Nucleic acid particle

Abstract

The present invention generally relates to nucleic acid particles, in particular nucleic acid-lipid particles, comprising targeting moieties that are bound to the particles, and to pharmaceutical compositions containing the nucleic acid particles and their uses in medicine.

Description

[0001] NUCLEIC ACID PARTICLE

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to nucleic acid particles, in particular nucleic acid-lipid particles, comprising targeting moieties that are bound to the particles, and to pharmaceutical compositions containing the nucleic acid particles and their uses in medicine.

[0004] BACKGROUND TO THE INVENTION

[0005] 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.

[0006] 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 I 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).

[0007] Lipid nanoparticles (LNPs) have demonstrated huge potential as delivery technology for nucleic acids for treating a wide range of conditions, such as in cancer immunotherapy, gene therapy, and the treatment of infectious diseases. Polyplex particles, based on cationic polymers (e.g., PEI, protamine, etc.), are also known to have potential as delivery technology for nucleic acids. However, targeted delivery of nucleic acids (e.g., to particular cell types), and particularly targeted delivery of nucleic acids comprising a mixture of DNA and RNA, is still a challenge in the field.

[0008] Thus, there is a need for improved cell-specific delivery of nucleic acid particles. SUMMARY OF THE INVENTION

[0009] The present invention provides a nucleic acid particle (e.g., a nucleic acid-lipid particle) comprising:

[0010] (i) one or more particle forming components (e.g., lipid particle forming components);

[0011] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0012] (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different; wherein at least one targeting moiety is non-covalently bound to the nucleic acid particle.

[0013] In another aspect, the present invention provides a nucleic acid particle (e.g., a nucleic acid- lipid particle) comprising:

[0014] (i) one or more particle forming components (e.g., lipid particle forming components);

[0015] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0016] (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0017] In some embodiments, the nucleic acid particle comprises at least three targeting moieties, wherein the third targeting moiety is capable of binding to a third target, wherein each target is different.

[0018] In some embodiments, the nucleic acid particle comprises 2-10 targeting moieties, wherein each targeting moiety is capable of binding to a different target. In some embodiments, the nucleic acid particle comprises 2-5 targeting moieties; wherein each targeting moiety is capable of binding to a different target.

[0019] In some embodiments, each targeting moiety is non-covalently bound to the nucleic acid particle.

[0020] In some embodiments, the one or more particle forming components further comprise: a compound of Formula (A): L-X1-P-X2-B (A) wherein: P is absent or comprises a polymer;

[0021] L comprises (i) a hydrophobic moiety, or (ii) a moiety comprising a negative charge, attached to B when P is absent or to a first end of the polymer P when present;

[0022] 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;

[0023] X1 is absent or a first linking moiety; and

[0024] X2 is absent or a second linking moiety.

[0025] In some embodiments, the nucleic acid particle further comprises one or more compound(s) of Formula (I):

[0026] B’-X3-B” (I) wherein

[0027] B’ comprises a moiety capable of non-covalently binding to B;

[0028] X3 is absent or a linking moiety; and

[0029] B” comprises a targeting moiety; wherein the targeting moiety is non-covalently bound to the nucleic acid particle through the non-covalent binding of B’ of the compound of Formula (I) to B of the compound of Formula (A).

[0030] In some embodiments, B comprises a peptide tag or a moiety binding to a peptide tag and the nucleic acid particle further comprises one or more compound(s) of the formula:

[0031] B’-X3-B” wherein

[0032] B’ comprises a moiety binding to B;

[0033] X3 is absent or a linking moiety; and

[0034] B” comprises a moiety binding to a cell surface antigen.

[0035] In some embodiments, each targeting moiety is non-covalently bound to a separate compound of Formula (A).

[0036] In some embodiments, L comprises a hydrophobic moiety. In such embodiments, the nucleic acid particle may be a nucleic acid lipid particle, such as a lipid nanoparticle (LNP), a lipoplex (LPX) or a lipidated polyplex (LPLX). Typically, the hydrophobic moiety is incorporated into the particle, for example through hydrophobic interactions. In some embodiments, the hydrophobic moiety comprises a lipid. In some embodiments, the lipid is a phospholipid. In some embodiments, the hydrophobic moiety comprises a moiety selected from the group consisting of: DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleylphosphatidylethanolamine). In some embodiments, the hydrophobic moiety comprises a DSPE moiety.

[0037] In some embodiments, L comprises a moiety comprising a negative charge. In such embodiments, the nucleic acid particle may be a nucleic acid polymer particle, such as a polyplex (PLX). Typically, the moiety comprising a negative charge is incorporated into the particle, for example through an ionic (charge-charge) interaction with the positively charged polymer particle. In some embodiments, the moiety comprising a negative charge comprises a polymer, optionally wherein the polymer has a net negative charge. In some embodiments, the polymer comprises one or more ionizable carboxy groups, optionally wherein the polymer comprises a polyglutamic acid moiety. In some embodiments, the particle has a positive charge and the moiety comprising a negative charge is incorporated into the particle through the negative charge interacting with an opposite positive charge in the particle.

[0038] In some embodiments, P is a polymer, preferably a hydrophilic polymer. In some embodiments, the polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), or poly-2-(2-(2-(N-methylamino)-(ethoxy)ethoxy)acetic acid (pMAEEA) and combinations thereof.

[0039] In some embodiments, the binding moiety B comprises:

[0040] (a) a peptide tag;

[0041] (b) a moiety capable of binding to a peptide tag; or

[0042] (c) an ALFA-tag.

[0043] In some embodiments, the B’ of Formula (I) comprises:

[0044] (a) a moiety capable of binding to a peptide tag;

[0045] (b) a peptide tag; or

[0046] (c) an anti-ALFA binding domain.

[0047] In some embodiments, the binding moiety B comprises an ALFA tag. In some embodiments, the B’ comprises an anti-ALFA binding domain. In some embodiments, the one or more lipid particle forming components comprises a cationic or cationically ionizable lipid. In some embodiments, the nucleic acid-lipid particle is a lipid nanoparticle (LNP) or a lipoplex (LPX).ln some embodiments, the particle is a lipid nanoparticle (LNP).

[0048] In some embodiments, the one or more particle forming components comprises a cationic polymer. In some embodiments, the nucleic acid particle is a polyplex (PLX) or a lipidated polyplex (LPLX).

[0049] In some embodiments, the cationic or cationically ionizable lipid is selected from the group consisting of:

[0050] 7, 7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide); BNT-51 ;

[0051] BNT-52; [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315);

[0052] 1.2-dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z, 12Z)-octadeca-9, 12-dien-1 -yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4- (dimethylamino)butanoate (HY501);

[0053] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8, 1 -diyl) bis(2- hexyldecanoate) (EA-405);

[0054] (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2-hexyldecanoate) (HY- 405);

[0055] 1.2-dioleoyl-3 trimethylammonium propane (DOTAP);

[0056] 1.2-dioleoyl-3-dimethylammomium propane (DODAP); and

[0057] 1.2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA);

[0058] BHD-C2C2-PipZ

[0059] BODD-C2C2-Pyr

[0060] 7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1 -sulfonamide (BL-207); or a mixture of any thereof.

[0061] In some embodiments, the particle forming composition further comprises one or more additional lipids. In some embodiments, the one or more additional lipids are selected from: (a) a neutral or zwitterionic lipid, such as a neutral or zwitterionic phospholipid, preferably wherein the neutral or zwitterionic phospholipid is selected from the group consisting of: distearoylphosphatidylcholine (DSPC); dioleoylphosphatidylcholine (DOPC); dimyristoylphosphatidylcholine (DMPC); dipalmitoylphosphatidylcholine (DPPC); palmitoyloleoyl-phosphatidylcholine (POPC); dioleoylphosphatidylethanolamine (DOPE);

[0062] 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG);

[0063] N-palmitoyl-D-erythro-sphingosylphosphorylcholine (SM); or a mixture of any thereof;

[0064] (b) a steroid, preferably wherein the steroid is cholesterol; and / or

[0065] (c) 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(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid; and a poly(2- methylaminoethoxy ethoxy acetic acid) (pmAEEA)-conjugated lipid; or a mixture of any thereof.

[0066] In some embodiments, at least one, or each, targeting moiety is selected from the list consisting of: a cytokine, an antibody or fragment thereof, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody and a single chain variable domain. In some embodiments, the targeting moiety is a nanobody. In some embodiments, the targeting moiety is a cytokine. In some embodiments, the cytokine is IL7.

[0067] In some embodiments, all of the targeting moieties are independently selected from the list consisting of: a cytokine, an antibody or fragment thereof, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody and a single chain variable domain. In some embodiments, each targeting moiety is independently a nanobody. In some embodiments, at least one targeting moiety is a nanobody and at least one targeting moiety is a cytokine. In some embodiments, the cytokine is IL7.

[0068] In some embodiments, the, or each, targeting moiety is capable of binding to T cells, B cells, NK cells, monocytes, macrophages, mast cells, basophils, eosinophils or dendritic cells. In some embodiments, the, or each, targeting moiety is capable of binding to T cells. In some embodiments, the, or each, targeting moiety is capable of activating T cells.

[0069] In some embodiments, the, or each, targeting moiety is independently selected from the list consisting of a: CD3 binding domain, CD2 binding domain, CD7 binding domain, CD4 binding domain CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain, CD5 binding domain, CD19 binding domain, CD20 binding domain, IgM binding domain, IgD binding domain. In some embodiments, at least one targeting moiety is a CD3 binding domain.

[0070] In some embodiments, the first targeting moiety is a CD3 binding domain, and the second targeting moiety is capable of binding to T cells, and optionally is selected from a CD2 binding domain, CD7 binding domain, CD4 binding domain, CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain, CD5 binding domain.

[0071] In some embodiments, the first targeting moiety is a CD3 binding domain, and the second or each further targeting moiety is independently selected from the list consisting of a: CD2 binding domain, CD7 binding domain, CD4 binding domain, CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain, and CD5 binding domain.

[0072] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises IL7. In some embodiments, the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain.

[0073] In some embodiments, the first target and second target are both expressed on the surface of CD4 T cells. In some embodiments, the first target and second target are both expressed on the surface of CD8 T cells.

[0074] In some embodiments, the ratio of first targeting moiety to second targeting moiety is in the range of about 10:1 to about 1:10.

[0075] In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of about 9:1 , about 7:3, about 1:1, about 3:7; or about 1 :9. In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of 9:1, 7:3, 1:1, 3:7; or 1:9. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 9:1. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety may be about 1:1:1. In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety is in the range of about 3 to 8:1 to 5: 1 to 5.

[0076] In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety may be about 6: 1:3, about 6:3:1, about 4:3:3 or about 1 :1 :1.

[0077] Suitably, the ratio of first targeting moiety to second targeting moiety to third targeting moiety may be about 6:1 :3. In some embodiments, the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain.

[0078] In some embodiments, the one or more nucleic acid molecules comprise RNA. In some embodiments, the one or more nucleic acid molecules comprise DNA. In some embodiments, the one or more nucleic acid molecules comprise RNA and DNA. In some embodiments, the RNA is mRNA.

[0079] The present invention also provides a nucleic acid particle comprising:

[0080] (i) one or more particle forming components;

[0081] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0082] (ii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different; wherein at least one targeting moiety is a nanobody.

[0083] The present invention also provides a nucleic acid particle comprising:

[0084] (i) one or more particle forming components;

[0085] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0086] (ii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different; wherein the ratio of first targeting moiety to second targeting moiety is 9:1, 7:3, 1:1 , 3:7; or The present invention also provides a composition comprising two or more nucleic acid particles, wherein each nucleic acid particle comprises:

[0087] (i) one or more particle forming components;

[0088] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0089] (iii) a targeting moiety, wherein the targeting moiety of a first nucleic acid particle is capable of binding to a first target and the targeting moiety of a second nucleic acid particle is capable of binding to a second target, wherein the first and second targets are different.

[0090] The present invention also provides a pharmaceutical composition comprising (i) the nucleic acid particle according to the invention or the composition according to the invention; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0091] The present invention also provides a method for treating a disease which comprises the step of administering the nucleic acid particle according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention to a subject in need thereof.

[0092] The present invention also provides use of the nucleic acid particle according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, in the manufacture of a medicament for treating a disease.

[0093] The present invention also provides the nucleic acid particle according to the invention, the composition according to the invention, or the pharmaceutical composition according to the invention, for use as a medicament in the treatment of a disease.

[0094] In some embodiments, the disease is an autoimmune disease or a cancer.

[0095] In some embodiments, the cancer is an immuno-oncology B cell malignancy or an immuno oncology solid cancer.

[0096] BRIEF DESCRIPTION OF THE FIGURES

[0097] Figure 1 : CD3 and CD7 overview of flow cytometric transfection data of LNPs at day 1.

[0098] In a) and b), the Thy1.1 mRNA expression percentage of unfunctionalized, single- functionalized (either aCD3-LNPs or aCD7-LNPs), and dual-functionalized LNPs with aCD3:aCD7 ratios of 9:1 , 7:3, 1 :1 , 3:7, and 1 :9, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in c) and d), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. In e) and f), the early activation of T cells by the MFI of CD69 for SM102-LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. The cell counts of viable CD4+, CD8+, CD14+ and CD19+ cells for all SM102-LNP and BHD-C2C2-PipZ LNP samples is demonstrated in g) and h), respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum. The BM abbreviation refers to a benchmark SM-102 LNP formulation differing in molar percentage composition with same lipids and cargo. WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0099] Figure 2: CD3 and CD7 overview of flow cytometric transfection data of LNPs at day 4.

[0100] In a) and b), the Thy1.1 mRNA expression percentage of unfunctionalized, singlefunctionalized (either aCD3-LNPs or aCD7-LNPs), and dual-functionalized LNPs with aCD3:aCD7 ratios of 9:1 , 7:3, 1 :1 , 3:7, and 1 :9, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in c) and d), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. In e) and f), the npVenus DNA expression percentage (left y axis) and the cell counts (right y axis) of unfunctionalized, single-functionalized (either aCD3-LNPs or aCD7-LNPs), and dualfunctionalized LNPs with aCD3:aCD7 ratios of 9:1 , 7:3, 1 :1 , 3:7, and 1 :9, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in g) and h), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA). The BM abbreviation refers to a benchmark SM-102 LNP formulation differing in molar percentage composition with same lipids and cargo. WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0101] Figure 3: CD3 and CD7 overview of T cell activation and cell counts of LNPs at day 4. In a) and b), the late activation of T cells by the MFI of CD25 for SM102-LNPs and BHD-C2C2- PipZ LNPs, respectively, is shown for unfunctionalized, single-functionalized (either aCD3- LNPs or aCD7-LNPs), and dual-functionalized LNPs with aCD3:aCD7 ratios of 9:1 , 7:3, 1 :1 , 3:7, and 1 :9. The cell counts of viable CD4+, CD8+, CD14+ and CD19+ cells for all SM102- LNP and BHD-C2C2-PipZ LNP samples is demonstrated in c) and d), respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA). The BM abbreviation refers to a benchmark SM-102 LNP formulation differing in molar percentage composition with same lipids and cargo. WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0102] Figure 4: Physicochemical properties of CD3 and CD7 LNPs. In a) and b), the DLS measurements of size (left y axis) and PDI (right y axis) of SM-102 LNPs and BHD-C2C2- PipZ LNPs, respectively, are shown. Visualized are unfunctionalized LNPs (LNP-ALFA), the single functionalized LNPs (either aCD3 or aCD7), and the dual-functionalized LNPs with aCD3:aCD7 ratios of 9:1 , 7:3, 1 :1 , 3:7, and 1 :9. Multimodal PDI values are set to 1.0. The detection of free cargo for all samples by agarose gel electrophoresis using GelRed is depicted in c).

[0103] Figure 5: CD3 and CD2 overview of flow cytometric transfection data of LNPs at day 1.

[0104] In a) and b), the Thy1.1 mRNA expression percentage of unfunctionalized, singlefunctionalized (either aCD3-LNPs or aCD2-LNPs), and dual-functionalized LNPs with aCD3:aCD2 ratios of 9:1 , and 7:3, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in c) and d), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. The cell counts of viable CD4+, CD8+, CD14+ and CD19+ cells for all SM102-LNP and BHD-C2C2-PipZ LNP samples is demonstrated in e) and f), respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum. WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0105] Figure 6: CD3 and CD2 overview of flow cytometric transfection data of LNPs at day 4.

[0106] In a) and b), the Thy1.1 mRNA expression percentage of unfunctionalized, singlefunctionalized (either aCD3-LNPs or aCD2-LNPs), and dual-functionalized LNPs with aCD3:aCD2 ratios of 9:1 and 7:3, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in c) and d), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. In e) and f), the npVenus DNA expression percentage (left y axis) and the cell counts (right y axis) of unfunctionalized, single-functionalized (either aCD3-LNPs or aCD2-LNPs), and dual-functionalized LNPs with aCD3:aCD2 ratios of 9:1 and 7:3, of SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively, is shown. Moreover, the mean fluorescence intensity (MFI) for each sample is plotted in g) and h), for SM-102 LNPs and BHD-C2C2-PipZ LNPs, respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA). The BM abbreviation refers to a benchmark SM-102 LNP formulation differing in molar percentage composition with same lipids and caro. WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0107] Figure 7: CD3 and CD2 overview of T cell activation and cell counts of LNPs at day 4 In a) and b), the late activation of T cells by the MFI of CD25 for SM102-LNPs and BHD-C2C2- PipZ LNPs, respectively, is shown for unfunctionalized, single-functionalized (either aCD3- LNPs or aCD2-LNPs), and dual-functionalized LNPs with aCD3:aCD2 ratios of 9:1 and 7:3. The cell counts of viable CD4+, CD8+, CD14+ and CD19+ cells for all SM102-LNP and BHD- C2C2-PipZ LNP samples is demonstrated in c) and d), respectively. All data show the results of one hPBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA). WO = without, negative control with only hPBMCs (human peripheral blood mononuclear cells).

[0108] Figure 8: Physicochemical properties of CD3 and CD2 LNPs. In a) and b), the DLS measurements of size (left y axis) and PDI (right y axis) of SM-102 LNPs and BHD-C2C2- PipZ LNPs, respectively, are shown. Visualized are unfunctionalized LNPs (LNP-ALFA), the single functionalized LNPs (either aCD3 or aCD2), and the dual-functionalized LNPs with aCD3:aCD2 ratios of 9:1 and 7:3. Multimodal PDI values are set to 1.0. The detection of free cargo for all samples by agarose gel electrophoresis using GelRed is depicted in c).

[0109] Figure 9: Overview of flow cytometric transfection data of aCD3 and aCD2 dualfunctionalized mRNA LNPs at day 1. In a) the Thy1.1 mRNA expression percentage of single-functionalized (either aCD3-LNPs or aCD2-LNPs) and tandem-functionalized LNPs (tandem 1 - 4) is shown. Moreover, the geometric mean fluorescence intensity (gMFI) for each sample is plotted in b). In c) the early activation of T cells by the MFI of CD69 is shown. The cell counts of viable CD4+, CD8+, CD14+ and CD19+ cells are demonstrated in d). All data show the results of one human PBMC donor with an LNP treatment of 250 ng mRNA per 0.3E06 PBMCs in 50% pooled, clotted human serum.

[0110] Figure 10: Physicochemical properties of CD3 and CD2 dual-functionalized mRNA LNPs. In a), the DLS measurements of size (left y axis) and PDI (right y axis) of unfunctionalized (LNP-ALFA), single-functionalized (either aCD3-LNPs or aCD2-LNPs), and dual-functionalized (aCD3-aCD2-LNPs). BHD-C2C2-PipZ mRNA LNPs are shown.

[0111] Figure 11 : CD3-CD2 tandem overview of flow cytometric transfection data of LNPs at day 4. In a), the npVenus DNA expression percentage (left y axis) and the cell counts (right y axis) of single-functionalized (either aCD3-LNPs or aCD2-LNPs) and tandem-functionalized LNPs (tandem 1 - 4) is shown. Also, the MFI of the npVenus DNA expression for each sample is plotted in b). In c), the late activation of T cells by the MFI of CD25 is shown for CD4+ and CD8+ T cells. All data show the results of one human PBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA).

