Blood cell-based delivery of therapeutic agents

Abstract

A blood cell-based therapeutic agent delivery system is disclosed. A blood cell-based therapeutic agent delivery vehicle is useful for delivering a therapeutic agent into a recipient organ cell. The blood cell-based therapeutic agent delivery vehicle is made by a method comprising encapsulating the therapeutic agent into a lipid nanoparticle to produce a therapeutic agent-containing lipid nanoparticle, and transfecting the therapeutic agent- containing lipid nanoparticle into a blood cell by incubating the therapeutic agent-containing lipid nanoparticle with the blood cell to form a transfected blood cell. The administration of the transfected blood cell causes the transfected blood cell to be in close proximity to or direct contact with the recipient organ cell, thereby causing a transfer of the therapeutic agent into the recipient organ cell. A composition for a blood cell-based therapeutic agent delivery vehicle for use in delivering a therapeutic agent into a recipient organ cell is also disclosed.

Description

BLOOD CELL-BASED DELIVERY OF THERAPEUTIC AGENTSThis invention was made with Government support under W81XWH-20-2-0041 awarded by the Medical Research and Development Command. The government has certain rights in the invention.Cross-Reference to Related Applications

[0001] This application claims priority from US application No. 63 / 546861 filed 1 November 2023 and entitled DELIVERY OF EXOGENOUS RNA FROM PLATELETS TO CELLS which is hereby incorporated herein by reference for all purposes. For purposes of the United States of America, this application claims the benefit under 35 U.S.C. §119 of US application No. 63 / 546861 filed 1 November 2023 and entitled DELIVERY OF EXOGENOUS RNA FROM PLATELETS TO CELLS which is hereby incorporated herein by reference for all purposes.Field

[0002] The invention pertains to the delivery of therapeutic agents, in particular those that use blood cells as a delivery vehicle.Background

[0003] Many pre-existing RNA delivery strategies rely on introduction of foreign viral vectors, whereas blood cells such as platelets are cells which exist naturally in circulation. RNA delivery tools such as lipid or polymeric nanoparticles are difficult to target, whereas blood cells such as platelets are stimulus responsive and thus naturally target to specific tissues. Several commercial groups are leveraging other properties of megakaryocytes and platelets for drug delivery. For example, STRM Bio is investigating megakaryocyte (platelet precursor cells) derived extracellular vesicles (EVs), which are small fragments, as a drug delivery vehicle. However, preparation of loaded EVs requires that cultured stem cells be modified to contain the desired RNA, matured, and manipulated to release EVs using specialized equipment.

[0004] Major improvements have been made in RNA gene therapy, which has largelybeen enabled by lipid nanoparticle (LNP) delivery technology. However, current nanomedicine approaches such as intravenous infusion of RNA-LNPs are limited by high hepatic accumulation of LNPs with low levels of RNA delivery into extrahepatic tissue. The natural hepatic targeting of RNA-LNP is beneficial for diseases of hepatocellular origin - exemplified by the approved siRNA-LNP drug, Onpattro™, which silences neurodegenerative protein production in the liver - but is problematic for developing LNP therapies for extra-hepatic diseases. Therefore, developing new therapeutic platforms to achieve RNA delivery into extrahepatic tissues will enable the treatment of previously untargetable diseases.

[0005] The inventors have recognised a general need for a system for delivering therapeutic agents into recipient organ cells and tissues, in particular, for delivering exogenous RNA agents throughout the human body into both hepatic and extrahepatic tissues in normal and diseased states.Summary

[0006] This application has a number of aspects. These include, without limitation:• compositions for a blood cell-based therapeutic agent delivery vehicle for use in delivering a therapeutic agent into a target recipient organ cell;• compositions of lipid nanoparticles that are useful in transferring the loaded therapeutic agents into blood cells and / or the target recipient organ cells;• methods of preparing a therapeutic agent-containing lipid nanoparticle loaded blood cell for use as a therapeutic agent delivery vehicle;• use of the therapeutic agent-containing lipid nanoparticle loaded blood cell in the delivery of the therapeutic agent into recipient organ cells and / or hepatic and extra-hepatic tissues;• use of the therapeutic agent-containing lipid nanoparticle loaded blood cell in the treatment of hepatic and extra-hepatic diseases and / or conditions.

[0007] In some embodiments, the blood cell-based therapeutic agent delivery vehicle is made by a method comprising encapsulating the therapeutic agent into a lipid nanoparticle to produce a therapeutic agent-containing lipid nanoparticle, andtransfecting the therapeutic agent-containing lipid nanoparticle into a blood cell by incubating the therapeutic agent-containing lipid nanoparticle with the blood cell to form a transfected blood cell. The administration of the transfected blood cell causes the transfected blood cell to be in close proximity to or in direct contact with the recipient organ cell, thereby transferring the therapeutic agent into the recipient organ cell.

[0008] In some embodiments, the composition for a blood cell-based therapeutic agent delivery vehicle for use in delivering a therapeutic agent into a recipient organ cell comprises a blood cell being transfected with a therapeutic agent-containing lipid nanoparticle.

[0009] In some embodiments, the therapeutic agent comprises a nucleic acid molecule, such as an RNA molecule and / or DNA molecule. In some embodiments, the RNA molecule is a small interfering RNA (siRNA), short hairpin RNA, messenger RNA (mRNA), small activating RNA, amplifying RNA, circular RNA (cirRNA), microRNA (miRNA), or guide RNA (gRNA).

[0010] In some embodiments, the blood cell comprises one or more of red blood cells, white blood cells, and platelets. The blood cell may be in the form of whole blood. In some embodiments, the blood cell comprises a precursor cell, such as megakaryocytes. The therapeutic agent-containing lipid nanoparticle may be transfected into the precursor cell. The transfected precursor cell may be further differentiated to produce platelets.

[0011] In some embodiments, the formulation of the lipid nanoparticle comprises one or more ionizable cationic lipids (ICL), one or more helper lipids, one or more sterol, and one or more polyethylene glycol (PEG)-lipid conjugates. In some example embodiments, the formulation of the lipid nanoparticle comprises ALC-0315 and / or SM-102, DOPC and / or POPC, cholesterol, and DMG-PEG. In further example embodiments, the formulation of the lipid nanoparticle comprises about 50 mole% of ALC-0315 and / or SM-102, about 10 mole% of DOPC and / or POPC, about 38.5% ofcholesterol, and about 1.5 mole% of DMG-PEG.

[0012] The transfected blood cells may be caused to deliver the therapeutic agentcontaining lipid nanoparticle to suitable recipient cells such as tumor cells, endothelial cells, epithelial cells, muscle cells, nerve cells, liver cells, kidney cells, blood cells (e.g., white blood cells and / or red blood cells), skin cells, pancreatic cells, heart cells, lung cells, intestinal cells, immune cells, etc. The delivery of the therapeutic agent into the recipient organ cells is useful in the treatment of hepatic and extra-hepatic diseases and conditions. Such hepatic and extra-hepatic diseases and conditions may include but are not limited to one or more of cancer, hemorrhage, thrombosis, sepsis, inflammation, tissue damage.

