Nanoparticles and uses thereof

Abstract

The present disclosure relates generally to nanoparticles and compositions comprising the same, and their use for transfecting transfection-recalcitrant cells, wherein the nanoparticle is tethered to an antigen-binding moiety having binding specificity for an antigen expressed on the surface of the cell, such as cluster of differentiation 2 (CD2) or cluster of differentiation 7 (CD7), wherein the nanoparticle comprises at least an ionisable lipid and a sterol.

Description

NANOPARTICLES AND USES THEREOF TECHNICAL FIELD

[0001] This application claims priority from Australian Provisional Patent Application No. 2024900557 filed 4 March 2024, the entire contents of each of which are incorporated herein by cross-reference.

[0002] The present disclosure relates generally to nanoparticles and compositions thereof, including their use in the targeted delivery of agents, such as nucleic acid-based therapeutics, in particular to transfection-recalcitrant cells. BACKGROUND

[0003] The success of therapeutic methods that rely on the delivery of exogenous agents, including proteins, nucleic acids and small molecules into the cell, is at least partly dependent on the efficiency of transfer, delivery and / or incorporation of the agents into a cell. Similarly, in research settings, which require the introduction genetic information or exogenous agents into cell or tissue models, high-efficiency introduction genetic information or exogenous agents is desirable.

[0004] Cell transfection typically refers to the transfer of nucleic acids into eukaryotic cells using non-viral methods, but the use of the term transfection has since evolved to encompass the transfer of proteins, peptides and other small molecules. Transfection is considered preferable to viral-based transduction, as it avoids the need for culture, preparation and the use of suitable viral vectors.

[0005] However, some cells are difficult to transfect or are transfection-recalcitrant, demonstrating low transfection efficiency and / or high transfection-related toxicity. Chemical transfection methods are most commonly used, in comparison to physical transfection methods (e.g., electroporation, gene gun) or viral transduction methods. Chemical transfection methods are often more convenient, insofar as they typically do not require specialised equipment or viral culture. While physical transfection methods can be useful for providing increased gene transfer efficiency, they are typically associated with harsher conditions, with undesirable loss of cell viability and / or changes in cell characteristics.

[0006] Many cells and tissues, in particular those relevant for therapeutic targeting, can be fragile, low in number, or resistant to the introduction of foreign material, such that therapeutic targeting and / or manipulation of these cells can be severely hampered by low gene transfer efficiency or conditions that would otherwise result in high cell death or cellular differentiation.

[0007] An example of a hard-to-transfect cell type is T cells, which form a critical part of the adaptive immune response against viruses and cancers. In addition, T cells can be subject to disease themselves, in the form of haematological malignancies or T cell-tropic viral infections. Indeed, human immunodeficiency virus (HIV) primarily infects CD4+ T cells and integrates its genome into the host cell DNA, thereby establishing a life-long infection. An important barrier to finding a cure for HIV is the persistence of latently infected, resting CD4+ T cells harbouring replication-competent virus, which can rebound upon activation and re-establish viremia in the absence of antiretroviral treatment. Attempts to eradicate this latent reservoir with systemic, small-molecule therapeutics have suffered from dose-limiting toxicities in off-target cells. In this regard, it has been suggested that the use of targeted nanoparticle-based drug delivery, such as lipid nanoparticles (LNP), could provide more potent and less toxic avenues towards an HIV cure.

[0008] LNP have the potential to provide a safe, stable and effective alternative for the delivery of therapeutic payloads, including nucleic acid, such as antisense therapies and mRNA vaccines. Delivery of such nucleic acid-based therapeutics using safe, stable and effective delivery systems is important to prevent degradation of the nucleic acid-based therapeutics, promote cellular uptake and avoid non-targeted or off-site effects and toxicity. However, many such LNP have shown varied ability to transfect cells, in particular hard-to- transfect or transfection-recalcitrant cells, including resting T cells.

[0009] Accordingly, there remains an urgent need for improved targeted delivery of agents to cells, in particular to transfection-recalcitrant cells.SUMMARY

[0010] The present invention is predicated, at least in part, on the inventors' surprising finding that CD2- and CD7-targeted nanoparticles, including LNP, provide improved targeted delivery and transfection of transfection-recalcitrant or difficult to transfect cells, in particular immune cells, such as T cells.

[0011] Thus, in an aspect disclosed herein, there is provided a nanoparticle for transfecting a transfection-recalcitrant cell, wherein the nanoparticle is tethered to an antigen-binding moiety having binding specificity for an antigen expressed on the surface of the cell, wherein the cell surface antigen is cluster of differentiation 2 (CD2) or cluster of differentiation 7 (CD7). In one embodiment, the transfection-recalcitrant cell is an immune cell.

[0012] In an embodiment, the immune cell is selected from the group consisting of a T cell, an NKT cell and an NK cell. In an embodiment, the immune cell is a T cell. In an embodiment, the T cell is a resting T cell. In an embodiment, the T cell is a memory T cell. In an embodiment, the T cell is a circulating T cell. In an embodiment, the T cell is a tissue- resident T cell.

[0013] In an embodiment, the nanoparticle is a lipid nanoparticle (LNP).

[0014] In an embodiment, the LNP comprises a sterol and an ionisable lipid.

[0015] In an embodiment, the sterol comprises less than 10 mol % cholesterol.

[0016] In an embodiment, the ionisable lipid has a structure of Formula (I):or a salt, solvate or isomer thereof, wherein: R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR", -YR" and -R"M'R';R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14alkenyl, -R*YR", -YR" and -R*OR", or R2and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, - (CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2and unsubstituted C1-6alkyl, where Q is selected from a C3-6carbocycle, 5- to 14-membered heterocycle, -OR, - O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, - C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, - N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, - N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, - N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and - C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; each R6is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; R8is selected from the group consisting of C3-6carbocycle and 5- to 14-membered heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H;each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13.

[0017] In an embodiment, the ionisable lipid is SM-102, or a salt, solvate or isomer thereof.

[0018] In an embodiment, the sterol is a C-24 alkyl phytosterol having a structure of Formula (III):or a stereoisomer thereof, wherein: each is individually a single bond or a double bond; and R is a C1-6 alkyl.

[0019] In an embodiment, R is a C1-4 alkyl, C1-3 alkyl, or C1-2 alkyl. In another embodiment, embodiment, the C-24 alkyl phytosterol is selected from the group consisting of , , ,, and any combination thereof.

[0020] In another embodiment, the sterol is β-sitosterol. In another embodiment, the sterol consists of β-sitosterol.

[0021] In an embodiment, the LNP composition comprises a phospholipid. In another embodiment, the LNP composition further comprises a PEG lipid. In an embodiment, the PEG lipid is DMG-PEG. In another embodiment, the PEG lipid is DSPE-PEG.

[0022] In another aspect, the present disclosure provides an LNP composition comprising SM-102, DSPC, β-sitosterol and DMG-PEG. In another aspect, the present disclosure provides an LNP composition comprising SM-102, DSPC, β-sitosterol and DSPE-PEG. In an embodiment the LNP composition comprises from about 20 mol% to about 80 mol% SM- 102, from about 5 mol% to about 30 mol% DSPC, about 30 mol% to about 50 mol% β- sitosterol, no more than 10% mol cholesterol, and about 0.5 mol% to about 3 mol% DMG- PEG. In an embodiment the LNP composition comprises from about 20 mol% to about 80 mol% SM-102, from about 5 mol% to about 30 mol% DSPC, about 30 mol% to about 50 mol% β-sitosterol, no more than 10% mol cholesterol, and about 0.5 mol% to about 3 mol% DSPE-PEG.

[0023] In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG. In an embodiment, the LNP composition does not comprise cholesterol. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% cholesterol and about 1.5 mol% DSPE- PEG. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DSPE-PEG. In an embodiment, the LNP composition does not comprise cholesterol. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% cholesterol and about 1.5 mol% DMG- PEG.

[0024] In an embodiment, the nanoparticle composition further comprises an agent to be delivered to the transfection-recalcitrant cell.

[0025] In another aspect, the present disclosure provides a pharmaceutical composition comprising a nanoparticle according to the present disclosure and an agent.

[0026] In one embodiment, the agent is a protein, a small-molecule drug, or a nucleic acid.

[0027] In another aspect, the present disclosure provides a method of delivering an agent to a cell, the method comprising contacting the cell with the nanoparticle or pharmaceutical composition according to the present disclosure. In another aspect, the present disclosure provides a method of expressing an exogenous nucleic acid in a cell, the method comprising contacting the cell with the nanoparticle or pharmaceutical composition according to the present disclosure, wherein the agent is an exogenous nucleic acid. In an embodiment, the exogenous nucleic acid is an mRNA encoding the polypeptide, and the mRNA is capable of being translated in the cell to produce the polypeptide.

[0028] In an embodiment, the cell is a transfection-recalcitrant cell. In an embodiment, the cell or the transfection-recalcitrant cell is a quiescent cell, a primary cell, an immune cell, a stem cell or a neuronal cell. In an embodiment, the immune cell is a lymphoid cell or a myeloid cell. In an embodiment, the immune cell is T cell, B cell, monocyte, and / or dendritic cell. In another embodiment, the cell or transfection-recalcitrant cell is a resting T cell or a Natural Killer cell.

[0029] In another aspect, the present disclosure provides a method of transfecting resting T cells, the method comprising contacting the resting T cell with the nanoparticle composition or pharmaceutical composition according to the present disclosure. In another aspect, the present disclosure provides a method of transfecting a circulating immune cell, the method comprising contacting a circulating immune cell with the nanoparticle composition or pharmaceutical composition according to the present disclosure. In an embodiment, the circulating immune cell is a circulating T cell. In another aspect, there is provided a method of transfecting a tissue-resident immune cell, the method comprising contacting a tissue- resident immune cell with the nanoparticle composition or pharmaceutical composition according to the present disclosure. In an embodiment, the tissue-resident immune cell is a tissue-resident T cell. In another aspect, there is provided a method of transfecting a naive immune cell, the method comprising contacting a naive immune cell with the nanoparticlecomposition or pharmaceutical composition according to the present disclosure. In an embodiment, the naive immune cell is a naive T cell.

[0030] In another aspect, the present disclosure provides a method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the nanoparticle composition or the pharmaceutical composition according to the present disclosure, wherein the agent is capable of treating or preventing the disease.

[0031] In an embodiment, the disease is a HIV infection, the method comprising administering to the subject an effective amount of the nanoparticle composition or the pharmaceutical composition according to the present disclosure, wherein the agent is capable of a. permanently inactivating or silencing the HIV genome; or b. reversing HIV latency to activate HIV transcription, to allow targeting of the activated HIV infected cells with concurrent antiretroviral therapy.

[0032] In another embodiment, the HIV infection is a latent HIV infection.

[0033] In another aspect, the present disclosure provides use of the nanoparticle composition or the pharmaceutical composition according to the present disclosure in the manufacture of a medicament for treating or preventing for treating or preventing a disease in a subject, wherein the agent is capable of treating or preventing the disease.

[0034] In an embodiment, the disease is an HIV infection, wherein the agent is capable of a. permanently silencing the HIV genome; or b. reversing HIV latency to activate HIV transcription, to allow targeting of the activated HIV infected cells with concurrent antiretroviral therapy.

[0035] In another embodiment, the agent is capable of reversing HIV latency to activate HIV transcription, to allow expression of viral proteins and recognition by HIV-specific immune clearance and or virus mediated cytolysis.BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Embodiments of the invention will now be described with reference to the following Figures, which are intended to be exemplary only, and in which:

[0037] Figure 1 shows the expression patterns on primary CD4+ T cells of surface receptors included in screening library. (a) Breadth of receptor expression measured as the percentage of live CD4+ T cells expressing the receptor of interest. (b) Receptor density on cells positive for receptor expression, represented by the absolute number of anti-receptor antibodies bound per cell (antibody binding capacity). Values shown as mean ± SEM of n=4 donors. CTLA-4, CXCR6 and CCR9 were excluded from further analysis. ICM, immune checkpoint markers; CytoR, cytokine receptors.

[0038] Figure 2 shows that receptor internalization kinetics can be assessed using a dual staining approach. (a) Schematic overview of the receptor internalization assay. The receptor of interest is stained with a fluorescent primary antibody (red), then kept on ice or incubated at 37°C to allow receptor internalization. Receptors present on the cell surface are then dually labelled with a secondary antibody, upon which receptor internalization can be detected by a downward shift in the fluorescent correlation between the primary (x-axis) and secondary (y-axis) fluorescent signals on flow cytometry. (b-i) Representative results of the receptor internalization assay for a non-internalizing (CD4, b-e) and strongly internalizing (transferrin receptor, TfR, f-i) receptor as negative and positive controls, respectively. (b, f) Raw fluorescent signals of primary and secondary antibody staining over time. The secondary antibody signal decreases over time in the presence of receptor internalization, while the primary antibody signal remains constant irrespective of receptor internalization kinetics. (c, g) Overlay of representative dot plots of the primary and secondary antibody signals at baseline (0 min, black) and 180 min (red), showing a downward shift over time in the presence of receptor internalization. (d, h) Ratio of secondary antibody signal to primary antibody signal over time as a newly computed parameter per cell, quantifying any receptor internalization. The baseline ratio at 0 min reflects the ratio of the respective degrees of labelling of the primary and secondary antibodies used. (f, i) Receptor internalization score over time, calculated by normalizing the values in (d) or (h) to the corresponding baseline (0 min timepoint). n=3, values shown as mean ± SEM. (j) Representative confocal microscopy images of dual receptor staining at various timepoints. At baseline (0 min), nointernalization of the primary antibody-receptor complexes (red) is observed, and all receptors are dually stained with the secondary antibody (green). At later timepoints, primary antibody-receptor complexes can be observed within the cytoplasm, while the secondary antibody selectively co-stains receptors present on the cell surface.

[0039] Figure 3 shows that CCR5, CD7 and CD2 exhibit potent receptor internalization in CD4+ T cells. (a-b) Internalization score of the selection of receptors expressed sufficiently on CD4+ T cells after 180 min (a) and kinetics over time (b). n=4, values shown as mean ± SEM as either error bars (a) or shading (b). Significance was determined through one-way ANOVA and Dunnett’s multiple comparisons test using CD4 as reference. ns p > 0.05 (not shown), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

[0040] Figure 4 shows that dual staining of targeted nanoparticles allows for simultaneous assessment of association and internalization. (a) Schematic overview of the nanoparticle internalization assay. Nanoparticles conjugated with fluorescently labelled, primary antibodies are added to target cells and incubated for various durations, after which nanoparticles on the cell surface are co-stained using a secondary antibody. After harvest, the percentage nanoparticle association is first determined (1). Within cells found positive for nanoparticle association, the median fluorescent intensity (MFI) of the Ab@SPION signal was then extracted and converted into a measure of the average absolute number of nanoparticles associated per cell (2). Simultaneously, the ratio of secondary (2°) to primary (1°) antibody signal, representing surface-bound nanoparticles and total nanoparticles, respectively, was extracted to calculate a nanoparticle internalization score (3). The first timepoint harvested was used as baseline for nanoparticle internalization. (b-d) Jurkat T cells were incubated with each of six targeting Ab@SPIONs or untargeted (isotype) control particles and nanoparticle association and internalization were assessed over time. (b) Percentage of live cells demonstrate association of either Ab@SPION at each timepoint. (c) Absolute number of each Ab@SPIONs associated per cell of cells found positive for nanoparticle association in (b). (d) Internalization score of each Ab@SPIONs over time in cells found positive for nanoparticle association in (a), calculated by normalizing the ratio of secondary to primary antibody signal to the first timepoint assessed (1 hour). n=3, values shown as mean ± SEM.

[0041] Figure 5 shows that targeting of CD2 and CD7 induces nanoparticle uptake in total CD4+ T cells. Primary CD4+ T cells were incubated with nanoparticles targeting CCR5, CD2, CD3 or CD7, or untargeted (isotype) control particles and nanoparticle association and internalization were assessed over time. (a) Percentage of live cells demonstrating association of either Ab@SPION at each timepoint. (b) Absolute number of each Ab@SPIONs associated per cell of cells found positive for nanoparticle association in (a). (c) Internalization score of each Ab@SPIONs over time in cells found positive for nanoparticle association in (a), calculated by normalizing the ratio of secondary to primary antibody signal to the first timepoint assessed (1 hour). An internalization score for isotype@SPIONs could not reliably be determined due to the negligible percentage of cells found positive for nanoparticle association, and was therefore excluded from analysis. n=4, values shown as mean ± SEM.

[0042] Figure 6 shows that receptor targeting can enhance nanoparticle association to T cells in the presence of off-target cells. (a-c) PBMC were incubated with untargeted control nanoparticles (isotype) (a) or nanoparticles targeted to CD2 (b) or CD7 (c) and nanoparticle- cell interactions with various PBMC subsets were assessed after 1 hour. For each nanoparticle, association was measured as the percentage of live cells that demonstrate association of the Ab@SPION (bars, grey), as well as the absolute number of each Ab@SPIONs associated per cell of cells found positive for nanoparticle association (symbols, red). n=4, values shown as mean ± SEM. (d,e) Summary of data in a-c, shown as a heat map with differences of means of each Ab@SPION compared to untargeted control particles for each PBMC subset. (d) difference in percentage nanoparticle association; (e) difference in absolute number of Ab@SPIONs associated per cell. Significance was determined through one-way ANOVA and Bonferroni’s multiple comparisons test using untargeted control particles as reference. ns p > 0.05 (not shown), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Mono, monocyte; NK, natural killer; DC, dendritic cell.

[0043] Figure 7 shows that CD2 and CD7-mediated T cell targeting is maintained in whole blood. (a-c) Untargeted control nanoparticles (isotype) (a) or nanoparticles targeted to CD2 (b) or CD7 (c) were incubated with whole blood and nanoparticle-cell interactions with various leukocyte subsets were assessed after 1 hour. For each nanoparticle, association was measured as the percentage of live cells that demonstrate association of the Ab@SPION(bars, grey), as well as the absolute number of each Ab@SPIONs associated per cell of cells found positive for nanoparticle association (symbols, red). n=4, values shown as mean ± SEM. (d,e) Heat map showing a summary of data in a-c, presented as differences of means of each Ab@SPION compared to untargeted control particles for each WBC subset. (d) difference in percentage nanoparticle association; (e) difference in absolute number of Ab@SPIONs associated per cell. Significance was determined through one-way ANOVA and Bonferroni’s multiple comparisons test using untargeted control particles as reference. ns p > 0.05 (not shown), * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Mono, monocyte; NK, natural killer; DC, dendritic cell; Gran, granulocyte.

[0044] Figure 8 shows that CD2- and CD7-targeting increases LNP-mediated delivery of functional mRNA to T cells. (a-b) Isolated CD4+ T cells were incubated with varying doses of DiD-labelled, mScarlet mRNA-LNPs targeted to CD2, CD7 or control LNPs for 1 hour, after which unbound LNPs were washed off. LNP association (a) and transfection efficiency (b) were determined after 24 hours. (c, d) Additionally, CD4+ T cells were continuously incubated with 60ng DiD-labelled, mScarlet mRNA-LNPs targeted to CD2, CD7 or control LNPs for 24 hours and LNP association (c) and transfection efficiency (d) were compared to results from (a, b). N=7, values shown as mean ± SEM. (e-h) PBMCs were incubated with 200ng naked (non-functionalized, e) mScarlet mRNA-LNPs, untargeted control LNPs (isotype, f) or LNPs targeted to CD2 (g) or CD7 (h) for 1 hour, after which unbound LNPs were washed off. Transfection efficiency was determined in each PBMC subset (from left to right, Monocytes; CD4 T cells; CD8 T cells; B cells and NK cells) after 24hours. N=6, values shown as mean ± SEM.

[0045] Figure 9 shows that receptor internalization occurs in both resting and activated CD4+ T cells. (a-b) Activated CD4+ T cells were identified by flow cytometry through staining for CD69 and HLA-DR after the dual staining procedure to detect receptor internalization. Representative histograms showing the distribution of the ratio of secondary (2°) to primary (1°) antibody (Ab) signals per cell, as a raw measure of CD7 (a) or CD2 (b) internalization, for resting (top, red) and activated (bottom, blue) cells. Overlays depict the baseline ratio (filled curve) versus the ratio observed at the final timepoint of 180 min. Note that a lower ratio of secondary to primary antibody compared to baseline indicates receptor internalization has occurred.

[0046] Figure 10 shows the generation and characterization of Ab@SPIONs. (a) 100 nm superparamagnetic iron oxide particles (SPIONs) coated in protein G were decorated with Alexa Fluor 647-labelled targeting antibodies through interaction between protein G and the Fc region of the antibodies. Excess unbound antibody was washed away and Ab@SPIONs were sonicated prior to use. (b) Size measurement of resulting Ab@SPIONs and unconjugated SPIONs as determined through dynamic light scattering. (c-d) Visualization of antibody conjugation to SPIONs through cryo-EM; the lighter area surrounding the SPIONS in (d) but not (c) indicates the addition of a protein layer.

[0047] Figure 11 shows the expression patterns on Jurkat T cells of a subset of the receptor library. (a) Breadth of receptor expression measured as the percentage of live Jurkat T cells expressing the receptor of interest. Values shown as mean ± SEM of n=3. (b) Receptor density on cells positive for receptor expression, represented by the absolute number of anti- receptor antibodies bound per cell (antibody binding capacity). For receptors not shown in this graph, no expression was detected.

