RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/347,051 filed on May 31 , 2022, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to lipid nanoparticles and more particularly to lipid nanoparticles that deliver RNA to a subject.

BACKGROUND OF THE INVENTION

Many cancers show metastasis and poor clinical outcome due to drug resistance: around 50-60% of colorectal cancer (CRC) patients develope unresectable liver and lung metastases and respond poorly to chemo/targeted therapy ¹ ; Nearly 20% of castration-resistant prostate cancer (CRPC) patients become resistant to androgen signaling targeted therapy and acquire a more aggressive metastatic neuroendocrine PCa (NEPC) phenotype, more than 80% of which present with liver metastasis and with a median survival of less than one year from diagnosis ²³ . Hence, there is an urgent need to identify novel druggable targets and new therapeutic modalities beyond traditional chemotherapy.

RNA platforms are an exciting addition to the toolkit of targeted gene therapies. They function through the delivery of curative RNAs to the cell cytosol, where they regulate expression level of critical disease-related RNA transcripts or proteins. ⁴⁵ This delivery is mediated by vehicles that protect RNAs from their otherwise rapid degradation in viva, Lipid nanoparticles (LNP) are one such vehicle. LNPs protect RNAs by housing RNAs within their core while circulating through the body. Upon reaching cells, LNPs are uptaken into endosomes, where they dissociate and subsequently release RNA into cytosols. This stabilization of LNPs has yielded clinical success in delivering siRNA (Onpattro®, 2018), ⁶ mRNA (COVID vaccines, 2020), ⁷ and CRISPR gene editing tools (Phase I trial, 2021). ⁸ However, current LNPs are still bottlenecked by extremely low RNA endosomal escape efficiency (e.g. 1-2% for siRNA delivered) after LNP endocytosis. ^{9 10} Consequently, >95% of RNAs are degraded in the lysosomes or being exocytosed, ¹¹ severely impairing their therapeutic efficacy. Thus, novel LNP systems are needed that boost RNA cytosolic delivery and improve RNA therapeutics to reach their potential.

Scavenger receptor class B type I (SR-B1) is an integral membrane glycoprotein receptor that plays a crucial role in the metabolism of high-density lipoprotein (HDL) and permits the direct cytosolic influx of the lipid core with no corresponding lysosomal degradation. ¹² SR-B1 is upregulated in many cancer cell lines. ¹³ For example, SR-B1 high expression was observed in PCa versus benign prostate, as well as in NEPC versus CRPC, ¹⁴ and elevated SR-B1 has been associated to PCa aggressiveness. ¹⁵ Besides, increased SRB1 expression in liver metastasis from CRC and breast cancer have also been reported. ^{16 17} Due to its upregulation in many cancer cell lines and capacity to induce cytosolic cargo delivery, SRBI becomes a promising surface receptor for targeted cancer therapy with RNA interference. Previously, our lab reported that by incorporating Apolipoprotein A-1 (ApoA-1) mimetic peptide R4F into HDL-like nanoparticles, enhanced cytosolic siRNA delivery and in vivo oncogene knockdown have been observed ^{18 19} . However, this delivery platform is limited by extra cholesterol modification on siRNA to enable siRNA incorporation onto nanoparticle’s lipid membrane via cholesterol overhang, which makes hydrophilic siRNA double strands facing outwards of nanoparticle surface, thus less protected and highly detachable during in vivo circulation.

SUMMARY OF THE INVENTION

In an aspect, there is provided a lipid nanoparticle for the delivery of RNA to a subject, the lipid nanoparticle comprising: (a) at least one phospholipid; (b) an ionisable or cationic lipid; (c) a PEG-lipid; (d) at least one peptide, the peptide comprising an amino acid sequence capable of forming at least one amphipathic a-helix; and (e) the RNA; wherein the components a), b), c), d) and e) associate to form the lipid nanoparticle.

In a further aspect, there is provided a method of delivering RNA to a subject, the method comprising administering to the subject the lipid nanoparticle described herein. In a further aspect, there is provided the lipid nanoparticle described herein, for use in the delivery of RNA to a subject.