[0013] Further aspects and example embodiments are illustrated in the accompanying drawings and / or described in the following description.Brief Description of the Drawings

[0014] The accompanying drawings illustrate non-limiting example embodiments of the invention.

[0015] Figure 1 A is a schematic diagram illustrating the transfection of exogenous reporter mRNA into platelet cells using LNPs and subsequent co-incubation with HEK-293 cells.

[0016] Figure 1 B are flow cytometry dot blots showing fluorescent RNA uptake during platelet transfection.

[0017] Figure 1C are widefield fluorescence images of HEK-293 cells 12h postincubation with platelets in the presence or absence of cytochalasin D. Platelets were transfected with HaloTag mRNA for 5 or 35 minutes, and HaloTag protein expression was detected in HEK-293 cells by staining with JFX646. Platelets were labeled for CD41 a. Magnification: 20x; scale bar: 100 pm.

[0018] Figure 1 D is a plot of normalized luminescence in HEK-293 cells (RLU I pg protein) as a function of platelet agonist (ADP) concentration (pM). NanoLuciferaseexpression was measured in HEK-293 cells following 12h of co-incubation with platelets transfected with NanoLuciferase mRNA for five minutes or 35 minutes. Expression was measured in the presence of endocytosis inhibitors (dynasore and cytochalasin D) as well as in the presence of ADP. The platelets were co-incubated in direct contact or separated by a 0.4 pm pore size membrane.

[0019] Figure 2 are representative histological images of lacerated kidneys. mRNA- LNP treated platelets contained a fluorescent lipid in the LNP (Dil). Sections were stained with an antibody for platelet CD61 and a nuclear stain (DAPI). Scale bar = 20 pm.

[0020] Figure 3A is a schematic diagram illustrating the extra-hepatic sites that RNA transfected platelets may naturally accumulate, and the variety of cell and tissue types that they may interact with.

[0021] Figure 3B is a schematic diagram illustrating example applications of transfusable platelets with customizable RNA payloads for the treatment of extrahepatic conditions.

[0022] Figure 4A is a plot of normalized luminescence in HEK-293 cells (RLU I pg protein) as a function of transfection time (min). Platelets were transfected for five or 35 minutes using LNPs that were formulated with ionizable lipids ALC-0315, KC2, or SM-102. The LNPs were then co-incubated with HEK-293 cells for 16 hours.NanoLuc luminescent signal was detected in HEK-293 cells and normalized to total protein levels.

[0023] Figure 4B is a plot showing the raw luminescence (A.U.) values for platelets that were transfected for five minutes or 60 minutes using LNPs formulated with ionizable lipid ALC-0315 and either assayed alone or after co-incubation with HEK 293 cells. n=3 technical replicates.

[0024] Figure 5A is a schematic diagram showing exogenous reporter mRNA transfection into platelets using LNPs and subsequent co-incubation with HEK-293 cells.

[0025] Figure 5B is a plot showing NanoLuc expression in various cell lines following co-incubation with platelets that were transfected for 0, 5 (Quick Transfect (QT)), and 60 min.

[0026] Figure 5C is a plot showing NanoLuc expression in HEK-293 cells after 12 hours co-incubation with transfer-platelets and treated with endocytic inhibitors, dynasore and cytochalasin D.

[0027] Figure 5D is a correlation plot between platelet-LNP uptake and the amount of NanoLuc expression in HEK-293 cells.

[0028] Figure 5E is a plot showing NanoLuc expression in HEK-293 cells following co-incubation with activated NanoLuc-loaded platelets, with and without membrane separation. The platelets were activated by a variety of agonists, including ADP (10 pM), thrombin (1 U / mL), collagen-related-peptide (CRP, 20 pg / mL), TRAP-6 (10 pM), and U46619 (10 pM) to stimulate extracellular vesicle production, which is a potential mechanism of transfer, n = 4.

[0029] Figure 6 is a plot of FLuc Luminescence (A.U.) in FLuc mutated HEK-293 as a function of platelets that were transfected for five minutes or 60 minutes using LNPs formulated with ionizable lipids ALC-0315 encapsulating mRNA encoding for an adenine base editor 8E (AB8E) and guide RNA to target the editing to a mutated firefly luciferase gene in the cell’s genome. Luciferase signal indicates successful transfer of the mRNA and guide RNA as well as successful editing at 24h and 48h. n=3 technical replicates.

[0030] Figure 7A is a schematic diagram showing the transfusion of mice with mRNA- LNP loaded transfer platelets.

[0031] Figure 7B are images taken by an in vivo imaging system (I VIS) of mice that were transfused with Firefly luciferase (FLuc) transfer- pl ate lets (PLTs), or control mRNA transfer-PLTs, and given laparotomy 18 hours post-transfusion. Scale on bottom right, n = 3.

[0032] Figure 8 is a plot of percentage of Cy5-mRNA uptake as a function of cellpopulation, in particular platelets, CD11 b / c+, CD3+, and B cells.

[0033] Figure 9A is plot of median fluorescence intensity of enhanced green fluorescence protein (EGFP) as a function of mRNA dose (pg / mL) in MKs treated with mRNA-LNP containing the ionizable lipids ALC-0315 or SM-102.

[0034] Figure 9B is a plot of median fluorescence intensity of enhanced green fluorescence protein (EGFP) as a function of treatment, in particular MKs treated with mRNA-LNP comprising 10 mol% DSPC (LNP-DSPC) or 40 mol% egg sphingomyelin (LNP-ESM), or without LNP (No LNP) (n=2).

[0035] Figure 10A are representative histograms of PLPs derived from MKs treated with LNP encoding for either non-EGFP (dark grey) or EGFP mRNA (light grey).

[0036] Figure 10B is a plot illustrating corresponding quantification of the median fluorescence intensity of PLPs derived from MKs treated with LNP encoding for either nonEGFP (dark grey) or EGFP mRNA (light grey). PLP were collected from MKs 72 hours post-transfection (n=1).

[0037] Figure 10C is a plot illustrating corresponding quantification of the percentage of PLPs derived from MKs treated with LNP encoding for either non-EGFP (dark grey) or EGFP mRNA (light grey). PLP were collected from MKs 72 hours post-transfection (n=1).Detailed Description

[0038] Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive sense.

[0039] Aspects of the invention pertain to a blood cell-based therapeutic delivery system. The blood cells are loaded with a therapeutic agent that is encapsulated in alipid nanoparticle or LNP. The loaded blood cells containing the therapeutic agentcontaining lipid nanoparticle may be transferred into recipient organ cells, thereby transferring the therapeutic agent into such recipient organ cells.

[0040] Proof of concept experiments demonstrate that platelets that are loaded with LNPs containing exogenous mRNA (mRNA-LNPs) resulted in successful transfer of the exogenous mRNA into human culture cells in vitro upon co-incubation, and that the loaded platelets resulted in successful localization to tissues that are naturally accessed by circulating platelets.Definitions

[0041] “Cationic lipid” is a protonatable tertiary amine (e.g., pH titratable) head group, C16 to C18 alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds.

