Lipid nanoparticles and uses thereof

Abstract

The present disclosure relates generally to lipid nanoparticles (LNPs) and compositions comprising the same, and their use in delivery of agents, such as nucleic acid-based therapeutics, in particular to transfection recalcitrant cells and / or to lung tissue.

Description

LIPID NANOPARTICLES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from Australian Provisional Patent Application No.2023901544 filed 18 May 2023 and Australian Provisional Patent Application No.2023903795 filed 24 November 2023, the entire contents of each of which are incorporated herein by cross-reference. TECHNICAL FIELD

[0002] The present disclosure relates generally to lipid nanoparticles (LNPs) and compositions comprising the same, and their use in delivery of agents, such as nucleic acid- based therapeutics, in particular to transfection recalcitrant cells and / or to lung tissue. BACKGROUND

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

[0004] Lipid nanoparticles (LNPs) 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 LNPs have shown varied ability to efficiently transfect hard- to-transfect or transfection-recalcitrant cells. For example, immune cells such as resting T cells, are extremely difficult to transfect.

[0005] Therapeutic targeting of immune cells (e.g., T cells) is important for treatment and prevention of cancer, autoimmune diseases and viral infections such as HIV. Resting T-cells are a reservoir for HIV and the major barrier to a cure. Therefore, it is desirable to be able to deliver therapeutic payloads to resting T cells as a strategy for treating HIV infection.T cells are also of interest in the development of chimeric antigen receptor (CAR)-based cellular immunotherapeutics. As T cells are also susceptible to disease, including in haematological malignancies or T cell-tropic viral infections, targeting of affected T cells with a therapeutic payload such as antisense nucleic acid and mRNA molecules, also has clinical relevance and potential if such payloads can be delivered to T cells. While increasing the number of LNPs has been shown to improve transfection efficiency, it is often associated with deleterious cell toxicity and can increase cost due to the need for higher dosage administration, which can limit their therapeutic use.

[0006] In another example, various pulmonary diseases such as chronic obstructive pulmonary disease (COPD), are well recognised as having a genetic component. Gene therapies, including CRISPR / Cas-based gene editing and mRNA-based gene replacement technologies, have demonstrated significant potential for the treatment and prevention of genetic diseases by modifying the genes responsible for particular disease states. Nucleic acid-based therapeutics, such as mRNA vaccines, have also been demonstrated to be particularly useful in preventing or ameliorating the symptoms of respiratory infections, such as COVID-19. Therefore, it is also desirable to be able to deliver nucleic acid-based therapeutics, such as CRISPR / Cas-based gene editing, mRNA-based gene replacement technologies and mRNA vaccines, directly to the lungs. While LNPs have previously been developed for the delivery of mRNAs vaccine against COVID-19 (e.g., WO 2017 / 049245), there remain few LNPs that are capable of targeted delivery of the mRNA to the lungs.

[0007] Accordingly, there remains an urgent need for improved delivery of agents, such as nucleic acid-based therapeutics, to hard-to-transfect or transfection-recalcitrant cells and / or to lung tissue. SUMMARY

[0008] The present invention is predicated, at least in part, on the inventors' surprising findings that certain LNPs provide improved delivery of agents, such as nucleic acid-based therapeutics, to transfection-recalcitrant (or “transfection-resistant”) cells or difficult to transfect (or “hard-to-transfect”) cells and / or provide targeted delivery to lung tissue.

[0009] In one aspect, the present disclosure provides a lipid nanoparticle (LNP) for delivering an agent a transfection-recalcitrant cell, a sterol, wherein the sterol comprises less than 10 mol% cholesterol, wherein the ionizable 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'; R2and R3are independently selected from the group consisting of H, C1-14alkyl, 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 C3-6 carbocycle; 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 C6-14 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 C1-3 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 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.

[0010] In an embodiment, the ionisable lipid is SM-102, or a salt, solvate or isomer thereof.

[0011] In an embodiment, the sterol is a C-24 alkyl phytosterol having a structure of Formula (III):(III) or a stereoisomer thereof, wherein: each is individually a single bond or a double bond; and R is a C1-6alkyl.

[0012] In an embodiment, R is a C1-4alkyl, C1-3alkyl, or C1-2alkyl. In another embodiment, embodiment, the C-24 consisting of , ,,, , and any combination thereof.

[0013] In another embodiment, the sterol is β-sitosterol. In another embodiment, the sterol consists of β-sitosterol.

[0014] In an embodiment, the LNP comprises a phospholipid. In another embodiment, the LNP further comprises a PEG lipid.

[0015] In another aspect, the present disclosure provides an LNP comprising SM-102, DSPC, β-sitosterol and DMG-PEG. In an embodiment the LNP 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.

[0016] In another aspect, the present disclosure provides an LNP 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 does not comprise cholesterol.

[0017] In another aspect, the present disclosure provides a composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid according to the present disclosure and an agent.

[0018] In one embodiment, the agent is a protein, a small-molecule drug, or a nucleic acid.

[0019] In another aspect, the present disclosure provides a method of delivering an agent to a cell, the method comprising contacting the cell with a composition comprising an LNPcomprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid according to the present disclosure, and the agent. 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 a composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid according to the present disclosure, and the agent, 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.

[0020] 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.

[0021] In another aspect, the present disclosure provides a method of transfecting resting T cells, the method comprising contacting the resting T cell with a composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid according to the present disclosure and an agent.

[0022] 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 a pharmaceutical composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid according to the present disclosure and a therapeutic agent, wherein the therapeutic agent is capable of treating or preventing the disease.

[0023] In an embodiment, the disease is a HIV infection, the method comprising administering to the subject an effective amount of a pharmaceutical composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid according to the present disclosure, 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.

[0024] In another embodiment, the HIV infection is a latent HIV infection.

[0025] In another aspect, the present disclosure provides use of a pharmaceutical composition comprising an LNP comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid according to the present disclosure and a therapeutic agent 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.

[0026] 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.

[0027] 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.

[0028] In one aspect, the present disclosure provides an LNP for delivering a therapeutic agent to a lung tissue, the LNP comprising an ionizable lipid and a quaternary ammonium lipid, wherein the ionizable lipid has a structure of Formula (I) as defined above.

[0029] In another aspect, the present disclosure provides an LNP comprising SM-102, DOTAP, DSPC, cholesterol and DMG-PEG.

[0030] In another aspect, the present disclosure provides an LNP comprising SM-102, DOTAP, DSPC, β-sitosterol and DMG-PEG.

[0031] In another aspect, the present disclosure provides a pharmaceutical composition comprising an LNP comprising an ionizable lipid and a quaternary ammonium lipid according to the present disclosure, and a therapeutic agent.

[0032] In another aspect, the present disclosure provides a method of delivering a therapeutic agent to a lung tissue, the method comprising contacting the lung tissue with thepharmaceutical composition comprising an LNP comprising an ionizable lipid and a quaternary ammonium lipid according to the present disclosure, and a therapeutic agent.

[0033] In another aspect, the present disclosure provides a method of producing a polypeptide in a lung tissue of a subject, the method comprising administering to the subject the pharmaceutical composition comprising an LNP comprising an ionizable lipid and a quaternary ammonium lipid according to the present disclosure, and a therapeutic agent, wherein the therapeutic agent is an mRNA encoding the polypeptide, and the mRNA is capable of being translated in the cell to produce the polypeptide.

[0034] In another aspect, the present disclosure provides a method of treating or preventing a pulmonary disease in a subject, the method comprising administering to the subject an effective amount of the pharmaceutical composition comprising an LNP comprising an ionizable lipid and a quaternary ammonium lipid according to the present disclosure, and a therapeutic agent, wherein the therapeutic agent is capable of treating or preventing the pulmonary disease.

[0035] In another aspect, the present disclosure provides use of the pharmaceutical composition comprising an LNP comprising an ionizable lipid and a quaternary ammonium lipid according to the present disclosure, and a therapeutic agent in the manufacture of a medicament for treating or preventing a pulmonary disease in a subject, wherein the therapeutic agent is capable of treating or preventing the pulmonary disease. 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 fluorescent images of HEK293T cells following transfection with Cas13b-GFP mRNA-loaded Formulation Comparator 1 (A) and Formulation mLNP (B). Formulation mLNP showed greater transfection rate compared to Formulation Comparator 1. The images were taken at 24 hours post-transfection.

[0038] Figure 2 shows transfection of Calu-3 cells with Cas13b-GFP mRNA packaged in Formulation Comparator 1 (SM102 LNP comprising cholesterol) and Formulation mLNP(mSM102 LNP comprising b-sitosterol) (A-B).24 h post-transfection, Formulation mLNP showed enhanced transfection rate (B) with minimal cytotoxicity (C).

[0039] Figure 3 shows Formulation mLNP effectively delivered Cas13b-GFP mRNA to hard-to-transfect B lymphoblastoid cell line. Fluorescent images of hard-to transfect human B lymphoblastoid cell line (Raji cells) transfected with various amounts of Cas13b-GFP mRNA at 24 and 48 hours post-transfection (A). Flow cytometry data is shown in (B-C).

[0040] Figure 4 shows Formulation mLNP effectively delivered Cas13b-GFP mRNA to hard-to-transfect Nasopharyngeal carcinoma cell. Fluorescent images of C666-1 transfected with various amounts of Cas13b-GFP mRNA at 24 and 48 hours post-transfection (A). Flow cytometry data is shown in (B).

[0041] Figure 5 shows Formulation mLNP effectively delivered Cas13b-GFP mRNA to a hard-to-transfect Jurkat-derived daughter cell line (J-Lat A2). Fluorescent images of J-Lat A2 cells transfected with various amounts of Cas13b-GFP mRNA at 24 and 48 hours post- transfection (A). Flow cytometry data is shown in (B-C).

[0042] Figure 6 shows that Jurkat T cells are not susceptible to transfection with lipofectamine. A Jurkat-derived daughter cell line which contains an integrated HIV promoter which controls expression of a green fluorescent protein (J-Lat A2) was transfected with mCherry mRNA at different doses using Lipofectamine 3000, leading to minimal mCherry expression. Means of duplicate measurements of single experiment.

[0043] Figure 7 shows the schematic overview of experimental set-up for Example 4. Total CD4+T cells are isolated from PBMC, then either stimulated with plate-bound anti-CD3 (αCD3) and soluble anti-CD28 (αCD28) or rested for 72 hours, both in the presence of 10 U / mL IL-2. Then, cells are incubated with LNPs of the present invention or control / comparator LNPs for another 72 hours prior to harvest for analyses.

[0044] Figure 8 shows a comparison of transfection efficiency between resting and stimulated T cells. mCherry expression (% mCherry), LNP association (DiD MFI) and viability (% viability) were measured using flow cytometry in unstimulated CD4+T cells and stimulated T cells after 72 hours of transfection with mCherry mRNA encapsulated by Formulation Comparator 2.

[0045] Figure 9 shows that Formulation mLNP (mSM102 LNP comprising β-sitosterol) potently deliver mRNA to unstimulated T cells with minimal toxicity. (A-D) Unstimulated CD4+T cells were treated with doses of 100 ng to 500 ng (A, B) or 1.6 ng to 100 ng (C, D) of mCherry mRNA delivered by Formulation mLNP. mCherry expression is shown in (A, C) and viability (B, D) as assessed after 72 hours. Data points represent mean ± SEM of n=2 donors. Data in (a, b) and (c, d) represent independent experiments, but were performed using the same n=2 donors.

[0046] Figure 10 shows that CRISPRa induces potent latency reversal in J-Lat A2 cells with minimal toxicity when delivered using Formulation mLNP (mSM102 LNP comprising β- sitosterol). (A) Jurkat T cells were treated with equal doses of mCherry-LNP using either Formulation Comparator 2 (open circles) or Formulation mLNP (closed circles) formulation. Protein expression was determined through flow cytometry after 24 hours. Datapoints indicate mean ± SEM of n=2 independent experiments. (B) dCas9-VP64 mRNA, MS2-p65- HSF1 mRNA and HIV targeting gRNA were co-encapsulated into either Formulation Comparator 2 (open circles) or Formulation mLNP (closed circles) LNPs at a 1:1:1 ratio and administered to J-Lat A2 cells at various doses. After 24 hours, reactivation of LTR- mediated transcription was determined through measuring GFP expression using flow cytometry.

[0047] Figure 11 shows that Formulation mLNP (comprising SM-102 and β-sitosterol) is capable of efficient delivery of a large nucleic acid payload (i.e. blue fluorescent protein- tagged Cas13b encoding plasmid DNA) into HEK293T cells, when compared to Comparator Formulation 3 (comprising MC3 and β-sitosterol).