In a further aspect, there is provided a use of the lipid nanoparticle described herein, in the preparation of a medicament for treating a disease or condition, wherein the RNA treats said disease or condition. In a further aspect, there is provided a use of the lipid nanoparticle described herein, in the preparation of a medicament for treating a disease or condition wherein the lipid nanoparticle treats said disease or condition.

In a further aspect, there is provided a pharmaceutical composition comprising the lipid nanoparticle described herein in a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF FIGURES

These and other features of the preferred embodiments of the invention will become more apparent in the following detailed description in which reference is made to the appended drawings wherein:

Figure 1 shows (A) Schematic illustration of siRNA encapsulation, LNP synthesis and R4F modification. (B) Transmission electronic microscopy images of R4F-LNP. (C) Number-based size distribution of R4F-LNP measured by dynamic light scattering. (D) Circular dichroism spectrum of conventional LNP and R4F-LNP, affirming the secondary structures assembled on the R4F-LNP.

Figure 2 shows (A) Intracellular delivery profile of R4F-LNP and conventional LNP in PC3-Luc6 at 6 and 24 h post-incubation. R4F-LNP displayed stronger cytosolic distribution of siRNA signal. Fluorescence was pseudocolored in green for FAM- labelled siRNA, Magenta for porphyrin-lipid. Scale bar: 20 pm. (B) Intracellular delivery profile of R4F-LNP in Idl(mSR-BI) (high SR-B1 expressing, top panel) cells and ldlA-7 (low SR-B1 expression, bottom panel) cells. R4F-LNP were more effectively uptaken by SR-B1 (+) vs SR-B1 (-) cells. Fluorescence was pseudocolored in blue for Hoechst 33342, green for FAM-labelled siRNA, and Magenta for porphyrin-lipid. Images on the most right hand are the overlay of previous channels. Scale bar: 40 pm.

Figure 3 shows in vitro luciferase knockdown by R4F-LNP and conventional LNP. (A) PC3-Luc6 cells were seeded in 96-well plates and treated for 48 hours with the indicated doses of siLuc or siCtrl in different formulations. The bioluminescence was captured by Xenogene imaging system. (B) Normalized bioluminescence expression from each treatment group based on cell viability. **: p < 0.0021 ; ****: p < 0.0001. (C) Viability of cells treated by each formulation.

Figure 4 shows cell uptake of R4F-LNP and convention LNP measured by flow cytometry. PC3-Luc6 were incubated with both formulations for 6 and 24 h before analyzed by flow cytometer for single cell fluorescence from porphyrin-lipid (A) and FAM-siRNA (B). After 24 h incubation, no significant difference in cellular uptake was observed between formulations.

Figure 5 shows (A) Experimental design of R4F-LNP siRNA treatment in mouse liver metastasis model. (B) Representative images of the NOD/SCID mouse livers harvested 16 days after treatment with R4F-LNP loaded with different sequences. Quantification of the liver weight at the experimental endpoint in mouse liver metastasis model was shown on the right.

DETAILED DESCRIPTION

In the following description, numerous specific details are set forth to provide a thorough understanding of the invention. However, it is understood that the invention may be practiced without these specific details.

In this study, utilizing siRNA as a model RNA therapeutics, we developed a novel peptide-functionalized lipid nanoplatform which facilitates RNA cytosolic delivery through SRBI-mediated uptake pathway to address the RNA endosomal escape challenge of LNP. We incorporated ApoA-1 mimetic R4F peptide (Ac- FAEKFKEAVKDYFAKFWD) into the FDA-approved LNP formulation to enable clinically used LNP formulation with cytosolic siRNA delivery functions without changing its high, stable siRNA loading capacity (R4F-LNP; Figure 1A). The RNAs are encapsulated in the core of lipid nanoparticles and further shielded by a R4F peptide network thereby being well protected during in vivo circulation. We demonstrated that R4F-LNP improved siRNA delivery over original LNP formulation with a 3~4-fold enhancement in luciferase reporter gene knockdown efficacy. Confocal microscopy imaging revealed that R4F-LNP significantly altered siRNA intracellular delivery profile, showing a transition from classic organelle-oriented endocytosis to direct cytosolic distribution. Furthermore, we have demonstrated the therapeutic potency of R4F-LNP in an in vivo colorectal cancer (CRC) liver metastasis model by targeting a novel CRC gene target we identified recently. ²⁰ Overall, this R4F-LNP holds great potential to enhance the efficacy of RNA therapeutics by delivering nucleic acids directly to cell cytosols which provides an effective therapeutic option for undruggable disease targets.