[0048] Figure 12 shows that receptor internalization kinetics in Jurkat T cells vary significantly. (a-b) Internalization score of a selection of receptors expressed on Jurkat T cells after 180 min (a) and kinetics over time (b). n=3, values shown as mean ± SEM. SEM is shown as either error bars (a) or shading (b). Significance was determined through one- way ANOVA and Dunnett’s multiple comparisons test using CD4 as reference. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

[0049] Figure 13 shows that potential target receptors express various levels of specificity towards T cells. Expression of CD2 and CD7 was determined in the various PBMC subsets studied in association assays. (a-b) Breadth of receptor expression measured as the percentage of live cells expressing, CD2 (a) or CD7 (b). (c-d) Receptor density on cells positive for receptor expression, represented by the absolute number of anti-CD2 (c) or anti- CD7 (d) antibodies bound per cell (antibody binding capacity). Values shown as mean ± SEM of n=4 donors. Mono, monocyte; NK, natural killer; DC, dendritic cell.

[0050] Figure 14 shows the distribution of cell subsets in PBMC (a) and whole blood (b). In all nanoparticle association studies, the proportion of monocytes, T cells, B cells, NK cells and dendritic cells of total leukocytes was determined. Cell subsets were identified as follows within single, live cells: granulocytes (CD66b+), T cells (CD66b- / CD3+), monocytes (CD66b- / CD14+), NK cells (CD66b- / CD3- / CD14- / CD56+), B cells (CD66b- / CD3- / CD14- / CD19+), dendritic cells (DC) ((CD66b- / CD3- / CD14- / CD56- / CD19- / HLA- DR+). Values shown as mean ± SEM of n=4 donors. Mono, monocyte; NK, natural killer; DC, dendritic cell.

[0051] Figure 15 shows conjugation of CD2- and CD7-targeting antibodies in non- controlled orientation increases LNP association with monocytes.PBMCs were incubated with varying doses of naked (non-functionalized) DiD-labelled mRNA-LNPs, untargeted control LNPs or LNPs targeted to CD2 or CD7 for 1 hour, after which unbound LNPs were washed off. LNP association was determined in CD4+T cells and monocytes after 24hours. N=3, values shown as mean ± SEM.Figure 16 shows that targeting CD2 or CD7 enables mRNA delivery to T cells in vivo. Human immune system mice received a 10 µg dose of naked (a, e), isotype control (b, f), CD2-targeted (c, f) or CD7-targeted (d, h) mScarlet-LNP intravenously. After 16 hours, the percentage mScarlet expression was determined in circulating (blood, a-d) or spleen-resident (spleen, e-h) human immune cell subsets. N=3 (naked, isotype) or n=4 (CD2, CD7), values shown as mean ± SEM.Figure 17 shows mScarlet expression in human and mouse immune cells in blood, spleen and lymph node after in vivo mScarlet-LNP administration. (a-d) Human immune system mice received a 10 µg dose of naked (a), isotype control (b), CD2-targeted (c) or CD7-targeted (d) mScarlet- LNP intravenously as in Figure 5. After 16 hours, the percentage mScarlet expression was determined in lymph node-resident human lymphocyte subsets. (e-g) Simultaneously, the mScarlet expression was determined in residual mouse immune cells in blood (e), spleen (f) and lymph node (g) after administration of naked, isotype control, CD2- or CD7-targeted mScarlet LNPs. N=3 (naked, isotype) or n=4 (CD2, CD7), values shown as mean ± SEM. DEFINITIONS

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the presentdisclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice the present invention.

[0055] As used herein, the term “alkyl” or “alkyl group” means a linear or branched, saturated hydrocarbon including one or more carbon atoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms), which is optionally substituted. The notation “C1-14 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-14 carbon atoms, “C1-6 alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-6 carbon atoms, “C1-3alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-3 carbon atoms, and the like. Unless otherwise specified, an alkyl group described herein refers to both unsubstituted and substituted alkyl groups. Examples of suitable alkyl groups may include, but are not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl, 2-butyl, isobutyl, tert-butyl, and the like.

[0056] As used herein, the term “alkenyl” or “alkenyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one double bond, which is optionally substituted. Unless indicated otherwise, the stereochemistry about each double bond may be independently cis or trans, or E or Z, as appropriate. The notation “C5-20 alkenyl” means an optionally substituted linear or branched hydrocarbon including 5-20 carbon atoms and at least one carbon-carbon double bond, “C2-18alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-18 carbon atoms and at least one carbon-carbon double bond, “C2-6 alkenyl” means an optionally substituted linear or branched hydrocarbon including 2-6 carbon atoms and at least one carbon-carbon double bond, and the like. An alkenyl group may include one, two, three, four, or more carbon-carbon double bonds. For example, C18 alkenyl may include one or more double bonds. A C18 alkenyl group including two double bonds may be a linoleyl group. Unless otherwise specified, an alkenyl group described herein refers to both unsubstituted and substituted alkenyl groups.

[0057] As used herein, the term “alkynyl” or “alkynyl group” means a linear or branched hydrocarbon including two or more carbon atoms (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, or more carbon atoms) and at least one carbon-carbon triple bond, which is optionally substituted. The notation “C2-14alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond, “C2-14alkynyl” means an optionally substituted linear or branched hydrocarbon including 2-14 carbon atoms and at least one carbon-carbon triple bond. An alkynyl group may include one, two, three, four, or more carbon-carbon triple bonds. For example, C18 alkynyl may include one or more carbon-carbon triple bonds. Unless otherwise specified, an alkynyl group described herein refers to both unsubstituted and substituted alkynyl groups.

[0058] As used herein, the term “carbocycle” or “carbocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings of carbon atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty membered rings. The notation “C3-14carbocycle” means a carbocycle including a single ring having 3-14 carbon atoms, “C3-6carbocycle” means a carbocycle including a single ring having 3-6 carbon atoms, and the like. Carbocycles may include one or more carbon-carbon double or triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl or aryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl, cyclohexyl, phenyl, naphthyl and 1,2-dihydronaphthyl groups. The term “cycloalkyl” as used herein means a non-aromatic carbocycle and may or may not include any double or triple bond. Unless otherwise specified, carbocycles described herein refers to both unsubstituted and substituted carbocycle groups, i.e., optionally substituted carbocycles.

[0059] As used herein, the term “heterocycle” or “heterocyclic group” means an optionally substituted mono- or multi-cyclic system including one or more rings, where at least one ring includes at least one heteroatom. Heteroatoms may be, for example, nitrogen, oxygen, or sulphur atoms. Rings may be three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen membered rings. Heterocycles may include one or more double or triplebonds and may be non-aromatic or aromatic (e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocycles include imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl and isoquinolyl groups. The term “heterocycloalkyl” as used herein means a non-aromatic heterocycle and may or may not include any double or triple bond. Unless otherwise specified, heterocycles described herein refers to both unsubstituted and substituted heterocycle groups, i.e., optionally substituted heterocycles.

[0060] As used herein, a “biodegradable group” (denoted M and M’) is a group that may facilitate faster metabolism of a lipid in a mammalian entity. A biodegradable group may be selected from the group consisting of, but is not limited to, -C(O)O-, -OC(O)-, -C(O)N(R’)- , -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, an aryl group and a heteroaryl group. As used herein, an “aryl group” is an optionally substituted carbocyclic group including one or more aromatic rings. Examples of aryl groups include phenyl and naphthyl groups. As used herein, a “heteroaryl group” is an optionally substituted heterocyclic group including one or more aromatic rings. Examples of heteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl, oxazolyl and thiazolyl. Both aryl and heteroaryl groups may be optionally substituted. For example, M and M’ can be selected from the non-limiting group consisting of optionally substituted phenyl, oxazole and thiazole. In the formulas herein, M and M’ can be independently selected from the list of biodegradable groups above. Unless otherwise specified, aryl or heteroaryl groups described herein refers to both unsubstituted and substituted groups, i.e., optionally substituted aryl or heteroaryl groups.

[0061] Alkyl, alkenyl and cyclyl (e.g., carbocyclyl and heterocyclyl) groups may be optionally substituted unless otherwise specified. Optional substituents may be selected from the group consisting of, but are not limited to, a halogen atom or “halo” group (e.g., a chloride, bromide, fluoride, or iodide group), a carboxylic acid (e.g., -C(O)OH), an alcohol (e.g., a hydroxyl, -OH), an ester (e.g., -C(O)OR -OC(O)R), an aldehyde (e.g.,-C(O)H), acarbonyl (e.g., -C(O)R, alternatively represented by C=O), an acyl halide (e.g.,-C(O)X, in which X is a halide selected from bromide, fluoride, chloride and iodide), a carbonate (e.g., -OC(O)OR), an alkoxy (e.g., -OR), an acetal (e.g.,-C(OR)2R””, in which each OR are alkoxy groups that can be the same or different and R”” is an alkyl or alkenyl group), a phosphate (e.g., P(O)43-), a thiol (e.g., -SH), a sulfoxide (e.g., -S(O)R), a sulfinic acid (e.g., -S(O)OH), a sulfonic acid (e.g., -S(O)2OH), a thial (e.g., -C(S)H), a sulfate (e.g., S(O)42-), a sulfonyl (e.g., -S(O)2-), an amide (e.g., -C(O)NR2, or -NIC(O)R), an azido (e.g., -N3), anitro (e.g.,- NO2), a cyano (e.g., -CN), an isocyano (e.g., -NC), an acyloxy (e.g.,-OC(O)R), an amino (e.g., -NR2, -NRH, or -NH2), a carbamoyl (e.g., -OC(O)NR2, -OC(O)NRH, or -OC(O)NH2), a sulfonamide (e.g., -S(O)2NR2, -S(O)2NRH, -S(O)2NH2,I(R)S(O)2R, -N(H)S(O)I - N(R)S(O)2H, or -N(H)S(O)2H), an alkyl group, an alkenyl group and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any of the preceding, R is an alkyl or alkenyl group, as defined herein. In some embodiments, the substituent groups themselves may be further substituted with, for example, one, two, three, four, five, or six substituents as defined herein. For example, a C1-6alkyl group may be further substituted with one, two, three, four, five, or six substituents as described herein.

[0062] As used herein, the tern “alkoxy” or “alkoxyl group” means a chemical substituent of formula -OR, where R is an alkyl group as defined herein (e.g., C1-6 alkyl or C1-3 alkyl), unless otherwise specified. Exemplary alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like. In some embodiments, the alkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein (e.g., hydroxyl or alkoxy).

[0063] As used herein, the term "isomer" means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer, or diastereomer of a compound. Compounds may include one or more chiral centers and / or double bonds and may thus exist as stereoisomers, such as double- bond isomers (i.e., geometric E / Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (-)) or cis / trans isomers). The present disclosure encompasses any and all isomers of the lipids or other compounds described herein, including stereomerically pure forms (e.g.,geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereomeric mixtures of compounds and means of resolving them into their component enantiomers or stereoisomers are well-known.

[0064] Unless otherwise specified, the indefinite articles “a”, “an” and “the” as used herein, include plural aspects. Thus, for example, reference to “an agent” includes a single agent, as well as two or more agents; reference to a “composition” or “formulation” includes a single composition or formulation, as well as two or more compositions or formulations; and so forth.

[0065] As used herein, the “about”, as applied to one or more values, refer to a value that is similar to a stated reference value. In certain embodiments, the tem “about” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). In a particular embodiment, the term “about” means ±10% of the recited value.

[0066] Throughout this specification and the claims that 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 integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0067] The term “consisting of” means “consisting only of”, that is, including and limited to the integer or step or group of integers or steps, and excluding any other integer or step or group of integers or steps.

[0068] The term “consisting essentially of” means the inclusion of the stated integer or step or group of integers or steps, but other integer or step or group of integers or steps that do not materially alter or contribute to the working of the invention may also be included.

[0069] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0070] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that prior art forms part of the common general knowledge.

[0071] Other definitions are provided throughout the specification. DETAILED DESCRIPTION

[0072] The present disclosure is predicated, at least in part, on the inventors' surprising findings that CD2- and CD7-targeted nanoparticles, provide improved targeted delivery and transfection of transfection-recalcitrant or difficult to transfect cells. In particular, the present inventors have surprisingly found that targeting CD2 or CD7 on the surface of immune cells (in particular primary immune cells, such as T cells), provides far better internalisation, including of CD2- or CD7-targeted nanoparticles, in these cells in comparison to other cell surface antigens.

[0073] Thus, in an aspect disclosed herein, there is provided a nanoparticle for transfecting a transfection-recalcitrant cell, wherein the nanoparticle is tethered to an antigen-binding moiety having binding specificity for an antigen expressed on the surface of the cell, wherein the cell surface antigen is cluster of differentiation 2 (CD2) or cluster of differentiation 7 (CD7). Nanoparticles

[0074] As used herein, the term “nanoparticle” refers to a small, non-viral particle that can encapsulate or associate with an agent, including a therapeutic agent (e.g., a protein, a drug, a nucleic acid molecule) for the delivery of that agent to a cell. Suitable nanoparticles will be familiar to persons skilled in the art, illustrative examples of which include lipid nanoparticles (LNP), polymeric nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA dendrimers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes (see, for example, WO 2023 / 196188, Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; and Neshat et al. (2020) Current Opin. Biotechnol. 66:1-10., the entire contents of which are incorporated herein by reference).

[0075] The production of nanoparticles and the incorporation of agents therein (e.g., nucleic acid molecules) will be well known to persons skilled in the art. Suitable production methods are described elsewhere herein, including in WO 2023 / 196188, Riley and Vermerris Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., (2020) NanoImpact 20, 100261; and Neshat et al. (2020) Current Opin. Biotechnol. 66:1- 10., the entire contents of which are incorporated herein by reference. Suitable methods of incorporating agents, including nucleic acid molecules, in a nanoparticle are described, for example, in Teo et al. (Advanced Drug Delivery Reviews (2016) 98, 41); Bochicchio et al. (Pharmaceutics (2021) 13, 198); Mahzabin and Das (IJPSR (2021) 12(1), 65); and Teixeira et al., (2017; Prog. Lipid Res. Oct;68:l-l l); Nii and Ishii (International Journal of Pharmaceutics (2005) 298, 198); and Chen et al. (Journal of Controlled Release (2018) 286, 46), the entire contents of which are incorporated herein by reference.

[0076] In an embodiment, the nanoparticle is a lipid nanoparticle (LNP).

[0077] In an embodiment, the LNP comprises a sterol and an ionisable lipid.

[0078] In an embodiment, the sterol comprises less than 10 mol % cholesterol.

[0079] In an embodiment, the ionisable lipid has a structure of Formula (I).

[0080] As used herein, the term “ionisable lipid” refers to a lipid molecule that is neutral (i.e., uncharged) at physiological pH (e.g., from about 7.35 to about 7.45 for humans), but which is protonated (i.e., becomes positively charged) at lower pH. As used herein, the term “cationic lipid” refers to a lipid molecule that is permanently positively charged (i.e., regardless of pH). Typically, cationic and ionisable lipids comprise three main chemical functional domains: a hydrophilic head group, a hydrophobic domain (or “tail”) and a linker domain that tethers the cationic head group and hydrophobic tail domain. The hydrophilic head group of an ionisable lipid comprises at least one functional group that is neutral at physiological pH and it protonated at lower pH (e.g., an amine group), whereas hydrophilic head group of a cationic lipid comprises at least one (e.g., one, two, three, or more) permanently positively charged functional group (e.g., an ammonium group) at physiological pH.

[0081] Suitable ionisable lipids will be familiar to persons skilled in the art, illustrative examples of which are disclosed in WO / 2017 / 049245, the entire contents of which is incorporated herein by reference. In an embodiment, the ionisable lipid comprises a structure of Formula (I):or a salt, solvate or isomer thereof, wherein: R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2- 14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4is selected from the group consisting of a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, where Q is selected from a C3-6carbocycle, 5- to 14-membered heterocycle, -OR, -O(CH2)nN(R)2, - C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2)nOR, - N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, - C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4 and 5; each R5is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H;each R6 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14aryl group and a 5- to 14-membered heteroaryl group; R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; R8 is selected from the group consisting of C3-6 carbocycle and 5- to 14-membered heterocycle; R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18alkyl, C2-18 alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14alkyl and C3-14alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12 alkenyl; each Y is independently a C3-6carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13.

[0082] In an embodiment of Formula (I), when R4is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5-, 6- or 7-membered heterocycloalkyl when n is 1 or 2.

[0083] In an embodiment of Formula (I):R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2- 14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4is selected from the group consisting of a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, - CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, - N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, - C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O and S which is substituted with one or more substituents selected from oxo (=O), OH, amino, mono- or di-alkylamino and C1-3 alkyl, and each n is independently selected from 1, 2, 3, 4 and each R5is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3alkenyl and H; M and M' are independently selected from -C(O)0-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; R8 is selected from the group consisting of C3-6 carbocycle and 5- to 14-membered heterocycle;R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6alkenyl, C3-6carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0084] In an embodiment of Formula (I): R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2- 14alkenyl, -R*YR", -YR" and -R*OR", or R2and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2and unsubstituted C1-6alkyl, where Q is selected from a C3-6carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, - CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2,-N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, - N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, - N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4 and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is -(CH2)nQ in which n is 1 or 2, or (ii) R4 is -(CH2)nCHQR in which n is 1, or (iii) R4 is -CHQR and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R5 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; each R6is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14aryl group and a 5- to 14-membered heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; R8 is selected from the group consisting of C3-6 carbocycle and 5- to 14-membered heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R' is independently selected from the group consisting ofC1-18 alkyl, C2- 18 alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14alkyl and C3-14 alkenyl;each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0085] In an embodiment of Formula (I): R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2- 14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4is selected from the group consisting of a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, where Q is selected from a C3-6carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, - CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, - N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, - N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, - C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4 and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; each R6is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H;M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14aryl and a 5- to 14-membered heteroaryl group; R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; R8 is selected from the group consisting of C3-6 carbocycle and 5- to 14-membered heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0086] In an embodiment of Formula (I): R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2- 14alkenyl, -R*YR", -YR" and -R*OR", or R2and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle;R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2 and n is selected from 3, 4 and 5; each R5is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18alkyl, C2-18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0087] In an embodiment of Formula (I): R1is selected from the group consisting of C5-30alkyl, C5-20alkenyl, -R*YR", -YR" and -R"M'R';R2 and R3 are independently selected from the group consisting of C1-14 alkyl, C2- 14alkenyl, -R*YR", -YR" and -R*OR", or R2and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4 is selected from the group consisting of -(CH2)nQ, -(CH2)nCHQR, -CHQR and - CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4 and 5; each R5is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2- 3 alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14aryl group and a 5- to 14-membered heteroaryl group; R7is selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18alkyl, C2-18 alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C1- 12 alkenyl; each Y is independently a C3-6carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0088] In an embodiment, the ionisable lipid has a structure of Formula (IA):or a salt, solvate or isomer thereof, wherein: p is an integer from 1 to 5; m is an integer from 5 to 9; M1is a bond or M'; R4 is unsubstituted C1-3 alkyl or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, - NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, - NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14-membered heteroaryl or 5- to 14-membered heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O-, -S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R2and R3are independently selected from the group consisting of H, C1-14alkyl and C2-14 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18 alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14alkyl and C3-14alkenyl;each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12alkenyl; each Y is independently a C3-6 carbocycle.

[0089] In an embodiment of Formula (IA): p is an integer from 1 to 5; m is an integer from 5 to 9; M1 is a bond or M′; R4 is unsubstituted C1-3 alkyl or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, - NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, - NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14-membered heteroaryl or 5- to 14-membered heterocycloalkyl; M and M′ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R′)-, - P(O)(OR′)O-, -S-S-, an aryl group, and a 5- to 14-membered heteroaryl group; and R2 and R3 are both C1-14 alkyl or C2-14 alkenyl, R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; and R′ is a linear alkyl.

[0090] In an embodiment of Formula (IA): R4is -(CH2)nQ, in which Q is OH, wherein n is an integer from 1 to 5;M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O- and -S-S-; and R2 and R3 are each individually C1-14 alkyl or C2-14 alkenyl, each optionally substituted with one or more substituents selected from halo, OH, unsubstituted C1-3 alkyl and unsubstituted C1-3alkoxy; and R' is a C1-18linear alkyl, optionally substituted with one or more substituents selected from halo, OH and unsubstituted C1-3 alkoxy.

[0091] In an embodiment of Formula (IA), n is an integer from 2 to 4.

[0092] In an embodiment of Formula (IA), R2and R3are the same. In a particular embodiment, R2 and R3 are both C8 alkyl.

[0093] In an embodiment of Formula (IA), R2 and R3 are different.

[0094] In an embodiment, the ionisable lipid has a structure of Formula (II):or a salt, solvate or isomer thereof, wherein: p is an integer from 1 to 5; M1 is a bond or M'; R4 is unsubstituted C1-3 alkyl or -(CH2)nQ, in which n is 2, 3 or 4, and Q is OH, - NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, - NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14- membered heteroaryl or 5- to 14-membered heterocycloalkyl;M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O-, -S-S-, a C6-14aryl group and a 5- to 14-membered heteroaryl group; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl and C2-14 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18 alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12alkenyl; and each Y is independently a C3-6carbocycle.