[0048] Figure 12 shows mRNA encoding HIV Tat exon 1 delivered by mLNP is a potent activator of HIV transcription. (a) J-Lat 10.6 cells were treated for 24h with indicated doses of mLNP encapsulating mRNA encoding HIV Tat exon 1 (Tat-LNP) or mCherry (mCherry- LNP) as control. Reactivation of HIV LTR-mediated transcription was determined after 24hr by measuring GFP expression. Treatment with PMA / ionomycin was included as a positive control. Mean ± SEM, n=3. (b-i) CD4+ T cells from people living with HIV on suppressive ART were treated with 200ng Tat-LNPs, mCherry-LNPs per 105cells or PMA / PHA as a positive control. After 48hr (^^) or 72hr (●), expression of HIV transcripts TAR (b) LongLTR (c), Pol (d), PolyA (e) and Tat-Rev (f) representing transcription initiation,proximal elongation, distal elongation, completion and splicing, respectively, was determined using digital RT-PCR. Data was normalized to RNA input, then presented as fold-change induction compared to non-treated (NT) control. Significance was determined using a one-tailed Wilcoxon signed-rank test, * p<0.05, ** p<0.01, ns non-significant. Concurrently, cellular toxicity (g) and expression of cellular activation markers CD25 (h), CD69 (i) and HLA-DR (j) were determined using flow cytometry. Lines represent median of n=8 donors. Where datapoints are missing, cell input was insufficient to perform an accurate measurement.

[0049] Figure 13 shows that mLNP are able to deliver the CRISPR activation machinery to activate transcription in CD4+ T cells from PLWH. (a) Schematic overview of the dCas9- synergistic activation mediator (SAM) CRISPR activation system, consisting of a catalytically inactive (dead, d)Cas9 fused to transcriptional activator domain VP64, a gRNA and a further transcriptional activation MS2-p65-HSF1 fusion protein that is recruited to the Cas9-gRNA complex via MS2-binding to stem-loop structures in the gRNA scaffold. (b) J- Lat 10.6 cells were treated for 24h with indicated doses of CRISPRa-mLNP containing one of four HIV LTR-targeting gRNAs (L, O, B, C) or scrambled control gRNA. Reactivation of HIV LTR-mediated transcription was determined after 24hr by measuring GFP expression. Treatment with PMA / ionomycin was included as a positive control. Mean ± SEM, n=3. (c-k) CD4+ T cells from people living with HIV on suppressive ART were treated with 200ng CRISPRa-mLNP containing gRNAs L and O (CRISPRa-LNP L+O) or a scrambled gRNA control (CRISPRa-LNP scr) per 105cells or PMA / PHA as a positive control. After 48hr (^^) or 72hr (●), expression of HIV transcripts TAR (b) LongLTR (c), Pol (d), PolyA (e) and Tat-Rev (f) representing transcription initiation, proximal elongation, distal elongation, completion and splicing, respectively, was determined using digital RT- PCR. Data was normalized to RNA input, then presented as fold-change induction compared to non-treated (NT) control. Significance was determined using a one-tailed Wilcoxon signed-rank test, * p<0.05, ** p<0.01, ns non-significant. Concurrently, cellular toxicity (h) and expression of cellular activation markers CD25 (i), CD69 (j) and HLA-DR (k) were determined using flow cytometry. Lines represent median of n=8 donors. Where datapoints are missing, cell input was insufficient to perform an accurate measurement.

[0050] Figure 14 shows optimisation of CRISPR activation potency in cell line models of HIV latency. (a) J-Lat A2 cells were simultaneously treated with three mLNP formulations containing dCas9-VP64 mRNA, MS2-p65-HSF1 mRNA and gRNA L or scrambled control gRNA, respectively. The gRNA-mLNP dose was kept constant at 64ng per 105cells; dCas9- VP64-mLNP was dosed at either 64ng (dark blue / grey) or 8ng (light blue / grey) and the MS2- p65-HSF1-mLNP dose was titrated to identify the relative ratio of the CRISPRa machinery components that yielded the highest potency (gRNA L, blue) with lowest non-specific activation (gRNA scr, grey). After 24hr, reactivation of HIV LTR-mediated transcription was determined by measuring GFP using flow cytometry. PMA / ionomycin treatment was included as a positive control. Mean ± SEM, n=2. (b) J-Lat A2 cells were treated with sub- optimal doses of CRISPRa-LNPs containing gRNA L, O, or scrambled (scr), a 1:1 mixture of two CRISPRa-LNPs containing gRNA L and O, respectively (L+O), or CRISPRa-LNPs containing a 1:1 mixture of gRNAs L and O (L+O co-packaged). Potency to reactivate HIV LTR-mediated transcription was determined using flow cytometry and compared using ratio paired t test. * p<0.05, ** p<0.01. Mean ± SEM, n=5.

[0051] Figure 15 shows that mLNP co-encapsulating CRISPR activation machinery can be used to induce expression of endogenous genes in T cells, with a peak response after 6 days of treatment. (a-c) mLNP formulation was used to encapsulate the CRISPR activation machinery (CRISPRa-LNP) including a gRNA targeting the endogenous gene encoding CD25 (CD25 CRISPRa-LNP) or a scrambled control gRNA (scr CRISPRa-LNP). (a) Jurkat T cells were treated for 24hr with CRISPRa-LNPs or PMA / ionomycin as positive control. Induction of CD25 expression was measured by surface stain using flow cytometry. (b,c) CD4+ T cells from HIV-negative donors were treated with indicated doses of CRISPRa- LNPs per 105cells for 72hr (b) or 6 days (c), after which CD25 expression was determined using flow cytometry. Treatment with PMA / ionomycin was included as a positive control. Dotted line represented average baseline CD25 expression in the absence of treatment. Mean ± SEM, n=4 independent experiments (a) or n=5-6 donors (b,c).

[0052] Figure 16 shows that CRISPRa-mLNP-mediated induction of expression of endogenous genes is non-toxic in T cell lines and primary T cells. (a-c) mLNP formulation was used to encapsulate the CRISPR activation machinery (CRISPRa-LNP) including a gRNA targeting the endogenous gene encoding CD25 (CD25 CRISPRa-LNP) or ascrambled control gRNA (scr CRISPRa-LNP) as in Figure 19. These were used to treat Jurkat T cells for 24hr (a) or primary CD4+ T cells for 72hr (b) or 6 days (c), after which viability was determined using flow cytometry. An mCherry mRNA-LNP control was included to assess the transfection efficiency at 72hr and 6 days of culture (d,e). Means ± SEM for n=4 independent experiments (a) or n=5-6 donors (b-e). Dotted lines represent baseline viability in the absence of LNP treatment.

[0053] Figure 17 shows a schematic of the in vivo study of lung-targeting of Formulation 1 and Comparative Formulations 3 and 4 loaded with Luciferase mRNA and labelled with lipophilic dye (DiD) in accordance with Example 13.

[0054] Figure 18 shows quantification of function of Comparative Formulation 3 and 4 in accordance with Example 13. Total photon flux per sec (Function) in lung, liver, and spleen. Each dot represents one mouse.

[0055] Figure 19 shows a schematic of in vivo study of lung-targeting of Formulation 2 and Comparative Formulation 5 loaded with Luciferase mRNA and labelled with lipophilic dye (DiD) in accordance with Example 13.

[0056] Figure 20 shows quantification of function of Comparative Formulation 5 in accordance with Example 13. Total photon flux per sec (Function) in lung, liver, and spleen. Each dot represents one mouse.

[0057] Figure 21 shows expression of tdTomato in lung tissues of Cre-LoxP tdTomato mouse after intravenous administration of DOTAP-SM102 Formulation 1. Mice received Cre recombinase encoding mRNA-LNP at 0.25 mg / kg and were sacrificed after 48 hr. A. DiD signal and tdTomato expression in lung, spleen, and liver were visualized using IVIS imaging, with quantification presented in terms of radiant efficiency (p / sec² / sr / µW / cm²). B. Confocal images of lung tissues, ranging from left to right: PBS and DOTAP-SM-102 LNP at 40X magnification demonstrating delivery to lung epithelial cells. Scale bar 50 µm. DEFINITIONS

[0058] 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.

[0059] 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, “C1-6alkyl” means an optionally substituted linear or branched, saturated hydrocarbon including 1-6 carbon atoms, “C1-3 alkyl” 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.

[0060] 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.

[0061] 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 “C2-14 alkynyl” 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, C18alkynyl 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.

[0062] 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, “C3-6 carbocycle” 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.

[0063] 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 triple bonds 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.

[0064] 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.

[0065] 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), a carbonyl (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.

[0066] 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).

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety.

[0074] 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.

[0075] Other definitions are provided throughout the specification. DETAILED DESCRIPTION

[0076] The present disclosure relates to lipid nanoparticles (LNPs), and compositions comprising the same, and their use in delivery of agents, such as nucleic acid-based therapeutics. In some embodiments, the LNPs may be particularly suitable for targeted delivery of an agent to hard-to-transfect or transfection recalcitrant cells In someembodiments, the LNPs may be particularly suitable for targeted delivery of an agent to lung tissue. Hard-to-transfect or transfection-recalcitrant cells

[0077] The present inventors have discovered that certain LNPs may provide improved delivery of agents, particularly nucleic acid-based agents (including “gene editing” agents), to cells, including hard-to-transfect cells or transfection-recalcitrant cells. Suitable LNPs for delivering agents to hard-to-transfect cells or transfection-recalcitrant cells in accordance with the present invention comprise an ionizable lipid and a sterol, wherein the sterol comprises less than 10 mol% cholesterol. Suitable ionizable lipids and sterols are described elsewhere herein.

[0078] Cell transfection has traditionally referred 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. 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, in that 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. 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.

[0079] Such LNPs may be particularly suitable for treating or preventing human immunodeficiency virus (HIV). Whilst antiretroviral therapy (ART) can block HIV replication and ameliorate disease, it is not curative and treatment is life-long. The majorbarrier to HIV cure is the persistence of latently infected, resting T cells harbouring replication-competent virus, which can rebound upon activation and re-establish viremia in the absence of antiretroviral treatment (ART). One approach towards an HIV cure is the “shock and kill” approach, which aims to reactivate HIV transcription using latency- reversing agents (LRAs), with the goal of subsequently inducing death through viral cytopathic effects or immune-mediated clearance of the infected cell. Traditionally, the ‘shock’ is attempted through small, hydrophobic compounds that target different cellular pathways to activate transcription. These first-generation LRAs can be administered systemically and will passively cross the plasma membranes to exert their effect intracellularly. Several LRAs have demonstrated potent induction of unspliced HIV RNA in vitro, ex vivo and in clinical trials, yet to date, no clinical trial of an LRA alone has shown a reduction in the size of the latent HIV reservoir. It has been speculated that this is due to most first-generation LRAs only increasing the initiation of HIV transcription (as measured by unspliced HIV RNA), but failing to overcome subsequent blocks in transcription elongation, completion and splicing (Zerbato et al.2019 Current Opinion in Virology 38, 1- 9). Secondly, first-generation LRAs activate transcription in a non-HIV-specific manner, meaning their potency to reactivate HIV is unavoidably coupled to activation of host gene transcription. Therefore, new LRAs are needed that have greater potency, lower toxicity, and greater specificity for the HIV provirus. Targeted lung delivery

[0080] The present inventors have also discovered that LNPs comprising certain combinations of ionizable lipids and cationic lipids provide improved and / or targeted delivery of agents, particularly nucleic acid-based agents (including “gene editing” agents), to lung tissue. Suitable ionizable lipids and cationic lipids are described elsewhere herein.

[0081] Such LNPs may be particularly suitable for treating or preventing pulmonary diseases, including chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, lung cancer and respiratory infections (e.g., COVID-19). Such pulmonary diseases are a leading cause of death worldwide. Indeed, COPD alone causes around 3 million deaths worldwide each year, while nearly 7 million deaths worldwide have been attributed to COVID-19 to date. While early intervention can be critical to treating or slowing theprogression of pulmonary diseases, treatment options remain limited and are, in some cases, associated with prohibitive side effects. Ionizable lipids

[0082] The LNPs in accordance with the present invention may comprise an ionizable lipid. As used herein, the term “ionizable 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. Typically, ionizable 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 ionizable lipid comprises at least one functional group that is neutral at physiological pH and it protonated at lower pH (e.g., an amine group).

[0083] The LNPs in accordance with the present invention may comprise an ionizable lipid as disclosed in WO / 2017 / 049245. In particular, the ionizable lipid may have 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'; R2and R3are independently selected from the group consisting of H, C1-14alkyl, 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 C3-6 carbocycle;R4is selected from the group consisting of a C3-6carbocycle, -(CH2)nQ, - (CH2)nCHQR, -CHQR, -CQ(R)2and unsubstituted C1-6alkyl, 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, C2-3alkenyl 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; 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; R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -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 C1-18alkyl, 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 C1-12alkyl 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.

[0084] In an embodiment of Formula (I), when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when 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.

[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'; R2and R3are independently selected from the group consisting of H, C1-14alkyl, 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 C3-6 carbocycle; 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, C2-3alkenyl 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)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 C6-14aryl 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 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 C3-6carbocycle; 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.

[0086] 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';R2and R3are independently selected from the group consisting of H, C1-14alkyl, 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 C3-6 carbocycle; 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 C3-6 carbocycle, 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) R4is -(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 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; 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, C2-6alkenyl, 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 ofC1-18alkyl, C2-18 alkenyl, -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- 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): R1 is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" and -R"M'R'; R2and R3are independently selected from the group consisting of H, C1-14alkyl, 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 C3-6 carbocycle; 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 C3-6 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)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 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 C6-14 aryl 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, C2-6alkenyl, 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 C1-18alkyl, 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; andm is an integer from 5 to 13, or a salt, solvate or isomer thereof.

[0088] 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, C2-14 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 -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2and n is selected from 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 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; 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- 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 C1- 12alkenyl; 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.

