WO2025228975A1

WO2025228975A1 - Particles, compositions and methods

Info

Publication number: WO2025228975A1
Application number: PCT/EP2025/061701
Authority: WO
Inventors: Karl KLINGER; Arne BILLMEIER; Meike GANGLUFF; Bernard Manuel HARYADI; Mario SAUCEDO-ESPINOSA; Andrew Kaiser
Original assignee: Biontech SE; Biontech Delivery Technologies GmbH
Current assignee: Biontech SE; Biontech Delivery Technologies GmbH
Priority date: 2024-04-29
Filing date: 2025-04-29
Publication date: 2025-11-06
Anticipated expiration: 2026-10-29

Abstract

The present disclosure relates generally to nucleic acid-lipid particles, in particular functionalised nucleic acid-lipid particles, methods for producing them, and to pharmaceutical compositions containing them and their uses in medicine.

Description

PARTICLES, COMPOSITIONS AND METHODS

Technical Field

Background to the Invention

Genetic engineering of immune cells enables novel immunotherapies that are tailored to their specific target. For example, T-cells can be modified to express a chimeric antigen receptor (CAR-T-cells) or a T cell receptor (TCR) that enables them to recognize and destroy cancer cells.

Specificity engineered-T-cells are usually produced from isolated T-cells, either by viral vectors or by non-viral transposon-based systems. The latter require transduction of transposon DNA into the nucleus, which can be achieved by electroporation of activated, dividing cells. However, this is accompanied by considerable toxicity. Moreover, engineering of non-activated / resting T cells is very difficult to achieve meaning that an additional step of activating the T cells is required to achieve some level of T cell engineering: see An, Jing et al. (2024): Nature Biomedical Engineering 8 (2), pp. 149-164 (DOI: 10.1038/s41551-023- 01073-7); and Hamilton, J.R. et al. (2024): In vivo human T cell engineering with enveloped delivery vehicles. In Nature Biotechnology. Jan 11 (DOI: 10.1038/s41587-023-02085-z).

Lipid nanoparticles (LNPs) have demonstrated huge potential as delivery technology for nucleic acid vaccines for treating a wide range of conditions, such as in cancer immunotherapy, gene therapy, and the treatment of infectious diseases.

Typically, LNPs contain cationic or cationically ionizable lipids, helper lipids (typically phospholipids) and a steroid (typically cholesterol). A standard LNP formulation known in the art typically comprises 47.5 mol% ionizable lipid, 40.5 mol% cholesterol, and 10 mol% phospholipid, all expressed as a percentage of the total lipids present in the lipid mixture composition.

WO 2009/127060 describes nucleic acid-lipid particle formulations comprising an interfering

RNA, such as small interfering RNA (siRNA), microRNA (miRNA) or asymmetrical interfering RNA (aiRNA), and their use in delivering nucleic acids. The lipid mixtures present in these formulations contain higher percentages of cationically ionizable lipids than those present in the standard commercial LNP formulation - the maximum proportion of ionizable lipid being 70 mol%. However, these formulations contain a lower ratio of phospholipid to cholesterol than those of the present invention - the maximum ratio in the examples disclosed therein being 0.418. Moreover, this document only specifically discloses the use of these lipid particle compositions to formulate siRNA, miRNA or aiRNA and provides no teaching that the compositions can successfully formulate and transfect DNA or non-interfering RNA.

WO 2023/148276 and WO 2023/148277 generally describe functionalised nucleic acid-lipid particles and their use in targeted delivery of payload to cells. However, neither document specifically discloses an LNP formulation having a cationically ionizable lipid in a proportion higher than the 47.5 mol% present in the standard commercial formulation referred to above. In addition, the formulations disclosed therein also have a lower ratio of phospholipid to cholesterol than those of the present invention - the maximum ratio in the examples disclosed therein being 0.261.

An LNP-based transfection system could provide a cheaper and less toxic alternative platform for DNA delivery. However, while RNA transfection with LNPs is well established and is effective with little toxicity, DNA transfection and stable engineering of T cell poses a considerable obstacle. An LNP formulation capable of delivery and transfection of DNA would therefore be desirable.

Summary of the Invention

In a first aspect of the invention, there is provided a nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) a steroid (e.g., cholesterol); and

(ii) a nucleic acid which is selected from DNA, a non-interfering RNA, or a mixture thereof; wherein the molar ratio of phospholipid to the steroid (e.g., cholesterol) is 0.5 to 3.0.

In a second aspect of the invention, there is provided a nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid in an amount of 50 to 75 mol% of the total lipids present in the lipid mixture composition,

(b) a phospholipid; and

(c) a steroid (e.g., cholesterol); and

(ii) a nucleic acid which is selected from DNA, a non-interfering RNA, or a mixture thereof.

In some embodiments of the first and second aspects, the lipid mixture composition further comprises:

(d) 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; and

X2 is absent or a second linking moiety.

(d) a compound of Formula (A’):

L-X1-P-X2-B1 (A’) wherein:

P is absent or comprises a polymer;

L comprises a hydrophobic moiety attached to Bi when P is absent or to a first end of the polymer P when present;

Bi comprises a binding moiety comprising a polymer, the binding moiety Bi 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; and

X2 is absent or a second linking moiety.

In some embodiments of the first and second aspects, the nucleic acid-lipid particle is a functionalised nucleic acid-lipid particle (as defined herein).

In a third aspect of the invention, there is provided a method of producing the nucleic acid- lipid particle of the first or second aspect, the method comprising mixing:

(a) a cationically ionizable lipid; (b) a phospholipid; and

(c) a steroid (e.g., cholesterol); and optionally

(d) a compound of formula (A) as defined herein; and

(ii) a nucleic acid; to form the nucleic acid-lipid particle.

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) a steroid (e.g., cholesterol); and optionally

(d) a compound of formula (A’) as defined herein; and

(ii) a nucleic acid; to form the nucleic acid-lipid particle.

The compounds of formulae (A) or (A’) are also referred to herein as a “targeting compounds”. Typically, the hydrophobic moiety (L) of compounds of formulae (A) or (A’) is incorporated into the particle, such that the binding moiety (B) or (Bi) of the compound of formula (A) is then oriented on the particle surface. The particle may be already functionalised where B or Bi is a moiety binding to a cell surface antigen on target cells. Otherwise, functionalisation of the particle is possible, for example by interacting a “docking compound” as defined herein, such as a compound of formula (I) or formula (T), as defined herein, with the binding moiety (B) or (Bi) (e.g., a peptide tag, polymer or a moiety binding to a peptide tag) of the compounds of formulae (A) or (A’), wherein the compound of formula (I) or formula (T) comprises a moiety B” binding to a cell surface antigen on target cells. In some embodiments, the targeting compound is a lipid bound to a targeting ligand.

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

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

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

In another embodiment of the above aspects, the binding moiety Bi is a polymer. In a fourth aspect of the invention, there is provided a functionalised nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) a steroid (e.g., cholesterol); and

(d) a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined herein; wherein the molar ratio of phospholipid to the steroid (e.g., cholesterol) is 0.5 to 3.0;

(ii) a nucleic acid; and

(iii) a docking compound.

In a fifth aspect of the invention, there is provided a functionalised nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid, in an amount of 50 to 75 mol% of the total lipids present in the lipid mixture composition;

(b) a phospholipid;

(c) a steroid (e.g., cholesterol); and

(d) a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined herein;

(ii) a nucleic acid; and

(iii) a docking compound.

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) a steroid (e.g., cholesterol); and

(ii) a nucleic acid; and

(iii) a docking compound.

In a sixth aspect of the invention, there is provided a method of forming a functionalised nucleic acid-lipid particle of the fourth or fifth aspects, the method comprising:

(a) forming a nucleic acid-lipid particle according to the method of the fourth or fifth aspects; and (b) mixing the nucleic acid-lipid particle with the docking compound, such that the docking compound interacts with the nucleic acid-lipid particle.

In one embodiment of the fourth, fifth and sixth aspects, the docking compound is a compound of formula (I):

B’-X3-B” (I) wherein B’ comprises a moiety binding to B of the compound of formula (A) or Bi of the compound of formula (A’) as defined herein;

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a functionalised nucleic acid-lipid particle according to the fourth or fifth aspect and a pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a functionalised nucleic acid-lipid particle according to the fourth or fifth aspect for use as a medicament.

In a ninth aspect of the invention, there is provided a functionalised nucleic acid-lipid particle according to the fourth or fifth aspect for use in treating a disease involving an antigen.

In a tenth aspect of the invention, there is provided a functionalised nucleic acid-lipid particle according to the fourth or fifth aspect for use in treating a disease characterized by the presence of diseased cells expressing an antigen.

In an eleventh aspect of the invention, there is provided a functionalised nucleic acid-lipid particle according to the fourth or fifth aspect for use in treating cancer.

In a twelfth aspect of the invention, there is provided a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) a steroid (e.g., cholesterol); and optionally

(d) a targeting compound, preferably a compound of formula (A) or formula (A’) as defined herein; wherein the molar ratio of phospholipid to the steroid (e.g., cholesterol) is 0.5 to 3.0. In a thirteenth aspect of the invention, there is provided a lipid mixture composition comprising:

(a) a cationically ionizable lipid in an amount of 50 to 75 mol% of the total lipids in the composition,

(b) a phospholipid;

(c) a steroid (e.g., cholesterol); and optionally

(d) a targeting compound, preferably a compound of formula (A) or formula (A’) as defined herein.

In a fourteenth aspect of the invention, there is provided a method of producing the lipid- mixture composition according to the twelfth or thirteenth aspect, the method, comprising mixing:

(a) a cationically ionizable lipid,

(b) a phospholipid; and

(c) a steroid (e.g., cholesterol); and optionally

(d) the targeting compound; to form the lipid mixture composition.

Advantages and Surprising Findings

The present inventors have surprisingly found that changing the formulation of the lipid mixture composition, by increasing the phospholipid: steroid (e.g., phospholipid:cholesterol) ratio to a higher value than in the compositions disclosed in WO2019/127060 (particularly, though not exclusively, while maintaining the N/P ratio constant), and/or by increasing the percentages of cationically ionizable lipids compared with standard commercial LNP formulations, led to a several-fold increase in DNA transfection. In particular, the present inventors have observed that these formulations also showed excellent potency for RNA delivery and maintained good T cell viability. The inventors have also found that the marked improvement of DNA delivery caused by increasing the phospholipid: steroid (e.g., phospholipid:cholesterol) ratio and/or by increasing the percentages of cationically ionizable lipids compared with standard commercial LNP formulations, was consistent across a variety of different lipids tested.

Brief Description of the Figures

Figure 1 shows the results of in vitro transfection studies of the functionalised nucleic acid- lipid particles of the invention (samples 2-1 through 2-20, as listed in Table 1) on peripheral blood mononuclear cells (PBMCs). Depicted are (a) the percentages of Thy 1.1- expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD 19+ B cells) out of all single and alive cells (y- axis) at a dose of 100 ng Thyl.l-RNA (b) the percentages of Venus-expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y-axis) and total count of Venus-positive T cells (stars; right x-axis) at a dose of 40 ng Venus-DNA; (c) the same at 200 ng Venus-DNA; (d) cell counts of overall cell subtypes (CD 14+ Monocytes; CD 19+ B cells, CD4+ T cells, CD8+ T cells) at day 4 after treatment with LNPs at a dose of 100 ng Thy 1.1/200 ng Venus.

Figure 2 shows Dynamic Light Scattering (DLS) data of the samples of Figure 1, before and after functionalisation and after one freeze-thaw cycle at -80°C.

Figure 3 shows the results of agarose gel electrophoresis of the samples from Figure 1.

Figure 4 shows the results of in vitro testing of the functionalised nucleic acid-lipid particles of the invention (samples 3-1 to 3-6, as listed in Table 2) on PBMCs after one freeze/thaw cycle. Depicted are (a) the percentages of Thy 1.1- expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD 19+ B cells) out of all single and alive cells (y-axis) at a dose of 100 ng Thyl.l-RNA (b) the percentages of Venus-expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y-axis) and total count of Venus-positive T cells (stars; right x-axis) at a dose of 200 ng Venus-DNA;; (c) cell counts of overall cell subtypes (CD14+ Monocytes; CD19+ B cells, CD4+ T cells, CD8+ T cells) at day 4 after treatment with LNPs at a dose of 100 ng Thy 1.1/200 ng Venus.

Figure 5 shows the results of in vitro testing of the functionalised nucleic acid-lipid particles of the invention (samples 4-1 to 4-4, as listed in Table 3) on PBMCs. Depicted are (a) the percentages of Venus-expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y-axis) and total count of Venus-positive T cells (stars; right x-axis) at a dose of 200 ng Venus-DNA.

Figure 6 shows the results of in vivo testing of a frozen and then thawed LNP formulation (Sample ID: 2-12) in human CD3EDG transgenic mice. Depicted are (a) the results of ex vivo organ bioluminescence imaging; (b) cell-type specific transfection; (c) T cell activation (monitored by CD69 expression levels) and (d) systemic cytokine levels.

Figure 7 shows the in vitro NLuc DNA expression in primary T cells for a functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2-1 (see table in Example 2).

Figure 8 shows the in vitro NLuc DNA expression in primary T cells for functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2-1 (see table in Example 2). Figure 9 shows the in vitro NLuc DNA expression in primary T cells for functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2- 1 (see table in Example 2).

Figure 10 shows DLS data of the samples of Figure 4 before and after functionalisation and after one freeze-thaw cycle at -80°C.

Figure 11 shows DLS data of the samples of Figure 5 before and after functionalisation and after one freeze-thaw cycle at -80°C.

Figure 12: targeted gene integration in T cells using LNP -formulated CRISPR-Cas9 or Zine- Finger Nuclease (ZFN) -mediated gene editing, with nanoplasmid DNA templates.

Figure 13: in vitro CRISPR-Cas9-mediated targeted gene insertion in B cells using LNPs.

Figure 14: targeted in vivo delivery using ahCD3-LNPs (A) % Thyl. l positive cells (B) mean fluorescence intensity (MFI) of Thyl.l positive cells.

Figure 15: in vivo CAR-T cell generation in mice with T cell expansion, by ahCD3-LNPs mediated delivery of CAR DNA and Sleeping Beauty (SB) transposase RNA mixed payload. Figure 16: in vivo CAR-T cell generation in PBMC-engrafted mice by ahCD3-LNPs mediated delivery of CAR DNA and SB transposase RNA payload.

Detailed Description

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.

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; Beyer/Walter, "Lehrbuch der Organischen Chemie", S. Hirzel Verlag Stuttgart, 1988; Carey/Sundberg, "Organische Chemie", VCH, 1995; March, "Advanced Organic Chemistry", John Wiley & Sons, 1985; Rompp 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.

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.

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.

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.

Definitions

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.

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 .

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.

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.

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.

"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.

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.

As used in the present disclosure, "mol % of the lipid mixture composition" 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 composition, multiplied by 100. In this context, in some embodiments, the term "total lipid" and/or “total lipid mixture” includes lipids and lipid-like material.

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 C1-40 alkyl (such as Ce-4o alkyl, Ce-so alkyl, Ce-2o alkyl, or C10-20 alkyl), C2-40 alkenyl (such as Ce-4o alkenyl, Ce-so alkenyl, or Ce-2o alkenyl) having 1, 2, or 3 double bonds, aryl, and aryl(Ci.e 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.

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-e). 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.

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, neopentyl, 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.

The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, z.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 (z.e., 1,1-ethylene,

1.2-ethylene), propylene (z.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-iso- butylene, 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.

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 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 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.

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 carboncarbon 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), z.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, z.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- 1,2-diyl, vinylidene (also called ethenylidene), 1 -propen- 1,2-diyl, 1 -propen- 1,3 -diyl, l-propen-2,3-diyl, allylidene, 1-buten- 1,2-diyl, l-buten-l,3-diyl, l-buten-l,4-diyl, l-buten-2,3-diyl, l-buten-2,4-diyl, l-buten-3,4- diyl, 2-buten- 1,2-diyl, 2-buten-l,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.

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 carboncarbon 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.

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 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, 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, 1-propyn- 1 ,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.

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.

The term "aryl" refers to a monoradical of an aromatic cyclic hydrocarbon. 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).

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 quatemized 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.

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, 1H- indazolyl, benzimidazolyl, benzoxazolyl, indoxazinyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl, benzodiazinyl, quinoxalinyl, quinazolinyl, benzotriazinyl, pyridazinyl, phenoxazinyl, thiazolopyridinyl, pyrrolothiazolyl, 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 “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).

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.

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

“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 R.?, 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 Ci-e 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.

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

“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.

“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 CMO alkyl group.

“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.

“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 C1-6 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. “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.

“Sulfonamide” means the group -S(=O)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 C1-6 alkyl group.

“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 C1-6 alkyl group.

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.

“List A” substituents are selected from the group consisting of C1-6 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’, - -X¹C(=X¹)X¹R’, wherein X¹ is independently selected from O, S, NH and N(CH₂); and each R’ is independently selected from the group consisting ofH, 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-3 alkyl), -N(CI-3 alkyl)₂, - NHS(O)₂(C1 3 alkyl), -S(O)₂NH_2.z(Ci ₃ alkyl)_z, -C(=O)OH, -C(=O)O(C1.₃ alkyl), -C(=O)NH₂_ _z(Ci.₃ alkyl)_z, -NHC(=0)(Cl-3 alkyl), -NHC(=NH)NH_Z-2(C1-3 alkyl)_z, and -N(CI-₃ alkyl)C(=NH)NH_2.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, -NH_2-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.

The term “DNA template” may refer to a DNA payload suitable for delivery as part of a gene editing tool (e.g. as described herein). For example, the DNA payload may be integrated into a target cell genome following cleavage by e.g. a nuclease of a gene editing tool. Suitably, the DNA template may encode a polypeptide. Suitably, a DNA template may be referred to as a ‘transgene’ .

Nucleic Acid

The lipid particle compositions of the present application (both when functionalised and prior to functionalisation) 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 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. In one embodiment, the active ingredient is nucleic acids comprising DNA.

In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is mRNA.

In one embodiment, the nucleic acid is a non-interfering RNA. In one embodiment, an interfering RNA may be understood as RNA that may elicit RNA interference (RNAi) and produce a gene silencing effect. In one embodiment, a non-interfering RNA may be any RNA molecule that does not have this RNAi effect. In one embodiment, a non-interfering RNA is any RNA that is not a siRNA, a miRNA or an aiRNA. In one embodiment, the nucleic acid is not an siRNA. In one embodiment, the nucleic acid is not an aiRNA. In one embodiment, the nucleic acid is not a miRNA. In one embodiment, the RNA may be in a form selected from an mRNA, a circular RNA, a self-replicating RNA (saRNA), a trans-amplifying RNA (taRNA), a replicon, or mixtures thereof.

In one embodiment, the nucleic acid is DNA. In one embodiment, the DNA may be in a form selected from a plasmid, minicircle, nanoplasmid, transposon, linear DNA, or mixtures thereof. In one embodiment, the nucleic acid comprises DNA.

In one embodiment, the nucleic acid is a mixture of DNA and mRNA. In one embodiment, the nucleic acid is a mixture of DNA and a non-interfering RNA. In one embodiment, the nucleic acid is a mixture of DNA and RNA. In one embodiment, the nucleic acid is a mixture of nanoplasmid DNA and mRNA. In one embodiment, the nucleic acid comprises one or more nanoplasmid DNAs and one or more mRNAs.

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.

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-C1-4 alkylguanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyluracil, 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-pseudouridine (mlT).

RNA

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 -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 analogues 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 analogues 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 analogues thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), singlestranded 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 RNA may be mRNA, saRNA, taRNA, or mixtures thereof. The RNA is preferably mRNA. In some instances, the RNA is not siRNA. In some instances, the RNA is not miRNA. In some instances, the RNA is not aiRNA.

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

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.

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

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

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.

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 (i.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™).

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 (\|/) or N(l)-methyl -pseudouridine (m h|i). 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 (\|i), Nl-methyl-pseudouridine (ml\|/), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (\|/) or Nl-methyl-pseudouridine (ml\|/).

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.

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.

DNA

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 analogues 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 analogues 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.

Pharmaceutically active peptides or polypeptides

"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.

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), N1 -methyl -pseudouridine (ml\|/), 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.

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), tumour 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, CD19), antigen receptor accessory molecules (e.g., CD-3y, CD3-5, CD-3a, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds such as tumour-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.

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 functionalised 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.