In an aspect therefore, there is provided a lipid nanoparticle for the delivery of RNA to a subject, the lipid nanoparticle comprising: at least one phospholipid; an ionisable or cationic lipid; a PEG-lipid; at least one peptide, the peptide comprising an amino acid sequence capable of forming at least one amphipathic a-helix; and the RNA; wherein the components a), b), c), d) and e) associate to form the lipid nanoparticle.

Suitable scaffold peptides may be selected from the group consisting of Class A, H, L and M a-helices or a fragment thereof. Suitable scaffold peptides may also comprise a reversed peptide sequence of the Class A, H, L and M amphipathic a-helices or a fragment thereof, as the property of forming an amphipathic a-helix is determined by the relative position of the amino acid residues within the peptide sequence.

In one embodiment, the scaffold peptide has an amino acid sequence comprising consecutive amino acids of an apolipoprotein, preferably selected from the group consisting of apoB-100, apoB-48, apoC, apoE and apoA.

The "amino acids" used in this invention, and the term as used in the specification and claims, include the known naturally occurring protein amino acids, which are referred to by both their common three letter abbreviation and single letter abbreviation. See generally Synthetic Peptides: A User's Guide, G A Grant, editor, W.H. Freeman & Co., New York, 1992, the teachings of which are incorporated herein by reference, including the text and table set forth at pages 11 through 24. As set forth above, the term "amino acid" also includes stereoisomers and modifications of naturally occurring protein amino acids, non-protein amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, constructs or structures designed to mimic amino acids, and the like. Modified and unusual amino acids are described generally in Synthetic Peptides: A User's Guide, cited above; Hruby J, Al-obeidi F and Kazmierski W: Biochem J 268:249-262,1990; and Toniolo C: Int J Peptide Protein Res 35:287-300,1990; the teachings of all of which are incorporated herein by reference.

"Alpha-helix" is used herein to refer to the common motif in the secondary structure of proteins. The alpha helix (a-helix) is a coiled conformation, resembling a spring, in which every backbone N-H group donates a hydrogen bond to the backbone C=O group of the amino acid four residues earlier. Typically, alpha helices made from naturally occurring amino acids will be right handed but left handed conformations are also known.

“Amphipathic” is a term describing a chemical compound possessing both hydrophilic and hydrophobic properties. An amphipathic alpha helix is an often-encountered secondary structural motif in biologically active peptides and proteins and refers to an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix.

Examples of small amphipathic helix peptides include those described in WO 09/073984.

Methods for detecting and characterizing protein domains with putative amphipathic helical structure are set forth in Segrest, J. P. et al. in PROTEINS: Structure, Function, and Genetics (1990) 8:103-117, the contents of which are incorporated herein by reference. Segrest et al. have identified seven different classes of amphipathic helices and have identified peptides/proteins associated with each class. Of the seven different classes there are four lipid-associating amphipathic helix classes (A, H, L, and M). Of these, Class A, the designated apolipoprotein class, possesses optimal properties for forming phospholipid-based particles.

As used herein, “phospholipid” is a lipid having a hydrophilic head group having a phosphate group and hydrophobic lipid tail.

PEG-lipid or PEG lipid is also known as PEGylated lipid, and is a class of PEG derivatives that is attached with lipid moiety such as, but not limited to, DMG, DSPE, DPPE, or DMPE.

In some embodiments, the lipid nanoparticle is a monolayer particle comprising the phospholipid, the PEG-lipid and the peptide having encapsulated therein the RNA and ionisable lipid. In some embodiments, the at least one amphipathic a-helix or peptide is between 6 and 30 amino acids in length. Preferably, the at least one amphipathic a-helix or peptide is between 8 and 28 amino acids in length, between 10 and 24 amino acids in length, between 11 and 22 amino acids in length, between 14 and 21 amino acids in length, between 16 and 20 amino acids in length, or 18 amino acids in length.

