of a polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG). In some embodiments of formula (A10), the targeting compound is obtainable by reacting the thiol or cysteine reactive group of a reagent comprising an amphiphilic derivative of a polymer, e.g., a PEG reagent comprising a hydrophobic moiety (e.g., lipid), with a thiol or cysteine group of a compound comprising the primary targeting moiety or epitope tag. In some embodiments of formula (A10), the thiol or cysteine reactive group comprises a mal eimide group. In some embodiments of formula (A10), the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments of formula (A10), the compound comprising the primary targeting moiety or epitope tag comprises the formula HS(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag. In some embodiments of formula (A10), n is 2.

[0363] In some embodiments of formula (A10), the targeting compound comprises the reaction product of l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(polyethylene glycol)] with a compound comprising the formula HS(CH2)nC(O)-B, wherein n ranges from 1 to 5 and B comprises the primary targeting moiety or epitope tag. In some embodiments of formula (A10), n is 2.

[0364] In some embodiments, the targeting compound is of the following general formula (A10’): wherein B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0365] In some embodiments, the targeting compound is of the following general formula

[0366] (A10”) wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein n is 5 to 50, e.g., 5 to 25, e.g., 7 to 14, e.g., 10 to 25, e.g., 14 to 17, e.g., 8 or

[0367] 14, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0368] In some embodiments of formula (A10”), n is 8 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A10”), n is 14 and Rl and R2 are CH3(CH2)i6C(O)- (stearoyl). In some embodiments of formula (A10”), n is 8 and Rl and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A10”), n is 14 and Rl and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A10”), X2 is of the following general formula:

[0369] In some embodiments, the targeting compound (hydrophobic moiety having a binding moiety covalently attached thereto) comprises the following general formula (A10’”): wherein X2 is as described above, Ri and R2 are CH3(CH2)ieC(O)- (stearoyl) or CH3(CH2)i2C(O)- (myristoyl), polymer P comprises the following general formula: wherein s is 2 to 200, e.g., 5 to 100, e.g., 10 to 50, e.g., 15 to 40, e.g., 20 or 23, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein. In some embodiments of formula (A10’”), s is 20 and Ri and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A10’”), s is 20 and Ri and R2 are CH3(CH2)i2C(O)- (myristoyl).

[0370] In some embodiments of formula (A10’”), X2 comprises the following general formula:

[0371] In some embodiments of formula (A10’”), B comprises a moiety comprising the structure -N-peptide-C(O)-NH2, wherein peptide comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

[0372] The present disclosure provides in one aspect, a targeting compound as described above which is integrated in a particle (e.g., a particle as described herein) via a hydrophobic component (e.g., lipid component) of the targeting compound.

[0373] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (A10’”), the binding moiety B is selected from the group consisting of a moiety binding to a cell surface antigen, a peptide tag, and a moiety binding to a peptide tag.

[0374] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (A10’”), the binding moiety B comprises a peptide or polypeptide.

[0375] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (A10’”), the moiety binding to a cell surface antigen or to a peptide tag comprises an antibody or antibody-like molecule.

[0376] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (Al O’”), the cell surface antigen is characteristic for an immune effector cell.

[0377] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (A10’”), the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0378] In one embodiment of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”) or (A10’”), the peptide tag comprises an ALFA-tag.

[0379] In some embodiments, the targeting compound is of Formula (A20):

[0380] L-X1-P-X2-B (A20) wherein

[0381] P comprises a polymer;

[0382] L comprises a hydrophobic moiety attached to a first end of the polymer;

[0383] B comprises a binding moiety attached to a second end of the polymer;

[0384] XI is absent or a first linking moiety; and

[0385] X2 is absent or a second linking moiety.

[0386] In some embodiments of Formula (A20), XI comprises a carbonyl group.

[0387] In some embodiments of Formula (A20), X2 comprises the reaction product of a maleimide group with a thiol or cysteine group of a compound comprising the binding moiety.

[0388] In some embodiments of Formula (A20), the hydrophobic moiety is or is comprised in a lipid. In some embodiments of Formula (A20), the lipid comprises a phospholipid, e.g., l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

[0389] In some embodiments of Formula (A20), the polymer provides stealth property, extends circulation half-life and / or reduces non-specific protein binding or cell adhesion. In some embodiments of Formula (A20), the polymer comprises polyethylene glycol (PEG). The average molecular weight of the PEG may range from 200 to 10,000, preferably 500 to 5000, more preferably 1000 to 4000, most preferably 2000.

[0390] In some embodiments of Formula (A20), the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoyl-glycero- phosphoethanolamine-polyethylene glycol-conjugate (DSPE-PEG).

[0391] In some embodiments of Formula (A20), the binding moiety covalently attached to the hydrophobic moiety comprises a peptide, preferably the binding moiety comprises an ALFA-tag.

[0392] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag comprises the amino acid sequence -AA0- AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are:

[0393] AA0 is Pro or deleted;

[0394] AA1 is Ser, Gly, Thr, or Pro;

[0395] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0396] AA3 is Leu, He, or Vai;

[0397] AA4 is Glu or Gin;

[0398] AA5 is Glu or Gin;

[0399] AA6 is Glu or Gin;

[0400] AA7 is Leu, He, or Vai;

[0401] AA8 is Arg, Ala, Gin, or Glu;

[0402] AA9 is Arg, Ala, Gin, or Glu;

[0403] AA10 is Arg;

[0404] AA11 is Leu;

[0405] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0406] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0407] AA14 is Pro or deleted. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE (SEQ ID NO: 1), P SRLEEELRRRLTE (SEQ ID NO: 2), SRLEEELRRRLTEP (SEQ ID NO: 3), and P SRLEEELRRRLTEP (SEQ ID NO: 4).

[0408] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag comprises the cyclized amino acid sequence - AA0-AA1 -AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11 -AA12-AA13 - AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not XI and X2 are: AAO is Pro or deleted;

[0409] AA1 is Ser, Gly, Thr, or Pro;

[0410] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0411] AA3 is Leu, He, or Vai;

[0412] AA4 is Glu or Gin;

[0413] AA5 is Glu or Gin;

[0414] AA6 is Glu or Gin;

[0415] AA7 is Leu, He, or Vai;

[0416] AA8 is Arg, Ala, Gin, or Glu;

[0417] AA9 is Arg, Ala, Gin, or Glu;

[0418] AA10 is Arg;

[0419] AA11 is Leu;

[0420] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0421] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0422] AA14 is Pro or deleted. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), XI and X2 are separated by 2 or 3 amino acids.

[0423] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA10 is X2 or AA4 is XI and AA8 is X2.

[0424] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A 10”’) or (A20), an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of a. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12- AA13-AA14-, b. -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12- AA13-AA14-, c. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)- AA14-, d. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12- AA13-AA14-, e. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-X2)-AA13- AA14-, f. -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(Xl-Leu-AA12-X2)- AA14-, g. -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-AA9-X2)-Leu-AA12- AA13-AA14-, and h. -AA0-AAl-AA2-AA3-cyclo(Xl-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12- AA13-AA14-, wherein the side-chains of XI and X2 amino acid residues are connected covalently; AAO is Pro or deleted;

[0425] AA1 is Ser, Gly, Thr, or Pro; AA2 is Arg, Gly, Ala, Glu, or Pro;

[0426] AA3 is Leu, He, or Vai;

[0427] AA4 is Glu or Gin;

[0428] AA5 is Glu or Gin;

[0429] AA6 is Glu or Gin;

[0430] AA7 is Leu, He, or Vai;

[0431] AA8 is Arg, Ala, Gin, or Glu;

[0432] AA9 is Arg, Ala, Gin, or Glu;

[0433] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0434] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0435] AA14 is Pro or deleted.

[0436] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), XI and X2 are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2,3-triazole.

[0437] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of XI and X2 to the carboxyl group of a side-chain of the other of XI and X2 via an amide bond. The amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.

[0438] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of XI and X2 to the sulfhydryl group of a side-chain of the other of XI and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl- containing amino acids as Pen.

[0439] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), XI and X2 are, independently, selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when XI is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when XI is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0440] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), XI is Glu and X2 is Lys. In some embodiments, - cyclo(Glu - Lys)-, -c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E -

[0441] K- cyclo, or -cycloE — cycloK comprises the following structure:

[0442] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), XI is Lys and X2 is Glu. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), -cyclo(Lys- — Glu)-, -c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE comprises the following structure: In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), XI is Cys and X2 is Cys. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), -cyclo(Cys- - Cys)-, c(Cys - Cys)-, -cyclo(C - C)-, -c(C - C)-, -C — C- cyclo, or - cycloC - cycloC comprises the following structure:

[0443] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), the cyclized amino acid sequence is -Ser-Arg-Leu-Glu- cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 5). In some other embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu- Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 6). In yet some other embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu- Thr-Glu- (SEQ ID NO: 7). In still some other embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 8).

[0444] The cyclic peptides may have different cyclic bridging moieties forming the ring structure. Preferably, chemically stable bridging moieties are included in the ring structure such as, for example, an amide group, a lactone group, an ether group, a thioether group, a disulfide group, an alkylene group, an alkenyl group, or a 1,2,3- triazole. The following are examples illustrating the variability of bridging moieties in a peptide:

[0445] The targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20) may be comprised in the lipid mixture as described herein, as incorporated into the aqueous dispersion. The targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20) may not be comprised in the lipid mixture, and may instead be subsequently added to the lipid particles comprised in the dispersed phase of the aqueous dispersion. The targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), such as a peptide-conjugated lipid, may not be comprised in the lipid mixture, and may instead be subsequently added to the nucleic acid-lipid particles. Where the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20) such as a peptide- conjugated lipid, is added to the lipid particles comprised in the dispersed phase of the aqueous dispersion or to the nucleic acid-lipid particles, the amount of targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), such as a peptide- conjugated lipid, added may displace the corresponding amount of steroid (e.g., cholesterol) in the particle. The peptide-conjugated lipid is typically added to the particle at a final molar ratio of 0.1-0.3 mol%, optionally about 0.2 mol %, of the total lipid.

[0446] When the nucleic acid-lipid particles comprise targeting compounds, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (Al O’), (A10”), (Al O’”) or (A20), such as peptide-conjugated lipids, this allows for functionalization of the nucleic acid-lipid particles. For example, the binding moiety B that specifically binds to the peptide of the peptide-conjugated lipid may be bound to the nucleic acid-lipid particles, wherein the binding moiety may also bind to target cells (for example by specifically binding a target cell surface antigen). This may provide for targeted delivery of the nucleic acid comprised within the functionalized nucleic acid-lipid particles. The binding moiety that specifically binds to the peptide of the compound may be an ALFA-tag binding moiety. In one embodiment, the binding moiety that specifically binds to the peptide of the compound is other than an ALFA-tag binding moiety.

[0447] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20)„ an ALFA- tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.

[0448] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTX1SALNAMAMG (SEQ ID NO: 9), wherein XI is I or V, the CDR2 sequence AVSX2RGNAM (SEQ ID NO: 10), wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 11).

[0449] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20)„ an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG, wherein XI is I or V, the CDR2 sequence AVSX2RGNAM, wherein X2 is E, H, N, D, or S, and the CDR3 sequence LEDRVDSFHDY.

[0450] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (Al O’”) or (A20), an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMV AAVSERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVL EDRVDSFHDYWGQGTQVTVSS (SEQ ID NO: 12), an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence, or a fragment of said amino acid sequence or the amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to said amino acid sequence.

[0451] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above.

[0452] In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20)„ an ALFA-tag binding moiety comprises a bispecific antibody which targets ALFA-tag and a cell surface antigen. In some embodiments of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20)„ an ALFA- tag binding moiety comprises a moiety binding to a peptide comprising an ALFA-tag and a moiety targeting a cell surface antigen.

[0453] Preferably the binding moiety is a peptide, and the compositions contain described herein may also contain a peptide-conjugated lipid. In the present specification the term “peptide-conjugated lipid” in its broadest sense means a lipid or lipid-like material, as defined above (either in a broadest aspect or a preferred aspect) conjugated to a peptide. In this aspect “peptide” is synonymous with “polypeptide” and “protein”. In one embodiment the peptide comprises an ALFA-tag, (i.e., the peptide conjugated lipid may be an ALFA-conjugated lipid). Such peptide-conjugated lipids are described in more detail in WO 2023 / 148276.

[0454] In one embodiment, the targeting compound is not DSPE-PEG2k-Alfa. In one embodiment, the targeting compound is not DOPE-(G2SG2)2 Alfa. In one embodiment, the targeting compound is not DOPE-(G2SG2)4 Alfa. In one embodiment, the targeting compound is not DOPE-(pSar)20 Alfa. In one embodiment, the targeting compound is not DOPE-(pSar)10 Alfa. In one embodiment, the targeting compound is not DSPE-(pAEEA)14 Alfa.

[0455] Aqueous Dispersion

[0456] The present invention provides in one aspect, an aqueous dispersion having an aqueous mobile phase and a dispersed phase, the dispersed phase comprising a lipid mixture including a compound of Formula (A), as defined herein, in either its broadest aspect or a preferred aspect. In this specification the term “dispersion” in its broadest sense takes its usual meaning in chemistry as a system in which distributed particles of one material (the “dispersed phase”) are dispersed in a phase of another material (the “continuous phase” or the “mobile phase”).