[0042] “Encapsulation” or “encapsulating” means full encapsulation or partial encapsulation of a therapeutic agent such as nucleic acid into a lipid nanoparticle.

[0043] “Ionizable cationic lipid” or “ICL” refers to a cationic lipid that is protonated and positively charged (e.g., >50% protonated and positively charged) at low pH (e.g., pH 4) but remains neutral at physiological pH (e.g., about pH 7-7.4).

[0044] “Exogenous” refers to a messenger RNA (mRNA) or protein that is not naturally found in, or produced by, blood cells.

[0045] “Helper lipid” refers to any vesicle-forming lipid (e.g., bilayer-forming lipid), other than the ionizable cationic lipid, that may be added to form a lipid nanoparticle.

[0046] “Minimal activation of platelets” or “minimally activated” refers to maintaining a state of platelet activation which is less than the level seen in platelets treated with agonist thrombin, following preparation for transfection.

[0047] “Mole percent” or “mol %” means the percentage of the moles of a particular component relative to total moles of all components that are in a mixture.

[0048] “Polyethylene glycol-lipid conjugate” or “PEG-lipid” or “PEGylated lipids” refers to derivatives of polyethylene glycol (PEG) covalently attached to a lipid moiety.

[0049] “Neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Non-limiting examples of neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydrosphingomyelins, cephalins, and cerebrosides.

[0050] “Transfer- pl ate let” or (transfer-PLT) refers to a platelet that has been transfected with one or more nucleic acids or a lipid nanoparticle or a nucleic acidcontaining lipid nanoparticle, and which the platelet is administered to deliver the one or more nucleic acids or the lipid nanoparticle or the nucleic acid-containing lipid nanoparticle to a recipient organ cell.Example Embodiments

[0051] Aspects of the invention relate to a method of preparing a blood cell-based therapeutic agent delivery vehicle. The blood cell-based therapeutic agent delivery vehicle is useful for delivering a therapeutic agent into a recipient organ cell. The method comprises encapsulating a therapeutic agent into a lipid nanoparticle (LNP) to form a therapeutic agent-containing lipid nanoparticle (or therapeutic agent-containing LNP).

[0052] In some embodiments, the therapeutic agent comprises a nucleic acid. The therapeutic agent may comprise any suitable one or more types of nucleic acids. In some embodiments, the nucleic acid is a RNA molecule. In some embodiments, the RNA molecule comprises a messenger RNA (mRNA) molecule. The mRNA molecule may be selected based on the desired conditions, disorder and / or disease to target. The mRNA molecule may be selected based on the desired target cell type. Nonlimiting examples of desired conditions, disorder and / or disease to target include cancer, hemorrhage, thrombosis, sepsis, inflammation, tissue damage, etc. In some non-limiting examples, the desired condition, disorder and / or disease to target is cancer, the selected mRNA sequence may encode one or more pro-apoptotic proteins. In such examples, the one or more pro-apoptotic proteins may be targetedfor expression in tumor cells. In some other non-limiting examples, the desired condition, disorder and / or disease to target is blood vessel damage, the selected mRNA sequence may encode one or more pro-coagulant enzymes and / or anti- fibrinolytic peptides which may be targeted for expression in infiltrating immune cells and / or endothelial cells, desirably hemostasis. The selected mRNA sequence may encode one or more growth factors and / or metalloproteases, which may be targeted for expression in infiltrating immune cells and / or endothelial cells for tissue repair. In some other non-limiting examples, the desired condition, disorder and / or disease to target is thrombosis, the selected mRNA sequence may encode one or more uPa and / or plasmin, which may be targeted for expression in infiltrating immune cells and / or endothelial cells for reducing clotting. The selected mRNA sequence may encode NETosis regulators which may be targeted for expression into neutrophils for reducing off target inflammation which may cause unwanted clotting via the thrombo- inflammatory axis. In some other non-limiting examples, the selected mRNA sequence may encode pro- and / or anti-inflammatory RNAs, which may be targeted for expression in immune cells, for increasing the levels of specific cytokines to mount a robust immune reason against invading pathogens and for decreasing excessive cytokine levels to reduce unwanted inflammation which may undesirably result in in diseases such as arthritis and diabetes.

[0053] In some embodiments, the RNA molecule comprises a silencing RNA (“siRNA”) molecule. The siRNA molecule may be designed to reduce the expression of a target mRNA. In some non-limiting examples, the siRNA molecule is designed to target certain classical oncogenes when delivered into tumor cells.

[0054] The RNA agent may comprise short hairpin RNA, small activating RNA, selfamplifying RNA, circular RNA (circRNA), guide RNA (gRNA) or single guide RNA (sgRNA), and other suitable RNA molecules that may be encapsulated into a lipid nanoparticle.

[0055] In some embodiments, the nucleic acid is DNA.

[0056] The lipid nanoparticle may comprise one or more ionizable cationic lipids (ICL), one or more helper lipids, one or more sterol, and / or one or more polyethylene glycol(PEG)-lipid conjugate.

[0057] Any suitable one or more ionizable cationic lipids may be used to form the lipid nanoparticle. Non-limiting examples of suitable ionizable cationic lipids that may be used include DLin-KC2-DMA, 7-(4-(dipropylamino)butyl)-7-hydroxytridecane-1 ,13-diyl dioleate (CL4H6), heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6- (undecyloxy)hexyl)amino)octanoate (SM-102), ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), CL1 H6, CL15H6, CL1 D6, and combinations thereof. In some example embodiments, the one or more ionizable cationic lipids that may be used to form the lipid nanoparticle comprises ALC-0315. In some example embodiments, the one or more ionizable cationic lipids that may be used to form the lipid nanoparticle comprises SM- 102.

[0058] In some embodiments, the one or more ionizable cationic lipids are present in the lipid nanoparticle in an amount in the range from about 20 mole% to about 55 mole%, or any mole percent therebetween, for example the mole percent of the ionizable cationic lipid may be 20%, 25%, 30%, 35%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, or any value and / or range therebetween.

[0059] In some embodiments, the lipid nanoparticle comprises one or more helper lipids. A helper lipid is a vesicle-forming lipid which may be added to the one or more ionizable cationic lipid to enable or enhance the ability of the therapeutic agentcontaining LNP to transfect a platelet cell and / or the ability of the therapeutic agentcontaining LNP to be transferred from the transfected platelet cells to the desired target cells.

[0060] In some embodiments, the helper lipid comprises one or more neutral lipids. Non-limiting examples of suitable neutral lipid may include one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (S OPE), and 1 ,2-dielaidoyl- sn-glycero-3-phophoethanolamine (transDOPE), egg sphingomyelin (ESM), and any combinations thereof. In some example embodiments, the one or more helper lipids that may be used to form the lipid nanoparticle comprise DOPC. In some example embodiments, the one or more helper lipids that may be used to form the lipid nanoparticle comprise POPC.

[0061] In some embodiments, the one or more helper lipids are present in the lipid nanoparticle in an amount in the range of from about 5 mole% to about 40 mole%, or any mole percent therebetween, for example, the mole percent of the helper lipid may be 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, and 40%, or any value and / or range therebetween.