In some embodiments, the nucleic acid provides a gene editing reagent or tool (e.g., a transposon/transposase system (such as sleeping beauty or piggy bac), a large serine recombinase (LSR) (e.g., Bxbl and PhiC31), a Zinc-finger nuclease (ZFN) or CRISPR/Cas (or related) based system.

CRISPR/Cas is a target-specific technique that can introduce gene knock out or knock in depending on the double strand repair pathway. The targeting specificity of CRISPR/Cas is determined by the 20-nt sequence at the 5' end of the guide RNA (gRNA). The desired target sequence must precede the protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 base pairs in length that follows the DNA region targeted for cleavage by CRISPR/Cas. The PAM is required for a Cas nuclease to cut and is generally found 3-4 nucleotides downstream from the cut site. After base pairing of the gRNA to the target, Cas mediates a double strand break about 3-nt upstream of PAM.

ZFNs are engineered proteins with sequence-specific nuclease activity. Suitably, a ZFN protein comprises a DNA cleavage domain fused to a zinc-finger DNA-binding domain. The zinc-finger DNA-binding domain may be designed to bind a specific DNA sequence. Pairs of ZFN proteins are able to generate a DNA double-strand break. The introduction of a DNA double -strand break can introduce gene knock out or knock in depending on the double strand repair pathway. Suitably, for the nuclease-mediated ZFN and CRISPR/Cas (e.g. Cas9) tools, the choice of gene knock out or knock in may depend on the provision or lack of a donor template. To carry out gene knock in, a donor template may be provided comprising a transgene to be knocked in flanked by DNA homologous to the genomic DNA at the nuclease cut site.

LSRs are site-specific recombinases that are able to mediate integration of DNA at attachment sites (att sites). LSRs can be used to introduce gene knock out or knock in depending on the location of the att site and the composition of a donor vector carrying the complementary att site.

Transposons are transposable DNA elements that are able to integrate into the genome. Transposons in combination with transposase enzyme activity are able to introduce gene knock out or knock in. Transposon integration can disrupt endogenous gene expression. Transposons may comprise a DNA template to be knocked in, integrating the DNA template into the genome when the transposon integrates into the genome.

Such tools (e.g. transposase, gene editing tools like LSR, ZFN or CRISPR/Cas (e.g. Cas9)) for genomic integration/editing may be delivered as protein or coding nucleic acid (e.g. DNA or RNA). Such tools may comprise multiple separable elements (e.g. Cas enzyme and gRNA for CRISPR/Cas tools).

In some embodiments, the nucleic acid comprises a gene editing tool or comprises at least one element of a gene editing tool. The at least one element may be encoded as coding nucleic acid (DNA or RNA). In some embodiments, all elements of a gene editing tool may be encoded as RNA. In some embodiments, all elements of a gene editing tool may be encoded as DNA. In some embodiments, at least one element of a gene editing tool may be encoded as RNA and at least one further element of the gene editing tool may be encoded as DNA. In some embodiments, the DNA may be in a form selected from a plasmid, minicircle, nanoplasmid, transposon, linear DNA, or mixtures thereof.

In some embodiments, the gene editing tool may comprise a gene editing enzyme (e.g. a nuclease).

In some embodiments, the gene editing tool may comprise a gene editing enzyme (e.g. a nuclease) and a DNA template. In some embodiments, the nucleic acid provides a CRISPR/Cas (e.g. Cas9) gene editing tool. In some embodiments, the CRISPR/Cas (e.g. Cas9) gene editing tool may be encoded as DNA and/or RNA. In some embodiments, the CRISPR/Cas (e.g. Cas9) gene editing tool comprises a Cas enzyme (e.g. a Cas9 enzyme), a gRNA and optionally a DNA template. Suitably, the Cas is encoded by DNA or RNA. In some embodiments, the Cas and gRNA are encoded by RNA. In some embodiments, the Cas and gRNA are encoded by RNA. In some embodiments the DNA template is a DNA nanoplasmid.

In some embodiments, the nucleic acid provides a CRISPR/Cas (e.g. Cas9) gene editing tool encoded as RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is RNA.

In some embodiments, the nucleic acid provides a CRISPR/Cas (e.g. Cas9) gene editing tool as a mixture of DNA and RNA, wherein a Cas enzyme is encoded by mRNA, a gRNA is RNA, and a DNA template is a DNA in the form of a nanoplasmid.

In some embodiments, the nucleic acid provides a ZFN gene editing tool. In some embodiments, the ZFN gene editing tool may be encoded as DNA and/or RNA. In some embodiments, the ZFN gene editing tool comprises at least two ZFN proteins and optionally a DNA template. In some embodiments, the ZFN proteins are encoded by DNA or RNA. In some embodiments, the ZFN proteins are encoded by mRNA. In some embodiments the DNA template is a DNA nanoplasmid.

In some embodiments, the nucleic acid provides a ZFN gene editing tool encoded as RNA, wherein two ZFN proteins are encoded by mRNA. Suitably, the ZFN proteins are encoded by separate mRNAs.

In some embodiments, the nucleic acid provides a ZFN gene editing tool encoded as a mix of DNA and RNA, wherein two ZFN proteins are encoded by separate mRNAs and a DNA template is a DNA in the form of a nanoplasmid.

In some embodiments, the nucleic acid comprises (i) a DNA nanoplasmid comprising a DNA template, and (ii) an mRNA encoding an enzyme selected from a transposase (e.g., Sleeping Beauty transposase), a DNA integrase (e.g., a LSR) or a nuclease (e.g., a Zn-fmger nuclease or a Cas9 nuclease), wherein the enzyme is capable of directing cleavage of a target DNA, such that the DNA template of the DNA nanoplasmid can be inserted into the genome of the target cell. In some embodiments, the nucleic acid comprises (i) a DNA nanoplasmid comprising a DNA template, (ii) an mRNA encoding a Cas9 nuclease, and (iii) a guide RNA. In some embodiments, the nucleic acid comprises (i) a DNA nanoplasmid comprising a DNA template and (ii) an mRNA encoding a Sleeping Beauty transposase. In some embodiments, the DNA template encodes a chimeric antigen receptor (CAR).

Polymers

In some embodiments, the compositions described herein (including the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles), and components thereof, particularly although not exclusively the targeting compound and, if present, the grafted lipid, may contain polymers.

A "polymer" as used herein, is given its ordinary meaning, z.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds. The repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer. In some cases, the polymer is biologically derived, z.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer, for example targeting moieties. If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." The repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, z.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc. Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.

In some embodiments, the targeting compound comprises a polymer, defined as P herein when present according to Formula (A), or otherwise described in relation to moiety Bi according to Formula (A’)..

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.

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 (i.e. payload or cargo) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

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.

In some embodiments, the polymer for use herein is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), poly-2 - (2-(2-(N-methylamino)-ethoxy)ethoxy)acetic acid (pmAEEA) (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.

In some embodiments, the polymer is preferably PEG. 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.

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.

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

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

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.

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 10 and 200, between 10 and 190, between 10 and 180, between 10 and 170, between 10 and 160, between 10 and 150, between 10 and 140, between 10 and 130, between 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. 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.

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.

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.

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 10 and 150, between 10 and 140, between 10 and 130, between 10 and 120, between 10 and 110, between 10 and 100, between 10 and 90, between 10 and 80, or between 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.

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.

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.

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, Ri i 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. In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-(N-methylamino)ethoxy)ethoxy)acetic acid (pmAEEA), or a derivative thereof, as defined herein.

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

X¹¹ and X¹² taken together are optionally substituted amide, optionally substituted thioamide or ester;

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

In some embodiments,

(i) when X¹¹ is -C(O)- then X¹² is -NR¹-;

(ii) when X¹¹ is -NR¹- then X¹² is -C(O)-;

(iii) when X¹¹ is -C(S)- then X¹² is -NR¹-;

(iv) when X¹¹ is -NR¹- then X¹² is -C(S)-;

(v) when X¹¹ is -C(O)- then X¹² is -O-; or

(vi) when X¹¹ is -O- then X¹² is -C(O)-; wherein R¹ is hydrogen or Ci-s alkyl.

In some embodiments, X¹¹ is -C(O)- and X¹² is -NR¹-, wherein R¹ is hydrogen or Ci-s alkyl. In some embodiments, X¹¹ is -C(O)- and X¹² is -NR¹-, wherein R¹ is hydrogen or methyl. In some embodiments, X¹¹ is -C(O)- and X¹² is -NR¹-, wherein R¹ is hydrogen.

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

R¹ is hydrogen or Ci-s 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.

R¹ is hydrogen or Ci-s alkyl; and n is 1 to 100.

In some embodiments of the above formulas, R¹ is hydrogen or methyl. In some embodiments, R¹ is hydrogen.

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

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.

Targeting compounds

In some embodiments of all aspects of the disclosure, the lipid mixture composition comprises a targeting compound, as defined herein.

Typically, the targeting compound present in the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles of the present invention comprise a hydrophobic moiety, such as a lipid, conjugated to a binding moiety. In one embodiment, the lipid L has a binding moiety B or moiety Bi covalently attached thereto, optionally via polymer P (when present) and/or linking moieties XI and X2, comprises a compound of formula (A) or formula (A’), as described further herein.

In some embodiments, the targeting compound comprises a lipid bonded to a targeting ligand. In some embodiments, the lipid bonded to a targeting ligand is a compound of Formula (A): L-X1-P-X2-B (A) wherein:

P is absent or comprises a polymer, as defined herein;

XI is absent or a first linking moiety; and

X2 is absent or a second linking moiety.

In some embodiments, the lipid bonded to a targeting ligand is a compound of Formula (A’): L-X1-P-X2-B1 (A’) wherein:

P is absent or comprises a polymer, as defined herein;

XI is absent or a first linking moiety; and

X2 is absent or a second linking moiety.

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).

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.

In some embodiments, the binding moiety Bi of the targeting compound comprises a polymer, as described herein. In some embodiments, the binding moiety Bi of the targeting compound comprises PEG.

In some embodiments, the binding moiety Bi of the targeting compound comprises a polymer, as described herein, and is chemically linked, e.g., through a linker, to the hydrophobic moiety (e.g., lipid).

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, 5-tocopherol, a-tocotrienol, P-tocotrienol, y- tocotrienol, 5-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.

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 (pahnitoyloleyl-phosphatidylethanolamine), and mixtures thereof. 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.

In some embodiments, the targeting compound comprises a polymer, defined 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. In some embodiments, the polymer moieties P and/or Bi of the targeting compound is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA), poly-2-(2-(2-(N-methylamino)- ethoxy)ethoxy)acetic acid (pmAEEA) (including derivatives thereof), as defined herein.

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%.

In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.001 to 10 mol% of the total lipids present in the lipid mixture composition. In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.002 to 5 mol% of the total lipids present in the lipid mixture composition. In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.005 to 2 mol% of the total lipids present in the lipid mixture composition. In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.01 to 1 mol% of the total lipids present in the lipid mixture composition. In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.02 to 0.5 mol% of the total lipids present in the lipid mixture composition. In some embodiments, the targeting compound, such as the compound of formula (A) or formula (A’), is present in an amount of 0.04 to 0.25 mol% of the total lipids present in the lipid mixture composition.

In one embodiment of formula (A) or formula (A’), the hydrophobic moiety comprises a lipid. In one embodiment of formula (A) or formula (A’), the hydrophobic moiety comprises a phospholipid. In one embodiment of formula (A) or 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) or formula (A’), the hydrophobic moiety comprises a DSPE moiety. In one embodiment of formula (A) or formula (A’), P is absent. In one embodiment of formula (A) or formula (A’), P is a polymer. In one embodiment of formula (A) or formula (A’), P is a hydrophilic polymer. In one embodiment of formula (A) or formula (A’), P is selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA), and poly-2-(2-(2-(N- methylamino)ethoxy)ethoxy)acetic acid (pmAEEA), derivatives and combinations thereof.

In one embodiment of formula (A) or 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) or formula (A’), P comprises the following general formula: wherein n is 1 to 100.

In one embodiment of formula (A) or formula (A’), XI comprises a carbonyl group.

In one embodiment of formula (A) or 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) or formula (A’), the thiol or cysteine reactive group comprises a maleimide group.

In one embodiment of formula (A) or formula (A’), the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoylglycerylphosphoethanolaminepolyethylene glycol-conjugate (DSPE-PEG).

In some embodiments, the targeting compound is a compound of Formula (Al):

L-X1-P-X2-B (Al) wherein

P comprises a polymer;

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;

XI is absent or a first linking moiety; and

X2 is absent or a second linking moiety.

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

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.

In some embodiments of formula (Al), L comprises a lipid as described above. In some embodiments of formula (Al), L comprises DSPE (distearoylphosphatidyl -ethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (pahnitoyloleylphosphatidyl-ethanolamine) which may be linked to P by an amide group.

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.

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

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 distearoyl-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) . 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.

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.

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.

In some embodiments, the targeting compound is of the general formula (A2): L-X1-P-X2-B (A2) wherein L, XI, P and B are as described above and X2 comprises a thiosuccinimide moiety.

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).

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.

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 -(CtfilnCHs (stearyl), -(CE^isCfE (palmityl), and -(CH2)i3CH3 (myristyl).

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

In some embodiments of formula (A3), the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)acetic acid (pAEEA) or poly-2-(2-(2-(N-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)ISCH3 (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 R_t2 is H. In some embodiments of formula (A4), R_ti is H, and Rt2 is methyl. In some embodiments of formula (A4), R_ti and Rt2 are both H.

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

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-(N- methylamino)ethoxy)-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.

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 moieties include CH₃(CH₂)I₆C(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)I₆C(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 -HPO₃ (CH₂)_n- NH-, wherein n is 1 to 5, e.g., 2.

In some embodiments of formula (A5), the polymer P comprises poly-2-(2-(2- aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-(N-methylamino)ethoxy)- 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.

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.

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 moieties 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-(N-methylamino)-ethoxy)- 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 Rl and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A5’), n is 14 and Rl and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments of formula (A5’), n is 8 and Rl and R2 are CH3(CH2)i2C(O)- (myristoyl). In some embodiments of formula (A5’), n is 14 and Rl and R2 are CH₃(CH₂)i2C(O)- (myristoyl).

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 Rl and R2 are CH3(CH2)ieC(O)- (stearoyl). In some embodiments, s is 20 and Rl and R2 are CH₃(CH₂)i2C(O)- (myristoyl). In some embodiments of the above formulae (A), (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), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the compound comprising a thiol or cysteine group comprises the formula HS(CH2)nC(0)-, 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:

In some embodiments of the above formulae (A), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), X2 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.

In some embodiments of the targeting compound, such as the compound of the above formulae (A), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the binding moiety B or moiety Bi 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), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5) or (A5’), the binding moiety B or moiety Bi 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.

In one embodiment, the targeting compound is of the general formula (A 10):

L-X1-P-X2-B (A 10) wherein:

P comprises a polymer;

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

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;

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

In one embodiment, the targeting compound is of the general formula (A 10):

L-X1-P-X2-B (A 10) wherein:

P comprises a polymer;

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;

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 (A 10), L comprises a phosphatidylethanolamine which may be linked to P by an amide group.

In some embodiments of formula (A 10), 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 (A 10), X2 comprises a thiosuccinimide moiety.

In some embodiments of formula (A10), L comprises a lipid as described above. In some embodiments of formula (A 10), L comprises DSPE (distearoylphosphatidyl -ethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (pahnitoyloleylphosphatidyl-ethanolamine) which may be linked to P by an amide group.

In some embodiments of formula (A10), P comprises a polymer as described above. In some embodiments of formula (A 10), 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 (A 10), P comprises a polymer selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly (N-methylgly cine), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), and poly-2-(2-(2-(N-methylamino)ethoxy)ethoxy)acetic acid (pmAEEA) (including derivatives thereof). In some embodiments of formula (A10), P comprises polyethylene glycol (PEG); e.g., PEG as described above.

In some embodiments of formula (A 10), L-Xl-P comprises an amphiphilic derivative of a polymer as described above. In some embodiments of formula (A 10), 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 (A 10), the amphiphilic derivative of a polymer comprises a disteroyl-glycero- phosphoethanolamine-polyethyleneglycol-conjugate (DSPE-PEG) .

In some embodiments of formula (A 10), 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 (A 10), the thiol or cysteine reactive group comprises a maleimide group. In some embodiments of formula (A10), the PEG reagent comprises DSPE- PEG-maleimide. In some embodiments of formula (A 10), 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 (A 10), n is 2.

In some embodiments of formula (A 10), 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 (A 10), n is 2.

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.

In some embodiments, the targeting compound is of the following general formula (A 10”) wherein X2 is as described above, Ri and R2 are CHs(CH2)i6C(O)- (stearoyl) or CH₃(CH₂)12C(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 14, and B comprises an epitope tag, e.g., an ALFA-tag such as an ALFA-tag described herein.

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

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)i6C(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)I₆C(O)- (stearoyl). In some embodiments of formula (A10’”), s is 20 and Ri and R₂ are CH₃(CH₂)I₂C(O)- (myristoyl).

In some embodiments of formula (A 10”’), X2 comprises the following general formula:

In some embodiments of formula (A 10’”), 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.

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.

In one embodiment of the targeting compound, such as of any of the above formulae (A), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (AIO’), (AIO”) or (AIO’”), 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.

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

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

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

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), (AIO’), (AIO”) or (AIO’”), the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3. 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), (AIO’), (AIO”) or (AIO’”), the cell surface antigen is selected from the group consisting of CD4, CD8, CD3, CD7, CD2, CD28, IL7R, CD 127 and CD5.

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), (AIO’), (AIO”) or (AIO’”), the peptide tag comprises an ALFA-tag.

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

L-X1-P-X2-B (A20) wherein

P comprises a polymer;

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

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

XI is absent or a first linking moiety; and

X2 is absent or a second linking moiety.

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

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.

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., 1,2- distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

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.

In some embodiments of Formula (A20), the hydrophobic moiety having a binding moiety covalently attached thereto comprises a distearoylglycerylphospho-ethanolaminepolyethylene glycol-conjugate (DSPE-PEG). 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.

In some embodiments, the targeting compound is DSPE-PEG2k-ALFA, wherein ALFA is an ALFA-tag as defined herein.

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 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: AAO is Pro or deleted;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin;

AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu;

AA9 is Arg, Ala, Gin, or Glu;

AA10 is Arg;

AA11 is Leu;

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

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

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), 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 PSRLEEELRRRLTEP (SEQ ID NO: 4).

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 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;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin;

AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu;

AA9 is Arg, Ala, Gin, or Glu;

AA10 is Arg;

AA11 is Leu;

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

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

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.

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 AA 13 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.

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 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. -AAO-AA 1 -AA2-AA3-AA4-cyclo(Xl-AA6-AA7-X2)-AA9- Arg-Leu -AA12-AA13 -AA 14- 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 . -AAO-AA 1 -AA2- AA3 -AA4-AA5 -cyclo(X 1 -AA7- AA8-AA9-X2)-Leu- AA 12- AA 13 - AA14-, and h. -AAO-AA 1 -AA2- AA3 -cyclo(X 1 -AA5 -AA6-AA7-X2)-AA9-Arg-Leu- AA 12- AA 13 -AA 14- wherein the side-chains of XI and X2 amino acid residues are connected covalently;

AAO is Pro or deleted;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin;

AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu;

AA9 is Arg, Ala, Gin, or Glu;

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

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

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 connected covalently via an amide, disulfide, thioether, ether, ester, thioester, thioamide, alkylene, alkenylene, alkynylene, and/or 1,2, 3 -triazole.

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), 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.

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), 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.

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, independently, selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Om, 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, Om, DOm, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Om, 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.

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 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu - Lys)-, -c(Glu - Lys)-, - cyclo(E - K)-, -c(E - K)-, -E - K- cyclo, or -cycloE — cycloK 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”), (A10’”) 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 stmcture:

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 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:

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 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).

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:

The targeting compound, such as of any of the above formulae (A), (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. 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), (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 nucleic acid-lipid particles, the amount of targeting compound, such as of any of the above formulae (A), (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. When the nucleic acid-lipid particles comprise targeting compounds, such as of any of the above formulae (A), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’), (A10), (A10’), (A 10”), (A 10”’) or (A20), such as peptide-conjugated lipids, this allows for functionalisation of the nucleic acid-lipid particles. For example, the binding moiety B or moiety Bi 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 functionalised nucleic acid-lipid particles. The binding moiety that specifically binds to the peptide of the compound may be an ALFA-tag binding moiety.

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 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), Nb ALFA-nanobody .