[0095] In an embodiment of Formula (II): p is an integer from 1 to 5, M1is M′; R4 is -(CH2)nQ, in which Q is OH, and n is an integer from 1 to 5; M and M′ are independently selected from -C(O)O-, and -OC(O)-; R2and R3are both C1-14alkyl, or C2-14alkenyl; and R′ is a C1-C12 linear alkyl.

[0096] In an embodiment, the ionisable lipid has a structure of Formula (IIa), (IIb), (IIc) or (IId):or a salt, solvate or isomer thereof, wherein R4 is as described elsewhere herein.

[0097] In an embodiment, the ionisable lipid has a structure of Formula (IId):or a salt, solvate or isomer thereof, wherein R4 is as described elsewhere herein.

[0098] In an embodiment of Formulae (I), (IA), (II), (IIa), (IIb), (IIc), (IId) and, R4 is - (CH2)nQ, in which n is 2, 3 or 4, and Q is OH.

[0099] In an embodiment, the ionisable lipid has a structure of Formula (IIe):or a salt, solvate or isomer thereof, wherein n is 2, 3 or 4; and m, R', R", R2, R3, R5and R6are as described elsewhere herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0100] In an embodiment, the ionisable lipid is selected from the group consisting of:, or a salt, solvate or isomer thereof.

[0101] In a particular embodiment, the ionisable lipid has the following structure, also known as SM-102:or a salt, solvate or isomer thereof.

[0102] The ionisable lipids disclosed herein may be synthesised according to the methods described in WO 2017 / 049245, the entire contents of which are incorporated herein by reference, or any other suitable methods known in the art.

[0103] The nanoparticles disclosed herein may suitably comprise a sterol. Thus, in an embodiment, the nanoparticle comprises a sterol. Suitable sterols will be familiar to persons skilled in the art, illustrative examples of which include cholesterol and a phytosterol. In an embodiment, the sterol comprises less than 10 mol % cholesterol. In an embodiment, the sterol is a phytosterol. In another embodiment, the sterol is a combination of a phytosterol and cholesterol. In particular, the sterol may comprise a phytosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol % of the nanoparticle.

[0104] The phytosterol may be a C-24 alkyl phytosterol, a versatile group of phytosterols that are important for plant cell membrane dynamics.

[0105] The phytosterol may be a C-24 alkyl phytosterol having a structure of Formula (III)or a stereoisomer thereof, wherein: each is individually a single bond or a double bond; and R is a C1-6 alkyl.

[0106] In some embodiments of Formula (III), R is a C1-4alkyl, C1-3alkyl, or C1-2alkyl. In one embodiment of Formula (III), R is a C1-2alkyl.

[0107] In some embodiments, the phytosterol is selected from the group consisting of β- sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, ergesterol, and combinations thereof.

[0108] In one embodiment, the C-24 alkyl phytosterol is selected from the group consisting of , ,or any combination thereof.

[0109] In one embodiment, the C-24 alkyl phytosterol is stigmasterol. In one embodiment, the C-24 alkyl phytosterol is β-sitostanol. In one embodiment, the C-24 alkyl phytosterol is campesterol. In one embodiment, the C-24 alkyl phytosterol is brassicasterol. In an embodiment, the C-24 alkyl phytosterol is ergesterol.

[0110] In one embodiment, the C-24 alkyl phytosterol is β-sitosterol (beta-sitosterol). In an embodiment, the C-24 alkyl phytosterol is

[0111] In one embodiment the sterol consists of β-sitosterol.

[0112] Other suitable sterols, phytosterols, including C-24 alkyl phytosterol and processes for their preparation for use in the compositions disclosed herein will be apparent to those skilled in the art.

[0113] In an embodiment, the sterol comprises less than 10 mol % cholesterol. In other embodiments, the sterol comprises a phytosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol % of the nanoparticle composition. In some embodiments, the cholesterol will comprise less than 9 mol %, less than 8 mol %, less than 7 mol %, less than 6 mol %, less than 5 mol %, less than 4 mol %, less than 3 mol %, less than 2 mol %, or less than 1 mol % of the nanoparticle composition. In an embodiment, the nanoparticle does not comprise cholesterol. In an embodiment, the nanoparticle composition does not comprise cholesterol.

[0114] In some embodiments, the nanoparticle or nanoparticle composition comprise a phytosterol in an amount of at least 28.5 mol %, preferably at least 29.5 mol %, preferably at least 30.5 mol %, preferably at least 31.5 mol %, preferably at least 32.5 mol %, preferably at least 33.5 mol %, preferably at least 34.5 mol %, preferably at least 35.5 mol %, preferably at least 36.5 mol %, or more preferably at least 37.5 mol % of the nanoparticle / nanoparticle composition.

[0115] The nanoparticle or nanoparticle composition, as described herein, may suitably comprise an ionisable lipid of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein and a sterol, wherein the sterol wherein the sterol comprises less than 10 mol % cholesterol. In an embodiment, the nanoparticle or nanoparticle composition comprises a sterol, wherein the sterol comprises a phytosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol % of the nanoparticle / nanoparticle composition and an ionisable lipid of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein.

[0116] In some embodiments, the ionisable lipid is present in the nanoparticle / nanoparticle composition in an amount of about 5 mol% to about 70 mol%; preferably about 10 mol% to about 60 mol%, preferably about 10 mol% to about 50 mol%, preferably about 10 mol% to about 40 mol%, or more preferably about 10 mol% to about 30 mol% of the total lipids present in the nanoparticle or nanoparticle composition. In an embodiment, the ionisable lipid is present in an amount of about 50 mol% of the nanoparticle or nanoparticle composition.

[0117] In some embodiments, the sterol is present in the nanoparticle or nanoparticle composition in an amount of about 10 mol% to about 80 mol%, preferably about 20 mol% to about 70 mol%, preferably about 20 mol% to about 60 mol%, preferably about 30 mol% to about 50 mol%, or more preferably about 30 mol% to about 40 mol% of the nanoparticle or nanoparticle composition. In an embodiment, the sterol is present in the nanoparticle or nanoparticle composition in an amount of about 45 mol%. In an embodiment, the sterol is present in the nanoparticle or nanoparticle composition in an amount of about 40 mol%. In an embodiment, the sterol is present in the nanoparticle or nanoparticle composition in an amount of about 38.5 mol%.

[0118] In an embodiment, the sterol comprises cholesterol, wherein cholesterol comprises less than about 10 mol % of the nanoparticle or nanoparticle composition. In some embodiments, the cholesterol will comprise less than about 9 mol %, preferably less than about 8 mol %, preferably less than about 7 mol %, preferably less than about 6 mol %, preferably less than about 5 mol %, preferably less than about 4 mol %, preferably less than about 3 mol %, preferably less than about 2 mol %, or more preferably less than about 1 mol % of the nanoparticle or nanoparticle composition. The nanoparticle or nanoparticlecomposition may comprise phytosterol and cholesterol in a molar ratio of from about 10:1 to about 1:1. For example, the phytosterol and cholesterol may be in a molar ratio of from about 10:1 to about 1:1, preferably from about 9:1 to about 1:1, preferably from about 8:1 to about 1:1, preferably from about 7:1 to about 1:1, preferably from about 6:1 to about 1:1, preferably from about 5:1 to about 1:1, preferably from about 4:1 to about 1:1, preferably from about 3:1 to about 1:1 or more preferably from about 2:1 to about 1:1.

[0119] In an embodiment, cholesterol is not present in the nanoparticle. In an embodiment, cholesterol is not present in the nanoparticle composition.

[0100] In an embodiment, the sterol may comprise a phytosterol. In some embodiments, phytosterol is present in an amount of at least 28.5 mol %, preferably at least 29.5 mol %, preferably at least 30.5 mol %, preferably at least 31.5 mol %, preferably at least 32.5 mol %, preferably at least 33.5 mol %, preferably at least 34.5 mol %, preferably at least 35.5 mol %, preferably at least 36.5 mol %, preferably at least 37.5 mol % preferably at least 37.5 mol %, preferably at least 40 mol % of the nanoparticle, preferably at least 45 mol % or more preferably at least 50 mol % of the nanoparticle or nanoparticle composition. In an embodiment, phytosterol is present in an amount of about 38.5 mol% of the nanoparticle or nanoparticle composition.

[0101] Advantageously, the substitution or addition of one or more additional lipids (e.g., PEG lipids, phospholipids, structural lipids, including those disclosed in WO / 2017 / 049245) in the nanoparticle or nanoparticle composition, as described herein, may reduce or prevent complex aggregation, in particular of LNP, promote LNP stability, prolong circulation time and / or enhance targeted delivery of the agent (e.g., a therapeutic nucleic acid molecule) to the targeted cell or intracellular organelle. The amount or proportion of lipids and sterols may also be modified, to reduce or prevent complex aggregation, promote stability, prolong circulation time and / or enhance targeted delivery of the agent e.g., a therapeutic nucleic acid molecule to the targeted cell or intracellular organelle. The choice of additional lipid(s) and the relative molar ratio of lipids in the pharmaceutical compositions disclosed herein may depend on the characteristics of the additional lipid(s), the nature of the intended target cell or intracellular organelle, the characteristics of the agent to be delivered, the saturation of the alkyl chain(s), as well as the size, charge, pH, pKa, fusogenicity and / or toxicity of the additional lipid(s). For example, increasing the proportion of PEG lipid in the LNP resultsin increased circulation time and prevents complex aggregation. In an illustrative example, DMG-PEG can be replaced by another PEG lipid with a longer acyl chain (e.g. DSPE-PEG). A skilled person will be able to select one or more additional lipids, and modify their amount and / or proportions, as required.

[0102] In certain embodiments, the one or more additional lipid components may be present in the composition in a combined amount of up to 60 mol% of the total lipids present in the composition. For example, the one or more additional lipid components may be present in the nanoparticle or nanoparticle composition in an individual or combined amount of from about 0 mol% to about 60 mol%, preferably from about 0 mol% to about 50 mol%, preferably from about 0 mol% to about 40 mol%, preferably from about about 0 mol% to about 35 mol%, preferably from about about 0 mol% to about 30 mol%, or more preferably from about 0 mol% to about 25 mol% of the total lipids present in the nanoparticle or nanoparticle composition, as described herein.

[0103] The nanoparticle or nanoparticle compositions described herein may suitably comprise one or more non-cationic helper lipids. In an embodiment, the non-cationic helper lipid is a phospholipid. In an embodiment, the non-cationic helper lipid is a phospholipid substitute or replacement. The term "non-cationic helper lipid" typically refers to a lipid comprising at least one fatty acid chain of at least 8 carbons in length and at least one polar head group moiety. In an embodiment, the helper lipid is a phosphatidyl choline (PC). In an embodiment, the helper lipid is not a phosphatidyl choline (PC). In an embodiments, a non- cationic helper lipid is a non-phosphatidyl choline (PC) zwitterionic lipid, a DSPC analog, oleic acid, an oleic acid analog, or a 1,2-distearoyl-i77-glycero-3-phosphocholine (DSPC) substitute. In an embodiment, the non-cationic helper lipid is a phospholipid or a phospholipid substitute. In an embodiment, the phospholipid or phospholipid substitute can be, for example, one or more saturated or (poly)unsaturated phospholipids, or phospholipid substitutes, or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0104] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). A phospholipid or an analog or derivative thereof may include choline. A phospholipid or ananalog or derivative thereof may not include choline. Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid- containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0105] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0106] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0107] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidylglycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipid, such as sphingomyelin.

[0108] In some embodiment, the nanoparticle or nanoparticle compositions disclosed herein further comprise a phospholipid. Suitable phospholipids will be familiar to persons skilled in the art, illustrative examples of which include 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero- phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero- phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 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- glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (18:3 (cis) PC), 1,2-diarachidonoyl-sn-glycero-3-phosphocholine (DAPC), 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine (22:6 (cis) PC) 1,2-diphytanoyl-sn- glycero-3-phosphoethanolamine (4ME 16.0 PE), 1,2-distearoyl-sn-glycero-3- phosphoethanolamine (DSPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (PE(18:2 / 18:2), 1,2-dilinolenoyl-sn-glycero-3-phosphoethanol amine (PE 18:3 (9Z,12Z, 15Z), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE 18:3 (9Z,12Z, 15Z), 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine (22:6 (cis) PE), 1,2-dioleoyl-sn- glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, the phospholipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-Dioleoyl-sn-glycero-3- phosphocholine (DOPC) or combinations thereof. In an embodiment, the phospholipid is DMPE. In one embodiment, the phospholipid is DOPC. In an embodiment, the phospholipid is DSPC.

[0109] The nanoparticle or nanoparticle compositions disclosed herein may further comprise a PEG lipid. A PEG lipid is a lipid modified with polyethylene glycol.

[0110] Suitable PEG lipids will be familiar to persons skilled in the art, illustrative examples of which include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG- ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids.

[0111] In an embodiment, the PEG-lipid is selected from the group consisting of 1,2- dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn- glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG- disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG- diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), and PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In an embodiment, the PEG- lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. The PEG lipid may be a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. Other suitable PEGlipids and processes for their preparation are described, for example, in WO 2020 / 061284 and WO 2020 / 061295, the entire contents of which are incorporate herein by reference.

[0112] In an embodiment, the PEG-lipid is selected from a group consisting of PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid or combinations thereof. In an embodiment, the PEG lipid is PEG-DMG. In an embodiment, the PEG lipid is PEG-DSPE.

[0113] In an embodiment, the PEG-lipid is used to functionalise the LNP with the targeting moiety. In an embodiment, the PEG-lipid used to functionalise the LNP with the targeting moiety is DSPE-PEG. That is to say, in some embodiments the LNP comprises DSPE-PEG and DMG-PEG.

[0114] The nanoparticle or nanoparticle compositions described herein may further comprise one or more additional ionisable lipids. Suitable additional ionisable lipids will be familiar to persons skilled in the art, illustrative examples of which include 3- (didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2- (didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25- ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N- dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yl oxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca- 9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)) and (2 S)-2-({8-[(3β)-cholest- 5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan- 1-amine (Octyl-CLinDMA (2S)).

[0115] It is also contemplated that the nanoparticles disclosed herein may comprise one or more functionalized lipids. For example, a lipid may be functionalized with an alkyne group that, when exposed to an azide under appropriate reaction conditions, may undergo a cycloaddition reaction. In particular, a lipid bilayer may be functionalized in this fashion with one or more groups useful in facilitating membrane permeation, or imaging. Functional groups and conjugates useful for imaging and membrane permeation will be known to persons skilled in the art.

[0116] The nanoparticles and nanoparticle compositions described herein may further comprise quaternary ammonium lipids. Illustrative examples of suitable quaternary ammonium lipids include, but are not limited to, DOTAP, DOTMA, DDAB, DMRIE, DORI and other commercial available lipids that may be purchased from Avanti® Polar Lipids. Other suitable quaternary ammonium lipids and processes for their preparation will be apparent to those skilled in art and may include those disclosed, for example, in WO 2000 / 030444, WO 2011 / 141705, the entire contents of each of which are incorporated herein by reference.

[0117] The nanoparticle and nanoparticle compositions described herein may further comprise an adjuvant. Suitable adjuvants will be familiar to persons skilled in the art, illustrative examples of which include Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminium hydroxide and Pam3CSK4.

[0118] In an embodiment, the nanoparticle or nanoparticle composition described herein comprises SM-102; β-sitosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol % of the nanoparticle or nanoparticle composition; DSPC; and DMG-PEG. In an embodiment, the nanoparticle or nanoparticle composition described herein comprises SM-102, β-sitosterol, DSPC, and DMG-PEG. In an embodiment, the nanoparticle or nanoparticle composition described herein comprises from about 20 mol% to about 80 mol% SM-102, from about 5 mol% to about 30 mol% DSPC, about 30 mol% to about 50 mol% β-sitosterol, no more than 10% mol cholesterol, and about 0.5 mol% to about 3 mol% DMG- PEG. In an embodiment, the nanoparticle or nanoparticle composition described herein does not comprise cholesterol. In an embodiment, the nanoparticle or nanoparticle compositiondescribed herein comprises about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG.

[0119] In an embodiment, the nanoparticle or nanoparticle composition described herein comprises SM-102; β-sitosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol % of the nanoparticle or nanoparticle composition; DSPC; and DMG-PEG. In an embodiment, the nanoparticle described herein comprises SM-102, β-sitosterol, DSPC, and DMG-PEG. In an embodiment, the nanoparticle described herein comprises from about 20 mol% to about 80 mol% SM-102, from about 5 mol% to about 30 mol% DSPC, about 30 mol% to about 50 mol% β-sitosterol, no more than 10% mol cholesterol, and about 0.5 mol% to about 3 mol% DMG-PEG. In an embodiment, the nanoparticle described herein does not comprise cholesterol. In one embodiment, the nanoparticle described herein comprises about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG.

[0120] In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG. In an embodiment, the LNP composition does not comprise cholesterol. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% cholesterol and about 1.5 mol% DSPE- PEG. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DSPE-PEG. In an embodiment, the LNP composition does not comprise cholesterol. In another aspect, the present disclosure provides an LNP composition comprising 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% cholesterol and about 1.5 mol% DMG- PEG.

[0121] The nanoparticle described herein may comprise particles having an average diameter of about 1 μm or less. For example, the nanoparticle may have an average particle diameter of about 1 μm, preferably about 900 nm, preferably about 800 nm, preferably about 700 nm, preferably about 600 nm, preferably about 500 nm, preferably about 400 nm, preferably about 300 nm, preferably about 200 nm, preferably about 175 nm, preferably about 150 nm, preferably about 125 nm, preferably about 100 nm, preferably about 75 nm, preferably about 50 nm, or less. In an embodiment, the nanoparticle has an average particlediameter of from about 300 to about 50 nm. In an embodiment, the nanoparticle comprises an average diameter in the range of from about 10 nm to about 1000 nm. In some embodiments, the nanoparticle has an average diameter of from about 50 nm to about 500 nm, or preferably from about 50 nm to about 200 nm. In some embodiments, the nanoparticle has an average diameter of about 100 nm. The average diameter of the nanoparticle may be measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or any other suitable method known to persons skilled in the art.

[0122] The nanoparticle described herein are suitable for delivering one or more agents, including therapeutic agents, to a cell, in particular to a hard-to-transfect or transfection- recalcitrant cell. The nanoparticle described herein may be particularly suitable for delivery of one or more agents, particularly nucleic acid-based agents, to hard-to-transfect cells or transfection-recalcitrant cells.

[0123] The nanoparticle described herein may advantageously provide enhanced or improved delivery (i.e., transfection efficiency) of one or more agents to a cell, including hard-to-transfect cells or transfection-recalcitrant cells. The transfection efficiency can be measured as a proportion, or percentage of cells that demonstrate uptake of the nanoparticle and / or the agent to be delivered to the cells. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 1-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 5-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 10-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 15-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 25-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 35-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 45-100%. In someembodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 50-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 55-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 60-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 65-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 70-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 75-100%. In some embodiments, the nanoparticle or nanoparticle composition provides transfection efficiency of 80-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 85-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 90-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 10-100%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 50-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 60-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 70-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 80-90%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 50-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 60-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 70-80%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-70%. In some embodiments, the nanoparticle ornanoparticle composition provides a transfection efficiency of 30-70%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-70%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 50-70%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 60-70%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-60%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-60%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-60%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 50-60%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-50%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-50%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 40-50%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 20-40%. In some embodiments, the nanoparticle or nanoparticle composition provides a transfection efficiency of 30-40%.

[0124] In an embodiment, the nanoparticle or nanoparticle composition described herein provides enhanced or improved delivery of one or more agents to cells, including hard-to- transfect cells or transfection-recalcitrant cells by at least 5%, preferably by at least about 10%, preferably by at least about 20%, preferably by at least about 30%, preferably by at least about 40%, preferably by at least about 50%, preferably by at least about 100%, preferably by at least about 200%, preferably by at least about 300%, preferably by at least about 400%, preferably by at least about 500%, preferably by at least about 600%, preferably by at least about 700%, preferably by at least about 800%, preferably by at least about 900%, or by at least about preferably 1000% in comparison to comparator nanoparticle or nanoparticle composition, wherein the comparator does not comprise the antigen-binding moiety.

[0125] In another example, the enhanced delivery of an agent to cells, including hard-to- transfect cells or transfection-recalcitrant cells may involve delivery of at least 1.5 fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold more, or at least 10-fold more of the agent by nanoparticle or nanoparticle composition as described herein when compared to the comparator.

[0126] In other examples, the enhanced delivery of an agent to the cell by the nanoparticle or nanoparticle composition as described herein, including to hard-to-transfect cells or transfection-recalcitrant cells, can be measured in comparison to the delivery provided by commonly used / commercially available transfection compositions, including lipid-based transfection compositions, that will be familiar to persons skilled in the art. Illustrative examples of such commercially available transfection compositions (or transfection reagents) include liposome-based mediated transfection reagents such as Lipofectamine, Lipofectamine 2000, Lipofectamine 3000 Lipofectamine LTX (a mixture of DOSPA and DOPE). Other examples of commercially available transfection reagents are XtremeGENE (Roche); DharmaFECT3 (Dharmacon); FUGENE, ViaFect and TransFast (Promega).