[0062] The lipid nanoparticle may comprise one or more sterols. Non-limiting examples of sterols that may be used include cholesterol and / or a cholesterol derivative, such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, beta-sitosterol, fucosterol, or any combination thereof. In some example embodiments, the one or more sterols that may be used to form the lipid nanoparticle comprise cholesterol.

[0063] In some embodiments, the one or more sterols are present in the lipid nanoparticle in an amount in the range of from about 25 mole% to about 50 mole%, or any mole percent therebetween, for example, the mole percent of the sterol may be 25%, 27.5%, 30%, 32.5%, 35%, 36%, 37%, 38%, 38.5%, 39%, 39.5%, 40%, 42%, 44%, 46%, 48%, 50%, or any value and / or range therebetween.

[0064] In some embodiments, the lipid nanoparticle comprises one or more polyethylene glycol (PEG)-lipid conjugates. Non-limiting examples of suitable PEG- lipid conjugates that may be used to form the lipid nanoparticle include (2- hexyldecanoate),2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG2000), l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG2000), or PEG-1 , 2-distearoyl-rac-glycero-3- methylpolyoxyethylene 2000 (DSG-PEG). In some example embodiments, the one or more PEG-lipid conjugates that may be used to form the lipid nanoparticle comprise DMG-PEG.

[0065] In some embodiments, the one or more PEG-lipid conjugates are present in the lipid nanoparticle in an amount in the range of from about 0.5 mole% to about 3 mole%, or any mole percent therebetween, for example, the mole percent of polyethylene glycol (PEG)-lipid conjugate may be 0.5%, 1 %, 1.5%, 2%, 2.5%, 3%, or any value and / or range therebetween.

[0066] In some example embodiments, the formulation of the lipid nanoparticle comprises about 50 mole% of the one or more ionizable cationic lipids, about 10 mole% of the one or more helper lipids, about 38.5% of the one or more sterols, and about 1.5 mole% of the one or more PEG-lipid conjugates. In some example embodiments, the formulation of the lipid nanoparticle comprises about 50 mole% of ALC-0315 and / or SM-102, about 10 mole% of DOPC and / or POPC, about 38.5% of cholesterol, and about 1.5 mole% of DMG-PEG.

[0067] The therapeutic agent-containing LNP may be transfected into blood cells. The blood cells may comprise red blood cells (erythrocytes), white blood cells (one or more of neutrophils, eosinophils, basophils, monocytes and lymphocytes, and macrophages), platelets, and combinations thereof. In some embodiments, the blood cells comprises whole blood and / or whole blood component. In some embodiments, the whole blood comprises components such as one or more of platelet rich plasma (or platelets), red cell concentrate (or red cells), concentrated white blood cells (or Leukopak), leukoreduced whole blood, and non-leukoreduced whole blood.

[0068] In some embodiments, the blood cells comprise transfusable platelets, i.e., platelets that are ready for transfusion into patients. The term “platelets” includes native platelets and / or platelet-like particles (PLPs), i.e., particles that are capable of mimicking platelet functions. The platelets may be derived from donors. The plateletsmay be produced ex vivo and / or in vitro.

[0069] In some embodiments, the blood cells comprise precursor cells. In some example embodiments, the blood cells comprise megakaryocytes. Megakaryocytes are the precursor cells of platelets. Megakaryocytes may be generated in vitro from any suitable stem cells, for example, umbilical cord blood-derived hematopoietic stem cells, human pluripotent stem cells (hPSCs), human induced pluripotent stem cells (iPSCs), and non-hematopoietic sources such as fibroblasts. In some embodiments, the therapeutic agent-containing LNP are transfected into the blood cells comprising culture-derived megakaryocytes. Functional platelets containing the therapeutic agent derived from the cultured megakaryocytes that are transfected with the therapeutic agent-containing LNP may be produced. In some example embodiments, the therapeutic agent comprises exogenous mRNA. Platelets that are derived from the mRNA-LNP modified megakaryocytes containing the mRNA may be generally modified upon the delivery of the exogenous mRNA in the megakaryocytes and / or the platelets.

[0070] In some embodiments, the transfection comprises incubating the blood cells with the therapeutic agent-containing LNP for about 20-40 minutes, and in some embodiments, about 25 to 35 minutes, and in some embodiments, about 30-35 minutes. The transfection step may comprise separating excess LNPs from the transfected blood cells. The separation may be performed by any suitable separation methods, such as centrifugation, filtration, and the like.

[0071] The transfected blood cells may be caused to co-incubate with target cells. The co-incubation may comprise causing the transfected blood cells to be in close proximity to or in direct contact with the target cells, thereby causing the transfer of the therapeutic agent into the target cells. In some example embodiments, the transfected blood cells are transfused into a subject. The subject may be a mammal. The subject may be a human. In some embodiments, activation of the blood cells releases the therapeutic agent-containing LNP. The released therapeutic agentcontaining LNPs may be transferred into the target cells. In some embodiments, the therapeutic agent is released from the LNP within the transfected blood cells. Thereleased therapeutic agent may then be transferred into the target cells. In some embodiments, the target cells are located at tissue sites that are naturally accessed and / or accumulated by circulating blood cells.

[0072] Proof of concept experiments demonstrate that platelets that are loaded with lipid nanoparticles containing exogenous mRNA (mRNA-LNPs) resulted in successful transfer of the exogenous mRNA into human culture cells in vitro upon co-incubation, and that the loaded platelets resulted in successful localization to tissues that are naturally accessed by circulating platelets. The exogenous mRNA transfer results in functional protein expression from the delivered exogenous mRNA in the human culture cells.

[0073] Aspects of the invention pertain to the use of therapeutic agent-containing lipid nanoparticle loaded blood cells. The therapeutic agent-containing lipid nanoparticle loaded blood cells may be used to deliver the loaded therapeutic agent into recipient organ cells and / or tissues. The therapeutic agent-containing lipid nanoparticle loaded blood cells may be used to deliver the loaded therapeutic agent into the liver and / or extrahepatic tissues such as but are not limited to the spleen, bone marrow, lung and kidney. Non-limiting examples of the types of recipient organ cells which the therapeutic agent-containing lipid nanoparticle loaded blood cells may target include tumor cells, endothelial cells, epithelial cells, muscle cells, nerve cells, liver cells, kidney cells, blood cells (e.g., white blood cells and / or red blood cells), skin cells, pancreatic cells, heart cells, lung cells, intestinal cells, immune cells, etc.

[0074] The therapeutic agent-containing lipid nanoparticle loaded blood cells may be used in the treatment of various hepatic and extra-hepatic diseases and / or conditions. Such diseases and / or conditions include but are not limited to cancer, hemorrhage, thrombosis, sepsis, inflammation, tissue damage, etc. In some embodiments, the therapeutic agent-containing lipid nanoparticle loaded blood cells is used in the treatment of various diseases and / or conditions by delivering an RNA agent into the recipient organ cells. Such RNA agent may for example include siRNA and / or mRNA agents. The therapeutic agent-containing lipid nanoparticle loaded blood cells are customizable with the desired RNA payload in the treatment of the desired targeteddiseases and / or conditions.