[0089] 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 C1-12alkyl and C1-12alkenyl; 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.

[0090] In an embodiment, the ionizable 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 C6-14aryl 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, C2-18 alkenyl, -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- 12 alkenyl; each Y is independently a C3-6 carbocycle.

[0091] In an embodiment of Formula (IA): 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-, an aryl group, and a 5- to 14-membered heteroaryl group; and R2and R3are both C1-14alkyl or C2-14alkenyl, R8 is selected from the group consisting of C3-6 carbocycle and 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 heterocycle;each R is independently selected from the group consisting of C1-3alkyl, C2-3alkenyl, and H; and R′ is a linear alkyl.

[0092] 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 R2and R3are each individually C1-14alkyl or C2-14alkenyl, each optionally substituted with one or more substituents selected from halo, OH, unsubstituted C1-3alkyl and unsubstituted C1-3 alkoxy; and R' is a C1-18linear alkyl, optionally substituted with one or more substituents selected from halo, OH and unsubstituted C1-3alkoxy.

[0093] In an embodiment of Formula (IA), n is an integer from 2 to 4.

[0094] In an embodiment of Formula (IA), R2 and R3 are the same. In a particular embodiment, R2and R3are both C8alkyl.

[0095] In an embodiment of Formula (IA), R2 and R3 are different.

[0096] In an embodiment, the ionizable has a structure of Formula (II):or a salt, solvate or isomer thereof, wherein:p is an integer from 1 to 5; M1is 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 C6-14 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 C2-14 alkenyl; 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- 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; and each Y is independently a C3-6carbocycle.

[0097] 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; andR′ is a C1-C12 linear alkyl.

[0098] In an embodiment, the ionizable lipid has a structure of Formula (IIa), (IIb), (IIc) or (IId):or a salt, solvate or isomer thereof, wherein R4is as described elsewhere herein.

[0099] In an embodiment, the ionizable lipid has a structure of Formula (IId):or a salt, solvate or isomer thereof, wherein R4 is as described elsewhere herein.

[0100] 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.

[0101] In an embodiment, the ionizable 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, R5 and R6 are as described elsewhere herein. For example, each of R2and R3may be independently selected from the group consisting of C5-14alkyl and C5-14alkenyl.

[0102] In an embodiment, the ionizable lipid is selected from the group consisting of:,, , or a salt, solvate or isomer thereof.

[0103] In a particular embodiment, the ionizable lipid has the following structure, also known as SM-102:or a salt, solvate or isomer thereof.

[0104] The ionizable 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.Sterols

[0105] Certain LNPs in accordance with the present disclosure further comprise a sterol. In one embodiment of the LNPs in accordance with the present the disclosure, comprises a sterol, wherein the sterol comprises less than 10 mol% cholesterol. In an embodiment, the sterol may be 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 LNP.

[0106] The phytosterol may be a C-24 alkyl phytosterol, a versatile group of phytosterols that are important for plant cell membrane dynamics.

[0107] 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.

[0108] In some embodiments of Formula (III), R is a C1-4 alkyl, C1-3 alkyl, or C1-2 alkyl. In one embodiment of Formula (III), R is a C1-2alkyl.

[0109] In some embodiments, the phytosterol is selected from the group consisting of β- sitosterol, stigmasterol, β-sitostanol, campesterol, brassicasterol, ergesterol, and combinations thereof.

[0110] In one embodiment, the C-24 alkyl phytosterol is selected from the group consisting of, , , , or any combination thereof.

[0111] 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 iscampesterol. In one embodiment, the C-24 alkyl phytosterol is brassicasterol. In an embodiment, the C-24 alkyl phytosterol is ergesterol.

[0112] In one embodiment, the C-24 alkyl phytosterol is β-sitosterol (beta-sitosterol). In an embodiment, the C-24 is

[0113] In one embodiment the sterol consists of β-sitosterol.

[0114] 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. Cationic lipids

[0115] Certain LNPs in accordance with the present invention further comprise a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid molecule that is permanently positively charged (i.e., regardless of pH). Typically, cationic 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 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.

[0116] In particular, the cationic lipid (specifically, the permanently positively charged head group) may comprise quaternary ammonium moiety, referred to herein as a "quaternary ammonium lipid". The quaternary ammonium moiety may be a straight or branched chain quaternary ammonium moiety (e.g., trimethylammonium, choline) or a cyclic quaternary ammonium moiety (e.g., morpholinium, pyrrolidinium, oxazolidium and piperidium salts, among others). Examples of quaternary ammonium lipids may include, but are not limited to:1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N-1-(2,3-dioleoyloxy) propyl-N,N,N-trimethyl ammonium chloride (DOTMA); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium (DLRIE); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide (DMRIE); 1,2-palmitoyl-3-trimethylammonium propane (DPTAP); 1,2-distearoyl-3-trimethylammonium propane (DSTAP), 1,2-myristoyl-3-trimethylammonium propane (DMTAP); dimethyldioctadecyl ammonium bromide (DDAB); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium bromide (DORI) 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA); or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-tri substituted ammonium surfactants; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxyethyl)-2,3-dioleoyloxy-1,4-butane- diammonium iodide (Tfx-50); O,O'-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine (DC-6-14); 3-N-methyl-1,2-dioleylpropandiol morpholino chloride (MMET); and 3-N-methyl-1,2-dioleoylpropandiol morpholino chloride (MMES).

[0117] Quaternary ammonium lipids as described herein may be available from commercial sources (e.g., DOTAP, DOTMA, DDAB, DORI, among others may be purchased from Avanti® Polar Lipids) or they may be synthesised using any suitable methods known in the art. 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, inWO 2000 / 030444, WO 2011 / 141705, the entire contents of each of which are incorporated herein by reference.

[0118] In an embodiment, the quaternary ammonium lipid has a structure of Formula (IV):or a solvate or isomer thereof, wherein: m and n are independently an integer from 0 to 3; Y is C or NR4; R1 is selected from H, C1-3 alkyl, C1-3 alkyl-OR', C1-3 alkyl-N(R5)3, C(O)C1-3 alkyl- N(R5)3, C(O)OC1-3alkyl-N(R5)3, wherein when Y is C, R1is selected from C1-3alkyl- N(R5)3, C(O)C1-3alkyl-N(R5)3, C(O)OC1-3alkyl-N(R5)3; R2 and R3 are independently selected from C5-30 alkyl and C5-20 alkenyl; R4is absent or C1-3alkyl, wherein when R4is absent, R1is selected from C1-3alkyl- N(R5)3, C(O)C1-3alkyl-N(R5)3, C(O)OC1-3alkyl-N(R5)3; each R5 is independently C1-3 alkyl, or two R5 groups, together with the atom to which they are attached, form a 5- or 6-membered heterocyclic group and the remaining R5 is C1-3alkyl; Z' and Z'' are independently selected from -CH-, -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; each R' is independently selected from H and C1-3alkyl; andX- is a counterion.

[0119] In an embodiment of Formula (IV), Y is NR4.

[0120] In an embodiment of Formula (IV), Y is C and R1 is selected from C1-3 alkyl-N(R5)3, C(O)C1-3 alkyl-N(R5)3, C(O)OC1-3 alkyl-N(R5)3.

[0121] In an embodiment of Formula (IV), each R5is independently C1-3alkyl. In a particular embodiment, each R5is methyl.

[0122] In an embodiment of Formula (IV), two R5 groups, together with the atom to which they are attached, form a 5- or 6-membered heterocyclic group and the remaining R5is C1-3alkyl. In a particular embodiment, the heterocyclyl group is a morpholino group.

[0123] In an embodiment of Formula (IV), Y is C, R1 is C1-3 alkyl-N(R5)3 and each R5 is independently C1-3 alkyl.

[0124] In an embodiment of Formula (IV), Z' and Z'' are independently selected from -CH- ,-C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)- and-C(O)-.

[0125] In an embodiment of Formula (IV), m is 1 and n is 0.

[0126] In an embodiment, the ammonium has a structure of Formula (IVa):or a solvate or isomer thereof, wherein: m, n and p are independently an integer from 0 to 3; R2 and R3 are independently selected from C5-30 alkyl and C5-20 alkenyl;each R5is independently C1-3alkyl, or two R5groups, together with the atom to which they are attached, form a 5- or 6-membered heterocyclic group and the remaining R5 is C1-3 alkyl; Z' and Z'' are independently selected from -CH-, -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)- and-C(O)-; and X- is a counterion.

[0127] In an embodiment of Formula (IVa), each R5 is methyl.

[0128] In an embodiment of Formula (IVa), p is 1.

[0129] In an embodiment, the quaternary ammonium lipid has a structure of Formula (IVb):or a solvate or isomer thereof, wherein: m and n are independently an integer from 0 to 3; R1 and R4 are independently selected from C1-3 alkyl, C1-3 alkyl-OR', or R1 and R4, together with the atom to which they are attached, form a 5- or 6-membered heterocyclic group; R2 and R3 are independently selected from C5-30 alkyl and C5-20 alkenyl; Z' and Z'' are independently selected from -CH-, -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)- and-C(O)-; each R' is independently selected from H and C1-3 alkyl; and X- is a counterion.

[0130] In an embodiment of Formula (IVb), R1 and R4 are each methyl.

[0131] In another embodiment, the quaternary ammonium lipid has a structure of Formula (V):or salt, solvate or isomer thereof, wherein: m and n are independently an integer from 0 to 3; R1 is C1-3 alkyl or C1-3 alkyl-OR'; R2 and R3 are independently selected from C5-30 alkyl and C5-20 alkenyl; Z' and Z'' are independently selected from -CH-, -C(O)O-, -OC(O)-, -C(O)N(R')-, - N(R')C(O)- and -C(O)-; each R' is independently selected from H and C1-3 alkyl; and X- is a counterion.

[0132] In Formulae (IV), (IVa), (IVb) and (V), X- may be any suitable counterion, preferably a pharmaceutically acceptable counterion. In an embodiment, X is selected from F, Cl, Br and I.

[0133] In an embodiment of Formulae (IV) and (IVa), the quaternary ammonium lipid is selected from the group consisting of DOTAP, DOTMA or DMRIE. In a particular embodiment, the cationic lipid is DOTAP, the structure of which is as follows:

[0134] Other suitable quaternary ammonium lipids and processes for their preparation will be apparent to those skilled in the art. LNPs

[0135] Certain LNPs in accordance with the present invention may be suitable for delivering agents to hard-to-transfect cells or transfection-recalcitrant cells. Such LNPs may comprise an ionizable lipid of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein and a sterol, wherein the sterol comprises less than 10 mol% cholesterol. In an embodiment, the sterol comprises a phytosterol. In some embodiments, such LNPs may further comprise a cationic lipid and / or one or more additional lipid components as described herein. In an embodiment, cholesterol is not present in the LNP (or a composition comprising the same). In an embodiment, a cationic lipid is not present in the LNP. In a particular embodiment, DOTAP is not present in the LNP.

[0136] Certain LNPs in accordance with the present invention may be suitable for targeted delivery of agents to lung tissue. Such LNPs may comprise an ionizable lipid of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein and a quaternary ammonium lipid. In an embodiment, the LNP comprises a quaternary ammonium lipid of Formula (IV), (IVa), (IVb) or (V) as described herein and an ionizable lipid of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) as described herein. Such LNPs may further comprise a sterol and / or one or more additional lipid components as described herein.

[0137] In some embodiments, the ionizable lipid is present in the LNPs disclosed herein in an amount of about 5 mol% to about 70 mol%; about 10 mol% to about 60 mol%, or about 10 mol% to about 50 mol%, about 10 mol% to about 40 mol%, or about 10 mol% to about 30 mol% of LNPs of the present invention. In an embodiment, the ionizable lipid is present at 50 mol% of the LNPs of the present invention.

[0138] In some embodiments, the sterol is present in the LNPs disclosed herein in an amount of about 10 mol% to about 80 mol%, or about 20 mol% to about 70 mol%, about 20 mol% to about 60 mol%, or about 30 mol% to about 50 mol%, or about 30 mol% to about 40 mol% of the LNPs of the present invention. In one embodiment, the sterol comprises about 45 mol% of the LNPs of the present invention. In one embodiment, the sterol comprises about 40 mol% of the LNPs of the present invention. In one embodiment, the sterol comprises about 38.5 mol% of the LNPs of the present invention.

[0139] In some embodiments, the sterol may comprise cholesterol, wherein cholesterol comprises less than 10 mol% LNPs of the present invention. 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% LNPs of the present invention. In an embodiment, cholesterol is not present in the LNPs of the present embodiment. In some embodiments, the sterol is a phytosterol, 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. The LNP may comprise a phytosterol and cholesterol in a molar ratio of from about 10:1 to about 1:1. For example, the phytosterol and cholesterol may be present in the LNP in a molar ratio of about 10:1 to about 1:1, or about 9:1 to about 1:1, or about 8:1 to about 1:1, or about 7:1 to about 1:1, or about 6:1 to about 1:1 , or about 5:1 to about 1:1, or about 4:1 to about 1:1, or about 3:1 to about 1:1 or about, or about 2:1 to about 1:1.

[0140] In an embodiment, the sterol may comprise a phytosterol. In some embodiments, 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 LNPs of the present invention, at least 37.5 mol%, at least 40 mol% of the LNPs of the present invention, or at least at least 45 mol% of the LNPs of the present invention or at least 50 mol% of the LNPs of the present invention. In one embodiment, phytosterol comprises 38.5 mol% of the LNPs of the present invention.