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 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).

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.

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 amino acid sequence EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVS ERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHD YWGQGTQVTVSS (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. 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.

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), (AIO’), (AIO”), (AIO’”) 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’), (AIO), (AIO’), (AIO”), (AIO’”) 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.

Preferably the binding moiety is a peptide, and the compositions 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.

Particles

The present invention provides in one aspect a composition comprising particles as described herein. The particles are capable of delivering a nucleic acid payload to a target. 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 nano-sized compact structure.

According to the present disclosure, the term "particle" includes nanoparticles. The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming agent, e.g., at least one cationic or cationically ionizable lipid or a cationic polymer, wherein all three external dimensions of the particle are in the nanoscale, z.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

In some embodiments, the particles described herein have a size (such as a diameter) in the range of 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 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 in the range of about 20 to about 1500 nm, such as about 30 to about 1200 nm, about 40 to about 1100 nm, about 50 to about 1000 nm, about 60 to about 900 nm, about 70 to about 800 nm, about 80 to about 700 nm, about 90 to about 600 nm, or about 50 to about 500 nm or about 100 to about 500 nm, such as in the range of about 10 to about 1000 nm, about 15 to about 500 nm, about 20 to about 400 nm, about 25 to about 300 nm, about 30 to about 250 nm, about 40 to about 200 nm, about 45 to about 150 nm, or about50 to about 100 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 150 nm, such as from about 60 nm to about 120 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 60 nm to about 150 nm. In some embodiments, nucleic acid may be non-covalently 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). In preferred embodiments the nucleic acid is not accessible to small molecule probes such as dyes. A standard test for encapsulation uses the RiboGreen® assay protocol, which uses the RiboGreen® dye which is a fluorescent nucleic acid stain for quantitating intact nucleic acid. Under this assay protocol, in one embodiment the nucleic acids are non-accessible to RiboGreen® by 70% or more, preferably 80% or more, more preferably 90% or more and even more preferably 95% or more.

Lipid Nanoparticles

In one embodiment of the present disclosure, the composition is a lipid nanoparticle (LNP) composition. In the present disclosure, LNPs may be understood as oil-in-water emulsions. LNPs thus typically comprise a central complex of lipid and optionally a nucleic acid (e.g., DNA or RNA, preferably non-interfering RNA, such as mRNA, or mixtures thereof) embedded in a disordered, non-lamellar phase made of lipid.

Nucleic acid-lipid particles, such as nucleic acid-lipid nanoparticles (LNP) are obtainable from combining a nucleic acid with lipids as outlined in more detail below. The lipid mixtures 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.

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 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, such as in the range of about 10 to about 1000 nm, about 15 to about 500 nm, about 20 to about 400 nm, about 25 to about 300 nm, about 30 to about 250 nm, about 40 to about 200 nm, about 45 to about 150 nm, or about 50 to about 100 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 40 nm to about 150 nm, such as from about 60 nm to about 120 nm. In some embodiments, the particles described herein have a size (such as a diameter) in the range of from about 60 nm to about 150 nm.

Lipid Mixture Composition

The present invention provides in one aspect a composition as defined herein. The composition comprises a cationically ionizable lipid, a phospholipid, and cholesterol.

In one embodiment, the composition takes the form of a mixture of lipids including a cationically ionizable lipid, a phospholipid, and cholesterol, each as defined and exemplified herein. This composition, in the absence of any nucleic acid, is also referred to herein as “the lipid mixture composition”.

In one embodiment, there is provided a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) cholesterol; wherein the molar ratio of phospholipid to cholesterol is 0.5 to 3.0.

In one embodiment, there is provided a lipid mixture composition comprising:

(a) a cationically ionizable lipid in an amount of 50 to 75 mol% of the total lipids in the composition;

(b) a phospholipid;

(c) cholesterol.

In one embodiment, the lipid mixture composition includes a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined and exemplified herein. In one embodiment, the lipid mixture composition includes a grafted lipid, as defined and exemplified herein. In one embodiment, the lipid mixture composition includes a targeting compound, e.g. a compound of formula (A) or formula (A’), and a grafted lipid, each as defined and exemplified herein.

Method of Forming Lipid Mixture Composition

In a further aspect, the present invention provides methods for producing the lipid mixture composition of the invention.

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) cholesterol; and

(ii) a nucleic acid; to form the nucleic acid-lipid particle.

In one embodiment, a targeting compound, e.g. a compound of formula (A) or formula (A’) is included in the mixture.

In one embodiment, a grafted lipid is included in the mixture.

The mixing can be carried out using methods well known to those skilled in the art. Nucleic Acid-Lipid Particle

The present disclosure further provides a lipid particle comprising a lipid or lipid mixture composition, 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”.

In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is a noninterfering 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 a mixture of DNA and a non-interfering RNA. In one embodiment, the nucleic acid is a mixture of DNA and a mRNA. In one embodiment, the nucleic acid is a mixture of a DNA nanoplasmid and a mRNA. In one embodiment, the nucleic acid is a mixture of a DNA transposon and a mRNA encoding a transposase. In one embodiment, the nucleic acid is a mixture of a DNA transposon encoding a CAR or TCR, and a mRNA encoding a transposase. In one embodiment, the nucleic acid is not siRNA. In one embodiment, the nucleic acid not miRNA. In one embodiment, the nucleic acid is not aiRNA.

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 nano-sized compact structure. According to the present disclosure, the term "particle" includes nanoparticles.

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 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.

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.

Particles described herein may exhibit a polydispersity 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 polydispersity index in a range of about 0.01 to about 0.4 or about 0. 1 to about 0.3.

A nucleic acid-lipid particle (such as a functionalised 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 (such as a functionalised 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.

In some preferred embodiments, the nucleic acid-lipid particle is a functionalised nucleic acid-lipid particle, as defined herein.

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). 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 favourable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.

In some embodiments, the N/P ratio is 20: 1 to 2: 1. In some embodiments, the N/P ratio is 15: 1 to 2: 1. In some embodiments, the N/P ratio is 12: 1 to 2: 1. In some embodiments, the N/P ratio is 10: 1 to 2: 1. In some embodiments, the N/P ratio is 8: 1 to 2: 1. In some embodiments, the N/P ratio is 18 : 1 to 3 : 1. In some embodiments, the N/P ratio is 15 : 1 to 3 : 1. In some embodiments, the N/P ratio is 12: 1 to 3: 1. In some embodiments, the N/P ratio is 10: 1 to 3: 1. In some embodiments, the N/P ratio is 8: 1 to 3: 1. In some embodiments, the N/P ratio is 16: 1 to 8: 1. In some embodiments, the N/P ratio is 14: 1 to 10: 1. In some embodiments, the N/P ratio is about 12: 1.

Method of Forming Nucleic Acid-Lipid Particle

In a further aspect, the present disclosure provides methods for producing the nucleic acid- lipid particles as disclosed herein.

In one embodiment, there is provided a method of producing the nucleic acid-lipid particle as defined herein, comprising mixing:

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) cholesterol; and

(ii) a nucleic acid; to form the nucleic acid-lipid particle.

In one embodiment, there is provided a method of producing the nucleic acid-lipid particle as defined herein, the method comprising mixing:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) cholesterol; and

(d) a lipid bonded to a targeting ligand; and (ii) a nucleic acid; to form the nucleic acid-lipid particle.

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

As described in more detail below, in one embodiment, the method further comprises adding a docking compound, such as a compound of formula (I) or formula (I’) (as further described herein) to functionalise the nucleic acid-lipid particles. In some instances, the docking compound, such as the compound of formula (I) or formula (I’), may displace (i.e., replace) a corresponding portion of other lipids present in the nucleic acid-lipid particles.

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 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.

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

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.

In one embodiment, the nucleic acid-lipid particles are not subjected to any further purification steps. Primary targeting moiety

According to some embodiments of 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.

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, e.g., the compound of formula (A) or 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), (A’), (Al), (A2), (A2’), (A3), (A4), (A4’), (A5), (A5’) (A10), (A10’), (A10”), (A10’”) or (A20), the binding moiety B or moiety Bi comprises a moiety binding to a target on target cells (e.g., a moiety binding to a cell surface antigen on target cells).

In some embodiments, the moiety that binds to a target on target cells is comprised by a docking compound, e.g. a compound of formula (I) or formula (I’), which comprises a moiety that binds to the targeting compound, e.g. a compound of formula (A) or 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 preferably 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. In some embodiments, the binding moiety B of the targeting compound in formula (A’) is a polymer (e.g., PEG); and B’ of the docking compound comprises a moiety binding to B (e.g., a moiety binding to a polymer, e.g., an anti-PEG antibody or fragment thereof), 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. 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 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.

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. 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.

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. 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, CD4, CD7, CD2, CD28, IL7R, CD 127 and CD5, 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.

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.

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.

Docking compound

In some embodiments, a "docking compound", e.g. a compound of formula (I) or 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, e.g. a compound of formula (A) or 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.

In some embodiments, a docking compound comprises a "primary targeting moiety", as defined above, e.g., B” of the compound of formula (I) or 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.

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.

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 singledomain antibody such as a VHH.

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):

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)

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 antiprimary 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 primary target is a T cell antigen, e.g., CD3, such CD3e, CD4, CD8, or CD7. In some embodiments, the primary target is a T cell antigen, e.g., CD4, CD8, CD3, CD7, CD2, CD28, IL7R, CD 127 and CD5. 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. 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-CD7 VHH. For example, a suitable anti-CD7 VHH may comprise the following sequence:

QVQLVESGGGLVQPGGSLRLSCAASGYPYSSYCMGWFRQAPGQGLEAVAAIDSDGR TRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAARFGPMGCVDLSTLSF GHWGQGTLVTVSS (SEQ ID NO: XX). 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-CD7 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-CD7 DARPin.

Interacting moieties on the targeting compound and on the docking compound

In some embodiments, the moiety on the targeting compound (e.g. B of the compound of formula (A) or Bi of the compound of formula (A’), binding moiety covalently attached to a hydrophobic moiety) and the moiety on the docking compound (e.g. B7Bi ’ of the compound of formula (I) or formula (F), moiety binding to the binding moiety covalently attached to a hydrophobic moiety) interact with each other, e.g., non-covalently bind to each other.

In some embodiments, the moieties on the targeting compound and on the docking compound interacting with each other (e.g. B, formula (A) and B’, formula (I); or Bi, formula (A’) and Bi’ formula (I’)) bind to each other under physiological conditions.

In some embodiments, the moieties on the targeting compound and on the docking compound interacting with each other (e.g. B, formula (A) and B’, formula (I); or Bi, formula (A’) and Bi’ formula (I’)) are antibody/antigen systems. In some embodiments, the moiety of the targeting compound binding to the docking compound (e.g. 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 (e.g. B’, formula (I)) comprises a binder, e.g., an antibody or antibody fragment, binding to the peptide or protein.

In some embodiments, the moiety of the targeting compound binding to the docking compound (e.g. Bi, formula (A’)) comprises a polymer (e.g., PEG), and the moiety of the docking compound binding to the targeting compound (e.g. B , formula (I’)) comprises a binder, e.g., an antibody or antibody fragment, binding to the polymer (e.g., an anti-PEG antibody or fragment thereof).

In some embodiments, the moiety of the docking compound binding to the targeting compound (e.g. 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 (e.g. 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 (e.g. B, formula (A) and B’, formula (I)) comprise an epitope tag/binder system.

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.

Functionalised Nucleic Acid-Lipid Particle

Particles which are “functionalised” 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 “functionalised 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, e.g., B of the compound of formula (A) where B is a moiety binding to a target (e.g., a cell surface antigen) on target cell, or B” of the compound of formula (I) or formula (F). Accordingly, functionalised nucleic acid-lipid particles provide targeted delivery of the nucleic acid payload/active ingredient to particular target cells. The invention also provides functionalised nucleic acid-lipid particles as described herein.

In one embodiment, there is provided a functionalised nucleic acid-lipid particle comprising:

(i) a lipid mixture composition, as defined herein;

(ii) a nucleic acid;

(iii) a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined herein; and

(iv) a docking compound.

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) cholesterol; and

(d) a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined herein; and optionally

(e) a grafted lipid, as defined herein;

(ii) a nucleic acid; and

(iii) a docking compound.

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) cholesterol; and

(d) a targeting compound, e.g. a compound of formula (A) or formula (A’) as defined herein; wherein the molar ratio of phospholipid to cholesterol is 0.5 to 3.0;

(ii) a nucleic acid; and

(iii) a docking compound.

(i) a lipid mixture composition comprising:

(b) a phospholipid; (c) cholesterol; and

(ii) a nucleic acid; and

(iii) a docking compound.

In one embodiment, the docking compound is a compound of formula (I):

B’-X3-B” (I) wherein B’ comprises a moiety binding to B of the compound of formula (A) as defined herein;

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

In one embodiment, the functionalised nucleic acid-lipid particle comprises: 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):

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

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

In one embodiment, the docking compound is a compound of formula (I’):

Bi’-X3-B” (I’) wherein B comprises a moiety binding to Bi of the compound of formula (A’) as defined herein;

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

In one embodiment, the functionalised nucleic acid-lipid particle comprises: a compound of Formula (A’) as defined herein, wherein Bi comprises a polymer; and a compound of formula (F):

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen. In some exemplary functionalised particles, a nucleic acid-lipid particle, as described herein, comprises a targeting compound, e.g. 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) functionalises 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.

In some exemplary functionalised particles, a nucleic acid-lipid particle, as described herein, comprising a compound of formula (A) or formula (A¹), as defined herein, is contacted with a compound of formula (I) or 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), or that the moiety Bi ’ of the compound of formula (I’) interacts with the moiety Bi of the compound of formula (A’). Thus, in some embodiments, the compound of formula (I) or 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) or formula (T) 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) or formula (T) functionalises the particles such that, in use, a connection can be made between the compound of formula (I) or formula (T) 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.

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

B’-X3-B” (I) wherein:

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

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

Therefore, the invention additionally provides in a further aspect a functionalised nucleic acid-lipid particle, wherein the nucleic acid-lipid particle comprises a compound of formula (A’), which is functionalised by interacting therewith a compound of formula (I’): Bi’-X3-B” (I’) wherein B comprises a moiety binding to Bi of the compound of formula (A’) as defined herein;

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

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.

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

In one embodiment, the cell surface antigen is selected from the group consisting of CD4, CD8 and CD3. In one embodiment, the cell surface antigen is selected from the group consisting of CD4, CD8, CD3, and CD7. In one embodiment, the cell surface antigen is selected from the group consisting of CD4, CD8, CD3, CD7, CD2, CD28, IL7R, CD 127 and CD5.

In one embodiment, the cell surface antigen is selected from the group consisting of CD3, CD7, CD2, and IgD.

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

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

In one embodiment, Bi comprises a polymer and Bi ’ comprises a moiety binding to the polymer.

In one embodiment, the moiety binding to a peptide tag and/or moiety binding to the polymer comprises an antibody or antibody-like molecule.

Antibodies which bind to a polymer (e.g. polyethylene -glycol) are known in the art (see e.g.

Creative Biolabs [HPAB-0772LY-S(P)] and Abeam [PEG-B-47]). In one embodiment, the peptide tag comprises an ALFA-tag. In one embodiment, the peptide tag comprises a cyclized ALFA-tag, as defined herein.

In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1 :0.5 to 1: 16. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1:0.7 to 1: 12. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1:0.8 to 1:10. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1:0.9 to 1:9.

In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1 :2 to 2: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1 : 1.5 to 1.5: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1 : 1.2 to 1.2: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 1 :2 to 2: 1.

In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 2: 1 to 12: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 3: 1 to 10: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 4: 1 to 9: 1.

In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 3 : 1 to 6: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 4: 1 to 5: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 7: 1 to 10: 1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is 8: 1 to 9: 1.

In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1 :0.5 to 1:16. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1:0.7 to 1: 12. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I ) is 1:0.8 to 1: 10. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1:0.9 to 1:9.

In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1 :2 to 2: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1 : 1.5 to 1.5 : 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 1 : 1.2 to 1.2: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (F) is 1 :2 to 2: 1.

In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 2: 1 to 12: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 3: 1 to 10: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 4: 1 to 9: 1.

In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 3: 1 to 6: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 4: 1 to 5: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 7: 1 to 10: 1. In one embodiment, the ratio of the compound of formula (A’) to the compound of formula (I’) is 8: 1 to 9: 1.

Typically, the ratio of the compound of formula (A) to the compound of formula (I) is the molar ratio of the compound of formula (A) to the compound of formula (I). This ratio may also referred to as the “X/L” ratio, e.g., as in the Examples of the present application. For example, “X/L” may represent a molar ratio of ALFA-lipid (X) to aCD3-aALFA bispecific docking compound (L). In some aspects, the ratio of the compound of formula (A) to the compound of formula (I) (e.g., the “X/L” ratio) can provide a measure of the ligand loading. For example, lower numbers typically indicate a higher density of targeting ligand on the particle surface. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.1 to 12.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.1 to 10.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 1.0 to 10.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.5 to 8.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.5 to 6.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.7 to 5.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.6 to 1.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is about 0.8. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.8 to 1.2. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 0.9 to 1.1. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is about 1.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 4.0 to 4.8. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 4.2 to 4.6. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 4.3 to 4.5. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is about 4.4. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 7.0 to 10.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 8.0 to 9.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 1.0 to 3.0. In one embodiment, the ratio of the compound of formula (A) to the compound of formula (I) is from 1.5 to 2.5.

Another parameter that may be measured is the ratio of the weight of the compound of formula (I) to the total weight of the nucleic acid (i.e. the nucleic acid “payload” or “cargo”). This weight ratio may also referred to as the “w:w*” or “ligand-to-cargo ratio”, e.g., as mentioned in relation to the Examples of the present application (see, e.g., Figure 13). In some aspects, the ratio by weight of the compound of formula (I) to the total nucleic acid payload (e.g., the “w/w*” ratio) can provide a measure of ligand density. For example, when the nucleic acid content (w) remains constant, then higher numbers for w/w* typically indicate a relatively higher density or weight of targeting ligand on the particle surface. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.01 to 5.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.1 to 5.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.2 to 4.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.3 to 3.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.4 to 2.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.5 to 1.5. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.6 to 1.0. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.4 to 0.8. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.8 to 1.2. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is from 0.6 to 0.8. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is about 0.6. In one embodiment, the ratio by weight of the compound of formula (I) to the total nucleic acid payload is about 1.0.

In some embodiments, an ALFA-tag comprises the amino acid sequence -AA0-AA1-AA2- AA3 -AA4-AA5 -AA6-AA7-AA8-AA9-AA 10-AA 11 -AA 12-AA 13 -AA 14-, wherein the amino acids of AA0, AA1, AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11, AA12, AA13 and AA14 are: AAO is Pro or deleted;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin;

AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu;

AA9 is Arg, Ala, Gin, or Glu;

AA10 is Arg;

AA11 is Leu;

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

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

AA14 is Pro or deleted.

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

In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence (i.e., a cyclized ALFA-tag): -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;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin; AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu;

AA9 is Arg, Ala, Gin, or Glu;

AA10 is Arg;

AA11 is Leu;

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

AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

In some embodiments, XI and X2 are separated by 2 or 3 amino acids.

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.

In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of

-AAO-AA 1 -AA2-AA3 -AA4-cyclo(X 1 -AA6-AA7-AA8-X2)-Arg-Leu-AA 12-AA 13 -AA 14-, -AAO-AA 1 -AA2-AA3 -AA4-cyclo(X 1 -AA6-AA7-X2)-AA9-Arg-Leu-AA 12-AA 13 -AA 14-, -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(Xl-Arg-Leu-AA12-X2)-AA14-, -AAO-AA 1 -AA2-AA3 -AA4-AA5 -cyclo(X 1 -AA7-AA8-X2)-Arg-Leu-AA 12-AA 13 -AA 14-, -AAO-AA 1 -AA2-AA3 -AA4-AA5 -AA6-AA7-AA8-cyclo(X 1 -Arg-Leu-X2)-AA 13 -AA 14-, -AA0-AAl-AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(Xl-Leu-AA12-X2)-AA14-, -AAO-AA 1 -AA2-AA3 -AA4-AA5 -cyclo(X 1 -AA7-AA8-AA9-X2)-Leu-AA 12-AA 13 -AA 14-, and

-AAO-AA 1 -AA2-AA3 -cyclo(X 1 -AA5 -AA6-AA7-X2)-AA9-Arg-Leu-AA 12-AA 13 -AA 14-, wherein the side-chains of XI and X2 amino acid residues are connected covalently;

AAO is Pro or deleted;

AA1 is Ser, Gly, Thr, or Pro;

AA2 is Arg, Gly, Ala, Glu, or Pro;

AA3 is Leu, He, or Vai;

AA4 is Glu or Gin;

AA5 is Glu or Gin;

AA6 is Glu or Gin;

AA7 is Leu, He, or Vai;

AA8 is Arg, Ala, Gin, or Glu; AA9 is Arg, Ala, Gin, or Glu;

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

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

AA14 is Pro or deleted.