[0127] The nanoparticle or nanoparticle compositions disclosed herein may further comprise an agent to be delivered to the cell or the transfection-recalcitrant cell. The agent may be a therapeutic agent. In some embodiments, the agent may be a non-therapeutic agent.

[0128] The efficiency, delivery and / or rate of delivery of the nanoparticle to the cell may be measured by detecting or measuring the amount of the agent or product produced by the agent that is delivered by the nanoparticle or nanoparticle composition to the cell. The efficiency, delivery and / or rate of delivery of the agent by the nanoparticle or nanoparticle composition described herein may be measured by comparing the amount of the agent or of a product produced by the agent, present in a cell that has been transfected with the nanoparticle disclosed herein comprising the agent, to the amount of the agent or product produced by the agent, present in a cell that has been contacted with a comparator transfection composition or reagent comprising the same agent. For example, if the agent to be delivered is a protein or a small molecule drug, the level of protein or small molecule drug present in the cell that has been transfected with the nanoparticle described herein comprising the protein or small molecule drug or a comparator transfection composition / reagent herein comprising the protein or small molecule drug, can be measured andcompared. In another example, if the agent to be delivered is a nucleic acid molecule, the level of expression of the nucleic acid present in the cell that has been transfected with the nanoparticle comprising the nucleic acid molecule or a comparator transfection composition / reagent herein comprising the nucleic acid molecule, can be measured and compared. In an example, wherein the agent to be delivered is an mRNA, the level of mRNA or protein product of the mRNA present in cells that has been transfected with the nanoparticle comprising the mRNA or a comparator transfection composition herein comprising the mRNA, can be measured and compared.

[0129] The present disclosure also extends to pharmaceutical compositions comprising the nanoparticles described herein and one or more agents. The pharmaceutical compositions may suitably comprise cells transfected with the nanoparticles described herein and the one or more agents.

[0130] In an embodiment, the agent is a protein, a small-molecule drug, a nucleic acid molecule or a combination of any of the foregoing. In an embodiment, the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule, or a combination thereof.

[0131] In some embodiments, the agent is a nucleic acid-based agent. As used herein, the term "nucleic acid-based agent" refers to an agent comprising a nucleic acid sequence (or "polynucleotide") that, when delivered to a cell or organ, produces a polypeptide that brings about a desirable change in the cell, organ, or other bodily tissue or system. Such nucleic acid-based agents may include, but are not limited to deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), including messenger mRNA (mRNA), hybrids thereof, RNAi- inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, guide RNA (gRNA), etc. In an embodiment, the therapeutic and / or prophylactic is an RNA. RNAs useful in the compositions and methods described herein can be selected from the group consisting of, but are not limited to, shortmers, antagomirs, antisense, ribozymes, small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer- substrate RNA, small hairpin RNA (shRNA), transfer RNA (tRNA), self-amplifying RNA, long non-coding RNA, circular RNA, messenger RNA (mRNA), guide RNA (gRNA) and any combination thereof.

[0132] In an embodiment, the nucleic acid-based agent is an mRNA. The mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. The polypeptide encoded by an mRNA may have a therapeutic effect when expressed in the cell. The nucleic acid-based agent may be a guide RNA and / or encodes for a CRISPR-associated protein.

[0133] In another embodiment, the nucleic acid-based agent is an siRNA. An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may comprise a sequence that is complementary to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA.

[0134] In some embodiments, the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA, a small hairpin RNA (shRNA), a messenger RNA (mRNA), self-amplifying RNA, long non-coding RNA, circular RNA, a guide RNA (gRNA) and any combination thereof.

[0135] In some embodiments, the nucleic acid-based agent is an shRNA or a vector or plasmid encoding the same. An shRNA may be produced inside a target cell upon delivery of an appropriate construct to the nucleus. Constructs and mechanisms relating to shRNA are well known in the relevant art.

[0136] In another embodiment, the nucleic acid-based agent is messenger RNA (mRNA) together with a guide RNA (gRNA) to enable CRISPR-Cas genome editing. The “clustered regularly interspaced short palindromic repeat” (CRISPR) / “CRISPR-associated protein” (Cas) system (CRISPR / Cas system) evolved in bacteria and archaea as an adaptive immune system to defend against viral attack. The mechanisms of CRISPR-mediated gene editing would be known to persons skilled in the art and have been described, for example, by Doudna et al., (2014, Methods in Enzymology, 546). CRISPR-Cas genome editing systems may advantageously be used to generate, for example, a site-specific double strand break(DSB) or single strand break (SSB) within a double-stranded DNA (dsDNA) with Cas9 or a site specific break in RNA with Cas13. Once a DSB or SSB is detected in a cell, the DNA repair machinery will repair the break by "non-homologous end-joining" or "NHEJ" or "homology-directed repair" or "HDR". NHEJ is triggered to repair double-stranded breaks in which the break ends are directly ligated without the need for a homologous template. Due to the error-prone nature of this repair pathway, small insertions or deletions (INDELs) may be introduced at the target locus near the site of the initial cleavage, and such INDELs can cause frameshift mutations, promote internal ribosomal entry, convert pseudo-mRNAs into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (see, e.g., Tuladhar et al., 2019, Nature Communications, 10: 4056). Unpredicted large genome modifications can also be introduced, which can be more than several kilobases (see, e.g., Kosicki et al., 2018 Nature Biotechnology, 36: 765-771). By contrast, HDR accurately and precisely repairs DNA breaks using a homologous template to guide repair. The most common form of HDR is homologous recombination (HR), by which nucleotide sequences are exchanged between two similar or identical molecules of DNA.

[0137] The term “guide RNA” (or gRNA) refers to a RNA sequence that is complementary to a target nucleic acid sequence and directs a RNA-guided nuclease to the target nucleic acid sequence. gRNA typically comprises CRISPR RNA (crRNA) and a tracr RNA (tracrRNA). "crRNA" is a 17-20 nucleotide sequence that is complementary to the target nucleic acid sequence, while the "tracrRNA" provides a binding scaffold for the RNA- guided nuclease. crRNA and tracrRNA exist in nature as two separate RNA molecules, which has been adapted for molecular biology techniques using, for example, 2-piece gRNAs such as CRISPR tracer RNAs (cr:tracrRNAs).

[0138] In an embodiment described herein, the gRNA is a single-guide RNA (sgRNA). sgRNA typically refers to a single RNA sequence that comprises the crRNA fused to the tracrRNA. In an embodiment, the sgRNA comprises a sequence of at least 10 contiguous nucleotides that are complementary to a target nucleic acid sequence. Accordingly, the sgRNA comprises a sequence of at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28 , at least 29, or at least 30 nucleotides that are complementary to a target nucleic acid sequence. In an embodiment,the sgRNA comprises a sequence of at least 20 contiguous nucleotides that are complementary to a target nucleic acid sequence. Methods and tools for the design of gRNA and sgRNA would be known to persons skilled in the art, illustrative examples of which include CHOPCHOP, CRISPR Design, sgRNA Designer, Synthego and GT-Scan.

[0139] In an embodiment, the RNA-guided nuclease is a CRISPR-associated (Cas) endonuclease. Suitable Cas endonucleases would be known to persons skilled in the art, illustrative examples of which include Cas3, Cas9, Cas12 (e.g., Cas12a, Cas12b, Cas12c, Cas12d, Cas12e), Cas13 (e.g., Cas13a, Cas13b, Cas13c, Cas13d) and Cas14. In some embodiments, the Cas endonuclease is catalytically active. In an embodiment, the Cas endonuclease is catalytically inactive (dead Cas protein [dCas9] that is devoid of nucleolytic activity). In one embodiment, the dCas9 can be fused to protein domains that can induce the transcription of a targeted gene (i.e. CRISPR activation), see for example Konermann et al. 2015, Nature517, 583–588. In another embodiment, the dCas9 is used in CRISPR activation to is reverse viral latency, including HIV latency such as those described in Zhang et al. 2015 Sci. Rep. 5, 16277; Bialek et al. 2016 PLoS One 11, e0158294-e0158294 and Limsirichai et al.2016 Mol. Ther.24, 499-507. In an embodiment, the Cas endonuclease is a Cas13 nuclease. The type VI Cas13 nucleases are programmable RNA-guided targeting enzymes that exclusively degrade single-stranded RNAs (ssRNAs) with high efficacy and specificity. Cas13 systems have been deployed in a variety of applications including RNA knockdown (Abudayyeh et al., 2017, Nature, 550: 280-284), nucleic-acid detection (Gootenberg et al., 2017, Science, 356: 438-442), precise RNA base editing (Cox et al., 2017, Science, 358), live-cell RNA imaging (Yang et al., 2019, Molecular Cell, 76: 981- 997), and viral suppression (Blanchard et al., 2021, Nature Biotechnology, 39: 717-726). The target recognition process of Cas13 is guided by a single CRISPR RNA (crRNA) consisting of a direct repeat (DR) and a programmable spacer sequence. The DR sequence forms a highly ordered stem-loop structure that facilitates crRNA loading into Cas13 protein, whereas the spacer sequence mediates RNA target recognition through RNA-RNA base pairing. The efficiency and reversibility of RNA targeting with Cas13 represents a promising modality to specifically edit coding and non-coding transcriptomes without risking permanent alteration of the genome. Compared to classical eukaryotic RNA interference (RNAi), RNA knockdown with Cas13 in mammalian cells typically demonstrates superiorspecificity, attributable to its extended spacer sequence, making it highly attractive for targeting aberrant transcripts that drive various human genetic diseases, e.g., cancer.

[0140] Other suitable nucleic acid therapeutics are described in WO 2017 / 049245, the entire contents of which are incorporated herein by reference.

[0141] The agent may be encapsulated or partially encapsulated by the nanoparticle described herein, and / or it may be disposed on the surface of the nanoparticle (e.g., by coating, adsorption, covalent linkage or other means).

[0142] In an embodiment, the pharmaceutical composition is a vaccine composition.

[0143] The pharmaceutical compositions disclosed herein may further comprise one or more pharmaceutically acceptable excipients (e.g., carriers, diluents, etc.). Where an excipient is used, it must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the pharmaceutical formulation and not injurious to the subject. Such pharmaceutically acceptable excipients will be apparent to those skilled in the art and may depend on the intended formulation and / or mode of administration. For example, the excipients may include, but are not limited to solvents, cryoprotectants, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, waxes, butters, colouring agents, coating agents, flavourings and perfuming agents, or any combination thereof. In one embodiment, the one or more excipient comprises a cryoprotectant. Suitable cryoprotectants will be familiar to persons skilled in the art, an illustrative example of which includes sucrose. In an embodiment, the cryoprotectant is sucrose.

[0144] In some embodiments, the pharmaceutic compositions disclosed herein are formulated for parenteral administration, for example, by subcutaneous injection, intravenous injection, intraperitoneally, intramuscular injection, intrasternal injection or infusion. In a particular embodiment, the pharmaceutic compositions disclosed herein are formulated for intravenous injection. Injectable preparations may be formulated according to the known art, for example, suitable dispersing, wetting, suspending and / or solubilizing agents. Injectable preparations may comprise one or more inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers suchas ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and any combination thereof.

[0145] In other embodiments, the pharmaceutic compositions disclosed herein are formulated for intranasal administration. In some embodiments, the intranasal compositions disclosed herein may be prepared as pharmaceutically acceptable emulsions, microemulsions, solutions, or suspensions. In particular, the compositions disclosed herein may be prepared as aqueous solutions or suspensions. Where the formulations of the present invention are aqueous solutions or suspensions, the formulations may comprise water in an amount of greater than 50%, 60%, 70%, 80% or 90% by weight of the total composition. The intranasal compositions disclosed herein may further comprise a pharmaceutically acceptable co-solvent. Suitable co-solvents may include but are not limited to alcohols, polyvinyl alcohols, propylene glycol, polyethylene glycols and derivatives thereof, glycerol, sorbitol, polysorbates, ethanol, and combination thereof. In some embodiments, intranasal formulations suitable for use in the present invention may comprise one or more of a thickening agent, pH modifying agent, sensory agent, antioxidant, surfactant, adhesive, stabilizer, osmolarity adjusting agent, preservative, permeation enhancer, chelating agent, sweetening agent, flavouring agent, taste masking agent, colorant. Some agents or components of the intranasal formulation may have more than one function. For example, where ethanol is used as a sensory agent in the formulations disclosed herein, it may further function as a penetration enhancer and / or a co-solvent.

[0146] In some embodiments, the pharmaceutical compositions disclosed herein are sustained-release formulations.

[0147] General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0148] The pharmaceutical compositions disclosed herein may be refrigerated or frozen for storage and / or shipment. For example, the pharmaceutical compositions may be stored at a temperature of 4 °C or lower, e.g., from about -150 °C to about 4 °C, or from about -80 °C to about 0°C or from about -80 °C to about -20 °C (e.g., about 4°C, 0°C, -5 °C, -10 °C, -15 °C, -20 °C, -25 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, -130 °C or -150 °C). In an embodiment, the pharmaceutical composition is a solution that is refrigerated for storage and / or shipment at, for example, about -20° C, -30 °C, - 40 °C, -50 °C, -60 °C, -70 °C or -80 °C. Antigen-binding moiety

[0149] The term "antigen-binding moiety", as used herein, is intended to mean a molecule that has binding specificity for the target antigen. It will be understood that this term extends to immunoglobulins, immunoglobulin fragments and non-immunoglobulin-derived protein frameworks that exhibit antigen-binding activity. Illustrative examples of suitable antigen- binding moieties include antibodies and antigen-binding fragments thereof. Preferably, the antigen-binding moiety binds specifically to the antigen expressed on the surface of the cell so as to facilitate internalisation of the moiety and the nanoparticle to which it is tethered. The term "internalise" is understood to mean that, upon binding of the antigen-binding moiety to the cell surface antigen, the moiety and the cell surface antigen complex that forms will traverse the cell membrane and enter the cell cytoplasm.

[0150] In an embodiment, the antigen-binding moiety, as described herein, is conjugated to another molecule or moiety, including functional moieties (e.g., toxins), detectable moieties (e.g., fluorescent molecules, radioisotopes), small molecule drugs and polypeptides.

[0151] In an embodiment, the antigen-binding moiety is an immunoglobulin molecule, also referred to interchangeably herein as an antibody. The term “antibody” is understood to mean any antigen-binding molecule or molecular complex comprising at least one complementarity determining region (CDR) that binds specifically to, or interacts specifically with, the target antigen. The term “antibody” includes full-length immunoglobulin molecules comprising two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (which may be abbreviated as HCVR, VH or VH) and a heavy chain constant region. The heavy chain constant region typically comprises threedomains - CH1, CH2 and CH3. Each light chain comprises a light chain variable region (which may be abbreviated as LCVR, VL, VK, VKor VL) and a light chain constant region. The light chain constant region will typically comprise one domain (CL1). The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, also referred to as framework regions (FR). Each VHand VLtypically comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the FRs of the antibody, or the antigen-binding fragment thereof, may be identical to the FR of germline sequences of the target species (i.e., the species to which the antibody or antigen-binding fragment thereof, as described herein, will be administered). In some embodiments, the FR may be naturally or artificially modified. Whilst it is generally desirable that each of the FR sequences are identical to FR sequences derived from immunoglobulin molecules of the target species, including to minimize an immune response being raised against the binding molecule upon administration to a subject of the target species, in some embodiments, the antibody or antigen-binding fragment thereof may comprise one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in one or more FR from the target species. Preferably, where the antibody or antigen-binding fragment thereof comprises one or more amino acid residues across one or more of its FR sequences that would be foreign at a corresponding position in the target species, that "foreign" amino acid residue will not (i) adversely impact the binding specificity of the antibody or antigen- binding fragment thereof to the target antigen, and / or (ii) cause an immune response to be raised against the antibody of antigen-binding fragment thereof when administered to a subject of the target species.

[0152] Suitable antibodies include antibodies of any class, such as IgG, IgA, or IgM (including sub-classes thereof). There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, characterised by heavy-chain constant regions α, δ, ε, γ, and µ, respectively. Several antibody classes may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins will be well known to persons skilled in the art.

[0153] In an embodiment, the antigen-binding moiety comprises the VH and VL complementarity determining regions (CDR) of the anti-CD2 antibody of Clone 299812 (R&D Systems). In an embodiment, the antigen-binding moiety comprises the VH and VL regions of the anti-CD2 antibody of Clone 299812 (R&D Systems) or a CD2-binding fragment thereof. In an embodiment, the antigen-binding moiety comprises the VH and VL CDR of the anti-CD7 antibody of Clone 848438 (R&D Systems). In an embodiment, the antigen-binding moiety comprises the VH and VL regions of the anti-CD7 antibody of Clone 848438 (R&D Systems) or a CD7-binding fragment thereof.

[0154] As used herein, the term “complementarity determining region” (CDR) refers to the region of an immunoglobulin variable domain that recognizes and binds to the target antigen. Each variable domain may comprises up to three CDR sequences, identified as CDR1, CDR2 and CDR3. The amino acid sequence of each CDR is often defined by Kabat numbering (e.g., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the light chain variable domain and residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or by Chothia numbering (e.g., about residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) of the light chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) of the heavy chain variable domain; see Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).

[0155] The present disclosure extends to antigen-binding moieties that bind specifically to the target antigen of any species. The species is preferably human, but it would be understood that the nanoparticles disclosed herein may have utility in other species, including in primates and non-primates.

[0156] The terms “antigen-binding fragment”, “antigen-binding portion”, “antigen-binding domain”, “antigen-binding site” and the like are used interchangeably herein to refer to a part of an antigen-binding molecule that retains the ability to bind to the target antigen; that is, to CD2 or CD7, including native CD2 or CD7. These terms include naturally occurring, enzymatically obtainable, synthetic or genetically engineered (recombinant) polypeptides and glycoproteins that specifically bind to the target antigen to form a complex.

[0157] Antigen-binding fragments may be derived, for example, from naturally derived immunoglobulin molecules using any suitable method known to persons skilled in the art, illustrative examples of which include proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of nucleic acid sequences encoding antibody variable and optionally constant domains. Suitable nucleic acid sequences are known and / or are readily available from, e.g., commercial sources, DNA libraries (including, e.g., phage-antibody libraries), or can be synthesized. The nucleic acid sequences may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.

[0158] Non-limiting examples of suitable antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated CDR such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, one-armed antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g. monovalent nanobodies, bivalent nanobodies, etc.), and small modular immunopharmaceuticals (SMIPs), are also encompassed by the term “antigen-binding fragment,” as used herein.

[0159] In an embodiment, an antigen-binding fragment comprises at least one immunoglobulin variable domain. The variable domain may comprise an amino acid sequence of any suitable length or composition and will generally comprise at least one CDR which is adjacent to or in frame with one or more framework sequences. Where the antigen- binding fragment comprises a VHdomain and a VLdomain, the VHand VLdomains may be situated relative to one another in any suitable arrangement. For example, the variable region may be dimeric and contain VH-VH, VH-VL or VL-VL dimers. Alternatively, the antigen- binding fragment of an antibody may contain a monomeric VHor VLdomain.

[0160] In some embodiments, an antigen-binding fragment may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting configurations of variable and constant domains that may be found within an antigen-binding fragment include: (i) VH-CH1; (ii) VH-CH2; (iii) VH-CH3; (iv) VH-CH1-CH2; (v) VH-CH1-CH2- CH3, (vi) VH-CH2-CH3; (vii) VH-CL; (viii) VL-CH1; (ix) VL-CH2, (x) VL-CH3; (xi) VL-CH1- CH2; (xii) VL-CH1-CH2-CH3; (xiii) VL-CH2-CH3; and (xiv) VL-CL. In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to one another or may be linked by a full or partial hinge or linker region. A hinge region may consist of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids which result in a flexible or semi-flexible linkage between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, the antigen-binding fragment, as herein described, may comprise a homo-dimer or hetero-dimer (or other multimer) of any of the variable and constant domain configurations listed above in non-covalent association with one another and / or with one or more monomeric VHor VLdomains (e.g., by disulfide bond(s)). A multispecific antigen- binding molecule will typically comprise at least two different variable domains, wherein each variable domain is capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antigen-binding molecule format, including bispecific antigen-binding molecule formats, may be adapted for use in the context of an antigen-binding fragment of an antibody of the present disclosure using routine techniques available in the art.

[0161] The term “variable region” or “variable domain” refers to the domain of an immunoglobulin heavy or light chain that is involved in binding to the target antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native immunoglobulin molecule will generally have similar structures, with each domain comprising four conserved framework regions and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0162] As noted elsewhere herein, the antigen-binding moiety is tethered to the nanoparticle. By "tethered" is meant that the antigen-binding moiety is attached to the nanoparticle, either covalently or non-covalently, preferably to the external surface of the nanoparticle so as to allow the moiety to bind to its cell-surface target antigen. Methods of tethering or attaching the antigen-binding moiety to the nanoparticle will be familiar to persons skilled in the art, illustrative examples of which are described or discussed, for example, in Friedman et al. (Curr. Pharm. Des. 2013, 19(35):6315-6329); Ahmad et al. (Nanomaterials (Basel), 2022; 12(8):1333); Sanita et al. (Front. Mol. Biosci., 2022; 7:587012); and Seidu et al. (Pharmaceutics, 202; 14(5):1113), the entire contents of which are incorporated herein by reference. Methods of use

[0163] The present disclosure also extends to methods of delivering an agent to a hard-to- transfect cell or transfection-recalcitrant cell, the method comprising contacting the hard-to- transfect cell or the transfection-recalcitrant cell with the nanoparticle or nanoparticle composition described herein.