In some embodiments, the peptide is 2F, 4F, the reverse sequence of 2F, or the reverse sequence of 4F.

In some embodiments, the total lipid to peptide molar ratio is 0.5-10%.

In some embodiments, the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidic acid, phosphatidylglycerols and combinations thereof. Preferably, the phospholipid is selected from the group consisting of 1 ,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1 ,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), 1 ,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 ,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC), 1 ,2-diarachidoyl-sn- glycero-3-phosphatidylcholine (DAPC), 1 ,2-dilignoceroyl-sn-glycero-3- phosphatidylcholine(DLgPC), 1 ,2-dipalmitoyl-sn-glycero-3-[phosphor-rac-(1 -glycerol)] (DPPG) and combinations thereof.

In some embodiments, a headgroup of the ionisable or cationic lipid comprises an amine, guanidine or heterocyclic group. Preferably, the headgroup is a primary amine, secondary amine, tertiary amine, quarternary amine, guanidine, pryidinium or imdazolium.

In some embodiments, the ionisable or cationic lipid comprises 1-4 hydrophobic tails that are independently saturated or unsaturated.

In some embodiments, the ionisable or cationic lipid comprises a linker between the headgroup and tail comprising an ether, carbomate, ester, amide, disulfide, thiol, ketal, phosphate or urea.

In some embodiments, the ionisable or cationic lipid is L319, YSK12-C4, CL4H6, SM- 102, ALC-0315, Arcturus 10q, DLin-DMA-MC3, or ssPalmO-Phe. In some embodiments, the ionisable lipid is present at a 20-90% molar ratio in the lipid nanoparticle.

In some embodiments, the PEG-lipid has a carbon chain length of C14-C22.

In some embodiments, the PEG-lipid has a molecular weight ranging from about 1000 to about 5000.

In some embodiments, the PEG-lipid is 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG2000), N-(methoxypolyethylene glycol 5000 carbamoyl)-1 ,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000- DPPE), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(p olyethylene glycol)-2000 (DMPE-PEG2000), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy(polyethylene glycol)-2000 (DSPE-PEG2000), Polyoxyethylene 40 stearate (PEG40S) or combinations thereof.

In some embodiments, the PEG-lipid is present at a 0.5-10% molar ratio in the lipid nanoparticle

In some embodiments, the RNA is a therapeutic RNA. Preferably, the RNA is a siRNA, mRNA, shRNA, miRNA, tRNA, circRNA or saRNA.

In some embodiments, the lipid nanoparticle further comprises a sterol, a sterol ester, or combinations thereof. Preferably, the sterol or a sterol ester is cholesterol, cholesterol oleate or an unsaturated cholesterol-ester.

In some embodiments, the lipid nanoparticle comprises Dlin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000.

In some embodiments, the lipid nanoparticle further comprises a targeting or homing molecule.

“Targeting molecule” is any molecule that can direct the nanovesicle to a particular target, for example, by binding to a receptor or other molecule on the surface of a targeted cell. Targeting molecules may be proteins, peptides, nucleic acid molecules, saccharides or polysaccharides, receptor ligands or other small molecules. The degree of specificity can be modulated through the selection of the targeting molecule. For example, antibodies typically exhibit high specificity. These can be polyclonal, monoclonal, fragments, recombinant, or single chain, many of which are commercially available or readily obtained using standard techniques.

In some embodiments, the lipid nanoparticle further comprises a porphyrinphospholipid conjugate.

In some embodiments, in the lipid nanoparticle is 20-70 nm in diameter, 30-60 nm in diameter or 40-50 nm in diameter.

In a further aspect, there is provided a method of delivering RNA to a subject, the method comprising administering to the subject the lipid nanoparticle described herein.

In a further aspect, there is provided the lipid nanoparticle described herein, for use in the delivery of RNA to a subject.

In a further aspect, there is provided a use of the lipid nanoparticle described herein, in the preparation of a medicament for treating a disease or condition, wherein the RNA treats said disease or condition.

In a further aspect, there is provided a pharmaceutical composition comprising the lipid nanoparticle described herein in a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier ¹ ' means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the pharmacological agent.