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.

In some embodiments, a cyclized amino acid sequence described herein is generated by linking an amino group of a side-chain of one of X 1 and X2 to the carboxyl group of a sidechain 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.

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.

In some embodiments, XI and X2 are, independently, selected from the group consisting of Glu, DGlu, Asp, DAsp, Lys, DLys, hLys, DhLys, Om, DOm, Dab, DDab, Dap, DDap, Cys, DCys, hCys, DhCys, Pen, and DPen, with the proviso that when X 1 is Glu, DGlu, Asp, or DAsp, X2 is Lys, DLys, hLys, DhLys, Om, DOm, Dab, DDab, Dap, or DDap; when XI is Lys, DLys, hLys, DhLys, Om, 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.

In some embodiments, XI is Glu and X2 is Lys. In some embodiments, -cyclo(Glu -

Lys)-, -c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E - K- cyclo, or -cycloE — cycloK- comprises the following stmcture: 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 - cycloEcomprises the following structure:

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:

In some embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Glu-Glu- Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 13). In some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 14). In yet some other embodiments, the cyclized amino acid sequence is -Ser- Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 15). In still some other embodiments, the cyclized amino acid sequence is -Ser-Arg-Leu-Glu-Glu-Glu- Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 16). In some embodiments, the cyclized amino acid sequence is -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg- Leu-Thr-Glu- (SEQ ID NO: 17). In some other embodiments, the cyclized amino acid sequence is Pro-Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 18). In yet some other embodiments, the cyclized amino acid sequence is Pro-Ser-Arg- Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 19). In still some other embodiments, the cyclized amino acid sequence is Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu- Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 20).

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:

In some embodiments, an ALFA-tag binding moiety comprises an antibody or antibody fragment, e.g., a camelid VHH domain. In some embodiments, an ALFA-tag binding moiety comprises a single-domain antibody (sdAb), NbALFA-nanobody.

In some embodiments, 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: 10).

In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTX1SALNAMAMG (SEQ ID NO: 21, 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). In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence VTISALNAMAMG (SEQ ID NO: 9), the CDR2 sequence AVSERGNAM (SEQ ID NO: 10), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 11).

In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the CDR1 sequence GVTISALNAMAMG (SEQ ID NO: 22), the CDR2 sequence AVSERGNAM (SEQ ID NO: 10), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 11). In such embodiments, the CDRs are provided according to the definition by AbM used by Oxford Molecular’s AbM antibody modelling software (see, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer- Verlag, Heidelberg)).

An ALFA-tag binding moiety may comprise CDRs comprising the following sequences: (a) CDR1 - ALNAMAMG (SEQ ID NO: 23); CDR2 - AVSERGNTYYRDSVKG (SEQ ID NO: 24); CDR3 - LEDRVDSFHDY (SEQ ID NO: 11); or (b) CDR1 - ALNAMAMG (SEQ ID NO: 23); CDR2 - AVSERGNAMYRESVQG (SEQ ID NO: 25); CDR3 - LEDRVDSFHDY (SEQ ID NO: 11); according to Kabat annotation. An ALFA-tag binding moiety may comprise CDRs comprising the following sequences: (a) CDR1 - GVTISALNAMA (SEQ ID NO: 26); CDR2 - VSERGNT (SEQ ID NO: 27); CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 28); or (b) CDR1 - GVTISALNAMA (SEQ ID NO: 26); CDR2 - VSERGNA (SEQ ID NO: 29); CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 28); according to IMGT annotation.

An ALFA-tag binding moiety may comprise a humanized VHH. Suitably, a humanised VHH that is capable of binding to an ALFA-tag peptide may comprise (a) CDRs comprising the following sequences: CDR1 - GVTISALNAMAMG (SEQ ID NO: 22), CDR2 - AVSERGNTY (SEQ ID NO: 30), CDR3 - LEDRVDSFHDY (SEQ ID NO: 11), according to AbM definition; (b) CDRs comprising the following sequences: CDR1 - ALNAMAMG (SEQ ID NO: 23), CDR2 - AVSERGNTYYRDSVKG (SEQ ID NO: 24), CDR3 - LEDRVDSFHDY (SEQ ID NO: 11), according to Kabat annotation; or (c) CDRs comprising the following sequences: CDR1 - GVTISALNAMA (SEQ ID NO: 26), CDR2 - VSERGNT (SEQ ID NO: 27), CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 28), according to IMGT annotation. In some embodiments, an ALFA-tag binding moiety comprises a single domain antibody, e.g., a camelid VHH domain comprising the amino acid sequence

EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVS ERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHD YWGQGTQVTVSS (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. In some embodiments, the amino acid sequence comprises CDR1, CDR2 and CDR3 sequences as described above. In some embodiments, an ALFA-tag binding moiety comprises a VHH comprising an amino acid sequence (a)

EVQLVESGGGLVQPGGSLRLSCAASGVTISALNAMAMGWYRQAPGKRREMVAAVS ERGNTYYRDSVKGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCHVLEDRVDSFHD YWGQGTQVTVSS (SEQ ID NO: 32); or (b)

EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVS ERGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHD YWGQGTQVTVSS (SEQ ID NO: 12); or (c)

EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVS SRGNAMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHD YWGQGTQVTVSS (SEQ ID NO: 33).

In some embodiments, the epitope tag/binder system comprises an epitope tag comprising the sequence PDRVRAVSHWSS (SEQ ID NO: 34) (Spot-tag) and the binder comprises a singledomain antibody (sdAb, or nanobody) (Spot-nanobody (14.7 kD)) that specifically binds to the Spot-tag.

In some embodiments, following binding of the moieties on the targeting compound and on the docking compound interacting with each other (B of the compound of formula (A) and B’ of the compound of formula (I); or Bi of the compound of formula (A’) and Bi ’ of the compound of formula (I’)), a covalent connection is formed. In these embodiments, the system used herein may comprise a Tag/Catcher system forming a covalent bond, e.g., SpyTag/SpyCatcher forming an isopeptide bond.

The SpyTag/SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag spontaneously reacts with the protein SpyCatcher to form an intermolecular isopeptide bond between the pair. Using the Tag/Catcher pair, bioconjugation can be achieved between two recombinant proteins. The present disclosure provides in one aspect, a complex wherein a particle comprising a targeting compound (hydrophobic moiety having a binding moiety covalently attached thereto) is bound to a docking compound (compound comprising (i) a moiety binding to the binding moiety covalently attached to a hydrophobic moiety and (ii) a moiety targeting a cell surface antigen). Thus, the targeting compound and the docking compound comprise moieties interacting with each other.

In one embodiment, the compound of formula (I) or formula (T) comprises a bispecific antibody comprising a nanobody which binds to an ALFA-tag, and an anti-CD3 VHH. An exemplary anti-CD3 binding VHH, which may be used in the present invention, comprises the CDR1 sequence GRTYRGYSMA (SEQ ID NO: 35), the CDR2 sequence AIVWSDGNTY (SEQ ID NO: 36), and the CDR3 sequence KIRPYIFKIAGQYDY (SEQ ID NO: 37). An exemplary anti-CD3 binding VHH, which may be used in the present invention, comprises the CDR1 sequence GRTYRGYS (SEQ ID NO: 38), the CDR2 sequence IVWSDGNT (SEQ ID NO: 39), and the CDR3 sequence AAKIRPYIFKIAGQYDY (SEQ ID NO: 40), according to IMGT annotation. An exemplary anti-CD3 binding VHH, which may be used in the present invention, comprises the CDR1 sequence GYSMA (SEQ ID NO: 41), the CDR2 sequence AIVWSDGNTYYEDFVKG (SEQ ID NO: 42), and the CDR3 sequence KIRPYIFKIAGQYDY (SEQ ID NO: 37), according to Kabat annotation. An exemplary anti- CD3 binding VHH, which may be used in the present invention, may comprise (or consist of) the sequence:

EVQLVESGGGPVQAGGSLRLSCAASGRTYRGYSMAWFRQSPGKEREFVAAIVWSDG NTYYEDFVKGRFTISRDSAKNTLYLQMTNLKPEDTALYYCAAKIRPYIFKIAGQYDY WGQGTQVTVSS (SEQ ID NO: 43).

In one embodiment, the peptide tag comprises an ALFA-tag and the moiety binding to the peptide tag comprises a VHH domain comprising the CDR1 sequence VTISALNAMAMG (SEQ ID NO: 9), the CDR2 sequence AVSERGNAM (SEQ ID NO: 10), and the CDR3 sequence LEDRVDSFHDY (SEQ ID NO: 11).

In some embodiments, there is provided a nucleic acid-lipid particle wherein the nucleic acid provides a gene editing tool. In some embodiments, the gene editing tool is a gene editing tool for knocking-in a transgene. In some embodiments, the gene editing tool is a gene editing tool for knocking -out an endogenous gene. In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid; (iii) a targeting compound, e.g a compound of formula (A) as defined herein; and optionally (iv) a docking compound as defined herein, wherein the nucleic acid provides a gene editing tool.

As defined herein, the targeting compound or, optionally, the docking compound comprises a moiety capable of binding to a target (e.g., a cell surface antigen) on a target cell.

Suitably, the moiety may be capable of binding to CD3, CD7, CD4, CD2 or IgD on a target cell.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a gene editing tool, wherein the nucleic acid comprises RNA and/or DNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a DNA (e.g. a DNA template) and an mRNA encoding a gene editing enzyme; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a DNA nanoplasmid and an mRNA encoding a gene editing enzyme; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a CRISPR/Cas (e.g. Cas9) gene editing tool, wherein the nucleic acid comprises DNA and/or RNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a CRISPR/Cas (e.g. Cas9) gene editing tool encoded as RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as RNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound. In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a CRISPR/Cas9 gene editing tool, wherein the nucleic acid comprises a DNA and an RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as an RNA, and wherein a DNA template is comprised in a DNA nanoplasmid; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a ZFN gene editing tool, wherein the nucleic acid comprises DNA and/or RNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a ZFN gene editing tool, wherein the nucleic acid comprises RNA, wherein at least two ZFN proteins are encoded by mRNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein; (ii) a nucleic acid encoding a ZFN gene editing tool encoded as a mixture of DNA and RNA, wherein at least two ZFN proteins are encoded by mRNA, and wherein a DNA template is comprised in a DNA nanoplasmid; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein, wherein the cationically ionizable lipid is BL-207; (ii) a nucleic acid encoding a CRISPR/Cas (e.g. Cas9) gene editing tool encoded as RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as RNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition as defined herein, wherein the cationically ionizable lipid is BL-207; (ii) a nucleic acid providing a CRISPR/Cas (e.g. Cas9) gene editing tool encoded as a mixture of DNA and RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as an RNA, and wherein a DNA template is comprised in a DNA nanoplasmid; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition, as defined herein, wherein the cationically ionizable lipid is BL-207; (ii) a nucleic acid encoding a ZFN gene editing tool, wherein the nucleic acid comprises RNA, wherein at least two ZFN proteins are encoded by mRNA; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

In some embodiments, there is provided a nucleic acid-lipid particle comprising: (i) a lipid mixture composition as defined herein, wherein the cationically ionizable lipid is BL-207; (ii) a nucleic acid encoding a ZFN gene editing tool encoded as a mixture of DNA and RNA, wherein at least two ZFN proteins are encoded by mRNA, and wherein a DNA template is comprised in a DNA nanoplasmid; (iii) a targeting compound, e.g. a compound of formula (A) as defined herein; and optionally (iv) a docking compound.

Method of Forming Functionalised Nucleic Acid-Lipid Particle

In a further aspect, the present disclosure provides methods for producing the functionalised nucleic acid-lipid particles as disclosed herein. Generally, such methods comprise addition of the docking compound, e.g. the compound of formula (I) or formula (F) as defined herein, to a composition containing a nucleic acid-lipid particle as defined herein.

Therefore, in further aspects, there is provided a method of forming a functionalised nucleic acid-lipid particle comprising: a lipid bonded to a targeting ligand; and a docking compound; the method comprising:

(a) forming a nucleic acid-lipid particle according to the methods defined herein, and

(b) mixing the nucleic acid-lipid particle with the docking compound, such that the docking compound interacts with the nucleic acid-lipid particle.

In one embodiment, the composition containing the nucleic acid-lipid particle is a solution containing the nucleic acid-lipid particle. In one embodiment, the composition containing the nucleic acid-lipid particle is an aqueous solution containing the nucleic acid-lipid particle. In one embodiment, the docking compound, e.g. compound of formula (I) or formula (I’), is provided in a solution containing the docking compound, e.g. the compound of formula (I) or formula (I’). In one embodiment, the docking compound, e.g. the compound of formula (I) or formula (I’), is provided in an aqueous solution containing the docking compound, e.g. the compound of formula (I) or formula (I’).

In a further aspect of the invention, there is provided a method of forming a nucleic acid-lipid particle functionalised by interacting therewith a compound of formula (I): B’-X3-B” (I) wherein:

B’ comprises a moiety binding to B of the compound of formula (A) as defined herein, wherein B comprises a peptide tag or a moiety binding to a peptide tag;

X3 is absent or a linking moiety; and

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

(a) forming a nucleic acid-lipid particle according to the method as defined herein, and (b) mixing the nucleic acid-lipid particle with the compound of formula (I), such that the compound of formula (I) interacts therewith to produce the functionalised nucleic acid-lipid particle.

In one embodiment, there is provided a method of forming a functionalised 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) as defined herein, wherein B’ comprises a moiety binding to B; the method comprising:

(a) forming a nucleic acid-lipid particle according to the method as defined herein, 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.

In a further aspect of the invention, there is provided a method of forming a nucleic acid-lipid particle functionalised by interacting therewith a compound of formula (I’): Bi’-X3-B” (I’) wherein

Bi’ comprises a moiety binding to Bi of the compound of formula (A’) as defined herein;

X3 is absent or a linking moiety; and

(a) forming a nucleic acid-lipid particle according to the method as defined herein, and (b) mixing the nucleic acid-lipid particle with the compound of formula (I’), such that the compound of formula (I’) interacts therewith to produce the functionalised nucleic acid-lipid particle.

In one embodiment, there is provided a method of forming a functionalised nucleic acid-lipid particle comprising: a compound of Formula (A’) as defined herein, wherein Bi comprises a peptide tag or a moiety binding to a peptide tag; and a compound of formula (I’) as defined herein, wherein Bi’ comprises a moiety binding to Bi; the method comprising:

(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.

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

In one embodiment, the method comprises further subjecting the functionalised nucleic acid- lipid particle to one or more dilution steps. In one embodiment, the method further comprises a step of diluting the functionalised nucleic acid-lipid particles with a storage matrix. 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.

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

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

Lipids and Amphiphiles

The compositions (such as the nucleic acid-lipid particles and functionalised nucleic acid- lipid particles) of the invention also contain a mixture of lipids. The terms "lipid" and "lipid- like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and also one or more hydrophilic moieties or groups.

Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases.

Lipids may comprise a polar portion and an apolar (or non-polar) portion. The term “amphiphile” as used in this specification is broadly defined herein as a molecule comprising hydrophobic moieties and hydrophilic moieties and/or a polar and apolar portion. As cationic and cationically ionizable lipids both contain such groups, they are therefore amphiphiles. In this specification the term “cationic lipid” is therefore synonymous with “cationic amphiphile”.

Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and/or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophilic groups may comprise polar and/or charged groups and include at least one amine and optionally hydrophilic non-charged groups such as hydroxyl, carbohydrate, sulfhydryl, nitro or like groups.

The term "hydrophobic" as used herein with respect to a compound, group or moiety means that said compound, group, or moiety is not attracted to water molecules and, when present in an aqueous solution, excludes water molecules. In some embodiments, the term "hydrophobic" refers to any compound, group or moiety which is substantially immiscible or insoluble in aqueous solution. In some embodiments, a hydrophobic compound, group or moiety is substantially nonpolar.

Examples of hydrophobic groups are hydrocarbyl groups (as defined and exemplified above), such as alkyl, alkenyl and/or alkynyl groups and such groups substituted by one or more aryl, heteroaryl, or cycloalkyl groups (as defined and exemplified above). The hydrophobic group can have functional groups (e.g., ether, thioether, ester, dioxolane, halide, amide, sulfonamide, carbamate, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.

The hydrophobic moieties of a lipid may have between 24 and 60 carbon atoms and can be hydrocarbyls (as described and exemplified above, typically comprising alkyl, alkenyl or alkynyl groups as described and exemplified above). Another example are lipids wherein the hydrophobic moiety comprises a steroid moiety, such as a cholesteryl moiety.

The hydrophobic moieties of a lipid preferably have between 24 and 60 carbon atoms and can also be heterohydrocarbyls wherein the heteroatoms are selected from N, O or S forming one, two, three or four non-charged groups of ether, thioether, ester, amide, carbamate, sulfonamide and the like. The 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, provided that each such moiety has at least 6 carbon atoms. An example for segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the diacylglycerol or dialkylglycerol moiety wherein each of the acyl or alkyl comprise between 12 and 20 carbon atoms. An example for hydrophobic moieties wherein each segment is heterohydrocarbyl are the ester-branched moieties in lipids such as SM-102 or ALC-0315, as defined and exemplified below.

Cationic and Cationically Ionizable Lipids

In some embodiments, the compositions (such as the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention also contain a cationic lipid or cationically ionizable lipid, or a mixture of any thereof. In some embodiments, the compositions (such as the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention comprise a cationically ionizable lipid.

As used herein, the term “cationic lipid” means a lipid or lipid-like material, as defined herein, having a constitutive positive charge. In this context a “constitutive charge” means that the cationic lipid carries the positive charge at all physiological pH. The cationic lipids carrying constitutive charged cationic moieties are typically quaternary ammonium salts (as defined above) or salts of organic bases, such as nitrogen-containing bases. Typically, such organic bases are strong bases (i.e. bases which are completely protonated when dissolved in a solvent, such as but not limited to an aqueous solvent, such that the concentration of the unprotonated species is too low to be measured). In one embodiment, the cationic lipid is a monovalent cationic lipid.

In one embodiment, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

Examples of cationic lipids include, but are not limited to l,2-dialkyloxy-3- dimethylammonium propanes and l,2-dialkenyloxy-3-dimethylammonium propanes (each alkyl or alkenyl portion being as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as l,2-di-O-octadecenyl-3 -trimethylammonium propane (DOTMA),

1.2-diacyloxy-3 -dimethylammonium propanes (the alkyl or alkenyl part of each acyl portion being as defined and exemplified above and preferably having 12 to 20 carbon atoms), such as l,2-dioleoyl-3 -trimethylammonium propane (DOTAP) or l,2-dioleoyl-3- dimethylammonium-propane (DODAP); dimethyldioctadecylammonium (DDAB); dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE); l,2-dimyristoyl-sn-glycero-3- ethylphosphocholine (DMEPC), l,2-dimyristoyl-3 -trimethylammonium propane (DMTAP),

1.2-dioleyloxypropyl-3 -dimethyl -hydroxyethyl ammonium bromide (DORIE), and 2,3- dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA).

In some embodiments, the compositions (such as the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention do not contain a cationic lipid.

In one embodiment, the lipid mixture comprises a cationically ionizable lipid. As used herein, a "cationically ionizable lipid" refers to a lipid or lipid-like material which, depending on whether it is protonated or deprotonated, has a net positive charge or is neutral, z.e., a lipid which is not permanently cationic. Thus, depending on the pH of the composition in which the cationically ionizable lipid is solved, the cationically ionizable lipid is either positively charged or neutral.

Therefore, in some embodiments, the compositions (such as the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention contain a cationically ionizable lipid, or a mixture of any thereof. In some embodiments, the cationically ionizable lipid is not a permanently cationic lipid.

In some embodiments, the cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is capable of being protonated, preferably under physiological or slightly acidic conditions.