[0112] Figure 12: CD3-CD7 tandem overview of flow cytometric transfection data of LNPs at day 4. In a) the npVenus DNA expression percentage (left y axis) and the cell counts (right y axis) of single-functionalized (either aCD3-LNPs or aCD7-LNPs) and tandem-functionalized LNPs (tandem 5 - 7) is shown. Also, the gMFI of the npVenus DNA expression for each sample is plotted in b). In c), the late activation of T cells by the gMFI of CD25 is shown for CD4+ and CD8+ T cells. All data show the results of one human PBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA).

[0113] Figure 13: Overview of flow cytometric transfection data of triplet-functionalized LNPs at day 4. In a) the npVenus DNA expression percentage (left y axis) and the cell counts (right y axis) of unfunctionalized (LNP-ALFA), single-functionalized (aCD3-LNPs, aCD2-LNPs, aCD7-LNPs), and triplet-functionalized LNPs (aCD3-aCD2-aCD7-LNPs) are shown. The gMFI of the npVenus DNA expression for each sample is plotted in b). In c), the late activation of T cells is shown for CD4+ and CD8+ T cells using the gMFI of CD25. All data show the results of one representative human PBMC donor with an LNP treatment of 500 ng in 50% pooled, clotted human serum (100 ng Thy1.1 mRNA and 200 ng Venus DNA)

[0114] Figure 14: Targeted gene integration in T cells using LNP-formulated CRISPR-Cas9 or Zinc-Finger Nuclease (ZFN)-mediated gene editing, with nanoplasmid DNA templates.

[0115] 1 pg of LNPs functionalised using aCD3 and aCD7 docking compounds (in a molar ratio 7:3) and containing (i) Cas9-encoding mRNA and single guide RNA (sgRNA) targeting the T-cell receptor a constant (TRAC) locus or (ii) a pair of ZFN mRNAs targeting the TRAC locus, each with a nanoplasmid DNA template encoding Venus transgene were used to transfect 1 x 106 human T cells. In a) knock-out efficiency was assessed by downregulation of CD3 expression and in a)-c) knock-in efficiency was assessed by Venus expression.

[0116] DETAILED DESCRIPTION OF THE INVENTION

[0117] 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.

[0118] 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; Rdmpp 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.

[0119] 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.

[0120] 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.

[0121] DEFINITIONS

[0122] 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.

[0123] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0124] 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".

[0125] 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.

[0126] 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.

[0127] 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.

[0128] "Physiological pH" as used herein refers to a pH of about 7.5 or about 7.4. In some embodiments, physiological pH is from 7.3 to 7.5. In some embodiments, physiological pH is from 7.35 to 7.45. In some embodiments, physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0129] "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.

[0130] 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.

[0131] As used in the present disclosure, "mol % of the lipid mixture" is defined as the ratio of the number of moles of that particular lipid component to the total number of moles of all lipids in the lipid mixture, multiplied by 100. In this context, in some embodiments, the term "total lipid" and / or “total lipid mixture” includes lipids and lipid-like material. 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 C6-40 alkyl, C6-30 alkyl, C6-20 alkyl, or C10-20 alkyl), C2-40 alkenyl (such as C6-40 alkenyl, C6-30 alkenyl, or C6-20 alkenyl) having 1 , 2, or 3 double bonds, aryl, and aryl(C1-6 alkyl). In some embodiments, the hydrocarbyl group is optionally substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0132] 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 some embodiments, 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.

[0133] 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., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.

[0134] The term "alkyl" refers to a monoradical of a saturated straight or branched hydrocarbon. Preferably, the alkyl group comprises from 1 to 40, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkyl groups include methyl, ethyl, propyl, iso-propyl (also called 2- propyl or 1 methylethyl), butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, sec-pentyl, neo-pentyl, 1 ,2-dimethylpropyl, iso-amyl, n-hexyl, iso-hexyl, sec-hexyl, n-heptyl, iso-heptyl, n-octyl, 2- ethyl-hexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-undecyl, n-dodecyl, n-tridecyl, n- tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl, n- triacontyl, n-tetracontyl, and the like. A "substituted alkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the alkyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, 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.

[0135] The term "alkylene" refers to a diradical of a saturated straight or branched hydrocarbon. Preferably, the alkylene group comprises from 1 to 40, i.e., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40, carbon atoms, such as 1 to 30, such as 1 to 20 carbon atoms, such as 1 to 12 carbon atoms, such as 1 to 10 carbon atoms, such as 1 to 8 carbon atoms, such as 1 to 6 or 1 to 4 carbon atoms. Exemplary alkylene groups include methylene, ethylene (i.e., 1 ,1- ethylene, 1 ,2-ethylene), propylene (i.e., 1 ,1 -propylene, 1 ,2-propylene (-CH(CH3)CH2-), 2,2- propylene (-C(CH3)2-), and 1 ,3-propylene), the butylene isomers (e.g., 1 ,1-butylene, 1 ,2- butylene, 2,2-butylene, 1 ,3-butylene, 2,3-butylene (cis or trans or a mixture thereof), 1 ,4- butylene, 1 ,1 -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-1 ,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 some embodiments, 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.

[0136] 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 carboncarbon 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), i.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) carbon-carbon 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 (i.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 some embodiments, 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.

[0137] 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), i.e., 1 , 2, 3, 4, 5, or 6, carbon-carbon double bonds. Preferably, the alkenylene group comprises from 2 to 12 (such as 2 to 10) carbon atoms, i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 carbon atoms (such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 2 to 8 carbon atoms, such as 2 to 6 carbon atoms or 2 to 4 carbon atoms. Thus, in a preferred embodiment, the alkenylene group comprises from 2 to 12 (such as 2 to 10 carbon) atoms and 1 , 2, 3, 4, 5, or 6 (such as 1 , 2, 3, 4, or 5) carbon-carbon double bonds, more preferably 5 it comprises 2 to 8 carbon atoms and 1 , 2, 3, or 4 carbon-carbon double bonds, such as 2 to 6 carbon atoms and 1 , 2, or 3 carbon-carbon double bonds or 2 to 4 carbon atoms and 1 or 2 carbon-carbon double bonds. The carbon-carbon double bond(s) may be in cis (Z) or trans (E) configuration. Exemplary alkenylene groups include ethen-1 ,2-diyl, vinylidene (also called ethenylidene), 1 -propen-1 ,2-diyl, 1 -propen-1 ,3-diyl, 1-propen-2,3-diyl, allylidene, 1-buten-1 ,2- diyl, 1-buten-1 ,3-diyl, 1-buten-1 ,4-diyl, 1 -buten-2,3-diyl, 1 -buten-2,4-diyl, 1 -buten-3,4-diyl, 2- buten-1 ,2-diyl, 2-buten-1 ,3-diyl, 2-buten-1 ,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 some embodiments, 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 some embodiments, 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.

[0138] 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-1 ,2-diyl, 1-propyn- 1 ,2-diyl, 1 -propyn-1 ,3-diyl. In some embodiments, 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.

[0139] 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 some embodiments, 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.

[0140] The terms "cycloalkylene" and “cycloalkenylene” represents cyclic non-aromatic versions of "alkylene" and "alkenylene" with preferably 3 to 40, such as 3 to 30, such as 3 to 20, such as 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms. Exemplary cycloalkylene groups include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cycloheptylene. Exemplary cycloalkylenene groups include cyclopentenylene and cyclohexenylene.

[0141] 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 some embodiments, 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 (i.e., 2-, 3-, or 4-methoxyphenyl).

[0142] The term “heteroaliphatic” or “heteroaliphatic group”, as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic (“heterocyclic”) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term “nitrogen” also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1- 2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2-O-CH2-CH2-O-CH3, and the like.

[0143] 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, 1 H-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 some embodiments, 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.

[0144] The term "heterocyclyl" or "heterocyclic ring" means a cycloalkyl group as defined above in which from 1 , 2, 3, or 4 carbon atoms in the cycloalkyl group are replaced by heteroatoms of oxygen, nitrogen, silicon, selenium, phosphorus, or sulfur, preferably O, S, or N. A heterocyclyl group has preferably 1 or 2 rings containing from 3 to 10, such as 3, 4, 5, 6, or 7, ring atoms. Preferably, in each ring of the heterocyclyl group the maximum number of O atoms is 1 , the 5 maximum number of S atoms is 1 , and the maximum total number of O and S atoms is 2. The term "heterocyclyl" is also meant to encompass partially or completely hydrogenated forms (such as dihydro, tetrahydro or perhydro forms) of the above-mentioned heteroaryl groups. Exemplary heterocyclyl groups include morpholinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl (also called piperidyl), piperazinyl, di- and tetrahydrofuranyl, di- and tetrahydrothienyl, di- and tetrahydropyranyl, urotropinyl, lactones, lactams, cyclic imides, and cyclic anhydrides. A "substituted heterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of the heterocyclyl group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the heterocyclyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0145] The term “alkylcycloalkyl” means a cycloalkyl group, as defined above, which is substituted with an alkyl group, as defined above, the cycloalkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the alkylcycloalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0146] The term “cycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted cycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a cycloalkylalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the cycloalkylalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0147] The term “alkylcycloalkylalkyl” means an alkyl group, as defined above, which is substituted with a cycloalkyl group, as defined above, the alkyl portion being connected to the rest of the molecule and the cycloalkyl portion in turn being substituted with a further alkyl group. Each of the cycloalkyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylcycloalkylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a alkylcycloalkylalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or cycloalkyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the alkylcycloalkylalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0148] The term “alkylaryl” means an aryl group, as defined above, which is substituted with an alkyl group, as defined above, the aryl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylaryl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the alkylaryl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0149] The term “arylalkyl” means an alkyl group, as defined above, which is substituted with an aryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted arylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a arylalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or aryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the arylalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0150] The term “alkylheteroaryl” means a heteroaryl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heteroaryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheteroaryl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheteroaryl group, e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the alkylheteroaryl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0151] The term “heteroarylalkyl” means an alkyl group, as defined above, which is substituted with a heteroaryl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the aryl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heteroarylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heteroarylalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the heteroarylalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0152] The term “alkylheterocyclyl” means a heterocyclyl group, as defined above, which is substituted with an alkyl group, as defined above, the heteroaryl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted alkylheterocyclyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to an alkylheterocyclyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heteroaryl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the alkylheterocyclyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0153] The term “heterocyclylalkyl” means an alkyl group, as defined above, which is substituted with a heterocyclyl group, as defined above, the alkyl portion being connected to the rest of the molecule. Each of the heterocyclyl and alkyl portions of the group may take any of the broadest or preferred meanings recited above. A "substituted heterocyclylalkyl" means that one or more (such as 1 to the maximum number of hydrogen atoms bound to a heterocyclylalkyl group, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or up to 10, such as between 1 to 5, 1 to 4, or 1 to 3, or 1 or 2) hydrogen atoms of either the alkyl or heterocyclyl portions of the group are replaced with a substituent other than hydrogen (when more than one hydrogen atom is replaced the substituents may be the same or different). In some embodiments, the heterocyclylalkyl is substituted with one or more, such as 1 , 2 or 3, such as 1 or 2, such as 1 substituents selected from List A.

[0154] The term “organosulfuric acid” or “sulfate” means a compound of formula R-OSO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfate” is used when the group is deprotonated. Depending on the pH, the sulfate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonic acid group is typically deprotonated at physiological pH).

[0155] The term “sulfonic acid” or “sulfonate” means a compound of formula R-SO2-OH, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). The term “sulfonate” is used when the group is deprotonated. Depending on the pH, the sulfonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the sulfonate group is typically deprotonated at physiological pH).

[0156] 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).

[0157] The term “dicarboxylic acid” or “dicarboxylate” means a compound of formula HO2C-R’- CO2H, wherein R’ is alkylene or alkenylene group (all as defined above, either in a broadest aspect or a preferred aspect). The term “dicarboxylate” is used when the group is deprotonated. Depending on the pH, the dicarboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the dicarboxylic acid group is typically protonated at acidic or neutral pH and deprotonated at alkaline pH).

[0158] The term “hydroxy carboxylic acid” or “hydroxy carboxylate” means a compound of formula R- CO2H, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkyl heteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), which is substituted by one or more (preferably 1 to 5, such as 1 , 2 or 3) hydroxy groups. The term “hydroxy carboxylate” is used when the group is deprotonated. Depending on the pH, the hydroxy carboxylic acid may be protonated or deprotonated (in the anionic amphiphiles as defined below, the carboxylic acid group is typically protonated at acidic pH and deprotonated at neutral or alkaline pH).

[0159] 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, alkyl heteroaryl, 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 some embodiments, 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).

[0160] The term "thioester" as used herein means a bivalent linkage of the structure

[0161] -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.

[0162] The term “phosphate” means a compound of formula RO-P(=O)(OH)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphate group is typically deprotonated at physiological pH).

[0163] The term “phosphonate” means a compound of formula R-P(=O)(OH)2, wherein R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect). Depending on the pH, the phosphonate group may be protonated or deprotonated (in the anionic amphiphiles as defined below, the phosphonate group is typically deprotonated at physiological pH).

[0164] “Halo” means fluoro (-F), chloro (-CI), bromo (-Br) or iodo (-1).

[0165] “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.

[0166] A “quaternary ammonium” salt is a compound containing a group -N+R3, wherein each R is a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group. In contrast to some amines as defined above which are protonated only at certain pH, a quaternary ammonium salt carries a constitutive positive charge (as defined herein) at all pH.

[0167] “Hydroxyl” - means the group -OH. “Sulfhydryl” - means the group -SH. “Nitro” means the group -NO2.

[0168] “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, alkyl heteroaryl, 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 C1-10 alkyl group.

[0169] “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.

[0170] “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, alkyl heteroaryl, 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.

[0171] “Thioamide” means a bivalent linkage of the formula -C(=S)NR-, wherein R is hydrogen or a C1-6 alkyl group, wherein one moiety is connected to the rest of the molecule via the carbon atom and the other via the nitrogen atom.

[0172] “Hydroxylamide” means the group -C(=O)O-NR(R’), wherein R and R’ are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect).

[0173] “Sulfonyl” means the group -S(=O)2R, wherein R is a C1-13 alkyl or C2-3 alkenyl group (all as defined above, either in a broadest aspect or a preferred aspect), or a bivalent linkage of formula -S(=O)2- where each moiety is connected to the rest of the molecule via the sulfur atom. “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.

[0174] “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.

[0175] “Amidine” means the group -C(=NR)NR’R” wherein R, R’ and R” are each independently hydrogen or a hydrocarbyl or heterohydrocarbyl group, such as an alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, alkylaryl, arylalkyl, alkylarylalkyl, alkylheteroaryl, heteroarylalkyl, alkylheterocyclyl, or heterocyclylalkyl group (all as defined above, either in a broadest aspect or a preferred aspect), and is preferably an alkyl group, such as a C1-6 alkyl group.

[0176] “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, alkyl heteroaryl, 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.

[0177] 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. “Carbohydrate” means a compound having the empirical formula Cm(H20)n where m may or may not be different from n. The term “carbohydrate residue” or “carbohydrate moiety” defines a residue attached to another atom, where one hydrogen atom of the carbohydrate is replaced by a bond attached to the rest of the molecule. The carbohydrate moiety may be a monosaccharide moiety. The monosaccharide moiety may have the D- or L-configuration. Furthermore, the monosaccharide moiety may be an aldose or ketose moiety. Suitably, the monosaccharide moiety may have 3 to 8, preferably 4 to 6, more preferably 5 or 6, carbon atoms. In some embodiments, the monosaccharide moiety is a hexose moiety (i.e. it has 6 carbon atoms), examples of which include aldohexoses such as glucose, galactose, allose, altrose, mannose, gulose, idose and talose, and ketohexoses such as fructose and sorbose. Preferably, the hexose moiety is a glucose moiety.

[0178] In another embodiment, the monosaccharide moiety is a pentose moiety (i.e. it has 5 carbon atoms), such as ribose, arabinose, xylose or lyxose. Preferably, the pentose moiety is an arabinose or xylose moiety.

[0179] In another embodiment, the carbohydrate may be a higher saccharide (i.e. a di-, or oligosaccharide) comprising more than one monosaccharide moiety joined together by glycoside bonds. When the monosaccharide moieties are hexose moieties, the glycoside bonds may be 1-a,1'-a glycoside bonds, 1 ,2'-glycoside bonds (which maybe 1-a2’ or T-p-2' glycoside bonds), 1 ,3'-glycoside bonds (which may be 1-a-3' or 1-p-3'-glycoside bonds), 1 ,4'- glycoside bonds (which may be 1-a-4' or 1-p-4'-glycoside bonds), 1 ,6'-glycoside bonds (which may be 1-a-6' or 1-p-6'-glycoside bonds), or any combination thereof. In some embodiments, the higher saccharide comprises 2 monosaccharide units (i.e. is a disaccharide). Examples of suitable disaccharides include maltose, isomaltose, isomaltulose, lactose, sucrose, cellobiose, nigerose, kojibiose, trehalose and trehalulose. In another embodiment, the higher saccharide comprises 3 to 10 monosaccharide units (i.e. is an oligosaccharide) in a chain, which may be branched or unbranched. Preferably, the oligosaccharide comprises 3 to 8, more preferably 3 to 6, monosaccharide units. Examples of suitable oligosaccharides include maltodextrin, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, melezitose, cellotriose, cellotetraose, cellopentaose, cellohexaose and celloheptaose.

[0180] “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’, -N(R’)2, -S(O)0-2R’, -S(O)1-2OR’,

[0181] -OS(O)1-2R’, -OS(O)1-2OR’, -S(O)1-2N(R’)2, -OS(O)1-2N(R’)2, -N(R’)S(O)1-2R’, -N(R’)S(O)1-2OR’, -C(=X1)R’, -C(=X1)X1 R’, -X1C(=X1)R’, and -X1C(=X1)X1 R’, wherein X1 is independently selected from O, S, NH and N(CH3); and each R’ is independently selected from the group consisting of H, C1-4 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(C1-3 alkyl), -S(C1-3 alkyl), -NH2, -NH(C1-3 alkyl), -N(C1-3 alkyl)2, -NHS(O)2(C1-3 alkyl), - S(O)2NH2-z(C1-3 alkyl)z, -C(=O)OH, -C(=O)O(C1-3 alkyl), -C(=O)NH2-z(C1-3 alkyl)z, - NHC(=O)(C1-3 alkyl), -NHC(=NH)NHz-2(C1-3 alkyl)z, and -N(C1-3 alkyl)C(=NH)NH2-z(C1-3 alkyl)z, wherein each z is independently 0, 1 , or 2 and each C1-3 alkyl is independently methyl, ethyl, A1 , consisting of C1-3 alkyl, phenyl, halogen, -CF3, -OH, -OCH3, -SCH3, -NH2- z(CH3)z, -C(=O)OH, and -C(=O)OCH3, wherein z is 0, 1 , or 2 and C1-3 alkyl is methyl, ethyl, propyl or isopropyl. In some embodiments, List A substituents are selected from List A2, consisting of methyl, ethyl, propyl, isopropyl, halogen (such as F, Cl, or Br), and -CF3.

[0182] 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’.

[0183] NUCLEIC ACID PARTICLE

[0184] The present invention relates to particles that provide targeted delivery of a nucleic acid payload to target cells.

[0185] Lipid particles, such as lipid nanoparticles (LNPs), have demonstrated huge potential as delivery technology for therapeutic payloads (e.g. nucleic acid molecules) for treating a wide range of conditions. Polymer particles, such as polyplexes (PLXs), are also known to have potential as delivery technology for therapeutic payloads such as nucleic acids. In order to improve the targeting of particles and the delivery of payloads to specific target cells, ligands (e.g. antibodies or nanobodies) for specific target molecules (e.g. cell-specific receptors) can be provided on the surface of the particles. As used herein, such particles may be termed as “functionalized particles”, “functionalized lipid particles”, “functionalized nucleic acid particles” or “functionalized nucleic acid-lipid particles”.