As used herein, “therapeutically effective amount' refers to an amount effective, at dosages and for a particular period of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the pharmacological agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the pharmacological agent to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the pharmacological agent are outweighed by the therapeutically beneficial effects.

The advantages of the present invention are further illustrated by the following examples. The examples and their particular details set forth herein are presented for illustration only and should not be construed as a limitation on the claims of the present invention.

EXAMPLES

Methods and Materials

Materials: 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and 1 ,2- dimyristoyl-rac-glycero3-methoxy(poly(ethylene glycol))-2000 (DMG-PEG2000), were purchased from Avanti Polar Lipids (Alabaster, AL, USA). DLin-MC3-DMA was purchased from Nanosoft Polymers (NO, USA). The ApoA-1 mimetic R4F peptide, Ac- FAEKFKEAVKDYFAKFWD, was purchased from GL Biochem Ltd. (Shanghai, China). Porphyrin-lipid was synthesized by the previously reported methods. ²¹

Small Interfering RNA: Luciferase targeting siLuc and scrambled siCtrl were both purchased from Horizon Discovery (USA). siLuc (sense, 5'-GAU UAU GUC CGG UUA UGU AdTsdT-3'; antisense, 5'-UAC AUA ACC GGA CAU AAU CdTsdT-3'). FAM-siLuc (sense, 5'-FAM-GAU UAU GUC CGG UUA UGU AdTsdT-3'; antisense, 5'-UAC AUA ACC GGA CAU AAU CdTsdT-3'). siCtrl (sense, 5’-UUC UCC GAA CGU GUC ACG UdTsdT-3’; antisense, 5’-ACG UGA CAC GUU CGG AGA AdTsdT-3’). siRNA targeting ARHGEF2 was purchased from GenePharma (Shanghai, China). siARHGEF2 (sense, 5’-GGA UCU ACC UGU CAC UAC Utt-3’; antisense, 5’- AGUAGUGACAGGUAGAUCCag-3’).

Synthesis and Characterization of R4F-LNP: R4F-LNP was synthesized via a two- step process: 1) siRNA-loaded LNP formulations were formed using microfluidic rapid mixing method as previously reported. ²² Lipids were mixed in ethanol at a molar ratio of DLin-MC3-DMA I DSPC I Cholesterol I DMG-PEG2000 = 50/10/38.5/1.5. siRNA was dissolved in 25mM sodium acetate buffer (pH=4.0). The two phases were mixed through herringbone microfluidic chips (microfluidic ChipShop, Germany) at a volumetric flow rate ratio of 3:1 (aqueous to ethanol). The mixed solution was dialyzed against PBS 7.4 overnight. Afterwards LNPs were passed through 0.22 um filter and concentrated using centrifuge. For imaging studies, DSPC in the formulation was fully replaced by porphyrin-lipid and siRNA was fluorescein-labelled siRNA was used. Formulations were prepared with the same method as described above. 2) R4F- peptide was dissolved in PBS and added dropwise into pre-formed LNP solutions under gentle shaking at a lipid/R4F ratio of 3:1. The mixture was kept at 4 °C overnight. Next, the solution was centrifuged at 12000 rpm for 20 min and filtered with 0.22 um filter before use. The hydrodynamic size and dispersity of R4F-LNP was characterized with a Zetasizer Nano ZS (Malvern Instruments). The morphology of R4F-LNP was checked by Hitachi HT7800 electron microscopy with 2% uranyl acetate negative staining. siRNA encapsulation efficiency was measured by Ribogreen Assay based on manufacture’s protocol (Thermofisher). The circular dichroism spectrum was measured with Jasco J-815 CD spectrophotometer (Jasco, Easton, MD). Formulations diluted in PBS was subjected to scanning from 250 nm to 190 nm at 0.1 nm data pitch with background subtracted.

