Novel drug delivery system based on engineered red blood cell

Abstract

The present disclosure relates to a novel drug delivery system, which comprises a lipid particle and a modified red blood cell. The present disclosure also relates to preparation of the drug delivery system and therapeutic use thereof.

Description

A novel drug delivery system based on engineered red blood cells

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to PCT patent application No. PCT / CN2024 / 114536, filed on August 26, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a novel drug delivery system, its preparation and application, the drug delivery system comprising lipid particles and modified red blood cells. Background Technology

[0004] In recent years, with the rapid development of lipid nanoparticles (LNPs) as a delivery system, mRNA drugs have made significant progress in the fields of medicine and therapy. Biotechnology companies (such as Moderna and BioNTech) have demonstrated the great potential of mRNA-LNP drugs by developing two COVID-19 mRNA vaccines. mRNA-LNPs continue to show therapeutic potential in a range of applications, including viral vaccines, protein replacement therapies, and cancer immunotherapy.

[0005] However, mRNA delivery systems still face challenges in targeted delivery (Hou et al., 2021; Rohner et al., 2022). To achieve therapeutic effects, mRNA molecules must reach specific target cells and produce sufficient target proteins (protein expression levels need to be increased 50-1000 times compared to vaccine drugs). Existing delivery methods (lipid nanoparticle-based delivery systems) are severely limited in terms of tissue bioavailability, efficiency of delivery to target tissues, and endosome escape. Although targeted delivery of nucleic acid drugs is currently achieved by modifying LNP formulations or mounting antibodies on LNPs, the actual preparation processes are very complex and cannot truly achieve tissue / organ targeting (Cheng et al., 2020). For example, antibody-LNP conjugations mainly accumulate in the liver and cannot effectively reach immune organs, while adding cationic compounds (such as DOTAP) to LNPs can introduce safety risks (Cheng et al., 2020; Rurik et al., 2022).

[0006] Furthermore, mRNA-LNP drugs can easily trigger the body's innate immune response, leading to adverse reactions or side effects, which is also a potential toxicity issue (Vlatkovic, 2021). Expanding mRNA-LNP to the treatment of non-vaccine diseases requires higher doses and longer dosing cycles, which will increase the risks associated with mRNA-LNP.

[0007] Therefore, to achieve better clinical application prospects for mRNA drugs, it is necessary to increase the in vivo protein expression level of mRNA drugs while avoiding toxicity. Overcoming biological barriers in drug delivery (such as recognition and clearance by nucleases and mononuclear phagocytes in plasma), improving endosomal escape efficiency (current drugs have an endosomal escape efficiency of less than 1%), and further enhancing targeted delivery efficiency are essential (Gilleron et al., 2013). Furthermore, existing mRNA-LNP drugs, based on different delivery methods (such as subcutaneous injection, intramuscular injection, and intravenous injection), enter different solid tissues and organs, such as lymph nodes, muscle tissue, and the liver. However, entering other solid tissues remains very difficult. For example, in mobilizing the immune system, lymph node immune cells are few in number and dispersed, thus hindering the comprehensive activation of a stronger immune response, limiting their application in tumor immunology (Hou et al., 2021). Therefore, it is necessary to optimize the delivery methods of mRNA drugs to enable targeted delivery to tissues rich in immune cells and effective expression in the target tissue.

[0008] Therefore, there is a need in the field to develop new delivery systems that can deliver nucleic acids, such as mRNA, to tissues rich in immune cells and enable them to be successfully expressed in the target tissues or cells. Summary of the Invention

[0009] This invention develops a novel nucleic acid drug delivery system that utilizes the unique biological characteristics of red blood cells (naturally targeting peripheral immune organs, high biocompatibility, etc.) to deliver nucleic acid drugs to the spleen.