In one embodiment, the cationically ionizable lipid is a compound represented by formula (TL- I): zL¹-X¹-T¹

G-L³-I/

L²— X²- T²

TL-I or a pharmaceutically acceptable salt thereof, wherein:

L¹ and L² are each independently an optionally substituted C1-C30 aliphatic group;

L³ is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10- membered heteroaliphatic group;

X¹ and X² are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)₂N(R¹)-, - N(R’)S(O)2, -S(O)-, -S(O)₂-, -S(O)2C(R’)₂-, -OC(S)C(R¹)₂-, -C(R¹)₂C(S)O-, and -S-, wherein one or both of X¹ or X² is selected from -S(O)₂N(R¹)-, -N(R¹)S(O)₂, -S(O)-, - S(O)₂-, -S(O)₂C(R¹)₂-, -OC(S)C(R¹)₂-, -C(R¹)₂C(S)O-, and -S-; each R¹ is, independently, at each instance, optionally substituted Ci-C₂o aliphatic or H;

T¹ and T² are each independently an optionally substituted C3-C30 aliphatic;

G is -N(R²)C(S)N(R²)₂, -N⁺(R³)₃, -OH, -N(R²)₂, -N(R⁵)C(O)R³, -N(R⁵)S(O)₂R³, - N(R⁵)C(O)N(R³)₂, -CH(N-R²), -R⁴; or -S(O)₂R³; each R² is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic and OR³; or two instances of R² come together with the atoms to which they are attached to form an optionally substituted 4- to 12-membered heterocycle ring or an optionally substituted 4- to 12-membered heteroaryl ring; each R³ is, independently, at each instance, selected from the group consisting of H and optionally substituted C1-C10 aliphatic; and

R⁴ is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, Ce-Ci₂ aryl substituted with one or more of -(CH₂)o-e-OH or - (CH₂)O-6-N(R⁵)₂, or Cs-Cn cycloaliphatic substituted with one or more of oxo, -(CH₂)o-6- OH, or -(CH₂)_O.6-N(R⁵)₂; each R⁵ is independently selected from H and optionally substituted Ci-Ce aliphatic.

In some embodiments of formula (TL-I), L¹ and L² are each independently -(CH₂)₆.IO-. In some embodiments of formula (TL-I), X¹ and X² are each independently selected from a - S(O)₂N(R’)-, -N(R¹)S(O)₂, -S(O)-, -S(O)₂-, -S(O)₂C(R¹)₂-, -OC(S)C(R¹)₂-, -C(R¹)₂C(S)O-, and -S-.

In some embodiments of formula (TL-I), X¹ and X² are each -S(O)₂N(R¹)-, where each R¹ is independently R¹ is Ci-Cw aliphatic.

In some embodiments of formula (TL-I), T¹ and T² are each independently selected from optionally substituted C3-C₂o alkyl.

In some embodiments of formula (TL-I), T¹ and T² are each independently selected from:

In some embodiments of formula (TL-I), G is -N(R²)C(S)N(R²)₂ or -N(R⁵)S(O)₂R³.

In some embodiments of formula (TL-I), G is -N(H)C(S)N(R²)₂, where each R² is selected from optionally substituted Ci-Ce aliphatic and OH.

In some embodiments of formula (TL-I), G is -OH. In some embodiments of formula (TL-I), G is selected from:

In some embodiments of formula (TL-I), -L³-G is selected from:

In some embodiments of formula (TL-I), the compound is represented by Formula (TL-IIa):

TL-IIa or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (TL-I), the compound is represented by Formula (TL-IIc): TL-IIc or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (TL-I), the compound is represented by Formula (TL-IIIb):

(TL-IIIb) or a pharmaceutically acceptable salt thereof.

In some embodiments of formula (TL-I), the compound is represented by Formula (TL-IIIe):

Ille or a pharmaceutically acceptable salt thereof.

Thiolipid compounds of formula (TL-I) can be prepared according to W02025027089A1, the contents of which are incorporated herein by reference.

In one embodiment, the cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide)

octylsulfamoyl)hexyl)(2-hydroxyethyl)amino)octanoate (BL-200); dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylhexane- 1 -sulfonamide) (BL-209);

[(4-hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate) (ALC-0315); hydroxypropyl)azanediyl)-bis(nonane-

9, 1-diyl) bis(2-butyloctanoate) (ALC-0366);

2, 2-dilinoleyl-4-dimethylaminoethyl-[ 1,3] -dioxolane (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);

1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA); di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); bis- -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-l-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4- (dimethylamino)butanoate (HY 501);

2-(di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2);

4-((di-((9Z. l 2Z)-octadcca-9. l 2-dicn- l -yl)amino)oxy)- ' '-dimcthyl-4-oxobutan-4-aminc (HYAM-2);

((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8, 1 -diyl) bis(2- hexyldecanoate) (EA-405);

(2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8, 1 -diyl) bis(2 -hexyldecanoate) (HY-405);

BODD-C2C2-Pyr bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD- C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4- Pyr); or a mixture of any thereof.

In one embodiment, the cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide) (BNT-51); heptadecan-9-yl 8- { (2 -hydroxyethyl) [6-oxo-6-(undecyloxy)hexyl] amino } -octanoate) (SM- 102);

7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane- 1 -sulfonamide (BL-207);

6,6'-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylhexane-l-sulfonamide) (BL-209);

BODD-C2C4-PipZ;

BHD-C2C2-PipZ;

BODD-C2C2-DMA;

BODD-C2C2-Pyr; bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD- C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4- Pyr); or a mixture of any thereof.

BODD-C2C4-PipZ; BHD-C2C2-PipZ;

BODD-C2C2-DMA;

BODD-C2C2-Pyr; bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD- C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4- Pyr);

[(4-hydroxybutyl)azanediyl]di(hexane-6, 1-diyl) bis(2-hexyldecanoate) (ALC-0315); (3-hydroxypropyl)azanediyl)bis(nonane-9, 1-diyl) bis(2-butyloctanoate) (ALC-0366); or a mixture of any thereof.

In one embodiment, the cationically ionizable lipid is 7,7’-((4-hydroxybutyl)azanediyl)bis(N- hexyl-N-octylheptane-1 -sulfonamide) (BNT-51). In one embodiment, the cationically ionizable lipid is BNT-52. In one embodiment, the cationically ionizable lipid is BNT-76. In one embodiment, the cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl] amino} -octanoate) (SM-102). In one embodiment, the cationically ionizable lipid is 7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane- 1 -sulfonamide (BL-207). In one embodiment, the cationically ionizable lipid is 6,6'-((4-(3,3-dimethylthio- ureido)-butyl)azanediyl)bis(N-hexyl-N-octylhexane-I-sulfonamide) (BL-209). In one embodiment, the cationically ionizable lipid is BODD-C2C4-PipZ. In one embodiment, the cationically ionizable lipid is BHD-C2C2-PipZ. In one embodiment, the cationically ionizable lipid is BODD-C2C2-DMA. In one embodiment, the cationically ionizable lipid is BODD- C2C2-Pyr. In one embodiment, the cationically ionizable lipid is bis(2 -hexyldecyl) 3, 3 '-((4- (4-methylpiperazin-I-yl)butyl)azanediyl)dipropionate (BHD-C2C4-PipZ). In one embodiment, the cationically ionizable lipid is bis(2 -octyldodecyl) 3,3'-((4-(pyrrolidin-l- yl)butyl)azanediyl)dipropionate (BODD-C2C4-Pyr). In one embodiment, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl] di (hexane-6, 1-diyl) bis(2-hexyldecanoate) (ALC-0315). In one embodiment, the cationically ionizable lipid is ((3- hydroxypropyl)azanediyl)bis(nonane-9,I-diyl) bis(2 -butyloctanoate) (ALC-0366).

In one embodiment, the cationically ionizable lipid is present in an amount of 50 to 75 mol% of the total lipids in the lipid mixture. In one embodiment, the cationically ionizable lipid is present in an amount of 55 to 70 mol% of the total lipids in the lipid mixture. In one embodiment, the cationically ionizable lipid is present in an amount of 58 to 65 mol% of the total lipids in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture composition in the absence of the nucleic acid, and the lipid mixture component of both the nucleic acid-lipid particle and the functionalised nucleic acid-lipid particle. Additional Lipids

The lipid mixture in the compositions (including the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention may further comprise one or more additional lipids. In one embodiment, the one or more additional lipids comprise a grafted lipid, as defined and exemplified below. The lipid mixture in the compositions (including the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention may comprise a cationically ionizable lipid, a phospholipid, a steroid, and a grafted lipid, each as defined herein.

Phospholipid

The compositions (including nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) of the present invention also comprise a phospholipid. In one embodiment, the phospholipid may be zwitterionic (i.e. it carries both a positive and a negative charge, so that it is neutral at a pH ranging around neutral).

In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins. The hydrocarbyl portion of the acyl moieties of phospholipids is as defined above, but is preferably an alkyl group (as defined above) having 6 to 40, preferably 8 to 24, carbon atoms or an alkenyl group (as defined above) having 6 to 40, preferably 14 to 22, carbon atoms and 1 to 6 carbon-carbon double bonds. The acyl parts of the phospholipids may be the same or different. In one embodiment, the acyl moieties are saturated fatty acid moieties having 8 to 24 carbon atoms (including the acyl carbon), preferably selected from the group consisting of lignoceroyl, behenoyl, arachidoyl, stearoyl, palmitoyl, myristoyl, lauroyl, decanoyl and octanoyl moieties. In a specific embodiment, neutral phospholipids have a T_m of 30°C or higher and are selected from di-stearoyl or di-palmitoyl or stearoyl-palmitoyl moieties. In one embodiment, the acyl moieties are unsaturated fatty acid moieties having 14 to 22 carbon atoms (including the acyl carbon), preferably selected from the group consisting of oleoyl, linoyl, and lineoyl moieties.

Examples of such phospholipids include diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine (DLPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), stearoyloleylphosphatidylcholine (SOPC), palmitoyloleoylphosphatidylcholine (POPC), diphytanoylphosphatidylcholine (DPyPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1 -oleoyl -2 -cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-10-glycero-3 -phosphocholine (Cl 6 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidyl-ethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl- phosphatidylethanolamine (DPyPE), l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG), l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (DPPG), 1 -palmitoyl -2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE), N-palmitoyl-D-erythro- sphingosylphosphorylcholine (SM), and further phosphatidyl-ethanolamine lipids with different hydrophobic chains.

In some embodiments, the phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); dioleoylphosphatidylethanolamine (DOPE);

Diphytanoylphosphatidylethanolamine (DPyPE) l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG); N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM); stearoyloleylphosphatidylcholine (SOPC); and diphytanoylphosphatidylcholine (DPyPC); or a mixture of any thereof.

In some embodiments, the phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylethanolamine (DOPE); and N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM); or a mixture of any thereof.

I l l In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).

Thus, in some embodiments, the compositions described herein comprise a compound of formula (A) or formula (A¹), as defined herein, a cationically ionizable lipid (as defined herein), cholesterol and a phospholipid. In some embodiments, the compositions described herein comprise a compound of formula (A) or formula (A¹), as defined herein, a cationically ionizable lipid, cholesterol and a phospholipid selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DOPE, DPyPC, DPyPE, SOPC and SM, or a mixture of any thereof.

In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 5 mol % to about 30 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 15 mol % to about 30 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is present in the lipid mixture in an amount of about 10 mol % to about 22 mol % of the total lipids present in the lipid mixture.

In one embodiment, the phospholipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 5 mol % to about 30 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is a phosphatidylcholine and is present in the lipid mixture in an amount of about 15 mol % to about 30 mol % of the total lipids present in the lipid mixture.

In one embodiment, the phospholipid is DSPC and is present in the lipid mixture in an amount of about 5 mol % to about 30 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is DSPC and is present in the lipid mixture in an amount of about 15 mol % to about 30 mol % of the total lipids present in the lipid mixture. In one embodiment, the phospholipid is DSPC and is present in the lipid mixture in an amount of about 10 mol % to about 22 mol % of the total lipids present in the lipid mixture.

The term “lipid mixture” in this context applies to the lipid mixture composition in the absence of the nucleic acid, and the lipid mixture component of both the nucleic acid-lipid particle and the functionalised nucleic acid-lipid particle.

Steroid The compositions (including the nucleic acid-lipid particles and functionalised nucleic acid- lipid particles) of the present invention also comprise a steroid. In one embodiment, the steroid is cholesterol, or a derivative thereof. In one embodiment, the steroid may be selected from the group consisting of: cholesterol, cholesterol sulfate, a cholesterol ester (such as cholesteryl acetate), and a phytosterol (such as sitosterol, fucosterol, or campesterol). In one embodiment, the steroid is cholesterol, or an ester thereof. In one preferred embodiment, the steroid is cholesterol.

In one embodiment, the steroid is a cholesterol ester. In this specification, the term “cholesterol ester” takes its normal meaning in the art as meaning an ester of general formula R-C(=O)-O-Chol, where Choi is a cholesteryl residue (the oxygen atom being present at the 3 -position of the sterol residue) and R is an organic group. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester (i.e. the organic group R is an alkyl group), the alkanoyl part having from 2 to 30 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 20 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 10 carbon atoms. In one embodiment, the cholesterol ester is a cholesterol alkanoyl ester, the alkanoyl part having from 2 to 6 carbon atoms. Examples of such cholesterol esters include cholesterol acetate, cholesterol propionate, cholesterol butyrate, cholesterol valerate, cholesterol caproate, cholesterol enanthate, cholesterol caprylate, cholesterol pelargonate, cholesterol caprate, cholesterol undecanoate, cholesterol laurate, cholesterol myristate, cholesterol palmitate, cholesterol stearate, and cholesterol arachidate. In one embodiment, the cholesterol ester is cholesterol acetate.

In one embodiment, the steroid (e.g., cholesterol) is present in an amount ranging from about 10 mol % to about 40 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid (e.g., cholesterol) is present in an amount ranging from about 15 mol % to about 35 mol % of the total lipids present in the lipid mixture. In one embodiment, the steroid (e.g., cholesterol) is present in an amount ranging from about 10 mol % to about 30 mol % of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture composition in the absence of the nucleic acid, and the lipid mixture component of both the nucleic acid-lipid particle and the functionalised nucleic acid- lipid particle.

Phospholipid / Steroid Ratio As described herein, the compositions of the present invention contain a phospholipid and a steroid (as defined above). In one embodiment, the compositions of the present invention contain a phospholipid and cholesterol.

In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.2 to 3.0, optionally 0.7 to 2.8, such as 0.2 to 1.0. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.5 to 0.7, optionally 0.55 to 0.65. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.8 to 2.6, optionally 0.8 to 2.2, such as 1.8 to 2.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.9 to 1.1, optionally 1.9 to 2.1.

In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.2 to 3.0. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.6 to 3.0. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.7 to 2.8. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.6 to 3.0. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.2 to 1.0. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.3 to 0.9. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.5 to 0.7. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.55 to 0.65.

In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.8 to 2.6. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 1.0 to 2.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.8 to 2.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.8 to 1.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 1.8 to 2.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 0.9 to 1.1. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to cholesterol is from 1.9 to 2.1.

In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.5 to 0.7. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.55 to 0.65.

In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.8 to 1.2. In some embodiments of the lipid mixtures of the present invention, the molar ratio of phospholipid to steroid is from 0.95 to 1.05.

Grafted Lipids

The compositions described herein (including the nucleic acid-lipid particles and functionalised nucleic acid-lipid particles) may also contain a grafted lipid. In the present specification the term “grafted 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 polymer, as defined herein (either in a broadest aspect or a preferred aspect).

In one embodiment, the grafted lipid is capable of acting as a stealth lipid. In this specification the term “stealth lipid” means a stealth polymer (as defined below) conjugated to a lipid (as defined herein). In this specification the term “stealth polymer” means a polymer (as defined above) having the following features: (a) polar (hydrophilic) functional groups; (b) hydrogen bond acceptor groups, and (c) no net charge. In some embodiments, a stealth polymer is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a stealth polymer can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

In one embodiment, the grafted lipid is a polyethylene-glycol conjugated lipid (also known as a PEG-lipid or PEGylated lipid), as defined herein. The term "PEGylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. PEGylated lipids are known in the art. The PEG-lipid may comprise 5-1000, 5-500, 5-100, 5-50, 8-1000, 8-500, 8-100, 8-50, 10-1000, 10-500, 10-100, or 10-50, ethylene glycol repeating units, which may be consecutive.

In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein each of R¹² and R¹³ is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.

In some embodiments of this formula, each of R¹² and R¹³ is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

In some embodiments of this formula, R¹² and R¹³ are identical. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 16 carbon atoms.

In some embodiments of this formula, R¹² and R¹³ are different. In some embodiments, one of R¹² and R¹³ is a straight alkyl chain containing 12 carbon atoms and the other of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms.

In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

In some embodiments of this formula, w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol. In some embodiments, each of R¹² and R¹³ is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-l-O- (co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoylxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate, and the like.

In some embodiments, the PEG-conjugated lipid (pegylated lipid) is or comprises 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159). In some embodiments, the pegylated lipid has the following structure:

In some embodiment, the PEG-conjugated lipid (pegylated lipid) is a pegylated ceramide.

Such conjugated lipids comprise a ceramide moiety of formula: wherein R is a fatty acid residue, typically a Cg.30 alkyl group or a Cg.30 alkenyl group, preferably a C12-20 alkyl group, more preferably a C14-18 alkyl group, most preferably a Cig alkyl group, wherein the oxygen atom on the carbon atom next to the nitrogen-bearing carbon atom is conjugated to a PEG portion, as defined and exemplified above. One typical example of the class of PEG ceramides is a PEG2000 ceramide, where the PEG portion is a PEG2000 portion. One especially preferred example is a C 16 PEG2000 ceramide, having the following structure: In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a DMG-PEG 2000, e.g.,

In some embodiments, the PEG-conjugated lipid (pegylated lipid) has the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50. In some embodiments, the PEG-conjugated lipid (pegylated lipid) is PEG2000-C-DMA which preferably refers to 3-

N-[(co-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine

(MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000) .

In some embodiments, nucleic acid particles described herein may comprise one or more PEG-conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

Other examples of grafted lipids include poly(sarcosine) (pSar)-conjugated lipids, poly(oxazoline) (POX)-conjugated lipids; poly(oxazine) (POZ)-conjugated lipids, poly(vinyl pyrrolidone) (PVP)-conjugated lipids; poly(/V-(2-hydroxypropyl)-methacrylamide) (pHPMA)- conjugated lipids; poly(dehydroalanine) (pDha)-conjugated lipids; poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipids and poly(2 -methylaminoethoxy ethoxy acetic acid) (pm AEEA) -conjugated lipids.

In one embodiment, the grafted lipid is a polysarcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid. The term "sarcosinylated lipid" refers to a molecule comprising both a lipid portion and a polysarcosine (poly(N-methylglycine) portion, the polysarcosine portion having the repeating unit shown below: wherein x refers to the number of sarcosine units. The polysarcosine may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 sarcosine units, preferably from 15 to 50 sarcosine units, more preferably from 20 to 30 sarcosine units, even more preferably 21 to 25 sarcosine units.

In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (Ce-so alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group. In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (C12-20 alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group. In one embodiment, the grafted lipid comprises a polysarcosine portion (as defined and exemplified above) the carbonyl terminus of which is bonded to a (Ci4alkyl)amine (as defined and exemplified above), and the amino terminus of which is optionally bonded to an acetyl group.

In one embodiment, the grafted lipid is a polyoxazoline (POX)-conjugated and/or a polyoxazine (POZ) -conjugated lipid and/or a POX/POZ-conjugated lipid, also referred to herein as a conjugate of a POX and/or POZ polymer and one or more hydrophobic chains or as oxazolinylated and/or oxazinylated lipid or POX and/or POZ-lipid. The term "oxazolinylated lipid" or "POX-lipid" refers to a molecule comprising both a lipid portion and a polyoxazoline portion, the polyoxazoline portion (pOx) having the repeating unit shown below. The term "oxazinylated lipid" or "POZ-lipid" refers to a molecule comprising both a lipid portion and a polyoxazine portion, the polyoxazine (pOz) portion having the repeating unit shown below. The term "oxazolinylated/ oxazinylated lipid" or "POX/POZ-lipid" or "POXZ -lipid" refers to a molecule comprising both a lipid portion and a portion of a copolymer of polyoxazoline and polyoxazine, i.e. a polymer having both the pOx and pOz repeating units shown below: wherein x refers to the number of pOx and/or pOz units. The total number of pOx and/or pOz repeating units in the polymer may comprise from 2 to 200, from 2 to 100, from 5 to 200, from 5 to 100, from 10 to 200, from 10 to 100, optionally from 5 to 80, preferably from 10 to 70 pOx and/or pOz units.