[0457] In one embodiment, there is provided an aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein: P is absent or comprises a polymer;

[0458] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0459] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0460] XI is absent or a first linking moiety; and

[0461] X2 is absent or a second linking moiety.

[0462] In one embodiment, the dispersion is a solid-liquid dispersion, in which the dispersed phase is solid and the mobile phase is a liquid. In one embodiment, the dispersion is a liquid-liquid dispersion, in which the dispersed phase and the mobile phase are both liquids.

[0463] In one embodiment, the dispersion is a colloid. The term "colloid" as used herein describes a stable mixture in which the dispersed particles do not settle out. Typically, the dispersed particles have at least in one direction a dimension roughly between 1 nm and 1 pm, or in such a system discontinuities are found at distances of that order.

[0464] In one embodiment, the dispersion is a suspension. The term “suspension” as used herein is a heterogeneous dispersion of larger particles in a medium. Unlike solutions and colloids, if left undisturbed for a long periods of time, the suspended particles may settle out of the mixture. The use of the terms “colloid” and “suspension” is sometimes overlapping or synonymous, with colloids in some instances being considered a sub-type of suspensions.

[0465] In one embodiment, the mobile phase is a solution. The term “solution” as used herein is a homogeneous mixture comprising a solvent which is typically water and solutes which can be salts, buffers, tonifiers and the like, as long as these materials are molecularly distributed within the solvent. The mobile phase may comprise solutes, as described further herein.

[0466] In one embodiment, the dispersed phase comprises a lipid mixture including a cationic or cationically ionisable lipid, as defined herein. In one embodiment, the aqueous dispersion is substantially free of inorganic cations.

[0467] In one embodiment, the aqueous dispersion is substantially free of organic solvents.

[0468] In one embodiment, the aqueous dispersion is substantially free of nucleic acids. In one embodiment, the aqueous dispersion is substantially free of RNA.

[0469] In one embodiment, the aqueous dispersion comprises a cryoprotectant, as defined and exemplified below.

[0470] In one embodiment, the aqueous dispersion comprises an anion of an aqueous acid, as defined and exemplified below.

[0471] In one embodiment, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and a cationic or cationically ionisable lipid; and the aqueous mobile phase comprises an anion of an aqueous acid; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA, and wherein the aqueous mobile phase comprises a cryoprotectant.

[0472] In one embodiment, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and a cationic or cationically ionisable lipid; and the aqueous mobile phase comprises an anion of an aqueous acid; wherein: the concentration of the aqueous acid is at least 6mM; and the aqueous mobile phase is substantially free of inorganic cations, organic solvents and RNA.

[0473] In one embodiment, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and a cationic or cationically ionisable lipid; and the aqueous mobile phase comprises malate anion or a succinate anion; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA. In one embodiment, the aqueous dispersion has a maximum pH of about 6.0, for example such as a maximum pH between 5.8 to 6.0. In one embodiment, the aqueous dispersion has a maximum pH of about 4.5, for example such as a maximum pH between 4.2 to 4.8. In one embodiment, the aqueous dispersion has a maximum pH of 4.5. The aqueous dispersion may have a pH of less than 4.5.

[0474] In one embodiment, the aqueous dispersion has a pH of from about 2.5 to about 6.0, optionally from about 2.5 to about 5.5, optionally from about 2.5 to about 5.0. In one embodiment, the aqueous dispersion has a pH of from about 2.5 to about 4.5. The aqueous dispersion may have a pH of 2.5 to 4.5. The aqueous dispersion may have a pH of 2.5 to 3.5 or 3.5 to 4.5. The aqueous dispersion may have a pH of 4.0 to 4.5. The aqueous dispersion may have a pH of about 4.5.

[0475] In one embodiment, the aqueous mobile phase comprises an anion of an aqueous acid. The acid may be any inorganic or organic acid which is at least partially miscible with water, and is capable of being at least partially deprotonated in water to produce the anion (i.e. the conjugate base) of the acid. It will therefore be understood by the skilled person that, depending on the pH and the strength of the acid, the aqueous mobile phase may contain both the undissociated acid and its corresponding anion in varying proportions. Strong acids are fully or largely deprotonated in water, so that the species in aqueous solution is mainly (in some embodiments completely) the anion of the acid. In contrast, weak acids are not fully deprotonated in water, so that the species in aqueous solution will comprise a mixture of undissociated acid and its conjugate base, the relative amounts of each depending on the pH.

[0476] In one embodiment, the anion is an acetate anion. In one embodiment, the anion is a malate anion. In one embodiment, the anion is a succinate anion. In one embodiment, the anion is a citrate anion.

[0477] Furthermore, the aqueous acid may undergo an acid-base reaction with a cationically ionisable lipid to produce the cationically ionisable lipid in its charged form and the acid in its anionic form. The extent to which such a reaction occurs depending on factors such as the basicity of the cationically ionisable lipid (when present in neutral form) and the pH.

[0478] Additionally, the anion of the aqueous acid may interact with the constitutively charged head group of a cationic lipid to form a lipid salt. It is expected that interactions between the anion of the aqueous acid and the cationic lipid will promote the formation of stable lipid particles. For example, formation of a lipid salt between the anion of the aqueous acid and a cationic lipid may affect the shape factor kappa (K) (i.e., the volume ratio between the polar and apolar section of a lipid; K = molecular volume (head, polar) / molecular volume (tail, apolar)) of the cationic lipid and promote formation of lipid nanoparticle structures (see, e.g., W02008 / 043575, W02009 / 047006, Siepi et al., Biophys J 2011, 100, 2412-2421). For example, the lipid salt may have a shape factor K of less than 0.25, optionally less than 0.15.

[0479] In one embodiment, the aqueous acid is an inorganic acid. Examples of suitable inorganic acids include hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydriodic acid, nitric acid, sulphuric acid and phosphoric acid.

[0480] In one embodiment, the aqueous acid is a water-soluble organic acid. Examples of suitable inorganic acids include sulfonic acids, carboxylic acids, dicarboxylic acids, hydroxy carboxylic acids (all as defined herein) or amino acids.

[0481] In one embodiment, the water-soluble organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid and citric acid, or combinations thereof. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, oxalic acid and citric acid. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, and succinic acid. The water-soluble organic acid may be selected from the group consisting of acetic acid, malic acid, maleic acid, succinic acid, ascorbic acid, and oxalic acid. The water- soluble organic acid may be selected from the group consisting of malic acid, maleic acid, succinic acid, ascorbic acid, and oxalic acid and citric acid. The water-soluble organic acid may be selected from the group consisting of malic acid, maleic acid, succinic acid, ascorbic acid, and oxalic acid.

[0482] In one embodiment, the water-soluble weak organic acid is acetic acid. In one embodiment, the water-soluble weak organic acid is malic acid. In one embodiment, the water-soluble weak organic acid is succinic acid. In one embodiment, the water- soluble weak organic acid is citric acid.

[0483] In one embodiment, the aqueous dispersion is substantially free of acetate buffers and citrate buffers. The aqueous dispersion may be substantially free of acetate buffers. The aqueous dispersion may be substantially free of citrate buffers. The aqueous mobile phase may be substantially free of citrate buffers. The aqueous dispersion and / or the aqueous mobile phase may be substantially free of a citrate buffer containing about 10 mM citrate, about 150 mM NaCl, pH of about 4.5. The aqueous dispersion may be substantially free of buffering agents. The aqueous dispersion may be substantially free of an acetate buffer, a citrate buffer, a phosphate buffer, and / or a tris buffer. The aqueous dispersion may be substantially free of a buffering agent selected from the group consisting of ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. The aqueous dispersion may be substantially free of a buffering agent selected from the group consisting of ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, tri s(hydroxymethyl)aminom ethane (tris), sodium phosphate, and HEPES.

[0484] In one embodiment, the aqueous dispersion is substantially free of inorganic cations. Such inorganic cations are thought to affect the colloidal stability of the lipid dispersion and reduce the stability of the formulations. In one embodiment the aqueous dispersion is substantially free of inorganic cations such as ammonium, sodium and / or potassium ions.

[0485] In one embodiment, the aqueous dispersion is substantially free of organic solvents. For example, the aqueous dispersion may be substantially free of water-soluble organic solvents, such as Ci-4 alcohols (e.g. isopropanol or ethanol), ketones (e.g. acetone), or mixtures thereof; and / or apolar organic solvents, such as hydrocarbons such as pentane or hexane; chlorinated hydrocarbons such as di chloromethane or chloroform; or mixtures thereof. In one embodiment, the aqueous dispersion is substantially free of organic solvents including isopropanol, ethanol, and / or acetone.

[0486] In one embodiment, the concentration of the aqueous acid is at least 6mM. In one embodiment, the concentration of the aqueous acid is in the range of 1 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 20 mM. In one embodiment, the concentration of the aqueous acid is in the range of 2.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 5.5 to 10 mM. In one embodiment, the concentration of the aqueous acid is in the range of 6 to 10 mM. It will be understood in this context that this concentration includes both the undissociated acid and its conjugate base.

[0487] In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between about 20: 1 and about 1 :20. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between about 10: 1 and about 1 : 10. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between about 5: 1 and about 1 :5. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between about 3 : 1 and about 1 :3. It will be understood in this context that the moles of cationic or cationically ionisable lipid includes both the unionised lipid and its conjugate acid, and the moles of aqueous acid includes both the undissociated acid and its conjugate base.

[0488] When the acid is a strong acid, in one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 : 10 and 10: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :5 and 5:1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :3 and 3: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :2 and 2: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 : 1.5 and 1.5: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 : 1.2 and 1.2: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of 1 : 1.

[0489] When the acid is a weak acid, in one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :20 and 5: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 : 10 and 2.5: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :6 and 1.5:1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :4 and 1.25: 1. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of between 1 :3 and 1 : 1.33. In one embodiment, the cationic or cationically ionisable lipid and the anions of aqueous acid are present in a molar ratio of 1 :2.

[0490] In some instances, the aqueous dispersion comprises a dispersed phase comprising lipid particles. In some instances, the lipid particles of the dispersed phase are lipid nanoparticles. In some instances, the lipid particles of the dispersed phase are not liposomes. In some instances, the aqueous dispersion comprises a dispersed phase comprising lipid particles having a size (i.e., a diameter) of from about 20nm to about 500nm, from about 20nm to about 200nm, from about 30nm to about 180nm, from about 40nm to about 120nm, or preferably from about 40nm to about 80nm. In some instances, the aqueous dispersion comprises a dispersed phase comprising lipid particles having a size (i.e., a diameter) of not more than about 200nm.

[0491] In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of anions of aqueous acids. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain an anion of an aqueous acid. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of acetate ions. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain acetate ions. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of malate ions. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain malate ions. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of succinate ions. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain succinate ions.

[0492] In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of a cryoprotectant. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain a cryoprotectant.

[0493] In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of cationic lipids. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain a cationic lipid.

[0494] In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of amino acids. In one embodiment of the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain an amino acid.

[0495] Storage Matrix - Cryoprotectants and other ingredients

[0496] In one embodiment, the aqueous dispersion (typically containing pre-LNPs) also contains a storage matrix. In this specification, the term “storage matrix” when used in its broadest sense typically covers any substance typically used to aid storage and improve the shelf-life of the aqueous dispersion. The storage matrix is typically added to the aqueous dispersion after the filtration (e.g., TFF) / dialysis step.

[0497] In one embodiment, the storage matrix comprises a cryoprotectant. In this specification, the term “cryoprotectant” when used in its broadest sense means any substance capable of protecting a composition from damage caused by freezing and / or by ice formation. Examples of cryoprotectants include glycols (i.e. alcohols containing at least two hydroxy groups, such as glycerol and propylene glycol) and carbohydrates, as defined and exemplified herein.

[0498] In one embodiment, the cryoprotectant is a carbohydrate. In one embodiment, the cryoprotectant is a monosaccharide or disaccharide. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose, lactose and glucose, or a mixture of any thereof. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, trehalose and glucose, or a mixture of any thereof. Preferably, the cryoprotectant is sucrose.

[0499] When the aqueous dispersion also contains a storage matrix which is a carbohydrate, typically, this is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 10% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 10% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 15% to about 25% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 18% to about 22% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 20% (w / v).

[0500] In one embodiment, the storage matrix is glucose and is present in a concentration of about 1% to about 15% (w / v). In one embodiment, the storage matrix is glucose and is present in a concentration of about 2% to about 10% (w / v). In one embodiment, the storage matrix is glucose and is present in a concentration of about 4% to about 8% (w / v). In one embodiment, the storage matrix is glucose and is present in a concentration of about 5% (w / v). In one embodiment, the storage matrix is glucose and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is glucose and is present in a concentration of about 10% (w / v).

[0501] In one embodiment, the storage matrix is provided in one or more water-soluble weak organic acids. In one embodiment, the water-soluble weak organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid and succinic acid. In one embodiment, the water-soluble weak organic acid is acetic acid.

[0502] In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 0.5 mM to 50 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 1 mM to 25 mM. In one embodiment, the concentration of the water- soluble weak organic acid used in the storage matrix is from about 2.5 mM to 10 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 4 mM to 6 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is about 5 mM.

[0503] In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 0.5 mM to 50 mM.