[0141] In some embodiments, the cationic lipid (e.g., a quaternary ammonium lipid) is present in the LNPs disclosed herein in an amount of about 10 mol% to about 80 mol%, orabout 20 mol% to about 70 mol%, about 30 mol% to about 60 mol%, or about 40 mol% to about 60 mol% of the LNPs of the present invention.

[0142] In embodiments in which the LNP comprises an ionizable lipid and a quaternary ammonium lipid, the ionizable lipid and the quaternary ammonium lipid may be present in a molar ratio of from about 1:10 to about 10:1. For example, the ionizable lipid and the quaternary ammonium lipid may be in a molar ratio of from about 1:10 to about 10:1, or about 1:5 to about 5:1, or about 1:2 to about 2:1, or about 1:2 to 1:1. In an embodiment, the molar ratio of ionizable lipid to quaternary ammonium lipid is about 1:2. In some embodiments, the ionizable lipid is present in the LNP in an amount of about 5 mol% to about 60 mol%, or about10 mol% to about 50 mol%, about 10 mol% to about 40 mol%, or about 10 mol% to about 30 mol% of LNPs of the present invention. In some embodiments, the quaternary ammonium lipid is present in the LNP in an amount of about 10 mol% to about 80 mol%, or about 20 mol% to about 70 mol%, about 30 mol% to about 60 mol%, or about 40 mol% to about 60 mol% of the LNPs of the present invention.

[0143] The LNPs disclosed herein may further comprise one or more additional lipid components, such as a non-cationic helper lipid, phospholipid, polyethylene glycol (PEG-) lipid, or any combination thereof. Suitable additional lipid components and processes for their preparation will be apparent to those skilled in the art. Advantageously, the use of one or more additional lipids (e.g., PEG-lipids, phospholipids, structural lipids) in LNPs of the present invention may reduce or prevent complex aggregation, promote LNP stability, prolong circulation time and / or enhance targeted delivery of the agent (e.g., a therapeutic nucleic acid sequence) 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). A skilled person will be able to select one or more additional lipids accordingly.

[0144] 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 LNPs of the present invention. For example, the one or more additional lipid components may be present in theLNP in an individual or combined amount of about 0 mol% to about 60 mol%, 0 mol% to about 50 mol%, 0 mol% to about 40 mol%, or about 0 mol% to about 35 mol%, about 0 mol% to about 30 mol%, or about 0 mol% to about 25 mol% of the LNPs of the present invention.

[0145] The LNPs disclosed herein may further comprise one or more non-cationic helper lipids. In some embodiments, the non-cationic helper lipid is a phospholipid. In some embodiments, the non-cationic helper lipid is a phospholipid substitute or replacement. The term non-cationic helper lipid 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 one embodiment, the helper lipid is a phosphatidyl choline (PC). In one embodiment, the helper lipid is not a phosphatidyl choline (PC). In some 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 one embodiment, the non-cationic helper lipid is a phospholipid or a phospholipid substitute. In some embodiments, 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.

[0146] 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 an analog 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. 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. A fatty acid moiety can be selected, for example, from the non-limitinggroup 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.

[0147] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phosphosphingolipids, such as sphingomyelin. In some embodiment, the LNPs disclosed herein may comprise a phospholipid selected from the non-limiting group consisting of 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 one embodiment, the phospholipid is DMPE. In one embodiment, the phospholipid is DOPC. In one embodiment, the phospholipid is DSPC.

[0148] The LNPs disclosed herein may further comprise a PEG-lipid. A PEG-lipid is a lipid modified with one or more polyethylene glycol (PEG) groups. Such lipids may also be referred to as “PEGylated” lipids. Suitable PEG-lipids may comprise a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid, e.g., having one or more alkyl chains (or "tails") of 6-20 carbons in length. Non-limiting examples of PEG-lipids 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. In some embodiments, the PEG-lipid includes, but not limited to 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), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one 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 PEG lipids and processes for their preparation are described, for example, in WO 2020 / 061284 and WO 2020 / 061295, among others. In some embodiments, 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 one embodiment, the PEG lipid is PEG-DMG.

[0149] The LNPs cdisclosed herein may further comprise one or more additional ionizable lipids. Non-limiting examples of suitable additional ionizable lipids may 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)).

[0150] It is also contemplated that the LNPs 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, cellular recognition, or imaging. The surface of a nanoparticle composition may also be conjugated with one or more useful antibodies. Functional groups and conjugates useful in targeted cell delivery, imaging and membrane permeation are well known in the art.

[0151] In some embodiments, the LNPs disclosed herein comprise SM-102; β-sitosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol% of the LNP; DSPC; and DMG-PEG. In some embodiments, the LNP disclosed herein comprises SM-102, β- sitosterol, DSPC, and DMG-PEG. In some embodiments, the LNP disclosed 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 another embodiment, the LNP disclosed herein does not comprise cholesterol. In one embodiment, the LNP disclosed herein comprises about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β- sitosterol and about 1.5 mol% DMG-PEG.

[0152] In some embodiments, the LNPs disclosed herein comprise SM-102; β-sitosterol and optionally, cholesterol, wherein cholesterol comprises less than 10 mol% of the composition; DSPC; and DMG-PEG. In some embodiments, the LNPs disclosed herein comprising SM- 102, β-sitosterol, DSPC, and DMG-PEG. In some embodiments, the LNPs disclosed hereincomprises 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 another embodiment, the LNPs disclosed herein does not comprise cholesterol. In one embodiment, the LNPs disclosed herein comprises about 50 mol% SM-102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG-PEG.

[0153] The LNPs disclosed herein may comprise particles having an average size of 1 μm or less. For example, the LNPs may have an average particle size of 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, or shorter. In one embodiment, the LNP may have an average particle size of 300-50 ng. The particle size of an LNP may be measured, for example, by dynamic light scattering (DLS), transmission electron microscopy, scanning electron microscopy, or any other suitable method known in the art. LNP compositions

[0154] The LNPs disclosed herein may be provided in a composition comprising one or more additional active or inert components (also referred to herein as an “LNP composition”). The LNPs composition may comprise an agent as disclosed herein. The agent may be a therapeutic agent or a non-therapeutic agent. The agent may be encapsulated or partially encapsulated in the LNP, and / or it may be disposed on the surface of the LNP (e.g., by coating, adsorption, covalent linkage or other means). As used herein, the term “pharmaceutical composition” refers to an LNP composition comprising a therapeutic agent.

[0155] In some embodiments, The LNP compositions (including pharmaceutical compositions) disclosed herein may comprise an agent to be delivered to a cell (e.g., a hard- to-transfect or transfection-recalcitrant cell) or to a tissue (e.g., lung tissue).

[0100] The efficiency, delivery and / or rate of delivery of an LNP composition to the target cell or tissue may be measured by detecting or measuring the amount of the agent or product produced by the agent that is delivered by the LNP composition, in the cell or tissue. The efficiency, delivery and / or rate of delivery of the LNP compositions disclosed herein can be measured by comparing the amount of the agent or product produced by the agent, present in a cell or tissue that has been contacted with the LNP composition disclosed hereincomprising the agent, to the amount of the agent or product produced by the agent, present in a cell or tissue that has been contacted with a comparator transfection composition comprising the 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 cells or tissues that has been contacted with LNP compositions disclosed herein comprising the protein or small molecule drug or a comparator transfection composition herein comprising the protein or small molecule drug, can be measured and compared. In another example, if the agent to be delivered is a nucleic acid, the level of nucleic acid present in cells or tissues that has been contacted with LNP compositions disclosed herein comprising the nucleic acid or a comparator transfection composition herein comprising nucleic acid, 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 or tissues that has been contacted with LNP compositions disclosed herein comprising the mRNA or a comparator transfection composition herein comprising the mRNA, can be measured and compared.

[0156] In one embodiment, the pharmaceutical compositions may comprise cells or tissues treated with an LNP composition as disclosed herein and one or more agents (including therapeutic or non-therapeutic agents).

[0157] In some embodiments, the agent is protein, a small-molecule drug, or a nucleic acid or any combinations thereof. In some embodiments, the nucleic acid agent is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) or any combinations thereof. The nucleic acid agent may be a therapeutic nucleic acid.

[0158] 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 consistingof, 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.

[0159] In a particular 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. In an embodiment, the nucleic acid-based agent is a guide RNA and / or encodes for a CRISPR-associated protein. In an embodiment, the nucleic acid-based agent is a guide RNA. In an embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA In an embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA B (SEQ ID NO:1), C (SEQ ID NO:2), L (SEQ ID NO:3) or O (SEQ ID NO:4), or an HIV LTR-targeting gRNA having at least 70% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid- based agent is an HIV LTR-targeting gRNA having at least 75% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA having at least 80% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA having at least 85% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA having at least 90% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA having at least 95% sequence identity to any one of SEQ ID NOs 1-4. In an embodiment, the nucleic acid-based agent encodes for a CRISPR- associated protein. In another embodiment, the nucleic acid-based agent encodes for a CRISPR activation (CRISPRa) system protein. In another embodiment, the nucleic acid- based agent encodes for a dCas9-SAM CRISPRa system protein. Example of dCas9-SAM CRISPRa system proteins would be familiar to those skilled in the art, including those in Konermann, et al.2015 Nature 517:583.

[0160] 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 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.

[0101] In some embodiments, the RNA is selected from the group consisting of a small interfering RNA (siRNA), an asymmetrical interfering RwNA (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. In an embodiment, the RNA is a guide RNA. In an embodiment, the nucleic acid-based agent is an HIV LTR-targeting gRNA. In an embodiment, the guide RNA is HIV LTR-targeting gRNA B (SEQ ID NO:1), C (SEQ ID NO:2), L (SEQ ID NO:3) or O (SEQ ID NO:4), or a HIV LTR-targeting gRNA having at least 70% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having at least 75% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having at least 80% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having at least 85% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having at least 90% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having at least 95% sequence identity to any one of SEQ ID NOs 1-4. In another embodiment, the nucleic acid-based agent is a HIV LTR-targeting gRNA having the sequence of any one of SEQ ID NOs 1-4 or a sequence having at least 70% sequence identity thereto.

[0161] 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.

[0162] 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. The dCas9-synergistic activation mediator (dCas9-SAM) CRISPR activation (CRISPRa) system has been proposed as a robust, HIV-specific latency-reversal agent. The dCas9-SAM CRISPRa system consists of a catalytically inactive (dead) Cas9 (dCas9) fused to a multimer of C-terminal herpes virus transcriptional activation domain 16 (VP64) that is guided to the genomic target site by a gRNA. The gRNA is modified to contain minimal hairpin aptamers that allow the recruitment of p65 and heat shock factor 1 (HSF1) trans-activators through binding of the bacteriophage protein MS2. This recruitment of multiple copies of the MS2- p65-HSF1 fusion protein that synergize with dCas9-VP64 has been shown to greatly enhance the potency of CRISPRa (Konermann, et al. 2015 Nature 517:583). In anotherembodiment, the nucleic acid-based agent encodes for a dCas9-SAM CRISPRa system protein.

[0163] 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).

[0164] 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.

[0165] 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 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), preciseRNA 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 superior specificity, attributable to its extended spacer sequence, making it highly attractive for targeting aberrant transcripts that drive various human genetic diseases, e.g., cancer.

[0166] Other suitable nucleic acid therapeutics are described in WO 2017 / 049245, the entire contents of which are incorporated herein by reference.

[0167] In some embodiments, the LNP compositions (e.g., pharmaceutical compositions) disclosed herein may comprise an adjuvant. Suitable adjuvants may include, but are not limited to Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(I:C), aluminum hydroxide and Pam3CSK4.

[0168] 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, 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.

[0169] 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 such as 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.

[0170] In other embodiments, the pharmaceutical compositions disclosed herein, particularly pharmaceutical compositions suitable for targeted lung delivery, 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.

[0171] In some embodiments, the pharmaceutical compositions disclosed herein are sustained-release formulations.

[0172] In an embodiment, the pharmaceutical composition is a vaccine.

[0173] 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).

[0174] 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. Methods of use

[0175] LNP compositions comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein may be useful in a method for delivering the 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 LNP composition. Such LNP compositions may be useful in the delivery of an exogenous nucleic acid to a hard-to-transfect cell or transfection-recalcitrant cell. The exogenous nucleic acid may be a DNA or an RNA. The exogenous nucleic acid may be a PNA (Peptide nucleic acid) or an LNA (locked nucleic acid). The exogenous nucleic acid may be further modified. The exogenous nucleic acid may be modified to improve its stability or half-life. The exogenous nucleic acid may be modified to improve its resistance to nuclease degradation. The exogenous nucleic acid may be modified to alter its binding specificity to its molecular target. The exogenous nucleic acid may be modified to alter its affinity for its molecular target. The exogenous nucleic acid may be modified to have a dye, a molecular label or a detectable label. Examples of nucleic acid modifications include thosedescribed 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 a suitable modifications (e.g., modifications to oligonucleotide backbone, sugars, bases and 5' phosphate) depending on the intended application.