[0010] Red blood cells naturally accumulate in the spleen and are cleared by phagocytes within it. They are considered excellent delivery vehicles targeting the spleen, enabling the targeted delivery of immunotherapies and effectively activating the immune system (Buffet et al., 2011). The spleen, one of the largest lymphoid organs in the human body, is rich in immune cells and serves as an important reservoir for them. The spleen is divided into red pulp and white pulp, containing a large number of antigen-presenting cells and lymphocytes, respectively, playing crucial roles in infection, tumor immunity, and autoimmune diseases (Bronte and Pittet, 2013; Lewis et al., 2019). Red blood cells naturally accumulate in the spleen and are cleared by phagocytes within it. They are considered excellent delivery vehicles targeting the spleen, enabling the targeted delivery of immunotherapies and effectively activating the immune system (Buffet et al., 2011). Red blood cells have the advantage of being concentrated in the spleen and interact closely with the immune cells within it.

[0011] Although some red blood cell products carrying drugs, such as small molecules or peptides, especially antibodies, have entered clinical research in recent years, the mechanism of action of some of these products is to enter the spleen and activate its immune cells (McArdel et al., 2021). However, unlike RNA, the function of these delivered small molecules or peptides is limited by the function of the delivered drug itself, and they cannot achieve diversified gene expression in the immune cells of the spleen, thereby achieving targeted modification of the immune cells in the spleen. Moreover, the existing red blood cell delivery methods are mainly non-covalently coupled (Ukidve et al., 2020; Zhao et al., 2021). In addition, the coupling of red blood cells with LNPs in previous studies is generally non-covalently coupled. Moreover, even for these small molecules or proteins that can directly exert their effects on target cells after being delivered to the spleen, the existing red blood cell delivery methods still have two major problems: (1) low drug coupling efficiency, that is, low drug loading on the surface of red blood cells; (2) high risk of drug off-target, as lipid nanoparticles detach before reaching the target organ and target cells with the red blood cells, resulting in low delivery efficiency.

[0012] Therefore, this invention is the first to develop such a red blood cell delivery system, which uses a specific covalent coupling process that can not only successfully deliver nucleic acid drugs to the spleen, but also enable them to be successfully expressed in the spleen, thereby achieving efficient and safe targeted delivery of nucleic acid drugs and expanding the application of nucleic acid drugs in a variety of diseases.

[0013] In summary, this invention covalently links nucleic acid drugs, such as mRNA drugs (mRNA-LNP), to erythrocytes, utilizing the natural distribution of erythrocytes to efficiently deliver the nucleic acid drugs to antigen-presenting cells (such as macrophages and dendritic cells) in the spleen. Erythrocytes then efficiently deliver the nucleic acid drugs they carry into the cells through phagocytosis. Depending on the target of the carried nucleic acid drug, such as the mRNA drug, immune activation or immunosuppression can be achieved.

[0014] Furthermore, the erythrocyte delivery system of the present invention can bypass the endosome-lysosome pathway, efficiently enter myeloid cells through phagocytosis of erythrocytes, and release mRNA in the cytoplasm, thereby improving protein expression efficiency.

[0015] In some implementations, without being bound by theory, the nucleic acid drug delivery system of the present invention has at least one or more of the following technical effects: (1) Red blood cells themselves do not have a nucleus, which greatly reduces safety risks compared with other cell therapies; (2) Red blood cells have high biocompatibility and long circulation time in vivo, which can effectively protect the carried nucleic acid drugs from being cleared and enhance targeted delivery efficiency; (3) Red blood cells carrying mRNA drugs can bypass the endosomal-lysosomal pathway and release mRNA in the cytoplasm, thereby improving protein expression efficiency; and / or (4) Red blood cells naturally target the spleen, a natural immune organ (containing nearly 1 / 3 of the body's immune cells), and fully interact with the immune cells in the spleen, which can give full play to the huge potential of the carried mRNA drugs in immune regulation. Furthermore, in this invention, mRNA-LNP is covalently coupled to the surface of erythrocyte membrane through enzymatic catalysis or chemical reaction, enabling a higher drug loading capacity of mRNA-LNP to be coupled to the surface of erythrocyte (approximately 10 times higher than previous technologies). At the same time, the coupled drug is more stable and is metabolized as erythrocytes are cleared, avoiding non-specific off-target effects.