[0075] The invention is further described with reference to the following specific examples, which are not meant to limit the invention, but rather to further illustrate it.EXAMPLES

[0076] A method of the type discussed herein was used to prepare the platelets that are loaded with lipid nanoparticles containing exogenous mRNA (mRNA-LNPs). Figure 1A is a schematic diagram illustrating transfection of exogenous reporter mRNA into platelet cells using LNPs and subsequent co-incubation with HEK-293 cells.Transfected platelets transfer functional exogenous mRNA to HEK-293 cells in vitro

[0077] In these experiments, to ensure that the mRNA could successfully be loaded into the platelets using LNPs, the mRNA in the LNP was pre-stained with SYTO RNASelect Green, a membrane-permeable dye which binds RNA. Flow cytometric analysis showed that platelets can be effectively loaded with exogenous RNA using LNP systems, as shown by an increase in fluorescence over time, with a maximum after 2 hours of transfection (Figure 1 B).

[0078] RNA transfer was then quantified by measuring reporter protein expression in culture cells after incubation with platelets loaded with mRNA. Platelets were first transfected for 30 minutes with LNPs containing mRNA encoding for HaloTag, a fluorescent protein. After transfection, the platelets were centrifuged and washed to remove excess LNPs, a process which took roughly five minutes (total transfection time: 35 minutes). The transfected platelets were then co-incubated with Human Embryonic Kidney 293 (HEK 293) cells for 12 hours. As a negative control to account for residual LNPs remaining after platelet washing, platelets were also transfected with an identical dose of mRNA-LNPs, but immediately conducted the centrifugation and wash before significant RNA uptake could occur (total transfection time: five minutes). By limiting the transfection time, this ensured that any observed RNA transfer was from platelets, rather than from contaminating LNPs. As another control, the uptake of exogenous content was blocked by pre-treating HEK 293 cells with 5mM cytochalasin D (CytD), an inhibitor of actin-dependent endocytosis. Results showed that HEK 293 cells were negative for HaloTag protein when co-incubated with platelets that were immediately washed after transfection, suggesting low residual LNP contamination. However, the HEK 293 cells that were incubated with platelets loaded with LNP for 35 minutes had high levels of HaloTag protein expression as shown by widefield microscopy (Figure 1C). Furthermore, pretreatment of HEK 293 cells with CytD also did not result in any detectable HaloTag expression, suggesting that the transfer of HaloTag mRNA is actin dependent. The levels of expression achieved in these experiments were from using non-optimized lipid formulations. The inventors expect that loading at least 25-fold more mRNA into each platelet will results in even lower platelet activation levels. Overall, these results confirm that RNA-loaded platelets can transfer reporter mRNA to HEK 293 cells in vitro to induce functional protein expression, and that the transfer can be prevented by inhibiting actin-dependent endocytosis.

[0079] Platelet derived extracellular vesicles (PEVs) is a mechanism of endogenous RNA transfer from platelets to other cells and are released after activation with physiological agonists such as ADP. To assess whether PEVs were contributing to exogenous mRNA transfer from platelets, NanoLuciferase (NLuc) mRNA loaded platelets were again co-incubated with HEK293 cells. However, a trans-well containing a 0.4-micron pore size polyester membrane was included to determine if the transfer required direct contact, or occurred through PEVs. Generally, PEVs are under 250 nm in size, and thus would be expected to freely cross the membrane. ADP was included at different concentrations to induce PEV generation. Again, the HEK 293 cells were pre-treated with endocytosis inhibitors to probe for potential uptake mechanisms. High levels of NLuc expression were observed in platelets that were allowed to directly interact with HEK 293 cells, but such was not observed in HEK 293 cells that were separated from platelets by a membrane (Figure 1 D). This was valid even in the presence of ADP at both physiological and supraphysiological concentrations, which suggests that the transfer of LNP loaded cargo is not reliant on PEV release. Pre-treatment of HEK293 using 80 mM dynasore — a dynamin inhibitor — and 5 mM CytD both decreased NLuc expression, indicating that mRNA transfer is likely occurring through clathrin-mediated endocytosis, as it is both adynamin and actin-dependent process. The similar results shown with NLuc compared to HaloTag also shows that platelets can successfully transfer a variety of different mRNA sequences. Exogenously loaded mRNA may differ in localization from endogenous RNA which supports that the transfer is not mediated through PEV release. The experimental results also support that the transfer does not require platelet activation, and instead requires direct interaction of the target cells with the mRNA loaded platelets.Rat platelets transfected mRNA-LNP accumulate at damages vasculature in wounds

[0080] In these experiments, the right kidney of the rats was injured, and the rats were resuscitated with platelets which were either untreated (normal platelets) or transfected with NLuc mRNA using a rat-optimized LNP labeled with the lipophilic tracer Dil. At the termination of the experiment, the histology of the kidneys was assessed to determine if the transfected platelets accumulated within the injury and clot. Histological images showed that Dil was concentrated in the kidney wounds, indicating that mRNA-loaded platelets localized to the injured tissue (Figure 2). This shows that the transfusable platelets successfully localize to tissues that are naturally accessed by circulating platelets, and that this technology could extend to other tissues where platelets accumulate.

[0081] The experimental results support that the delivery system discussed herein allows for easily customizable RNA payloads, and therefore can be easily adapted for a broad set of conditions such as but not limited to cancer, hemorrhage, thrombosis, sepsis, inflammation, and tissue damage among others (Figure 3A). The target cell type for each condition, as well as the proposed RNA cargo, is summarized in Figure 3B.ALC-DOPC LNP enable platelet RNA transfer to cells in vitro

[0082] To test whether LNP composition may have an effect on whether RNA can be transferred into platelets and into other cells, NanoLuc luciferase mRNA was formulated into several LNP formulations that only differed by the ionizable lipid used, which were ALC-0315, KC2, or SM-102. In these experiments, the LNP formulationthat was used was 50% ionizable lipid, 10% DOPC (as the helper lipid), 38.5% cholesterol and 1.5% PEG-DMG2000. The LNP that was formulated with ALC-0315 led to the greatest signal in HEK-293 cells (Figure 4A). To ensure that the signal did not come from platelets expressing NanoLuc, ALC-0315 LNP transfected platelets alone were assessed in a subsequent study. No significant signal was detected in the platelets alone compared to the cells that were co-incubated with NanoLuc transfer platelets (Figure 4B). mRNA-loaded transfer platelets can transfer mRNA into a variety of cell types and transfer is contact dependent