In one embodiment, the grafted lipid is a poly(vinyl pyrrolidone) (PVP)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of a poly(vinyl pyrrolidone) (PVP) conjugated to a lipid. The term “poly(vinyl pyrrolidone)” or “PVP” means a polymer having a vinyl pyrrolidine repeating unit, i.e. the repeating unit shown below. pVP

In one embodiment, the grafted lipid is a poly(/V-(2-hydroxypropyl)methacrylamide) (pHPMA)-conjugated lipid. In one embodiment, the lipid nanoparticle composition is substantially free (as defined above, either in its broadest aspect of a preferred aspect) of poly(/V-(2-hydroxypropyl)methacrylamide) (pHPMA) conjugated to a lipid. The term “poly(JV-(2-hydroxypropyl)-methacrylamide” or “pHPMA” means a polymer having the repeating unit shown below.

In one embodiment, the grafted lipid is a poly(dehydroalanine) (pDha)-conjugated lipid. The term “pDha” means a polymer having the repeating unit shown below. pDha In one embodiment, the grafted lipid is an amphiphilic oligoethylene glycol (OEG)- conjugated lipid. Examples of amphiphilic oligoethylene glycol (OEG) -conjugated lipids include poly(aminoethyl-ethylene glycol acetyl) (pAEEA) and/or poly(methylaminoethyl- ethylene glycol acetyl) (pmAEEA). The terms “pAEEA” and “pmAEAA” means a polymer having the repeating unit shown below: pAEEA pmAEEA wherein x refers to the total number of pAEEA and/or pmAEEA units in the polymer. The total number of pAEEA and/or pmAEEA repeating units in the polymer may comprise from 1 to 100, from 5 to 50, from 5 to 25, preferably from 7 to 14.

In one embodiment, the one or more additional lipids comprise a grafted lipid, preferably 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) (P VP) -conjugated lipid; a poly(/V-(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.

In one embodiment, the grafted lipid is a poly(aminoethoxy ethoxy acetic acid) (pAEEA)- conjugated lipid as defined above, either in its broadest aspect or a preferred aspect.

In one embodiment, the grafted lipid is selected from the group consisting of polyethylene glycol (2000) - C16 ceramide, Ac-pAEEA14-DSPE, Ac-pAEEA14-a-tocopherol, Ac- pAEEA14-DMA and 2- [(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC- 0159). In one embodiment, the grafted lipid is selected from the group consisting of polyethylene glycol (2000) - C16 ceramide, Ac-pAEEA14-DSPE, Ac-pAEEA14-a- tocopherol, and Ac-pAEEA14-DMA. In one embodiment, the grafted lipid is a polyethylene glycol (2000) - C16 ceramide.

In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.5 to 10 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1.0 to 4.0 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1.5 to 4.0 mol% of the total lipids present in the lipid mixture. In one embodiment, the grafted lipid is present in the lipid mixture in an amount of 1 to 2.5 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture composition in the absence of the nucleic acid, and the lipid mixture component of both the nucleic acid-lipid particle and the functionalised nucleic acid-lipid particle.

Pharmaceutical Compositions

The functionalised nucleic acid-lipid particle compositions described herein are useful as or for preparing pharmaceutical compositions or medicaments for therapeutic or prophylactic treatments.

Therefore, in a further aspect, there is provided a pharmaceutical composition comprising a functionalised nucleic acid-lipid particle as defined herein and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a nucleic acid-lipid particle as defined herein and a pharmaceutically acceptable carrier.

The functionalised nucleic acid-lipid particle compositions or the nucleic acid-lipid particle compositions described herein may be administered in the form of any suitable pharmaceutical composition.

The term "pharmaceutical composition" relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a subject. In some embodiments, the therapeutically effective agent is or comprises the active ingredient, as described herein. In the context of the present disclosure, the pharmaceutical composition typically comprises a nucleic acid as described herein. In some embodiments, the therapeutically effective agent is or comprises a nucleic acid, 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. The pharmaceutical compositions of the present disclosure may be in in a frozen form or in a "ready-to-use form" (z.e., in a form, in particular a liquid form, which can be immediately administered to a subject, e.g., without any processing such as thawing, reconstituting or diluting). Thus, prior to administration of a storable form of a pharmaceutical composition, this storable form has to be processed or transferred into a ready-to-use or administrable form. E.g.. a frozen pharmaceutical composition has to be thawed. Ready to use injectables can be presented in containers such as vials, ampoules or syringes wherein the container may contain one or more doses.

In one embodiment, the pharmaceutical composition is lyophilized. In one embodiment, the pharmaceutical composition is spray dried. These techniques are well known to those skilled in the art.

The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".

The term "pharmaceutically acceptable" refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.

The term "pharmaceutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition. An effective amount of the particles or pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the particles or pharmaceutical compositions described herein may depend on various such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

In particular embodiments, a pharmaceutical composition of the present disclosure (e.g., an immunogenic composition, z.e., a pharmaceutical composition which can be used for inducing an immune response) is formulated as a single-dose in a container, e.g., a vial. In some embodiments, the immunogenic composition is formulated as a multi-dose formulation in a vial. In some embodiments, the multi-dose formulation includes at least 2 doses per vial. In some embodiments, the multi-dose formulation includes a total of 2-20 doses per vial, such as, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 doses per vial. In some embodiments, each dose in the vial is equal in volume. In some embodiments, a first dose is a different volume than a subsequent dose.

A "stable" multi-dose formulation preferably exhibits no unacceptable levels of microbial growth, and substantially no or no breakdown or degradation of the active biological molecule component(s). As used herein, a "stable" immunogenic composition includes a formulation that remains capable of eliciting a desired immunologic response when administered to a subject.

The pharmaceutical compositions of the present disclosure may contain buffers (in particular, derived from the nucleic acid (such as RNA or DNA) compositions with which the pharmaceutical compositions have been prepared), preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure, in particular the ready-to-use pharmaceutical compositions, comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The term "excipient" as used herein refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Examples of excipients, include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavouring agents, or colorants.

The term "diluent" relates a diluting and/or thinning agent. Moreover, the term "diluent" includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol and water.

The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition. A carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject. Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.

Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

In one embodiment, the compositions described herein, such as the pharmaceutical compositions or ready-to-use pharmaceutical compositions described herein, may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, intramuscularly or intratumourally. In certain embodiments, the (pharmaceutical) composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In another preferred embodiment, the (pharmaceutical) compositions, in particular the ready-to-use pharmaceutical compositions, are formulated for intramuscular administration.

Medical Uses and Methods of Treatment

The functionalised nucleic acid-lipid particles or nucleic acid-lipid particles, (i.e., those comprising a primary targeting moiety) and pharmaceutical composition thereof, may be used in methods for targeted delivery of payloads (i.e., active ingredients, e.g., nucleic acids) to cells. In some embodiments, the active ingredient (i.e., payload) comprises one or more nucleic acid(s) encoding an antigen receptor such as a T cell receptor (TCR) or chimeric antigen receptor (CAR). The particles, compositions and methods for targeted delivery of a nucleic acid encoding an antigen receptor described herein may be used for generating in vitro/ex vivo or in vivo immune effector cells genetically modified to express an antigen receptor. Genetic modification is achieved using particles described herein comprising nucleic acid encoding an antigen receptor for genetic modification and a docking compound binding to the particles via a connector compound, said docking compound comprising a targeting molecule for targeting immune effector cells. The particles may deliver the nucleic acid to cells in vitro/ex vivo as well as in vivo. Immune effector cells genetically modified to express an antigen receptor described herein are useful in the treatment of diseases wherein targeting cells such as diseased cells expressing an antigen such as a tumour antigen is beneficial. The target cells may express the antigen on the cell surface for recognition by a CAR or in the context of MHC for recognition by a TCR. The treatments described herein may provide for the selective eradication of such cells expressing an antigen, thereby minimizing adverse effects to normal cells not expressing the antigen. Immune effector cells genetically modified to express an antigen receptor, e.g., a CAR or TCR, targeting cells through binding to the antigen (or a procession product thereof) are provided to a subject such as by administration of genetically modified immune effector cells to the subject or generation of genetically modified immune effector cells in the subject.

In some embodiments, the immune effector cells are CD3+ T cells. In some embodiments, the docking compound described herein binds to the CD3 receptor on T cells. In some embodiments, the immune effector cells are CD8+ T cells. In some embodiments, the docking compound described herein binds to the CD8 receptor on T cells. In some embodiments, the immune effector cells are CD4+ T cells. In some embodiments, the docking compound described herein binds to the CD4 receptor on T cells. In some embodiments, the immune effector cells are CD7+ T cells. In some embodiments, the docking compound described herein binds to the CD7 receptor on T cells. The particles and compositions described herein and immune effector cells genetically modified to express an antigen receptor are, in particular, useful for the treatment of diseases characterized by diseased cells expressing an antigen to which the immune effector cells are directed. In some embodiments, the immune effector cells by means of a CAR have a binding specificity for disease-associated antigen when present on diseased cells. In some embodiments, the immune effector cells by means of a TCR have a binding specificity for a procession product of disease-associated antigen when presented on diseased cells. In some embodiments, a cell is genetically modified to stably express an antigen receptor on its surface. In some embodiments, a cell is genetically modified to transiently express an antigen receptor on its surface.

In one aspect, provided herein is a method for delivering a payload to a cell expressing a cell surface antigen, comprising adding to the cell a functionalised nucleic acid-lipid particle or a nucleic acid lipid particle comprising a primary targeting moiety, or pharmaceutical compositions thereof. In some embodiments, the payload comprises a nucleic acid. In some embodiments, the cell comprises a diseased cell. In some embodiments, the payload comprises a compound that is effective for treating the disease. In some embodiments, the cell surface antigen comprises a tumour antigen. In some embodiments, the cell comprises a tumour or cancer cell. In some embodiments, the cell comprises an immune effector cell. In some embodiments, the payload comprises a nucleic acid encoding an antigen receptor. In some embodiments, the cell surface antigen comprises a cell surface antigen on an immune effector cell.

In one aspect, provided herein is a method for preparing an immune effector cell genetically modified to express an antigen receptor, comprising adding to an immune effector cell a functionalised nucleic acid-lipid particle or a nucleic acid lipid particle comprising a primary targeting moiety, or pharmaceutical compositions thereof. In some embodiments, the cell described herein is present ex vivo. In some embodiments, the cell described herein is present in a subject and the method described herein comprises administering the composition described herein to the subject.

In one aspect, provided herein is a method for treating a subject comprising:

(i) preparing ex vivo an immune effector cell genetically modified to express an antigen receptor using a method comprising adding to an immune effector cell a functionalised nucleic acid-lipid particle or a nucleic acid lipid particle comprising a primary targeting moiety, or pharmaceutical compositions thereof;

(ii) administering the immune effector cell genetically modified to express an antigen receptor to the subject.

In one aspect, provided herein is a method for treating a subject comprising administering to the subject a functionalised nucleic acid-lipid particle or a nucleic acid lipid particle comprising a primary targeting moiety, or pharmaceutical compositions thereof.

In some embodiments, the nucleic acid encoding an antigen receptor is delivered to the immune effector cell. In some embodiments, delivering the nucleic acid encoding an antigen receptor to the immune effector cell generates an immune effector cell genetically modified to express an antigen receptor. In some embodiments, the cell surface antigen is characteristic for the immune effector cell. In some embodiments, the immune effector cell comprises a T cell. In some embodiments, the immune effector cell comprises a CD 8+ and/or CD4+ T cell. In some embodiments, the cell surface antigen comprises CD4 and/or CD8. In some embodiments, the cell surface antigen comprises CD3. In some embodiments, the antigen receptor targets an antigen associated with a disease, disorder or condition or cells expressing an antigen associated with a disease, disorder or condition. In some embodiments, the subject has a disease, disorder or condition associated with an antigen. In some embodiments, the antigen associated with a disease, disorder or condition comprises a tumour antigen. In some embodiments, the cells expressing an antigen associated with a disease, disorder or condition are tumour or cancer cells. In some embodiments, the disease, disorder or condition associated with an antigen comprises a tumour or cancer. In some embodiments, the method described herein is a method for treating or preventing cancer in a subject.

In some embodiments, the antigen associated with a disease, disorder or condition comprises an antigen of an infectious agent. In some embodiments, the infectious agent comprises a virus. In some embodiments, the disease, disorder or condition associated with an antigen is infection. In some embodiments, the method described herein is a method for treating or preventing an infection in a subject. In some embodiments, the method described herein further comprises administering to the subject an antigen targeted by the antigen receptor, a polynucleotide encoding the antigen, or a host cell genetically modified to express the antigen.

In some embodiments, the polynucleotide is RNA, DNA, or mixtures thereof. In some embodiments, the antigen receptor comprises a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the genetic modification is transient or stable. In some embodiments, the genetic modification takes place by a virus-based method, transposon-based method, or a gene editing-based method.

The primary target may be upregulated during a disease, e.g. infection or cancer. In diseased tissues, markers can differ from healthy tissue and offer unique possibilities for therapy, especially targeted therapy. In some embodiments, the primary target is a disease-associated antigen, such as a tumour antigen, a viral antigen, or a bacterial antigen. This allows diseased cells to be targeted by the particles and compositions described herein, e.g., for delivering an active ingredient. The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. Disease-associated antigens may be associated with infection by microbes, typically microbial antigens, or associated with cancer, typically tumours. In some embodiments, the primary target is a structure such as a protein present on the surface of a target cell such as a cell surface antigen or cell surface receptor the presence or amount of which is characteristic for certain cell types or organs as compared to others. In some embodiments, the primary target is a tumour antigen or tumour-associated antigen. In the context of the present disclosure, the tumour antigen is preferably associated with the cell surface of a cancer cell and is preferably not or only rarely expressed in normal tissues. Examples for tumour antigens include p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE- AI, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART- I/Melan-A, MC1R, Myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR- I, pl90 minor BCR-abL, Pml/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE and WT. Particularly preferred tumour antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

The term "immune effector cell" in the context of the present disclosure relates to a cell which exerts effector functions during an immune reaction. An "immune effector cell" in some embodiments is capable of binding an antigen such as an antigen presented by in the context of MHC on a cell or expressed on the surface of a cell and mediating an immune response. For example, immune effector cells comprise T cells (cytotoxic T cells, helper T cells, tumour infdtrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present disclosure, "immune effector cells" are T cells, preferably CD4+ and/or CD8+ T cells, most preferably CD8+ T cells.

The particles and compositions described herein may be used for in vitro or in vivo introduction of the payload into the target cell, depending on the location of the target cell. By "in vivo" it is meant, for example, in the targeting particles are administered to a living body of an animal. By "ex vivo" it is meant, for example, that cells are modified outside of the body. Such cells may be returned to a living body. For example, cells described herein, e.g., immune effector cells, may be genetically modified ex vivo/in vitro or in vivo in a subject being treated to express a peptide or polypeptide, e.g., an antigen receptor such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR) binding antigen or a procession product thereof, in particular when present on or presented by a target cell, e.g., an antigen presenting cell or a diseased cell. In some embodiments, modification to express a peptide or polypeptide, e.g., an antigen receptor, takes place in vivo. The cells may be endogenous cells of the patient or may have been administered to a patient. In some embodiments, modification to express a peptide or polypeptide, e.g., an antigen receptor, takes place ex vivo/in vitro. Subsequently, modified cells may be administered to a patient.

The term "T cell receptor" or "TCR" as used herein refers to a protein receptor on T cells that is composed of a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR consists of gamma and delta (y8) chains. In some embodiments, the TCR may be derived from any cell comprising a TCR, including a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell, for example. Each a, , y, and 5 chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR), followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. The TM region associates with the invariant subunits of the CD3 signalling apparatus. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (MHC).

As used herein, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or a complex of molecules which recognizes, i.e., binds to, a target structure (e.g. an antigen) on a target cell such as a cancer cell (e.g., by binding of an antigen binding domain to an antigen expressed on the surface of the target cell) and may confer specificity onto an immune effector cell such as a T cell expressing said CAR on the cell surface. A CAR comprises a target-specific binding element otherwise referred to as an antigen binding moiety or antigen binding domain that is generally part of the extracellular domain of the CAR. Specifically, the CAR may target an antigen on target cells, e.g., diseased cells such as tumour cells. In some embodiments, an antigen binding domain comprises a variable region of a heavy chain of an immunoglobulin (VH) with a specificity for the antigen and a variable region of a light chain of an immunoglobulin (VL) with a specificity for the antigen. In some embodiments, an immunoglobulin is an antibody. In some embodiments, said heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected via a peptide linker. Preferably, the antigen binding moiety portion in the CAR is a scFv. In some embodiments, an antigen binding domain comprises a VHH domain. The CAR is preferably designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In some instances, the CAR comprises a hinge domain which forms the linkage between the transmembrane domain and the extracellular domain. The cytoplasmic domain or otherwise the intracellular signalling domain of the CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. In some embodiments, the CAR comprises a primary cytoplasmic signalling sequence derived from CD3-zeta. Further, the cytoplasmic domain of the CAR may comprise the CD3-zeta signalling domain combined with a costimulatory signalling region. Suitable co-stimulation domains include CD28, CD137 (4-1BB), a member of the tumour necrosis factor receptor (TNFR) superfamily, CD 134 (0X40), a member of the TNFR-superfamily of receptors, and CD278 (ICOS), a CD28 -superfamily co-stimulatory molecule expressed on activated T cells. A CAR may comprise the above domains, together in the form of a fusion protein.

In some embodiments, the immune effector cell to be targeted is a T cell, in which case, the primary target may be a cell surface molecule on T cells, e.g., a T cell marker. As used herein, the term "T cell marker" refers to surface molecules on T cells which are specific for particular T cells. T cell markers suitable for use herein include, but are not limited to surface CD3, CD4, CD8, CD45RO or any other CD antigen specific for T cells, such as CD7, CD2, CD28, CD 127 and CD5. In some embodiments, the immune effector cell to be targeted is a B cell, in which case, the primary target may be a cell surface molecule on B cells, e.g., a B cell marker. As used herein, the term "B cell marker" refers to surface molecules on B cells which are specific for antigen-specific IgG-producing B cells. B cell markers suitable for use herein include, but are not limited to surface IgG, kappa and lambda chains, Ig-alpha (CD79alpha), Ig-beta (CD79beta), CD19, la, Fc receptors, B220 (CD45R), CD20, CD21, CD22, CD23, CD81 (TAPA-1), IgD or any other CD antigen specific for B cells.

The T cell marker or B cell marker for use herein may be selected from CD3, CD7, CD2 or IgD.

The functionalised nucleic acid-lipid particles or nucleic acid-lipid particles, and pharmaceutical compositions comprising them as described herein may be used in the therapeutic or prophylactic treatment of various diseases, in particular diseases in which provision of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, provision of an antigen or epitope which is derived from a virus may be useful in the treatment or prevention of a viral disease caused by said virus. Provision of a tumour antigen or epitope may be useful in the treatment of a cancer disease wherein cancer cells express said tumour antigen. Provision of a functional protein or enzyme may be useful in the treatment of genetic disorder characterized by a dysfunctional protein, for example in lysosomal storage diseases (e.g. mucopolysaccharidoses) or factor deficiencies. Provision of a cytokine or a cytokine-fusion may be useful to modulate tumour microenvironment. Therefore, in one aspect there is disclosed the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in medicine.

In one embodiment, there is provided a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein for use in delivery of a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) to a cell. In one embodiment, there is provided a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, for use in transfecting a cell with a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA). In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for delivery of a nucleic acid (such as an mRNA) to a cell. In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for delivery of a nucleic acid (such as an mRNA) to a T cell. In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for transfecting a cell with a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA). In one embodiment, there is provided use of a functionalised nucleic acid- lipid particle, or pharmaceutical composition as defined herein in the manufacture of a medicament for transfecting a T cell with a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA). In one embodiment, there is provided a method of delivery of a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) to a cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein. In one embodiment, there is provided a method of delivery of a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) to a T cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein. In one embodiment, there is provided a method of transfecting a cell with a nucleic acid (such as an mRNA), the method comprising adding to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein; and incubating the mixture of the composition and cells for a sufficient amount of time. In some embodiments, in particular those where the nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) encodes a pharmaceutically active protein, the mixture of the composition and cells is incubated for a time sufficient to allow the expression of the pharmaceutically active protein. In some embodiments, the sufficient amount of time is at least one hour (such at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 9 hours, at least about 12 hours) and/or up to about 48 hours (such as up to about 36 or up to about 24 hours). In some embodiments, incubating the mixture of the composition and cells is conducted in the presence of serum (such as human serum).