[0016] In some implementations, the present invention has successfully achieved one or more of the following aspects:

[0017] 1. The first covalent conjugation system of erythrocytes and mRNA-LNP drugs (mRNA-LNP-RBC) was constructed, and the optimal engineering connection method of mRNA-LNP-RBC was determined through screening and testing of various conjugation methods. This included the improvement and optimization of LNP formulation and the optimization of reaction conditions.

[0018] 2. The in vitro characterization of mRNA-LNP-RBC was completed, including the identification of its phenotype and the quantitative analysis of the carried mRNA.

[0019] 3. This study confirmed the advantages of mRNA-LNP-RBC over mRNA-LNP in lysosomal escape from cells. For example, it can bypass the endosomal-lysosomal pathway, efficiently enter myeloid cells through phagocytosis by erythrocytes, and release mRNA in the cytoplasm, thereby improving protein expression efficiency.

[0020] 4. The advantages of mRNA-LNP-RBC over mRNA-LNP drugs in gene delivery and expression in the spleen were confirmed.

[0021] 5. The superiority of mRNA-LNP-RBC over mRNA-LNP drugs in gene delivery and expression in CD11b+ myeloid cells, a group of immune cells in the spleen, was confirmed. Attached Figure Description

[0022] The following figures illustrate preferred embodiments of the invention. For illustrative purposes, the figures show currently preferred embodiments. However, it should be understood that the invention is not limited to the specific embodiments shown in the figures.

[0023] Figure 1 shows three process coupling pathways for mRNA-LNP-RBC.

[0024] Figure 2 shows the drug loading assay of mRNA-LNP-RBC (where the mRNA used was Luciferase).

[0025] Figure 3 shows the in vitro characterization results of mRNA-LNP-RBC (the mRNA used in these results is Luciferase). A, Flow cytometry results. B, Confocal imaging results.

[0026] Figure 4 shows the in vitro mRNA-LNP-RBC gene delivery efficiency study (the mRNA used in this result is Luciferase or GFP).

[0027] Figure 5 shows the in vitro mRNA-LNP-RBC gene delivery pathway study (the mRNA used in this result is Luciferase).

[0028] Figure 6 shows the in vivo tissue distribution and expression of Luc-LNP-RBC-1, Luc-LNP-RBC-2, and Luc-LNP-RBC-3.

[0029] Figure 7 shows the in vivo tissue distribution and expression of Luc-LNP and Luc-LNP-RBC-1.

[0030] Figure 8 shows the distribution and expression of GFP-LNP and GFP-LNP-RBC-1 in the spleen. A, GFP positivity rate in different cell populations within the spleen. B, Proportion of GFP+ positive cells. C, Distribution of GFP+ positive cells.

[0031] Figure 9 shows the in vivo pharmacokinetic study of Luc-mRNA-LNP-RBC-1. A. Percentage of Luc-mRNA-LNP-RBC-1 in peripheral blood. B. Content of Luc-mRNA-LNP on Luc-mRNA-LNP-RBC-1.

[0032] definition

[0033] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] As used herein, unless the context clearly indicates otherwise, the singular expressions “a,” “an,” and “the” cover the plural reference. Unless otherwise stated, nucleic acids are written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in the direction from amino to carboxyl. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described, as they can vary depending on the specific circumstances used by those skilled in the art.

[0035] As used herein, the term “and / or” means any one of the options or two or more of the options.

[0036] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps.

[0037] The terms “patient,” “individual,” and “subject” refer to any mammal that may use the treatments or compositions disclosed herein. Therefore, the methods and compositions disclosed herein can have medical and / or veterinary applications. In a preferred form, the mammal is a human.