Cell Culture: PC3-luc6 cells were purchased from Caliper LifeSciences and cultured in Ham's F-12K (Kaighn's) Medium (supplemented with 10% FBS). Chinese hamster ovary (CHO) Idl(mSR-BI) and ldlA-7 cells were gifts from Dr. Monty Krieger (Massachusetts Institute of Technology, Cambridge, MA). ldlA-7 cells were cultured in Hams F-12 medium (Gibco) supplemented with penicillin-streptomycin (1 v/v %), FBS (5 v/v%), and L-glutamine (2 mM). Idl(mSR-BI) cells were cultured under similar conditions as ldlA-7 with the addition of 300 ug/mL of G418 Geneticin. HCT116 were purchased from ATCC and cultured in McCoy's 5a Medium Modified with 10% FBS. All cell cultures were maintained in a 37 °C humidified incubator under 5% CO ₂ .

Confocal Microscopy and Cell-Uptake Studies: For confocal imaging studies, PC3- Iuc6, Idl(mSR-BI) and ldlA-7 cells were seeded into 8-well coverglass-bottom chambers (Nunc LabTek, Sigma-Aldrich, Rochester, NY) at a cell-seeding density of 2 x 10 ⁴ cells per well. After 48 h of incubation, R4F-LNP or pyro-LNP were added at a concentration of 4 pM based on porphyrin and incubated for 6 and 24 h. Cells were washed twice with culturing medium before imaging. Fluorescence images were captured by Stimulated emission depletion (STED) microscopy (Leica, Germany) using a 63x oil objective lens. Customized filter settings were used to collect signal from porphyrin-lipid (excitation: 660 nm; emission: 670 nm - 765 nm), and FAM-labelled siRNA (excitation: 488 nm; emission: 507-580 nm). Laser power and detector gain adjustment were kept consistent between the time points. For flow cytometry experiments, PC3-Luc6 cells were seeded at 4 x 10 ⁴ cells per well into 24-well plates for 48 h. Then cells were incubated with R4F-LNP or pyro-LNP at a concentration of 50 nM siRNA (1.6 pM porphyrin) for 6 or 24 h. Afterwards, the treated cells were washed and centrifuged before analyzed by cytoFLEX S (Beckman Coulter, USA). Porphyrin fluorescence was collected at APC channel while FAM fluorescence was collected at FITC channel.

In Vitro Luciferase Knockdown: PC3-Luc6 cells were seeded at 4 x 10 ³ cells per well into 96-well plates for 48 h. Then cells were incubated with R4F-LNP or pyro-LNP at different siRNA concentration for 48 h, after which the cells were washed twice with culturing medium and replaced with medium that contains 0.5 mg.mL ^-1 alamarBlue (Invitrogen) for viability measurement: cells were incubated for 2 h, after which fluorescence emission was collected using a CLARIOstar microplate reader (BMG LABTECH) (excitation of 540/8 nm and emission of 590/8 nm). Luciferase expression of PC3-Luc6 on the same plate was evaluated through bioluminescence: after alamarBlue assay, 5 pL D-luciferin solution (25 mM) was added into each well (100 pL medium), after which the bioluminescence was collected by IVIS Spectrum In Vivo Imaging System (PerkinElmer). Bioluminescence intensity of each well was further normalized by its viability before analysis.

In Vivo Liver Metastasis Treatment: The mouse model of liver metastasis was established by intrasplenic injection of HCT116 cells into NOD/SCID mouse. In the intrasplenic injection model, enrofloxacin in the drinking water as a prophylactic oral antibiotic was administered to mice 72 h prior to surgery. Mice were anesthetized with Buprenorphine Sustained-Release (SR). ~1 cm incision was made in the left upper abdominal wall and ~1 cm incision was made in the peritoneum to expose the mouse spleen. Moistened sterile cotton swab was used to gently exteriorize the spleen. HCT116 cells (0.75x10 ⁶ cells per mouse) were injected into each mouse with a 27G needle. After the spleen was returned to the abdominal cavity, the muscle layer and skin were closed, and subcutaneous fluid therapy was administered. The mice were sacrificed 3~4 weeks after intrasplenic injection. The mouse liver was excised and fixed for histological examination. The liver metastasis was assessed either by the number of visible liver metastatic nodules or the liver weight and the proportion of tumor metastases in the mouse liver when tumor nodules were indistinguishable. Results and Discussion