[0504] In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 1 mM to 25 mM. In one embodiment, the water-soluble weak organic acid used in storage matrix is acetic acid and the concentration of the acetic acid is from about 2.5 mM to 10 mM. In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 4 mM to 6 mM. In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is about 5 mM.

[0505] Method of Forming Aqueous Dispersion

[0506] In a further aspect, the present disclosure provides methods for producing the aqueous dispersion of the invention. In this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A), as defined above, either in its broadest aspect or a preferred aspect, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0507] In one embodiment, the aqueous mobile phase is substantially free of inorganic cations.

[0508] In one embodiment, the aqueous mobile phase is substantially free of organic solvents. In one embodiment, the aqueous mobile phase is substantially free of nucleic acids.

[0509] In one embodiment, the aqueous dispersion is substantially free of RNA.

[0510] In one embodiment, the aqueous mobile phase comprises a cryoprotectant, as defined and exemplified below.

[0511] In one embodiment, the aqueous mobile phase comprises an anion of an aqueous acid, as defined and exemplified below.

[0512] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0513] P is absent or comprises a polymer;

[0514] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0515] B comprises (i) a moiety capable of binding to a cell surface antigen, said moiety comprising a peptide or protein, or (ii) a moiety capable of binding to a peptide tag, said moiety comprising a peptide or protein; the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0516] XI is absent or a first linking moiety; and

[0517] X2 is absent or a second linking moiety; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0518] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A), as defined herein; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion; wherein (i) the aqueous mobile phase is substantially free of organic solvents; (ii) the aqueous mobile phase is substantially free of nucleic acids; (iii) the aqueous mobile phase is substantially free of inorganic cations; and / or (iv) the aqueous dispersion contains a cryoprotectant.

[0519] In one embodiment of this aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase comprising an anion of an aqueous acid; and a dispersed phase comprising a lipid mixture including a compound of Formula (A), as defined herein, the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0520] In one embodiment, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A): L-X1-P-X2-B (A) wherein:

[0521] P is absent or comprises a polymer;

[0522] L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;

[0523] B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;

[0524] XI is absent or a first linking moiety; and

[0525] X2 is absent or a second linking moiety the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion.

[0526] In an alternative aspect, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect, the method comprising: (a) mixing a lipid mixture, wherein the lipid mixture does not comprise the compound of formula (A), and the aqueous phase to produce a first intermediate aqueous dispersion, and

[0527] (b) mixing the first intermediate aqueous dispersion with the compound of formula (A) to produce the aqueous dispersion.

[0528] In both such methods, the methods may optionally further comprise mixing a compound of formula (I) as defined above, either in its broadest aspect or a preferred aspect, with the compound of formula (A). In such aspects, the resulting aqueous dispersion comprises (i) an aqueous mobile phase; and (ii) a dispersed phase comprising a lipid mixture including a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect, and (ii) a compound of formula (I) as defined above, either in its broadest aspect or a preferred aspect, wherein the compound of formula (I) interacts with the dispersed phase.

[0529] In other alternative aspects, there is provided a method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid (wherein the lipid mixture preferably does not comprise a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect), the method comprising: mixing the lipid mixture comprising a cationic or cationically ionizable lipid and the aqueous phase to produce the aqueous dispersion. In such alternative aspects, the compound of Formula (A) is added at later stages.

[0530] In some embodiments, the compound of Formula (A) is present in an amount of 0.01 to 10 mol%, optionally 0.05 to 5 mol%, of the lipid mixture. In some embodiments, the compound of Formula (A) is present in an amount of 0.1 to 2 mol%, optionally 0.1 to 1 mol%, of the lipid mixture.

[0531] A number of methods of making dispersions are known in the art and the skilled person would be readily capable of selecting a suitable method and applying this to make the dispersion compositions of the present invention.

[0532] In one embodiment, the aqueous phase is acidified. In one embodiment, the aqueous phase comprises an aqueous acid. In one embodiment, the aqueous acid is selected from the group consisting of acetic acid, malic acid and citric acid. In one embodiment, the aqueous acid is acetic acid.

[0533] In one embodiment, the aqueous phase contains an acidic buffer. As is known to the person skilled the art, an acidic buffer comprises a weak acid and its conjugate base. In one embodiment, the acidic buffer is an acetic acid / acetate buffer. In one embodiment, the acidic buffer is a citric acid / citrate buffer. In one embodiment, the acidic buffer is a phosphate buffer. In one embodiment, the acidic buffer is a malic acid / malate buffer. In one embodiment, the acidic buffer is a succinic acid / succinate buffer.

[0534] In one embodiment, the aqueous phase is substantially free of inorganic cations.

[0535] In one embodiment, the method comprises:

[0536] (a) mixing:

[0537] (i) a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid dissolved in a water-soluble organic solvent; and

[0538] (ii) an aqueous phase; the lipid mixture and / or the aqueous phase comprising the aqueous acid; to produce an intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid; and

[0539] (b) performing on the intermediate acidified aqueous lipid dispersion a dialysis or filtration step at a pH of about 2.5 to about 5.5, to remove the organic solvent and produce the aqueous dispersion.

[0540] In one embodiment, the dialysis or filtration step comprises tangential flow filtration.

[0541] In one embodiment, the method further comprises the step (c) subsequent to step (b):

[0542] (c) adding a cryoprotectant to the aqueous dispersion.

[0543] In one embodiment, the method comprises mixing: (i) a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid; and

[0544] (ii) an aqueous phase comprising an aqueous acid and a cryoprotectant; to produce the aqueous dispersion comprising an anion of the aqueous acid.

[0545] In one embodiment, the method comprises:

[0546] (a) mixing:

[0547] (i) a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid; and

[0548] (ii) an aqueous phase comprising an aqueous acid; to produce a first intermediate aqueous dispersion comprising an anion of the aqueous acid; and

[0549] (b) adding the cryoprotectant to the first intermediate aqueous dispersion to produce the aqueous dispersion.

[0550] In one embodiment, the method comprises:

[0551] (a) mixing:

[0552] (i) a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid dissolved in a water-soluble organic solvent; and

[0553] (ii) an aqueous phase; the lipid mixture and / or the aqueous phase comprising the aqueous acid; to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid;

[0554] (b) performing on the first intermediate acidified aqueous lipid dispersion a dialysis or filtration step at a pH of about 2.5 to about 5.5, to remove the organic solvent and produce a second intermediate aqueous dispersion; and

[0555] (c) adding a cryoprotectant to the second intermediate aqueous dispersion; to produce the aqueous dispersion comprising an anion of the aqueous acid. The addition of the cryoprotectant typically does not affect the pH, such that the pH of the aqueous dispersion comprising an anion of the aqueous acid is essentially the same as that of the second intermediate aqueous dispersion, i.e., about 2.5 to about 5.5.

[0556] In one embodiment, the method comprises: i) mixing a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid dissolved in a water-soluble organic solvent with an aqueous phase, wherein the lipid solution and / or the aqueous phase comprises an aqueous acid, to produce a first intermediate acidified aqueous lipid dispersion comprising an anion of the aqueous acid; ii) performing on the first intermediate acidified aqueous lipid dispersion a dialysis or filtration step at a pH of about 2.5 to about 5.5, or at a pH of about 6.5 to about 8.5, to remove the organic solvent and produce a second intermediate aqueous dispersion; and iii) adding a cryoprotectant to the second intermediate aqueous dispersion; to produce the aqueous dispersion; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA. The addition of the cryoprotectant typically does not affect the pH, such that the pH of the aqueous dispersion is essentially the same as that of the second intermediate aqueous dispersion, i.e., about 2.5 to about 5.5 or about 6.5 to about 8.5.

[0557] In one embodiment, the method comprises: i) preparing a solution of a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid dissolved in water-soluble and / or apolar organic solvents (preferably wherein such solvents are volatile); ii) evaporation of the organic solvent below atmospheric pressure, to provide the lipid mixture in the form of a film (or layer) of lipids, optionally a thin film, typically a homogenous thin film; iii) addition of an aqueous acid to the film (e.g., thin film) of lipid mixture, to produce the aqueous dispersion; and iv) diluting the aqueous dispersion with a cryoprotectant; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA. An exemplary suitable solvent for dissolving the lipid mixture in step i) may be, for example, a 1 : 1 mixture of methanol and dichloromethane.

[0558] This method is referred to herein as “the thin film method”.

[0559] In one embodiment of the above thin film method, the method further comprises the following step ii’) after step ii): ii’) reduction of the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably by pore size extrusion.

[0560] In one embodiment, the method comprises: i) preparing a solution of a lipid mixture comprising the compound of formula (A) and a cationic or cationically ionisable lipid dissolved in a non-polar water immiscible organic solvent, or if required for lipid solubility a mixture of a non-polar water immiscible organic solvent and a polar organic solvent; ii) adding an aqueous phase to produce a first intermediate composition including the lipid mixture, wherein the solution of the lipid mixture and / or the aqueous phase are acidified; iv) removal of the organic solvents by standard unit operations, such as evaporation or filtration (preferably by evaporation), below atmospheric pressure, to produce a second intermediate composition including the lipid mixture; v) sonicating the second intermediate composition, to produce the aqueous dispersion; and vi) diluting the aqueous dispersion with a cryoprotectant; wherein the aqueous dispersion is substantially free of inorganic cations, organic solvents and RNA.

[0561] This method is referred to herein as “the emulsification method”.

[0562] In one embodiment, the emulsification method further comprises the following step v’) after step v): v’) reduction of the particle size of the aqueous dispersion by standard unit operations such as extrusion, sonication, homogenization, preferably pore size extrusion.

[0563] In some embodiments of the methods of the invention, the lipid mixture containing the cationically ionisable lipid, and optionally the compound of formula (A) is introduced into the mixture in an organic solvent. Typically, the organic solvent is a water-soluble organic solvent.

[0564] In the methods of the invention, the organic solvent (e.g., the water-soluble organic solvent) may be selected from the lists of Class 2 and Class 3 solvents, as described in the FDA’s “Q3C - Tables and List Guidance for Industry”, June 2017, Revision 3 (see, e.g., https: / / www.fda.gov / media / 71737 / download). When the organic solvent is a water-soluble organic solvent, examples include Ci-4 alcohols (e.g. isopropanol or ethanol), ketones (e.g. acetone), or mixtures thereof. When the organic solvent is an apolar organic solvent, examples include hydrocarbons such as pentane or hexane; chlorinated hydrocarbons such as dichloromethane or chloroform; or mixtures thereof. The organic solvent (e.g., the water-soluble organic solvent) is preferably ethanol or isopropanol.

[0565] In one embodiment, the lipid mixture does not comprise phosphatidylserine.

[0566] In one embodiment, the mixing is carried out using a T-mixer or Y-mixer.

[0567] In one embodiment, the flow rate during mixing is at least 50 mL / min. The flow rate during mixing may be from about 50 mL / min to about 400 mL / min, optionally from about 100 mL / min to about 300 mL / min, optionally from about 150 mL / min to about 250 mL / min. optionally from about 50 mL / min to about 150 mL / min, optionally from about 70 mL / min to about 110 mL / min, such as about 90 mL / min. The term “flow rate” in this sense means the total flow rate of all of the components of the mixture.

[0568] The volume ratio of organic solvent to aqueous phase may be from about 1 :6 to about 6: 1, optionally from about 1 :2 to about 1 :6, optionally from about 1:2.5 to about 1 :4.5, optionally from about 1 :2 to about 1 :4, optionally about 1 :3.

[0569] The pH of the aqueous dispersion as produced according to any of the above methods may be about 2.5 to about 5.5, optionally about 2.5 to about 4.5. The pH of the aqueous dispersion as produced according to any of the above methods may be about 2.5 to about 3.5. The pH of the aqueous dispersion as produced according to any of the above methods may be about 3.5 to about 4.5. The pH of the aqueous dispersion as produced according to any of the above methods may be about 6.5 to about 8.5, optionally 6.8 to 8.5, further optionally about 7.0 to about 8.0.

[0570] Further Processing Steps

[0571] In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more further processing steps. In one embodiment, the method further comprises subjecting the aqueous dispersion to one or more further dilution or purification steps.

[0572] In one embodiment, the purification steps comprise a dialysis or filtration step.

[0573] In one embodiment, the dialysis or filtration step is performed at a pH of about 4.0 to about 5.0. In one embodiment, the dialysis or filtration step comprise tangential flow filtration. In one embodiment, the dialysis or filtration step is carried out using one or more water-soluble weak organic acids. In one embodiment, the water-soluble weak organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid and succinic acid. In one embodiment, the water-soluble weak organic acid is acetic acid.

[0574] In one embodiment, the concentration of the water-soluble weak organic acid used in the dialysis or filtration step is from about 0.5 mM to 50 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the dialysis or filtration step is from about 1 mM to 25 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the dialysis or filtration step is from about 2.5 mM to 10 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the dialysis or filtration step is from about 4 mM to 6 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the dialysis or filtration step is about 5 mM.

[0575] In one embodiment, the water-soluble weak organic acid used in the dialysis or filtration step is acetic acid and the concentration of the acetic acid is from about 0.5 mM to 50 mM. In one embodiment, the water-soluble weak organic acid used in the dialysis or filtration step is acetic acid and the concentration of the acetic acid is from about 1 mM to 25 mM. In one embodiment, the water-soluble weak organic acid used in the dialysis or filtration step is acetic acid and the concentration of the acetic acid is from about 2.5 mM to 10 mM. In one embodiment, the water-soluble weak organic acid used in the dialysis or filtration step is acetic acid and the concentration of the acetic acid is from about 4 mM to 6 mM. In one embodiment, the water-soluble weak organic acid used in the dialysis or filtration step is acetic acid and the concentration of the acetic acid is about 5 mM.