[0038] As used herein, "lipid particles" are compositions comprising one or more lipids and one or more therapeutic and / or preventative agents. Lipid particles are typically micrometer-sized or smaller, but can also be larger than micrometers and may comprise a lipid bilayer. Lipid particles encompass lipid nanoparticles (LNPs), liposomes (e.g., lipid vesicles), and cationic liposome complexes, with lipid nanoparticles being preferred. Lipid nanoparticles are spherical or polyhedral particles of nanometer (1-1000 nm) size with an electron-dense core. Due to the presence of ionizable lipids, LNPs can encapsulate negatively charged nucleic acids at the core of the particle through the mutual adsorption of positive and negative charges. For example, the diameter of lipid nanoparticle groups is approximately 50-500 nm, such as approximately 50-350 nm. Therapeutic or preventative agents, such as nucleic acids, can be encapsulated within the lipid portions of lipid nanoparticles or in an aqueous space encapsulated by some or all of the lipid portions of lipid nanoparticles, thereby protecting them from enzymatic degradation or other undesirable effects induced by host organism or cellular mechanisms, such as adverse immune responses.

[0039] "Lipid nanoparticles (LNPs)" refer to particles containing multiple (i.e., more than one) lipid molecules that are physically bound together by intermolecular forces. Lipid nanoparticles can be, for example, microspheres (including monolayer and multilayer vesicles, such as liposomes), dispersed phases in emulsions, micelles in suspensions, or internal phases.

[0040] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0041] "Antigen-binding fragment" refers to a molecule that is distinct from the intact antibody, contains a portion of the intact antibody, and binds the antigen that the intact antibody binds to. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dAb (domain antibody); linear antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; bivalent antibodies or fragments thereof; or camelid antibodies.

[0042] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA.

[0043] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any or a combination of many known antibody CDR assignment systems, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (on the World Wide Web imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)).

[0044] The term "drug loading" refers to the amount of drug carried per unit weight, unit volume, or single red blood cell. In this invention, the drug loading of red blood cells is typically measured in μg / mL.

[0045] The term "nucleic acid drug" refers to a drug or active agent containing nucleic acids that can be used for prevention, treatment, or diagnosis. Therefore, the term "nucleic acid drug" as used herein encompasses both drugs used for treatment or prevention and diagnostic agents used for diagnosis.

[0046] The term "treatment" refers to a clinical intervention intended to alter the natural course of a disease in an individual undergoing treatment. Desired therapeutic effects include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis.

[0047] The term "prevention" includes the suppression of the occurrence or development of a disease or condition, or symptoms of a particular disease or condition. In some implementations, subjects with a family history of cancer are candidates for preventative programs. Generally, in the context of cancer, the term "prevention" refers to the administration of a drug prior to the onset of signs or symptoms of cancer, particularly in subjects at risk of cancer.

[0048] The term "effective amount" refers to such an amount or dose of the modified red blood cells or composition of the present invention, which, when administered to a patient in a single or multiple doses, produces the desired effect in a patient requiring treatment or prevention. The effective amount can be readily determined by a physician skilled in the art by considering a variety of factors, such as: the species of the mammal; weight, age, and general health condition; the specific disease involved; the degree or severity of the disease; the individual patient's response; the specific drug administered; the mode of administration; the bioavailability characteristics of the administered formulation; the chosen dosing regimen; and the use of any concomitant therapies.

[0049] The term "therapeutic effective amount" refers to the amount that effectively achieves the desired therapeutic outcome at the required dose and for the required duration. The therapeutic effective amount of the modified red blood cells or compositions of the present invention can vary depending on various factors such as disease state, individual age, sex, and weight. Relative to an untreated subject, the "therapeutic effective amount" preferably inhibits measurable parameters (e.g., uric acid content, tumor growth rate, tumor volume, etc.) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%, and still more preferably at least about 80% or 90%.

[0050] The term "preventive effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because the prophylactic dose is administered to the subject before or at an early stage of the disease, the preventive effective dose will be less than the therapeutic effective dose.