Synthesis and Characterization of R4F-LNP. R4F-LNP was formulated by coating ApoA-1 mimetic peptide R4F onto the preformed LNP following a process illustrated in Figure 1A. Briefly, LNP nanoparticles were prepared through rapid mixing of siRNA and lipid components via a microfluidic system, followed by raising pH from 4.0 to 7.4 with dialysis process. The R4F peptides were then added dropwisely into the preformed LNPs in PBS condition with a R4F/lipid ratio of 1 :3 and subsequently incubated at 4°C overnight to form stable R4F-LNPS. The morphology of R4F-LNP was disclosed by transmission electronic microscopy imaging (Figure 1 B), showing spherical structures with homogenous size distribution at ~50 nm. The size of R4F- LNP was also confirmed by dynamic light scattering measurement, showing a monodispersed peak at 45.22 ± 2.18 nm, with a polydispersity index (PDI) of 0.146 (Figure 1C). The size of R4F-LNP is in line with that of the LNP (~ 40-50 nm) before adding R4F, ²³ suggesting a surface coating of R4F doesn’t change the particles size. Moreover, the circular dichroism (CD) spectra demonstrated a significantly enhanced signature peak in 200 -230 nm wavelength in R4F-LNP versus LNP, suggesting a stable secondary structure formed by peptide incorporation onto LNP surface (Figure 1 D). In addition, R4F-LNP demonstrated >95% siRNA encapsulation efficiency as determined by Ribogreen assay, indicating incorporation of R4F peptide into preformed LNP enabling stable and high payload of siRNA loading. siRNA Intracellular Delivery Profile. To investigate the cellular uptake profile of R4F- LNP, both R4F-LNP and conventional LNP were formulated with porphyrin-lipid dopped onto lipid bilayer and FAM dye labelled on siRNA as markers for lipids and siRNA. First, we compared the intracellular siRNA delivery pattern of both formulations in PC3-Luc6 cells using confocal microscopy. As shown in Figure 2A, conventional LNP treated cells (bottom panels) displayed well-colocalized FAM-siRNA and porphyrin-lipid signals with punctate pattern in cells after 6 h incubation, indicating LNP entrapment in acidic organelles (i.e. endosomes, lysosomes) by clathrin-mediated endocytosis as well as macropinocytosis. ¹⁰ In contrast, R4F-LNP treated cells showed strong siRNA fluorescence (FAM, green colour) in both cytosols and organelles. The FAM signal in the cytosols was well diffused. Interestingly, the porphyrin lipid fluorescence (magenta colour) was observed on cellular membrane in addition to co- localization with FAM-siRNA in the same organelles with a punctate pattern. These data suggested that two delivery pathways existed in R4F-LNP delivery: 1) Classic LNP endocytosis that directed nanoparticle entrapment in the organelles. 2) SR-B1 mediated uptake pathway after R4F incorporation onto LNP, which enabled direct siRNA delivery into cellular cytosol while leaving the lipid components on the cellular membrane. This delivery pattern is also consistent with our previous reported R4F- constrained HDL-like nanoparticles for cytosolic siRNA delivery. ^{18 192425} Besides, the confocal images at 24 h incubation also support the enhanced cytosolic delivery by R4F-LNP. Therefore, R4F incorporation on LNP membrane can alter the siRNA intracellular uptake pathway from organelles to cytosols, which can potentially improve siRNA therapeutic efficacy.

Next, we incubated R4F-LNP with Idl(mSR-BI) (SR-B1 overexpressing) and ldlA-7 (SR-B1 deficient) cells for 24 h to examine the SR-B1 -dependent cellular uptake. After 24 h incubation, cells were imaged under confocal microscopy. As displayed in Figure 2B, Idl(mSR-BI) cells demonstrated markedly higher uptake with stronger fluorescence in both FAM-siRNA and porphyrin-lipid channels when compared with ldlA-7 cells. Significant cytosolic diffusion of siRNA signal was also observed in the Idl(mSR-BI) cells, suggesting that R4F-LNP was mainly taken up through SR-B1 receptor mediated pathway.