[0576] In one embodiment, the method of the invention comprises dilution of the aqueous dispersion with a storage matrix, as defined and exemplified above. The storage matrix is typically added to the aqueous dispersion after the filtration (e.g., TFF) / dialysis step. In one embodiment, the storage matrix comprises a cryoprotectant, as defined and exemplified above.

[0577] In one embodiment, the dilution steps comprise addition of cryoprotectant, as defined and exemplified above. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose and mannitol. In one embodiment, the cryoprotectant is sucrose.

[0578] In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is a carbohydrate and is present in a concentration of about 10% (w / v).

[0579] In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the storage matrix is sucrose or trehalose and is present in a concentration of about 10% (w / v).

[0580] In one embodiment, the storage matrix is provided in one or more water-soluble weak organic acids. In one embodiment, the water-soluble weak organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid and succinic acid. In one embodiment, the water-soluble weak organic acid is acetic acid. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 0.5 mM to 50 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 1 mM to 25 mM. In one embodiment, the concentration of the water- soluble weak organic acid used in the storage matrix is from about 2.5 mM to 10 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is from about 4 mM to 6 mM. In one embodiment, the concentration of the water-soluble weak organic acid used in the storage matrix is about 5 mM.

[0581] In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 0.5 mM to 50 mM.

[0582] In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 1 mM to 25 mM. In one embodiment, the water-soluble weak organic acid used in storage matrix is acetic acid and the concentration of the acetic acid is from about 2.5 mM to 10 mM. In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is from about 4 mM to 6 mM. In one embodiment, the water-soluble weak organic acid used in the storage matrix is acetic acid and the concentration of the acetic acid is about 5 mM.

[0583] In one embodiment, the method further comprises adding peptide-conjugated lipid (as further described herein) to the lipid particles comprised in the dispersed phase of the aqueous dispersion. In some instances, the peptide-conjugated lipid may displace (i.e., replace) a corresponding portion of the steroid (e.g., cholesterol) in the lipid particles comprised in the dispersed phase of the aqueous dispersion.

[0584] In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of cationic lipids. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain a cationic lipid. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of anions of aqueous acids. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain an anion of an aqueous acid. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of acetate ions. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain acetate ions. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of malate ions. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain malate ions. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of succinate ions. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain succinate ions.

[0585] In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of a cryoprotectant. In one embodiment of the method of forming the aqueous dispersion as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture does not contain a cryoprotectant. Thus, the composition of the lipid particles comprised in the dispersed phase of the aqueous dispersion before addition of peptide-conjugated lipid may comprise a cationic or cationically ionizable lipid as described herein, a neutral or zwitterionic phospholipid as descried herein, a steroid as described herein; and optionally a grafted lipid as described herein, in a molar ratio of 20-70 mol% : 5-15 mol% : 20-60 mol% and optionally 0.5-10 mol%, respectively; preferably 40-60 mol% : 8-12 mol% : 30- 50 mol% and optionally 1.0-5 mol%, respectively. Following addition of the peptide- conjugated lipid, the peptide-conjugated lipid may comprise 0.05-1.0 mol%, optionally 0.1 to 0.5 mol%, preferably 0.1-0.3 mol% of the lipid particles comprised in the dispersed phase of the aqueous dispersion, with a corresponding reduction in the mol% of the steroid.

[0586] In one embodiment, the purification is carried out using aqueous phase essentially free of buffering agents.

[0587] In one embodiment, the purification comprises sterile filtration of the aqueous dispersion.

[0588] In one embodiment, the method further comprises storing the aqueous dispersion for 24 hours, 48 hours, 72 hours, 5 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months, 6 months, 9 months, 12 months, 18 months, 2 years, 3 years, or more. The aqueous dispersion may be stored at about 25°C, at about room temperature (e.g., 18-23°C), at about 4-8°C, at about 4°C, at about -20°C, or at about -80°C. The aqueous dispersion may be stored at about 4°C or at about -20°C.

[0589] In one embodiment, the method further comprises the step of freezing the aqueous dispersion, for example at a temperature between -15°C to -90°C, preferably at a temperature of from about -18° to about -25°C. In one embodiment, the freezing is carried out in acidic conditions. In one embodiment, the freezing is carried out at a pH of 2.0 to 6.5. In one embodiment, the freezing is carried out at a pH of 3.0 to 6.0. In one embodiment, the method further comprising the step of drying of the aqueous dispersion. In one embodiment, the drying is freeze drying (i.e., lyophilization) or spray drying.

[0590] In one embodiment, the aqueous dispersion is stable for at least 3 months at 4°C. In one embodiment, the aqueous dispersion is stable for at least 6 months at -20°C. Therefore, in one embodiment, there is provided an aqueous dispersion which is stable for at least 3 months at 4°C. In one embodiment, there is provided an aqueous dispersion which is stable for at least 6 months at -20°C. In this context “stable” may be understood to mean that the size (Zaverage) and / or size distribution and / or PDI of the particles in the aqueous dispersion after storage for the indicated time period at the indicated temperature is essentially equal to the size (Zaverage) and / or size distribution and / or PDI of the particles before storage and immediately after preparation. For example, the size (Zaverage) and / or size distribution and / or PDI of the particles in the aqueous dispersion may not change by more than 20%, optionally by more than 10%, preferably by more than 5%, during the indicated storage.

[0591] Nucleic Acid-Lipid Particle

[0592] The present disclosure further provides a lipid particle comprising a lipid or lipid mixture, as defined herein, and a nucleic acid. In one embodiment, there is provided a lipid particle obtained or obtainable by the methods defined herein. Such particles are also referred to herein as “nucleic acid-lipid particles”. When the nucleic acid is RNA, such particles are also referred to herein as “RNA-lipid particles”. When the nucleic acid is DNA, such particles are also referred to herein as “DNA-lipid particles”.

[0593] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA, saRNA, taRNA, or mixtures thereof. In one embodiment, the nucleic acid is mRNA. In one embodiment, the nucleic acid is DNA. In one embodiment, the nucleic acid is a mixture of RNA and DNA. In one embodiment, the nucleic acid is RNA which encodes for one or more personalized cancer antigens. In one embodiment, the nucleic acid is mRNA which encodes for one or more personalized cancer antigens. In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds. In some embodiments, a particle is a nucleic acid containing particle such as a particle comprising DNA, RNA or a mixture thereof. In some embodiments, the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids). In this context, the expression "amphiphilic substance" means that the substance possesses both hydrophilic and lipophilic properties. The envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic. Thus, the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic. In some embodiments, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nanosized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

[0594] In one embodiment of the present disclosure, the pre-formed lipid particles (i.e., the aqueous dispersion as described herein, which does not contain nucleic acid; also referred to as “empty particles”) are lipid nanoparticles (LNPs). In one embodiment of the present disclosure, the nucleic acid-lipid particle is a lipid nanoparticle (LNP). The function of the LNP is to stabilise and encapsulate the nucleic acid to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol. The LNPs and / or their lipid components may have adjuvant activity.

[0595] In the present disclosure, LNPs may be understood as oil-in-water emulsions in which the LNP core materials are preferably in liquid state and hence have a melting point below body temperature. LNPs thus typically comprise a central complex of lipid and optionally nucleic acid (e.g., mRNA, DNA or mixtures thereof) embedded in a disordered, non-lamellar phase made of lipid. This is in contrast to the structure of a liposome which comprises unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers surrounding an encapsulated aqueous lumen. In some

[0596] I l l instances, the pre-formed lipid particles and / or nucleic acid-lipid particles described herein are not liposomes. In some instances, the nucleic acid-lipid particles described herein are not lipoplexes.

[0597] Lipid nanoparticles (LNP) are obtainable from combining a nucleic acid with lipids. The lipids used for LNP formation typically do not form lamellar (bilayer) phases in water under physiological conditions. The LNPs typically do not comprise or encapsulate an aqueous core. The LNPs typically comprise a lipidic (or oily) core.

[0598] In some embodiments, the lipid nanoparticles described herein have an average size (such as a diameter) that in some embodiments ranges from about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and / or at most about

[0599] 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400 nm, at most about 1300 nm, at most about 1200 nm, at most about 1100 nm, at most about 1000 nm, at most about 950 nm, at most about 900 nm, at most about 850 nm, at most about 800 nm, at most about 750 nm, at most about 700 nm, at most about 650 nm, at most about 600 nm, at most about 550 nm, or at most about 500 nm), such as about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450 nm, from about 100 nm to about 400 nm, from about 100 nm to about 350 nm, from about 100 nm to about 300 nm, from about 100 nm to about 250 nm, from about 100 nm to about 200 nm, from about 150 nm to about 1000 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 150 nm to about 500 nm, from about 150 nm to about 450 nm, from about 150 nm to about 400 nm, from about 150 nm to about 350 nm, from about 150 nm to about 300 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 200 nm to about 700 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, from about 200 nm to about 450 nm, from about 200 nm to about 400 nm, from about 200 nm to about 350 nm, from about 200 nm to about 300 nm, or from about 200 nm to about 250 nm.

[0600] In one embodiment, the nucleic acid-lipid particles are stable for at least 3 months at 4°C. In one embodiment, the nucleic acid-lipid particles are stable for at least 6 months at -20°C.

[0601] Therefore, in one embodiment, there is provided a nucleic acid-lipid particle which is stable for at least 3 months at 4°C. In one embodiment, there is provided a nucleic acid-lipid particle which is stable for at least 6 months at -20°C.

[0602] In one embodiment, the integrity of the nucleic acid (e.g., RNA, DNA or mixtures thereof) in the nucleic acid-lipid particles does not decrease by more than 20% after storage of the nucleic acid-lipid particles for at least 3 months at 4°C. In one embodiment, the integrity of the nucleic acid in the nucleic acid-lipid particles does not decrease by more than 20% after storage of the nucleic acid-lipid particles for at least 6 months at -20°C.

[0603] Therefore, in one embodiment, there is provided a nucleic acid-lipid particle (e.g., RNA-lipid particle, DNA-lipid particle or RNA / DNA-lipid particle) wherein the integrity of the nucleic acid in the nucleic acid-lipid particles does not decrease by more than 20% after storage of the nucleic acid-lipid particles for at least 3 months at 4°C. In one embodiment, there is provided a nucleic acid-lipid particle wherein the integrity of the nucleic acid in the nucleic acid-lipid particles does not decrease by more than 20% after storage of the nucleic acid-lipid particles for at least 6 months at -20°C.

[0604] In one embodiment, the nucleic acid-lipid particles are capable of inducing comparable or higher (e.g., 0.5 fold, 2 fold, 5 fold, 100 fold) antibody and / or T-cell responses after administration in vivo as compared to nucleic acid-lipid particles made using a standard process.

[0605] Therefore, in one embodiment, there is provided a nucleic acid-lipid particle wherein, the nucleic acid-lipid particle is capable of inducing comparable or higher (e.g., 0.5 fold, 2 fold, 5 fold, 100 fold) antibody and / or T-cell responses after administration in vivo as compared to nucleic acid-lipid particles made using a standard process.

[0606] Particles described herein may exhibit a poly dispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05. By way of example, the particles can exhibit a poly dispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.

[0607] A nucleic acid-lipid particle can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid-lipid particle may be formed from at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the nucleic acid. Without intending to be bound by any theory, it is believed that the cationic or cationically ionizable compound combines together with the nucleic acid to form aggregates, and this aggregation results in colloidally stable particles.

[0608] In some embodiments, nucleic acid may be noncovalently associated with a particle. In some embodiments, the nucleic acid may be adhered to the outer surface of the particle (surface nucleic acid) and / or may be contained in the particle (encapsulated nucleic acid).

[0609] The N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles. In some embodiments of the present disclosure, the pre-formed lipid particles (i.e., the aqueous dispersion as described herein, which does not contain nucleic acid; also referred to as “empty particles”) are liposomes. Liposomes may be understood to be unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers surrounding an encapsulated aqueous lumen (i.e., enclosing a central aqueous compartment).

[0610] In some embodiments of the present disclosure, the nucleic acid (such as RNA or DNA or mixtures thereof), described herein may be present in lipoplex particles. In one embodiment of the present disclosure, the nucleic acid-lipid particle is a lipoplex. Lipoplexes (LPXs) are electrostatic complexes which are generally formed by mixing preformed liposomes comprising cationic lipid with anionic nucleic acid. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.

[0611] Nucleic acid lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the nucleic acid lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the nucleic acid lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the nucleic acid lipoplex particles have an average diameter of about 400 nm.

[0612] In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) cationic lipids.

[0613] In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) anions of aqueous acids. In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) acetate ions. In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) malate ions. In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) succinate ions.

[0614] In one embodiment of the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) a cryoprotectant.