[0186] Such particles may comprise, bind to or interact with, a compound comprising a targeting moiety (also referred to herein as a “targeting ligand”) that binds a target on target cells. Thus, a “functionalized nucleic acid 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 targeting moiety of the particle.

[0187] Usually, a receptor-specific ligand is chosen based on its affinity towards a specific cell type, e.g., selecting CD3 receptors that are found on the surface of T cells for modulating the immune system. Thus, immobilizing ligands on the surface of a particle may improve cellspecific targeting and internalization through receptor-mediated endocytosis followed by endosomal escape and delivery of the cargo into the cytosol.

[0188] However, this single-ligand functionalized particle approach often lacks a desirable cell activation and expansion, and, in the case of nucleic acid payloads, transfection of DNA.

[0189] The present inventors have surprisingly found that targeting of particles and delivery of payloads can be improved by utilising two or more targeting moieties, in particular where at least one targeting moiety is non-covalently bound to the particle.

[0190] Moreover, the present inventors have surprisingly found that such particles may be useful for targeting and activating T cells, such as CD8+ T cells.

[0191] Indeed, the use of multifunctionalized LNPs may enhance targeting of T cells by inducing both activation and proliferation, as well as improved delivery of the nucleic acid payload.

[0192] In one aspect, the present invention provides a nucleic acid particle comprising:

[0193] (i) one or more particle forming components;

[0194] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0195] In particular, the present invention provides a nucleic acid particle comprising:

[0196] (i) one or more particle forming components;

[0197] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0198] (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different; wherein at least one targeting moiety is non-covalently bound to the nucleic acid particle.

[0199] In some embodiments, the nucleic acid particle is a nucleic acid-lipid particle (such as an LNP, LPX or LPLX). In some embodiments, the nucleic acid particle is a nucleic acid-polymer particle (such as a PLX or a LPLX). In some embodiments, all of the targeting moieties are non-covalently bound to the nucleic acid particle.

[0200] In some embodiments, at least one targeting moiety is non-covalently bound to the nucleic acid particle and at least one targeting moiety is covalently bound to the nucleic acid particle. Such covalent binding of a targeting moiety to a particle may be known in the art, for example from WO2024 / 077232.

[0201] It will be understood that the terms “particle” may encompass the terms “nucleic acid particle”, “nucleic acid-lipid particle”, “lipid particle”, “functionalised particle”, “functionalised nucleic acid particle”, and “functionalised nucleic acid-lipid particle” as used herein.

[0202] Suitably, when the nucleic acid is RNA, such particles are also referred to herein as “RNA particles”. Suitably, when the nucleic acid is DNA, such particles are also referred to herein as “DNA particles”. Suitably, when the nucleic acid is a mixture of DNA and RNA, such particles are also referred to herein as “DNA-RNA particles” or “RNA-DNA particles”.

[0203] The nucleic acid particles enable the nucleic acid payload to be administered by functioning as delivery vehicles that protect the payload from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells. Indeed, in use the particles act to stabilise and encapsulate the payload to enable it to be delivered into a cell while facilitating its uptake into the cell and release into the cytosol.

[0204] Such delivery vehicles may complex or encapsulate the payload and include a range of materials, including polymers, lipids and mixtures thereof. In some embodiments, such delivery vehicles may form particles with the payload. The nucleic acid-lipid particle may be a functionalized LNP or a functionalized LPX. The nucleic acid particle may be a functionalized polyplex (PLX). The nucleic acid particle may be a functionalized lipidated-polyplex (LPLX).

[0205] The terms “interacts with” and “binds to” may be used interchangeably in this context.

[0206] 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 components. 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.

[0207] 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. Polydispersity index (PDI) may be calculated from the cumulant analysis using Dynamics 7.8.1.3 software.

[0208] A nucleic acid 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 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 colloidally stable particles.

[0209] The term "nanoparticle" relates to a nano-sized particle comprising at least one particle forming component, 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, i.e., at least about 1 nm and below about 1000 nm. Preferably, the size of a particle is its diameter.

[0210] In some embodiments, the particles described herein have a size (such as an average 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 10 to 1000 nm, 15 to 500 nm, 20 to 450 nm, 25 to 400 nm, 30 to 350 nm, 40 to 300 nm, 50 to 250 nm, 60 to 200 nm, 70 to 150 nm, or 80 to 150 nm. In some embodiments, the particles described herein have a size (such as an average diameter) in the range of from about 40 nm to about 300 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.

[0211] In some embodiments, nucleic acid may be noncovalently associated with a particle. In some embodiments, the nucleic acid may be contained in the particle (encapsulated nucleic acid).

[0212] The N / P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the nucleic acid. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged. The N / P ratio, where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N / P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, nucleic acid is considered to be completely bound to nanoparticles.

[0213] 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.

[0214] Lipid Nanoparticle

[0215] In some embodiments, the nucleic acid-lipid particle is a lipid nanoparticle (LNP). In some embodiments, the one or more particle forming components in LNPs comprises a cationic or cationically ionizable lipid, as defined and exemplified herein. In some embodiments, the one or more particle forming components in LNPs comprises (i) a cationic or cationically ionizable lipid, (ii) a neutral lipid such as a phospholipid, (iii) a grafted lipid, and (iv) a steroid such as cholesterol, as defined and exemplified herein.

[0216] 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 may comprise a central complex of lipid and a nucleic acid payload, (e.g., RNA (such as 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 nucleic acid-lipid particles described herein are not liposomes. In some instances, the nucleic acid-lipid particles described herein are not lipoplexes.

[0217] Lipid nanoparticles (LNP) are obtainable from combining a payload, such as 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.

[0218] 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 40 nm (e.g., at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, or at least about 100 nm) and / or at most about 1000 nm (e.g., at most about 900 nm, at most about 800 nm, at most about 700 nm, at most about 600 nm, or at most about 500 nm), such as 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 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. In some embodiments, the lipid nanoparticles described herein have an average size (such as a diameter) that in some embodiments ranges from about 60 to about 250 nm. In some embodiments, the lipid nanoparticles described herein have an average size (such as a diameter) that in some embodiments ranges from about 80 to about 180 nm.

[0219] Lipoplexes

[0220] In some embodiments, the nucleic acid-lipid particle of the present disclosure is a lipoplex (LPX). In such particles, the one or more particle forming components comprises a cationic or cationically ionizable lipid. In LPX particles, the one or more particle forming components may comprise (i) a cationic lipid, and (ii) a neutral lipid, such as a phospholipid. Lipoplexes (LPX) are electrostatic complexes which are generally formed by mixing preformed particles containing cationic or cationically ionizable lipid with an anionic payload (such as a nucleic acid). Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact nucleic acid lipoplexes.

[0221] Polyplexes

[0222] In some embodiments, the nucleic acid particle of the present disclosure is a polyplex (PLX). In such particles, the one or more particle forming components comprises a cationic polymer, as defined and exemplified below. Typically, such a cationic polymer is capable of electrostatically condensing a negatively charged payload (such as a nucleic acid) into particles. Polyplexes can also contain anionic polymers. Polyplexes can also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.) as described below.

[0223] Lipidated Polyplexes

[0224] In some embodiments, the nucleic acid particle of the present disclosure is a lipidated polyplex (LPLX). In such particles, the one or more particle forming components comprises a cationic polymer, as defined and exemplified below. Typically, the cationic polymer used for LPLX formation comprises a hydrophobic portion to facilitate interaction with the lipid component of the LPLX (e.g., Viromers), as further described below. LPLX can also contain neutral (e.g., hydrophilic) polymers, such as stealth polymers (e.g., PEG, pSar, pAEEA, etc.), as described below. Lipidated polyplexes also contain lipids, as defined and exemplified below.

[0225] “TARGETING COMPOUND” - COMPOUND INTEGRATED INTO THE PARTICLE

[0226] As described herein, at least one targeting moiety of the nucleic acid particle according to the invention is non-covalently bound to the nucleic acid particle.

[0227] It will be understood that at least one targeting moiety may be non-covalently bound to a molecule comprising a portion capable of inserting / integrating into the nucleic acid particle. For example, the functionalized nucleic acid particles described herein may comprise (i) a molecule comprising the targeting moiety, and (ii) a molecule comprising a portion capable of inserting / integrating into the nucleic acid particle. In such cases, the interaction or binding between the molecule comprising targeting moiety and the molecule comprising a portion capable of inserting / integrating into the nucleic acid particle is non-covalent (e.g., an affinitybased interaction). For example, the interaction or binding between the molecule comprising targeting moiety and the molecule comprising a portion capable of inserting / integrating into the nucleic acid particle may have a micromolar, nanomolar or picomolar binding affinity (for example as measured by surface plasmon resonance). For example, the interaction or binding between the molecule comprising targeting moiety and the molecule comprising a portion capable of inserting / integrating into the nucleic acid particle may have a binding affinity of from about 0.1 to about 1000 pM, or from about 1.0 to about 500 pM, such as from about 10 to about 200 pM.

[0228] It will be understood that the term “bound” may also mean “conjugated”, or “associated with”. In some embodiments, each targeting moiety is non-covalently bound to the nucleic acid particle.

[0229] It will also be understood that one or more targeting moiety may be covalently bound to the nucleic acid particle, provided that at least one targeting moiety is non-covalently bound to the nucleic acid particle. It will be understood that a targeting moiety which is covalently bound to the nucleic acid particle may comprise a targeting moiety that is covalently conjugated to a molecule comprising a portion capable of inserting / integrating into the nucleic acid particle. For example, a targeting moiety that is covalently conjugated to a lipid, wherein the lipid is incorporated (i.e. , inserted or integrated) into the nucleic acid particle would be understood to be a targeting moiety that is covalently bound to the nucleic acid particle.

[0230] In some embodiments, at least one targeting moiety is non-covalently bound to the nucleic acid particle and at least one targeting moiety is covalently bound to the nucleic acid particle.

[0231] It will be understood that the, or each, targeting moiety may be capable of being non- covalently bound to a molecule present on the surface of the nucleic acid particle.

[0232] In some embodiments, the molecule present on the surface of the nucleic acid particle comprises a lipid component and a peptide component. In some embodiments, the lipid component and peptide component are linked by a spacer. In some embodiments, the peptide component is a binding moiety.

[0233] In some embodiments, the molecule present on the surface of the nucleic acid particle comprises a compound of Formula (A) as described herein.

[0234] In some embodiments, the molecule present on the surface of the nucleic acid particle is a compound of Formula (A) as described herein.

[0235] As such, in some embodiments, the, or each, targeting moiety and the compound of Formula (A) are capable of binding to each other.

[0236] In some embodiments, a compound comprising the targeting moiety further comprises a moiety that is capable of binding to the compound of Formula (A). In some embodiments, compound of Formula (A) comprises the formula:

[0237] L-X1-P-X2-B (A) wherein:

[0238] P is absent or comprises a polymer;

[0239] L comprises (i) a hydrophobic moiety, or (ii) a moiety comprising a negative charge, attached to B when P is absent or to a first end of the polymer P when present;

[0240] 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;

[0241] X1 is absent or a first linking moiety; and

[0242] X2 is absent or a second linking moiety.

[0243] In some embodiments, each targeting moiety is non-covalently bound to a separate compound of Formula (A).

[0244] In some embodiments, each targeting moiety is non-covalently bound to a separate binding moiety B of compound of Formula (A).

[0245] In some embodiments, a compound comprising the targeting moiety further comprises a moiety that is capable of binding to the binding moiety B of Formula (A).

[0246] In some embodiments, a compound comprising each targeting moiety further comprises a moiety that is non-covalently bound to a separate compound of Formula (A).

[0247] In some embodiments, a compound comprising each targeting moiety further comprises a moiety that is non-covalently bound to a separate binding moiety B of compound of Formula (A).

[0248] The compound of formula (A) is also referred to herein as a “targeting compound”. A compound comprising a targeting moiety that further comprises a moiety that is capable of non-covalently binding to the binding moiety B of a compound of Formula (A) is also referred to herein as a “docking compound”. Typically, the hydrophobic moiety (L) or the moiety comprising a negative charge (L) of the compound of formula (A) is incorporated into the particle, such that the binding moiety (B) of the compound of formula (A) is then oriented on the particle surface.

[0249] Functionalization of the particle is possible, for example by interacting a “docking compound” as defined herein, such as a compound of formula (I), as defined herein, with the binding moiety (B) (e.g., a peptide tag or a moiety binding to a peptide tag) of the compound of formula (A), wherein the compound of formula (I) comprises a moiety B” binding to a cell surface antigen on target cells. In such cases the docking compound comprises the targeting moiety and the targeting moiety is considered to be non-covalently bound to the nucleic acid particle, in that the binding of the targeting moiety to the particle is mediated by the non-covalent interaction between the docking compound and the targeting compound.

[0250] Functionalization of the particle is also possible, for example, where the binding moiety (B) of the compound of formula (A) is a targeting moiety. In such cases, the targeting moiety is considered to be covalently bound to the nucleic acid particle, in that the targeting moiety is covalently bound to moiety that is integrated (i.e. , inserted or incorporated) into the particle. In some embodiments, the targeting compound is a lipid bound to a targeting moiety.

[0251] It will therefore be understood that the targeting compound is capable of interacting with a docking compound.

[0252] Generally, the targeting compound is incorporated into the particle through the hydrophobic moiety or the moiety comprising a negative charge, i.e., it forms an integral part of the particle, and the binding moiety of the targeting compound is covalently attached to the hydrophobic moiety or the moiety comprising a negative charge in a manner such that it is available for binding to a docking compound.

[0253] In some embodiments, the binding moiety B comprises a peptide or protein.

[0254] In some embodiments, the binding moiety B comprises a peptide or protein and is chemically linked, e.g., through a linker, to the hydrophobic moiety (e.g., lipid) or the moiety comprising a negative charge. In some embodiments, the binding moiety B is:

[0255] (a) a peptide tag;

[0256] (b) a moiety capable of binding to a peptide tag; or

[0257] (c) an ALFA-tag.

[0258] In some embodiments, binding moiety B comprises a peptide tag.

[0259] In some embodiments, the binding moiety B is a moiety capable of binding to a peptide tag.

[0260] In some embodiments, the binding moiety B is an ALFA-tag.

[0261] In some embodiments, binding moiety B comprises a peptide tag and the compound comprising the targeting moiety further comprises a moiety that is capable of binding to the peptide tag. In some embodiments, the peptide tag comprises an ALFA-tag, as defined herein.

[0262] In some embodiments, the compound comprising the targeting moiety further comprises a moiety that comprises an antibody or antibody-like molecule capable of binding to the peptide tag.

[0263] In some embodiments, the compound comprising the targeting moiety further comprises a moiety that comprises an antibody or antibody-like molecule capable of binding to the ALFA tag.

[0264] In some embodiments, the compound comprising the targeting moiety further comprises a moiety that comprises an anti-ALFA binding domain, as defined herein.

[0265] In alternative embodiments, binding moiety B is a targeting moiety.

[0266] In some embodiments, the targeting compound described herein comprises a hydrophobic component (e.g., lipid component) which allows it to be anchored in the particle.

[0267] In some embodiments, the hydrophobic moiety comprises a lipid.

[0268] In some embodiments, the hydrophobic moiety comprises a phospholipid. In some embodiments, the hydrophobic component comprises a moiety selected from a vitamin E compound (which may be a-tocopherol, p-tocopherol, y-tocopherol, b-tocopherol, a- tocotrienol, p-tocotrienol, y-tocotrienol, b-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.

[0269] 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.

[0270] In some embodiments, the targeting compound described herein has as a hydrophobic group (e.g., lipid) a glycerophospholipid.

[0271] In some embodiments, the phospholipid is selected from the group consisting of DSPE (distearoylphosphatidylethanolamine), DPPE (dipalmitoylphosphatidylethanolamine), DOPE (dioleoylphosphatidylethanolamine), and POPE (palmitoyloleyl-phosphatidylethanolamine), and mixtures thereof.

[0272] In some embodiments, the phospholipid is a DSPE moiety.

[0273] 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.

[0274] In some embodiments, the targeting compound comprises a polymer, defined as P herein when present. In some embodiments, the hydrophobic moiety (e.g., lipid) of the targeting compound and the binding moiety of the targeting compound are connected (typically covalently) through the polymer.

[0275] In some embodiments, P is a hydrophilic polymer.

[0276] In some embodiments, P is selected from the group consisting of poly(ethylene 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.

[0277] “DOCKING COMPOUND” - COMPOUND COMPRISING THE TARGETING MOIETY

[0278] It will be understood that the targeting moiety may be comprised within a compound that also comprises a moiety that is capable of binding to the nucleic acid particle.

[0279] It will be understood that each targeting moiety may be comprised within a separate compound, wherein each compound also comprises a moiety that is capable of binding to the to the nucleic acid particle

[0280] It will be also understood that the, or each, targeting moiety may be independently comprised within a separate compound that comprises a moiety that is capable of being non-covalently bound to a molecule present on the surface of the nucleic acid particle.

[0281] In some embodiments, the nucleic acid particle may comprise:

[0282] (i) at least one targeting moiety that is comprised within a compound that comprises a moiety that is capable of being non-covalently bound to a molecule present on the surface of the nucleic acid particle; and

[0283] (ii) at least one targeting moiety that is capable of being covalently bound to a molecule present on the surface of the nucleic acid particle.

[0284] The molecule present on the surface of the nucleic acid particle may be the compound of Formula (A).

[0285] As such, the, or each, targeting moiety may be independently comprised within a separate compound that comprises a moiety that is capable of being non-covalently bound to the compound of Formula (A).

[0286] In some embodiments, a compound comprising a targeting moiety further comprises a moiety that is capable of binding to the binding moiety B of Formula (A).

[0287] In some embodiments, a compound comprising a targeting moiety further comprises a moiety that is non-covalently bound to a separate compound of Formula (A). In some embodiments, a compound comprising a targeting moiety further comprises a moiety that is non-covalently bound to a separate binding moiety B of compound of Formula (A).

[0288] In some embodiments, the nucleic acid particle further comprises one or more compound(s) of Formula (I):

[0289] B’-X3-B” (I) wherein

[0290] B’ comprises a moiety capable of non-covalently binding to B of the compound of formula (A) as defined herein;

[0291] X3 is absent or a linking moiety; and

[0292] B” comprises a targeting moiety; wherein the targeting moiety is non-covalently bound to the nucleic acid particle through the non-covalent binding of B’ of the compound of Formula (I) to B of the compound of Formula (A).

[0293] In some embodiments, B” is capable of binding to a cell surface antigen.

[0294] In some embodiments where there are two or more targeting moieties non-covalently bound to the nucleic acid particle, each targeting moiety may be independently comprised within a separate compound of Formula (I).

[0295] It will be understood that compound of Formula (I) may be termed a “docking compound”, as defined herein.

[0296] It will be understood that functionalization of the nucleic acid is possible by the interaction of the “docking compound” as defined herein with the “targeting compound” as defined herein.

[0297] It will be understood that functionalization of the nucleic acid is possible by the interaction of B’ of the compound of formula (I) with the binding moiety (B) of the compound of formula (A).

[0298] In some embodiments, a "docking compound", e.g. a compound of formula (I), is used to form a connection between a primary target, e.g., a target cell or an antigen on target cells, and a targeting compound, e.g. a compound of formula (A), which is integrated into the nucleic acid 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.

[0299] 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 targeting moiety of the docking compound are linked to each other, preferably by a covalent linkage.

[0300] 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, the docking compound comprises a cytokine binding to a primary target and a single-domain antibody such as a VHH binding to a targeting compound. 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 singledomain antibody such as a VHH. In some embodiments, the binding domain binding to a primary target comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a primary target comprises a single-domain antibody such as a VHH. In some embodiments, the binding domain binding to a targeting compound comprises a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody. In some embodiments, the binding domain binding to a targeting compound comprises a single-domain antibody such as a VHH.