[0051] The term "pharmaceutical composition" refers to a composition that exists in a form that allows for the biological activity of the active ingredient contained therein, and does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition. The term "sorting enzyme receptor motif" refers to a motif comprising oligoglycine as an intermediate in the formation of an amine nucleophile attacking sorting enzyme and a recognition motif, and ultimately covalently coupled to the recognition motif. In one embodiment, the sorting enzyme receptor motif is located at the N-terminus or C-terminus of the polypeptide.

[0052] The term "sorting enzyme recognition motif" refers to a polypeptide that is cleaved by a sorting enzyme molecule and is capable of forming a covalent bond with the sorting enzyme after cleavage. In one embodiment, the sorting enzyme recognition motif comprises LPXTG / A, where X is any amino acid. In one embodiment, sorting enzyme cleavage occurs between T and G / A.

[0053] The term "connector" refers to a bifunctional or multifunctional molecule that can link (conjugate) two molecules or entities together, typically possessing two reactive functions. Connectors used in conjugates can be broadly classified as either non-cleavable or cleavable. They can also be classified as straight-link connectors and branched connectors based on whether they are branched. Branched connectors may contain branched units, each of which can be conjugated to at least one drug or molecule.

[0054] The term "red blood cell" or "RBC" refers to the most common type of blood cell and the primary carrier of oxygen in vertebrates, transported through the bloodstream and circulatory system. The cytoplasm of red blood cells is rich in hemoglobin, an iron-containing biomolecule that binds oxygen, giving cells and blood their red color. The cell membrane, composed of proteins and lipids, provides essential properties for physiological cellular function, such as deformability and stability, while allowing passage through the circulatory system, particularly the capillary network. In humans, mature red blood cells are flexible, oval-shaped, biconcave discs. They lack a nucleus and most organelles to maximize the space for hemoglobin; they can be viewed as bags for hemoglobin, with the plasma membrane acting as a sac. Adults produce approximately 2.4 million new red blood cells per second. About 84% of the cells in the human body are 20–30 trillion red blood cells. Nearly half the volume of blood (40% to 45%) is composed of red blood cells.

[0055] The term "adult natural red blood cells" refers to mature natural red blood cells obtained directly from the blood of animals, especially humans (such as adults or children).

[0056] The terms "blood products" or "blood preparations" used herein are interchangeable and refer to products made from (human) blood for medical use. Blood products include whole blood preparations, component blood preparations, plasma preparations, or leukocyte-reduced blood preparations.

[0057] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (such as immunosuppressants or immune agonists).

[0058] As used herein, the term "immunomodulator" refers to a natural or synthetic active agent or drug that inhibits or modulates an immune response, such as an immunomodulatory protein. An immune response can be a humoral or cellular response. Immunomodulators include immunosuppressants or immune agonists. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0059] The term "cytotoxic agent" is used in this invention to refer to substances that inhibit or prevent cell function and / or cause cell death or damage.

[0060] "Chemotherapy agents" include chemical compounds that are useful in treating cancer or immune system diseases.

[0061] The term "small molecule drug" refers to low-molecular-weight compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0062] The terms “tumor” and “cancer” are used interchangeably in this article to cover both solid tumors and hematologic malignancies.

[0063] The term “cancer” refers to or describes a physiological disorder in mammals that is typically characterized by unregulated cell growth.

[0064] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used in this article.

[0065] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0066] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the active ingredient contained therein to be biologically effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0067] The terms "drug combination" or "combination product" are used interchangeably to refer to non-fixed or fixed combinations, including but not limited to pillboxes and pharmaceutical compositions. The term "non-fixed combination" means that active ingredients (e.g., (i) the lipid particle-erythrocyte conjugate of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level of two or more active ingredients. In some embodiments, the lipid particle-erythrocyte conjugate and other therapeutic agents of the present invention used in the drug combination are administered at levels not exceeding those achieved when used alone. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components produces an effect greater than that achieved by using any one component alone in treating a disease or condition. The components may each be in separate formulations, and their formulations may be the same or different.

[0068] The term "combination therapy" refers to the administration of two or more therapeutic agents or modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential use of each type of therapeutic agent or modality at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of combination therapy in treating the condition or symptom described herein.