[0615] Method of Forming Nucleic Acid-Lipid Particle

[0616] In a further aspect, the present disclosure provides methods for producing the nucleic acid-lipid particles as disclosed herein. Generally, such methods comprise addition of the aqueous dispersion as described herein to a composition containing a nucleic acid. In one embodiment, the composition containing the nucleic acid is a solution containing the nucleic acid. In one embodiment, the composition containing the nucleic acid is an aqueous solution containing the nucleic acid. Therefore, the invention provides a method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion according to the method as defined herein, either in its broadest aspect or a preferred aspect; and ii) mixing the aqueous dispersion with an aqueous solution comprising a nucleic acid, to produce the nucleic acid-lipid particle.

[0617] In one aspect, the method comprises: i) preparing an aqueous dispersion, as defined herein, according to any of the methods defined herein; and ii) mixing the aqueous dispersion with an aqueous solution comprising the nucleic acid, to produce the nucleic acid-lipid particle.

[0618] In one embodiment, the method of forming the nucleic acid-lipid particle comprises: i) preparing an aqueous dispersion as defined herein according to any of the methods defined herein; and ii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid, to produce the nucleic acid-lipid particle.

[0619] In one embodiment, the method of forming the nucleic acid-lipid particle comprises: i) preparing an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid (wherein the lipid mixture preferably does not comprise a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect), by mixing the lipid mixture and the aqueous phase; and ii) mixing the aqueous dispersion with (a) an aqueous solution comprising nucleic acid and an aqueous solution comprising a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect, or with (b) an aqueous solution comprising nucleic acid and a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect, to produce the nucleic acid-lipid particle.

[0620] In one embodiment, the method of forming the nucleic acid-lipid particle comprises: i) preparing an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid (wherein the lipid mixture preferably does not comprise a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect), by mixing the lipid mixture and the aqueous phase; and ii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid, to produce an intermediate nucleic acid-lipid particle; and iii) mixing the intermediate nucleic acid-lipid particle with an aqueous solution comprising a compound of Formula (A) as defined above, either in its broadest aspect or a preferred aspect, to produce the nucleic acid-lipid particle.

[0621] In all of the above methods, the methods may optionally further comprise mixing a compound of formula (I) as defined above, either in its broadest aspect or a preferred aspect, with the compound of formula (A), such that the compound of formula (I) already interacts with the formed nucleic acid-lipid particles, such that the formed particles are functionalized nucleic acid-lipid particles, as further described herein.

[0622] In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing the nucleic acid is about 3: 1 to about 1 :3. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing the nucleic acid is about 2: 1 to about 1 :2. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing the nucleic acid is about 1.5: 1 to about 1 : 1.5. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing the nucleic acid is about 1.2: 1 to about 1 : 1.2. In one embodiment, the volume ratio of the aqueous dispersion to the aqueous solution containing the nucleic acid is about 1 : 1.

[0623] In one embodiment, the aqueous solution containing the nucleic acid also contains one or more buffering agents. In one embodiment, the aqueous solution containing the nucleic acid is provided in a buffer comprising an anionic group / moiety. In one embodiment, the aqueous solution containing the nucleic acid is provided in a buffer comprising a sulfonic acid group / moiety, or derivatives thereof. In one embodiment, the aqueous solution containing the nucleic acid is provided in a buffer which is a zwitterionic acid buffer. In one embodiment, the aqueous solution containing the nucleic acid is provided in a buffer which comprises an amino group / moiety and a sulfonic acid group / moiety, or derivatives thereof. In one embodiment, the aqueous solution containing the nucleic acid is provided in a buffer which is selected from the group consisting of 4-(2-hy droxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), 2- morpholin-4-ylethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[4-(2-hydroxy- ethyl)piperazin-l-yl]propane-l -sulfonic acid (HEPPS), 2-(bis(2-hydroxy- ethyl)amino)ethanesulfonic acid (BES), N-cyclohexyl-2-aminoethanesulfonic acid (CHES), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), N-(2-acetamido)-2- aminoethanesulfonic acid (ACES), 2-(bis(2 -hydroxy ethyl)amino)ethane sulfonic acid (BES), and 2-{[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-l- sulfonic acid (TES), or a mixture of any thereof. In one embodiment, the aqueous solution containing the nucleic acid is provided in 4-(2-hydroxy-ethyl)-l- piperazineethanesulfonic acid (HEPES).

[0624] The buffer may be present in any concentration to enable it to perform its buffering function. In one embodiment, the buffer is present in a concentration of about 0.1 mM to about 1 M. In one embodiment, the buffer is present in a concentration of about 1 mM to about 100 mM. In one embodiment, the buffer is present in a concentration of about 5 mM to about 20 mM. In one embodiment, the buffer is present in a concentration of about 10 mM.

[0625] In one embodiment, the buffer is HEPES and is present in a concentration of about 0.1 mM to about 1 M. In one embodiment, the buffer is HEPES and is present in a concentration of about 1 mM to about 100 mM. In one embodiment, the buffer is HEPES and is present in a concentration of about 5 mM to about 20 mM. In one embodiment, the buffer is HEPES and is present in a concentration of about 10 mM.

[0626] In one embodiment, the aqueous solution containing the nucleic acid also contains a chelator. The function of the chelator is to chelate divalent cations (such as Mg2+, Ca2+and Zn2+), and protect the RNA from hydrolysis and degradation by enzymes which require a divalent cations as cofactor. Examples of suitable chelators include ethylenediamine-A, A, A’, A’-tetraacetic acid (EDTA), ethylene glycol-bis (P- aminoethyl ether)-A, A, A’, A’-tetraacetic acid (EGTA), or a mixture thereof. In one embodiment, the chelator is present in a concentration of about 0.001 mM to about 10 mM. In one embodiment, the chelator is present in a concentration of about 0.01 mM to about 1 mM. In one embodiment, the chelator is present in a concentration of about 0.1 mM.

[0627] In one embodiment, the buffering agent is 4-(2-hydroxy-ethyl)-l- piperazineethanesulfonic acid (HEPES), optionally in combination with ethylenediaminetetraacetic acid (EDTA) or an acceptable salt thereof.

[0628] The aqueous solution containing the nucleic acid may have a pH of from about 6.5 to about 8.5, optionally from about 6.8 to about 7.5. The aqueous solution containing the nucleic acid may have a pH of about 7.0.

[0629] In one embodiment, the aqueous dispersion (typically containing pre-LNPs) is introduced into the mixture in an aqueous solution containing a water-soluble weak organic acid. The water-soluble weak organic acids may be any of those defined and exemplified above. Preferably, the water-soluble weak organic acid is acetic acid.

[0630] In one embodiment, the concentration of the water-soluble weak organic acid is from about 0.5 mM to 50 mM. In one embodiment, the concentration of the water-soluble weak organic acid is from about 1 mM to 25 mM. In one embodiment, the concentration of the water-soluble weak organic acid is from about 2.5 mM to 10 mM. In one embodiment, the concentration of the water-soluble weak organic acid is from about 4 mM to 6 mM. In one embodiment, the concentration of the water- soluble weak organic acid is about 5 mM.

[0631] In one embodiment, the water-soluble weak organic acid is acetic acid and the concentration of the acetic acid is from about 0.5 mM to 50 mM. In one embodiment, the water-soluble weak organic acid is acetic acid and the concentration of the acetic acid is from about 1 mM to 25 mM. In one embodiment, the water-soluble weak organic acid is acetic acid and the concentration of the acetic acid is from about 2.5 mM to 10 mM. In one embodiment, the water-soluble weak organic acid is acetic acid and the concentration of the acetic acid is from about 4 mM to 6 mM. In one embodiment, the water-soluble weak organic acid matrix is acetic acid and the concentration of the acetic acid is about 5 mM.

[0632] In one embodiment, the aqueous dispersion (typically containing pre-LNPs) is introduced into the mixture in an aqueous solution containing a carbohydrate. In one embodiment, the carbohydrate is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the carbohydrate is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the carbohydrate is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the carbohydrate is present in a concentration of about 10% (w / v).

[0633] In one embodiment, the carbohydrate is sucrose or trehalose and is present in a concentration of about 1% to about 30% (w / v). In one embodiment, the carbohydrate is sucrose or trehalose and is present in a concentration of about 2% to about 20% (w / v). In one embodiment, the carbohydrate is sucrose or trehalose and is present in a concentration of about 5% to about 15% (w / v). In one embodiment, the carbohydrate is sucrose or trehalose and is present in a concentration of about 8% to about 12% (w / v). In one embodiment, the carbohydrate is sucrose or trehalose and is present in a concentration of about 10% (w / v).

[0634] In one embodiment, the storage matrix is provided in one or more water-soluble weak organic acids. In one embodiment, the water-soluble weak organic acid is selected from the group consisting of acetic acid, malic acid, maleic acid and succinic acid. In one embodiment, the water-soluble weak organic acid is acetic acid.

[0635] In one embodiment, the method of forming the nucleic acid-lipid particle comprises: i) mixing a lipid mixture comprising a compound of formula (A) and a cationic or cationically ionisable lipid dissolved in a water-soluble organic solvent with an aqueous phase, wherein the lipid solution and / or the aqueous phase is acidified, to produce an intermediate acidified aqueous lipid dispersion; ii) performing on the intermediate dispersion a dialysis or filtration step at a pH of about 2.5 to about 5.5 (preferably about 2.5 to about 4.5), or at a pH of about 6.5 to about 8.5 (preferably about 7.5 or about 8.5), to remove the organic solvent and produce an aqueous dispersion, wherein the aqueous dispersion is substantially free of acetate buffers, citrate buffers, organic solvents and RNA, and wherein the aqueous dispersion comprises a cryoprotectant; iii) mixing the aqueous dispersion with a solution comprising a nucleic acid in an organic solvent, to produce the nucleic acid-lipid particle.

[0636] Preferably the organic solvent is a water-soluble organic solvent. Examples of suitable organic solvents include as Ci-4 alcohols (e.g. isopropanol or ethanol), ketones (e.g. acetone), or mixtures thereof. In one embodiment, the organic solvent is selected from isopropanol, ethanol, acetone, and mixtures thereof. Preferably the organic solvent is ethanol.

[0637] In some embodiments, the flow rate during mixing may be from about 200 mL / min to about 600 mL / min, optionally from about 300 mL / min to about 500 mL / min, optionally from about 350 mL / min to about 400 mL / min. The term “flow rate” in this sense means the total flow rate of all of the components of the mixture.

[0638] In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle to one or more further processing steps.

[0639] As described in more detail below, in one embodiment, the method further comprises adding a compound of formula (I) (as further described herein) to functionalize the nucleic acid-lipid particles. In some instances, the compound of formula (I) may displace (i.e., replace) a corresponding portion of the steroid (e.g., cholesterol) in the nucleic acid-lipid particles. The composition of the nucleic acid-lipid particles before addition of compound of formula (I) may comprise the compound of formula (A), a cationic or cationically ionizable lipid as described herein, a neutral or zwitterionic phospholipid as descried herein, a steroid as described herein and optionally a grafted lipid as described herein, in a molar ratio of 0.1-2 mol%: 20-70 mol% : 5-15 mol% : 20-60 mol% and optionally 0.5-10 mol%, respectively; optionally 0.1-2 mol%: 40-60 mol% : 8-12 mol% : 30-50 mol% and optionally 1.0-5 mol%, respectively. Following addition of the compound of formula (I), the peptide-conjugated lipid may comprise 0.05-1.0 mol%, optionally 0.1 to 0.5 mol%, preferably 0.1-0.3 mol% of the lipid in the nucleic acid-lipid particles, with a corresponding reduction in the mol% of the steroid.

[0640] In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle to one or more purification steps. In one embodiment, the purification step comprises a dialysis or filtration step. In one embodiment, the dialysis or filtration step comprises tangential flow filtration. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particle to a filtration or dialysis step. In one embodiment, the method does not comprise subjecting the nucleic acid-lipid particle to a tangential flow filtration step.

[0641] In one embodiment, the method comprises further subjecting the nucleic acid-lipid particle to one or more dilution steps. In one embodiment, the one or more dilution steps comprise addition of cryoprotectant. In one embodiment, the cryoprotectant is selected from the group consisting of sucrose, glycerol, trehalose, lactose, glucose and mannitol. In one embodiment, the cryoprotectant is sucrose.

[0642] In one embodiment, the purification step is carried out using aqueous phase essentially free of buffering agents.

[0643] In one embodiment, the method further comprises the step of sterile filtration of the nucleic acid-lipid particle.

[0644] In one embodiment, the method further comprises the step of drying of the nucleic acid-lipid particle. In one embodiment, the drying is freeze drying. In one embodiment, the drying is spray drying.

[0645] In one embodiment, the one or more purification steps for the nucleic acid-lipid particle do not comprise a tangential flow filtration step.

[0646] In one embodiment, the nucleic acid-lipid particles are not subjected to any further purification steps. In one embodiment, the method further comprises a step of diluting the lipid particles with a storage matrix. In one embodiment, the storage matrix comprises one or more buffering agents. In one embodiment, the buffering agent or mixture thereof has a pH of 4.5 to 8.5. In one embodiment, the buffering agent is selected from the group consisting of 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES), tris- (hydroxymethyl)aminomethane (Tris), histidine, triethanolamine, or a mixture of any thereof. In one embodiment, the buffering agent is a mixture of HEPES and Tris. Preferred molar ratios of HEPES:Tris are between 100: 1 to 1 : 100, preferably 10:1 to 1 : 10.

[0647] In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) cationic lipids.

[0648] In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) anions of aqueous acids. In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) acetate ions. In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) malate ions. In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) succinate ions.