[0301] 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.

[0302] 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, cytokine 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, cytokine 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):

[0303] 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) Cytokine (a targeting compound)-optional linker-VHH (a primary target) VHH (a primary target)-optional linker-Cytokine (a targeting compound) Cytokine (a primary target)-optional linker-scFv (a targeting compound) scFv (a targeting compound)-optional linker-Cytokine (a primary target) peptide (bound by targeting compound)-optional linker-Cytokine (a primary target) Cytokine (a primary target)-optional linker-peptide (bound by targeting compound)

[0304] 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, cytokine) wherein the antibody portion binds to a primary target, e.g., a cell surface antigen on target cells.

[0305] 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.

[0306] 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., PEG, and the other specificity binds to a primary target, e.g., a cell surface antigen on target cells. Antibodies which bind to a polymer (e.g. PEG) are known in the art (see e.g. Creative Biolabs [HPAB-0772LY-S(P)] and Abeam [PEG-B-47]).

[0307] 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, cytokine or DARPin.

[0308] In some embodiments, the moiety targeting a primary target of the docking compound is selected from the group consisting of an anti-primary target VHH, an anti-primary target scFv, an anti-primary target cytokine (e.g., where the primary target is the cytokine receptor), an anti-primary target DARPin, and / or the moiety binding to a targeting compound of the docking compound is an NbALFA-nanobody (NbALFA). In some embodiments, the docking compound has a structure selected from the group consisting of NbALFA x anti-primary target VHH and NbALFA x anti-primary target scFv, NbALFA x anti-primary target cytokine, NbALFA x antiprimary target DARPin.

[0309] In some embodiments, the primary target is a T cell antigen, e.g., CD3 (such as CD3e), CD2, CD7, CD4, CD8, CD28, IL7 receptor, CD127, or CD5. In some embodiments, the target is a B cell antigen, e.g., CD19, CD20, IgM, or IgD. 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.

[0310] 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.

[0311] 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.

[0312] 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-CD2 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-CD2 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-CD2 DARPin.

[0313] 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. 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. 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-CD28 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-CD28 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-CD28 DARPin.

[0314] In some embodiments, the docking compound comprises a cytokine such as interleukin 7 (IL7).

[0315] In some embodiments, the moiety on the targeting compound (e.g. B of the compound of formula (A), binding moiety covalently attached to a hydrophobic moiety) and the moiety on the docking compound (e.g. B’ of the compound of formula (I), moiety binding to the binding moiety covalently attached to a hydrophobic moiety) interact with each other, e.g., non- covalently bind to each other.

[0316] 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)) bind to each other under physiological conditions.

[0317] 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)) are antibody / antigen systems.

[0318] 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.

[0319] 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.

[0320] 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.

[0321] 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.

[0322] The ALFA-tag may be defined as described below.

[0323] PEPTIDE TAG, SUCH AS ALFA-TAG

[0324] In some embodiments at least one targeting moiety may be non-covalently bound to a peptide tag (e.g., a peptide tag comprising an epitope for a high affinity binder, e.g., an epitope tag) which is covalently conjugated to a portion capable of inserting / integrating into the nucleic acid particle (e.g., a lipid). For example, the non-covalent binding (e.g., between the targeting compound on the surface of the particle, and the docking compound comprising the targeting moiety may be an epitope tag / binder system). In some embodiments, binding moiety B of the compound of Formula A, which is comprised in the particle of the present invention, comprises a peptide tag. Alternatively, in some embodiments, binding moiety B’ of the compound of Formula I, which non-covalently binds to the particle of the present invention, comprises a peptide tag.

[0325] A peptide tag may comprise from about 5 to about 50 amino acids. In some embodiments, the peptide tag comprises from about 8 to about 30 amino acids. In some embodiments, the peptide tag comprises from about 10 to about 20 amino acids.

[0326] In some embodiments at least one targeting moiety may be non-covalently bound to an ALFA- tag which is covalently conjugated to a portion capable of inserting / integrating into the nucleic acid particle (e.g., a lipid). In some embodiments, binding moiety B (of the compound of Formula A) of the particle of the present invention comprises an ALFA-tag. Alternatively, in some embodiments, binding moiety B’ of the compound of Formula I, which non-covalently binds to the particle of the present invention, comprises an ALFA-tag.

[0327] In some embodiments, an ALFA-tag comprises the amino acid sequence:

[0328] -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:

[0329] AAO is Pro or deleted;

[0330] AA1 is Ser, Gly, Thr, or Pro;

[0331] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0332] AA3 is Leu, lie, or Vai;

[0333] AA4 is Glu or Gin;

[0334] AA5 is Glu or Gin;

[0335] AA6 is Glu or Gin;

[0336] AA7 is Leu, lie, or Vai;

[0337] AA8 is Arg, Ala, Gin, or Glu;

[0338] AA9 is Arg, Ala, Gin, or Glu;

[0339] AA10 is Arg;

[0340] AA11 is Leu;

[0341] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0342] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and

[0343] AA14 is Pro or deleted.

[0344] In some embodiments, an ALFA-tag comprises a sequence selected from the group consisting of SRLEEELRRRLTE (SEQ ID NO: 10), PSRLEEELRRRLTE (SEQ ID NO: 11), SRLEEELRRRLTEP (SEQ ID NO: 12), and PSRLEEELRRRLTEP (SEQ ID NO: 13).

[0345] In some embodiments, an ALFA-tag comprises the cyclized amino acid sequence -AA0-AA1- AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-AA10-AA11-AA12-AA13-AA14-, wherein the side-chains of any two of the amino acids of AAO, AA1 , AA2, AA3, AA4, AA5, AA6, AA7, AA8, AA9, AA10, AA11 , AA12, AA13 and AA14 (X1 , 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 X1 and X2 are:

[0346] AAO is Pro or deleted; AA1 is Ser, Gly, Thr, or Pro;

[0347] AA2 is Arg, Gly, Ala, Glu, or Pro;

[0348] AA3 is Leu, lie, or Vai;

[0349] AA4 is Glu or Gin;

[0350] AA5 is Glu or Gin;

[0351] AA6 is Glu or Gin;

[0352] AA7 is Leu, lie, or Vai;

[0353] AA8 is Arg, Ala, Gin, or Glu;

[0354] AA9 is Arg, Ala, Gin, or Glu;

[0355] AA10 is Arg;

[0356] AA11 is Leu;

[0357] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0358] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

[0359] In some embodiments, X1 and X2 are separated by 2 or 3 amino acids.

[0360] In some embodiments, AA5 is X1 and AA9 is X2, AA5 is X1 and AA8 is X2, AA9 is X1 and AA13 is X2, AA6 is X1 and AA9 is X2, AA9 is X1 and AA12 is X2, AA10 is X1 and AA13 is X2, AA6 is X1 and AA10 is X2 or AA4 is X1 and AA8 is X2.

[0361] In some embodiments, an ALFA-tag comprises a cyclized amino acid sequence selected from the group consisting of

[0362] -AA0-AA1 -AA2-AA3-AA4-cyclo(X1 -AA6-AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, -AA0-AA1 -AA2-AA3-AA4-cyclo(X1 -AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-AA12-X2)-AA14-, -AA0-AA1 -AA2-AA3-AA4-AA5-cyclo(X1 -AA7-AA8-X2)-Arg-Leu-AA12-AA13-AA14-, -AA0-AA1-AA2-AA3-AA4-AA5-AA6-AA7-AA8-cyclo(X1-Arg-Leu-X2)-AA13-AA14-, -AA0-AA1 -AA2-AA3-AA4-AA5-AA6-AA7-AA8-AA9-cyclo(X1 -Leu-AA12-X2)-AA14-, -AA0-AA1-AA2-AA3-AA4-AA5-cyclo(X1-AA7-AA8-AA9-X2)-Leu-AA12-AA13-AA14-, and -AA0-AA1 -AA2-AA3-cyclo(X1 -AA5-AA6-AA7-X2)-AA9-Arg-Leu-AA12-AA13-AA14-, wherein the side-chains of X1 and X2 amino acid residues are connected covalently;

[0363] AAO is Pro or deleted;

[0364] AA1 is Ser, Gly, Thr, or Pro;

[0365] AA2 is Arg, Gly, Ala, Glu, or Pro; AA3 is Leu, lie, or Vai;

[0366] AA4 is Glu or Gin;

[0367] AA5 is Glu or Gin;

[0368] AA6 is Glu or Gin;

[0369] AA7 is Leu, lie, or Vai;

[0370] AA8 is Arg, Ala, Gin, or Glu;

[0371] AA9 is Arg, Ala, Gin, or Glu;

[0372] AA12 is Thr, Ser, Asp, Glu, Pro, Ala, or deleted;

[0373] AA13 is Glu, Lys, Pro, Ser, Ala, Asp, or deleted; and AA14 is Pro or deleted.

[0374] In some embodiments, X1 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 X1 and X2 to the carboxyl group of a side-chain of the other of X1 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 X1 and X2 to the sulfhydryl group of a side-chain of the other of X1 and X2 via a disulfide bond. Sulfhydryl group-containing amino acids include cysteine and other sulfhydryl-containing amino acids as Pen.

[0375] In some embodiments, X1 is Glu and X2 is Lys. In some embodiments, -cyclo(Glu - Lys)-

[0376] , -c(Glu - Lys)-, -cyclo(E - K)-, -c(E - K)-, -E - K- cyclo, or -cycloE— -cycloK- comprises the following structure: In some embodiments, X1 is Lys and X2 is Glu. In some embodiments, -cyclo(Lys - Glu)-,

[0377] -c(Lys - Glu)-, -cyclo(K - E)-, -c(K - E)-, -K - E- cyclo, or cycloK - cycloEcomprises the following structure:

[0378] In some embodiments, X1 is Cys and X2 is Cys. In some embodiments, -cyclo(Cys - Cys)-

[0379] , c(Cys - Cys)-, -cyclo(C - C)-, -c(C - C)-, -C- — C- cyclo, or -cycloC — cycloC- comprises the following structure:

[0380] In some embodiments, the cyclized amino acid sequence is one selected from the group consisting of -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 14), -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 15), -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 16), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Glu)- (SEQ ID NO: 17), -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 18), -Ser-Arg-Leu-Glu-cyclo(Asp-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 19), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Lys-Arg-Leu-Thr-Asp)- (SEQ ID NO: 20), -Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 21), -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 22), -Pro-Ser-Arg-Leu-Glu-cyclo(DGIu-Glu-Leu-Arg-Lys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 23), -Pro-Ser-Arg-Leu-Glu-cyclo(Glu-Glu-Leu-Arg-DLys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 24), -Pro-Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu-(SEQ ID NO: 25), -Pro-Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 26), -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-DCys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 27), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)- (SEQ ID NO: 28), -Pro-Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 29), -Pro-Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 30), -Pro-Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu- (SEQ ID NO: 31), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-cyclo(Cys-Arg-Leu-Thr-Cys)- (SEQ ID NO: 32), -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Arg-Cys)-Arg-Leu-Thr-Glu- (SEQ ID NO: 33), -Ser-Arg-Leu-Glu-cyclo(Cys-Glu-Leu-Cys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 34), -Ser-Arg-Leu-Glu-Glu-Glu-Leu-Arg-Arg-cyclo(Cys-Leu-Thr-Cys)- (SEQ ID NO: 35), -Ser-Arg-Leu-Glu-Glu-cyclo(Cys-Leu-Arg-Arg-Cys)-Leu-Thr-Glu- (SEQ ID NO: 36), -Ser-Arg-Leu-Glu-cyclo(Lys-Glu-Leu-Arg-Glu)-Arg-Leu-Thr-Glu- (SEQ ID NO: 37), and -Ser-Arg-Leu-cyclo(Glu-Glu-Glu-Leu-Lys)-Arg-Arg-Leu-Thr-Glu- (SEQ ID NO: 38).

[0381] 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: 14). 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: 15). 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: 16). 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: 17).

[0382] 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:

[0383]

[0384] Other suitable peptide tags (e.g., which have high affinity interactions with respective binding domains) may be used, for example a Spot-tag, e.g., comprising the sequence PDRVRAVSHWSS (SEQ ID NO: 39).

[0385] BINDING DOMAIN (ANTI-ALFA BINDING DOMAIN)

[0386] In some embodiments a molecule comprising a targeting moiety may comprise a binding domain (such as B’ of the compound of Formula I) which is capable of non-covalently binding to a peptide tag which is covalently conjugated to a portion capable of inserting / integrating into the nucleic acid particle (e.g., a lipid). In some embodiments, B’ of the compound of Formula I comprises an anti-ALFA binding domain that is capable of binding to an ALFA tag peptide (e.g., where B of the compound of Formula A is an ALFA-tag peptide). Alternatively, in some embodiments, B of the compound of Formula A comprises an anti-ALFA binding domain that is capable of binding to an ALFA tag peptide (e.g., where B’ of the compound of Formula I is an ALFA-tag peptide).

[0387] In some embodiments, the anti-ALFA binding domain comprises an antibody or antibody fragment that is capable of binding to an ALFA tag peptide. In some embodiments, the anti- ALFA binding domain comprises a camelid VHH domain that is capable of binding to an ALFA tag peptide. In some embodiments, the anti-ALFA binding domain comprises a single-domain antibody (sdAb), i.e. a NbALFA-nanobody. In some embodiments, the antibody or antibody fragment, the VHH, or the sdAb is humanised. Suitably, the anti-ALFA binding domain comprises a sdAb (preferably a VHH) comprising:

[0388] (i) CDRs with the following sequences:

[0389] CDR1 - GVTISALNAMAMG (SEQ ID NO: 40)

[0390] CDR2 - AVSERGNTY (SEQ ID NO: 41)

[0391] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42); or

[0392] (ii) CDRs with the following sequences:

[0393] CDR1 - GVTISALNAMAMG (SEQ ID NO: 40)

[0394] CDR2 - AVSERGNAM (SEQ ID NO: 43)

[0395] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42); or

[0396] (iii) CDRs with the following sequences:

[0397] CDR1 - GVTISALNAMAMG (SEQ ID NO: 40)

[0398] CDR2 - AVSSRGNAM (SEQ ID NO: 44)

[0399] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42);

[0400] (according to AbM definition) optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.

[0401] The CDRs of the moiety which is capable of specifically binding to an ALFA tag may also be provided according to IMGT or Kabat annotation.

[0402] Suitably, the anti-ALFA binding domain may comprise:

[0403] (i) CDRs comprising the following sequences:

[0404] CDR1 - ALNAMAMG (SEQ ID NO: 45)

[0405] CDR2 - AVSERGNTYYRDSVKG (SEQ ID NO: 46)

[0406] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42); or

[0407] (ii) CDRs comprising the following sequences:

[0408] CDR1 - ALNAMAMG (SEQ ID NO: 45)

[0409] CDR2 - AVSERGNAMYRESVQG (SEQ ID NO: 47)

[0410] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42); according to Kabat annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.

[0411] Suitably, the anti-ALFA binding domain may comprise:

[0412] (i) CDRs comprising the following sequences:

[0413] CDR1 - GVTISALNAMA (SEQ ID NO: 48) CDR2 - VSERGNT (SEQ ID NO: 49)

[0414] CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 50); or

[0415] (ii) CDRs comprising the following sequences:

[0416] CDR1 - GVTISALNAMA (SEQ ID NO: 48)

[0417] CDR2 - VSERGNA (SEQ ID NO: 51)

[0418] CDR3 - HVLEDRVDSFHDY (SEQ ID NO: 50); according to IMGT annotation, optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations.

[0419] It is understood that an anti-ALFA binding domain in which 1 , 2 or 3 mutations have been introduced to one, two, or all three of the CDR sequences must still capable of specifically binding to the ALFA tag.

[0420] The anti-ALFA binding domain may comprise CDRs consisting of the sequences provided above.

[0421] Suitably, the ALFA tag may be an ALFA tag peptide as described herein.

[0422] Suitably, the anti-ALFA binding domain may comprise or consist of the VHH sequence of any one of SEQ ID NO: 52-54 or a variant having at least 80% identity thereto.

[0423] Suitably, the anti-ALFA binding domain may comprise or consist of the VH H sequence of SEQ ID NO: 52 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. Suitably the VHH may comprise a sequence shown as SEQ ID NO: 52.

[0424] Suitably, the anti-ALFA binding domain may comprise or consist of the VH H sequence of SEQ ID NO: 53 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. Suitably the VHH may comprise a sequence shown as SEQ ID NO: 53.

[0425] Suitably, the anti-ALFA binding domain may comprise or consist of the VH H sequence of SEQ ID NO: 54 or a variant having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity thereto. Suitably the VHH may comprise a sequence shown as SEQ ID NO: 54. SEQ ID NO: 52

[0426] EVQLVESGGGLVQPGGSLRLSCAASGVTISALNAMAMGWYRQAPGKRREMVAAVSERGN TYYRDSVKGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCHVLEDRVDSFHDYWGQGTQV TVSS

[0427] SEQ ID NO: 53

[0428] EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGERRVMVAAVSERGN AMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQV TVSS

[0429] SEQ ID NO: 54

[0430] EVQLQESGGGLVQPGGSLRLSCTASGVTISALNAMAMGWYRQAPGEERVMVAAVSSRGN AMYRESVQGRFTVTRDFTNKMVSLQMDNLKPEDTAVYYCHVLEDRVDSFHDYWGQGTQV TVSS

[0431] Suitably, the variant maintains the ability to bind to an ALFA tag. Suitable variants are described herein.

[0432] The present invention also provides the use of a humanised sbAb as described above, preferably a VHH as described above, to couple a targeting moiety to an ALFA-tag, suitably wherein the ALFA-tag is incorporated into a particle, preferably a lipid nanoparticle.

[0433] Accordingly, the present humanized sdAb may be suitable for use in a docking compound. For example, the present humanized sdAb may be used in a docking compound comprising a targeting moiety capable of binding any target of interest; for example any cell surface target of interest.

[0434] In some embodiments, the peptide tag comprises an ALFA-tag (e.g., comprising the sequence SRLEEELRRRLTE) and the moiety binding to the peptide tag comprises a VHH domain comprising CDRs with the following sequences:

[0435] CDR1 - GVTISALNAMAMG (SEQ ID NO: 40),

[0436] CDR2 - AVSERGNTY (SEQ ID NO: 41) and

[0437] CDR3 - LEDRVDSFHDY (SEQ ID NO: 42). COVALENTLY BOUND TARGETING MOIETIES

[0438] In one aspect, the present invention provides a nucleic acid particle comprising:

[0439] (i) one or more particle forming components;

[0440] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0441] (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0442] In some embodiments, each of the first and second targeting moieties is covalently bound to the nucleic acid particle.

[0443] Suitably the nucleic acid particle may comprise at least three targeting moieties, wherein the third targeting moiety is capable of binding to a third target, wherein each target is different.

[0444] Suitably, the nucleic acid particle comprises 2-10 targeting moieties, wherein each targeting moiety is capable of binding to a different target. In some embodiments, the nucleic acid particle comprises 2-5 targeting moieties; wherein each targeting moiety is capable of binding to a different target.

[0445] In some embodiments, each targeting moiety is covalently bound to the nucleic acid particle.

[0446] Suitable means of covalent attachment are described herein. Such covalent binding of a targeting moiety to a particle may be known in the art, for example from WO2024077232.

[0447] In some embodiments (e.g. embodiments of Formula (A)), 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 (A), the thiol or cysteine reactive group comprises a maleimide group.

[0448] In some embodiments (e.g. embodiments of Formula (A)), a polymer (e.g. P of Formula(A)) may provide a stealth property, thereby extending circulation half-life and / or reducing nonspecific protein binding or cell adhesion. In some embodiments of Formula (A), P comprises a polymer selected from the group consisting of poly(ethylene 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) (including derivatives thereof). In some embodiments of Formula (A), P comprises poly(ethylene glycol) (PEG); e.g., PEG as described above.