In some embodiments, there is provided a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, for use in delivery of a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) to a cell. In some embodiments, the gene editing tool is a transposon, a LSR, a CRISPR/Cas9 or a ZFN gene editing tool. In some embodiments, the gene editing tool is a CRISPR/Cas9 gene editing tool or a ZFN gene editing tool. In some embodiments, there is provided a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, for use in gene editing of a cell. In some embodiments, there is provided use of a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, in the manufacture of a medicament for gene editing a cell. In some embodiments, there is provided a method of gene editing a cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool. In some embodiments, there is provided a method of gene editing a cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool; and incubating the mixture of the composition and cells for a sufficient amount of time.

In some embodiments, there is provided a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, for use in a method of gene editing in a subject in need thereof, wherein said method comprises administering the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, to the subject. In some embodiments, there is provided use of a functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool, for the manufacture of a medicament for gene editing in a subject in need thereof. In some embodiments, there is provided a method of gene editing in a subject in need thereof, the method comprising administering to the subject the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool.

In some embodiments there is provided a method of carrying out a gene knock-out in a cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool. In some embodiments there is provided a method of carrying out a gene knock-in of a transgene in a cell, the method comprising administering to the cell the functionalised nucleic acid-lipid particle, or pharmaceutical composition as defined herein, wherein the nucleic acid encodes a gene editing tool that includes a DNA template encoding the transgene to be knocked-in.

Suitably, the gene editing, gene knock-in and/or gene knock-out may be performed ex vivo or in vivo.

The cell may be any cell capable of receiving nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) to produce a therapeutic effect. In one embodiment, the cell is a liver cell. In one embodiment, the cell is a spleen cell. In one embodiment, the cell is a lung cell. In one embodiment, the cell is a T cell.

In one embodiment, there is provided a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein, for use in treating a disease treatable by a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA). In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for treating a disease treatable by a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA). In one embodiment, there is provided a method of treating a disease treatable by a nucleic acid (such as an mRNA, or a mixture of mRNA and DNA) in a subject in need thereof, the method comprising administering to the subject a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

In one embodiment, there is provided a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in a prophylactic and/or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein in the manufacture of a medicament for a prophylactic and/or therapeutic treatment of a disease involving an antigen. In one embodiment, there is provided a method of prophylactic and/or therapeutic treatment of a disease involving an antigen in a subject in need thereof, the method comprising administering to the subject a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein. In one embodiment, there is provided a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein for use in inducing an immune response. In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for inducing an immune response.

In one embodiment, there is provided a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein, for use in treating cancer. In one embodiment, there is provided use of a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein, in the manufacture of a medicament for treating cancer. In one embodiment, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a functionalised nucleic acid-lipid particle or a pharmaceutical composition as defined herein.

The term "infectious disease" refers to any disease which can be transmitted from individual to individual or from organism to organism, and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, a viral disease, a bacterial disease, or a parasitic disease, which diseases are caused by a virus, a bacterium, and a parasite, respectively. In this regard, the infectious disease can be, for example, sexually transmitted diseases (e.g., chlamydia, gonorrhoea, or syphilis), SARS, coronavirus diseases (e.g., COVID- 19), acquired immune deficiency syndrome (AIDS), measles, chicken pox, cytomegalovirus infections, herpes simplex virus (e.g., HSV-1, HSV-2), hepatitis (such as hepatitis B or C), influenza (flu, such as human flu, swine flu, dog flu, horse flu, and avian flu), HPV infection, shingles, rabies, common cold, gastroenteritis, rubella, mumps, anthrax, cholera, diphtheria, foodbome illnesses, leprosy, meningitis, peptic ulcer disease, pneumonia, sepsis, septic shock, tetanus, tuberculosis, typhoid fever, urinary tract infection, Lyme disease, Rocky Mountain spotted fever, chlamydia, pertussis, tetanus, meningitis, scarlet fever, malaria, trypanosomiasis, Chagas disease, leishmaniasis, trichomoniasis, dientamoebiasis, giardiasis, amoebic dysentery, coccidiosis, toxoplasmosis, sarcocystosis, rhinosporidiosis, and balantidiasis.

In some embodiments, the functionalised nucleic acid-lipid particle or a pharmaceutical composition described herein may be used in the therapeutic or prophylactic treatment of an infectious disease.

In the present context, the term "treatment", "treating" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the subject is suffering, such as administration of the therapeutically effective compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of an individual for the purpose of combating the disease, condition or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.

The term "therapeutic treatment" relates to any treatment which improves the health status and/or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, arrest or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, decrease the frequency or severity of symptoms in an individual, and/or decrease the recurrence in an individual who currently has or who previously has had a disease.

The terms "prophylactic treatment" or "preventive treatment" relate to any treatment that is intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventive treatment" are used herein interchangeably.

The terms "individual" and "subject" are used herein interchangeably. They refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate), or any other non-mammal-animal, including birds (chicken), fish or any other animal species that can be afflicted with or is susceptible to a disease or disorder (e.g., cancer, infectious diseases) but may or may not have the disease or disorder, or may have a need for prophylactic intervention such as vaccination, or may have a need for interventions such as by protein replacement. In many embodiments, the individual is a human being. Unless otherwise stated, the terms "individual" and "subject" do not denote a particular age, and thus encompass adults, elderlies, children, and newborns. In embodiments of the present disclosure, the "individual" or "subject" is a "patient".

The term "patient" means an individual or subject for treatment, in particular a diseased individual or subject.

In some embodiments of the disclosure, the aim is to provide protection against an infectious disease by vaccination. In some embodiments of the disclosure, the aim is to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, to a subject, in particular a subject in need thereof.

In some embodiments of the disclosure, the aim is to provide a protein replacement therapy, such as production of erythropoietin, Factor VII, Von Willebrand factor, p-galactosidase, Alpha-N-acetylglucosaminidase, to a subject, in particular a subject in need thereof.

In some embodiments of the disclosure, the aim is to modulate/reprogram immune cells in the blood.

In some embodiments, the compositions described herein, which contain mRNA encoding a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof (in the following simply "SARS- CoV-2 S nucleic acid compositions" which explicitly include SARS-CoV-2 S RNA compositions), following administration to a subject, induce an antibody response, in particular a neutralizing antibody response, in the subject that targets a panel of different S protein variants such as SARS-CoV-2 S protein variants, in particular naturally occurring S protein variants. In some embodiments, the panel of different S protein variants comprises at least 5, at least 10, at least 15, or even more S protein variants. In some embodiments, such S protein variants comprise variants having amino acid modifications in the RBD domain and/or variants having amino acid modifications outside the RBD domain. In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets VOC-202012/01. In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets 501 ,V2. In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "Cluster 5". In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "B. 1.1.28" . In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets "B. 1.1.248" . In some embodiments, the SARS-CoV-2 S nucleic acid compositions described herein following administration to a subject induce an immune response (cellular and/or antibody response, in particular a neutralizing antibody response) in the subject that targets the Omicron (B.1. 1.529) variant.

A person skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective reaction to a disease is produced by immunizing a subject with an antigen or an epitope, which is immunologically relevant with respect to the disease to be treated. Accordingly, pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Pharmaceutical compositions described herein are thus useful in a prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.

The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example, for therapeutic or prophylactic reason.

Examples

Example 1 - General method - preparation of functionalised LNPs by a fluid-path system

Ethanolic solutions of a cationically ionisable lipid (lOOmM), cholesterol (50mM), a phospholipid (25mM), a stealth lipid (sLip; 25mM) and a targeting compound (a lipid carrying an ALFA-tag (PSRLEEELRRRLTE, SEQ ID NO: 2; ImM) were prepared, mixed in the desired ratio and diluted with additional ethanol to obtain a volume that allows a 3: 1 volume ratio of aqueous nucleic phase and ethanolic lipid phase, and was loaded into a syringe. In a second syringe, a mixture of one or several nucleic acids (total nucleic acid concentration 0.15 g/L) and an acidifier (50mM) in water was loaded (typically, this comprised at least one reporter DNA and one reporter RNA). Both syringes were loaded into a microfluidic device (Precision Nanosystems Nanoassemblr Ignite®) for mixing. The product fraction was loaded into a dialysis cassette (Slide-A-Lyzer™, 10,000 MWCO) and dialyzed against the formulation buffer. To balance volume changes during dialysis, the product is up-concentrated by centrifugal fdtration; cryoprotectant (trehalose, to a final concentration of 10% (w/w)) and formulation buffer (HEPES 20Mm; pH 6) are added to achieve the desired final concentration. The obtained ALFA-tagged LNPs (‘parent LNP’) are subsequently mixed with the desired amount of a docking compound (bispecific construct comprising an anti-ALFA-tag VHH (SEQ ID NO: 12) and aCD3 VHH (SEQ ID NO: 43)) construct and formulation buffer and incubated for 1 h at room temperature. Example 2

Applying the method described generally in example 1, samples having the lipid composition shown in Table 1 below (all % being expressed as mol% of the total lipids in the lipid mixture composition) were prepared with a cargo mix containing 20% Thy 1.1 RNA and 40% Venus DNA, and a final overall cargo concentration of 0. 1 g/L.

* Sample IDs 2-12 and 2-12.1 have the identical composition but were prepared as two separate batches.

Example 3

Applying the method described generally in Example 1, samples having the compositions described in Table 2 were prepared with a cargo mix containing 20% Thy 1.1 RNA and 40% Venus DNA, and a final overall cargo concentration of 0. 1 g/L. Samples marked “f ’ were frozen and stored at -80°C for a minimum of 12 h.

Example 4

Applying the method described generally in example 1, samples having the lipid compositions shown in Table 3 were prepared with a cargo mix containing 20% Thy 1. 1 RNA and 40% Venus DNA, and a final overall cargo concentration of 0. 1 g/L.

Example 5 - General Method - preparation of functionalised LNP by a liquid-dispensing system

Ethanolic solutions of a cationically ionisable lipid (50mM), cholesterol (40mM), a phospholipid (33mM), a stealth lipid (4mM) and a targeting compound (a lipid carrying an ALFA-tag (PSRLEEELRRRLTE, SEQ ID NO:2; 0.5mM) were prepared. The different lipid solutions were dispensed in predefined ratios into a 96-well plate in the following order: cationically ionisable lipid, cholesterol, phospholipid, stealth lipid, and the targeting compound. Enough ethanol was then added to complete a final volume of 33 pL. A mixture of one or several nucleic acids in an acidic solution were added to a final nucleic acid concentration of 55 ng/pL. The nucleic acid solution completed the final volume of 200 pL. Aliquots were taken and further diluted stepwise using buffer at neutral pH, generating ALFA-tagged LNPs with a final nucleic acid concentration of 5 ng/pL. After dilution, the docking compound construct (bispecific construct comprising an anti-ALFA-tag VHH (SEQ ID NO: 12) and aCD3 VHH (SEQ ID NO: 43)) was dispensed, and particles were incubated for 1 h at room temperature. Typically, the obtained LNPs were used without further purification.

Example 6 - Transfection studies - in vitro testing on peripheral blood mononuclear cells (PBMCs)

For transfection studies, either 1 pL or 5 pL of respective thawed formulations were prediluted in 50 pL X-Vivol5 in an ultra-low adhesion 96 well plate. 0.3e6 thawed human PBMCs were diluted in 50 pL clotted pooled human serum (PHS) and added to the nanoparticle dilution. After 30 min of incubation (37°C, 5 % CO2) 100 pL X-Vivol5 + 5 v% PHS + 100 U/ml IL-2 were added and PBMCs were further incubated for 3 days (37°C, 5% CO2). PBMCs were analysed for cell type-specific Thyl.l-RNA transfection or Venus-DNA transfection by flow cytometry.

Figure 1 shows the results of in vitro testing on PBMCs. Depicted are (a) the percentages of Thy 1.1- expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD 19+ B cells) out of all single and alive cells (y-axis) at a dose of 100 ng Thyl.l-RNA (b) the percentages of Venus- expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y- axis) and total count of Venus-positive T cells (stars; right x-axis) at a dose of 40 ng Venus- DNA; (c) the same at 200 ng Venus-DNA; (d) cell counts of overall cell subtypes (CD 14+ Monocytes; CD 19+ B cells, CD4+ T cells, CD8+ T cells) at day 4 after treatment with LNPs at a dose of 100 ng Thy 1.1/200 ng Venus.

Example 7 - Dynamic light scattering (DLS) - Size and polydispersity index analysis

The measurement was carried out in water (1:20 dilution). Size analysis was performed by dynamic light scattering (DLS) using a DynaPro Plate Reader II (Wyatt, Dembach, Germany). LNP formulations were diluted 1:20 in water and 100 pL of diluted sample were measured in triplicate in a 96-well plate. From the measurements, size (Z_aVerage), and polydispersity index (PDI) were calculated from the cumulant analysis using Dynamics 7.8. 1.3 software (settings: 10 Acquisitions each 3 sec).

Figure 2 shows DLS data of the samples of Figure 1 , before and after functionalisation and after one freeze-thaw cycle at -80°C. All samples were within the desired size and PDI range.

Figure 10 shows DLS data of the samples of Figure 4 before and after functionalisation and after one freeze-thaw cycle at -80°C. All samples were within the desired size and PDI range.

Figure 11 shows DLS data of the samples of Figure 5 before and after functionalisation and after one freeze-thaw cycle at -80°C. All samples were within the desired size and PDI range.

Example 8 - Agarose gel electrophoresis

10 pL of sample were mixed with 2 pL of loading dye (Thermo Fisher DNA loading buffer, 6x) and added into the pockets of a 1% agarose gel stained with GelRed Nucleic Acid Gel Stain (Biotium, Hayward, CA, USA). The gel was run at 80 V, 500 mA, 50 W for 40 minutes. Gel images were taken on a Chemidoc XRS imaging system (Bio-Rad, Berkeley, CA, USA).

Figure 3 shows the results of agarose gel electrophoresis of the samples from Figure 1. The gel shows the absence of any free DNA or RNA in all prepared formulations, indicating full encapsulation.

Example 9 - Transfection studies

For transfection studies, either 1 pU or 5 pU of respective freshly prepared or thawed formulations were pre-diluted in 50 pU X-Vivol5 in an ultra-low adhesion 96 well plate. 0.3e6 thawed human PBMCs were diluted in 50 pU clotted PHS and added to the nanoparticle dilution. After 30 min of incubation (37 °C, 5 % CO2) 100 pU X-Vivol5 + 5% PHS + 100 U/ml IU-2 were added and PBMCs were further incubated for 3 days (37°C, 5% CO2). PBMCs were analysed for cell type-specific Thy 1.1 -RNA transfection or Venus-DNA transfection by flow cytometry.

Figure 4 shows the results of in vitro testing on PBMCs - (samples 3-1 to 3-6; lipids BU-207, BU-209, SM102: freeze/thaw. Depicted are (a) the percentages of Thy 1.1- expressing cell subtypes (CD4+ T cells, CD8+ T cells, CD 19+ B cells) out of all single and alive cells (y-axis) at a dose of 100 ng Thyl.l-RNA; (b) the percentages of Venus-expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y-axis) and total count of Venuspositive T cells (stars; right x-axis) at a dose of 200 ng Venus-DNA; (c) cell counts of overall cell subtypes (CD14+ Monocytes; CD19+ B cells, CD4+ T cells, CD8+ T cells) at day 4 after treatment with UNPs at a dose of 100 ng Thy 1. 1/200 ng Venus

Example 10 - In vitro testing on PBMCs - samples 4-1 to 4-4 (lipid BHD-C2C2-PipZ)

For transfection studies, either 1 pU or 5 pU of respective thawed formulations were pre-diluted in 50 pU X-Vivol5 in an ultra-low adhesion 96 well plate. 0.3e6 thawed human PBMCs were diluted in 50 pU clotted PHS and added to the nanoparticle dilution. After 30 min of incubation (37°C, 5% CO₂) 100 pU X-Vivol5 + 5% PHS + 100 U/ml IU-2 were added and PBMCs were further incubated for 3 days (37°C, 5 % CO2). PBMCs were analysed for cell type-specific Thyl.l-RNA transfection or Venus-DNA transfection by flow cytometry.

Figure 5 shows the results of in vitro testing on PBMCs of these additional lipids. Depicted are (a) the percentages of Venus-expressing cell subtypes (CD4+ T cells, CD8+ T cells) out of all single and alive cells (left y-axis) and total count of Venus-positive T cells (stars; right x-axis) at a dose of 200 ng Venus-DNA.

Example 11 - In vivo LNP testing in hCD3EDGtg mice - in vivo efficacy study

Frozen LNP formulation was thawed (Sample ID: 2-12.1, see table in Example 2) and adjusted to 5 pg in 100 pl in respective formulation buffer for i.v. tail vein injection into 3 female B6- hCD3EDG transgenic mice. Non-injected mice were used as control. 18 h after injection serum was collected to evaluate the systemic cytokine levels in multiplex assay. Furthermore, the LNP distribution was analysed by ex vivo organ bioluminescence imaging. Activation and cell typespecific transfection was assessed by analysing the expression of the delivered Thyl. 1-RNA in spleen, brachial lymph nodes and blood via flow cytometry.

Figure 6 shows the results of testing of LNPs in administered in vivo to hCD3EDGtg mice in this study. The organs were extracted from the mice after administration for imaging.

Depicted are (a) the results of ex vivo organ bioluminescence imaging; (b) cell-type specific transfection; (c) T cell activation (monitored by CD69 expression levels) and (d) systemic cytokine levels.

This study in B6-hCD3EDG transgenic mice to allow successful binding of the anti-human CD3 targeting ligand to mouse T cells. Functionalised LNPs formulated with high ionisable lipid content (59.67% of SM-102) show efficient drainage to spleen and lymph nodes after i.v. injection (Figure 6a). In line with in vitro data (depicted in Figure 1 above), the functionalised LNPs show specific T cell transfection in spleen, lymph nodes and blood (Figure 6b). Upon injection of functionalised LNPs, T cell in spleen, lymph nodes and blood are activated (Figure 6c) and systemic cytokine levels of IL2 and IFN-y increase (Figure 6d). In summary, CD3- targeting LNPs with high ionisable lipid content are stable and highly effective in vivo.

Example 12 - In vitro NLuc DNA expression in primary T cells - functionalised lipids

Applying the method described generally in Example 5, unfunctionalised LNP were made from a phospholipid (DSPC), cholesterol, a stealth lipid (C16Cer-PEG2k), a lipid carrying an ALFA-tag, a cationically ionisable lipid selected from BNT-51, BHD-C2C2-PipZ, BODD- C2C2-DMA, BODD-C2C2-Pyr, BDH-C2C4-PipZ, BODD-C2C4-PipZ, DND-C2C4-PipZ, BODD-C2C4-Pyr or SM-102, and functionalised with an anti-human CD3 targeting ligand. For transfection, 5 pL of the respective freshly prepared formulations were pre-diluted in 50 pL X-Vivol5 in an ultra-low adhesion 96 well plate. 0.75e5 isolated human T cells were diluted in 50 pL clotted PHS and added to the nanoparticle dilution. After 30 min of incubation (37 °C, 5 % CO2) 100 pL X-Vivol5 + 5% PHS + 100 U/ml IL-2 were added and T cells were further incubated for 5 days (37°C, 5% CO2). T cells were analysed for NLuc- DNA transfection using a luminescence assay.

Figure 7 shows the in vitro NLuc DNA expression in primary T cells for functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2-1 (see table in Example 2).

Example 13 - In vitro NLuc DNA expression in primary T cells - functionalised lipids

Applying the method described generally in Example 5, unfunctionalised LNP were made from cholesterol, a stealth lipid (C16Cer-PEG2k), a lipid carrying an ALFA-tag, a cationically ionisable lipid selected from BNT-51, BNT-52, BODD-C2C2-DMA, BODD- C2C4-Pyr or SM-102, a phospholipid selected from DSPC, sphingomyelin (SPML) or DOPE, and functionalised with an anti-human CD3 targeting ligand.

For transfection, 5 pL of the respective freshly prepared formulations were pre-diluted in 50 pL X-Vivol5 in an ultra-low adhesion 96 well plate. 0.75e5 isolated human T cells were diluted in 50 pL clotted PHS and added to the nanoparticle dilution. After 30 min of incubation (37 °C, 5 % CO2) 100 pL X-Vivol5 + 5% PHS + 100 U/ml IL-2 were added and T cells were further incubated for 5 days (37°C, 5% CO2). T cells were analysed for NLuc-DNA transfection using a luminescence assay.