[0649] In one embodiment of the method of forming the nucleic acid-lipid particle as defined herein, the dispersed phase of the aqueous dispersion comprises a lipid mixture including a compound of formula (A), as defined herein, and the lipid mixture is substantially free (as defined herein) of (e.g., does not contain) a cryoprotectant.

[0650] Primary targeting moiety

[0651] According to the disclosure, a nucleic acid payload (i.e., active ingredient) is delivered specifically to a target cell by providing a moiety that binds to a target on target cells, e.g., an antigen on target cells, thus targeting particles comprising the nucleic acid payload to the target cells.

[0652] In some embodiments, the moiety that binds to a target on target cells is comprised by a compound which is an integral part of a particle carrying the payload, i.e., the compound of formula (A) or the targeting compound, as defined above. In these embodiments, the targeting compound comprises a binding moiety that binds to a target on target cells. In such embodiments of the targeting compound, such as the compound of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’) (A10), (A10’), (A10”), (A10’”) or (A20), the binding moiety B comprises a moiety binding to a target on target cells, (e.g., a moiety binding to a cell surface antigen on target cells).

[0653] In some embodiments, the moiety that binds to a target on target cells is comprised by a compound (i.e., docking compound, compound of formula (I)) further comprising a moiety that binds to a compound (i.e., targeting compound, compound of formula (A)) which is an integral part of a particle carrying the payload and comprising a moiety for binding to the docking compound. In these embodiments, the targeting compound itself preferably does not comprise a moiety that binds to a target on target cells. Rather, the targeting compound comprises a binding moiety that forms the binding partner for a docking compound which binds to target cells. In such embodiments, the binding moiety B of the targeting compound, such as of any of the above formulae (A), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A10”), (A10’”) or (A20), is a peptide tag, or a moiety binding to a peptide tag; and B’ of the docking compound comprises a moiety binding to B (a moiety binding to a peptide tag, or a peptide tag, respectively), and B” of the docking compound comprises a moiety binding to a cell surface antigen. The target on target cells is also referred to herein as "primary target". In some embodiments, a primary target is a cell surface antigen on target cells.

[0654] A "primary targeting moiety" as used herein relates to the part of the targeting compound or docking compound which binds to a primary target, e.g., a cell surface antigen on target cells (e.g., B” of the compound of formula (I); or B of the compound of formula (A), wherein B is a moiety binding to a target (e.g., a cell surface antigen) on target cells). Such targeting moieties are typically moieties that have affinity for cell surface targets. These moieties can be any peptide or protein (e.g. antibodies or antibody fragments) binding to the primary target. Particular embodiments of suitable primary targeting moieties for use herein include cell surface antigen binding moieties, such as antibodies, antibody fragments and DARPins. Other examples of primary targeting moieties are peptides or proteins which bind to a receptor.

[0655] A primary targeting moiety preferably binds with high specificity and / or high affinity and the bond with the primary target is preferably stable within the body.

[0656] In order to allow specific targeting of primary targets, the primary targeting moiety of the targeting compound or docking compound can comprise compounds including but not limited to antibodies, antibody fragments, e.g. Fab2, Fab, scFV, VHH domains, and other proteins or peptides.

[0657] According to some embodiments, the primary target is a cell surface antigen such as a T cell antigen, e.g., CD3, such as CD3e, CD8 or CD4, and suitable primary targeting moieties include but are not limited to, peptides and polypeptides targeting the cell surface antigen, e.g., antibodies, antibody fragments and DARPins.

[0658] According to some embodiments, the primary target is a receptor and suitable primary targeting moieties include but are not limited to, the ligand of such a receptor or a part thereof which still binds to the receptor, e.g., a receptor binding peptide in the case of receptor binding protein ligands.

[0659] Other examples of primary targeting moieties of protein nature include interferons, e.g. alpha, beta, and gamma interferon, interleukins, and protein growth factors, such as transforming growth factor (TGF), or platelet-derived growth factor (PDGF). According to some embodiments, the primary target and primary targeting moiety are selected so as to result in the specific or increased targeting of certain cells. This can be achieved by selecting primary targets with cell-specific expression. For example, T cell antigens, e.g., those described herein, may be expressed in T cells while they are not expressed or expressed in a lower amount in other cells.

[0660] Docking compound

[0661] In some embodiments, a "docking compound" (i.e., a compound of formula (I)) is used to form a connection between a primary target, e.g., a target cell or an antigen on target cells, and a targeting compound (i.e., a compound of formula (A)) which is integrated into a particle comprising a nucleic acid payload to be delivered to a target cell. In some embodiments, a connection between a primary target, e.g., a target cell or an antigen on target cells, and a docking compound is a non-covalent connection. In some embodiments, a connection between a docking compound and a targeting compound is a non-covalent or covalent connection. In some embodiments, the targeting compound comprises a binding moiety for binding to the docking compound which is covalently attached to a hydrophobic moiety (e.g., lipid). The hydrophobic moiety (e.g., lipid) forms part of said particle.

[0662] In some embodiments, a docking compound comprises a "primary targeting moiety", as defined above, (e.g., B” of the compound of formula (I)) e.g., a moiety targeting a cell surface antigen on target cells, that is capable of binding to the primary target of interest, e.g., a cell surface antigen on target cells. In some embodiments, a "primary targeting moiety" as used herein relates to the part of the docking compound which binds to a primary target.

[0663] The docking compound further comprises a group which serves as a binding partner for a respective binding moiety of a targeting compound. The portion of the targeting compound comprising the hydrophobic moiety (e.g., lipid) (having a binding moiety for the docking compound covalently attached) integrates into a particle carrying a payload and thus forms a connection between the particle and the docking compound. The moiety of the docking compound binding to the targeting compound and the primary targeting moiety are linked to each other, preferably by a covalent linkage.

[0664] According to some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody. In some embodiments, the docking compound comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound. In some embodiments, the docking compound comprises an antibody or antibody fragment binding to a primary target and an antibody or antibody fragment binding to a targeting compound. In some embodiments, at least one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, each binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, at least one binding domain comprises a single-domain antibody such as a VHH. In some embodiments, each binding domain comprises a single-domain antibody such as a VHH. In some embodiments, one binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody and the other binding domain comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a primary target comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a targeting compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a targeting compound comprises a single-domain antibody such as a VHH.

[0665] In some embodiments, the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a binding domain binding to a targeting compound. In some embodiments, the docking compound comprises a fusion protein which comprises a binding domain binding to a primary target and a peptide (e.g., an epitope tag, such as ALFA) binding to a targeting compound. In some embodiments, the docking compound comprises a single peptide chain. In some embodiments, the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., antibody or antibody fragment, binding to a targeting compound. In some embodiments, the single peptide chain comprises a portion, e.g., antibody, antibody fragment or DARPin, binding to a primary target and a portion, e.g., a peptide (such as an epitope tag), binding to a targeting compound (e.g., which comprises an antibody or antibody fragment capable of binding to said peptide). In some embodiments, the antibody fragments are VHH, scFv, or a mixture thereof. In different embodiments, the docking compound comprises one of the following structures (from N- to C-terminus):

[0666] VHH (a targeting compound)-optional linker- VHH (a primary target) VHH (a primary target)-optional linker- VHH (a targeting compound) VHH (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker- VHH (a targeting compound) VHH (a primary target)-optional linker-scFv (a targeting compound) scFv (a targeting compound)-optional linker- VHH (a primary target) scFv (a targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-scFv (a targeting compound) peptide (bound by targeting compound)-optional linker- VHH (a primary target) VHH (a primary target)-optional linker-peptide (bound by targeting compound) peptide (bound by targeting compound)-optional linker-scFv (a primary target) scFv (a primary target)-optional linker-peptide (bound by targeting compound)

[0667] In some embodiments, the docking compound comprises a peptide portion (optionally wherein the peptide is an epitope tag, e.g., an ALFA-tag) and an antibody portion (e.g., which may be an antibody, antibody fragment, DARPin, VHH, scFv, nanobody) wherein the antibody portion binds to a primary target, e.g., a cell surface antigen on target cells. In some embodiments, the docking compound comprises a bispecific molecule, such as a bispecific polypeptide, e.g., a bispecific antibody, wherein one specificity binds to an epitope tag, e.g., an ALFA-tag, and the other specificity binds to a primary target, e.g., a cell surface antigen on target cells. In some embodiments, the specificity which binds to an epitope tag is an antibody or antibody fragment such as an NbALFA-nanobody (NbALFA). In some embodiments, the specificity which binds to a primary target is an antibody, antibody fragment or DARPin. In some embodiments, the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target DARPin, an anti-primary target VHH and an anti-primary target scFv and / or the moiety binding to a targeting compound of the docking compound is an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound has a structure selected from the group consisting of NbALFA x anti-primary target DARPin, NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv. In some embodiments, the primary target is a T cell antigen, e.g., CD3, such CD3e, CD4 or CD8. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD3 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti- CD3 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD4 DARPin. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 VHH. In some embodiments, the docking compound comprises a bispecific antibody comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 scFv. In some embodiments, the docking compound comprises a bispecific molecule comprising a nanobody which binds to an epitope tag, e.g., an ALFA-tag, and an anti-CD8 DARPin.

[0668] In one embodiment, the docking compound is not aCD8-DARPin X NbAlfa. In one embodiment, the docking compound is not aCD3-VHH X NbAlfa. In one embodiment, the docking compound is not aCD8-DARPin X Spycatcher. In one embodiment, the docking compound is not algM-VHH X NbAlfa. Interacting moieties on the targeting compound and on the docking compound

[0669] In some embodiments, the moiety on the targeting compound (B of the compound of formula (A), binding moiety covalently attached to a hydrophobic moiety) and the moiety on the docking compound (B’ of the compound of formula (I), moiety binding to the binding moiety covalently attached to a hydrophobic moiety) interact with each other, e.g., non-covalently bind to each other.

[0670] In some embodiments, the moieties on the targeting compound and on the docking compound interacting with each other (B, formula (A) and B’, formula (I)) bind to each other under physiological conditions.

[0671] In some embodiments, the moieties on the targeting compound and on the docking compound interacting with each other (B, formula (A) and B’, formula (I)) are antibody / antigen systems.

[0672] In some embodiments, the moiety of the targeting compound binding to the docking compound (B, formula (A)) comprises a peptide or protein, e.g., a peptide tag, and the moiety of the docking compound binding to the targeting compound (B’, formula (I)) comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.

[0673] In some embodiments, the moiety of the docking compound binding to the targeting compound (B’, formula (I)) comprises a peptide or protein, e.g., a peptide tag, and the moiety of the targeting compound binding to the docking compound (B, formula (A)) comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein. In some embodiments, the moieties on the targeting compound and on the docking compound interacting with each other (B, formula (A) and B’, formula (I)) comprise an epitope tag / binder system.

[0674] As used herein, an "epitope tag" refers to a stretch of amino acids to which an antibody or proteinaceous molecule with antibody -like function can bind. In some embodiments, the epitope tag comprises an ALFA-tag. In some embodiments, the epitope tag / binder system comprises an ALFA-tag and an ALFA- specific single-domain antibody (sdAb), NbALFA-nanobody. The ALFA-tag may be defined as described below.

[0675] Functionalized Nucleic Acid-Lipid Particle

[0676] Particles which are “functionalized” as described herein, comprise, bind to or interact with, a compound comprising a primary targeting moiety that binds a target on target cells. Thus, a “functionalized nucleic acid-lipid particle” may be understood as a particle that exhibits preferential interaction with target cells expressing or exhibiting a particular primary target as defined herein (such as a marker or antigen, preferably on the cell surface) which is preferentially recognized by the primary targeting moiety of the particle (i.e., B of the compound of formula (A) where B is a moiety binding to a target (e.g., a cell surface antigen) on target cells); or B” of the compound of formula (I)). Accordingly, functionalized nucleic acid-lipid particles provide targeted delivery of the nucleic acid payload / active ingredient to particular target cells. The invention also provides functionalized nucleic acid-lipid particles as described herein.

[0677] In one embodiment, there is provided a functionalized nucleic acid-lipid particle comprising: a compound of Formula (A) as defined herein, wherein B comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I):

[0678] B’-X3-B” (I) wherein B’ comprises a moiety binding to B;

[0679] X3 is absent or a linking moiety; and

[0680] B” comprises a moiety binding to a cell surface antigen.

[0681] In some exemplary functionalized particles, a nucleic acid-lipid particle, as described herein, comprises a compound of formula (A), wherein the moiety B of the compound of formula (A) is a moiety binding to a target (e.g., a cell surface antigen) on target cells. In such embodiments, the compound of formula (A) functionalizes the particles such that, in use, a connection can be made between moiety B of the compound of formula (A) and a primary target, e.g., a target cell or an antigen on target cells, to enable the nucleic acid payload comprised in the nucleic acid-lipid particle to be delivered to a target cell.

[0682] In some exemplary functionalized particles, a nucleic acid-lipid particle, as described herein, comprising a compound of formula (A), is contacted with a compound of formula (I), as defined herein, such that the moiety B’ of the compound of formula (I) interacts with the moiety B of the compound of formula (A). Thus, in some embodiments, the compound of formula (I) interacts with, or binds to, the nucleic acid-lipid particle, as described herein. The terms “interacts with” and “binds to” may be used interchangeably in this context. The compound of formula (I) may covalently or non-covalently (preferably non-covalently) interact with, or bind to, the nucleic acid-lipid particle. In such embodiments, the interaction between the nucleic acid- lipid particle and the compound of formula (I) functionalizes the particles such that, in use, a connection can be made between the compound of formula (I) and a primary target, e.g., a target cell or an antigen on target cells, to enable the nucleic acid payload comprised in the nucleic acid-lipid particle to be delivered to a target cell.