[0164] As used herein, the term “contacting”, e.g. in methods comprising “contacting” the cell with the nanoparticle, nanoparticle composition or pharmaceutical compositions disclosed herein, means that the cell is brought into contact with, or is otherwise exposed to the nanoparticle or nanoparticle composition as disclosed herein. Without being bound by theory or node of application, upon contact of the nanoparticle to the cell, the nanoparticle is taken up by the cell (internalised).

[0165] The nanoparticle or nanoparticle compositions disclosed herein may be useful in the delivery of an exogenous nucleic acid sequence or molecule (i.e., a transgene) to a cell, in particular to a hard-to-transfect cell or transfection-recalcitrant cell. The exogenous nucleic acid sequence may be a DNA or an RNA sequence. The exogenous nucleic acid sequence may be a PNA (Peptide nucleic acid) or an LNA (locked nucleic acid) sequence. The exogenous nucleic acid sequence may be further modified, for example, to improve its stability or half-life, such as by improving its resistance to nuclease degradation. The exogenous nucleic acid sequence may be modified to alter its binding specificity to its molecular target. The exogenous nucleic acid sequence may be modified to alter its affinity for its molecular target. The exogenous nucleic acid sequence may be modified to have adye, a molecular label or a detectable label. Examples of nucleic acid modifications include those described in Adachi et al., 2021 Biomedicines 9:550 and Robert et al., 2020 Nature Reviews Drug Discovery 19:673. A skilled person will be able to readily select suitable modifications (e.g., modifications to oligonucleotide backbone, sugars, bases and 5' phosphate) depending on the intended application.

[0166] In one embodiment, the exogenous nucleic acid sequence is an mRNA sequence. In an embodiment, the exogenous nucleic acid sequence is a non-coding functional RNA sequence that is not translated into a polypeptide. The functional RNA maybe a transfer RNA (tRNA), ribosomal RNA (rRNA), as small RNAs such as microRNAs, siRNAs, piRNAs, snoRNAs, snRNAs, or scaRNAs. The functional RNA may be a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA, a small hairpin RNA (shRNA), self- amplifying RNA, long non-coding RNA, circular RNA or a guide RNA (gRNA).

[0167] In another aspect of the present invention, the nanoparticle or nanoparticle compositions described herein may be useful in a method of expressing an exogenous nucleic acid sequence (transgene) in a transfection-recalcitrant cell, method comprising contacting the cell with the nanoparticle or nanoparticle composition described herein, wherein the agent is an exogenous nucleic acid sequence or molecule. The term "expressed", “expression” or "expressing" as used herein, typically refers to any step involved in the production of an RNA molecule or a polypeptide, including transcription, post- transcriptional modification, translation, and post-translational modification. Expression of an exogenous nucleic acid can include transcription of DNA to produce an RNA molecule, wherein the exogenous nucleic acid is a DNA molecule. Expression of an exogenous nucleic acid can include translation of an mRNA to produce a polypeptide, wherein the exogenous nucleic acid is an mRNA molecule.

[0168] In an embodiment, the exogenous nucleic acid sequence is an mRNA sequence encoding the polypeptide, and the mRNA is capable of being translated in the cell to produce the polypeptide.

[0169] The term "transfection" or "transfect" and the like, has traditionally referred to the transfer of nucleic acids into eukaryotic cells using non-viral methods, but has since to evolved to include the transfer of proteins, peptides and other small molecules.

[0170] The terms "hard-to-transfect cell" or "transfection-recalcitrant cell" as used herein refers to cells that are resistant to the introduction of exogenous molecules or show low uptake and / or retention of exogenous molecules, in particular when transfected using chemical methods. A hard-to-transfect cell of a transfection-resistant cell is a cell or cell type that has been tested with multiple different transfection reagents or methods, with low and unworkable rates of transfection efficiency. Improving transfection efficiency is important when there are only small numbers of cells to be transfected, or the cells are fragile or susceptible to cellular differentiation (stem cells). Some hard-to-transfect cells or transfection-resistant cells may have unusual membrane compositions which reduce uptake under chemical transfection methods. Some hard-to-transfect cells or transfection-resistant cells may lack or have altered endocytic machinery or pathways, which may minimise uptake or retention of the exogenous materials under chemical transfection methods. Some hard-to-transfect cells or transfection-resistant cells may lack or have altered endocytic machinery or pathways, which may result in inefficient expression or processing of the exogenous materials under chemical transfection methods. Some hard-to-transfect cells or transfection-resistant cells may have normal uptake mechanisms but have evolved natural defence mechanisms that quickly breakdown and destroy endosomal contents, or exogenous substances. Hard-to-transfect cells or transfection-resistant cells would be known or easily recognisable to the persons skilled in the art, for at least some of the reasons provided above (see for example, Rahimmanesh et al.2020; Ali et al.2021; and Wang and Tian 2022).

[0171] The nanoparticle or nanoparticle compositions described herein advantageously provide enhanced or improved delivery (i.e., transfection efficiency) of one or more agents to cells, including hard-to-transfect cells or transfection-recalcitrant cells, in particular to transfection-recalcitrant T cells. The transfection efficiency can be measured as a proportion, or percentage of cells that demonstrate uptake of the nanoparticle to be delivered to the cells. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 1-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 5-100%. Insome embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 10-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 15-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 25-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 35-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 40-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 45-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 50-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 55-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 60-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 65-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 70-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 75-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 80-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 85-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 90-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 10-100%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 40-90%. In some embodiments, the nanoparticle or nanoparticlecomposition described herein provides a transfection efficiency of 50-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 60-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 70-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 80-90%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 40-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 50-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 60-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 70-80%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-70%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-70%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 40-70%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 50-70%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 60-70%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-60%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-60%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 40-60%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 50-60%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-50%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-50%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides atransfection efficiency of 40-50%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 20-40%. In some embodiments, the nanoparticle or nanoparticle composition described herein provides a transfection efficiency of 30-40%.

[0172] In some embodiments, the hard-to-transfect cell or transfection-resistant cell is a quiescent cell, a primary cell, an immune cell, a stem cell or a neuronal cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a quiescent cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is an immune cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a stem cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a neuronal cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a resting T-cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a Natural Killer T (NKT) cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a Natural Killer (NK) cell. In an embodiment, the hard-to-transfect cell or transfection- resistant cell is a macrophage. In an embodiment, the hard-to-transfect cell or transfection- resistant cell is a latently viral-infected cell. In an embodiment, the hard-to-transfect cell or transfection-resistant cell is a latently HIV-infected cell.

[0173] In some embodiments, the hard-to-transfect cell or transfection-resistant cell may be an in vitro hard-to-transfect cell or transfection-resistant cell. In some embodiments, the hard-to-transfect cell or transfection-resistant cell may be an in vivo hard-to-transfect cell or transfection-resistant cell. In some embodiments, the hard-to-transfect cell or transfection- resistant cell may be an in situ hard-to-transfect cell or transfection-resistant cell. In some embodiments, the hard-to-transfect cell or transfection-resistant cell may be contacted with the composition or the pharmaceutical composition disclosed herein in vivo. In some embodiments, the hard-to-transfect cell or transfection-resistant cell may be contacted with the composition or the pharmaceutical composition disclosed herein in vitro. In some embodiments, the hard-to-transfect cell or transfection-resistant cell may be contacted with the composition or the pharmaceutical composition disclosed herein in situ. In an embodiment, the nanoparticle or nanoparticle compositions described herein may be contacted with the hard-to-transfect cell or transfection-resistant cell within a subject using varied routes of administration (e.g., subcutaneous, intracutaneous, intravenous,intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique). Such in vivo, in situ and in vitro contacting methods are known in the art and would be familiar to the person skilled in the art. The selection of routes of administration may depend on the location of the hard-to-transfect cell or transfection-resistant cell that is to be transfected. For example, if the hard-to-transfect cell or transfection-resistant cell is located in the lungs, a suitable administration route of the composition may be inhalation or intranasal administration.

[0174] The present invention also provides use of the nanoparticle described herein in the manufacture of a medicament for delivering an agent to a cell, including an immune cell. In an embodiment, the cell is a hard-to-transfect cell or transfection-recalcitrant cell. In an embodiment, the hard-to-transfect cell or transfection-recalcitrant cell is an immune cell.

[0175] The present invention also provides use of the nanoparticle described herein in the manufacture of a medicament for expressing an exogenous nucleic acid molecule (transgene) in a cell. In an embodiment, the cell is a hard-to-transfect cell or transfection- recalcitrant cell. In an embodiment, the hard-to-transfect cell or transfection-recalcitrant cell is an immune cell.

[0176] The present invention also provides use of the nanoparticle described herein in the manufacture of a medicament for delivering of an exogenous nucleic acid molecule (transgene) to a cell. In an embodiment, the cell is a hard-to-transfect cell or hard-to-transfect cell or transfection-recalcitrant cell. In an embodiment, the hard-to-transfect cell or transfection-recalcitrant cell is an immune cell.

[0177] In an embodiment, the immune cell is selected from the group consisting of a T cell, an NKT cell and an NK cell. In an embodiment, the immune cell is a T cell. In an embodiment, the T cell is a circulating T cell. In another embodiment, the T cell is a tissue- resident T cell. In an embodiment, the immune cell is a memory T cell. In an embodiment, the T cell is a resting T cell. In an embodiment, the T cell is a naive T cell. In an embodiment, the T cell is a CD4+ T cell. In an embodiment, the T cell is a CD8+ T cell. In another embodiment, the T cell is a Tumour-infiltrating T cell. In another embodiment, the T cell is a regulatory T cell (T reg cell).

[0178] T lymphocytes are maintained in an inactivated (resting) state for most of their lives and switch to the proliferating state once stimulated. A T cell may be stimulated or activated, after which, the T cell may return to a resting state. That is, a resting T cell is a T cell that is not in an activated state. In an embodiment, a resting T cell is a T cell that is not a proliferating T cell. In another embodiment, a resting T cell is not a stimulated T cell. In another embodiment, the resting T cell is an unstimulated T cell. Resting T cells can be characterised by the absence of expression of activation markers including HLA-DR, CD25 and CD69. (see, for example, Siliciano et al. 2002; Saleh et al. 2007 and Cameron et al. 2010).

[0179] In some embodiments, the cell or resting T cell may be an in vitro cell or resting T cell. In some embodiments, the cell or resting T cell may be an in vivo cell or resting T cell. In some embodiments, the cell or resting T cell may be an in situ cell or resting T cell. In some embodiments, the cell may be an in vitro cell. In some embodiments, the cell may be an in vivo cell. In some embodiments, the cell may be an in situ cell. In some embodiments, the circulating T cell may be an in vitro circulating T cell. In some embodiments, the circulating T cell may be an in vivo circulating T cell. In some embodiments, the cell may be an in situ circulating T cell. In some embodiments, the circulating T cell may be an in vitro tissue-resident T cell. In some embodiments, the circulating T cell may be an in vivo tissue-resident T cell. In some embodiments, the cell may be an in situ tissue-resident T cell. In some embodiments, the circulating T cell may be an in vitro memory T cell. In some embodiments, the memory T cell may be an in vivo circulating T cell. In some embodiments, the cell may be an in situ memory T cell. In some embodiments, the circulating T cell may be an in vitro resting T cell. In some embodiments, the memory T cell may be an in vivo resting T cell. In some embodiments, the cell may be an in situ resting T cell. In some embodiments, the naive T cell may be an in vitro naive T cell. In some embodiments, the naive T cell may be an in vivo naive T cell. In some embodiments, the naive T cell may be an in situ naive T cell. In some embodiments, the CD4+ T cell may be a CD4+ T cell. In some embodiments, the CD4+ T cell may be an in vivo CD4+ T cell. In some embodiments, the CD4+ T cell may be an in situ CD4+ T cell. In some embodiments, the CD8+ T cell may be an in vitro CD8+ T cell. In some embodiments, the CD8+ T cell may be an in vivo CD8+ T cell. In some embodiments, the CD8+ T cell may be an in situ CD8+ T cell. In some embodiments, the Tumour-infiltrating T cell may be an in vitro Tumour-infiltrating T cell.In some embodiments, the Tumour-infiltrating T cell may be an in vivo Tumour-infiltrating T cell. In some embodiments, the Tumour-infiltrating T cell may be an in situ Tumour- infiltrating T cell. In some embodiments, the T reg cell may be an in vitro T reg cell. In some embodiments, the T reg cell may be an in vivo T reg cell. In some embodiments, the T reg cell may be an in situ T reg cell. In some embodiments, the cell or resting T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the cell or resting T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the cell or resting T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the circulating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the circulating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the circulating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the tissue- resident T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the tissue-resident T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the tissue-resident T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the memory T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the memory T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the memory T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the naive T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the naive T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the naive T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the CD4+ cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the CD4+ T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the CD4+ T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In someembodiments, the CD8+ cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the CD8+ T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the CD8+ T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the Tumour-infiltrating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the Tumour-infiltrating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the Tumour-infiltrating T cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. In some embodiments, the T reg cell may be contacted with the nanoparticle or nanoparticle composition described herein in vitro. In some embodiments, the T reg cell may be contacted with the nanoparticle or nanoparticle composition described herein in vivo. In some embodiments, the T reg cell may be contacted with the nanoparticle or nanoparticle composition described herein in situ. The nanoparticle or nanoparticle composition may be contacted with the cell or T cell within a subject using any suitable route of administration (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique). The nanoparticle or nanoparticle composition may be contacted with the circulating T cell, the tissue-resident T cell, the memory T cell, the resting T cell, the naive T cell, the CD4+ T cell, the CD8+ T cell, the Tumour-infiltrating T cell and / or the T reg within a subject using any suitable route of administration (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique).Such in vivo and in vitro contacting methods are known in the art and would be familiar to the person skilled in the art.

[0180] In some embodiments, the cell or resting T cell is purified from a subject, for delivery of the agent, before being administered to the subject.

[0181] In certain embodiments, the nanoparticle or nanoparticle compositions described herein may provide enhanced (e.g., 1.1 fold, 1.5 fold, 2 fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more) delivery of the agent to the cell compared to a comparator nanoparticle or nanoparticle composition (e.g., a nanoparticle that does not include a CD2- or CD7-binding moiety).

[0182] In another aspect disclosed herein, there is provided a method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the nanoparticle or nanoparticle composition described herein. It is to be understood that the type of disease for which such methods may provide therapeutic or prophylactic benefit to a subject in need thereof be useful will depend on the type of agent to be transfected to the cell. For example, if a disease is associated with an upregulation expression of a certain factor in an immune cell, treatment of that disease may be accomplished by using the nanoparticles described herein to transfect the cells with an agent that reduces, inhibits, abrogates or otherwise decreases the expression of that factor in the transfected cell. Conversely, if a disease is associated with the downregulation of a certain factor in an immune cell, treatment of that disease may be accomplished by using the nanoparticles described herein to transfect the cells with an agent that enhances, promotes, initiates or otherwise increases the expression of that factor in the transfected cell. Such diseases may include, but are not limited to, cancer (e.g., ovarian cancer, breast cancer, lymphomas, thyroid cancer, pancreatic cancer, bowel cancer, renal cancer, skin cancer, prostate cancer, hepatocellular carcinoma, small cell and non-small cell lung cancer), infectious diseases, inflammation, autoimmune diseases, HIV, autoimmune diseases (e.g., diabetes, myasthenia gravis, autoimmune thyroiditis, systemic lupus erythematosus, graft- versus-host disease, and autoimmune vasculitis, Rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, Graves' disease, Sjögren’s disease, Hashimoto's disease and celiac disease). Other diseases may include, but are not limited to, cystic fibrosis, asthma, pneumonia, pulmonary fibrosis, COPD, bronchiectasis, sarcoidosis, pulmonary hypertension, emphysema, alpha-1 antitrypsin deficiency, aspergillosis, bronchiolitis, bronchitis, pneumoconiosis, Legionnaire’s disease, pertussis, pulmonary embolism, tuberculosis, a coronavirus infection (e.g., SARS-CoV-1 or SARS-CoV-2, MERS-CoV), influenza infection, paramyxovirus infection and / or any other disease caused by a pathogen or an infectious pathogen.

[0183] In an embodiment, the disease to be treated is a T cell mediated disease or disorder. In some embodiments, the T cell mediated disease or disorder is an autoimmune disease. Illustrative examples of a T cell mediated disease or disorder include Type 1 diabetes, rheumatoid arthritis, multiple sclerosis, coeliac disease, systemic lupus erythematosus (SLE), Crohn’s Disease, T1D, autoimmune hepatitis, Sjögren’s syndrome, Autoimmune thyroid disease, inflammatory bowel disease, ulcerative colitis, Sjögren’s syndrome and Myasthenia gravis. In some embodiments, the T cell mediated disease or disorder is HIV infection. In some embodiments, the T cell mediated disease or disorder is T cell lymphoma. In an embodiment, the T cell mediated disease or disorder is T cell leukemia.

[0184] In an embodiment, the disease to be treated is a viral infection. The viral infection may be an acute, chronic, reactivated, persistent or latent viral infection. Examples of viruses known to cause latent infections, which include herpes simplex viruses, varicella zoster virus, HSV-1, HSV-2, VZV, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, HIV virus, Human T-cell leukemia virus -1 (HTLV-1), BK virus, parvovirus and adenovirus. In one embodiment, the viral infection is a HIV infection. In another embodiment the vital infection is a latent HIV infection. In another embodiment, the viral infection is a latent HIV infection.

[0185] In an embodiment, the disease is characterised by aberrant immune function. In some embodiments, the disease is characterised by abnormal viral integration or viral re- activation. In some embodiments, the disease is characterised by dysfunctional or aberrant protein or polypeptide activity. A skilled person will be able to readily select a suitable agent depending on the disease.

[0186] Depending on the intended therapeutic application, the nanoparticle or nanoparticle composition described herein can be used to deliver agents to hard-to transfect diseased cells or hard-to-transfect cell that is a healthy (undiseased) cell.

[0187] In some embodiments, the nanoparticle or nanoparticle composition described herein can be used to deliver immune modulating agents to hard-to transfect cells, such as T cells, in particular for therapeutic applications, for example, applications in which T cells can be used to target diseased cells or to induce immune tolerance. In an embodiment, thenanoparticle or nanoparticle composition described herein can be used to deliver agents for expression of genes that modulate T cell function and activity. In an embodiment, the nanoparticle or nanoparticle composition described herein can be used to deliver gene- editing agents (e.g. nucleic acids encoding CRISPR machinery), including to modify the expression of genes that modulate T cell function and activity. In an embodiment, the nanoparticle or nanoparticle composition described herein can be used to deliver nucleic acids encoding CARs to T cells to generate CAR-T cells.In an embodiment, the nanoparticle or nanoparticle composition described herein can be used to deliver nucleic acid encoding CARs to T cells to generate CAR-T cells recognising specific disease-related epitopes, to enhance the immune response against diseased cells. In some examples, the nanoparticle or nanoparticle composition described herein can be used to deliver nucleic acid encoding CARs to T cells to generate CAR-T cells recognising specific cancer-related epitopes, to enhance the immune response against cancer cells. In another example, the nanoparticle or nanoparticle composition described herein can be used to deliver nucleic acids encoding CARs to T cells to generate CAR-T cells to mediate tolerance to specific antigens in the context of inducing tolerance in autoimmune disease, or to induce tolerance in the context of organ transplantation and transplantation tolerance.

[0188] Thus, the present invention also extends to a method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the nanoparticle or nanoparticle composition comprising an agent, described herein, wherein the agent is capable of treating or preventing the disease. In an embodiment, the agent is a nucleic acid molecule capable of treating or preventing the disease. In an embodiment, the agent is a nucleic acid molecule encoding a polypeptide that is capable of treating the disease.

[0189] In an embodiment, there is provided a method of treating HIV infection in a subject, the method comprising administering to the subject an effective amount of the nanoparticle or nanoparticle composition, as disclosed herein, wherein the agent is capable of permanently silencing the HIV genome; or reversing HIV latency to re-activate HIV infection, to allow targeting of the re-activated HIV infected cells with concurrent antiretroviral therapy. In some embodiments, the HIV infection is a latent infection. In an embodiment, the agent is a nucleic acid capable of treating HIV infection. In an embodiment,the agent is a nucleic acid encoding a polypeptide that is capable of treating HIV infection. In an embodiment, the agent encodes for a CRISPR-Cas system protein or a guide RNA.

[0190] In an embodiment, the agent is capable of knocking out, mutating or otherwise inactivating CCR5 in a cell. See, for example, Xu et al.2019. In another embodiment, the agent is capable of disrupting the HIV proviral genome by targeting the HIV LTR or protein encoding sequences. See for example Das et al.2019; Panfil et al.2018; Dash et al.2019; Lebbink et al.2017; Kaminski et al.2016).