[0449] In some embodiments of Formula (A), L-X1-P comprises an amphiphilic derivative of a polymer, as described herein. In some embodiments of Formula (A), the amphiphilic derivative of a polymer comprises a conjugate of distearoyl-glycero-phosphoethanolamine (DSPE) and a polymer. In some embodiments of Formula (A), the amphiphilic derivative of a polymer comprises a disteroyl-glycero-phosphoethanolamine-polyethyleneglycol-conjugate (DSPE- PEG).

[0450] In some embodiments of Formula (A), 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 (A), the thiol or cysteine reactive group comprises a maleimide group.

[0451] In some embodiments of Formula (A), the PEG reagent comprises DSPE-PEG-maleimide. In some embodiments of Formula (A), 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.

[0452] In some embodiments of Formula (A), the targeting compound comprises the reaction product of 1 ,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 (A), n is 2.

[0453] In some embodiments, the targeting compound comprises the following general Formula (A1) wherein B comprises the binding moiety, and PEG is polyethylene glycol, as defined above (either in its broadest aspect or a preferred aspect).

[0454] In some exemplary embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain, wherein the first targeting moiety and the second targeting moiety are covalently bound to the nucleic acid particle.

[0455] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the first targeting moiety and the second targeting moiety are covalently bound to the nucleic acid particle.

[0456] In some embodiments, the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain, wherein the first targeting moiety, the second targeting moiety and the third targeting moiety are covalently bound to the nucleic acid particle.

[0457] TARGETING MOIETIES

[0458] The nucleic acid particle according to the invention may comprise two or more targeting moieties which are capable of trafficking the particle to specific targets (e.g. cellular targets) for delivery of the nucleic acid payload.

[0459] A nucleic acid particle comprising two or more targeting moieties can be termed a “multifunctionalised nucleic acid particle”. An LNP comprising two or more targeting moieties can be termed a “multifunctionalised LNP”. By using two or more targeting moieties, enhanced targeting of target cells can be achieved, resulting in improved transfection of target cells (e.g. immune cells), along with improved DNA delivery. In the case of target immune cells, such as T cells, improved activation and proliferation of the cells can also be achieved using two or more targeting moieties.

[0460] Accordingly, the nucleic acid particle according to the invention may comprise two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0461] In some embodiments, the nucleic acid particle comprises two targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein each targeting moiety is different and wherein the first and second targets are different.

[0462] In some embodiments, the nucleic acid particle comprises at least three targeting moieties, at least four targeting moieties, at least five targeting moieties, at least six targeting moieties, at least seven targeting moieties, at least eight targeting moieties, at least nine targeting moieties, or at least ten targeting moieties, wherein each targeting moiety is different and is capable of binding to a different target.

[0463] In some embodiments, the nucleic acid particle comprises 2-10 targeting moieties, wherein each targeting moiety is different and is capable of binding to a different target.

[0464] In some embodiments, the nucleic acid particle comprises 2-5 targeting moieties, wherein each targeting moiety is different and is capable of binding to a different target.

[0465] In some embodiments, the nucleic acid particle comprises at least three targeting moieties, wherein the third targeting moiety is capable of binding to a third target, wherein each target is different. In other words, the third target is different to the first target and the second target.

[0466] In some embodiments, the nucleic acid particle comprises at least four targeting moieties, wherein the fourth targeting moiety is capable of binding to a fourth target, wherein each target is different. In other words, the fourth target is different to the first target, the second target, and the third target.

[0467] In some embodiments, the nucleic acid particle comprises at least five targeting moieties, wherein the fifth targeting moiety is capable of binding to a fifth target, wherein each target is different. In other words, the fifth target is different to the first target, the second target, the third target, and the fourth target.

[0468] In some embodiments, the nucleic acid particle comprises three targeting moieties, wherein the third targeting moiety is capable of binding to a third target, wherein each target is different. In other words, the third target is different to the first target and the second target.

[0469] In some embodiments, the nucleic acid particle comprises four targeting moieties, wherein the fourth targeting moiety is capable of binding to a fourth target, wherein each target is different. In other words, the fourth target is different to the first target, the second target, and the third target.

[0470] In some embodiments, the nucleic acid particle comprises five targeting moieties, wherein the fifth targeting moiety is capable of binding to a fifth target, wherein each target is different. In other words, the fifth target is different to the first target, the second target, the third target, and the fourth target.

[0471] In some embodiments, the target is a cell-surface molecule, such as a cell-surface receptor.

[0472] It will be understood that the, or each, targeting moiety may be any type of targeting moiety that can be directed against any cell-specific marker of interest.

[0473] In some embodiments, at least one, or each, targeting moiety is independently selected from the list consisting of: an antibody or fragment thereof, a peptide, a cytokine, a receptor, a Designed Ankyrin Repeat Protein (DARPin) (for example as described in WO2021130225A1), and a cell surface molecule.

[0474] In some embodiments, the antibody or fragment thereof may be selected from the list consisting of: a single-chain variable fragment (scFv), a Fab, a F(ab)’2, a Fv, a single domain antibody, a nanobody, a VHH antibody, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, a chimeric antibody or fragment thereof, a bifunctional antibody, and a bispecific antibody.

[0475] In some embodiments, the at least one targeting moiety is selected from the list consisting of: an antibody or fragment thereof, a cytokine, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody, a DARPin, and a single chain variable domain.

[0476] In some embodiments, the at least one targeting moiety comprises a nanobody.

[0477] In some embodiments, the at least one targeting moiety comprises a cytokine. In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is IL7. In some embodiments, the two targeting moieties are independently selected from the list consisting of: an antibody or fragment thereof, a cytokine, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody, a DARPin, and a single chain variable domain.

[0478] In some embodiments, the two targeting moieties each independently comprise a nanobody.

[0479] In some embodiments, the two targeting moieties each independently comprise a cytokine.

[0480] In some embodiments, at least one of the two targeting moieties is a nanobody and at least one of the two targeting moieties is a cytokine.

[0481] In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is IL7.

[0482] In some embodiments, all of the targeting moieties are independently selected from the list consisting of: an antibody or fragment thereof, a cytokine, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody and a single chain variable domain.

[0483] In some embodiments, all of the targeting moieties each independently comprise a nanobody.

[0484] In some embodiments, all of the targeting moieties each independently comprise a cytokine.

[0485] In some embodiments, at least one of the targeting moieties is a nanobody and at least one of the targeting moieties is a cytokine.

[0486] In some embodiments, the cytokine is an interleukin. In some embodiments, the cytokine is IL7.

[0487] In some embodiments, one or more, or each, targeting moiety may comprise a heavy chain variable (VH) domain. In some embodiments, one or more, or each, targeting moiety may comprise a light chain variable (VL) domain. In some embodiments, one or more, or each, targeting moiety may comprise a VH domain and a VL domain.

[0488] In some embodiments, the VH domain may comprise one or more complementarity determining regions (CDRs). In some embodiments, the VH domain may comprise one, two or three CDRs. In some embodiments, the VH domain may comprise three CDRs. It will be understood that CDRs of the VH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the VH domain may be termed HCDR1 , HCDR2 and HCDR3 respectively.

[0489] In some embodiments, the VL domain may comprise one or more CDRs. In some embodiments, the VL domain may comprise one, two or three CDRs. In some embodiments, the VL domain may comprise three CDRs. It will be understood that CDRs of the VL domain may be termed LCDRs. It will also be understood that each of the three CDRs of the VL domain may be termed LCDR1 , LCDR2 and LCDR3 respectively.

[0490] In some embodiments, one or more, or each, targeting moiety may comprise a non-human antibody, a chimeric antibody, a humanised antibody or a fully human antibody.

[0491] In some embodiments, one or more, or each, targeting moiety may comprise a humanised antibody.

[0492] In some embodiments, one or more, or each, targeting moiety may comprise a full-length, classical antibody. In some embodiments, the antibody may be an IgG, IgM, IgD, IgE or IgA molecule.

[0493] In some embodiments, one or more, or each, targeting moiety may comprise an IgG molecule. In some embodiments, the antibody may be an lgG1 molecule. In some embodiments, the antibody may be an lgG2 molecule. In some embodiments, the antibody may be an lgG3 molecule. In some embodiments, the antibody may be an lgG4 molecule.

[0494] In some embodiments, one or more, or each, targeting moiety may comprise a monoclonal antibody or fragment thereof. In some embodiments, one or more, or each, targeting moiety may comprise a humanised monoclonal antibody or fragment thereof.

[0495] In some embodiments, one or more, or each, targeting moiety may comprise a VHH antibody or fragment thereof. In some embodiments, one or more, or each, targeting moiety may comprise a humanised VHH antibody or fragment thereof. As used herein, “antibody” may refer to a protein or polypeptide having an antigen binding site or antigen-binding domain which comprises at least one complementarity determining region (CDR).

[0496] In some embodiments, the antibody may comprise 6 CDRs. It will be understood that such an antibody may be a classical antibody molecule. In some embodiments, the antibody may comprise 3 CDRs and have an antigen binding site which is equivalent to that of a single domain antibody (sdAb). A sdAb (i.e. a nanobody) may be defined as an antibody fragment comprising of a single monomeric variable antibody domain. The sdAb may be a single chain variable domain which may be a heavy chain variable (VH) domain or light chain variable (VL) domain, having 3 CDRs. sdAbs have been engineered from heavy-chain antibodies found in camelids to produce variable heavy chain domain antibodies (VHHs). SdAbs have also been engineered from heavy-chain antibodies called immunoglobulin new antigen receptor (IgNAR) found in cartilaginous fishes to produce variable new antigen receptor antibodies (VNARs).

[0497] Suitably, the sdAb may be variable heavy chain domain antibody (VHH), a heavy chain variable (VH) domain, a light chain variable (VL) domain, or a variable new antigen receptor antibody (VNAR). Preferably, the sdAb may be a VHH.

[0498] The sdAb may be a Humabody® (Crescendo Biologies). A Humabody is an antibody produced by a transgenic mouse that produces heavy-chain-only antibodies with fully human VH domains, without VL domains.

[0499] The sdAb may be non-human, humanised or fully human. Suitably, the sdAb may be a humanised sdAb. Suitably, the sdAb may be a fully human sdAb.

[0500] The remainder of the polypeptide may be any sequence which provides a suitable scaffold for the antigen binding site and displays it in an appropriate manner for it to bind the antigen.

[0501] “Heavy chain variable region” or “VH” refers to the fragment of the heavy chain of an antigenbinding domain or antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. “Light chain variable region” or “VL” refers to the fragment of the light chain of an antigen-binding domain or antibody that contains three CDRs interposed between framework regions.

[0502] “Complementarity determining region” or “CDR” with regard to an antigen-binding domain or antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain of the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs (heavy chain CDRs 1 , 2 and 3 and light chain CDRs 1 , 2 and 3, numbered from the amino to the carboxy terminus).

[0503] A number of definitions of the CDRs are commonly in use. The Kabat definition is based on sequence variability and is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). The ImMunoGeneTics information system (IMGT) (see http: / / www.imgt.org) can also be used. According to this system, a complementarity determining region (CDR-IMGT) is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG). Other definitions of the CDRs have also been developed, such as the Chothia, the AbM and the contact definitions (see http: / / www.imgt.org). The CDRs of the sdAbs according to the present invention may be defined using any suitable system, such as any suitable system known in the art.

[0504] "Humanised antibody" may refer to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. “Humanised antibody” may also refer to a sdAb which has been modified to increase the level of sequence homology to human variable domains and / or to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR). Non-limiting examples of antibody humanisation methods include CDR grafting, and resurfacing (i.e. replacing surface residues to obtain a “more human” surface. For example, in order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modelling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanised antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back- mutations to the non-human amino acid sequence, and, optionally, fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back- mutations, may be introduced to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. Humanisation of non-human therapeutic antibodies is performed to minimise its immunogenicity in man while such humanised antibodies at the same time maintain the specificity and binding affinity of the antibody of non- human origin. Exemplary methods for humanisation of VHHs are described in Vincke et al. (Journal of Biological Chemistry; 2009; 284(5); 3273-3284) and Rossotti et al. (FEBS; 2021 ; doi:10.1111 / febs / 15809).

[0505] In some embodiments, one or more, or each, targeting moiety is capable of binding to T cells, B cells, NK cells, monocytes, macrophages, mast cells, basophils, eosinophils or dendritic cells.

[0506] In some embodiments, one or more, or each, targeting moiety is capable of binding to T cells. In some embodiments, one or more, or each, targeting moiety is capable of specifically binding to a cell surface marker expressed on T cells. In some embodiments, one or more, or each, targeting moiety is capable of activating T cells. In some embodiments, one or more, or each, targeting moiety is capable of activating CD8+ T cells. In some embodiments, one or more, or each, targeting moiety is capable of binding to and activating T cells. In some embodiments, one or more, or each, targeting moiety is capable of binding to and activating CD8+ T cells.

[0507] It will also be understood that activation of T cells may encompass expansion and / or proliferataion of T cells. Without wishing to be bound by theory, it will be understood that T cell activation may be determined based on expression of CD69, CD25 and / or IL-2 production.

[0508] In some embodiments, one or more, or each, targeting moiety is capable of binding to B cells. In some embodiments, one or more, or each, targeting moiety comprises a B cell targeting domain.

[0509] In some embodiments, one or more, or each, targeting moiety comprises a T cell targeting domain. In some embodiments, one or more, or each, targeting moiety comprises a T cell activating domain. In some embodiments, one or more, or each, targeting moiety comprises a T cell targeting domain and a T cell activating domain.

[0510] In some embodiments (e.g., where T cells are to be targeted), at least one targeting moiety is selected from the list consisting of a: CD3 binding domain, CD2 binding domain, CD7 binding domain, CD4 binding domain, CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain and CD5 binding domain.

[0511] In some embodiments (e.g., where B cells are to be targeted), at least one targeting moiety is selected from the list consisting of a: CD19 binding domain, CD20 binding domain, IgM binding domain, and IgD binding domain.

[0512] In some embodiments, at least one targeting moiety is a cytokine. In some embodiments, at least one targeting moiety is IL7. In some embodiments, at least one targeting moiety is capable of binding to an IL7 receptor.

[0513] In some embodiments, each targeting moiety is independently selected from the list consisting of a: CD3 binding domain, CD2 binding domain, CD7 binding domain, CD4 binding domain CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain, and CD5 binding domain. In some embodiments, each targeting moiety is independently selected from the list consisting of a: CD3 binding domain, CD2 binding domain, and CD7 binding domain.

[0514] In some embodiments, each targeting moiety is independently selected from the list consisting of a: CD19 binding domain, CD20 binding domain, IgM binding domain, and IgD binding domain.

[0515] In some embodiments, (i) at least one of the targeting moieties is selected from the list consisting of a: CD2 binding domain, CD7 binding domain, CD4 binding domain, CD8 binding domain, CD28 binding domain, cytokine (e.g., IL7), CD127 binding domain, and CD5 binding domain; and

[0516] (ii) at least one of the targeting moieties is a CD3 binding domain.

[0517] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain. In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain. In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises IL7. In some embodiments, the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain.

[0518] In some embodiments, the, or each, target is expressed on CD4 T cells or CD8 T cells. In some embodiments, the first target and second target are both expressed on the surface of CD4 T cells. In some embodiments, the third target, fourth and fifth target are all expressed on the surface of CD4 T cells. In some embodiments, the first target and second target are both expressed on the surface of CD8 T cells. In some embodiments, the third target, fourth and fifth target are all expressed on the surface of CD8 T cells.

[0519] In some embodiments, the target is independently selected from the list consisting of: CD3, CD2, CD7, CD4, CD8, CD28, IL7R, CD127 and CD5.

[0520] In some embodiments, the target is independently selected from the list consisting of: CD19, IgM, IgD and CD20.

[0521] It will be understood that the ratio of the two or more (e.g. three, four, five etc.) targeting moieties can be altered depending on the desired target cell. As used herein, it will be understood that the “ratio” refers to the molar ratio.

[0522] In some embodiments, the ratio of first targeting moiety to second targeting moiety is in the range of about 10:1 to about 1 :10.

[0523] In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of about 9:1 , about 7:3, about 3:1 , about 1 :1 , about 1 :3, about 3:7; or about 1 :9.

[0524] In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of about 9:1 , about 7:3, about 1 :1 , about 3:7; or about 1 :9. In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of 9:1 , 7:3, 3:1 , 1 :1 , 1 :3, 3:7; or 1 :9.

[0525] In some embodiments, the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of 9:1 , 7:3, 1 :1 , 3:7; or 1 :9.

[0526] In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 9:1. In some embodiments, the ratio of first targeting moiety to second targeting moiety is 9: 1. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the ratio of first targeting moiety to second targeting moiety is 7:3. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 1 :1. In some embodiments, the ratio of first targeting moiety to second targeting moiety is 1 :1. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 3:7. In some embodiments, the ratio of first targeting moiety to second targeting moiety is 3:7. In some embodiments, the ratio of first targeting moiety to second targeting moiety is about 1 :9. In some embodiments, the ratio of first targeting moiety to second targeting moiety is 1 :9.

[0527] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 9:1. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 9:1.

[0528] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 7:3.

[0529] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 9:1. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 9:1. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 7:3.

[0530] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 1:1. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 1 :1.

[0531] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 3:7. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 3:7.

[0532] In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 1:9. In some embodiments, the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 1 :9.

[0533] In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 7:3.

[0534] In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 1 :1. In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 1 :1.

[0535] In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 3:7. In some embodiments, the first targeting moiety comprises a CD2 binding domain and the second targeting moiety comprises a CD7 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 3:7.

[0536] In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 7:3. In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 7:3.

[0537] In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 1:1. In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 1 :1.

[0538] In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is about 3:7. In some embodiments, the first targeting moiety comprises a CD4 binding domain and the second targeting moiety comprises a CD8 binding domain, wherein the ratio of first targeting moiety to second targeting moiety is 3:7.

[0539] In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety may be about 1 :1:1. In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety is in the range of about 3 to 8:1 to 5: 1 to 5.

[0540] In some embodiments, the ratio of first targeting moiety to second targeting moiety to third targeting moiety may be about 6: 1:3, about 6:3:1, about 4:3:3 or about 1:1 :1. In some embodiments, the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain, wherein the ratio of the first targeting moiety to second targeting moiety to third targeting moiety is about 6:1 :3.

[0541] TANDEM TARGETING MOLECULE

[0542] Without wishing to be bound by theory, it will be understood that in an alternative embodiment, the two or more targeting moieties may be combined within a single molecule (e.g. a single polypeptide). Such a molecule may be termed “a tandem targeting molecule”.

[0543] In some embodiments, at least three targeting moieties, at least four targeting moieties, at least five targeting moieties, at least six targeting moieties, at least seven targeting moieties, at least eight targeting moieties, at least nine targeting moieties, or at least ten targeting moieties, may be combined within a single molecule (i.e. a tandem targeting molecule).

[0544] In some embodiments, the nucleic acid particle comprises a tandem targeting molecule comprising a combination of the two or more targeting moieties, wherein the tandem targeting molecule comprises:

[0545] (i) a moiety capable of non-covalently binding to B of Formula (A));

[0546] (ii) a first targeting moiety;

[0547] (iii) a second targeting moiety; and optionally

[0548] (iv) one or more further targeting moieties; wherein each targeting moiety is different and binds to a different target.

[0549] In some embodiments, the tandem targeting molecule comprise the structure B’-X3-B1-X4-B2 (II) wherein

[0550] B’ is the same B’ of Formula (I) (i.e. B’ comprises a moiety capable of non-covalently binding to B of Formula (A));

[0551] X3 is absent or a linking moiety;

[0552] B1 comprises a first targeting moiety;

[0553] X4 is absent or a linking moiety;

[0554] B2 comprises a second targeting moiety; wherein X3 and X4 may be the same or different; wherein each targeting moiety is different and binds to a different target; wherein the targeting moieties are non-covalently bound to the nucleic acid particle through the non-covalent binding of B’ of the compound of Formula (II) to B of the compound of Formula (A).