[0683] Therefore, the invention additionally provides in a further aspect a functionalized nucleic acid-lipid particle, wherein the nucleic acid-lipid particle comprises a compound of formula (A), which is functionalized by interacting therewith a compound of formula (I):

[0684] B’-X3-B” (I) wherein:

[0685] B’ comprises a moiety binding to B, wherein B comprises a peptide tag or a moiety binding to a peptide tag;

[0686] X3 is absent or a linking moiety; and

[0687] B” comprises a moiety binding to a cell surface antigen.

[0688] In one embodiment, the compound of the formula B’-X3-B” comprises a peptide or polypeptide. In one embodiment, the moiety binding to a cell surface antigen comprises an antibody or antibody-like molecule.

[0689] In one embodiment, the cell surface antigen is characteristic for an immune effector cell.

[0690] In one embodiment, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3.

[0691] In one embodiment, B comprises a peptide tag and B’ comprises a moiety binding to the peptide tag.

[0692] In one embodiment, B’ comprises a peptide tag and B comprises a moiety binding to the peptide tag.

[0693] In one embodiment, the moiety binding to a peptide tag comprises an antibody or antibody-like molecule.

[0694] In one embodiment, the peptide tag comprises an ALFA-tag.

[0695] In some embodiments, an ALFA-tag comprises the amino acid sequence -AA0-AA1- AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are:

[0696] AAO is Pro or deleted;

[0697] AA1 is Ser, Gly, Thr, or Pro;

[0698] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0699] A A3 is Leu, He, or Vai;

[0700] AA4 is Glu or Gin;

[0701] AA5 is Glu or Gin;

[0702] AA6 is Glu or Gin;

[0703] AA7 is Leu, He, or Vai;

[0704] AA8 is Arg, Ala, Gin, or Glu;

[0705] AA9 is Arg, Ala, Gin, or Glu; AA10 is Arg;

[0706] AA1 1 is Leu;

[0707] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0708] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0709] AA14 is Pro or deleted.

[0710] In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE (SEQ ID NO: 1), P SRLEEELRRRLTE (SEQ ID NO: 2), SRLEEELRRRLTEP (SEQ ID NO: 3), and P SRLEEELRRRLTEP (SEQ ID NO: 4).

[0711] In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence - AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13- AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 (XI, X2) are connected covalently; and wherein the amino acids of AAO, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 which are not XI and X2 are:

[0712] AAO is Pro or deleted;

[0713] AA1 is Ser, Gly, Thr, or Pro;

[0714] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0715] A A3 is Leu, He, or Vai;

[0716] AA4 is Glu or Gin;

[0717] AA5 is Glu or Gin;

[0718] AA6 is Glu or Gin;

[0719] AA7 is Leu, He, or Vai;

[0720] AA8 is Arg, Ala, Gin, or Glu;

[0721] AA9 is Arg, Ala, Gin, or Glu;

[0722] AA10 is Arg;

[0723] AA1 1 is Leu;

[0724] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0725] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0726] AA14 is Pro or deleted. In some embodiments, Xl and X2 are separated by 2 or 3 amino acids.

[0727] In some embodiments, AA5 is XI and AA9 is X2, AA5 is XI and AA8 is X2, AA9 is XI and AA13 is X2, AA6 is XI and AA9 is X2, AA9 is XI and AA12 is X2, AA10 is XI and AA13 is X2, AA6 is XI and AA10 is X2 or AA4 is XI and AA8 is X2.

[0728] In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-X2)-Arg-Leu-AA12-AA13- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-X2)-AA13- AA14-,

[0729] - AAO- AA 1 - AA2- AA3 - AA4- AA5 - AA6- AA7- AA8- AA9-cy clo(X 1 -Leu- AA 12-X2)- AA14-, -AA0-AAl-AA2-AA3-AA4-AA5-cyclo(Xl-AA7-AA8-AA9-X2)-Leu-AA12-AA13- AA14-, and -AA0-AAl-AA2-AA3-cyclo(Xl-AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13- AA14-, wherein the side-chains of XI and X2 amino acid residues are connected covalently; AAO is Pro or deleted;

[0730] AA1 is Ser, Gly, Thr, or Pro;

[0731] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0732] A A3 is Leu, He, or Vai;

[0733] AA4 is Glu or Gin;

[0734] AA5 is Glu or Gin;

[0735] AA6 is Glu or Gin;

[0736] AA7 is Leu, He, or Vai; AA8 is Arg, Ala, Gin, or Glu;

[0737] AA9 is Arg, Ala, Gin, or Glu;

[0738] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0739] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0740] AA14 is Pro or deleted.

[0741] In some embodiments, XI and X2 in the peptides disclosed herein are connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and / or 1,2, 3 -triazole.

[0742] In some embodiments, a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of XI and X2 to the carboxyl group of a side-chain of the other of XI and X2 via an amide bond. The amino group of the side chain of an amino acid that possesses a pendant amine group, e.g., lysine or a lysine derivative, and the carboxyl group of the side chain of an acidic amino acid, e.g., aspartic acid, glutamic acid or a derivative thereof, can be used to generate a cyclized amino acid sequence via an amide bond.

[0743] In some embodiments, a cyclized amino acid sequence described herein is generated by linking a sulfhydryl group of a side-chain of one of XI and X2 to the sulfhydryl group of a side-chain of the other of XI and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.

[0744] In some embodiments, XI and X2 are, independently, selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when XI is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Orn, DOrn, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Orn, DOm, Dab, DDab, Dap, or DDap, X2 is Glu, DGlu, Asp, or DAsp; and when XI is Cys, DCys, hCys, DhCys, Pen, or DPen, X2 is Cys, DCys, hCys, DhCys, Pen, or DPen.

[0745] In some embodiments, XI is Glu and X2 is Lys. In some embodiments, -cyclo(Glu — — Lys)-, -c(Glu - Lys)-, -cyclo cyclo, or - cycloE — cycloK- comprises the following structure:

[0746] In some embodiments, XI is Lys and X2 is Glu. In some embodiments, -cyclo(Lys — — Glu)-, -c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloE- comprises the following structure:

[0747] In some embodiments, XI is Cys and X2 is Cys. In some embodiments, -cyclo(Cys — — Cys)-, c(Cys - Cys)-, -cyclo(C - C)-, -c(C - C)-, -C — C- cyclo, or - cycloC - cycloC- comprises the following structure:

[0748] Particular cyclized amino acid sequences of the above-identified generic formulas include, for example, -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 13), -Ser- Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 14), -Ser-Arg- Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 15), -Ser-Arg-Leu- Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 16), -Ser-Arg-Leu-Glu- cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 17), -Ser-Arg-Leu-Glu- cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 18), -Ser-Arg-Leu-Glu-Glu- Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)- (SEQ ID NO: 19), -Ser-Arg-Leu-Glu-cyclo(Glu- Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 20), -Pro-Ser-Arg-Leu-Glu-cyclo(Glu- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 21), -Pro-Ser-Arg-Leu-Glu-cyclo(DGlu- Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 22), -Pro-Ser-Arg-Leu-Glu-cyclo(Glu- Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 23), -Pro-Ser-Arg-Leu-Glu-cyclo(Lys- Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu- (SEQ ID NO: 24), -Pro-Ser-Arg-Leu-cyclo(Glu-Glu- Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 25), -Pro-Ser-Arg-Leu-Glu-cyclo(Cys- Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 26), -Pro-Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)- (SEQ ID NO: 27), -Pro-Ser-Arg-Leu-Glu-cyclo(Cys- Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 28), -Pro-Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 29), -Pro-Ser-Arg-Leu-Glu-Glu- cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu- (SEQ ID NO: 30), -Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)- (SEQ ID NO: 31), -Ser-Arg-Leu-Glu-cyclo(Cys-Glu- Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 32), -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu- Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 33), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg- cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 34), -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg- Cys)-Leu-Thr-Glu- (SEQ ID NO: 35), -Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-Arg-DLys)- Arg-Leu-Thr-Glu- (SEQ ID NO: 36), -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg- Leu-Thr-Glu- (SEQ ID NO: 37), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGlu)-Arg-Leu- Thr-Glu- (SEQ ID NO: 38), -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGlu)-Arg-Leu- Thr-Glu- (SEQ ID NO: 39), -Pro-Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-Arg-DLys)-Arg- Leu-Thr-Glu- (SEQ ID NO: 40), -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Glu)-Arg- Leu-Thr-Glu- SEQ ID NO: 41, -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DGlu)-Arg- Leu-Thr-Glu- SEQ ID NO: 42), -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DGlu)-Arg- Leu-Thr-Glu- SEQ ID NO: 43, -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu- Thr-Glu- (SEQ ID NO: 44), -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu- Thr-Glu- SEQ ID NO: 45), -Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr- Glu- (SEQ ID NO: 46), -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 47), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 48), -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 49), -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-Asp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 50), -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr- Glu- (SEQ ID NO: 51), -Pro-Ser-Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr- Glu- (SEQ ID NO: 52), -Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-DLys)-Arg-Leu-Thr- Glu- (SEQ ID NO: 53), -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-DAsp)-Arg-Leu-Thr- Glu- (SEQ ID NO: 54), -Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Arg-DAsp)-Arg-Leu- Thr-Glu- (SEQ ID NO: 55), -Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr- Glu- (SEQ ID NO: 56), -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 57), -Ser-Arg-Leu-Glu-cyclo(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 58), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 59), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 60), -Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 61), -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 62), -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 63), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 64), -Ser- Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 65), -Ser-Arg- Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 66), -Ser-Arg-Leu- Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 67), -Ser-Arg-Leu- Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 68), -Ser-Arg-Leu- Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 69), -Pro-Ser-Arg-Leu- Glu-cyclo(Glu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 70), -Pro-Ser-Arg-Leu- Glu-cyclo(DGlu-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 71); -Pro-Ser-Arg- Leu-Glu-cyclo(Lys-Glu-Leu-Glu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 72), -Pro-Ser-Arg- Leu-Glu-cyclo(Lys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 73), -Pro-Ser-Arg- Leu-Glu-cyclo(DLys-Glu-Leu-DGlu)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 74), -Pro-Ser- Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 75), -Pro-Ser- Arg-Leu-Glu-cyclo(Asp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 76), -Pro-Ser- Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 77), -Pro-Ser- Arg-Leu-Glu-cyclo(Lys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 78), -Pro-Ser- Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 79), -Pro-Ser- Arg-Leu-Glu-cyclo(DAsp-Glu-Leu-DLys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 80), -Pro- Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-DAsp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 81), - Pro-Ser-Arg-Leu-Glu-cyclo(DLys-Glu-Leu-Asp)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 82), - Ser-Arg-Leu-Glu-Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 83), -Ser- Arg-Leu-Glu-Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 84), -Ser-Arg- Leu-Glu-Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 85), -Ser-Arg-Leu- Glu-Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 86), -Ser-Arg-Leu-Glu- Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 87), -Ser-Arg-Leu-Glu-Glu- cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 88), -Ser-Arg-Leu-Glu-Glu- cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 89), -Ser-Arg-Leu-Glu-Glu- cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 90), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(Asp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 91), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(Lys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 92), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(DAsp-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 93), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(Lys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 94), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(Asp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 95), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(DLys-Leu-Arg-Asp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 96), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(DLys-Leu-Arg-DAsp)-Arg-Leu-Thr-Glu- (SEQ ID NO: 97), -Pro-Ser-Arg-Leu- Glu-Glu-cyclo(DAsp-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 98), -Ser-Arg-Leu- Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 99), -Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 100), -Ser-Arg-Leu-Glu-Glu- Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu- (SEQ ID NO: 101), -Ser-Arg-Leu-Glu-Glu- cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 102), -Pro-Ser-Arg-Leu-Glu- cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 103), -Pro-Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 104), -Pro-Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Cys)-Glu- (SEQ ID NO: 105), -Pro-Ser-Arg-Leu-Glu- Glu-cyclo(Cys-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 106), -Ser-Arg-Leu-Glu- cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 107), -Ser-Arg-Leu-Glu- cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 108), -Ser-Arg-Leu-Glu- cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 109), -Ser-Arg-Leu-Glu- cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 110), -Ser-Arg-Leu-Glu- cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 111), -Ser-Arg-Leu-Glu- cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 112), -Ser-Arg-Leu-Glu- cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 113), -Ser-Arg-Leu-Glu- cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 114), -Ser-Arg-Leu-Glu- cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 115), -Ser-Arg-Leu- Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 116), -Ser-Arg-Leu- Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 117), -Ser-Arg-Leu- Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 118), -Ser-Arg-Leu- Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 119), -Pro-Ser-Arg- Leu-Glu-cyclo(DCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 120), -Pro-Ser- Arg-Leu-Glu-cyclo(DCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 121), -Pro- Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 122), -Pro- Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 123), - Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 124), - Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 125), -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-hCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 126), -Pro-Ser-Arg-Leu-Glu-cyclo(hCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 127), -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 128), -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DhCys)-Arg-Arg-Leu-Thr- Glu- (SEQ ID NO: 129), -Pro-Ser-Arg-Leu-Glu-cyclo(DCys-Glu-Leu-DhCys)-Arg-Arg-Leu- Thr-Glu (SEQ ID NO: 130)-, -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-Cys)-Arg-Arg- Leu-Thr-Glu- (SEQ ID NO: 131), -Pro-Ser-Arg-Leu-Glu-cyclo(DhCys-Glu-Leu-DCys)-Arg- Arg-Leu-Thr-Glu- (SEQ ID NO: 132), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys- Leu-Thr-DCys)- (SEQ ID NO: 133), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys- Leu-Thr-Cys)- (SEQ ID NO: 134), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys- Leu-Thr-DCys)- (SEQ ID NO: 135), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys- Leu-Thr-Cys)- (SEQ ID NO: 136), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys- Leu-Thr-DCys)- (SEQ ID NO: 137), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(hCys- Leu-Thr-hCys)- (SEQ ID NO: 138), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys- Leu-Thr-hCys)- (SEQ ID NO: 139), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys- Leu-Thr-hCys)- (SEQ ID NO: 140), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys- Leu-Thr-hCys)- (SEQ ID NO: 141), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys- Leu-Thr-Cys)- (SEQ ID NO: 142), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys- Leu-Thr-DCys)- (SEQ ID NO: 143), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys- Leu-Thr-hCys)- (SEQ ID NO: 144), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys- Leu-Thr-DhCys)- (SEQ ID NO: 145), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DCys- Leu-Thr-DhCys)- (SEQ ID NO: 146),-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys- Leu-Thr-DhCys)- (SEQ ID NO: 147),-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen- Leu-Thr-Pen)- (SEQ ID NO: 148), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Pen-Leu- Thr-DPen)- (SEQ ID NO: 149), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu- Thr-Pen)- (SEQ ID NO: 150), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DPen-Leu- Thr-DPen)- (SEQ ID NO: 151), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu- Cys)-Glu- (SEQ ID NO: 152), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DCys-Arg-Leu- DCys)-Glu- (SEQ ID NO: 153), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu- DCys)-Glu- (SEQ ID NO: 154), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu- Cys)-Glu- (SEQ ID NO: 155), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu- DCys)-Glu- (SEQ ID NO: 156), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu- hCys)-Glu- (SEQ ID NO: 157), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu- hCys)-Glu- (SEQ ID NO: 158), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(hCys-Arg-Leu- DhCys)-Glu- (SEQ ID NO: 159), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(DhCys-Arg- Leu-hCys)-Glu- (SEQ ID NO: 160), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg- cyclo(Cys-Leu-Thr-DCys)- (SEQ ID NO: 161), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg- Arg-cyclo(DCys-Leu-Thr-Cys)- (SEQ ID NO: 162), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu- Arg-Arg-cyclo(DCys-Leu-Thr-DCys)- (SEQ ID NO: 163), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-