[0191] In another example, the nanoparticle or nanoparticle composition described herein can be used to deliver gene-editing agents (i.e., nucleic acids encoding CRISPR machinery) to T cells to generate CAR-T cells recognising specific HIV epitopes, to enhance the immune response against HIV-infected cells. See for example Ali et al. 2016; Anthony- Gonda et al. 2019; Liu et al. 2021). In another example, the nanoparticle or nanoparticle composition described herein can be used to deliver agents (i.e., nucleic acids encoding CRISPR machinery) that are capable of reactivate transcription of the HIV provirus without affecting host cell transcription. See for example Zhang et al 2015; Ji et al. 2016; Limsirichai et al.2016; Bialek et al.2016; Saayman et al.2016; Klinnert et al.2022; Zhang et al.2018).

[0192] In another example, the nanoparticle or nanoparticle composition described herein can be used to deliver agents capable of inhibiting, reducing or silencing HIV transcription / RNA levels; see for example Jin et al. 2019; Kessing et al. 2017; Mediouni et al. 2019; Ahlenstiel et al.2020; da Costa et al.2022; Olson et al.2020; Nguten et al.2021; Yin et al. 2020).

[0193] The present invention also provides use of the nanoparticle or nanoparticle composition comprising an agent, as described herein, in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the agent is capable of treating or preventing the disease. In an embodiment, the agent is a nucleic acid molecule capable of treating the disease. In an embodiment, the agent is a nucleic acid molecule encoding a polypeptide capable of treating the disease.

[0194] The present invention also provides use of a pharmaceutical composition comprising the nanoparticle or nanoparticle composition, as disclosed herein, and an agent for treating HIV infection in a subject, wherein the agent is capable of treating for treating HIV infection in a subject. In an embodiment, the agent is capable of permanently silencing the HIV genome; or reversing HIV latency to re-activate HIV infection, to allow targeting of the re- activated HIV infected cells with concurrent antiretroviral therapy.

[0195] The terms “treat”, “treating” or “treatment” with regard to a disease refers to alleviating or abrogating the cause and / or the effects of the disease. As used herein, the terms “treat”, “treatment” and “treating” refer to the inhibition, reduction or amelioration of the progression, severity and / or duration of the disease, or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of the disease (i.e., “managing” without “curing” the condition), resulting from the administration of one or more therapies (e.g., one or more agents such as a compound or composition as disclosed herein). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a disease described herein. In other embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a disease described herein, either physically by, e.g., stabilization of a discernible symptom or physiologically by, e.g., stabilization of a physical parameter, or both. As used herein, the terms “disease,” “disorder,” and “condition” may be used interchangeably. As used herein, inhibition, treatment, treating, and ameliorating are used interchangeably and refer to, e.g., stasis of symptoms, prolongation of survival, partial or full amelioration of symptoms, and partial or full eradication of a condition, disease or disorder.

[0196] The terms “preventing” and “prophylaxis” as used herein refer to administering a medicament in order to avert or forestall the appearance of one or more symptoms of a condition or a disease. The person of ordinary skill in the medical art recognises that the term “prevent” is not an absolute term. In the medical art, it is understood to refer to the prophylactic administration of a drug to substantially diminish the likelihood or seriousness of a condition, or symptom of the condition and this is the sense intended in this disclosure. As used in a standard text in the field, the Physician’s Desk Reference, the terms “prevent”, “preventing” and “prevention” with regard to a condition refer to averting the cause, effects, symptoms or progression of a condition prior to the condition fully manifesting itself.

[0197] The terms “administer”, “administering” or “administration” in reference to a pharmaceutical composition or formulation disclosed herein means introducing the pharmaceutical composition into the system of the subject in need of treatment. When the pharmaceutical composition is provided in combination with one or more other active agents, “administration” and its variants are each understood to include concurrent and / or sequential introduction of the pharmaceutical composition and the other active agents. The method of administration may be selected to target delivery (e.g., to specifically deliver) to a specific region or system of a body. For example, an administration may be parenteral (e.g., subcutaneous, intracutaneous, intravenous, intraperitoneal, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, transfusion, as well as any suitable infusion technique), cell or tissue transplantations, oral, trans- or intra-dermal, interdermal, rectal, intravaginal, topical (e.g. by powders, ointments, creams, gels, lotions, and / or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual, intranasal; by intratracheal instillation, bronchial instillation, and / or inhalation; as an oral spray and / or powder, nasal spray, and / or aerosol, and / or through a portal vein catheter.

[0198] In some embodiments, the subject in need of treatment or prevention of a disease is a mammal. The term “mammal” as used herein includes humans, primates, livestock animals (e.g., horses, cattle, sheep, pigs, donkeys), laboratory test animals (e.g., mice, rats, guinea pigs), companion animals (e.g., dogs, cats) and captive wild animals (e.g., kangaroos, deer, foxes). Preferably, the mammal is a human.

[0199] In some embodiments, the subject in need of treatment of a HIV infection is a primate. In some embodiments, the subject in need of treatment of a HIV infection is a human.

[0200] In an embodiment, the nanoparticles or nanoparticle compositions described herein are to be administered to the subject in need thereof so as to deliver a treatment effective amount of the agent. In some embodiments, a treatment effective amount is a therapeutically effective amount or a prophylactically effective amount. The term “therapeutically effective amount” as used herein means an amount of the pharmaceutical composition sufficient to deliver an amount of the agent sufficient to treat or alleviate the symptoms associated with a disease. The therapeutically effective amount to be administered will be governed by suchconsiderations, and is either, an incremental maximum tolerated dose, or the minimum amount, necessary to ameliorate, cure, or treat the condition or one or more of its symptoms. The term “prophylactically effective amount” refers to an amount effective in preventing or substantially lessening the chances of acquiring a disease or in reducing the severity of the disease before it is acquired or reducing the severity of one or more of its symptoms before the symptoms develop. Roughly, prophylactic measures are divided between primary prophylaxis (to prevent the development of a disease or symptom) and secondary prophylaxis (whereby the disease or symptom has already developed and the patient is protected against worsening of this process). Prophylaxis may include post-exposure prophylaxis (e.g., administering an effective amount of a pharmaceutical composition as disclosed herein to a subject known to have been exposed, for example, to a respiratory infection).

[0201] As used herein, the term “effective amount” relates to an amount of a pharmaceutical composition as disclosed herein which, when administered according to a desired dosing regimen, provides sufficient agent to achieve the desired therapeutic activity. For example, an effective amount of a pharmaceutical composition as disclosed herein may be an amount sufficient to inhibit, slow, interrupt, halt, prevent or arrest viral or bacterial growth or replication. Suitable effective amounts may depend on the age, gender, weight and general health of the patient and can be determined by the attending physician. Suitable dosages may lie within the range of about 0.1 ng per kg of body weight to 100 g per kg of body weight per dosage. The dosage may be in the range of 1 µg to 10 g per kg of body weight per dosage, such as is in the range of 1 mg to 1000 mg per kg of body weight per dosage. In one embodiment, the dosage may be in the range of 1 mg to 500 mg per kg of body weight per dosage. In another embodiment, the dosage may be in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage may be in the range of 1 mg to 200 mg per kg of body weight per dosage, such as up to 50 mg per kg body weight per dosage.

[0202] In certain embodiments, an effective amount of an agent for administration one or more times a day to a 70 kg adult human may comprise about 0.0001 mg to about 4000 mg, about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 200 mg, about 0.001 mg to about 1500 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of the agent per unit dosage form. In certain embodiments, formulations of the agent may be at dosage levels sufficient to deliver from about 0.001 mg / kg to about 100 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, and from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic effect. In certain embodiments, an effective amount of an agent for administration to a 70 kg adult human may comprise about 0.0001 mg to about 4000 mg, about 0.0001 mg to about 3000 mg, about 0.0001 mg to about 200 mg, about 0.001 mg to about 1500 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg, of an extract or compound per unit dosage form. In some embodiments, a single dose may be sufficient to treat or prevent the disease, which may be delivered in one or more aliquots to achieve the desired dose. In other embodiments, multiple doses may be required to treat or prevent the disease or condition and associated symptoms. Dosing may occur at intervals of minutes, hours, days, weeks, months or years or continuously over any one of these periods. The administered amount may be an amount sufficient to treat or alleviate the symptoms associated with the disease.

[0203] The amount of nanoparticle or nanoparticle composition, including the agent, administered per dose or the total volume of composition administered will depend on such factors as the nature and severity of the symptoms, the age, weight, and general health of the patient, as well as the mode of administration. It is to be recognised that relative amounts of excipients, solvents, diluents, salts, thickening agents, sensory agents, buffers, and / or any additional ingredients in a pharmaceutical composition as disclosed herein may also depending upon the identity, size, and / or condition of the subject treated, as well as the mode of administration. For example, in some embodiments, the dosage of agent required to achieve a therapeutically equivalent effect may be greater for one dosage form compared to another.

[0204] Pharmaceutical compositions comprising the nanoparticles or nanoparticle compositions with the agent, as described herein, may be administered in a single dose or a series of doses. Suitable dosage amounts and dosing regimens can be determined by the attending physician and may depend on the particular condition being treated, the severity of the condition as well as the general age, health and weight of the subject. It will be appreciated that dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered can be determined by a medical practitioner or person skilled in the art.

[0205] In certain embodiments, it is envisaged that the nanoparticles or nanoparticle compositions disclosed herein may be administered to a subject in need thereof as a substitute or replacement for other traditional medication for the treatment of the disease. In other embodiments, it is envisaged that the nanoparticles or nanoparticle compositions disclosed herein be administered to a subject in need thereof as a supplement or adjunct to traditional medication. In still other embodiments, it is envisaged that the nanoparticles or nanoparticle compositions disclosed herein may be administered to a subject in need thereof in the absence of adjunct therapy. Replacing traditional medication for the treatment of the disease with the nanoparticles or nanoparticle compositions disclosed herein may be advantageous, particularly where the traditional medication is associated with one or more adverse effects.

[0206] In other embodiments, the nanoparticles or nanoparticle composition, as described herein, may be administered to a subject in need thereof, together with one or more additional agents for a discrete period of time, to address specific symptoms of a disease. In still other embodiments, the subject in need thereof may be administered the nanoparticles or nanoparticle compositions, as described herein, and one or more additional agents (administered sequentially or in combination) for the duration of the treatment period. Such combination therapy may be particularly useful, for example, where an additive or synergistic therapeutic effect is desired. Where the active agents are provided in separate dosage formulations, the active agents may be administered separately or in conjunction. In addition, the administration of one active agent may be prior to, concurrent with, or subsequent to the administration of the other agent.

[0207] The phrase “combination therapy” as used herein, is to be understood to refer to administration of an effective amount, using a first amount of, for example, the nanoparticle or nanoparticle composition, as described herein, and a second amount of an additional suitable agent. An “effective amount” of the second agent will depend on the type of drug used. Suitable dosages are known for approved agents and can be adjusted by a person skilled in the art according to the condition of the subject, the type of condition(s) being treated and the amount of a compound or composition being used. In certain embodiments, the nanoparticle or nanoparticle composition and the additional agent are each administered in an effective amount (i.e., each in an amount that would be therapeutically effective if administered alone). In other embodiments, the nanoparticle or nanoparticle composition and the additional agent are each administered in an amount that alone does not provide a therapeutic effect (a sub-therapeutic dose). In yet other embodiments, the nanoparticle or nanoparticle composition can be administered in an effective amount, while the additional agent is administered in a sub-therapeutic dose. In still other embodiments, the nanoparticle or nanoparticle composition can be administered in a sub-therapeutic dose, while the additional agent is administered in an effective amount.

[0208] As used herein, the terms “in combination” or “co-administration” can be used interchangeably to refer to the use of more than one therapy (e.g., one or more prophylactic and / or therapeutic agents). The use of the terms does not restrict the order in which therapies (e.g., prophylactic and / or therapeutic agents) are administered to a person in need thereof. Co-administration encompasses administration of the pharmaceutical composition as disclosed herein and one or more additional agents in an essentially simultaneous manner, such as in a single pharmaceutical composition, for example, having a fixed ratio of first and second amounts, or as discrete dosage forms. In addition, such co-administration also encompasses use of each compound in a sequential manner in either order. When co- administration involves the separate administration of a first amount of a pharmaceutical composition as disclosed herein and a second amount of an additional agent, they are administered sufficiently close in time to have the desired therapeutic effect. For example, the period of time between each administration which can result in the desired therapeutic effect, can range from minutes to hours and can be determined taking into account the properties of each compound such as potency, solubility, bioavailability, plasma half-life, and kinetic profile.

[0209] In one or more embodiments where the nanoparticle or nanoparticle composition as described herein is administered in combination with an additional agent, the additional agent may be any agent that provides a desired treatment outcome. In particular, the additional agent may be selected from known agents for the treatment or prevention of the disease, including one or more symptoms thereof. Such agent will be known to those skilled in art.

[0210] By way of non-limiting example, known agents for the treatment of HIV include anti-HIV agents such as anti-HIV antibodies, HIV protease inhibitors, HIV non-nucleoside or non-nucleotide inhibitors of reverse transcriptase, HIV nucleoside or nucleotide inhibitors of reverse transcriptase, HIV integrase inhibitors, HIV non-catalytic site (or allosteric) integrase inhibitors, HIV entry inhibitors (e.g., CCR5 inhibitors, gp41 inhibitors (i.e., fusion inhibitors) and CD4 attachment inhibitors), CXCR4 inhibitors, gpl20 inhibitors, G6PD and NADH-oxidase inhibitors, HIV vaccines, HIV maturation inhibitors, latency reversing agents (e.g., histone deacetylase inhibitors, proteasome inhibitors, protein kinase C (PKC) activators, and BRD4 inhibitors), compounds that target the HIV capsid ("capsid inhibitors"; e.g., capsid polymerization inhibitors or capsid disrupting compounds, HIV nucleocapsid p7 (NCp7) inhibitors, HIV p24 capsid protein inhibitors), pharmacokinetic enhancers, immune- based therapies (e.g., PD-1 modulators, PD-Ll modulators, toll like receptors modulators, IL-15 agonists), HIV antibodies, bispecific antibodies and "antibody-like" therapeutic proteins (e.g., DARTs®, Duobodies®, Bites®, XmAbs®, TandAbs ®, Fab derivatives) including those targeting HIV gpl20 or gp41, combination drugs for HIV, HIV pl7 matrix protein inhibitors, IL-13 antagonists, Peptidyl- prolyl cis-trans isomerase A modulators, Protein disulfide isomerase inhibitors, Complement C5a receptor antagonists, DNA methyltransferase inhibitor, HIV vif gene modulators, Vif dimerization antagonists, HIV-1 viral infectivity factor inhibitors, TAT protein inhibitors, HIV-1 Nef modulators, Hck tyrosine kinase modulators, mixed lineage kinase-3 (MLK-3) inhibitors, HIV-1 splicing inhibitors, Rev protein inhibitors, Integrin antagonists, Nucleoprotein inhibitors, Splicing factor modulators, COMM domain containing protein 1 modulators, HIV Ribonuclease H inhibitors, Retrocyclin modulators, CDK-9 inhibitors, Dendritic ICAM-3 grabbing nonintegrin 1 inhibitors, HIV GAG protein inhibitors, HIV POL protein inhibitors, Complement Factor H modulators, Ubiquitin ligase inhibitors, Deoxycytidine kinase inhibitors, Cyclin dependent kinase inhibitors Proprotein convertase PC9 stimulators, ATPdependent RNA helicase DDX3X inhibitors, reverse transcriptase priming complex inhibitors, HIV gene therapy, PI3K inhibitors, and other drugs for treating HIV, and combinations thereof. In other embodiments, the additional agent is a latency reversing agent (LRA), e.g., a TLR8 agonist. The additional agents for the treatment of HIV include latency reversing agent (LRA) e.g., a TLR7 agonist. In other embodiments, the additional agent is a latency reversing agent (LRA), e.g., a TLR8 agonist. Examples of TLR agonists include but are not limited to Vesatolimod. In one embodiment, the additional agent is a TLR modulator. TLR modulators may include modulators of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR 10, TLR11, TLR 12, and TLR13. In other embodiments, the additional anti-HIV agent is comprised of one or more antiretroviral therapies (ARTs). In particular embodiments, the ART comprises one or more of a nucleoside reverse transcriptase inhibitor (NRTI), a non-nucleoside reverse transcriptase inhibitor (NNRTI), a protease inhibitor (PI), an entry inhibitor, or an HIV integrase inhibitor.

[0211] Where the nanoparticle or nanoparticle combination as described herein is administered in combination with an additional agent, the additional agent may be administered in any “effective amount” which provides the desired therapeutic activity, as described above. Suitable dosage amounts and dosing regimens of the additional agent can be determined by the attending physician and may depend on the particular condition being treated, the severity of the condition as well as the general age, health and weight of the subject. It will be appreciated that, unless otherwise specified, dose ranges as described herein provide guidance for the administration of the nanoparticle or nanoparticle compositions to an adult. The amount to be administered to can be determined by a medical practitioner or person skilled in the art.

[0212] The nanoparticles or nanoparticle compositions described herein may be contained in a kit. The suitable kit may include, for example, the nanoparticle or nanoparticle composition and the agent (e.g., therapeutic agent), each packaged or formulated individually, or packaged or formulated in combination. Thus, in an embodiment, the nanoparticle may be present in a first container, and the agent may be present in a fourth container. In another embodiment, the nanoparticle comprising the agent may be present in the same container. The container or containers may be placed within a package, and the package can optionally include administration or dosage instructions. The kits disclosedherein may suitably comprise the nanoparticle and agent in forms suitable for intranasal administration. The kits disclosed herein may comprise the nanoparticle and the agent in forms suitable for parenteral administration. The kits may optionally comprise instructions describing a method of using the kit in one or more of the methods described herein (e.g., for the treatment of the disease). The kit may optionally comprise a second pharmaceutical composition comprising one or more additional agents described herein for co-therapy use, and / or one or more pharmaceutically acceptable carriers, diluents, adjuvants and / or excipients.

[0213] Those skilled in the art will be aware that the invention described herein is subject to variations and modifications other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, methods, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0214] Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described. EXAMPLES Materials and methods A. Antibodies

[0215] Primary antibodies targeting the various receptors of interest were all purchased as mouse-anti-human Alexa Fluor 647-conjugated monoclonal antibodies. Anti-CCR4 (1G1, 557863), anti-CCR6 (11A9, 560466), anti-CCR7 (150503, 560816), anti-CD3 (UCHT1, 557706), anti-CD4 (RPA-T4, 557707), anti-PD-1 (AH12.1, 560838) and anti-transferrin receptor (OKT9, 566724) antibodies were purchased from BD Biosciences. Anti-CD25 (BC96, 302618), anti-CD5 (UCHT2, 300616), anti-CXCR3 (G025H7, 353712), anti- CXCR5 (J252D4, 356906) and anti-OX40 (OX40, 350018) antibodies were purchased from Biolegend. Anti-CCR5 (45529, FAB184R), anti-CD2 (299812, FAB18561R), anti-CD7(848438, FAB7579R), anti-CXCR4 (44716, FAB172R) and isotype control (133303, IC0041R) antibodies were purchased from R&D Systems. Primary antibodies that were stored in a buffer containing sodium azide were buffer-exchanged into Phosphate Buffered Saline (PBS) using Zeba™ Spin Desalting Columns, 7K MWCO (89882, Thermo Fisher Scientific). The secondary, PE-conjugated goat-anti-mouse antibody (P-852) was purchased from Thermo Fisher Scientific. B. Cell culture

[0216] The human Jurkat T lymphocyte cell line (clone JE6.1) was cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS), supplemented with 100 U / mL penicillin, 100 µg / mL streptomycin and 2 mM L-glutamine (RF10) in a humidified 37°C, 5% CO2 incubator. For primary cell experiments, peripheral blood mononuclear cells (PBMC) were isolated from buffy coats obtained from the Australian Red Cross Blood Service via Ficoll-Paque (GE Healthcare, Champaign, IL) density centrifugation. CD4+T cells were subsequently isolated through negative magnetic-activated cell sorting using a CD4+T cell isolation kit (130-096-533, Miltenyi Biotec). PBMC and isolated total CD4+T cells were frozen in FCS supplemented with 10% DMSO and stored in liquid nitrogen until use. Prior to usage, total CD4+ T cells were thawed and rested overnight in RF10 supplemented with 2 U / mL IL-2. Subsequent experimental incubations of total CD4+T cells were performed in the continued presence of 2 U / mL IL-2. C. Receptor internalization assay