[0083] Figures 5A and 5B support that human transfer-PLTs transferred functional NanoLuc mRNA into a variety of relevant human cell lines upon co-incubation, including human embryonic kidney cells (HEK-293), pancreatic cancer cells (AsPC- 1), and monocytes (THP-1). To account for residual LNP in the experiment, a ‘quick transfect’ (QT) control was included to limit LNP uptake into platelets but control for any expression in the HEK-293 cells caused by excess LNP. Co-incubation of HEK- 293 cells with QT platelets resulted in significantly lower reporter protein expression in recipient organ cells supporting that the bulk luminesce signal is derived from mRNA transferred out of transfected platelets (Figure 5C). Higher mRNA uptake into platelets was also directly proportional to NanoLuc expression in HEK293 cells after co-incubation (r = 0.7314, p < 0.0001), further suggesting that transferred mRNA is from platelets (Figure 5D). In a similar experiment, incubating platelets transfected with mRNA encoding HaloTag, a reporter protein highly specific to a fluorescent protein ligand, with HEK-293 cells for 30 minutes resulted in platelet-transfer mediated HaloTag expression as visualized by fluorescence microscopy (Figure 1C). This signal was lost when the endocytosis inhibitor, cytochalasin D, was included. To investigate the mechanism of mRNA-transfer, actin- and dynamin-dependent endocytosis inhibitors (cytochalasin D and dynasore, respectively) were included while transfected platelet were co-incubated with HEK-293 cells. The addition of endocytosis inhibitors prevented NanoLuc expression in HEK-293 cells suggesting that mRNA transfer occurs through actin- and / or dynamin-dependent mechanism (Figure 5C). To determine whether mRNA transfer occurs through contact-based orplatelet activation-dependent mechanisms, a trans-well system with a 0.4 pm pore size was used to block passage of platelets but allow permeation of platelet-derived EVs. NanoLuc expression was not observed in the HEK-293 cells in the trans-well system after platelets were stimulated with extracellular vesicle producing agonists (Figure 5E). This strongly suggests that mRNA is not transferred through PEVs even after platelet activation but is instead via direct contact between platelets and recipient organ cells.Transfected platelets can be used to transfer mRNA and guide RNA to get DNA editing in recipient organ cells after co-incubation

[0084] ALC-0315 LNPs were formulated to co-encapsulate a mRNA encoding for an adenine base editor 8E (ABE8E) and guide RNA that associates with the produced AB8E protein. It is believed that once synthesized in a cell, the AB8E protein-guide complex is capable of entering the nucleus and will correct a defective firefly luciferase gene present the recipient organ cell genome. Transfer platelets loaded with AB8E mRNA and guide RNA were capable of achieving editing of the defective firefly luciferase gene in specialize HEK-293 cells resulting in luminescence (Figure 6)Platelets transfer firefly luciferase mRNA in vivo and luminescence can be seen near the wound site

[0085] To determine if mRNA-LNP loaded platelets are capable of transferring mRNA in vivo, mice were transfused with Firefly luciferase (FLuc) mRNA loaded transfer platelets prior to injury by abdomen incision (laparotomy). Flue mRNA was used since the signal detectable in platelets is low compared to other cells and tissues which allows us to be confident that any detected FLuc signal is not coming from the transfected platelets, but rather the cells that have received transferred mRNA (Figure 6 (UT platelets)). The formulation that was used to load the platelets was ALC-0315 DOPC, similar to the one described previously. Eighteen hours after transfusion, recipient mice were imaged using an in vivo imaging system (I VIS) which is useful for detecting cell penetrating luminescence and can indicate where Flue is detected. Compared to control transfer platelets, luminescence was observed in FLuc-Transfer platelet receiving mice, and in some cases the signal was coming from areas near thewound site (Figures 7A and 7B). These preliminary results support that mRNA-LNP loaded transfer platelets can be used to deliver mRNA to other cells in vivo. mRNA can be transfected into several blood cell types directly in whole blood prior to blood transfusion

[0086] Rat whole blood was transfected with Cy5-labelled mRNA-LNP for 90 minutes at 37°C. Cy5-mRNA uptake was detected in several blood cell types including platelets (platelets), CD11 b / c+ cells (macrophages, monocytes, granulocytes, and dendritic cells), CD3+ cells (T-cells) and B Cells. n=4 (Figure 8). mRNA can be transfected into cord-blood derived megakaryocytes (MKs)

[0087] Cord-blood derived megakaryocytes (MKs) can be transfected with mRNA encoding for enhanced green fluorescent protein (EGFP) using LNP of varying compositions which results in the expression of exogenous EGFP protein. Figure 9A is a plot illustrating median fluorescence intensity of enhanced green fluorescence protein (EGFP) of MKs treated with mRNA-LNP containing either the ionizable lipids ALC-0315 or SM-102 (n=3) **P<0.01. Figure 9B is a plot illustrating median fluorescence intensity of enhanced green fluorescence protein (EGFP) of MKs treated with mRNA-LNP comprising 10 mol% DSPC (LNP-DSPC) or 40 mol% egg sphingomyelin (LNP-ESM), or without LNP (No LNP) (n=2).Cargo transfected into megakaryocytes ends up in platelet-like particles (PLPs)

[0088] Cargo transfected into megakaryocytes ends up in platelet-like particles (PLPs). Figure 10A are representative histograms of PLPs derived from MKs treated with LNP encoding for either nonEGFP (dark grey) or EGFP mRNA (light grey). Figures 10B and 10C are corresponding quantification of the median fluorescence intensity and percentage of PLPs derived from MKs treated with LNP encoding for either nonEGFP (dark grey) or EGFP mRNA (light grey) respectively. PLP were collected from MKs 72 hours posttransfection (n=1).

[0089] Further developing of the blood cell-based delivery system discussed herein requires in vivo validation. Next steps for developing the system include conductingadditional in vitro evaluation that transfer of mRNA from blood cells can occur into other human cell lines, and further characterizing transfer mechanisms. In addition, multiple mouse or rat models of extra-hepatic disease will be generated, which would assist with determining the distribution of mRNA throughout the body after transfusion with mRNA-LNP loaded blood cells. This is expected to be accomplished using mRNA encoding reporter proteins such as Firefly Luciferase (FLuc), NLuc, GFP, and HaloTag. After transfusion of mRNA-loaded blood cells, complete biodistribution studies of various tissues and organs to evaluate expression of the respective reporter protein will be performed. These results will be compared to the protein expression localization of direct mRNA-LNP injection to determine whether blood cells can alter the location of RNA delivery in these disease models.Interpretation of Terms

[0090] Unless the context clearly requires otherwise, throughout the description and the claims:• “comprise”, “comprising”, and the like are to be construed in an inclusive sense, as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”;• “connected”, “coupled”, or any variant thereof, means any connection or coupling, either direct or indirect, between two or more elements; the coupling or connection between the elements can be physical, logical, or a combination thereof;• “herein”, “above”, “below”, and words of similar import, when used to describe this specification, shall refer to this specification as a whole, and not to any particular portions of this specification;• “or”, in reference to a list of two or more items, covers all of the following interpretations of the word: any of the items in the list, all of the items in the list, and any combination of the items in the list; the singular forms “a”, “an”, and “the” also include the meaning of anyappropriate plural forms. These terms (“a”, “an”, and “the”) mean one or more unless stated otherwise;• “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes both (A and B) and (A or B);• “approximately” when applied to a numerical value means the numerical value ± 10%;• where a feature is described as being “optional” or “optionally” present or described as being present “in some embodiments” it is intended that the present disclosure encompasses embodiments where that feature is present and other embodiments where that feature is not necessarily present and other embodiments where that feature is excluded. Further, where any combination of features is described in this application this statement is intended to serve as antecedent basis for the use of exclusive terminology such as "solely," "only" and the like in relation to the combination of features as well as the use of "negative" limitation(s)” to exclude the presence of other features; and• “first” and “second” are used for descriptive purposes and cannot be understood as indicating or implying relative importance or indicating the number of indicated technical features.