[0749] Leu-Arg-Arg-cyclo(hCys-Leu-Thr-Cys)- (SEQ ID NO: 164), -Pro-Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-Arg-cyclo(hCys-Leu-Thr-DCys)- (SEQ ID NO: 165), -Pro-Ser-Arg-Leu-Glu-Glu- Glu-Leu-Arg-Arg-cyclo(hCys-Leu-Thr-hCys)- (SEQ ID NO: 166), -Pro-Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)- (SEQ ID NO: 167), -Pro-Ser-Arg-Leu-Glu- Glu-Glu-Leu-Arg-Arg-cyclo(DCys-Leu-Thr-hCys)- (SEQ ID NO: 168), -Pro-Ser-Arg-Leu- Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-hCys)- (SEQ ID NO: 169), -Pro-Ser-Arg-Leu- Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-Cys)- (SEQ ID NO: 170), -Pro-Ser-Arg- Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(DhCys-Leu-Thr-DCys)- (SEQ ID NO: 171), -Pro-Ser- Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Dh...

Claims

CLAIMS1. A method of forming an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula(A):L-X1-P-X2-B (A) wherein:P is absent or comprises a polymer;L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;XI is absent or a first linking moiety; andX2 is absent or a second linking moiety; the method comprising mixing the lipid mixture comprising the compound of formula (A) and the aqueous phase to produce the aqueous dispersion; wherein the aqueous dispersion is substantially free of organic solvents.

2. An aqueous dispersion comprising: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture including a compound of Formula(A):L-X1-P-X2-B (A) wherein:P is absent or comprises a polymer;L comprises a hydrophobic moiety attached to B when P is absent or to a first end of the polymer P when present;B comprises a binding moiety comprising a peptide or protein, the binding moiety B being attached to L when P is absent or to a second end of the polymer P when present;XI is absent or a first linking moiety; andX2 is absent or a second linking moiety;wherein the aqueous dispersion is substantially free of organic solvents.

3. A method according to claim 1, or an aqueous dispersion according to claim 2, wherein the aqueous dispersion is substantially free of a nucleic acid.

4. A method or an aqueous dispersion according to any preceding claim, wherein the aqueous dispersion is substantially free of inorganic cations.

5. A method or an aqueous dispersion according to any preceding claim, wherein the aqueous dispersion comprises an anion of an aqueous acid.

6. A method or aqueous dispersion according to any preceding claim, wherein the aqueous mobile phase comprises a cryoprotectant.

7. A method or aqueous dispersion according to any preceding claim, wherein the aqueous dispersion has a pH of 2.5 to 5.5.

8. A method or aqueous dispersion according to any preceding claim, wherein the aqueous dispersion has a pH of 2.5 to 4.5.

9. A method or aqueous dispersion according to any preceding claim, wherein the hydrophobic moiety comprises a lipid.

10. A method or aqueous dispersion according to claim 9, wherein the hydrophobic moiety comprises a phospholipid.

11. A method or aqueous dispersion according to claim 10, wherein the hydrophobic moiety comprises a moiety selected from the group consisting of: DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine), or a mixture thereof.

12. A method or aqueous dispersion according to claim 11, wherein the hydrophobic moiety comprises a DSPE moiety.

13. A method or aqueous dispersion according to any preceding claim, wherein P is a polymer.

14. A method or aqueous dispersion according to claim 13, wherein P is a hydrophilic polymer.

15. A method or aqueous dispersion according to claim 14, wherein P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2- (2-aminoethoxy)ethoxy)acetic acid (pAEEA), derivatives and combinations thereof.

16. A method or aqueous dispersion according to any preceding claim, wherein the binding moiety B is a moiety capable of binding to a cell surface antigen.

17. A method or aqueous dispersion according to any preceding claim, wherein the binding moiety B is a peptide tag.

18. A method or aqueous dispersion according to any preceding claim, wherein the binding moiety B is a moiety capable of binding to a peptide tag.

19. A method or aqueous dispersion according to claim 17 or claim 18, wherein the peptide tag comprises an ALFA-tag.

20. A method or aqueous dispersion according to any preceding claim, wherein the lipid mixture includes a cationic or cationically ionisable lipid.

21. A method or aqueous dispersion according to claim 20, wherein the lipid mixture includes a cationically ionisable lipid.

2. A method or aqueous dispersion according to claim 21, wherein the cationic or cationically ionisable lipid is selected from the group consisting of:7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide)1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA);2.2-dilinoleyl-4-dimethylaminoethyl-[l,3]-di oxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3- DMA);1.2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); di((Z)-non-2-en-l-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate(L319); bis-(2 -butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)- nonadecanedioate (A9);(heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}- octanoate) (L5); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}- octanoate) (SM-102);O- [N- { (9Z, 12Z)-octadeca-9, 12-dien- 1 -yl) } -N- { 7 -pentadecylcarbonyloxy octyl } - amino]4-(dimethylamino)butanoate (HY501 );2-(di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate(EA-2);4-((di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)oxy)-A,A-dimethyl-4-oxobutan- 4-amine (HYAM-2);((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1 -diyl) bis(2- hexyl decanoate) (EA-405);(2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2- hexyldecanoate) (HY-405);or a mixture of any thereof.

23. A method or aqueous dispersion according to any preceding claim, wherein the lipid mixture further comprises one or more additional lipids.

24. A method or aqueous dispersion according to claim 23, wherein the one or more additional lipids comprise a neutral or zwitterionic lipid.

25. A method or aqueous dispersion according to claim 24, wherein the neutral or zwitterionic lipid is a neutral or zwitterionic phospholipid.

26. A method or aqueous dispersion according to claim 25, wherein the neutral or zwitterionic phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); di ol eoy Iphosphati dy 1 ethanol amine (DOPE) ; l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG); N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); or a mixture of any thereof.

27. A method or aqueous dispersion according to claim 26, wherein the neutral or zwitterionic phospholipid is distearoylphosphatidylcholine (DSPC).

28. A method or aqueous dispersion according to any one of claims 23 to 27, wherein the one or more additional lipids comprise a steroid.

29. A method or aqueous dispersion according to claim 28, wherein the steroid is cholesterol.

30. A method or aqueous dispersion according to any one of claims 23 to 29, wherein the one or more additional lipids comprise a grafted lipid.

31. A method or aqueous dispersion according to claim 30, wherein the grafted lipid is selected from the group consisting of a poly(alkylene glycol)-conjugated lipid, a poly(sarcosinate)-conjugated lipid, a poly(oxazoline) (POX)-conjugated lipid; a poly(oxazine) (POZ)-conjugated lipid; a poly(vinyl pyrrolidone) (PVP)-conjugated lipid; a poly(A-(2-hydroxypropyl)-methacrylamide) (pHPMA)- conjugated lipid; a poly(dehydroalanine) (pDha)-conjugated lipid; a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2- methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipid; or a mixture of any thereof.

32. A method or aqueous dispersion according to claim 31, wherein the grafted lipid is a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid.

33. A method or aqueous dispersion according to any preceding claim, wherein the lipid mixture does not contain a cationic lipid.

34. A method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion according to the method of any one of claims 1 to 33; and ii) mixing the aqueous dispersion with an aqueous solution comprising a nucleic acid, to produce the nucleic acid-lipid particle.

35. A method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid, by mixing the lipid mixture and the aqueous phase; and ii) mixing the aqueous dispersion with an aqueous solution comprising nucleic acid, to produce an intermediate nucleic acid-lipid particle; and iii) mixing the intermediate nucleic acid-lipid particle with an aqueous solution comprising a compound of Formula (A) as defined in claim 1, to produce the nucleic acid-lipid particle.

36. A method of forming a nucleic acid-lipid particle, the method comprising: i) preparing an aqueous dispersion having: an aqueous mobile phase; and a dispersed phase comprising a lipid mixture comprising a cationic or cationically ionizable lipid, by mixing the lipid mixture and the aqueous phase; andii) mixing the aqueous dispersion with an aqueous solution comprising:(a) nucleic acid, and(b) a compound of Formula (A) as defined in claim 1, to produce the nucleic acid-lipid particle.

37. A method according to claim 35 or 36, wherein the lipid mixture in step i) does not comprise a compound of Formula (A) as defined in claim 1.

38. A method according to any one of claims 34 to 37, wherein the nucleic acid is RNA.

39. A method according to claim 38, wherein the RNA is mRNA.

40. A method according to claim 39, wherein the mRNA encodes for one or more personalized cancer antigens.

41. A lipid-nucleic acid particle, obtained or obtainable by the method of any one of claims 34 to 40.

42. A method of forming a functionalized nucleic acid-lipid particle comprising: a compound of Formula (A) as defined in claim 1, wherein B comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I):B’-X3-B” (I) wherein B’ comprises a moiety binding to B;X3 is absent or a linking moiety; andB” comprises a moiety binding to a cell surface antigen, the method comprising:(a) forming a nucleic acid-lipid particle according to the method of any one of claims 34 to 40, and(b) mixing the nucleic acid-lipid particle with the compound of formula (I), such that the compound of formula (I) interacts with the nucleic acid-lipid particle.

43. A functionalized nucleic acid-lipid particle comprising: a compound of Formula (A) as defined in claim 1, wherein B comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I):B’-X3-B” (I) wherein B’ comprises a moiety binding to B;X3 is absent or a linking moiety; andB” comprises a moiety binding to a cell surface antigen.

44. A functionalized nucleic acid-lipid particle according to claim 43 wherein B comprises a peptide tag and B’ comprises a moiety binding to the peptide tag.

45. A functionalized nucleic acid-lipid particle according to claim 43 wherein B’ comprises a peptide tag and B comprises a moiety binding to the peptide tag.

46. A functionalized nucleic acid-lipid particle according to claim 44 or 45, wherein the moiety binding to a peptide tag comprises an antibody or antibody-like molecule.

47. A functionalized nucleic acid-lipid particle according to any one of claims 44 to 46 wherein the peptide tag comprises an ALFA-tag.

48. A pharmaceutical composition comprising a functionalized nucleic acid-lipid particle according to any one of claims 43-47 and a pharmaceutically acceptable carrier.

49. A functionalized nucleic acid-lipid particle according any one of claims 43-47 for use as a medicament.

50. A functionalized nucleic acid-lipid particle according to any one of claims 43-47 for use in treating a disease involving an antigen.

51. A functionalized nucleic acid-lipid particle according to any one of claims 43-47 for use in treating a disease characterized by the presence of diseased cellsexpressing an antigen.

52. A functionalized nucleic acid-lipid particle according any one of claims 43-47 for use in treating cancer.

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