[0555] In some embodiments, the tandem targeting molecule comprise the structure B’-X3-B1-X4-B2-X5-B3 (III) wherein

[0556] B’ is the same B’ of Formula (I) (i.e. B’ comprises a moiety capable of non-covalently binding to B of Formula (A));

[0557] X3 is absent or a linking moiety;

[0558] B1 comprises a first targeting moiety;

[0559] X4 is absent or a linking moiety;

[0560] B2 comprises a second targeting moiety;

[0561] X5 is absent or a linking moiety;

[0562] B3 comprises one or more further targeting moieties; wherein X3, X4, and X5 may be the same or different; wherein each targeting moiety is different and binds to a different target; wherein the targeting moieties are non-covalently bound to the nucleic acid particle through the non-covalent binding of B’ of the compound of Formula (III) to B of the compound of Formula (A).

[0563] In some embodiments, the tandem targeting molecule may further comprise a third targeting moiety; wherein each targeting moiety is different and binds to a different target. In some embodiments, the tandem targeting molecule may further comprise a fourth targeting moiety; wherein each targeting moiety is different and binds to a different target. In some embodiments, the tandem targeting molecule may further comprise a fifth targeting moiety; wherein each targeting moiety is different and binds to a different target. In some embodiments, the nucleic acid particle comprises one or more tandem targeting molecules as defined above. In some embodiments, the nucleic acid particle comprises a compound of Formula (A) and: (a) two different compounds of Formula (I), (b) a compound of Formula (II), (c) three different compounds of Formula (I), or (d) a compound of Formula (I) and a compound of Formula (II). It will be understood that the, or each, targeting moiety may be any type of targeting moiety as described herein.

[0564] DELIVERY OF PAYLOADS

[0565] The nucleic acid particle according to the invention finds use in a variety of applications in which it is desired to introduce a nucleic acid payload into a target cell, and are particularly of interest where it is desired to express peptide or polypeptide encoded by a nucleic acid in a target cell into which the nucleic acid has been introduced. The agents described herein may be administered by in vitro or in vivo protocols.

[0566] Delivery of nucleic acid payloads using the methods and agents described herein can be used with a variety of target cells such that the nucleic acid payload is introduced into the target cells. The present disclosure may provide for in vitro or in vivo introduction of the payload into the target cell, depending on the location of the target cell.

[0567] For example, where the target cell is an isolated cell, the payload may be introduced directly into the cell under cell culture conditions permissive of viability of the target cell. Alternatively, where the target cell or cells are part of a multicellular organism, the targeting particles described herein may be administered to the organism or host in a manner such that the targeting particles are able to enter the target cell(s).

[0568] By "in vivo" it is meant in the targeting particles are administered to a living body of an animal. By "ex vivo" it is meant that cells are modified outside of the body. Such cells may be returned to a living body.

[0569] The route of administration of the targeting particles to the multicellular organism depends on several parameters, including the nature of the targeting particles. Of particular interest as systemic routes are vascular routes, by which the targeting particles are introduced into the vascular system of the host, e.g., an artery or vein, where intravenous routes of administration are of particular interest in some embodiments.

[0570] For administration, particles typically are present in a pharmaceutical preparation, e.g., comprising a pharmaceutically acceptable carrier, diluent and / or adjuvant, and include an effective amount of the payload. In some embodiments, the particles are administered in an aqueous delivery vehicle, e.g., a saline solution.

[0571] As such, in some embodiments, the particles are administered intravascularly, e.g., intraarterially or intravenously, employing an aqueous based delivery vehicle, e.g., a saline solution.

[0572] In some embodiments, the targeting particles are administered to a multicellular organism in an in vivo manner such that the payload is introduced into a target cell of the multicellular organism.

[0573] Nucleic acid payloads are typically administered under conditions sufficient for expression of the nucleic acid to occur. In some embodiments, the agents and methods described herein result in persistent expression of the nucleic acid payload, as opposed to transient expression. By persistent expression is meant that the expression of nucleic acid at a detectable level persists for an extended period of time, if not indefinitely, following administration of the nucleic acid payload. By extended period of time is meant at least 1 week, usually at least 2 months and more usually at least 6 months. By detectable level is meant that the expression of the nucleic acid is at a level such that one can detect the encoded protein in the mammal, e.g., in the serum of the mammal, at a therapeutic concentration.

[0574] In some embodiments, the above-described persistent expression is achieved with or without integration of the nucleic acid payload into the target cell genome of the host. In some embodiments, the nucleic acid introduced into the target cells integrates into the target cell genome, i.e. , one or more chromosomes of the target cell. In some embodiments, the nucleic acid is maintained episomally, e.g., it is an episomal vector that provides for persistent expression.

[0575] Accordingly, 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. For example, such cells may be genetically modified (e.g., using a transposon or CRISPR / Cas system, as described herein) ex vivo / in vitro or in vivo in a subject being treated, to express a transgene, an antibody, broadly-neutralizing antibodies (bNAbs), an antigen receptor such as a chimeric antigen receptor (CAR), a B cell receptor (BCR) 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.

[0576] In some embodiments, the methods and agents described herein are used to transfect immune effector cells with nucleic acid encoding an antigen receptor for generating immune effector cells genetically modified to express an antigen receptor.

[0577] NUCLEIC ACID PAYLOAD

[0578] The nucleic acid particle of the present invention comprises a nucleic acid payload comprising one or more nucleic acid molecules.

[0579] Typically, the nucleic acid particle described herein encapsulates the nucleic acid payload. 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.

[0580] In some embodiments, the nucleic acid molecules may be RNA. In some embodiments, the nucleic acid molecules may be mRNA. In some embodiments, the nucleic acid molecules may be DNA. In some embodiments, the nucleic acid molecules may be a mixture of RNA and DNA. In some embodiments, the nucleic acid molecules may be a mixture of mRNA and DNA.

[0581] In some embodiments, the nucleic acid payload may further comprise a therapeutic or diagnostic moiety.

[0582] In some embodiments, the nucleic acid particles allow the nucleic acid payload to be delivered to target cells to genetically modify the target cells and enable the target cells to express a biomolecule, e.g., peptide or protein, encoded by the nucleic acid.

[0583] In some embodiments, the target cells may be immune cells or immune effector cells. In some embodiments, the nucleic acid may be nucleic acid encoding an antigen receptor.

[0584] In some embodiments, the payload is a gene editing reagent or tool (e.g., transposon (such as sleeping beauty or piggy bac) or CRISPR / Cas (or related) based system). Such tools (e.g., transposase, gene editing tools like CRISPR / Cas9) for genomic integration / editing may be delivered as protein or coding nucleic acid (DNA or RNA). For example, the payload may comprise one or more nucleic acids (e.g. RNA or mRNA) encoding for a Cas endonuclease (e.g. Cas9) and a gRNA. The payload may comprise a peptide or protein comprising a Cas endonuclease (e.g., Cas9) function and one or more nucleic acids (e.g. RNA, mRNA, DNA or mixtures thereof) comprising a gRNA. Cas is an enzyme that uses CRISPR sequences as a guide to recognize and cleave specific strands of DNA that are complementary to the CRISPR sequence. Cas enzymes, together with CRISPR sequences, form the basis of a technology known as CRISPR-Cas that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.

[0585] 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.

[0586] In some embodiments, the DNA may be in a form selected from a plasmid, minicircle, nanoplasmid, transposon, linear DNA, or mixtures thereof.

[0587] In some embodiments, the nucleic acid is a non-interfering RNA. In some embodiments, an interfering RNA may be understood as RNA that may elicit RNA interference (RNAi) and produce a gene silencing effect. In some embodiments, a non-interfering RNA may be any RNA molecule that does not have this RNAi effect. In some embodiments, a non-interfering RNA is any RNA that is not a siRNA, a miRNA or an aiRNA. In some embodiments, the nucleic acid is not an siRNA. In some embodiments, the nucleic acid is not an aiRNA. In some embodiments, the nucleic acid is not a miRNA. In some embodiments, the RNA may be in a form selected from an mRNA, a circular RNA, a self-replicating RNA (saRNA), a transamplifying RNA (taRNA), a replicon, or mixtures thereof.

[0588] In some embodiments, the nucleic acid is mRNA, saRNA, taRNA, or mixtures thereof. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is a mixture of RNA and DNA. In some embodiments, the nucleic acid is a mixture of DNA and a non-interfering RNA. In some embodiments, the nucleic acid is a mixture of DNA and a mRNA. In some embodiments, the nucleic acid is a mixture of a DNA nanoplasmid and a mRNA. In some embodiments, the nucleic acid is a mixture of a DNA transposon and a mRNA encoding a transposase. In some embodiments, the nucleic acid is a mixture of a DNA transposon encoding a CAR or TCR, and a mRNA encoding a transposase. In some embodiments, the nucleic acid is not siRNA. In some embodiments, the nucleic acid not miRNA. In some embodiments, the nucleic acid is not aiRNA.

[0589] In some embodiments, the nucleic acid is a mixture of DNA and mRNA.

[0590] In some embodiments, the nucleic acid is a mixture of DNA and a non-interfering RNA.

[0591] 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.

[0592] 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.

[0593] 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(1)-alkyl-uracil, such as N7-C1-4 alkylguanine, N6-C1-4 alkyl-adenine, 5-C1-4 alkyl-cytosine, 5-C1-4 alkyl-uracil, and N(1)-C1-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl-cytosine, 5-methyl-uridine (m5U), pseudouridine (i ), and N1-methyl-pseudouridine (ml^P).

[0594] RNA

[0595] In some embodiments, 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 (II), cytidine (C) and guanosine (G). In preferred embodiments, the RNA contains all or a majority of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group. RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered / modified nucleotides (or modified nucleosides) can be referred to as analogs of naturally occurring nucleotides (nucleosides), and the corresponding RNAs containing such altered / modified nucleotides or nucleosides ( / .e., altered / modified RNAs) can be referred to as analogs of naturally occurring RNAs. A molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard ( / .e., naturally occurring) nucleotide residues or analogs thereof). "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), transamplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA. The active ingredient may be mRNA, saRNA, taRNA, or mixtures thereof. The active ingredient is preferably mRNA. In some instances, the active ingredient is not siRNA. mRNA

[0596] In some embodiments, the nucleic acid is mRNA.

[0597] 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.

[0598] In some embodiments, the mRNA relates to an RNA transcript which encodes a peptide, polypeptide or protein.

[0599] 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.

[0600] 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 ( / .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, MAXI script®), 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™).

[0601] 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 (ip) or N(1)-methyl-pseudouridine (m1 ip). 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 (ip), N1-methyl-pseudouridine (m1 ip), and 5-methyl-uridine (m5U). The modified nucleoside is preferably pseudouridine (ip) or N1-methyl-pseudouridine (m1 ip).

[0602] In some embodiments, 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 particles or lipid particles containing RNA as described herein may contain mRNA, saRNA, taRNA, or mixtures thereof. The particles or 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.

[0603] Inhibitory RNA

[0604] In some embodiments, the nucleic acid is an inhibitory RNA.

[0605] The term "inhibitory RNA" as used herein means RNA which selectively hybridizes to and / or is specific for a target mRNA, thereby inhibiting (e.g., reducing) transcription and / or translation thereof. Inhibitory RNA includes RNA molecules having sequences in the antisense orientation relative to the target mRNA. Suitable inhibitory oligonucleotides typically vary in length from five to several hundred nucleotides, more typically about 20 to 70 nucleotides in length or shorter, even more typically about 10 to 30 nucleotides in length. Examples of inhibitory RNA include antisense RNA, ribozyme, iRNA, siRNA and miRNA. In some embodiments of all aspects of the disclosure, the inhibitory RNA is siRNA.

[0606] The term "antisense RNA" as used herein refers to an RNA which hybridizes under physiological conditions to DNA comprising a particular gene or to mRNA of said gene, thereby inhibiting transcription of said gene and / or translation of said mRNA. The size of the antisense RNA may vary from 15 nucleotides to 15,000, preferably 20 to 12,000, in particular 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, 300 to 5,000 nucleotides, such as 15 to 2,000, 20 to 1 ,000, 25 to 800, 30 to 600, 35 to 500, 40 to 400, 45 to 300, 50 to 250, 55 to 200, 60 to 150, or 65 to 100 nucleotides.

[0607] By "small interfering RNA" or "siRNA" as used herein is meant an RNA molecule, preferably greater than 10 nucleotides in length, more preferably greater than 15 nucleotides in length, and most preferably 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length that is capable of binding specifically to a portion of a target mRNA. This binding induces a process, in which said portion of the target mRNA is cut or degraded and thereby the gene expression of said target mRNA inhibited. A range of 19 to 25 nucleotides is the most preferred size for siRNAs. Typically siRNAs comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. Without wishing to be bound by any theory, it is believed that the hairpin area of the siRNA molecule is cleaved intracellularly by the "Dicer" protein (or its equivalent) to form an siRNA of two individual base-paired RNA molecules.

[0608] As used herein, "target mRNA" refers to an RNA molecule that is a target for downregulation. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide as specified herein. In some embodiments, the pharmaceutically active peptide or polypeptide is one whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with a disease. In some embodiments, the target mRNA comprises an ORF encoding a pharmaceutically active peptide or polypeptide whose expression (in particular increased expression, e.g., compared to the expression in a healthy subject) is associated with cancer. According to the present disclosure, siRNA can be targeted to any stretch of approximately 19 to 25 contiguous nucleotides in any of the target mRNA sequences (the "target sequence"). Techniques for selecting target sequences for siRNA are given, for example, in Tuschl T. et al., "The siRNA User Guide", revised Oct. 11 , 2002, the entire disclosure of which is herein incorporated by reference. Further guidance with respect to the selection of target sequences and / or the design of siRNA can be found on the webpages of Protocol Online (www. protocolonline. com) using the keyword "siRNA". Thus, in some embodiments, the sense strand of the siRNA used in the present disclosure comprises a nucleotide sequence substantially identical to any contiguous stretch of about 19 to about 25 nucleotides in the target mRNA. siRNA can be obtained using a number of techniques known to those of skill in the art. For example, siRNA can be chemically synthesized or recombinantly produced. Preferably, siRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. Selection of other suitable promoters is within the skill in the art. Selection of plasmids suitable for transcribing siRNA, methods for inserting nucleic acid sequences for expressing the siRNA into the plasmid, and IVT methods of in vitro transcription of said siRNA are within the skill in the art.

[0609] The term "miRNA" (microRNA) as used herein relates to non-coding RNAs which have a length of 21 to 25 (such as 21 to 23, preferably 22) nucleotides and which induce degradation and / or prevent translation of target mRNAs. miRNAs are typically found in plants, animals and some viruses, wherein they are encoded by eukaryotic nuclear DNA in plants and animals and by viral DNA (in viruses whose genome is based on DNA), respectively. miRNAs are post- transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression or target degradation and gene silencing. miRNA can be obtained using a number of techniques known to those of skill in the art. For example, miRNA can be chemically synthesized or recombinantly produced using methods known in the art (e.g., by using commercially available kits such as the miRNA cDNA Synthesis Kit sold by Applied Biological Materials Inc.). Preferably, miRNA is transcribed from recombinant circular or linear DNA plasmids using any suitable promoter. DNA

[0610] In some embodiments, the nucleic acid is DNA.

[0611] Herein, the term "DNA" relates to a nucleic acid molecule which includes deoxyribonucleotide residues. DNA typically comprises the naturally occurring nucleic acids adenosine (dA), thymidine (dT), cytidine (dC) and guanosine (dG) ("d" represents "deoxy"). In preferred embodiments, the DNA contains all or a majority of deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide which lacks a hydroxyl group at the 2'- position of a p-D-ribofuranosyl group. DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and / or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA. A molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxy-ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard ( / .e., naturally occurring) nucleotide residues or analogs thereof). DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA. The cDNA may be obtained by reverse transcription of RNA. The DNA may comprise a plasmid, a nanoplasmid, a minicircle, a transposon, or linear DNA such as doggybone DNA.

[0612] Pharmaceutically active peptides or polypeptides

[0613] "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 ( / .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.

[0614] In some embodiments, the nucleic acid 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 particle or lipid particle comprising RNA and / or DNA may comprise one or more species of RNA and / or DNA, wherein each RNA and / or DNA encodes a different peptide or protein.

[0615] 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 (qj), N1-methyl-pseudouridine (m1i ), and 5-methyl-uridine); and / or codonoptimization), or (iii) both (i) and (ii).

[0616] 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.

[0617] Specific examples of pharmaceutically active peptides and proteins include, but are not limited to, cytokines, interferons, such as interferon-alpha (IFN-a), interferon beta (I FN|3) or interferongamma (IFN-y), interleukins, such as interleukin 2 (IL2), IL-4, IL7, IL-10, IL-11 , IL12, IL15, IL- 21 and IL23, colony stimulating factors, such as colony stimulating factor (CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor (TNF), erythropoietin (EPO), and bone morphogenetic protein (BMP); immunoglobulin superfamily members including antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD-3y, CD3-5, CD-3E, CD79a, CD79b), co-stimulatory or inhibitory molecules (e.g., CD28, CD80, CD86); other immunologically active compounds such as tumor- associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens.

[0618] 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 nucleic acid 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.

[0619] In some embodiments, the nucleic acid payload provides a gene editing reagent or tool (e.g., a transposon / transposase system (such as sleeping beauty or piggy bac), a Large serine recombinases (LSRs)(e.g., Bxb1 and PhiC31), Zinc-finger nucleases (ZFN) or CRISPR / Cas (or related) based system).

[0620] 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.

[0621] 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.

[0622] 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.

[0623] 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.

[0624] 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).

[0625] In some embodiments, the nucleic acid payload 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.

[0626] In some embodiments, the gene editing tool may comprise a gene editing enzyme (e.g. a nuclease).

[0627] In some embodiments, the gene editing tool may comprise a gene editing enzyme (e.g. a nuclease) and a DNA template.

[0628] In some embodiments, the nucleic acid payload provides a CRISPR / Cas (e.g. Cas9) gene editing tool. In some embodiments, the CRISPR / Cas (e.g. Cas9) gene editing tool may 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 comprised in a DNA nanoplasmid.

[0629] In some embodiments, the nucleic acid payload 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.

[0630] In some embodiments, the nucleic acid payload 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 comprised in a DNA nanoplasmid.

[0631] In some embodiments, the nucleic acid payload 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 comprised in a DNA nanoplasmid.

[0632] In some embodiments, the nucleic acid payload 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.

[0633] In some embodiments, the nucleic acid payload 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 comprised in a DNA nanoplasmid.

[0634] In some embodiments, the nucleic acid payload 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-finger 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 payload 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 payload 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).

[0635] EXEMPLARY PAYLOADS

[0636] In some embodiments, there is provided a nucleic acid particle comprising:

[0637] (i) one or more particle forming components;

[0638] (ii) a nucleic acid payload comprising one or more nucleic acid molecules; and

[0639] (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different, wherein the nucleic acid payload provides a gene editing reagent or tool.

[0640] In some embodiments, the nucleic acid payload 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.

[0641] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising one or more nucleic acid molecules, wherein the nucleic acid payload comprises a gene editing tool, wherein the nucleic acid payload comprises RNA and / or DNA, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0642] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising a DNA (e.g. a DNA template) and an mRNA encoding a gene editing enzyme, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0643] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising a DNA nanoplasmid and an mRNA encoding a gene editing enzyme, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0644] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload encoding a CRISPR / Cas (e.g. Cas9) gene editing tool, wherein the nucleic acid payload comprises DNA and / or RNA, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0645] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload 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, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0646] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload encoding a CRISPR / Cas (e.g. 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 RNA, and wherein a DNA template is comprised in a DNA nanoplasmid, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0647] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload encoding a ZFN gene editing tool, wherein the nucleic acid payload comprises DNA and / or RNA, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0648] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload encoding a ZFN gene editing tool, wherein the nucleic acid payload comprises RNA, wherein at least two ZFN proteins are encoded by mRNA, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0649] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload 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 and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different.