[0091] Words that indicate directions such as “vertical”, “transverse”, “horizontal”, “upward”, “downward”, “forward”, “backward”, “inward”, “outward”, “left”, “right”, “front”, “back”, “top”, “bottom”, “below”, “above”, “under”, and the like, used in this description and any accompanying claims (where present), depend on the specific orientation of the apparatus described and illustrated. The subject matter described herein may assume various alternative orientations. Accordingly, these directional terms are not strictly defined and should not be interpreted narrowly.

[0092] Where a range for a value is stated, the stated range includes all sub-ranges of the range. It is intended that the statement of a range supports the value being atan endpoint of the range as well as at any intervening value to the tenth of the unit of the lower limit of the range, as well as any subrange or sets of sub ranges of the range unless the context clearly dictates otherwise or any portion(s) of the stated range is specifically excluded. Where the stated range includes one or both endpoints of the range, ranges excluding either or both of those included endpoints are also included in the invention.

[0093] Certain numerical values described herein are preceded by "about". In this context, "about" provides literal support for the exact numerical value that it precedes, the exact numerical value ±5%, as well as all other numerical values that are near to or approximately equal to that numerical value. Unless otherwise indicated a particular numerical value is included in “about” a specifically recited numerical value where the particular numerical value provides the substantial equivalent of the specifically recited numerical value in the context in which the specifically recited numerical value is presented. For example, a statement that something has the numerical value of “about 10” is to be interpreted as: the set of statements:• in some embodiments the numerical value is 10;• in some embodiments the numerical value is in the range of 9.5 to 10.5; and if from the context the person of ordinary skill in the art would understand that values within a certain range are substantially equivalent to 10 because the values with the range would be understood to provide substantially the same result as the value 10 then “about 10” also includes:• in some embodiments the numerical value is in the range of C to D where C and D are respectively lower and upper endpoints of the range that encompasses all of those values that provide a substantial equivalent to the value 10.

[0094] Specific examples of systems, methods and apparatus have been described herein for purposes of illustration. These are only examples. The technology provided herein can be applied to systems other than the example systems described above.Many alterations, modifications, additions, omissions, and permutations are possible within the practice of this invention. This invention includes variations on described embodiments that would be apparent to the skilled addressee, including variations obtained by: replacing features, elements and / or acts with equivalent features, elements and / or acts; mixing and matching of features, elements and / or acts from different embodiments; combining features, elements and / or acts from embodiments as described herein with features, elements and / or acts of other technology; and / or omitting combining features, elements and / or acts from described embodiments.

[0095] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any other described embodiment(s) without departing from the scope of the present invention.

[0096] Any aspects described above in reference to apparatus may also apply to methods and vice versa.

[0097] Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, while processes or blocks are presented in a given order, alternative examples may perform routines having steps, or employ systems having blocks, in a different order, and some processes or blocks may be deleted, moved, added, subdivided, combined, and / or modified to provide alternative or subcombinations. Each of these processes or blocks may be implemented in a variety of different ways. Also, while processes or blocks are at times shown as being performed in series, these processes or blocks may instead be performed in parallel, simultaneously or at different times.

[0098] Various features are described herein as being present in “some embodiments”. Such features are not mandatory and may not be present in all embodiments. Embodiments of the invention may include zero, any one or any combination of two or more of such features. All possible combinations of such features are contemplated by this disclosure even where such features are shown in different drawings and / or described in different sections or paragraphs. This is limitedonly to the extent that certain ones of such features are incompatible with other ones of such features in the sense that it would be impossible for a person of ordinary skill in the art to construct a practical embodiment that combines such incompatible features. Consequently, the description that “some embodiments” possess feature A and “some embodiments” possess feature B should be interpreted as an express indication that the inventors also contemplate embodiments which combine features A and B (unless the description states otherwise or features A and B are fundamentally incompatible). This is the case even if features A and B are illustrated in different drawings and / or mentioned in different paragraphs, sections or sentences.

[0099] It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions, omissions, and sub-combinations as may reasonably be inferred. The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims

WHAT IS CLAIMED IS:1 . Use of a blood cell-based therapeutic agent delivery vehicle for delivering a therapeutic agent into a recipient organ cell, wherein the blood cell-based therapeutic agent delivery vehicle is made by a method comprising: encapsulating the therapeutic agent into a lipid nanoparticle to produce a therapeutic agent-containing lipid nanoparticle; and transfecting the therapeutic agent-containing lipid nanoparticle into a blood cell by incubating the therapeutic agent-containing lipid nanoparticle with the blood cell to form a transfected blood cell, wherein administration of the transfected blood cell causes the transfected blood cell to be in close proximity to, or in direct contact with the recipient organ cell, thereby transferring the therapeutic agent- into the recipient organ cell.

2. The use as defined in claim 1 , wherein the therapeutic agent comprises one or more nucleic acid molecules.

3. The use as defined in claim 2, wherein the nucleic acid molecule is an RNA molecule.

4. The use as defined in claim 3, wherein the RNA molecule is a small interfering RNA (siRNA), short hairpin RNA, messenger RNA (mRNA), self-amplifying RNA, small activating RNA, circular RNA (cirRNA), microRNA (miRNA), guide RNA (gRNA) or single guide RNA (sgRNA).

5. The use as defined in any one of the preceding claims, wherein the blood cell comprises one or more of red blood cells, white blood cells, and platelets.

6. The use as defined in any one of claims 1 to 5, wherein the blood cell is in the form of whole blood or whole blood component.

7. The use as defined in any one of claims 1 to 4, wherein the blood cell comprises a precursor cell.

8. The use as defined in claim 7, wherein the precursor cell comprises megakaryocytes.

9. The use as defined in any one of claims 1 to 4, wherein the blood cell comprises native platelets derived from one or more mammalian donors.

10. The use as defined in any one of claims 1 to 4, wherein the blood cell comprises platelets produced ex vivo and / or in vitro.11 . The use as defined in any one of the preceding claims, wherein the formulation of the lipid nanoparticle comprises one or more ionizable cationic lipids (ICL), one or more helper lipids, one or more sterol, and one or more polyethylene glycol (PEG)-lipid conjugates.

12. The use as defined in claim 11 , wherein the one or more ionizable cationic lipids (ICL) comprise DLin-KC2-DMA, CL4H6, SM-102, ALC-0315, CL1 H6, CL15H6, CL1 D6, or combinations thereof.