[0102] In one embodiment, the exogenous nucleic acid may be an mRNA. In an embodiment, the exogenous nucleic acid is a non-coding functional RNA 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). In an embodiment, the exogenous nucleic acid is a guide RNA and / or encodes for a CRISPR- associated protein. In an embodiment, the exogenous nucleic acid is a guide RNA. In an embodiment, the exogenous nucleic acid is HIV LTR B, C, L or O gRNA. In an embodiment, the exogenous nucleic acid encodes for a CRISPR-associated protein. In another embodiment, the exogenous nucleic acid encodes for a CRISPR activation (CRISPRa) system protein. In another embodiment, the exogenous nucleic acid encodes for a dCas9- SAM CRISPRa system protein. Example of dCas9-SAM CRISPRa system proteins would be familiar to those skilled in the art, including those in Konermann, et al. 2015 Nature 517:583.

[0176] LNP compositions comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein may be useful in a method expressing an exogenous nucleic acid in a transfection-recalcitrant cell, the method comprising contacting the cell with the LNP composition or pharmaceutical composition according to the present disclosure, wherein the agent is an exogenous nucleic acid. 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 anexogenous nucleic acid can include translation of an mRNA to produce a polypeptide, wherein the exogenous nucleic acid is an mRNA molecule.

[0177] 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.

[0178] 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. Transfection can be carried out using physical or chemical methods. Examples of physical methods include electroporation, sonoporation, magnetofection, microinjection, gene guns and impalefection, which generally function to transiently disrupt, permeabilise or puncture the cell membrane to allow uptake of the agent. Examples of chemical methods include the use of complexes of cyclodextrin, polymers, calcium phosphate, liposomes, dendrimers and nanoparticles (e.g., lipid nanoparticles), which fuse or merge with the cell membrane to deposit their cargo into the cell. Chemical transfection methods is the most commonly used method of transfecting, as in comparison to physical transfection methods or viral transduction methods, it is relatively easier in that it does not require specialised equipment or viral culture. Previously, where chemical methods of transfection do not provide satisfactory transfection efficiency, physical transfection methods were employed (Johnston and Tang, 2994 Methods Cell Biol 43:353; Stewart et al., 2018 Chem Review 118:7409). However physical transfection methods tend to be associated with harsher conditions, with undesirable loss of cell viability and / or changes in cell characteristics.

[0179] 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 uptakeunder 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).

[0180] The LNP compositions comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein may advantageously provide enhanced or improved delivery (i.e., transfection efficiency) of the one or more agents to cells, 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 LNP compositions and / or the agent to be delivered to the cells. In some embodiments, the LNP compositions provide a transfection efficiency of 1-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 5-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 10-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 15-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 25-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 35-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 45-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 50-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 55-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 60-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 65-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 70-100%. Insome embodiments, the LNP compositions provide a transfection efficiency of 75-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 80-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 85-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 90-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 10-100%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 50-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 60-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 70-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 80-90%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 50-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 60-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 70-80%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-70%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-70%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-70%. In some embodiments, the LNP compositions provide a transfection efficiency of 50-70%. In some embodiments, the LNP compositions provide a transfection efficiency of 60-70%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-60%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-60%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-60%. In some embodiments, the LNP compositions provide a transfection efficiency of 50-60%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-50%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-50%. In some embodiments, the LNP compositions provide a transfection efficiency of 40-50%. In some embodiments, the LNP compositions provide a transfection efficiency of 20-40%. In some embodiments, the LNP compositions provide a transfection efficiency of 30-40%.

[0181] The LNP compositions comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent may advantageously provide enhanced or improved delivery of one or more agents to cells, including hard-to-transfect cells or transfection-recalcitrant cells. In some embodiments, the LNP compositions provide 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 LNP compositions, wherein the comparator compositions do not comprise an ionizable lipid having a structure of Formula (I); and comprises less than 10 mol% cholesterol. In some embodiments, the LNP compositions provide 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 LNP compositions, wherein the comparator compositions do not comprise an ionizable lipid having a structure of Formula (I) and comprises more than 10 mol% cholesterol. In some embodiments, the LNP compositions provide 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 comparatorLNP compositions, wherein the comparator compositions do not comprise an ionizable lipid having a structure of Formula (I) and does not comprise a C-24 alkyl phytosterol, and wherein cholesterol comprises more than 10 mol% of the comparator composition. In another embodiment, the LNP compositions provide 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 LNP compositions, wherein the comparator compositions do not comprise SM- 102 and β-sitosterol, and comprises more than 10 mol% cholesterol.

[0182] 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 a LNP composition as disclosed herein when compared to comparator LNP compositions, wherein the comparator compositions do not comprise an ionizable lipid having a structure of Formula (I) and comprises more than 10 mol% cholesterol. 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 a LNP composition as disclosed herein when compared to comparator LNP compositions, wherein the comparator compositions do not comprise SM-102 and β- sitosterol, and comprises more than 10 mol% cholesterol.

[0183] In some examples, the enhanced delivery of an agent by a LNP composition as disclosed herein to cells, including hard-to-transfect cells or transfection-recalcitrant cells can be measured in comparison to the delivery provided by commonly used / commercially available lipid-based transfection compositions that would be familiar to persons skilled in the art. Examples of such commercially available 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).

[0184] 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 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.

[0185] 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 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 an embodiment, the LNP composition or pharmaceutical composition 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 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 isto 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.

[0186] The present invention also provides use of an LNP composition comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein in the manufacture of a medicament for delivering an agent to a cell. In an embodiment, the cell is a hard-to-transfect cell or transfection-recalcitrant cell.

[0187] The present invention also provides use of an LNP composition comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein in the manufacture of a medicament for expressing an exogenous nucleic acid in a cell. In an embodiment, the cell is a hard-to-transfect cell or transfection-recalcitrant cell.

[0188] The present invention also provides use of an LNP composition comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein in the manufacture of a medicament for delivering of an exogenous nucleic acid to a cell. In an embodiment, the cell is a hard-to-transfect cell or hard-to- transfect cell or transfection-recalcitrant cell.

[0189] In accordance with the present invention, the LNP compositions comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent as disclosed herein may be useful in methods for delivering the agent to cells, the method comprising contacting the cell with the LNP composition. In an embodiment, the cell is a resting T cell.

[0190] 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, CD25and CD69. (see, for example, Siliciano et al. 2002; Saleh et al. 2007 and Cameron et al. 2010).

[0191] 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 contacted with the composition or the pharmaceutical composition disclosed herein in vivo. In some embodiments, the cell or resting T cell may be contacted with the composition or the pharmaceutical composition disclosed herein in vitro. In an embodiment, the LNP composition or pharmaceutical composition may be contacted with the cell or T 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 and in vitro contacting methods are known in the art and would be familiar to the person skilled in the art.

[0192] In some embodiments, the cell or resting T cell is purified from a subject, for delivery of the agent, before being restored to the subject.

[0193] In certain embodiments, the LNP compositions and pharmaceutical compositions disclosed 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 or resting T cells compared to other comparator LNP compositions.

[0194] The LNP compositions (particularly pharmaceutical compositions) comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid an agent (particularly a therapeutic agent) as disclosed herein may be useful in treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the LNP composition. 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, 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.

[0195] In some embodiments, 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.

[0196] In some embodiments, 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.

[0197] Thus, the present invention also provides a method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of an LNP composition (particularly a pharmaceutical composition) comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent (particularly a therapeutic agent) as disclosed herein, wherein the agent is capable of treating or preventing the disease. In an embodiment, the agent is a nucleic acid capable of treating or preventing the disease. In an embodiment, the agent is an mRNA encoding a polypeptide that is capable of treating the disease.

[0198] Thus, present invention also provides a method of treating HIV infection in a subject, the method comprising administering to the subject an effective amount of an LNP composition or a pharmaceutical composition comprising an agent 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 an mRNA encoding a polypeptide that is capable of treating treating HIV infection. In an embodiment, the agent encodes for a CRISPR-Cas system protein or a guide RNA.

[0199] In an embodiment, the agent is a guide RNA and / or encodes for a CRISPR-associated protein. In an embodiment, the agent is a guide RNA. In an embodiment, the agent is HIV LTR B, C, L or O gRNA. In an embodiment, the agent is a nucleic acid that encodes for a CRISPR-associated protein. In another embodiment, the agent is a nucleic acid that encodes for a CRISPR activation (CRISPRa) system protein. In another embodiment, the agent is a nucleic acid that encodes for a dCas9-SAM CRISPRa system protein. Example of dCas9- SAM CRISPRa system proteins would be familiar to those skilled in the art, including those in Konermann, et al. 2015 Nature 517:583.In an embodiment, the agent is capable of reversing HIV latency to re-activate HIV infection, to allow targeting of the re-activated HIV infected cells with concurrent antiretroviral therapy. The dCas9-synergistic activation mediator (dCas9-SAM) CRISPR activation (CRISPRa) system has been proposed as a robust, HIV-specific latency-reversal agent, but to date, the delivery of this CRISPRa system has been limited to in vitro-based transfection systems with little clinical application. The dCas9-SAM CRISPRa system consists of a catalytically inactive (dead) Cas9 (dCas9) fused to a multimer of C-terminal herpes virus transcriptional activation domain 16 (VP64) that is guided to the genomic target site by a gRNA. The gRNA is modified to contain minimal hairpin aptamers that allow the recruitment of p65 and heat shock factor 1 (HSF1) trans- activators through binding of the bacteriophage protein MS2. This recruitment of multiple copies of the MS2-p65-HSF1 fusion protein that synergize with dCas9-VP64 has been shown to greatly enhance the potency of CRISPRa. In an embodiment, the agent is HIV LTR B, C, L or O gRNA. In an embodiment, the agent is a nucleic acid that encodes for a CRISPR-associated protein. In another embodiment, the agent is a nucleic acid that encodesfor a CRISPR activation (CRISPRa) system protein. In another embodiment, the agent is a nucleic acid that encodes for a dCas9-SAM CRISPRa system protein. Example of dCas9- SAM CRISPRa system proteins would be familiar to those skilled in the art, including those in Konermann, et al.2015 Nature 517:583.

[0200] 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).

[0201] In another example, the LNP compositions described herein comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid can be used to deliver gene-editing agents (i.e., nucleic acids encoding CRISPR machinery or CARs) 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 LNP compositions 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).

[0202] In another example, the LNP compositions described herein comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, and an ionizable lipid 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).

[0203] The present invention also provides use of an LNP composition (particularly a pharmaceutical composition) comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and an agent (particularly a thereapeutic agent) as disclosed 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 anembodiment, the agent is a nucleic acid capable of treating the disease. In an embodiment, the agent is an mRNA encoding a polypeptide capable of treating the disease.

[0204] The present invention also provides use of a pharmaceutical composition comprising a sterol, wherein the sterol comprises less than 10 mol% cholesterol, an ionizable lipid and a therapeutic agent as disclosed herein for treating HIV infection in a subject, wherein the therapeutic 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.

[0205] In accordance with the present invention, LNP compositions comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent, particularly a nucleic acid-based therapeutic agent, as disclosed herein may be suitable for delivery of the therapeutic agent to lung tissue. In certain embodiments, such LNP compositions may provide enhanced (e.g., 1.5 fold, 2 fold, 3-fold, 4-fold, 5-fold or more) delivery of the therapeutic agent to lung tissue compared to other LNP compositions. The lung tissue may be selected from one or more of: epithelium, endothelium, interstitial connective tissue, blood vessel, hematopoietic tissue, lymphoid tissue and pleura.

[0206] Thus, the present invention also provides a method of delivering a therapeutic agent to a lung tissue, the method comprising contacting the lung tissue with an LNP composition comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent as disclosed herein. The lung tissue may be contacted with the LNP composition in vivo or ex vivo, methods for which are well known in the art. In an embodiment, the LNP composition may be contacted with a lung tissue disposed within a subject using varied routes of administration (e.g., intravenous or intranasal).

[0207] The present invention also provides a method of producing a polypeptide in a lung tissue of a subject, the method comprising administering to the subject an LNP composition comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent as disclosed herein, wherein the therapeutic agent is an mRNA encoding the polypeptide, and the mRNA is capable of being translated in the cell to produce the polypeptide.

[0208] The pharmaceutical LNP compositions disclosed herein comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent may be useful in the treatment of a variety of pulmonary diseases. Such pulmonary diseases may include but are not limited to lung cancer (including small cell and non-small cell lung cancer), 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 transmitted by the respiratory route. In some embodiments, the pulmonary disease is characterised by dysfunctional or aberrant protein or polypeptide activity. A skilled person will be able to readily select a suitable therapeutic agent depending on the pulmonary disease.

[0209] Thus, present invention also provides a method of treating or preventing a pulmonary disease in a subject, the method comprising administering to the subject an effective amount of a pharmaceutical composition as disclosed herein comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent, wherein the therapeutic agent is capable of treating or preventing the pulmonary disease. In an embodiment, the therapeutic agent is an mRNA encoding a polypeptide that is capable of treating the pulmonary disease.

[0210] The present invention also provides use of a pharmaceutical composition as disclosed herein comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent in the manufacture of a medicament for treating or preventing a pulmonary disease in a subject, wherein the therapeutic agent is capable of treating or preventing the pulmonary disease. In an embodiment, the therapeutic agent is an mRNA encoding a polypeptide capable of treating the pulmonary disease.