[0650] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising one or more nucleic acid molecules, wherein the nucleic acid payload comprises a gene editing tool, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to CD3 and a second targeting moiety is capable of binding to CD7.

[0651] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising one or more nucleic acid molecules, wherein the nucleic acid payload comprises a gene editing tool, and (iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to CD3 and a second targeting moiety is capable of binding to CD2.

[0652] In some embodiments, there is provided a nucleic acid particle comprising (i) one or more particle forming components, (ii) a nucleic acid payload comprising one or more nucleic acid molecules, wherein the nucleic acid payload comprises a gene editing tool, and (iii) three or more targeting moieties, wherein a first targeting moiety is capable of binding to CD3, a second targeting moiety is capable of binding to CD2, and a third targeting moiety is capable of binding to CD7.

[0653] Suitably, at least one targeting moiety is non-covalently bound to the nucleic acid particle - as described herein. In some embodiments, the one or more particle forming components comprises one or more lipid particle forming components, wherein the one or more lipid particle forming components comprises a cationic or cationically ionizable lipid. Suitable cationic or cationically ionisable lipids are described herein.

[0654] PARTICLE FORMING COMPONENTS

[0655] The nucleic acid particle of the present invention comprises particle forming components.

[0656] It will be understood that the term “particle forming components” may encompass “lipid particle forming components”, as used herein.

[0657] In some embodiments, the one or more particle forming components comprise a cationic or cationically ionizable lipid, as defined and exemplified herein. In some embodiments, such particles are lipid nanoparticles (LNP) as defined herein. In some embodiments, such particles are lipoplexes (LPX) as defined herein.

[0658] In some embodiments, one or more particle forming components comprises a cationic polymer, as defined and exemplified herein. In some embodiments, particles formed from cationic polymers are polyplexes (PLX) as defined herein. In some embodiments, particles formed from cationic polymers are lipidated polyplexes (LPLX), as defined herein.

[0659] POLYMERS

[0660] In some embodiments, the particles (e.g. LNPs) of the present invention comprise polymers.

[0661] In the specification the term "polymer" is given its ordinary meaning, i.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, i.e., a biopolymer such as a protein. In some cases, additional moieties can also be present in the polymer.

[0662] Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense negatively charged nucleic acid into nanoparticles. These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture.

[0663] Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers for use as particle forming components herein. In addition, some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0664] If more than one type of repeat unit is present within the polymer, then the polymer is said to be a "copolymer." It is to be understood that the polymer being employed herein can 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, i.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.

[0665] In some embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and / or biologically, within a physiological environment, such as within the body.

[0666] In some embodiments, polymer may be protamine or polyalkyleneimine. In some embodiments, the particle forming components may comprise protamine or polyalkyleneimine. The term "protamine" refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish). In particular, the term "protamine" refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.

[0667] According to the disclosure, the term "protamine" as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.

[0668] In some embodiments, the polyalkyleneimine comprises polyethylenimine and / or polypropylenimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75 102to 107Da, preferably 1000 to 105Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.

[0669] Preferred according to the disclosure is linear polyalkyleneimine such as linear polyethyleneimine (PEI).

[0670] Stealth polymers

[0671] In some embodiments, the polymer is a hydrophilic polymer and the connector 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.

[0672] Consequently, the polymer component faces the outside of said particle and forms a protective hydrophilic shell surrounding the particle. In some embodiments, the polymer portion of the amphiphilic derivative contributes to conferring stealth properties on the particles. In some embodiments, the polymer portion of the amphiphilic derivative confers stealth properties on the particles. In some embodiments, the plasmatic half-life of the particles described herein is greater than 2 hours, e.g., between 3 and 10 hours. This characteristic advantageously allows the particles to accumulate at the target cells and to liberate therein their contents (payload) within reasonable amounts of time. The effectiveness of the targeted delivery described herein therefore increases as a result.

[0673] 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.

[0674] In some embodiments, the polymer is selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), polyoxazoline (POX), polyoxazine (POZ), and poly-2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA) (including derivatives thereof), as defined and exemplified below.

[0675] 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.

[0676] 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.

[0677] 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.

[0678] In some embodiments, the PEG comprises "PEG2k", also termed "PEG 2000", which has an average molecular weight of about 2000 Daltons.

[0679] In some embodiments, DSPE-PEG2000, DSPE-PEG3000 and DSPE-PEG5000 are used as the amphiphilic derivative of a polymer.

[0680] 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.

[0681] In some embodiments, a pSar comprises the structure of the following general formula: wherein s is the number of sarcosine units.

[0682] 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.

[0683] 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.

[0684] 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.

[0685] 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.

[0686] 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 some 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.

[0687] 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.

[0688] 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.

[0689] 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 Ri 1 may be selected from two different alkyl groups (such as methyl and ethyl) and not all Rn are the same alkyl. In any of the above embodiments, Rn preferably is methyl or ethyl, more preferably methyl. Thus, in some embodiments, each Rn is methyl or each Rn is ethyl. In some alternative embodiments, Rn is independently selected from methyl and ethyl for each repeating unit, wherein in at least one repeating unit Rn is methyl, and in at least one repeating unit Rn is ethyl. In some embodiments, the polymer comprises poly-2-(2-(2-aminoethoxy)ethoxy)-acetic acid (pAEEA) or poly-2-(2-(2-methylaminoethoxy)ethoxy)acetic acid (pMAEEA), or a derivative thereof, as defined herein.

[0690] In some embodiments, the polymer comprises the following general formula: wherein

[0691] X11and X12taken together are optionally substituted amide, optionally substituted thioamide or ester;

[0692] Y is -CH2-, -(CH2)2-, or -(CH2)3-; z is 2 to 24; and n is 1 to 100.

[0693] In some embodiments,

[0694] (i) when X11is -C(O)- then X12is -NR1-;

[0695] (ii) when X11is -NR1- then X12is -C(O)-;

[0696] (iii) when X11is -C(S)- then X12is -NR1-;

[0697] (iv) when X11is -NR1- then X12is -C(S)-;

[0698] (v) when X11is -C(O)- then X12is -O-; or

[0699] (vi) when X11is -O- then X12is -C(O)-; wherein R1is hydrogen or Ci-s alkyl.

[0700] In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen or Ci-s alkyl. In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen or methyl. In some embodiments, X11is -C(O)- and X12is -NR1-, wherein R1is hydrogen.

[0701] In some embodiments, Y is -CH2- or -(CH2)2-. In some embodiments, Y is -CH2-.

[0702] In some embodiments, the polymer comprises the following general formula: wherein

[0703] R1is hydrogen or Ci-s alkyl; z is 2 to 24; and n is 1 to 100.

[0704] In some embodiments of the above formulas, z is 2 to 10. In some embodiments, z is 2 to 7.

[0705] In some embodiments, z is 2 to 5. In some embodiments, z is 2 or 3. In some embodiments, z is 2.

[0706] In some embodiments, the polymer comprises the following general formula: wherein

[0707] R1is hydrogen or C1-8 alkyl; and n is 1 to 100.

[0708] In some embodiments of the above formulas, R1is hydrogen or methyl. In some embodiments, R1is hydrogen.

[0709] In some embodiments, the polymer comprises the following general formula: wherein n is 1 to 100.

[0710] 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.

[0711] 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%.

[0712] Cationic polymers

[0713] In some embodiments, the particles of the present invention, especially the polyplex (PLX) particles and lipidated-polyplex (LPLX) particles, comprise a cationic polymer, as a particle forming component. In this specification, the term “cationic polymer” means a polymer, as defined generally herein, which carries at least one cation, i.e. an ionic species having a positive charge. Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid. In some embodiments, cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g. by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.

[0714] In some embodiments, a “targeting compound” (e.g., a compound of Formula (A)) is incorporated into the particle comprising a cationic polymer through a negative charge in the moiety incorporating the connector compound into the particle interacting with a positive charge of the particle. For example, where the one or more particle forming components comprise a cationic polymer and further comprise a compound of Formula (A), L of the Compound of Formula (A) comprises a moiety comprising a negative charge.

[0715] In some embodiments, a targeting compound (e.g., the compound of Formula (A)) is incorporated into the particle comprising a cationic polymer through a moiety incorporating the connector compound into the particle comprising an anionic polymer. In some embodiments, the cationic polymer comprises one or more selected from the group consisting of cationic or polycationic peptides or proteins, including protamine, spermin or spermidine, poly-lysine, poly-arginine, cationic polysaccharides, including chitosan, cationic polymers, including poly(ethyleneimine), poly(propyleneimine), polybrene, polyallylamines, and polyvinylamine. In some embodiments, the polymer comprises a polyamidoamine (PAMAM) polymer.

[0716] In some embodiments, a cationic polymer is a homopolymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L- lysine, poly-L-arginine, poly-L-histidine, and poly(2-aminoethyl methacrylate), or a pharmaceutically acceptable salt thereof.

[0717] It is understood that polymers described herein can be linear or branched. In some embodiments, a cationic polymer is linear. In some embodiments, a cationic polymer is a linear polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2- aminoethyl methacrylate). In some embodiments, a cationic polymer is a branched polymer selected from poly(ethylenimine), poly(propylenimine), polybrene, polyallylamine, polyvinylamine, polyamidoamine, poly-L-lysine, poly-L-arginine, poly-L-histidine, and poly(2- aminoethyl methacrylate).

[0718] In some embodiments, the cationic polymer comprises poly(ethyleneimine). In some embodiments, the poly(ethyleneimine) is a linear polymer. In some embodiments, the poly(ethyleneimine) is a branched polymer. In some embodiments, the poly(ethyleneimine) has a mean molar mass between 1000 Da and 150000 Da, between 5000 Da and 100000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or of about 22500 Da. In some embodiments, the poly(ethyleneimine) has a mean molar mass between 22500 Da and 150000 Da.

[0719] In some embodiments, the nucleic acid particles of the present invention, especially the polyplex (PLX) particles and lipidated-polyplex (LPLX) particles, comprise a polyamine derivative, e.g., a carboxylated polyamine derivative, as particle forming component. Polyamines form polycations in solution, which facilitates the complex formation with polyanions such as nucleic acids.

[0720] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety.

[0721] In some embodiments, a polyamine derivative which is useful herein as delivery vehicle for polyanions comprises: a polyamine moiety comprising a plurality of amino groups; a plurality of carboxylated substituents comprising a carboxyl group bonded via a hydrophobic linker to amino groups of said polyamine moiety, wherein each of said carboxylated substituents comprises from 6 to 40 carbon atoms, preferably from 6 to 20 carbon atoms, and more preferably from 8 to 16 carbon atoms, and each of said hydrophobic linker may comprise from 1 to 3 heteroatoms selected from O, N, and S; and a plurality of hydrophobic substituents bonded to amino groups of said polyamine moiety, wherein each of said hydrophobic substituents comprises at least 2 carbon atoms, preferably from 6 to 40 carbon atoms, and may comprise from 1 to 3 heteroatoms selected from O, N, and S provided said hydrophobic substituent has at least 6 carbon atoms. In some embodiments, the polyamine derivative has a linear poly(ethyleneimine) moiety of from 2 to 500 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls.

[0722] In some embodiments, the polyamine derivative has a branched poly(ethyleneimine) moiety of from 0.5 to 200 kDa (in terms of number average molecular weight), the carboxylated substituents have from 10 to 16 carbon atoms and are n-alkylcarboxylic acids and the hydrophobic substituents have from 1 to 12 carbon atoms and are alkyls, preferably n-alkyls, and / or alkylarylalkyls.

[0723] In some embodiments, the particle forming components comprise a compound comprising the following formula:

[0724] The particles described herein, especially the polyplex (PLX) and lipopolyplex (LPLX) particles, may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0725] Anionic polymers

[0726] In some embodiments, the particles of the present disclosure, especially when in the form of a polyplex (PLX), contain an anionic polymer. In this specification, the term “anionic polymer” means a polymer, as defined generally herein, which carries at least one anion, i.e. an ionic species having a negative charge. In some embodiments, one or more particle forming components of the particles of the present disclosure comprise a cationic polymer (as described above), and further comprise a compound of Formula (A) (as described above), wherein L comprises an anionic polymer. In some embodiments, particularly where the particle is a polyplex, L of the compound of Formula (A) comprises an anionic polymer.

[0727] An anionic polymer described herein can be linear or branched, and comprises one or more anionic moieties or groups. In some embodiments, an anionic polymer is a polyanionic polymer, e.g., a polymer having one or more anionic groups. In some embodiments, an anionic group is a -CO2; a -OSO3; or a -OPC>32' group. In some embodiments, the anionic polymer is a homopolymer. In some embodiments, the anionic polymer is a heteropolymer.

[0728] In some embodiments, an anionic polymer is polyglutamic acid. In some embodiments, an anionic polymer is poly-L-glutamic acid. In some embodiments, an anionic polymer is poly aspartic acid. In some embodiments, an anionic polymer is poly-L-aspartic acid. In some embodiments, an anionic polymer is a polyphosphate. In some embodiments, particularly where the particle is a polyplex, L of the compound of Formula (A) comprises a polyglutamic acid.

[0729] In some embodiments, an anionic polymer is a homopolymer. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 150 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 10 to about 100 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 20 to about 100 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 20 to about 80 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 50 repeating monomeric units. In some embodiments, an anionic polymer is a homopolymer comprising about 100 repeating monomeric units.

[0730] In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 150 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 10 to about 100 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 100 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 20 to about 80 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 50 repeating units of glutamic acid. In some embodiments, an anionic polymer is a poly-L-glutamic acid homopolymer comprising about 100 repeating units of glutamic acid. In some embodiments, particularly where the particle is a polyplex, L of the compound of Formula (A) comprises a poly-L-glutamic acid homopolymer comprising about 100 repeating units of glutamic acid.

[0731] LIPIDS AND AMPHIPHILES

[0732] The nucleic acid-lipid particles of the invention also contain a mixture of lipids.

[0733] 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.

[0734] 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.

[0735] 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 both cationic and anionic lipids both contain such groups, they are therefore amphiphiles. In this specification the term “cationic lipid” is therefore synonymous with “cationic amphiphile” and the term “anionic lipid” is synonymous with “anionic amphiphile”.

[0736] 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 and may further include anionic groups such as phosphate, phosphonate, carboxylic acid, sulfate, sulfonate (all as defined and exemplified above) and other 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.

[0737] 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.

[0738] 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). The 24 to 60 carbon atoms can be segmented into two or more hydrophobic moieties, with each such moiety typically having at least 6 carbon atoms. An example for segmented hydrophobic moieties wherein each segment is hydrocarbyl are lipids comprising the DACA moiety as described in WO2011 / 003834 wherein each of the acyl or alkyl groups comprise between 12 and 20 carbon atoms. Another example are lipids wherein the hydrophobic moiety comprises a steroid moiety, such as a cholesteryl moiety.

[0739] 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-315, as defined and exemplified below. CATIONIC AND CATIONICALLY IONIZABLE LIPIDS

[0740] In some embodiments, the nucleic acid-lipid particles of the present invention also contain a cationic lipid or cationically ionizable lipid, or a mixture of any thereof.

[0741] 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 some embodiments, the cationic lipid is a monovalent cationic lipid.

[0742] In some embodiments, the cationic lipid contains a charged polar moiety selected from the group consisting of guanidinium, ammonium, imidazolium, pyridinium, amidinium, and piperazinium.

[0743] Examples of cationic lipids include, but are not limited to 1 ,2-dialkyloxy-3-dimethylammonium propanes and 1 ,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 1 ,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 1 ,2-dioleoyl-3- trimethylammonium propane (DOTAP) or 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP); dimethyldioctadecylammonium (DDAB); dioctadecyldimethyl ammonium chloride (DODAC), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE); 1 ,2- dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1 ,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-1-propanamium trifluoroacetate (DOSPA).

[0744] Further suitable cationic lipids are described in Sun and Lu, Pharmaceutical Research, 2023, https : / / do i . org / 10.1007 / s 11095-022-03460-2. In some embodiments, the lipid is 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, i.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.

[0745] 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.

[0746] In some embodiments, the cationic or cationically ionizable lipid is a compound represented by formula (TL-I):

[0747] TL-I or a pharmaceutically acceptable salt thereof, wherein:

[0748] L1and L2are each independently an optionally substituted C1-C30 aliphatic group;

[0749] L3is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10- membered heteroaliphatic group;

[0750] X1and X2are each independently selected from a bond, -OC(O)-, -C(O)O-, -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-, wherein one or both of X1or X2is 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 R1is, independently, at each instance, optionally substituted Ci-C2o aliphatic or H;

[0751] T1and T2are each independently an optionally substituted C3-C30 aliphatic;

[0752] 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), or-R4; each R2is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic or OR3; or two instances of R2come 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 R3is, independently, at each instance, selected from the group consisting of H and optionally substituted C1-C10 aliphatic; and R4is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, C6-C12 aryl substituted with one or more of -(CH2)o-6-OH or -(CH2)o-6- N(R5)2, or C3-C12 cycloaliphatic substituted with one or more of oxo, -(CH2)o-6-OH, or-(CH2)o- 6-N(R5)2; each R5is independently selected from H and optionally substituted Ci-Ce aliphatic.

[0753] In some embodiments of formula (TL-I), L1and L2are each independently -(CH2)e-io-.

[0754] In some embodiments of formula (TL-I), X1and X2are each independently selected from a - 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-.

[0755] In some embodiments of formula (TL-I), X1and X2are each -S(O)2N(R1)-, where each R1is independently R1is C1-C10 aliphatic.

[0756] In some embodiments of formula (TL-I), T1and T2are each independently selected from optionally substituted C3-C20 alkyl.

[0757] In some embodiments of formula (TL-I), T1and T2are 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.

[0758] In some embodiments of formula (TL-I), G is -N(H)C(S)N(R2)2, where each R2is selected from optionally substituted Ci-Ce aliphatic and OH.

[0759] In some embodiments of formula (TL-I), G is -OH.

[0760] In some embodiments of formula (TL-I), G is selected from:

[0761] In some embodiments of formula (TL-I), -L3-G is selected from:

[0762] In some embodiments of formula (TL-I), the compound is represented by Formula (TL-lla):

[0763] TL-lla or a pharmaceutically acceptable salt thereof. In some embodiments of formula (TL-I), the compound is represented by Formula (TL-llc):

[0764] TL-llc or a pharmaceutically acceptable salt thereof.

[0765] In some embodiments of formula (TL-I), the compound is represented by Formula (TL-lllb):

[0766] (TL-lllb) or a pharmaceutically acceptable salt thereof.

[0767] In some embodiments of formula (TL-I), the compound is represented by Formula (TL-llle):

[0768] Hie or a pharmaceutically acceptable salt thereof

[0769] In some embodiments of formula (TL-I), the compound is In some embodiments of formula (TL-I), the compound is 7,7’-((4- hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide) or a pharmaceutically acceptable salt thereof.

[0770] In some embodiments of formula (TL-I), the compound is 7 ,7’-((4-(3,3- dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide) or a pharmaceutically acceptable salt thereof.

[0771] Thiolipid compounds of formula (TL-I) can be prepared according to the following General

[0772] Schemes 1 , 2, and 3, as also described in PCT / EP2023 / 071270: General Scheme 1

[0773] General Scheme 3

[0774] In these schemes, a tail-linker moiety is any bivalent linker, such as an aliphatic or heteroaliphatic group; a tail-end is a hydrophobic group, e.g., an aliphatic group, a head group is a polar or cationic or ionizable head group, a tail junction is a biodegradable group, such as an ester, or a sulfur-containing moiety (e.g., thioether, sulfonyl, or sulfonamide), and a headtail junction is a central atom or functional group connecting a tail or tails to the head group (e.g., a tertiary amine group).