[0217] To assess T cell surface receptor internalization kinetics, a dual antibody staining approach was used based on prior work31. Jurkat T cells or primary total CD4+T cells were harvested, counted and washed once in cold PBS with 1% FCS and 1 µM EDTA (FACS wash buffer) prior to staining with titrated volumes of the respective primary antibodies for 30min, on ice, in the dark. Cells were kept on ice to inhibit any active endocytic processes during the binding of the primary antibodies. Next, excess primary antibody was washed off and cells were resuspended in cold culture media prior to incubation at 37°C for 0, 15, 30, 60, 90 or 180min. After incubation, cells were placed back on ice and kept cold throughout subsequent handling to inhibit further receptor internalization. A secondary antibody stain with PE-conjugated goat-anti-mouse antibody was then performed for 30min, on ice, in thedark to dually stain any receptor remaining on the cell surface, while any internalized receptors remained singly stained. All cells were stained with LIVE / DEAD™ Fixable Aqua Dead Cell Stain (L34957, Thermo Fisher Scientific) to allow gating on live cells during subsequent analysis. In primary cell experiments, cells were subsequently incubated with unlabelled IgG from murine serum (I5381, Sigma) to block any free anti-mouse epitope binding sites of the goat-anti-mouse secondary antibody, followed by staining against the activation markers HLA-DR (564364, BD Biosciences) and CD69 (564364, BD Biosciences) with mouse-anti-human antibodies. Cells were then fixed in PBS with 1% paraformaldehyde (PFA) prior to flow cytometry analysis using an LSR Fortessa II (BD Biosciences) or microscopy analysis (Zeiss LSM780 Confocal). D. Nanoparticle preparation

[0218] 100 nm Absolute Mag™ Protein G Magnetic Particles (WHM-X035, Creative Diagnostics) (1 mg / mL, 1.3 × 1011particles / mL) were functionalized with Alexa Fluor 647- labelled primary antibodies to generate fluorescently labelled, targeted nanoparticles (Ab@SPIONs). In brief, 10 µg of nanoparticles was mixed with 1 µg of primary antibody in 100 µL of 10mM phosphate buffer (PB), pH 7.4, supplemented with 0.05 mg / mL bovine serum albumin (BSA), vortexed briefly to mix, then incubated for 30min at room temperature. Excess unbound antibody was removed by immobilizing the nanoparticles using a magnet (EasyEights™ EasySep™ Magnet, Stemcell) and washing twice with 10 mM PB, pH 7.4, 0.5 mg / mL BSA, before resuspending in equal volumes of that same buffer to ensure equal nanoparticle concentration across all Ab@SPIONs. Antibody-functionalized nanoparticle solutions were then sonicated briefly using an ultrasonic bath. The hydrodynamic diameter and polydispersity index (PDI) of the resulting solutions, as measures of nanoparticle size and uniformity, respectively, were subsequently analysed using dynamic light scattering (DLS) using a Zetasizer (Nano-ZS, Malvern Analytical). Immediately prior to cell experiments, nanoparticle solutions were sonicated again to ensure a single-particle suspension. Antibody clones used correspond with those used in the receptor internalization assays.E. Nanoparticle internalization assay

[0219] Jurkat T cells or primary total CD4+T cells were harvested, counted and resuspended in RF10. Cells were then plated at 1.25 × 106cells / mL in 200 µL in round-bottom 96-well culture plate, after which respective targeted nanoparticles were added at a nanoparticle-to- cell ratio of 100:1. Cells were incubated for 24 or 48 hours at 37°C. To allow for simultaneous harvesting and analysis, the remaining timepoints were started “in reverse”; at 8, 4, 2 and 1 hours before harvesting in separate wells. For primary CD4+T cells, all timepoints were run using this reverse timecourse set-up. After incubation, cells were immediately pelleted (400 rcf, 4min) in a pre-cooled centrifuge (4°C) and kept cold throughout subsequent handling to inhibit further nanoparticle internalization. Unbound nanoparticles were washed off through three washes with cold PBS, followed by cell staining with LIVE / DEAD™ Fixable Aqua Dead Cell Stain and a secondary PE-conjugated goat- anti-mouse antibody as per previous. Cells were fixed in PBS with 1% paraformaldehyde (PFA) prior to flow cytometry analysis using an LSR Fortessa II (BD Biosciences). F. Nanoparticle association assays

[0220] The interaction of the various targeted nanoparticles with fresh whole blood cells and PBMC was studied as previously described68. Whole blood was collected from healthy volunteers after informed consent through microbleeds into NH sodium heparin collection tubes (454051, Greiner Bio-One). Fluorescently labelled, targeted nanoparticles were added at a ratio of 5 × 107particles per 200 µL of whole blood and incubated for 1 hour at 37°C. Cells were then transferred to ice and kept cold throughout subsequent handling to inhibit further nanoparticle association. Red blood cells were lysed through incubation with 1X Pharm Lyse™ solution (BD Biosciences) for 15min on ice, after which leukocytes were stained with LIVE / DEAD™ Fixable Blue Dead Cell Stain (L23105, Thermo Fisher Scientific) for 30min on ice, in the dark, followed by surface staining with anti-CD56-AF488 (B159, BD Biosciences), anti-CD19-PE (HIB19, BD Biosciences), anti-CD66b- PE / Dazzle594 (G10FS, Biolegend), anti-HLA-DR-Pacific Blue (L243, Pharmingen), anti- CD3-Brilliant Violet 510 (UCHT1, BD Biosciences) and anti-CD14-Brilliant Violet 785 (M5E2, Biolegend) for 30min on ice, in the dark. Cells were fixed in PBS with 1% paraformaldehyde (PFA) prior to flow cytometry analysis using an LSR Fortessa II (BD Biosciences). The following cell subsets were identified: granulocytes (CD66b+), T cells(CD66b- / CD3+), monocytes (CD66b- / CD14+), NK cells (CD66b- / CD3- / CD14- / CD56+), B cells (CD66b- / CD3- / CD14- / CD19+), dendritic cells (DC) ((CD66b- / CD3- / CD14- / CD56- / CD19- / HLA-DR+). PBMC were obtained as described above, thawed and plated at 4 × 105cells per well in 100 µL in flat-bottom 96-well culture plates. Subsequently, respective targeted nanoparticles were added at a nanoparticle to cell ratio of 100:1 and cells were incubated for 1 hour at 37°C. Cells were then phenotyped and analysed as described above. G. Absolute quantitation of fluorescence

[0221] Conversion of median fluorescent intensity (MFI) values of Alexa Fluor 647 fluorescence into absolute number of nanoparticles was performed as published previously (Cevaal et al. 2022 JoVE). Briefly, the absolute fluorescent intensity per nanoparticle was determined using a microplate reader (FLUOstar Omega, BMG Labtech) (Table 1). The fluorescence of cell samples measured on flow cytometry was converted to absolute fluorescence using quantitation beads (Quantum™ MESF 647 beads, Bangs laboratories). The degree of labelling of each antibody was determined using fluorescence spectroscopy (Fluorolog 3, HORIBA). The antibody binding capacity of cells was determined by dividing the absolute fluorescence by the degree of labelling of each corresponding antibody. Table 1. Fluorescent intensities of Ab@SPIONs. Experiment Nanoparticle Jurkat CD4+PBMC / WBC T cel Tls cells TfR@SPION 305 - - (MESF / nanoparticle) CD2@SPION 41.8 41.7 32.1 (MESF / nanoparticle) CD3@SPION 38.7 48.1 48.1 (MESF / nanoparticle) CD4@SPION 122 - - (MESF / nanoparticle) CD5@SPION 158 - - (MESF / nanoparticle)CD7@SPION 105 233 97.6 (MESF / nanoparticle) CXCR4@SPION 339 - - (MESF / nanoparticle) CCR5@SPION - 373 243 (MESF / nanoparticle) Isotype@SPION 190 358 175 (MESF / nanoparticle) CD, cluster of differentiation; CCR, C-C chemokine receptor; CTLA-4, cytotoxic T- lymphocyte–associated antigen 4; CXCR, C-X-C chemokine receptor; MESF, molecules of equivalent soluble fluorochrome; PBMC, peripheral blood mononuclear cells; SPION, superparamagnetic iron oxide nanoparticles; TfR, transferrin receptor; WBC, whole blood cells. H. Lipid nanoparticles

[0222] Lipid nanoparticles (LNP) encapsulating mScarlet reporter mRNA were synthesized through microfluidic mixing using a NanoAssemblr Spark or Benchtop (Precision Nanosystems). In brief, SM102 (Sapphire Bioscience), DSPC (Avanti Polar Lipids), ß- sitosterol (Sigma-Aldrich) and DMG-PEG2000 (Avanti Polar Lipids) were mixed at a molar ratio of 50:10:38.5:1.5, to which 0.2% DiD fluorescent dye (Invitrogen) was added. Formulations were performed at a flow rate ratio of 1.8 (aqueous) : 1 (organic) and N / P ratio of 6:1. LNP size and polydispersity were determined using dynamic light scattering. RNA encapsulation efficiency and total RNA concentration of the LNP solution were determined using a modified RiboGreen assay

[0223] To functionalize LNPs with targeting antibodies in upright orientation, LNPs were purified from ethanol using Amicon Ultra-2 centrifugal filters (30k MWCO). Anti-mouse IgG nanobodies were then conjugated to DSPE-PEG2000 in upright orientation using strain- promoted azide−alkyne cycloaddition (SPAAC) as described previously69. Nanobody- DSPE-PEG2000 conjugates were then post-inserted into the LNP, after which purified mouse anti-human antibodies targeting CD2 (clone RPA2.1, Biolegend), CD7 (clone 124- 1D1, Thermo Fisher) or isotype control antibodies (clone P3.6.2.8.1, Thermo Fisher) werethen captured in upright orientation onto the nanobody-conjugated LNP surface. To conjugate targeting antibodies to the LNP surface in non-controlled orientation, a DSPE- PEG2000-maleimide lipid was post-inserted into the LNP surface. Targeting antibodies were then functionalized with N-succinimidyl S-acetylthioacetate (SATA) using NHS- chemistry, followed by conjugation to the maleimide-functionalized LNP surface. I. Generation of Human Immune System (HIS) mice

[0224] As described in Arandjelovic et al.2023 Cell Rep. Med.4:101178, human cord blood CD34+ hematopoietic stem cells (Lonza, 80-90% purity) were thawed, resuspended and cultured in 1 mL of X-VIVO-10 media (Lonza) supplemented with 2% human serum albumin, 100 ng / mL human TPO (Peprotech), 100 ng / mL human Flt3L (Peprotech) and 300 ng / mL human SCF (Peprotech) at 37 °C, 5% CO2 for 3 days. Cells were then counted and resuspended in PBS. Newborn NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ mouse pups (NSG; The Jackson Laboratory) were sub-lethally irradiated (150 cGy) between 24 and 48 h after birth, and followed by injection of 35 µL 1x105 CD34+ HSCs into the temporal facial vein. Mandibular bleeds were performed at sixteen weeks of age to assess hematopoietic reconstitution by flow cytometry (Cytek Aurora). J. In vivo LNP treatment and analysis

[0225] HIS mice were injected intravenously with 10 µg targeted or control mScarlet-LNP. After 16 hours, mice were euthanised with blood, spleen and lymph nodes (salivary gland, axillary, cervical, mesenteric, brachial, pancreatic) collected. Whole blood samples were treated with ACK solution twice to lyse red blood cells. Spleen and lymph nodes are mashed through a 70 µm cell strainer into complete RPMI media to get single cell suspension. Splenocytes were treated with ACK solution once to lyse red blood cells. Cells from 200 µL blood, 20% of total cells from lymph nodes and 5% of total splenocytes were placed into a 96-well U bottom plate. Cells were then stained with Zombie UV viability dye (BioLegend) and anti-huCD45-BV786 (HI30), anti-huCD24-PacBlue (M5E2), anti-huCD19-BV510 (SJ25C1), anti-huCD3-PE / Cy7 (SK7), anti-huCD8-APC (RPA-T8), anti-huCD4-APC / H7 (RPA-T4) (all BD Biosciences) and anti-moCD45.1-FITC (A20-1, WEHI), in the presence of blocking reagent (Human BD Fc Block, Fc1 and Mouse BD Fc Block, 2.4G2, BD Biosciences) followed by flow cytometry (Cytek Aurora).K. Analysis

[0226] Flow cytometry analysis, compensation and cell subset gating was performed using FCS Express 7. Further analysis including conversion of relative fluorescence into absolute number of nanoparticles per cell and graphing was performed in Python, using the Pandas, Numpy, Matplotlib, Sklearn and Seaborn packages. Confocal images were processed using ImageJ.

[0227] To calculate the receptor or nanoparticle internalization scores, cells were gated on single cells, live cells, then cells expressing the receptor of interest (for calculation of receptor internalization) or positive for nanoparticle association (for calculation of nanoparticle internalization). Within FCS Express, a new parameter was then computed (“Ratio primary (1°) to secondary (2°) antibody”), which was defined as the fluorescent intensity of surface receptor expression or nanoparticle surface binding divided by the fluorescent intensity of total receptor expression or nanoparticle association. The mean of this new parameter was then extracted and normalized to the first timepoint analysed (baseline) to calculate the receptor or nanoparticle internalization scores using Equation 2:Example 1: Selection of library of potential target receptors

[0228] A library of potential target receptors was generated that consisted of a range of pan- T cell markers, chemokine receptors and immune checkpoint and activation markers. Receptors with low breadth of expression and low receptor density were excluded from further analysis (Figure 1). Example 2: Receptor internalization can be measured using a dual staining approach on flow cytometry

[0229] To assess the ability of the target receptors of the library to undergo receptor- mediated internalization upon binding with a targeting antibody, a high-throughput, flow cytometry-based internalization assay was designed based on previous work by Qureshi et al31. Initial assay development was performed using Jurkat T cells, an acute T cell leukemia-derived cell line, using CD71 (transferrin receptor, TfR – known to cycle extensively37,38) and CD4 (known to exhibit minimal endocytosis20,21) as positive and negative controls for receptor internalization, respectively. Receptor internalization could be tracked by staining a surface receptor of interest with a pH-stable, fluorescently labelled primary (1°) antibody (Figure 2a) on ice, which is endocytosed along with the receptor during the subsequent incubation period at 37°C. Any receptor-antibody complexes present on the cell surface after incubation could be co-stained with a fluorescently labelled secondary (2°) antibody (Figure 2a). In the absence of receptor internalization, a linear relationship between the primary and secondary antibody was observed, as all receptors labelled with the primary antibody remained available for labelling with the secondary antibody. Receptor internalization, in contrast, resulted in a reduced secondary antibody signal and as such, a reduced ratio of the secondary antibody signal to the primary antibody signal (Figure 2a). These phenotypes were observed for CD4 and TfR (Figures 2b-d and Figures 2f-h, respectively), with a receptor internalization score (IS) of 2.5±8.5 (CD4) and 52.8±6.8 (TfR) (Figures 2e and i). Using confocal microscopy, internalisation of the primary antibody signal was confirmed, whereas the secondary signal selectively co-stained receptor-antibody complexes that were present on the cell surface after incubation (Figure 2j). Example 3: CD2 and CD7 are highly expressed and internalized in resting and activated primary CD4+T cells

[0230] The receptor internalization kinetics was measured in Jurkat T cells and primary, total CD4+T cells. While CD5 receptor internalization was strong in Jurkat T cells, CD5 receptor internalization was not observed at all while CD3 receptor internalization was substantially reduced in total CD4+T cells (IS of 24.1±3.3) compared to Jurkat T cells (IS of 40.3±1.6) (Figure 3a and Figure 12). In contrast, CD2 and CD7 internalization was maintained in both cell models, with an IS of 56.6±4.3 and 61.8±4.0, respectively, in CD4+T cells after 180 min. CCR5 was identified as a receptor with potent receptor internalization kinetics with an IS of 71.3±7.6 in primary T cells (Figure 3a). Of these, CD7 and CCR5 internalized more rapidly than CD2 (Figure 3b). Interestingly, programmed cell death protein 1 (PD-1), a marker repeatedly shown to enrich for latent HIV in individuals both on and off antiretroviral treatment32-36, also showed potent internalization with an IS of46.3±7.2 after 180 min. CD2 and CD7, however, exhibited the highest expression and receptor density (Figure 1) of the receptors that were found to internalize most potently.

[0231] To confirm that the observed receptor internalization also occurred in resting CD4+T cells, phenotypic staining of the activation markers CD69 and HLA-DR was included in the receptor internalization assay. On average, 94% of total CD4+T cells were found to be resting (CD69_ / HLA-DR_) across all timepoints. While activated cells exhibited stronger receptor internalization than resting cells for both CD2 and CD7, substantial internalization was still observed in resting CD4+T cells, as evidenced by a stark reduction in the ratio of 2° antibody signal over 1° antibody signal compared to baseline (0 min; Figure 9). Examples 4: Receptor targeting enhances nanoparticle association and internalization in Jurkat T cells

[0232] This study sought to examine whether the receptors identified as strongly internalizing could be exploited to enhance internalization of targeted nanoparticles. To this end, this study employed 100 nm superparamagnetic iron oxide nanoparticles (SPIONs) coated in protein G for facile surface decoration with fluorescently labelled targeting antibodies (Figure 10). The receptor internalization assay was adapted to allow simultaneous assessment of the percentage of cells exhibiting nanoparticle association, the absolute number of nanoparticle association, as well as the degree of nanoparticle internalization over time relative to the first baseline timepoint (Figure 4a).

[0233] In the Jurkat T cells, nanoparticle targeting to any of the receptors tested resulted in enhanced breadth of nanoparticle association after 48 hours compared to untargeted nanoparticles, with strong differences in the percentage association between the various Ab@SPIONs (Figure 4b). Similarly, targeting any of the receptors tested resulted in a higher number of nanoparticles per cell compared to untargeted nanoparticles. The most efficient delivery of nanoparticles was achieved upon targeting CD3 (mean±SEM 119±8.1 nanoparticles per cell), CD5 (67±1) and CD2 (52.3±6.7) (Figure 4c), despite both CD7 and CXCR4 exhibiting receptor expression in a larger proportion of Jurkat T cells as well as at a higher receptor density (Figure 11). These findings potentially reflect the higher degree of internalization of CD3-, CD5- and CD2@SPIONs (Figure 4d) compared to their CD7 and CXCR4 counterparts resulting in particle accumulation within the intracellularcompartments as well as on the cell surface. Targeting CD2 induced the highest and most rapid internalization of SPIONs into Jurkat T cells (maximal IS of 85.7 with an IS of 56.6 reached within 4 hours), while CD4 targeting resulted in a negligible IS of 0.42 after 48 hours (Figure 4d). These findings are consistent with the internalization kinetics for these two receptors (Figure 12b), suggesting that targeting strongly internalizing receptors could indeed serve as a strategy towards enhancing the uptake of nanoparticles. Example 5: CD2 and CD7, but not CCR5 targeting enhances nanoparticle delivery to CD4+T cells

[0234] To examine whether nanoparticle uptake could be triggered similarly in total CD4+T cells, we generated Ab@SPIONs targeting CD2, CD7 and CCR5 – the receptors that exhibited the most potent receptor internalization (Figure 3). CD3@SPIONs were included as a control, as CD3 internalization remains one of the most well-described examples of receptor-mediated endocytosis in primary T cells39. Surprisingly, CCR5 targeting only resulted in nanoparticle association in, at best, 2.3% of CD4+T cells (1 hour), compared to 1.5% for untargeted control particles (Figure 5a), and a modest increase from 2.0±0.86 to 13.8±4.7 nanoparticles per cell (Figure 5b). This low breadth of T cell targeting using CCR5 is reflective of the low breadth of CCR5 expression in total CD4+T cells (Figure 2a), and the minimal increase in nanoparticles associated per cell may reflect the low receptor expression in CCR5+ / CD4+T cells (Figure 1). Furthermore, in those cells that associated with CCR5@SPIONs, minimal to no internalization was observed (Figure 5c). The discrepancy between CCR5 receptor internalization and CCR5@SPION internalization could mean that in our studies, CCR5 was internalized into endocytic vesicles that are too small to engulf a 100-120 nm nanoparticle.

[0235] Targeting CD2 and CD7, in contrast, resulted in a broad nanoparticle association in 98% and 68% of CD4+T cells, respectively, after one hour, which increased further to 99% and 91%, respectively, at 48 hours (Figure 5a). The behaviour of CD2@SPIONs differed strongly from that observed in Jurkat T cells, where CD2@SPION association at any timepoint did not exceed 5.9% (Figure 5a). These findings potentially reflect the higher CD2 receptor density on primary CD4+compared to Jurkat T cells (Figure 1b and Figure 11b), suggesting the expression level of CD2 on Jurkat T cells may be insufficient to recruit CD2@SPIONs. While targeting CD2 resulted in a higher dose of nanoparticles per cell(99.6±14.4) compared to CD7 (21.1±7.2) (Figure 5b), both receptors triggered high internalization of those nanoparticles associated with IS of 78.4±2.8 and 66.0±5.1 for CD2 and CD7, respectively, both exceeding internalization achieved with CD3 targeting (Figure 5c). Nanoparticle internalization of untargeted control nanoparticles could not reliably be determined due to the low percentage of CD4+T cells that exhibited nanoparticle association (Figure 5a) and was as such excluded from further analysis. Overall, these findings corresponded closely to the internalization behaviour observed in the receptor internalization assay of Example 3, above, suggesting that CD2 and CD7 are promising candidates for T cell targeting. Example 6: CD2 and CD7 targeting enhances specific association of nanoparticles with T cells in the presence of bystander cells

[0236] Targeting a receptor that enhances nanoparticle internalization in T cells will benefit T cell targeting in vivo specifically when the chosen receptor exhibits a degree of specificity to the T cell compartment. This study therefore sought to determine the expression of CD2 and CD7 in PBMC and assess whether targeting these receptors could enhance nanoparticle association to T cells during a 1-hour co-culture with PBMC. CD2 and CD7 were expressed broadly on T cells with negligible expression in phagocytes, although NK cells were found to express both receptors at similar levels and receptor densities to T cells (Figure 13). The data show that untargeted control particles exhibited strong association with phagocytic cells such as monocytes, B cells and DCs (79%, 44% and 23%, respectively, compared to 3% association with T cells) (Figure 6a), despite T cells comprising the largest proportion of PBMCs (Figure 14a). In contrast, targeting either CD2 or CD7 resulted in increased nanoparticle association with T cells of 68% (p < 0.0001) and 24% (p = 0.06), respectively, compared to isotype control particles (Figure 6b,c).