[0069] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the progression or severity of existing symptoms, conditions, illnesses, or diseases.

[0070] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0071] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0072] Invention Details

[0073] This invention relates to a novel lipid particle-erythrocyte conjugate comprising lipid particles and modified erythrocytes, wherein the lipid particles are covalently coupled to the membrane surface of the modified erythrocytes, optionally via a linker.

[0074] In some embodiments, one or more lipid particles are coupled to red blood cells. In some embodiments, each red blood cell contains 10 to 10,000 lipid particles.

[0075] I. Lipid particles

[0076] In some embodiments, the lipid particles are lipid nanoparticles (LNPs).

[0077] In some embodiments, the size of the lipid nanoparticles is about 1 to about 2,500 nm, for example, about 50 to about 600 nm in one embodiment, about 50 to about 400 nm in one sub-implementation, and about 50 to about 250 nm in another sub-implementation, preferably about 50 to about 150 nm in one sub-implementation.

[0078] In one embodiment, the LNP comprises one or more lipids selected from the group consisting of ionizable lipids, accessory lipids, steroids, and PEG lipids.

[0079] In one embodiment, ionizable lipids suitable for the lipid particles of the present invention include, but are not limited to, 3-(bisdodecylamino)-N1,N1,4-tridecyl-1-piperazine ethylamine (KL10), N1-[2-(bisdodecylamino)ethyl]-N1,N4,N4-tridecyl-1,4-piperazine diethylamine (KL22), 14,25-bistridecyl-15,18,21,24-tetraaza-octacosane (KL25), and 1,2-dilinolenicooxy-N,N-dimethylaminopropane (DLi). n-DMA), 2,2-dilinolenic-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) (abbreviated as MC3), 2,2-dilinolenic-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-diolenic-oxy-N,N-dimethylaminopropane (DODMA), 2 -({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA), (2R)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]propyl-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8 -[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanedioate (L319), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}9-heptadecanedioate (SM102) and / or combinations thereof.

[0080] In one embodiment, the ionizable lipid is selected from MC3 or SM102.

[0081] In one embodiment, the auxiliary lipids suitable for the lipid particles of the present invention include, but are not limited to, 1,2-dilinoleoyl-sn-glycerol-3-phosphate choline (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), and 1,2-distearateoyl-sn-glycerol-3-phosphate choline (DLPC). DSPC), 1,2-di-undecanoyl-sn-glycerol-3-phosphate choline (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccinoyl-sn-glycerol-3-phosphate choline (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphate choline (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycerol-3-phosphate choline, 1,2-disarachidonicoyl-sn-glycerol-3-phosphate choline, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphate choline), 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanyl-sn-glycerol-3-phosphate ethanolamine (ME) 16.0PE), 1,2-distearate-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docosahexaenooyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin and / or combinations thereof.

[0082] In one embodiment, the assisting lipid is selected from DSPC.

[0083] In one embodiment, the steroids suitable for the lipid particles of the present invention include, but are not limited to, cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol and / or combinations thereof.

[0084] In one embodiment, the steroid is selected from cholesterol.

[0085] In one embodiment, the PEG lipids suitable for the lipid particles of the present invention include, but are not limited to, 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (DMG-PEG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-distearylglycerol (PEG-DSG), PEG-dispalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycineamide (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and / or combinations thereof.

[0086] In one embodiment, the PEG lipid is selected from DMG-PEG, preferably DMG-PEG2000.

[0087] II. Modified red blood cells

[0088] The lipid particles in this invention can form conjugates with any suitable cell or membrane structure.

[0089] The membrane structure can be any biological membrane structure, preferably selected from erythrocyte membrane, macrophage membrane, stem cell membrane, neutrophil membrane, hematopoietic stem cell membrane, erythrocyte progenitor cell membrane, platelet membrane, reticulocyte membrane, and mature erythrocyte membrane.