[0775] Therefore, as described herein, the present disclosure provides a compound represented by

[0776] Formula (TL-IV):

[0777] L4-X3-T3

[0778] G1-L6-N'

[0779] 'L5-X4-T4

[0780] TL-IV or a pharmaceutically acceptable salt thereof, the method comprising: contacting a compound represented by Formula (TL-V)

[0781] G1-L6-NH2

[0782] TL-V with a compound represented by one of Formulae (TLVIa)-(TL-Vlc)

[0783] TL-Vla TL-Vlb TL-VIc

[0784] (TL-VI) and a compound represented by one of Formula (TL-VI la)-(TL-Vllc)

[0785] TL-Vlla TL-Vllb TL-Vllc in the presence of a reducing agent, wherein: each of L4and L5are each independently an optionally substituted C1-C30 aliphatic group;

[0786] L6is a bond, optionally substituted C1-C10 aliphatic group, or optionally substituted 2- to 10- membered heteroaliphatic group;

[0787] X3and X4are each independently selected from a bond, -OC(O)-, -C(O)O-, -S(O)2N(R40)-, - N(R40)S(O)2, -S(O)-, -S(O)2-, -S(O)2C(R40)2-, -OC(S)C(R40)2-, -C(R40)2C(S)O-, or -S-, wherein one or both of X3or X4is selected from -S(O)2N(R40)-, -N(R40)S(O)2, -S(O)-, -S(O)2-, - S(O)2C(R40)2-, -OC(S)C(R40)2-, -C(R40)2C(S)O-, or -S-; each R40is, independently, at each instance, optionally substituted Ci-C2o aliphatic or H;

[0788] T3and T4are each independently an optionally substituted C3-C2o aliphatic;

[0789] G1is -N(R6)C(S)N(R6)2, -OH, -N(R6)2, -N(R9)C(O)R7, -N(R9)S(O)2R7, -N(R9)C(O)N(R7)2, - CH(N-R7), or-R8; each G2is independently O or N2; each G3is independently halogen (e.g., Cl, Br, or I), -OTs, or OTf; each R6is, independently, at each instance, selected from the group consisting of H, optionally substituted Ci-Ce aliphatic or OR7; or two instances of R6come 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 R7is, independently, at each instance, selected from the group consisting of H and optionally substituted Ci-Ce aliphatic;

[0790] R8is optionally substituted 4- to 12-membered heterocycle, optionally substituted 4- to 12 membered heteroaryl, Ce-Ci2aryl substituted with one or more of -(CH2)o-6-OH or -(CH2)o-6- N(R9)2, or C3-Ci2cycloaliphatic substituted with one or more of oxo, -(CH2)o-6-OH, or-(CH2)o- 6-N(R9)2; and each R9is independently selected from H and optionally substituted Ci-Ce aliphatic.

[0791] In some embodiments, a reducing agent is NaBH3CN or NaBH(OCOCH3)3. In some embodiments, a reducing agent is NaBH3CN. In some embodiments, a reducing agent is NaBH(OCOCH3)3.

[0792] In some embodiments, a compound as described herein further comprises a compound represented by Formula (TL-Vla) or the compound represented by Formula (TL-Vlla), wherein G1is O,

[0793] TL-Vla TL-Vlla by contacting a compound represented by Formula (TL-VIII) or a compound represented by

[0794] Formula (TL-IX):

[0795] TL-VIII TL-IX with an oxidizing agent.

[0796] In some embodiments, an oxidizing agent is DMSO, PCC, or DMP. In some embodiments, an oxidizing agent is DMSO, and the method further comprises contacting the compound represented by Formula VIII or IX and DMSO with a sulfur trioxide pyridine complex (SOa’pyridine).

[0797] In some embodiments, a compound described herein further comprises the compound represented by Formula (TL-VIII) or (TL-IX):

[0798] TL-VIII TL-IX by contacting a compound represented by Formula (TL-X) or a compound represented by Formula (TL-XI):

[0799] Yi Y,

[0800] TL-X TL-XI with a compound represented by Formula (TL-XII) or a compound represented by Formula (TL-XIII):

[0801] TL-XII TL-XIII in the presence of H2O2 and SOCh; and wherein each Y1is halogen.

[0802] In some embodiments, the cationic or cationically ionizable lipid is selected from the group consisting of:

[0803] 7, 7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide) (BNT-51);

[0804] 7, 7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide)

[0805] [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315);

[0806] ((3-hydroxypropyl)azanediyl)-bis(nonane-9, 1 -diyl) bis(2-butyloctanoate)

[0807] 1.2-dioleoyloxy-3-dimethylaminopropane (DODMA);

[0808] 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);

[0809] 1.2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA); di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319); b / s-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)nonanamido)-nonadecanedioate (A9);

[0810] (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);

[0811] O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-

[0812] (dimethylamino)butanoate (HY501);

[0813] 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA-2);

[0814] 4-((di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)oxy)- / V, / \ / -dimethyl-4-oxobutan-4-amine

[0815] (HYAM-2);

[0816] ((2-(4-(dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8, 1 -diyl) bis(2- hexyldecanoate) (EA-405);

[0817] (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1 -diyl) bis(2-hexyldecanoate) (HY-

[0818] 405); palmitoyl-oleoyl-nor-arginine (PONA); guanidino-di[(heptadecyl)methyl]carboxylic acid (GUADACA);

[0819] 4-methylpyridinium-di(heptadecyl)methylcarboxylic acid (MPDACA);

[0820] 1.2-dioleoyl-3 trimethylammonium propane (DOTAP);

[0821] 1.2-dioleoyl-3-dimethylammomium propane (DODAP);

[0822] 1.2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); BODD-C2C4-PipZ (bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate)

[0823] BHD-C2C2-PipZ (bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate)

[0824] BODD-C2C2-DMA (bis(2-octyldodecyl) 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate)

[0825] BODD-C2C2-1 Me-Pyr (bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2- yl)ethyl)azanediyl)dipropionate)

[0826] BODD-C2C2-Pyr (bis(2-octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate)

[0827] 7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1 -sulfonamide 0 (BL-207) or a mixture of any thereof.

[0828] In some embodiments, the cationically ionizable lipid is 7,7’-((4-hydroxybutyl)azanediyl)bis(N- hexyl-N-octylheptane-1-sulfonamide) (BNT-51). In some embodiments, the cationically ionizable lipid is [(4-hydroxybutyl)azanediyl]-di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC- 315). In some embodiments, the cationically ionizable lipid is 1 ,2-dioleoyloxy-3- dimethylaminopropane (DODMA). In some embodiments, the cationically ionizable lipid is 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA). In some embodiments, the cationically ionizable lipid is heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (D-Lin-MC3-DMA). In some embodiments, the cationically ionizable lipid is 1 ,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA). In some embodiments, the cationically ionizable lipid is di((Z)-non-2-en-1-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319). In some embodiments, the cationically ionizable lipid is b / s-(2-butyloctyl) 10-(N-(3-(dimethylamino)propyl)-nonanamido)- nonadecanedioate (A9). In some embodiments, the cationically ionizable lipid is (heptadecan- 9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)octyl]amino}-octanoate) (L5). In some embodiments, the cationically ionizable lipid is heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino}-octanoate) (SM-102). In some embodiments, the cationically ionizable lipid is O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}- amino]4-(dimethylamino)butanoate (HY501). In some embodiments, the cationically ionizable lipid is 2-(di-((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl 4-(dimethylamino)butanoate (EA- 2).

[0829] In some embodiments, the cationically ionizable lipid is BODD-C2C4-PipZ. In some embodiments, the cationically ionizable lipid is BHD-C2C2-PipZ. In some embodiments, the cationically ionizable lipid is BODD-C2C2-DMA. In some embodiments, the cationically ionizable lipid is BODD-C2C2-1Me-Pyr. In some embodiments, the cationically ionizable lipid is BODD-C2C2-Pyr.

[0830] In some embodiments, the cationically ionizable lipid is selected from those described generally and specifically in WO 2018 / 087753.

[0831] In some embodiments, the cationically ionizable lipid is 4-((di-((9Z,12Z)-octadeca-9,12-dien- 1-yl)amino)oxy)- / V, / \ / -dimethyl-4-oxobutan-4-amine (HYAM-2). In some embodiments, the cationically ionizable lipid is ((2-(4-(dimethylamino)butanoyl)-oxy)ethyl)-azanediylbis(octane

[0832] 8.1-diyl) bis(2-hexyldecanoate) (EA-405). In some embodiments, the cationically ionizable lipid is (2-(4-(dimethylamino)butanoyl)-oxy)azanediylbis-(octane 8,1-diyl) bis(2- hexyldecanoate) (HY-405). In some embodiments, the cationically ionizable lipid is O-[N- {(9Z, 12Z)-octadeca-9, 12-dien-1 -yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4- (dimethylamino)butanoate (HY501).

[0833] In some embodiments, the cationic or cationically ionizable lipid is selected from the group consisting of:

[0834] 7, 7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide) (BNT-51); 7,7’- ((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1 -sulfonamide) (BNT-52)J(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315);

[0835] 1 .2-dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta-6,9,28,31-tetraen-19-yl- 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo- 6-(undecyloxy)hexyl]amino}-octanoate) (SM-102) ; O-[N-{(9Z, 12Z)-octadeca-9, 12-dien-1 -y I)}- N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); ((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); 1 ,2-dioleoyl-3 trimethylammonium propane (DOTAP); 1 ,2-dioleoyl-3- dimethylammomium propane (DODAP); 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); BHD-C2C2-PipZ (bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate); BODD-C2C2-1Me-Pyr (bis(2-octyldodecyl) 3,3'-((2-(1 - methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate); or a mixture of any thereof.

[0836] In some embodiments, the cationic or cationically ionizable lipid is ((3- hydroxypropyl)azanediyl)-bis(nonane-9,1-diyl) bis(2-butyloctanoate) (ALC-0366).

[0837] In some embodiments, the cationic or cationically ionisable lipid is 7,7’-((4- hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1 -sulfonamide (BL-207).

[0838] In some embodiments, the cationic or cationically ionisable lipid is present in an amount of 20 to 70 mol% of the total lipids present in the lipid mixture. In some embodiments, the cationic or cationically ionisable lipid is present in an amount of 30 to 60 mol% of the total lipids present in the lipid mixture. In some embodiments, the cationic or cationically ionisable lipid is present in an amount of 40 to 50 mol% of the total lipids present in the lipid mixture. The term “lipid mixture” in this context applies to the lipid mixture component of both the aqueous dispersion and the nucleic acid-lipid particle.

[0839] ADDITIONAL LIPIDS

[0840] The nucleic acid-lipid particles of the present invention may further comprise one or more additional lipids. In some embodiments, the one or more additional lipids comprise a grafted lipid, as defined and exemplified below. The 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.

[0841] PHOSPHOLIPID

[0842] The nucleic acid-lipid particles of the present invention may also comprise a phospholipid. In some embodiments, 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).

[0843] 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 some embodiments, 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 Tmof 30°C or higher and are selected from di-stearoyl or di-palmitoyl or stearoyl-palmitoyl moieties. In some embodiments, 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.

[0844] 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), 1 ,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), 1-hexadecyl-sn-10-glycero-3-phosphocholine (C16 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), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPG), 1 ,2-dipalmitoyl- sn-glycero-3-phospho-(1'-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.

[0845] 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) 1 ,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.

[0846] 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. In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0847] Thus, in some embodiments, the particles described herein comprise a compound of formula (A), as defined herein, a cationically ionizable lipid (as defined herein), cholesterol and a phospholipid. In some embodiments, the particles described herein comprise a compound of formula (A), 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.

[0848] In some embodiments, 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 some embodiments, 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 some embodiments, 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.

[0849] In some embodiments, 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 some embodiments, 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.

[0850] In some embodiments, 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 some embodiments, 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 some embodiments, 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.

[0851] The term “lipid mixture” as used herein applies to the mixture of lipids in the nucleic acid-lipid particle.

[0852] STEROID The nucleic acid-lipid particles of the present invention may also comprise a steroid. In some embodiments, the steroid is cholesterol, or a derivative thereof. In some embodiments, 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 some embodiments, the steroid is cholesterol, or an ester thereof. In one preferred embodiment, the steroid is cholesterol.

[0853] In some embodiments, 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 some embodiments, 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 some embodiments, 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.

[0854] The term “lipid mixture” as used herein applies to the mixture of lipids in the nucleic acid-lipid particle.

[0855] PHOSPHOLIPID / STEROID RATIO

[0856] As described herein, the particles of the present invention may contain a phospholipid and a steroid (as defined above). In some embodiments, the particles of the present invention contain a phospholipid and cholesterol.

[0857] In some embodiments, 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, the molar ratio of phospholipid to steroid is from 0.5 to 0.7, optionally 0.55 to 0.65. In some embodiments, 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, the molar ratio of phospholipid to steroid is from 0.9 to 1.1 , optionally 1.9 to 2.1.

[0858] In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.2 to 3.0. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.6 to 3.0. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.7 to 2.8. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.6 to 3.0. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.2 to 1.0. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.3 to 0.9. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.5 to 0.7. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.55 to 0.65.

[0859] In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.8 to 2.6. In some embodiments, the molar ratio of phospholipid to cholesterol is from 1.0 to 2.2. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.8 to 2.2. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.8 to 1.2. In some embodiments, the molar ratio of phospholipid to cholesterol is from 1.8 to 2.2. In some embodiments, the molar ratio of phospholipid to cholesterol is from 0.9 to 1.1. In some embodiments, the molar ratio of phospholipid to cholesterol is from 1.9 to 2.1.

[0860] GRAFTED LIPIDS

[0861] The the 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 below (either in a broadest aspect or a preferred aspect”).

[0862] In some embodiments, 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.

[0863] In some embodiments, the grafted lipid is a polyethylene-glycol conjugated lipid (also known as a PEG-lipid or PEGylated lipid). 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 R12and R13is 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.

[0864] In some embodiments of this formula, each of R12and R13is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.

[0865] In some embodiments of this formula, R12and R13are identical. In some embodiments, each of R12and R13is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R12and R13is a straight alkyl chain containing 16 carbon atoms.

[0866] In some embodiments of this formula, R12and R13are different. In some embodiments, one of R12and R13is a straight alkyl chain containing 12 carbon atoms and the other of R12and R13is a straight alkyl chain containing 14 carbon atoms.

[0867] In some embodiments of this formula, w has a mean value ranging from 40 to 50, such as a mean value of 45.

[0868] 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 R12and R13is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.

[0869] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as 1-(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-1-O- (o-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as o-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N- (o methoxy(polyethoxy)ethyl)carbamate, and the like.

[0870] 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:

[0871] In some embodiment, the PEG-conjugated lipid (pegylated lipid) is a pegylated ceramide.

[0872] Such conjugated lipids comprise a ceramide moiety of formula: wherein R is a fatty acid residue, typically a Ce-3o alkyl group or a Ce-3o alkenyl group, preferably a C12-20 alkyl group, more preferably a C14-18 alkyl group, most preferably a C16 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 C16 PEG2000 ceramide, having the following structure: In some embodiments, the PEG-conjugated lipid (pegylated lipid) is a DMG-PEG 2000, e.g.,

[0873] 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 poly(ethylene glycol)2000)carbamoyl]-1 ,2-dimyristyloxy- propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3- bis(tetradecyloxy)propylcarbamate (2000).

[0874] In some embodiments, 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.

[0875] 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)-methacryla...

Claims

1. CLAIMS1. A nucleic acid-lipid particle comprising:(i) one or more lipid particle forming components;(ii) a nucleic acid payload comprising one or more nucleic acid molecules; and(iii) two or more targeting moieties, wherein a first targeting moiety is capable of binding to a first target and a second targeting moiety is capable of binding to a second target, wherein the first and second targets are different; wherein at least one targeting moiety is non-covalently bound to the nucleic acid-lipid particle.

2. The nucleic acid-lipid particle according to claim 1, wherein each targeting moiety is non-covalently bound to the nucleic acid-lipid particle.

3. The nucleic acid-lipid particle according to claim 1 or claim 2, wherein the one or more lipid particle forming components further comprise: 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;X1 is absent or a first linking moiety; andX2 is absent or a second linking moiety.

4. The nucleic acid-lipid particle according to claim 3, wherein the nucleic acid-lipid particle comprises one or more compound(s) of Formula (I):B’-X3-B” (I) whereinB’ comprises a moiety capable of non-covalently binding to B;X3 is absent or a linking moiety; andB” comprises a targeting moiety;wherein the targeting moiety is non-covalently bound to the nucleic acid-lipid particle through the non-covalent binding of B’ of the compound of Formula (I) to B of the compound of Formula (A).

5. The nucleic acid-lipid particle according to claim 3 or claim 4, wherein:(a) 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), andPOPE (palmitoyloleylphosphatidylethanolamine); most preferably a DSPE moiety; and / or(b) P is a polymer, preferably a hydrophilic polymer, more preferably selected from the group consisting of poly(ethylene glycol) (PEG), polysarcosine (pSar) (poly(N-methylglycine), poly- 2-(2-(2-aminoethoxy)ethoxy)acetic acid (pAEEA), or poly-2-(2-(2-(N-methylamino)- (ethoxy)ethoxy)acetic acid (pMAEEA) and combinations thereof.

6. The nucleic acid-lipid particle according to any of claims 3-5, wherein the binding moiety B comprises:(a) a peptide tag;(b) a moiety capable of binding to a peptide tag; or(c) an ALFA-tag; optionally wherein the binding moiety B comprises an ALFA tag; further optionally wherein B’ comprises an anti-ALFA binding domain.

7. The nucleic acid-lipid particle according to any of claims 1-6, wherein the one or more lipid particle forming components comprises a cationic or cationically ionizable lipid, optionally wherein the nucleic acid-lipid particle is a lipid nanoparticle (LNP).

8. The nucleic acid-lipid particle of claim 7, wherein the cationic or cationically ionizable lipid is selected from the group consisting of:1 ,2-dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102);O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(1- methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr);7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1-sulfonamide (BL-207); or a mixture of any thereof.

9. The nucleic acid-lipid particle according to one of claims 1-8, wherein at least one, or each, targeting moiety is selected from the list consisting of: a cytokine, an antibody or fragment thereof, a Fab, a F(ab)’2, a Fv, a single chain Fv (ScFv), a nanobody and a single chain variable domain; optionally wherein the targeting moiety is a nanobody.

10. The nucleic acid-lipid particle according to one of claims 1-9, wherein the, or each, targeting moiety is capable of binding to T cells, B cells, NK cells, monocytes, macrophages, mast cells, basophils, eosinophils or dendritic cells; optionally wherein the, or each, targeting moiety is capable of binding to T cells; further optionally wherein the, or each, targeting moiety is capable of activating T cells.

11. The nucleic acid-lipid particle according to one of claims 1-10, wherein the, or each, targeting moiety is independently selected from the list consisting of a: CD3 binding domain, CD2 binding domain, CD7 binding domain, CD4 binding domain, CD8 binding domain, CD28 binding domain, IL7, CD127 binding domain, and CD5 binding domain; optionally wherein:(i) the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD2 binding domain;(ii) the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises a CD7 binding domain;(iii) the first targeting moiety comprises a CD3 binding domain and the second targeting moiety comprises IL7; or(iv) the first targeting moiety comprises a CD3 binding domain, the second targeting moiety comprises a CD2 binding domain, and the third targeting moiety comprises a CD7 binding domain.

12. The nucleic acid-lipid particle according to one of claims 1-11, wherein the ratio of first targeting moiety to second targeting moiety is selected from the list consisting of 9:1, 7:3, 1 :1, 3:7; or 1 :9.

13. The nucleic acid-lipid particle according to one of claims 1-12, wherein the one or more nucleic acid molecules comprise:(i) RNA;(ii) DNA; or(iii) RNA and DNA; optionally wherein the RNA is mRNA.

14. A pharmaceutical composition comprising (i) the nucleic acid-lipid particle according to one of claims 1-13; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

15. The nucleic acid-lipid particle according to one of claims 1-13, or the pharmaceutical composition according to claim 14, for use as a medicament in the treatment of a disease; optionally wherein the disease is an autoimmune disease or a cancer; further optionally wherein the cancer is an immuno-oncology B cell malignancy or an immuno-oncology solid cancer.

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