[0237] The absolute number of nanoparticles per cell in T cells was greatest upon CD2 targeting at 86±17 nanoparticles / cell, which exceeded the dose of CD2@SPIONs found to associate with any other cell type (Figure 6b) and was significantly higher than the number of nanoparticles per T cell in the absence of targeting (p < 0.0001) (Figure 6e). CD7 targeting did not significantly increase the number of nanoparticles per T cell, yet surprisingly did lead to significantly greater nanoparticles per monocyte (p < 0.0001) (Figure 6e). As expected, targeting either CD2 or CD7 led to increases in the percentage of NK cellsexhibiting nanoparticle association as well as the number of nanoparticles per NK cell compared to untargeted control particles, yet these increases only reached statistical significance for CD2 targeting (p < 0.0001 and p = 0.0034, respectively) (Figures 6d,e). Nonetheless, the targeting effect of CD2@SPIONs was greater towards T cells than towards NK cells (Figures 6b,d,e). These findings indicate the potent targeting capability of CD2- and CD7-nanoparticles to T cells in the presence of bystander cells. Example 7: T cell targeting capabilities are largely maintained in whole human blood ex vivo

[0238] When introduced into biological fluids, proteins and other biomacromolecules adsorb onto the nanoparticle surface. This biomolecular corona may change the physicochemical properties of the nanoparticle, affecting both targeting and stealth characteristics. This study therefore sought to assessed the T cell targeting capabilities of the various targeted nanoparticles in the presence of blood serum by incubating the nanoparticles with whole blood and measuring association with various leukocyte subsets, including granulocytes (Figure 14b), as described previously40. The distribution of nanoparticle association in the absence of targeting remained largely unchanged, with T cell association being practically absent and monocytes and B cells accounting for the majority of nanoparticle association observed (Figure 7a). Consistent with previous findings, it was found that CD2 targeting in the presence of whole blood led to association with each leukocyte subtype assessed. However, association with T cells appeared most potent with 98% of T cells associating with an average of 151±31 nanoparticles per cell (Figure 7b), thus yielding significantly higher association breadth (p < 0.0001) as well as potency (p < 0.0001) with T cells compared to untargeted control particles (Figures 7d,e). CD2 targeting furthermore significantly reduced nanoparticle association to B cells (p = 0.0038). While CD2 targeting significantly increased the percentage of association to NK cells (p < 0.0001) as well as the number of nanoparticles per NK cell (p < 0.0001), these effects were less pronounced than the targeting effect towards T cells (Figure 7b,d,e). The T cell targeting capacity of CD7@SPIONs in whole blood showed a similar trend to CD2@SPIONs, significantly increasing the percentage of T cells associated with nanoparticles (p = 0.0136). Despite CD2 and CD7 exhibiting similar receptor expression patterns in T cells, CD7@SPION association with T cells reached only 37% (Figure 7c). CD7 targetingappeared to more specifically increase NK cell association compared to untargeted control (p < 0.0001). These findings suggest that targeting CD7 and, in particular, CD2, leads to pronounced association, and thus potentially internalization, with T cells in biologically relevant conditions. Example 8: Lipid nanoparticles targeted to CD2 or CD7 exhibit greater delivery of functional mRNA to primary T cells in the presence of bystander cells

[0239] To evaluate whether the enhanced nanoparticle internalization observed with CD2- and CD7-targeted nanoparticles translates to enhanced delivery of a functional therapeutic cargo, fluorescently labelled, targeted lipid nanoparticles (LNPs) encapsulating an mScarlet reporter mRNA were generated. To minimize the effects of Fc-tail-mediated interactions between the targeting antibody and Fc-receptor-expressing off-target cells, targeting antibodies were conjugated onto the LNP surface in an upright orientation. Compared to isotype-targeted control LNPs, CD2- and CD7-targeted LNPs exhibited a significantly greater LNP association with isolated primary CD4+T cells (<0.0001 for both receptors) (Figure 8a). This increased LNP association coincided with an increased transfection efficiency, that reached statistical significance for CD2 (p=0.0034) and is trending towards significance for CD7 (p=0.067) (Figure 8b). While the degree of association and transfection efficiency of untargeted control LNPs increased with increasing duration of LNP-T cell co- culture, the observed association of targeted LNPs occurred within the first hour of co- culture (Figures 8c,d). These findings confirm an increased speed of nanoparticle association and, likely, internalization in the presence of antibodies targeting CD2 or CD7.

[0240] A further study was undertaken to assess the ability of CD2- or CD7-targeted LNPs to transfect T cells in the presence of bystander PBMCs. In the absence of targeting, mRNA was primarily delivered and expressed in phagocytes, as expected, with minimal transfection of T cells (<1% mScarlet+ T cells for naked and isotype control LNPs, respectively) (Figures 8e,f). In contrast, CD2- and CD7-targeting increased the transfection of T cells in the presence of PBMC to 24.9±11% and 37.6±12% respectively. Functionalizing the LNPs with targeting antibodies in upright orientation did not significantly alter the transfection efficiency of monocytes or B cells (Figures 8g,h). In contrast, functionalization with targeting antibodies in non-controlled orientation led to a stark increase in association withmonocytes, despite demonstrating ability to enhance association with CD4+ T cells (Figure 15).

[0241] The present inventors have surprisingly identified CD2 and CD7 as promising candidates for enhanced, targeted nanoparticle association to, and internalization by, T cells in vitro, as summarised in Table 2. Table 2. Summary T cell targeting potential of key receptors based on all parameters assessed.The performance of the key receptors in this study – CD2, CD3, CD7, and CCR5 – across the various assays performed was scored to identify the most promising candidate for successful T cell targeting. For each parameter, scoring indicates absence (-) or presence (+) of beneficial effect of the respective receptor towards T cell targeting, with (++) or (+++) indicating stronger effects than (+). Parameter assessed CD2 CD3 CD7 CCR5 Receptor expression (CD4+T cells) +++ +++ +++ + Receptor density (CD4+T cells) +++ +++ +++ + Receptor specificity to target cell ++ +++ ++ - Receptor internalization in target cell +++ + +++ +++ Targeted nanoparticle association inmonoculture Targeted nanoparticle internalization in- monoculture Specificity of nanoparticle association +++ + + - Specificity of nanoparticle association (in- serum) Potency of transfection in monoculture +++ n / a +++ n / a (LNPs)Specificity of transfection (LNPs) +++ n / a +++ n / a Example 9: Efficient and selective mRNA delivery to T cells in vivo

[0242] To validate whether CD2- or CD7-targeted LNPs enabled mRNA delivery to T cells in vivo, a Human Immune System (HIS) mouse model based on NOD.Cg-PrkdcscidIl2rgtm1Wjl / SzJ (NSG) mice reconstituted with human cord blood hematopoietic stem cells (see Arandjelovic et al.2023 Cell Rep. Med.4:101178). Antibodies targeting human CD2 or CD7 were conjugated to a modified LNP formulation (50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5% DSPE-PEG) optimised to exhibit high immune evasion (stealth) and prolonged circulation half-life (Chen et al. bioRxiv preprint https: / / doi.org:10.1101 / 2024.08.07.607101). The targeted LNPs, encapsulating an mScarlet reporter mRNA, were administered by i.v. injection.

[0243] After 16 hour treatment with non-targeted LNPs, minimal mScarlet expression (< 3%) was observed in all circulating or spleen-resident human immune cells (Figure 16a,b,e,f). Upon functionalisation of the LNPs with anti-CD2 antibodies, 33±6.3% of circulating CD4+T cells and 27±1.1% of spleen-resident CD4+T cells expressed mScarlet. CD7-targeted LNPs yielded 32±4.2% and 37±6.0% mScarlet expression in circulating and spleen-resident CD4+T cells, respectively. CD8+T cells were transfected at similar efficiencies to the CD4+T cells. Expression of mScarlet was restricted to the T cell compartment with < 5% of B cells and monocytes expressing mScarlet in both blood and spleen (Figure 16c,d,g,h). mScarlet expression was also observed only in T cells within lymph-nodes after administration of CD2- or CD7-targeted LNPs, albeit at lower levels than in blood and spleen (Figure 17a-d). 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Claims

CLAIMS 1. A nanoparticle for transfecting a transfection-recalcitrant cell, wherein the nanoparticle is tethered to an antigen-binding moiety having binding specificity for an antigen expressed on the surface of the cell, wherein the cell surface antigen is cluster of differentiation 2 (CD2) or cluster of differentiation 7 (CD7).

2. The nanoparticle according to claim 1, wherein the transfection-recalcitrant cell is an immune cell.

3. The nanoparticle according to claim 2, wherein the immune cell is selected from the group consisting of a T cell, an NKT cell and an NK cell.

4. The nanoparticle according to claim 2 or claim 3, wherein the immune cell is a T cell.

5. The nanoparticle according to claim 4, wherein the T cell is selected from the group consisting of a resting T cell, a memory T cell, a circulating T cell, a tissue-resident T cell and a naive T cell.

6. The nanoparticle according to any one of claims 1 to 5, wherein the nanoparticle is a lipid nanoparticle (LNP).

7. The nanoparticle according to claims 6, wherein the LNP comprises: a. an ionisable lipid; and b. a sterol, wherein the sterol comprises less than 10 mol % cholesterol; wherein the ionisable lipid has a structure of Formula (I):or a salt, solvate or isomer thereof, wherein: R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2- 14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3, together with the atom to which they are attached, form a 5- to 14-membered heterocycle or C3-6carbocycle; R4is selected from the group consisting of a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2 and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, 5- to 14-membered heterocycle, -OR, -O(CH2)nN(R)2, - C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -O(CH2)nOR, - N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, - N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, - C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, and each n is independently selected from 1, 2, 3, 4 and 5; each R5is independently selected from the group consisting of C1-3alkyl, C2-3 alkenyl and H; each R6 is independently selected from the group consisting of C1-3 alkyl, C2-3alkenyl and H; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, - S-S-, a C6-14aryl group and a 5- to 14-membered heteroaryl group;R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; R8is selected from the group consisting of C3-6carbocycle and 5- to 14-membered heterocycle; R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and 5- to 14-membered heterocycle; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br and I; and m is an integer from 5 to 13.

8. The nanoparticle according to claim 7, wherein the ionisable lipid has a structure of Formula (IA):or a salt, solvate or isomer thereof, wherein: p is an integer from 1 to 5; m is an integer from 5 to 9; M1 is a bond or M'; R4is unsubstituted C1-3alkyl or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, - NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, - NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14-membered heteroaryl or 5- to 14-membered heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O-, -S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl and C2-14alkenyl; each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl and H; each R' is independently selected from the group consisting of C1-18 alkyl, C2- 18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14 alkenyl; each R* is independently selected from the group consisting of C1-12alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle.

9. The nanoparticle according to claim 8, wherein: p is an integer from 1 to 5; m is an integer from 5 to 9; M1 is a bond or M′;R4 is unsubstituted C1-3 alkyl or -(CH2)nQ, in which Q is OH, -NHC(S)N(R)2, - NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2, - NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14-membered heteroaryl or 5- to 14-membered heterocycloalkyl; M and M′ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R′)-, - P(O)(OR′)O-, -S-S-, an aryl group, and a 5- to 14-membered heteroaryl group; and R2 and R3 are both C1-14 alkyl or C2-14 alkenyl, R8 is selected from the group consisting of C3-6 carbocycle and heterocycle; R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -OR, -S(O)2R, - S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; and R′ is a linear alkyl.

10. The nanoparticle according to claim 9, wherein: R4 is -(CH2)nQ, in which Q is OH, wherein n is an integer from 1 to 5; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O- and -S-S-; and R2 and R3 are each individually C1-14 alkyl or C2-14 alkenyl, each optionally substituted with one or more substituents selected from halo, OH, unsubstituted C1-3alkyl and unsubstituted C1-3 alkoxy; and R' is a C1-18 linear alkyl, optionally substituted with one or more substituents selected from halo, OH and unsubstituted C1-3alkoxy.

11. The nanoparticle according to claim 10, wherein the ionisable lipid has a structure of Formula (II):or a salt, solvate or isomer thereof, wherein: p is an integer from 1 to 5; M1 is a bond or M'; R4is unsubstituted C1-3alkyl or -(CH2)nQ, in which n is 2, 3 or 4, and Q is OH, - NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, - NHC(=NR9)N(R)2, -NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, 5- to 14- membered heteroaryl or 5- to 14-membered heterocycloalkyl; M and M' are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R')-, - P(O)(OR')O-, -S-S-, a C6-14 aryl group and a 5- to 14-membered heteroaryl group; R2and R3are independently selected from the group consisting of H, C1-14alkyl and C2-14alkenyl; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl and H; each R' is independently selected from the group consisting of C1-18alkyl, C2-18alkenyl, -R*YR", -YR" and H; each R" is independently selected from the group consisting of C3-14 alkyl and C3- 14alkenyl;each R* is independently selected from the group consisting of C1-12 alkyl and C2- 12alkenyl; and each Y is independently a C3-6 carbocycle.

12. The nanoparticle according to claim 11, wherein: p is an integer from 1 to 5, M1is M′; R4 is -(CH2)nQ, in which Q is OH, and n is an integer from 1 to 5; M and M′ are independently selected from -C(O)O-, and -OC(O)-; R2and R3are both C1-14alkyl, or C2-14alkenyl; and R′ is a C1-C12 linear alkyl.

13. The nanoparticle according to claim 6, wherein the ionisable lipid has a structure of Formula (IIa), (IIb), (IIc) or (IId):or a salt, solvate or isomer thereof.

14. The nanoparticle according to claim 6, wherein the ionisable lipid has a structure of Formula (IIe):or a salt, solvate or isomer thereof, wherein n is 2, 3 or 4.

15. The nanoparticle according to claim 6, wherein the ionisable lipid has a structure of Formula (IId):or a salt, solvate or isomer thereof.

16. The nanoparticle according to claim 6, wherein the ionisable lipid is selected from the group consisting of: ,or a salt, solvate or isomer thereof.

17. The nanoparticle according to claim 6, wherein the ionisable lipid is SM-102:or a salt, solvate or isomer thereof.

18. The nanoparticle according to any one of claims 6 to 17, wherein the sterol comprises about 38.5 mol % of the LNP composition.

19. The nanoparticle according to any one of claims 6 to 18, wherein cholesterol comprises less than 9 mol %, less than 8 mol %, less than 5 mol %, less than 4 mol %, less than 3 mol %, less than 2 mol % or less than 1 mol % of the LNP composition.

20. The nanoparticle according to any one of claims 6 to 19, wherein the sterol is a phytosterol.

21. The nanoparticle according to claim 20, wherein the phytosterol comprises at least 28.5 mol %, at least 29.5 mol %, at least 30.5 mol %, at least 31.5 mol %, at least 32.5 mol %, at least 33.5 mol %, at least 34.5 mol %, at least 35.5 mol %, at least 36.5 mol %, at least 37.5 mol % of the LNP composition.

22. The nanoparticle according to claim 20 or claim 21, wherein the phytosterol is a C- 24 alkyl phytosterol having a structure of Formula (III):or a stereoisomer thereof, wherein: each is individually a single bond or a double bond; and R is a C1-6alkyl.

23. The nanoparticle according to claim 22, wherein R is a C1-4 alkyl, C1-3 alkyl, or C1- 2 alkyl.

24. The nanoparticle according to claim 22 or claim 23, wherein R is a C1-2alkyl.

25. The nanoparticle according to any one of claims 22 to 24, wherein the C-24 alkyl phytosterol is selected from the group consisting of ,,, , and any combination thereof.

26. The nanoparticle according to claim 25, wherein the C-24 alkyl phytosterol is β- sitosterol:

27. The nanoparticle of any one of claims 6 to 26, wherein the sterol or phytosterol consists of β-sitosterol.

28. The nanoparticle according to any one of claims 6 to 27, further comprising a phospholipid.

29. The nanoparticle according to claim 28, wherein the phospholipid is selected from a group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2- Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and 1,2-Dioleoyl-sn- glycero-3-phosphocholine (DOPC).

30. The nanoparticle of claim 29, wherein the phospholipid is DSPC.

31. The nanoparticle according to any one of claims 6 to 30, further comprising a PEG lipid.

32. The nanoparticle of claim 31, wherein the PEG-lipid is selected from a group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG- modified diacylglycerols, PEG-modified dialkylglycerols, PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, a PEG-DSPE lipid and combinations of any of the foregoing.

33. The nanoparticle according to claim 32, wherein the PEG lipid is DMG-PEG or DSPE-PEG.

34. The nanoparticle according to any one of claims 6 to 33 comprising SM-102, β- sitosterol, DSPC and DMG-PEG.

35. The nanoparticle according to any one of claims 6 to 34, comprising from about 20 mol% to about 80 mol% SM-102, from about 5 mol% to about 30 mol% DSPC, about 30 mol% to about 50 mol% β-sitosterol, no more than 10% mol cholesterol, and about 0.5 mol% to about 3 mol% DMG-PEG.

36. The nanoparticle according to any one of claims 6 to 30, wherein the LNP does not comprise cholesterol.

37. The nanoparticle according to any one of claims 6 to 36, comprising about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG.

38. The nanoparticle according to any one of claims 1 to 37, wherein the nanoparticle comprises a therapeutic agent.

39. A pharmaceutical composition comprising the nanoparticle according to any one of claims 1 to 38.

40. The nanoparticle or pharmaceutical composition according to claim 38 or claim 39, wherein the therapeutic agent is protein, a small-molecule drug, or a nucleic acid molecule.

41. The nanoparticle or pharmaceutical composition according to claim 40, wherein the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.

42. The nanoparticle or pharmaceutical composition according to claim 41, wherein the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA (aiRNA), a microRNA (miRNA), a Dicer-substrate RNA, a small hairpin RNA (shRNA), a messenger RNA (mRNA), a guide RNA (gRNA), self-amplifying RNA, long non-coding RNA, circular RNA and any combination thereof.

43. The nanoparticle or pharmaceutical composition according to claim 42, wherein the RNA is an mRNA.

44. The nanoparticle or pharmaceutical composition according to claim 43, wherein the RNA is a gRNA.

45. A method of delivering an agent to an immune cell, the method comprising contacting the immune cell with the nanoparticle or the pharmaceutical composition according to any one of claims 38 to 44.

46. A method of expressing a transgene in an immune cell, the method comprising contacting the immune cell with the nanoparticle according to claim 38 or the pharmaceutical composition according to claim 39, wherein the therapeutic agent comprises a transgene.

47. The method according to claim 45 or claim 46, wherein the immune cell is a transfection-recalcitrant immune cell.

48. The method according to claim 47, wherein the transfection-recalcitrant immune cell is a quiescent immune cell, a primary immune cell, or a stem cell.

49. The method according to claim 47 or claim 48, wherein the transfection-recalcitrant immune cell is a T cell.

50. The method according to claim 49, wherein the T cell is selected from the group consisting of an unstimulated or a resting T cell, a memory T cell, a circulating T cell, a tissue-resident T cell and a naive t cell.

51. The method according to claim 47 or claim 48, wherein the transfection-recalcitrant immune cell is an NK cell.

52. A method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the nanoparticle or pharmaceutical composition according to any one of claims 38 to 44, wherein the therapeutic agent is capable of treating or preventing the disease.

53. The method according to claim 52, wherein the disease is an HIV infection, wherein the therapeutic agent is capable of: a. permanently inactivating or silencing the HIV genome; or b. reversing HIV latency to re-activate HIV infection, to allow targeting of the re-activated HIV infected cells with concurrent antiretroviral therapy.

54. The method according to claim 53, wherein the HIV infection is a latent HIV infection.

55. The method according to any one of claim 52 to 54, wherein the therapeutic agent is a protein, a small-molecule drug, or a nucleic acid molecule.

56. The method according to claim 55, wherein the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule.

57. The method according to claim 56, wherein the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RNA(aiRNA), a microRNA (miRNA), a Dicer-substrate RNA, a small hairpin RNA (shRNA), a messenger RNA (mRNA), a guide RNA (gRNA, self-amplifying RNA, long non-coding RNA, circular RNA and any combination thereof.

58. The method of claim 55 or claim 56, wherein the nucleic acid is a guide RNA and / or encodes for a CRISPR-associated protein.

59. Use of the nanoparticle according to any one of claims 38 to 44 in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the therapeutic agent is capable of treating or preventing the disease.

60. The use according to claim 59, wherein the disease is an HIV infection, wherein the agent is capable of a. permanently silencing the HIV genome; or b. reversing HIV latency to re-activate HIV infection, to allow targeting of the re-activated HIV infected cells with concurrent antiretroviral therapy.