[0211] The present invention also provides use of a pharmaceutical composition as disclosed herein comprising an ionizable lipid, a quaternary ammonium lipid and a therapeutic agent for treating a pulmonary disease in a subject, wherein the therapeutic agent is capable of treating the pulmonary disease. In an embodiment, the therapeutic agent is an mRNA encoding a polypeptide capable of treating the pulmonary disease.

[0212] When treating a pulmonary disease, itranasal administration of the LNP composition may be preferred. The intranasal formulations disclosed herein may be administered to a person in need thereof by any suitable intranasal delivery method. Suitable methods for intranasal administration would be well known to a person skilled in the art. The intranasal formulations disclosed herein can be administered as a spray or drop. Accordingly, suitable commercial packages containing the intranasal formulation can be in any spray container known in the art. In one or more embodiments, the formulations disclosed herein may be administered via a spray device or container. Spray devices may be single unit dose systems or multiple dose systems, for example comprising a bottle, a pump and / or an actuator. Such spray devices are available commercially, for example, from Nemera, Aptar, Bespak and Becton-Dickinson. In still other embodiments, the formulations disclosed herein may be administered via an electrostatic spray device, such as described in U.S. Pat. No.5,655,517. Other suitable means for administering formulations intranasally in accordance with the invention include via a dropper, a syringe, a squeeze bottle, and any other means known in the art for applying liquids to the nasal mucosa in an accurate and repeatable fashion.

[0213] The spray devices used to administer the intranasal formulation can range from single-use metered-dose spray devices, multiple-use metered dose nasal spray devices and are not limited to spraying the solutions into each naris but can be administered as a gentle liquid stream from a plunger, syringe or the like or as drops from a unit-dose or multi-dose squeeze bottle, or other means known in the art for applying liquids to the nasal mucosa in an accurate fashion.

[0214] In one or more embodiments, a spray device suitable for use with the invention may typically deliver a volume of liquid in a single spray actuation in the range of from 0.01 to 0.15 mL. A typical dosing regimen for a nasal spray product may be in the range of one spray into a single nostril (naris) to two sprays into each nostril (naris). Repeat dosing of the same nostril (naris) may also be undertaken. It is recognised that the dosing schedule, including a repeat dosing schedule, may be modified to obtain a desired pharmacokinetic profile. Further, the dosing schedule may be modified to achieve a rapid reduction in severity, preferably cessation, of symptoms of a pulmonary disease. In some cases, incremental increases in repeat dosing may be required to achieve a reduction in severity or cessation of symptoms the viral infection. For example, it may be necessary to increase eachrepeat dose by 25%, 50%, 75%, 100%, 150% or 200% in order to achieve a reduction in severity or cessation of symptoms of the pulmonary disease.

[0215] 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.

[0216] 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.

[0217] 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 activeagents, “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.

[0218] 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.

[0219] 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.

[0220] The pharmaceutical compositions disclosed 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 such considerations, 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 thedisease 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).

[0221] 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.

[0222] 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 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 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 about0.1 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.

[0223] The amount of pharmaceutical composition, specifically 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.

[0224] Pharmaceutical compositions comprising an LNP composition as disclosed herein and an agent 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 toan adult. The amount to be administered can be determined by a medical practitioner or person skilled in the art.

[0225] In certain embodiments, it is envisaged that the pharmaceutical 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 pharmaceutical 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 pharmaceutical 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 pharmaceutical compositions disclosed herein may be advantageous, particularly where the traditional medication is associated with one or more adverse effects.

[0226] In other embodiments, a pharmaceutical composition as disclosed 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 treated with a pharmaceutical composition as disclosed 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.

[0227] 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, a pharmaceutical composition as disclosed 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 pharmaceutical composition and the additional agent are each administered in an effectiveamount (i.e., each in an amount that would be therapeutically effective if administered alone). In other embodiments, the pharmaceutical 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 pharmaceutical composition can be administered in an effective amount, while the additional agent is administered in a sub- therapeutic dose. In still other embodiments, the pharmaceutical composition can be administered in a sub-therapeutic dose, while the additional agent is administered in an effective amount.

[0228] 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.

[0229] In one or more embodiments where the pharmaceutical composition as disclosed 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.

[0230] 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-nucleosideor 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, ATP dependent 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.

[0231] By way of non-limiting example, known therapeutic agents for the treatment of COPD include bronchodilators (e.g., albuterol, ipratropium, levalbuterol, aclidinium, arformoterol, formoterol, indacaterol, tiotropium, salmeterol and umeclidinium), inhaled steroids (e.g., fluticasone and budesonide), bronchodilator combinations (e.g., aclidinium- formoterol, albuterol-ipratropium, formoterol-glycopyrrolate, glycopyrrolate-indacaterol, olodaterol-tiotropium and umeclidinium-vilanterol), bronchodilator-steroid combinations (e.g., fluticasone-vilanterol, fluticasone-umeclidinium-vilanterol, formoterol-budesonide, salmeterol-fluticasone), which may be suitable for use in combination with the pharmaceutical combinations of the present invention. Non-limiting examples of known therapeutic agents for the treatment of cystic fibrosis include ivacaftor, lumacaftor and elexacaftor–tezacaftor–ivacaftor, which may be suitable for use in combination with the pharmaceutical combinations of the present invention. Non-limiting examples of known therapeutic agents for the treatment of asthma include inhaled corticosteroids (e.g., fluticasone, budesonide, beclomethasone and ciclesonide), leukotriene modifiers (e.g., montelukast, zafirlukast and zileuton), long-acting beta agonists (LABAs; e.g., salmeterol and formoterol), long-acting muscarinic antagonists (LAMAs), theophylline, short-acting beta agonists (e.g., albuterol and bevalbuterol), ipratropium, oral corticosteroids (e.g., prednisone and methylprednisolone) and biologics (e.g., benralizumab, dupilumab, mepolizumab, omalizumab, reslizumab and tezepelumab-ekko), which may be suitable for use in combination with the pharmaceutical combinations of the present invention.

[0232] Other suitable additional therapeutic agents suitable for use with the pharmaceutical compositions of the present invention will be apparent to those skilled in the art depending on the disease to be treated.

[0233] Where a pharmaceutical combination as disclosed 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 provided pharmaceutical compositions to an adult. The amount to be administered to can be determined by a medical practitioner or person skilled in the art.

[0234] The LNPs or LNP compositions as disclosed herein may be contained in a kit. The kit may include, for example, the components of the LNP or LNP composition (e.g., an ionizable lipid, sterol and / or cationic lipid), each packaged or formulated individually, or packaged or formulated in combination. Thus, in an embodiment, an ionizable lipid may be present in a first container and a sterol and / or cationic lipid may be present in a second container. In another example, the kit may include the LNP and any additional components or agents, each packaged or formulated individually, or packaged or formulated in combination. Thus, in an embodiment, the LNP may be present in a first container (optionally together with other components of an LNP composition as disclosed herein) and the agent may be present in a second container. The container or containers may be placed within a package, and the package can optionally include administration or dosage instructions. The kits disclosed herein may comprise the components of the LNPs or LNP compositions in forms suitable for intranasal administration. The kits disclosed herein may comprise the components of the LNPs or LNP compositions 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.

[0235] 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 compoundsreferred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

[0236] 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 Example 1. Preparation of lipid nanoparticles for delivering agents to transfection- recalcitrant cells

[0237] 10 mM stocks of ionizable amino lipid SM-102, helper lipid DSPC, sterol lipids (cholesterol or β-sitosterol), PEG-lipid DMG-PEG, and lipophilic dye (DiD) were dissolved in ethanol (Ethanol phase). mRNA was dissolved in acetic buffer, pH 4 (Aqueous phase). At a flow ratio of 1:3 ethanol:aqueous phases (NanoAssemblr Ignite) or 1:1.8 (NanoAssemblr Spark) and the total flow rate 12 ml / min, the solutions were combined in the microfluidic system NanoAssemblr (Precision Nanosystems, Vancouver BC). The mixed material was then immediately diluted 1 in 4 with phosphate buffer saline (PBS) after leaving the micromixer outlet. Nitrogen to phosphate ratio was 6:1.

[0238] The particle size and polydispersity index (PdI) of the LNPs were determined by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Panalytical, UK). Samples were diluted in deionized (DI) water and transferred to disposable low volume plastic cuvettes. Samples were analysed at 25 °C with refractive indices of liposomes as materials and water as the bulk diluent to measure the hydrodynamic particle sizes and PdI.

[0239] The RNA encapsulation efficiency was determined using the Quant-it™ RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA). 1 µL of LNP were diluted in 1:100 in TE buffer (10 mM Tris-HCl, 20 mM EDTA).50 µL of the diluted LNPs were then incubated with 50 uL 0.2% Triton™ X-100 (Sigma-Aldrich) or TE buffer in a 96-well fluorescent plate. The plate was incubated for 30 min at room temperature to allow particles to disrupt by 0.2% Triton™ X-100 and liberate RNA before adding 100 μL of RiboGreen® reagent solution in TE buffer (1:2000 v / v) to each well. Fluorescence was measured using FLUOstar Omega (BMG LABTECH, Aylesbury, UK). The RNA concentration was calculated basedon ribosomal RNA standard curve. The RNA encapsulation efficiency was calculated by comparing the RNA concentrations in the presence and absence of Triton X-100. The formula used was: RNA encapsulation % = (Total RNA-Free RNA) / (Total RNA concentration) × 100.

[0240] The LNPs in accordance with the present invention (mLNP), and comparative formulations, were prepared as shown in Table 1. Table 1. LNP formulations 3

[0241] The LNPs were formulated using Nanoassemblr Ignite microfluidic devices at settings shown in Table 2.Table 2. Microfluidic settings (NanoAssemblr Ignite)

[0242] The LNP Formulations were loaded mRNA.

[0243] The physicochemical properties of Formulation mLNP loaded with Cas13b-GFP mRNA is shown in Table 3. Table 3. Physicochemical properties of an exemplary mLNP of the present disclosure nExample 2. Transfection of primary cells, hard-to-transfect cells, transfection-recalcitrant cell

[0244] The mLNP described in Table 1 was tested on several cell lines to assess transfection efficiency and potential toxicity. Table 4 provides a list of cells tested that demonstrate at least 90% transfection efficiency by GFP mRNA-loaded mLNP. Table 4. Cell lines and primary cells that were efficiently transfected (≥80%) by mRNA- loaded mLNP.

[0245] When HEK293T cells (a common laboratory cell line known to be amenable to transfection) transfected with Cas13b-GFP mRNA-loaded mLNPs showed greater transfection rates when compared to HEK293T cells transfected with Cas13b-GFP mRNA- loaded Formulation Comparator 1 (Figure 1).

[0246] Formulation mLNP also showed improved transfection rates in lung adenocarcinoma cell line, Calu-3, with minimal effect on cell toxicity, when compared to Formulation Comparator 1 (which comprised 38.5% cholesterol). (Figure 2 A-B).

[0247] The mLNPs were tested in other cells including THP-1 (Human leukemia monocytic cell line); SUP-B15 (B lymphoblast cell line); C666-1 (Nasopharyngeal carcinoma cell line); Raji (B lymphoblastoid cell line); HT144 , SK-MEL-2, CHL-1, WM-266-4, MEWO (Melanoma cell lines); RK) (colorectal cancer cell line) and primary, unstimulated T cells (primary immune cells), which are known to be transfection recalcitrant cell lines. Surprisingly, the Formulation mLNP demonstrated at least 80%-90% transfection efficiency in these cells.

[0248] Figure 3 demonstrates the high transfection rate of Raji cells (hard-to-transfect B lymphoblastoid cell line), when using Formulation mLNP of the present disclosure.

[0249] Figure 4 demonstrates the high transfection rate of C666-1 (hard-to-transfect Nasopharyngeal carcinoma cell line), when using Formulation mLNP of the present disclosure.Example 3. Transfection of HIV-latently infected cells

[0250] Figure 5 demonstrates the high transfection rate of J-Lat-A2, when using Formulation mLNP of the present disclosure. J-Lat-A2 is a Jurkat-derived cell line that has been latently infected with a GFP-tagged HIV-1 strain (Jordan et al., 2003 EMBO J. 22:1868).

[0251] In contrast, commercially available Lipofectamine 3000, even when loaded with high concentrations of mCherry mRNA, did not provide appreciable levels of transfection, as can be seen by minimal mCherry expression (Figure 6). Example 4. Transfection of resting T cells

[0252] Formulation mLNP of the present disclosure was tested for their ability to transfect stimulated CD4+T cells and unstimulated CD4+T cells, when compared to a control formulation comprising 50% DLin-MC3-DMA ionizable lipid, 10% DSPC, 38.5% cholesterol, 1.5% DMG-PEG (Formulation Comparator 2).

[0253] Figure 7 provides a schematic of the experimental set-up. Total CD4+T cells are isolated from human HIV negative PBMCs. The CD4+T cells were stimulated with plate- bound anti-CD3 (αCD3) and soluble anti-CD28 (αCD28) or rested for 72 hours, both in the presence of 10 U / mL IL-2.