In Vitro Luciferase Knockdown: After the investigation of the cytosolic siRNA delivery profile of R4F-LNP, we validated its influence on siRNA knockdown efficacy. Luciferase targeting siRNA (siLuc) and scramble control siRNA (siCtrl) were encapsulated into R4F-LNP and conventional LNP, respectively. Formulations were subsequently incubated with PC3-Luc6 cells to validate the level of luciferase gene knockdown. Bioluminescence intensity was decreased in a dose-dependent manner in the cells treated by both R4F-LNP and conventional LNP while negligible signal reduction was observed in LNP-siCtrl treated wells (Figure 3A, B). Notably, enhanced bioluminescence signal reduction was achieved by R4F-LNP treatment when compared to the conventional LNP across all siRNA concentrations applied. Specifically, R4F-LNP caused ~70% decrease of bioluminescence expression in PC3- Luc6 cells at 10 nM siLuc concentration whereas only ~20% knockdown was achieved by conventional LNP without R4F coating, resulting in 3-4 fold therapeutic enhancement. Given no obvious difference in cell viability caused by R4F-LNP and conventional LNP, the significantly enhanced luciferase knockdown by R4F-LNP is possibly contributed by cytosolic delivery of siRNA that was demonstrated in previous confocal imaging studies.

To further validate whether there’s enhancement of total siRNA uptake amount by R4F-LNP, which can also contribute to improved siRNA efficacy, we performed a flow cytometry experiment to quantitatively compare the uptake difference between R4F- LNP and conventional LNP in PC3-Luc6 cells. Both single cell fluorescent signals from porphyrin-lipid (Figure 4A) and FAM-siRNA (Figure 4B) were collected and quantified. The results demonstrated that there’s a slight enhancement of delivery by R4F-LNP compared to conventional LNP at short/early time incubation (6h). When cells were treated for 24 h, no significant difference in uptake amount was noticed. Given that the in vitro luciferase knockdown study shown in Figure 3 was conducted with 48h incubation, the improvement in siRNA knockdown efficacy by R4F-LNP is mainly due to the intracellular distribution of siRNA into cytosol, rather than the enhancement of delivery amount, thus demonstrating the importance of cytosolic delivery for siRNA efficacy.

In Vivo Liver Metastasis Treatment: After in vitro validation for the improved RNAi efficacy and cytosolic delivery by R4F-LNP, next we performed in vivo gene knockdown experiment to evaluate therapeutic potential of R4F-LNP. Recently, we have identified a novel therapeutic gene target for drug-resistant CRC, ARHGEF2, which is a highly upregulated m ⁶ A reader in CRC that promotes CRC tumorigenesis and metastasis. ²⁰ As liver metastasis from CRC showed upregulated SR-B1 level, therefore, R4F-LNP with SR-B1 targeting ability and cytosolic siRNA delivery feature is promising for treating CRC-induced liver metastasis. The mouse liver metastasis model was established by injecting 7.5x10 ⁵ HCT116 cells intrasplenically into each NOD/SCID mouse. At twelve days after surgery, the mice were randomly defined into two groups and received 1.2 mg/kg of R4F-LNP siCtrl or R4F-LNP siARHGEF2-1 intravenously, every four days for total four doses (at day12, day 16, day20, day24) (Figure 5A). The results showed that R4F-LNP siARHGEF2-1 significantly reduced colorectal liver metastases compared to the R4F-LNP siCtrl treated group as evidenced by significantly reduced liver weight by two-fold (P <0.05, student’s t-test; Figure 5B), which is an indicator of liver metastasis level

Discussion: In summary, this study first demonstrates the therapeutic potential of applying R4F peptide to FDA-approved lipid nanoparticle formulations for enhancing cytosolic delivery of siRNA therapeutics. The R4F-LNP exhibited a combined delivery pattern enabling direct delivery of a portion of siRNA into cancer cell cytosol via a SR- B1 mediated internalization pathway. A 3~4-fold enhancement of RNAi therapeutic efficacy by R4F-LNP was demonstrated on an in vitro luciferase expressing prostate cancer cells, highlighting the significance of cytosolic RNA delivery. Using a mice liver metastasis model, R4F-LNP also proved its therapeutic potential for treating drugresistant cancers by delivering RNA interference to novel genetic targets. Overall, this study demonstrated the feasibility of using R4F-LNP to improve siRNA efficacy through cytosolic delivery, which advances undruggable diseases treatment using RNA interference and holds great potentials for enhancing efficacy of mRNA-based gene therapy.

Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

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