13. The use as defined in claim 11 or 12, wherein the one or more helper lipids comprise one or more neutral lipids.

14. The use as defined in any one of claims 11 to 13, wherein the one or more helper lipids comprise DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, S OPE, transDOPE, ESM, or combinations thereof.

15. The use as defined in any one of claims 11 to 14, wherein the one or more sterol comprise cholesterol and / or a cholesterol derivative.

16. The use as defined in any one of claims 11 to 15, wherein the one or more PEG-lipid conjugates comprise DMG-PEG2000, DSPE-PEG2000, DSG-PEG, ALC-0159, or combinations thereof.

17. The use as defined in any one of claims 11 to 16, wherein the one or more ionizable cationic lipids are present in the lipid nanoparticle in an amount in the range from about 20 mole% to about 55 mole%,18. The use as defined in any one of claims 11 to 17, wherein the one or more helper lipids are present in the lipid nanoparticle in an amount in the range of from about 5 mole% to about 40 mole%.

19. The use as defined in any one of claims 11 to 18, wherein the one or more sterols are present in the lipid nanoparticle in an amount in the range of from about 25 mole% to about 50 mole%.

20. The use as defined in any one of claims 11 to 19, wherein the one or more PEG-lipid conjugates are present in the lipid nanoparticle in an amount in the range of from about 0.5 mole% to about 3 mole%.21 . The use as defined in any one of claims 11 to 20, wherein the formulation of the lipid nanoparticle comprises ALC-0315 and / or SM-102, DOPC and / or POPO, cholesterol, and DMG-PEG.

22. The use as defined in any one of claims 11 to 21 , wherein the formulation of the lipid nanoparticle comprises about 50 mole% of ALC-0315 and / or SM-102, about 10 mole% of DOPC and / or POPC, about 38.5% of cholesterol, and about 1.5 mole% of DMG-PEG.

23. The use as defined in any one of the preceding claims, wherein the recipient organ cell comprises one or more of tumor cells, endothelial cells, epithelial cells, muscle cells, nerve cells, liver cells, kidney cells, blood cells, skin cells, pancreatic cells, heart cells, lung cells, intestinal cells, and immune cells.

24. The use as defined in any one of the preceding claims, wherein the delivery of the therapeutic agent into the recipient organ cell is useful in the treatment of hepatic and extra-hepatic diseases and conditions.

25. The use as defined in claim 24, wherein the hepatic and extra-hepaticdiseases and conditions comprise one or more of cancer, hemorrhage, thrombosis, sepsis, inflammation, tissue damage.

26. A composition for a blood cell-based therapeutic agent delivery vehicle for use in delivering a therapeutic agent into a recipient organ cell, the composition comprising a blood cell being transfected with a therapeutic agent-containing lipid nanoparticle.

27. The composition as defined in claim 26, wherein the lipid nanoparticle comprises one or more ionizable cationic lipids (ICL), one or more helper lipids, one or more sterol, and one or more polyethylene glycol (PEG)-lipid conjugates.

28. The composition as defined in claim 26 or 27, wherein the one or more ionizable cationic lipids (ICL) comprise DLin-KC2-DMA, CL4H6, SM-102, ALC- 0315, CL1 H6, CL15H6, CL1 D6, or combinations thereof.

29. The composition as defined in claim 27 or 28, wherein the one or more helper lipids comprise one or more neutral lipids.

30. The composition as defined in any one of claims 27 to 29, wherein the one or more helper lipids comprise DSPC, DOPC, DPPC, DOPG, DPPG, DOPE, POPC, POPE, DOPE-mal, DPPE, DMPE, DSPE, S OPE, transDOPE, ESM, or combinations thereof.31 . The composition as defined in any one of claims 27 to 30, wherein the one or more sterol comprise cholesterol and / or a cholesterol derivative.

32. The composition as defined in any one of claims 27 to 31 , wherein the one or more PEG-lipid conjugates comprise DMG-PEG2000, DSPE-PEG2000, DSG- PEG, ALC-0159, or combinations thereof.

33. The composition as defined in any one of claims 27 to 32, wherein the one or more ionizable cationic lipids are present in the lipid nanoparticle in an amount in the range from about 20 mole% to about 55 mole%,34. The composition as defined in any one of claims 27 to 33, wherein the one or more helper lipids are present in the lipid nanoparticle in an amount in the range of from about 5 mole% to about 40 mole%.

35. The composition as defined in any one of claims 27 to 34, wherein the one or more sterols are present in the lipid nanoparticle in an amount in the range of from about 25 mole% to about 50 mole%.

36. The composition as defined in any one of claims 27 to 35, wherein the one or more PEG-lipid conjugates are present in the lipid nanoparticle in an amount in the range of from about 0.5 mole% to about 3 mole%.

37. The composition as defined in any one of claims 27 to 36, wherein the formulation of the lipid nanoparticle comprises ALC-0315 and / or SM-102, DOPC and / or POPO, cholesterol, and DMG-PEG.

38. The composition as defined in any one of claims 27 to 37, wherein the formulation of the lipid nanoparticle comprises about 50 mole% of ALC-0315 and / or SM-102, about 10 mole% of DOPC and / or POPC, about 38.5% of cholesterol, and about 1.5 mole% of DMG-PEG.

39. The composition as defined in any one of claims 26 to 38, wherein the therapeutic agent comprises one or more nucleic acid molecules.

40. The composition as defined in claim 39, wherein the nucleic acid molecule is an RNA molecule.41 . The composition as defined in claim 40, wherein the RNA molecule is a small interfering RNA (siRNA), short hairpin RNA, messenger RNA (mRNA), selfamplifying RNA, small activating RNA, circular RNA (cirRNA), microRNA (miRNA), guide RNA (gRNA) or single guide RNA (sgRNA).

42. The composition as defined in any one of claims 26 to 41 , wherein the blood cell comprises one or more of red blood cells, white blood cells, and platelets.

43. The composition as defined in any one of claims 26 to 42, wherein the bloodcell is in the form of whole blood.

44. The composition as defined in any one of claims 26 to 41 , wherein the blood cell comprises a precursor cell.

45. The composition as defined in claim 44, wherein the precursor cell comprises megakaryocytes.

46. The composition as defined in any one of claims 26 to 41 , wherein the blood cell comprises native platelets derived from one or more mammalian donors.

47. The composition as defined in any one of claims 26 to 41 , wherein the blood cell comprises platelets produced ex vivo and / or in vitro.

48. The composition as defined in any one of claims 26 to 47, wherein the recipient organ cell comprises one or more of tumor cells, endothelial cells, epithelial cells, muscle cells, nerve cells, liver cells, kidney cells, blood cells, skin cells, pancreatic cells, heart cells, lung cells, intestinal cells, and immune cells.

49. The composition as defined in any one of claims 26 to 48, wherein the delivery of the therapeutic agent into the recipient organ cell is useful in the treatment of hepatic and extra-hepatic diseases and conditions.

50. The composition as defined in claim 49, wherein the hepatic and extra-hepatic diseases and conditions comprise one or more of cancer, hemorrhage, thrombosis, sepsis, inflammation, tissue damage.

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