[0090] In the human body, red blood cells (RBCs) constitute the largest proportion of blood cells in the circulatory system. Unlike other blood cells, RBCs lack a nucleus and are flexible, capable of changing shape to adapt to blood vessels. Their primary function is to supply oxygen to the body. Key protein markers on the surface of RBCs allow them to circulate in the body for extended periods without being cleared by macrophages, resulting in a long half-life. This characteristic makes them excellent candidates for drug carriers. Mature, enucleated red blood cells contain no genetic material, thus exhibiting good safety profiles compared to other gene and cell therapies. In some embodiments, the cells suitable for use in this invention are red blood cells (e.g., mature red blood cells). Unless otherwise stated or clearly implied from the context, when referring to red blood cells in this invention, it generally refers to mature red blood cells.

[0091] In some embodiments, the cells suitable for use in this invention are red blood cells that can be obtained from eukaryotic organisms, such as mammals, such as primates, like chimpanzees or humans; cattle; dogs; cats; rodents, such as guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish, preferably obtained from humans. In some embodiments, the red blood cells are human red blood cells, such as natural human red blood cells.

[0092] In some embodiments, the RBCs of the present invention are natural adult red blood cells. In some embodiments, the RBCs are mature red blood cells isolated from human (e.g., adult or child) blood, or mature red blood cells differentiated from stem cells (e.g., pluripotent stem cells such as induced pluripotent stem cells).

[0093] In some embodiments, the invention contemplates the use of autologous red blood cells isolated from an individual, which, after in vitro modification, are administered to the individual. In some embodiments, the invention contemplates the use of immunocompatible red blood cells that have the same blood type as the individual to whom the cells will be administered (e.g., at least with respect to the ABO blood group system, and in some embodiments, with respect to the D blood group system) or may be compatible with it. In some embodiments, the RBCs are mature red blood cells having the same blood type as the subject to be administered.

[0094] In some embodiments, the red blood cells suitable for use in this invention are modified red blood cells. For example, this invention provides a method for engineering adult natural red blood cells so that the natural red blood cells can be used to efficiently deliver therapeutic drugs.

[0095] In some embodiments, the red blood cells suitable for use in this invention are modified red blood cells that contain nucleophilic or electrophilic groups.

[0096] In some embodiments, the red blood cells suitable for use in this invention are red blood cells treated with a reducing agent, such as red blood cells treated with a thiol reducing agent.

[0097] In some embodiments, RBCs are mature red blood cells (e.g., natural adult red blood cells) obtained by treatment with a thiol reducing agent, which contain cysteine ​​residues containing a thiol group. In some embodiments, the thiol reducing agent is tris(2-carboxyethyl)phosphine (TCEP).

[0098] In some embodiments, the red blood cells suitable for use in this invention are red blood cells treated with a reducing agent, such as red blood cells treated with a thiol reducing agent, obtained by a method comprising:

[0099] (i) Separating and concentrating red blood cells from (human) whole blood, optionally by filtering out white blood cells; or obtaining red blood cells from stem cells such as pluripotent stem cells such as induced pluripotent stem cells;

[0100] (ii) Treating red blood cells with a reducing agent such as a thiol reducing agent (e.g., TCEP) to chemically modify the surface of the red blood cells;

[0101] (iii) Collect and concentrate the modified red blood cells.

[0102] In some implementations, the surface chemical modification in (ii) includes the following steps:

[0103] The thiol reducing agent is mixed with concentrated red blood cells, wherein the concentration of the reducing agent is between 0.1 mM and 50 mM, for example, 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM; preferably about 5.0 mM.

[0104] III. Lipid particle-erythrocyte conjugate

[0105] III-1. Directly covalently coupled lipid particle-erythrocyte conjugates and their preparation methods

[0106] In some embodiments, one or more lipid particles are directly covalently coupled to erythrocytes to obtain a lipid particle-erythrocyte conjugate. In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: lipid particle-erythrocyte.