[0254] In experiments where the stimulated and unstimulated T cells were incubated with Formulation Comparator 2 packaged with 125ng or 500ng of mCherry encoding mRNA or control solutions (untreated, vehicle controls [0.45% or 1.8% EtOH for 125ng or 500ng conditions, respectively], empty LNPs), cells were incubated for 72 hours prior to harvest. The formulations were labelled with lipophilic dye (DiD). Cells were then analysed for mCherry expression, LNP association and viability using flow cytometry. The results shown in Figure 8 (N=4) indicated that Formulation Comparator 2 was capable of transfecting stimulated CD4+T cells at about 30% efficiency, but not unstimulated CD4+T cells (which showed less than 1% transfection efficiency when 125ng of mRNA was used, and about 2% transfection efficiency when 500ng mRNA was used). This difference in transfection efficiency between stimulated and unstimulated CD4+T cells may in part due to thedecreased LNP association (10 fold) seen in unstimulated T cells. There was no significant change in cell viability.

[0255] In contrast, when similar experiments were performed using unstimulated T cells incubated with Formulation mLNP (50% SM-102 ionizable lipid, 10% DSPC, 38.5% β- sitosterol, 1.5% DMG-PEG (compared to an untreated control) packaged with varying amounts of mCherry encoding mRNA, for 72 hours prior to harvest. Cells were then analysed for mCherry expression, and viability using flow cytometry.

[0256] The results shown in Figure 9 indicated that Formulation mLNP was capable of transfecting unstimulated CD4+T cells efficiently. At high doses of mRNA (100-500ng), more than 80% of the unstimulated T cells demonstrated detectable mCherry expression (Figure 9A). This high transfection efficiency was accompanied by some loss in cell viability, though it is notable that at 100ng-500ng mRNA dose, cells remained 60-70% viable, when compared to ~80% viability in the untreated control (Figure 9B). In an independent experiment, testing transfection efficiency using lower doses of mRNA (1.6 - 100ng of mRNA), a dose-dependent transfection efficiency could be observed (Figure 9C). Notably, these lower mRNA doses were accompanied by improvements in cell viability (Figure 9D).

[0257] The data indicated that an LNP formulation comprising SM-102 ionizable lipid, DSPC, β-sitosterol and DMG-PEG provided significantly improved transfection efficiency of unstimulated T cells, over formulations comprising DLin-MC3-DMA ionizable lipid, DSPC, cholesterol and DMG-PEG. Example 5. Potent latency reversal in J-Lat A2 cells

[0258] In light of the data above, the inventors sought to understand whether Formulation mLNP comprising SM-102 ionizable lipid, DSPC, β-sitosterol and DMG-PEG was capable of transfecting a cell that contains an integrated copy of the HIV promoter. Transfection of latently infected cells may be useful for introducing the nucleic acids necessary to induce latency reversal in these cells.

[0259] Formulation mLNP and Formulation Comparator 2 were used to deliver a range of mCherry mRNA doses to Jurkat cells. mCherry expression was determined using flowcytometry after 24 hours. The results indicate that both formulations provided a dose- dependent transfection efficiency, however Formulation mLNP resulted in stronger mCherry fluorescence than Formulation Comparator 2 (Figure 10A).

[0260] Formulation mLNP and Formulation Comparator 2 were used to deliver dCas9- VP64 mRNA, MS2-p65-HSF1 mRNA and HIV-targeting gRNA to J-Lat-A2 cells (Jurkat- derived cell line latently infected with a GFP-tagged HIV-1 strain (Jordan et al., 2003 EMBO J. 22:1868)) to target the GFP-tagged HIV-1 integrated into genome of J-Lat A2. dCas9- VP64 mRNA, MS2-p65-HSF1 mRNA and HIV-targeting gRNA were co-encapsulated into Formulation Comparator 2 (open circles) or Formulation mLNP (closed circles) LNPs at a 1:1:1 ratio and administered to J-Lat A2 cells at various doses. After 24 hours, the cells were analysed for GFP expression using flow cytometry, as a proxy for reactivation of LTR- mediated transcription. Cells that had been transfected with dCas9-VP64 mRNA, MS2-p65- HSF1 mRNA and HIV-targeting gRNA were co-encapsulated into Formulation mLNP, resulted in high proportion (>80%) of GFP-expressing cells (Figure 10B), suggesting potent latency reversal had occurred in J-Lat A2 cells, indicative of high transfection efficiency. In contrast, less than 10% of cells transfected with dCas9-VP64 mRNA, MS2-p65-HSF1 mRNA and HIV-targeting gRNA co-encapsulated into Formulation Comparator 2 demonstrated GFP expression. Example 6: Formulation mLNP provides improved transfection efficiency

[0261] HEK293T cells were transfected with blue fluorescent protein-tagged Cas13b encoding plasmid DNA, and loaded in either Formulation mLNP or Formulation Comparator 3 (MC3 LNP containing equivalent amount of β-sitosterol), Formulation mLNP (SM-102 LNP containing 38.5% β-sitosterol) demonstrated greater delivery of blue fluorescent protein-tagged Cas13b encoding plasmid DNA. Figure 11 shows an example where the cells were treated with equal doses of plasmid-LNP (2 µg / mL) and fluorescent images were taken at 96 hrs post-treatment. Example 7: mLNP efficiently deliver mRNA encoding HIV Tat to reverse HIV latency in human CD4+ T cells from people living with HIV.

[0262] CD4+T cells derived from people living with HIV (PLWH) on suppressive ART were treated with an equivalent dose of 200ng per 105cells Tat mRNA-mLNP or controlmCherry mRNA-mLNP for 72hr. To assess reactivation of transcription ex vivo, the expression of various HIV transcripts was measured to quantify the degree to which blocks in transcription elongation, completion and splicing have been overcome. After a single dose of Tat mRNA-mLNP, all measured HIV transcripts were found to be significantly upregulated compared to non-treated control cells, suggesting treatment with Tat exon 1 delivered by the highly-efficient mLNP formulation is able to induce and overcome blocks in transcription initiation (TAR transcript), proximal and distal elongation (LongLTR and Pol transcripts, respectively), completion of transcription (PolyA transcripts) and splicing (Tat-Rev transcripts) (Figure 12b-f). Treatment with Tat mRNA-mLNP or control mCherry mRNA-mLNP resulted in minimal overall toxicity (Figure 12g).

[0263] The induction seen upon treatment with Tat mRNA-mLNP exceeded that observed in cells stimulated with general T cell mitogens PMA and PHA (considered the golden standard to reverse HIV latency in vitro and ex vivo). However, in contrast to PMA / PHA treatment, Tat mRNA-mLNP did not induce generalized cellular activation as observed by minimal changes to the expression of activation markers CD25, CD69 and HLA-DR compared to non-treated control cells (Figure 12h-j). Example 8: mLNP is capable of delivering CRISPR activation machinery

[0264] The dCas9-synergistic activation mediator (dCas9-SAM) CRISPR activation (CRISPRa) system has been proposed as a robust, HIV-specific latency-reversal agent, but the delivery of this CRISPRa system has been limited to in vitro-based transfection systems with little clinical application. The dCas9-SAM CRISPRa system uses a catalytically inactive (dead) Cas9 (dCas9) fused to a multimer of C-terminal herpes virus transcriptional activation domain 16 (VP64) that is guided to the genomic target site by a gRNA. The gRNA is modified to contain minimal hairpin aptamers that allow the recruitment of p65 and heat shock factor 1 (HSF1) trans-activators through binding of the bacteriophage protein MS2 (Figure 13a). Recruitment of multiple copies of the MS2-p65-HSF1 fusion protein that synergize with dCas9-VP64 has been shown to greatly enhance the potency of CRISPRa (Konermann et al., 2015 Nature 517: 583-588).

[0265] To generate CRISPRa-mLNPs, four hairpin-modified gRNAs (B, C, L and O) (see Table 6) targeting the HIV LTR region upstream of the transcription start site were designedbased on previous work (Zhang et al.2015 Sci. Rep.5: 16277; and Tantale et al.2021 Nat Commun 12: 4503). ingCGG UGC UUU UUingCGG UGC UUU UU

[0266] Using gRNA L, the relative dose of the three RNAs that comprise the dCas9-SAM CRISPRa machinery: an mRNA encoding dCas9-VP64, an mRNA encoding MS2-p65- HSF1 and the gRNA was tested. A relative ratio of 0.8 : 0.00625 : 1 (dCas9-VP64 : MS2- p65-HSF1 : gRNA) yielded a balance between on-target potency and non-specific background reactivation in J-Lat 10.6 cells (Figure 14a). Using the optimized ratio, we formulated CRISPRa-LNPs co-encapsulating the two mRNAs and one of the HIV LTR- targeting gRNAs or a scrambled control gRNA, and found that all four CRISPRa-LNPs could induce LTR-mediated transcription in J-Lat 10.6 cells above scrambled control (Figure 13b). These findings demonstrate that the dCas9-SAM CRISPRa machinery can be co- encapsulated into LNP and activate transcription of a target gene in T cell lines when delivered by mLNP. Example 9: HIV LTR-targeting CRISPRa-LNPs reverse latency in CD4+T cells from PLWH ex vivo

[0267] To assess the potency of HIV LTR-targeting CRISPRa-LNPs ex vivo, CD4+T cells from ART-suppressed PLWH were treated for 72hr with an equivalent dose of 200ng CRISPRa-LNPs per 105cells, analogous to Tat mRNA-LNP treatment in J-Lat cells of Figure 12. We used CRISPRa-LNPs co-encapsulating a 1:1 ratio of gRNA L and gRNA O (CRISPRa-LNP L+O), which was found to exceed the potency of either gRNA alone (Figure 14b) and was hypothesized to increase the sequence coverage of HIV subtype B LTR sequences found in our leukapheresis cohort. In following of Tat mRNA-LNP treatment, CRISPRa-LNP L+O treatment was found to significantly induce the abundance of allmeasured HIV transcripts but multiply-spliced Tat-Rev, which only showed a trend towards increase (Figure 13c-g). The strength of latency-reversal was attenuated compared to Tat mRNA-LNP treatment, with a maximum fold-increase over non-treated of 2.0 (IQR 0.87- 3.39) for Pol transcripts, and did not exceed that of PMA / PHA treatment. Non-specific reactivation of transcription using a scrambled gRNA was not observed. Treatment with CRISPRa-LNPs resulted in comparably minimal toxicity as treatment with Tat or mCherry mRNA-LNPs, and analogously did not result in generalized cellular activation (Figure 13h- k). Example 10: CRISPRa-LNP treatment can be optimized to enhance potency

[0268] To identify bottlenecks and strategies to enhance CRISPRa potency, a model system was developed, in which CRISPRa was targeted to the promotor of an endogenous gene, cd25, to induce overexpression of the CD25 receptor on the cell surface. The CD25-targeting gRNA was validated in Jurkat T cells, which express negligible CD25 at baseline. 24hr treatment with CD25 CRISPRa-LNPs induced surface CD25 expression in up to 30% of cells, while minimal to no background activation was observed with scrambled gRNA CRISPRa-LNP or mCherry mRNA-LNP controls (Figure 15a and Figure 16a). This potency was replicated in non-stimulated HIV-negative CD4+T cells with an average increase in CD25 expression from 8.5% at baseline to 14% after 72hr treatment with CD25 CRISPRa- LNPs (Figure 15b and Figure 16b). Importantly, these findings confirm, to the first of our knowledge, the successful delivery of the dCas9-SAM CRISPRa machinery to non- stimulated T cells in vitro.

[0269] To enhance this activity in primary T cells, we increased the treatment duration of CD25 CRISPRa-LNPs to 6 days and found an increased potency of up to 25% cf.8.7% at baseline (Figure 15c and Figure 16c). This enhanced potency was not the result of an increased transfection efficiency after 6 days of treatment (Figure 16d,e). Example 11: Preparation of lipid nanoparticles for lung targeting

[0270] 10 mM stocks of permanently charged cationic lipid DOTAP, ionizable amino lipid SM-102, helper lipid DSPC, sterol lipid (cholesterol or β-sitosterol), PEG-lipid DMG-PEG, and lipophilic dye (DiD) were dissolved in ethanol (Ethanol phase) at 50 / 25 / 5 / 19.2 / 0.85 / 0.1 molar % ratio, respectively. mRNA was dissolved in acetic buffer, pH 4 (Aqueous phase).At a flow ratio of 1:3 ethanol:aqueous phases and the total flow rate 12 ml / min, the solutions were combined in the microfluidic system NanoAssemblr Ignite (Precision Nanosystems, Vancouver BC). The mixed material was then immediately diluted 1 in 4 with phosphate buffer saline (PBS) after leaving the micromixer outlet. The weight to weight ratio of total lipids to mRNA was set to 20 / 1 for each formulation.

[0271] The particles were washed three times with PBS (pH 7.4) and concentrated using 100 kDa Amicon ultra centrifugal filter units (Merck) at 3,000 × g for 10 min to remove ethanol and ensure complete buffer exchange.

[0272] The particle size and polydispersity index (PdI) of the LNPs were determined by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Panalytical, UK). Samples were diluted in deionized (DI) water and transferred to disposable low volume plastic cuvettes. Samples were analysed at 25 °C with refractive indices of liposomes as materials and water as the bulk diluent to measure the hydrodynamic particle sizes and PdI.