Figure 8 shows the in vitro NLuc DNA expression in primary T cells for functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2-1 (see table in Example 2).

Example 14 - In vitro NLuc DNA expression in primary T cells - functionalised lipids

Applying the method described generally in Example 5, unfunctionalised LNP were made from a cationically ionisable lipid (BNT-52), cholesterol, a phospholipid (DSPC), a lipid carrying an ALFA-tag, a stealth lipid selected from C16Cer-PEG2k, Ac-pAEEA14-DMA, Ac-pAEEA14-DSPE or Ac-pAEEA14-tocopherol, and functionalised with an anti-human CD3 targeting ligand.

For transfection, 5 pL of the respective freshly prepared formulations were pre-diluted in 50 pL X-Vivol5 in an ultra-low adhesion 96 well plate. 0.75e5 isolated human T cells were diluted in 50 pL clotted PHS and added to the nanoparticle dilution. After 30 min of incubation (37 °C, 5 % CO2) 100 pL X-Vivol5 + 5% PHS + 100 U/ml IL-2 were added and T cells were further incubated for 5 days (37°C, 5% CO2). T cells were analysed for NLuc- DNA transfection using a luminescence assay.

Figure 9 shows the in vitro NLuc DNA expression in primary T cells for functionalised LNP formulated at different phospholipid-to-cholesterol ratios (hLip/Chol) and cationically ionisable lipid content (iLip). The in vitro expression is presented as the fold change with respect to the composition in sample ID: 2- 1 (see table in Example 2).

The data in figures 7, 8 and 9 demonstrate that functionalised LNP with an expression superior to sample ID: 2-1 can be obtained with materials comprising cholesterol, a phospholipid, and a stealth lipid plus various cationically ionisable lipids (Figure 7), or cholesterol, a cationically ionisable lipid and a stealth lipid plus various phospholipids (Figure 8), or cholesterol, a cationically ionisable lipid, and a phospholipid plus various stealth lipids (Figure 9).

Example 15 - CRISPR-Cas9 or Zinc-finger nuclease (ZFN)-mediated targeted knock-in insertion in T cells using LNPs in vitro

The efficiency of CRISPR-Cas9 and ZFN-mediated gene editing combined with nanoplasmid DNA templates, co-delivered via LNPs was evaluated. To prepare the CRISPR-LNPs, Cas9- encoding mRNA and single guide RNA (sgRNA) targeting the T-cell receptor a constant (TRAC) locus, were co-formulated with a nanoplasmid DNA template encoding a Venus transgene, in a 1: 1: 1 weight ratio, in LNPs. The formulation was performed according to a method similar to that described in Example 1. The LNPs had the following composition: BL- 207/Chol/DSPC/C16Cer-PEG2k/DSPE-PEG2k-ALFA (in the molar ratios 59.67/24/14.33/1.8/0.2). The LNPs were functionalized in order to specifically target T cells. ZFN-LNPs were similarly formulated using the same LNP composition, to co-deliver a pair of ZFN mRNAs, also targeting the TRAC locus, together with a nanoplasmid DNA template encoding Venus transgene, in the same 1: 1: 1 weight ratio. As a control, CRISPR- and ZFN- LNPs were also formulated without Nanoplasmid DNA template. A 1 pg dose of the LNPs was used to transfect 1 x 10^A6 human T cells. Transfected cells were then analysed by flow cytometry at various time points to measure both knock-out efficiency (assessed by downregulation of CD3 expression) and knock-in efficiency (assessed by Venus expression)(Figure 12). Both the CRISPR- and ZFN-LNPs were able to efficiently facilitate targeted gene integration in human T cells, demonstrating potent delivery of the mixed DNA/RNA payloads using the LNP formulation of the invention (Figure 12).

Example 16 CRISPR-Cas9-mediated targeted knock-in insertion in B cells using LNPs in vitro

Freshly isolated human B cells were treated with LNPs encapsulating Cas9 mRNA+sgRNA+ Venus nanoplasmid DNA. Two different LNP compositions were compared: (1) “BL-207” comprising BL-207/Chol/DSPC/C16Cer-PEG2k/DSPE-PEG2k-ALFA (molar ratios 59.67/24/14.33/1.8/0.2), and (2) “C12” comprising: DODMA/Chol/DOPE/C16Cer- PEG2k/DSPE-PEG2K-ALFA (molar ratios 40.0/48.0/10.0/1.8/0.2). The LNPs were functionalised using an algD docking compound. LNP treatment was performed on 1 million isolated B cell in 100 pl media containing 50% human serum at 37°C for 30 mins, following which the cells were diluted with media containing human serum supplemented with IL4 and IL21. The cells were then co-cultured with CD40 ligand secreting 3T3 irradiated feeder cells. Cells were regularly split onto fresh feeder cells every 3 days. To follow the expression of the Venus reporter DNA, cells were analysed via FACs for the expression of the B cell marker CD20 and Venus.

DNA delivery was measured by the expression of the reporter DNA Venus. Some Venus positive B cells were observed for the BL-207 LNPs by Day 6. The percentage and the MFI of the Venus positive cells increased steadily from day 6 to day 10 suggesting that the DNA was integrated in the genome and was propagated as the cells divided and proliferated (Figure 13). These data demonstrate that the BL-207 LNPs, formulated according to the invention, were able to effectively co-deliver a mixed RNA and DNA payload (Figure 13). In contrast, no Venus positive cells were observed in the B cells treated with the C12 LNP formulations.

Example 17 - In vivo targeted delivery of DNA/RNA mixed payloads using the LNP formulations for in vivo CAR-T cell generation.

For the following in vivo experiments, BL-207-based ahCD3-LNPs were prepared according to a method similar to that specified in Example 1. The LNPs had the following composition: 59.67 mol% BL-207, 24 mol% Choi, 14.33 mol% DSPC, 1.8 mol% C16Cer-PEG2k, 0.2 mol% DSPE-PEG2k-ALFA, N/P ratio = 12, X/L 4.4. The LNPs were formulated with a nucleic acid payload of a DNA nanoplasmid encoding a chimeric antigen receptor (CAR), an mRNA encoding a sleeping beauty (SB) transposase (SB100X) and an mRNA encoding a Thy 1.1 reporter (weight ratio 9:9:2). Unless otherwise indicated, the LNPs were injected at a 90 pg/kg dose i.v. into the tail vein of B41CD3EDG mice. This mouse strain expresses extracellular human CD3 domains on the surface of T cells and NKT cells, which allows effective targeting with ahCD3-LNPs. As a negative control, ahCD3-LNPs without the CAR cargo were injected.

A. In vivo RNA delivery to mouse splenocytes following i.v. injection using BL-207 -based ahCD3-LNPs (Figure 14).

Mice were analysed to confirm whether the tested formulation was effective for payload delivery in vivo. Mice were sacrificed 18 hours after ahCD3-LNP injection and splenocytes were analysed for the expression of the delivered Thyl. 1 RNA via flow cytometry. The LNP formulation demonstrated effective and selective delivery of RNA to CD4 and CD 8 positive T cells (Figure 14A). Additionally, the mean fluorescence intensity (MFI) data indicated effective uptake of the LNPs specifically into cells that expressed extracellular human CD3 domains (including NK cells, CD4+ T cells and CD8+ T cells) (Figure 14B). These data demonstrate that LNP formulations having the above composition are functional and show highly effective delivery of the payloads also in vivo.

B. In vivo CAR T cell generation in mice following single injection of BL-207 -based ahCD3- LNPs (Figure 15).

Mice were also analysed to determine whether the tested LNP formulation was effective in delivering the mixed payloads at sufficient quantities for the integration of the CAR construct from the DNA nanoplasmid into the T cell genome through the SB transposase activity and subsequent expression of the CAR. Following administration of the ahCD3-LNPs, mice were treated according to a proprietary regimen to allow expansion of generated CAR T cells. CAR T cell frequencies in blood were monitored by weekly blood draws (Figure 15A). On day 30, mice were sacrificed and splenocytes were analysed for CAR-T cell frequencies (Figure 15B). The ahCD3-LNP formulation allows efficient DNA delivery in vivo, resulting in the formation of a stable CAR-T cell population over time in the mice.

C. In vivo CAR T cell generation in PBMC-engrafted NSG mice following single injection of BL-207-based ahCD3-LNPs (Figure 16). A different mouse model was used to test whether the LNP formulation was effective in delivering the mixed payloads to T cells at sufficient quantities for integration and expression of the CAR construct, also without an T cell expansion protocol. NSG mice reconstituted with human PBMCs were injected with BL-207-based ahCD3-LNPs at a 100 pg/kg dose i.v. into the tail vein. On day 7 after ahCD3-LNP injection mice were sacrificed and splenocytes and blood were analysed for CAR-T cell populations (Figure 16). These data demonstrate that the ahCD3-LNP formulation allows efficient DNA delivery in vivo, resulting in the formation of a stable CAR-T cell population over time in PBMC-NSG mice, even without CAR specific expansion.

The embodiments of the disclosure described above are intended to be merely exemplary, numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

1. A nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid; and

(c) cholesterol; and

(ii) a nucleic acid which is selected from DNA, RNA, or a mixture thereof; wherein the molar ratio of phospholipid to cholesterol is 0.5 to 3.0 wherein the particle comprises a moiety that binds to a target on target cells.

2. A nucleic acid-lipid particle according to claim 1, wherein the nucleic acid-lipid particle is a functionalised nucleic acid-lipid particle.

3. A nucleic acid-lipid particle according to claim 1 or 2, wherein the cationically ionizable lipid is present in an amount of 50 to 75 mol%, preferably 55 to 70 mol%, of the total lipids in the lipid mixture composition.

4. A nucleic acid-lipid particle according to any one of claims 1-3, wherein the molar ratio of phospholipid to cholesterol is 0.5 to 1.2.

5. A nucleic acid-lipid particle according to any one of claims 1-4, wherein the molar ratio of phospholipid to cholesterol is from 0.5 to 0.7, optionally 0.55 to 0.65.

6. A nucleic acid-lipid particle according to any one of claims 1-4, wherein the molar ratio of phospholipid to cholesterol is from 0.8 to 1.2, optionally 0.95 to 1.05.

7. A nucleic acid-lipid particle according to any one of claims 1-6, wherein the cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-l-sulfonamide)

hydroxybutyl )azancdiyl )bis(N.N-dioctyl heptane- 1 -sulfonamide (BL-207); octylsulfamoyl)hexyl)(2-hydroxyethyl)amino)octanoate (BL-200); dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylhexane- 1 -sulfonamide) (BL- 209);

2.2-dilinoleyl-4-dimethylaminoethyl-[ 1,3] -dioxolane (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);

1.2-dioleoyloxy-3 -dimethylaminopropane (DODMA); di((Z)-non-2-en- 1 -yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); bis- l -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);

2-(di-((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethyl 4-(dimethylamino)butanoate (EA- 2);

BODD-C2C2-Pyr ; bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-l-yl)butyl)azanediyl)dipropionate (BHD- C2C4-PipZ); bis(2-octyldodecyl) 3,3'-((4-(pyrrolidin-l-yl)butyl)azanediyl)dipropionate (BODD-C2C4- Pyr); or a mixture of any thereof.

8. A nucleic acid-lipid particle according to any one of claims 1-7, wherein the phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); dioleoylphosphatidylethanolamine (DOPE); diphytanoylphosphatidylethanolamine (DPyPE) l,2-di-(9Z-octadecenoyl)-sn-glycero-3 -phosphocholine (DOPG); N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM); stearoyloleylphosphatidylcholine (SOPC); and diphytanoylphosphatidylcholine (DPyPC); or a mixture of any thereof; preferably wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

9. A nucleic acid-lipid particle according to any one of claims 1-8, wherein the phospholipid is present in an amount of 5 to 30 mol%, preferably 10 to 22 mol% of the total lipids in the lipid mixture composition.

10. A nucleic acid-lipid particle according to any one of claims 1-9, wherein the cholesterol is present in an amount of 10 to 40 mol%, preferably 20 to 30 mol%, of the total lipids in the lipid mixture composition.

11. A nucleic acid-lipid particle according to any one of claims 1-10, wherein the moiety that binds to a target on target cells is a peptide, a protein, an antibody, an antibody fragment, or a DARPin capable of specifically binding to a target on target cells.

12. The nucleic acid-lipid particle according to claim 11, wherein the antibody fragment is a VHH, a sdAb, an scFv, a Fab or a Fab2.

13. The nucleic acid-lipid particle according to any one of claims 1-12, wherein the target on target cells is a cell surface antigen or a receptor.

14. The nucleic acid-lipid particle according to any one of claims 1-13, wherein the target cells are immune cells, optionally T cells or B cells.

15. The nucleic acid-lipid particle according to any one of claims 1-14, wherein the moiety that binds to a target on target cells is covalently or non-covalently attached to the particle.

16. A nucleic acid-lipid particle according to any one of claims 1-15, wherein the lipid mixture composition further comprises:

(d) a compound of Formula (A):

L-X1-P-X2-B (A) wherein:

P is absent or comprises a polymer;

B comprises a binding moiety, 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; and

X2 is absent or a second linking moiety.

17. A nucleic acid-lipid particle according to claim 16, wherein the compound of Formula (A) is present in an amount of 0.01 to 1 mol% of the total lipids present in the lipid mixture composition; preferably wherein the compound of Formula (A) is present in an amount of 0.02 to 0.5 mol% of the total lipids present in the lipid mixture composition; more preferably wherein the compound of Formula (A) is present in an amount 0.04 to 0.25 mol% of the total lipids present in the lipid mixture composition.

18. A nucleic acid-lipid particle according to claim 16 or 17, wherein the hydrophobic moiety comprises a lipid, preferably a phospholipid, more preferably a moiety selected from the group consisting of:

DSPE (distearoylphosphatidylethanolamine),

DPPE (dipalmitoylphosphatidylethanolamine),

DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine); most preferably a DSPE moiety.

19. A nucleic acid-lipid particle according to any one of claims 16-18, wherein P is a polymer.

20. A nucleic acid-lipid particle according to any one of claims 16-19, wherein the binding moiety B is:

(a) a peptide or protein;

(b) a moiety capable of binding to a cell surface antigen;

(c) a peptide tag;

(d) a moiety capable of binding to a peptide tag;

(e) an ALFA-tag; or

(f) a polymer.

21. A nucleic acid-lipid particle according to any one of claims 16-20, wherein the polymer is a hydrophilic polymer, more preferably selected from the group consisting of polyethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), or poly-2-(2-(2-(N-methylamino)-(ethoxy)ethoxy)acetic acid (pMAEEA) and combinations thereof.

22. A nucleic acid-lipid particle according to any one of claims 1-21, wherein the lipid mixture composition further comprises:

(e) a grafted lipid, preferably 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; more preferably a polyethylene glycol-conjugated lipid; most preferably a polyethylene glycol (2000) - C16 ceramide.

23. A nucleic acid-lipid particle according to any one of claims 1-22, wherein the grafted lipid is present in the lipid mixture in an amount of 0.2 to 5 mol% of the total lipids present in the lipid mixture; preferably wherein the grafted lipid is present in the lipid mixture in an amount of 1.0 to 4.0 mol% of the total lipids present in the lipid mixture; more preferably wherein the grafted lipid is present in the lipid mixture in an amount of 1.5 to 4.0 mol% of the total lipids present in the lipid mixture.

24. A nucleic acid-lipid particle according to any one of claims 1-23, wherein the nucleic acid is a mixture of DNA and mRNA.

25. A nucleic acid-lipid particle according to any one of claims 1-24, wherein the nucleic acid comprises:

(i) a DNA nanoplasmid and an mRNA;

(ii) DNA and an mRNA encoding a gene editing enzyme; or

(iii) a DNA nanoplasmid and an mRNA encoding a gene editing enzyme.

26. A nucleic acid-lipid particle of claim 25, wherein the gene editing enzyme is selected from the group consisting of a Large Serine Recombinase (LSR), a Zinc -Finger nuclease, a Cas enzyme, atransposase, and combinations thereof.

27. A nucleic acid particle of claim 26, wherein the nucleic acid encodes a CRISPR/Cas gene editing tool, wherein the nucleic acid comprises an RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as RNA.

28. A nucleic acid particle of claim 26 or 27, wherein the nucleic acid encodes a CRISPR/Cas gene editing tool, wherein the nucleic acid comprises a DNA and an RNA, wherein a Cas enzyme is encoded by a mRNA and a gRNA is provided as RNA, and wherein a DNA template is comprised in a DNA nanoplasmid.

29. A nucleic acid particle of claim 26, wherein the nucleic acid encodes a ZFN gene editing tool, wherein the nucleic acid comprises an RNA, wherein at least two ZFN nucleases are encoded by mRNA.

30. A nucleic acid particle of claim 26 or 29, wherein the nucleic acid encodes a ZFN gene editing tool, wherein the nucleic acid comprises a DNA and an RNA, wherein at least two ZFN nucleases are encoded by mRNA, and wherein a DNA template is comprised in a DNA nanoplasmid.

31. A nucleic acid-lipid particle according to any one of claims 1-30, further comprising: (iii) a docking compound which is a compound of formula (I):

B’-X3-B” (I) wherein B’ comprises a moiety binding to B of the compound of formula (A);

X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

32. A nucleic acid-lipid particle according to claim 31, wherein:

(a) B comprises a peptide tag and B’ comprises a moiety binding to the peptide tag; or

(b) B’ comprises a peptide tag and B comprises a moiety binding to the peptide tag; or

(c) B comprises a polymer and B’ comprises a moiety binding to the polymer.

33. A nucleic acid-lipid particle according to claim 32, wherein:

(i) the moiety binding to a peptide tag comprises an antibody or antibody-like molecule;

(ii) wherein the peptide tag comprises an ALFA-tag; and/or

(iii) wherein the polymer comprises PEG.

34. A nucleic acid-lipid particle according to any one of claims 31-33, wherein the ratio of the compound of formula (A) to the compound of formula (I) is 1:0.5 to 1: 16.

35. A functionalised nucleic acid-lipid particle comprising:

(i) a lipid mixture composition comprising:

(a) a cationically ionizable lipid;

(b) a phospholipid;

(c) cholesterol; and

(d) a compound of formula (A) as defined in any one of claims 16-34; wherein the molar ratio of phospholipid to cholesterol is 0.5 to 3.0;

(ii) a nucleic acid; and

(iii) a docking compound which is a compound of formula (I):

B’-X3-B” (I) wherein B’ comprises a moiety binding to B of the compound of formula (A); X3 is absent or a linking moiety; and

B” comprises a moiety binding to a cell surface antigen.

36. A functionalised nucleic acid-lipid particle of claim 35, wherein:

(i) the nucleic acid is as defined in any one of claims 24-30; and/or

(ii) the docking compound of formula (I) is as defined in any one of claims 31-34.

37. A nucleic acid-lipid particle according to any one of claims 13-34, or a functionalised nucleic acid-lipid particle according to claim 35 or 36, wherein the cell surface antigen is selected from the group consisting of: CD3, CD7, CD2, CD4, CD8, and IgD.

38. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is CD3.

39. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is CD7.

40. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is CD2.

41. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is CD4.

42. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is CD8.

43. A nucleic acid-lipid particle or a functionalised nucleic acid-lipid particle according to claim 37, wherein the cell surface antigen is IgD.

44. A pharmaceutical composition comprising the nucleic acid-lipid particle of any one of claims 1-34 or claims 37 to 43 or the functionalised nucleic acid-lipid particle of claim 35 to 43.

45. The nucleic acid-lipid particle of any one of claims 1-34 or claims 37 to 43, the functionalised nucleic acid-lipid particle of claim 35 to 43, or the pharmaceutical composition of claim 44, for use in therapy.

46. The nucleic acid-lipid particle of any one of claims 1-34 or claims 37 to 43, the functionalised nucleic acid-lipid particle of claim 35 to 43, or the pharmaceutical composition of claim 44, for use in a method for treating or preventing cancer in a subject.

47. The nucleic acid-lipid particle, functionalised nucleic acid-lipid particle, or pharmaceutical composition, for use according to claim 45 or 46, wherein the nucleic acid encodes an antigen receptor such as a T cell receptor (TCR) or chimeric antigen receptor (CAR); and wherein the particle comprises a moiety that binds to a target on immune effector cells such as T cells or B cells.