[0273] The RNA encapsulation efficiency was determined using the Quant-it™ RiboGreen RNA Assay Kit (Invitrogen, Waltham, MA, USA).1 µL of LNP were diluted in 1:1000 in TE buffer (10 mM Tris-HCl, 20 mM EDTA).50 µL of the diluted LNPs were then incubated with 50 uL 0.2% Triton™ X-100 (Sigma-Aldrich) or TE buffer in a 96-well fluorescent plate. The plate was incubated for 30 min at room temperature to allow particles to disrupt by 0.2% Triton™ X-100 and liberate RNA before adding 100 μL of RiboGreen® reagent solution in TE buffer (1:2000 v / v) to each well. Fluorescence was measured using FLUOstar Omega (BMG LABTECH, Aylesbury, UK). The RNA concentration was calculated based on ribosomal RNA standard curve. The RNA encapsulation efficiency was calculated by comparing the RNA concentrations in the presence and absence of Triton X-100. The formula used was: RNA encapsulation % = (Encapsulated RNA-Free RNA) / (Total RNA concentration) × 100.

[0274] Two formulations of lung-targeting LNPs in accordance with the present invention, and two comparative formulations, were prepared as shown in Table 7.Table 7. LNP formulations ive n 5 ) ) ol %) G)

[0275] The LNPs were formulated using Nanoassemblr Ignite microfluidic devices at settings shown in Table 2 of Example 1.

[0276] Formulations 1 and 2, and Comparative Formulations 3 to 4, were loaded with Luciferase mRNA. The physicochemical properties of Formulation 1 and Formulation 2 loaded with Firefly Luciferase mRNA are shown in Table 8.Table 8. Physicochemical properties of Formulation 1 and Formulation 2 loaded with Firefly Luciferase mRNA 02 2Example 12: In vivo study of lung-targeting

[0277] Formulation 1, Comparative Formulation 3 and Comparative Formulation 4, loaded with Luciferase mRNA and labelled with lipophilic dye (DiD), were injected intravenously into mice at the dose 3 µg / mouse (100 µL).100 µL of PBS was injected as a control. The mice were incubated for 7 h prior to sacrifice and organ harvesting. Liver, lung, spleen, kidney, heart and brain were harvested and the DiD signal (LNP distribution) and Fluc (LNP function) were monitored on IVIS imaging system.

[0278] Formulation 1, Comparative Formulation 3 and Comparative Formulation 4 showed accumulation in the liver, but only Comparative Formulation 3 induced expression of luciferase in the liver (Figure 18). Similarly, Formulation 1, Comparative Formulation 3 and Comparative Formulation 4 showed accumulation in the spleen, but only Comparative Formulation 3 induced expression of luciferase in the spleen (Figure 18).

[0279] Only the DOTAP-containing LNPs (Formulation 1 and Comparative Formulation 4) were accumulated in the lungs. Notably, only mice dosed with Formulation 1 showed bright luciferase signal in the lungs (Figure 18).

[0280] Partial accumulation of tested LNP formulations was observed in the kidneys, whilst the heart and brain remained untouched by the LNPs. However, no expression of luciferase was observed in these organs.Example 13: In vivo study of Formulation 2

[0281] Formulation 2 and Comparative Formulation 5 loaded with Luciferase mRNA and labelled with lipophilic dye (DiD) were injected intravenously into mice at the dose 3 µg / mouse (100 µL). 100 µL of PBS was injected as a control. The mice were incubated for 7 h prior to sacrifice and organ harvesting. Liver, lung, spleen, kidney, heart and brain were harvested and the DiD signal (LNP distribution) and Fluc (LNP function) were monitored on IVIS imaging system (Figure 19).

[0282] Like Formulation 1, Formulation 2 was localised in the spleen, liver and lungs (DiD signal) but luciferase was selectively expressed in the lungs only (Figure 20).

[0283] Comparative Formulation 5 were localised and functioned in two organs (spleen and liver) and showed no effect in the lungs (Figure 20). Example 14: Cellular distribution in lung tissue following intravenous delivery of LNPs

[0284] The mRNA delivery efficacy following intravenous administration of DOTAP- SM102 Formulation 1 was examined. The Ai9 mouse model was used for this purpose. The Ai9 strain is a Cre reporter tool strain that has a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven red fluorescent protein variant (tdTomato) - all inserted into the Gt(ROSA)26Sor locus. Ai9 mice would express robust tdTomato fluorescence following Cre-mediated recombination.

[0285] Cre recombinase encoding mRNA was encapsulated into DiD-labelled LNPs and administered by IV at a dosage of 0.25 mg / kg. Initial lung-specific tdTomato expression, driven by Cre activity, was validated using IVIS. There was no expression of tdTomato in the liver and spleen (Figure 21A), nor in other organs, including the kidneys, heart, and brain.

[0286] Upon lung sectioning, tdTomato expression was observed in nearly all alveolar cells (Figure 21B). The substantial tdTomato expression in lung epithelial cells indicates the effective penetration of LNPs across blood vessels, facilitating the entry and mRNA delivery to airway epithelial cells (Figure 21B).References 1. Ahlenstiel et al.2020 Frontiers in Cellular and Infection Microbiology 10: 424. 2. Ali et al.2016 J. Virol.90, 6999-7006. 3. Ali et al.2021 ACS Nano 15, 3736-3753. 4. Anthony-Gonda, K. et al.2019 Sci. Transl. Med.11. 5. Bialek, J. K. et al.2016 PLoS One 1: e0158294-e0158294 6. Cameron et al.2010 PNAS 107(39):16934-9. 7. da Costa et al.2022 Retrovirology 19: 12. 8. Das et al.2019 Curr. Opin. Virol.38: 81-88. 9. Dash et al.2019 Nat. Commun.10: 2753-2753. 10. Ji et al.2016 Mol. Ther.24: 508-521. 11. Jin et al.2019 mBio 10. 12. Kaminski et al.2016 Sci. Rep.6: 22555. 13. Kessing et al.2017 Cell Rep.21: 600-611. 14. Klinnert et al.2022 J. Gen. Virol.103: 1-10. 15. Konermann, S. et al.2015 Nature 517, 583. 16. Lebbinket al.2017 Sci. Rep.7, 41968. 17. Limsirichai et al.2016 Mol. Ther.24: 499-507. 18. Liu. et al.2021 J. Clin. Invest.131. 19. Mediouni et al.2019 FASEB J.33: 8280-8293. 20. Nguyen et al.2020 Viruses 12 21. Olson et al.2021 Viruses 13.3107-3107. ci.15(5):437-446. . . :727-8. 03. -1247. :219-224. leic Acids 21:147-155. Virology 38, 1-9. s 11: 78.

Claims

CLAIMS 1. A lipid nanoparticle (LNP) for delivering an agent to a transfection-recalcitrant cell, wherein the LNP comprises: a. an ionizable lipid; and b. a sterol, wherein the sterol comprises less than 10 mol% cholesterol; wherein the ionizable 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 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; 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; R9is selected from the group consisting of H, CN, NO2, C1-6alkyl, -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 C1-18alkyl, 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 C1-12alkyl 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.

2. The LNP according to claim 1, wherein the ionizable 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'; 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 C6-14aryl 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 C1-3alkyl, C2-3alkenyl 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 C3-14alkyl 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.

3. The LNP according to claim 2, 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-, an aryl group, and a 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, C2-6alkenyl, C3-6carbocycle 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.

4. The LNP according to claim 2, 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 C1-3 alkyl and unsubstituted C1-3alkoxy; and R' is a C1-18 linear alkyl, optionally substituted with one or more substituents selected from halo, OH and unsubstituted C1-3 alkoxy.

5. The LNP according to claim 1, wherein the ionizable lipid has a structure of Formula (II):or a salt, solvate or isomer thereof, wherein: p is an integer from 1 to 5; M1is 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 C1-3alkyl, C2-3alkenyl 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- 12 alkenyl; and each Y is independently a C3-6carbocycle.

6. The LNP according to claim 5, 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-C12linear alkyl.

7. The LNP according to claim 1, wherein the ionizable lipid has a structure of Formula (IIa), (IIb), (IIc) or (IId):or a salt, solvate or isomer thereof.

8. The LNP according to claim 1, wherein the ionizable lipid has a structure of Formula (IIe):or a salt, solvate or isomer thereof, wherein n is 2, 3 or 4.

9. The LNP composition according to claim 1, wherein the ionizable lipid has a structure of Formula (IId):or a salt, solvate or isomer thereof.

10. The LNP according to claim 1, wherein the ionizable lipid is selected from the group consisting of: , ,,or a salt, solvate or isomer thereof.

11. The LNP according to any one of claims 1 to 10, wherein the ionizable lipid is SM- 102:or a salt, solvate or isomer thereof.

12. The LNP according to any one of claims 1 to 11, wherein the sterol comprises about 38.5 mol% of the LNP composition.

13. The LNP according to any one of claims 1 to 12, 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.

14. The LNP to any one of claims 1 to 13, wherein the sterol is a phytosterol.

15. The LNP according to claim 14, 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.

16. The LNP according to claim 14 or claim 15, wherein the phytosterol is a C-24 alkyl phytosterol having a structure of Formula (III):(III) or a stereoisomer thereof, wherein: each is individually a single bond or a double bond; and R is a C1-6 alkyl.

17. The LNP according to claim 16, wherein R is a C1-4alkyl, C1-3alkyl, or C1-2alkyl.

18. The LNP according to claim 16 or claim 17, wherein R is a C1-2alkyl.

19. The LNP according to any one of claims 16-18, wherein the C-24 alkyl phytosterol is selected from the of ,,, , , and any combination thereof.

20. The LNP according to claim 19, wherein the C-24 phytosterol is β-sitosterol:

21. The LNP of any one of claims 1-20, wherein the sterol or phytosterol consists of β- sitosterol.

22. The LNP according to any one of claims 1 to 21, further comprising a phospholipid.

23. The LNP according to claim 22, 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).

24. The LNP of claim 23, wherein the phospholipid is DSPC.

25. The LNP according to any one of claims 1 to 24, further comprising a PEG lipid.

26. The LNP of claim 25, 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 or combinations thereof.

27. The LNP according to claim 26, wherein the PEG lipid is PEG-DMG.

28. The LNP according to any one of claims 1 to 27 comprising SM-102, β-sitosterol, DSPC and DMG-PEG.

29. The LNP according to any one of claims 1 to 28, 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.

30. The LNP according to any one of claims 1 to 29, wherein the composition does not comprise cholesterol.

31. The LNP according to any one of claims 1 to 30, comprising about 50 mol% SM- 102, about 10 mol% DSPC, about 38.5 mol% β-sitosterol and about 1.5 mol% DMG- PEG.

32. A composition comprising an LNP according to any one of claims 1 to 31 and an agent to be delivered to a cell.

33. The composition according to claim 32, wherein the agent is protein, a small- molecule drug, or a nucleic acid.

34. The composition according to claim 33, wherein the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

35. The composition according to claim 34, 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.

36. The composition n according to claim 35, wherein the RNA is an mRNA.

37. The composition of claim 36, wherein the mRNA encodes for a CRISPR-associated protein.

38. The composition of claim 37, wherein the mRNA encodes for a dCas9-SAM protein.

39. The composition according to claim 36, wherein the RNA is a gRNA.

40. The composition of claim 40, wherein the gRNA is an HIV LTR-targeting gRNA.

41. A method of delivering an agent to a cell, the method comprising contacting the cell with the composition according to any one of claims 32 to 40.

42. A method of expressing an exogenous nucleic acid in a cell, the method comprising contacting the cell with the composition according to claim 32-40, wherein the agent is an exogenous nucleic acid.

43. The method according to claim 41 or claim 42, wherein the cell is a transfection- recalcitrant cell.

44. The method according to claim 43, wherein the transfection-recalcitrant cell is a quiescent cell, a primary cell, an immune cell, a stem cell or a neuronal cell.

45. The method according to claim 43 or claim 44, wherein the transfection-recalcitrant cell is a T-cell.

46. The method according to claim 45, wherein the T cell is an unstimulated or a resting T cell.

47. The method according to claim 43 or claim 43, wherein the transfection-recalcitrant cell is a Natural Killer cell.

48. A method of treating or preventing a disease in a subject, the method comprising administering to the subject an effective amount of the composition according to any one of claims 32 to 40, wherein the composition is a pharmaceutical composition and the agent is a therapeutic agent capable of treating or preventing the disease.

49. The method of claim 48, 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.

50. The method of claim 49, wherein the HIV infection is a latent HIV infection.

51. The method according to any one of claim 48 to 50, wherein the agent is a protein, a small-molecule drug, or a nucleic acid.

52. The method according to claim 51, wherein the nucleic acid is a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).

53. The method according to claim 52, 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.

54. The method of claim 52 or claim 53, wherein the nucleic acid is a guide RNA and / or encodes for a CRISPR-associated protein.

55. The method of claim 54, wherein the guide RNA is an HIV LTR-targeting gRNA.

56. The method of claim 54, wherein the CRISPR-associated protein is a dCas9-SAM protein.

57. Use of the composition according to any one of claims 32 to 40 in the manufacture of a medicament for treating or preventing a disease in a subject, wherein the composition is a pharmaceutical composition and the agent is a therapeutic agent capable of treating or preventing the disease.

58. The use of claim 57, 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.

59. A lipid nanoparticle (LNP) for delivering a therapeutic agent to a lung tissue, the LNP comprising an ionizable lipid and a quaternary ammonium lipid, wherein the ionizable lipid is as defined in claim 1.

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