[0107] In some implementations, red blood cells contain nucleophilic groups and lipid particles contain electrophilic groups, wherein the nucleophilic groups react with the electrophilic groups to form covalent bonds.

[0108] In some implementations, the thiol groups of cysteine ​​residues on erythrocyte membrane proteins can form chemical bonds with reactive functional groups of lipid particles to prepare lipid particle-erythrocyte conjugates.

[0109] Nucleophilic groups on erythrocyte membrane proteins include, but are not limited to: (i) N-terminal amino groups, (ii) side-chain amino groups, such as the amino group of lysine, (iii) side-chain thiol groups, such as the thiol group of cysteine, and (iv) glycosyl or amino groups (in the case of glycosylation), such as amino groups on the side chain. The amino, thiol, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on lipid particles selected from: (i) active esters, such as NHS esters, HOBt esters, halocarbamates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

[0110] In some embodiments, the electrophilic group on the lipid particles is maleimide, and the nucleophilic group on the erythrocyte membrane protein is a side-chain thiol group, such as a thiol group after cysteine ​​reduction (e.g., a thiol group of free cysteine, or a thiol group exposed after disulfide bond reduction).

[0111] In another embodiment, a lipid containing an electrophilic group is covalently coupled to a membrane protein on a red blood cell, such as a membrane protein on a red blood cell containing a nucleophilic group, such as a thiol group, preferably a mature red blood cell, such as an adult natural red blood cell. In another embodiment, a lipid containing maleimide is linked to a thiol group on a red blood cell, preferably a mature red blood cell, such as an adult natural red blood cell.

[0112] In some embodiments, RBCs are mature red blood cells (e.g., natural adult red blood cells derived from stem cells such as pluripotent stem cells such as iPSCs) obtained by treatment with a reducing agent such as a thiol reducing agent as described above, and contain cysteine ​​residues containing thiol groups.

[0113] In some embodiments, lipid particles, such as LNP, comprise lipids containing maleimide groups (MAL), and optionally one or more other lipids suitable for lipid particles. In some embodiments, the amount of the lipids containing maleimide groups in the lipid particles is 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%).

[0114] In one embodiment, the lipids containing maleimide groups suitable for the lipid particles of the present invention include, but are not limited to, ionizable lipids containing maleimide groups, auxiliary lipids containing maleimide groups, steroids containing maleimide groups, and / or PEG lipids containing maleimide groups.

[0115] In one embodiment, the maleimide-containing lipid suitable for the lipid particles of the present invention is a PEG lipid containing a maleimide group.

[0116] In one embodiment, the lipids containing maleimide groups suitable for the lipid particles of the present invention include DSPE-PEG lipids containing maleimide groups, preferably DSPE-PEG(2000) containing maleimide groups, such as DSPE-PEG(2000)-MAL (DSPE-PEG2000-Maleimide).

[0117] In one embodiment, the lipid particles of the present invention, such as LNP, comprise DSPE-PEG(2000)-MAL, and optionally one or more other lipids suitable for lipid particles. In some embodiments, the DSPE-PEG(2000)-MAL content in the lipid particles, such as LNP, is 0.1%-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%).

[0118] In one embodiment, the DSPE-PEG(2000)-MAL of the present invention has the following molecular structure:

[0119] In some embodiments, the lipid particles of the present invention, such as LNP, are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-Maleimide; DMG-PEG2000.

[0120] In some embodiments, the lipid particles of the present invention, such as LNP, are composed of the following five components: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-Maleimide; and DMG-PEG2000. In some embodiments, the lipid particles of the present invention, such as LNP, are composed of the following five components: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-Maleimide; and DMG-PEG2000, wherein the molar ratio of MC3 or SM102 is 40-60%, cholesterol is 30-45%, DSPC is 5-15%, DSPE-PEG2000-Maleimide is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and DMG-PEG2000 is 0. 1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%); for example, MC3 or SM102 molar ratio is approximately 50%, cholesterol = approximately 38.5%, DSPC = approximately 10%, DSPE-PEG2000-Maleimide = approximately 0.75%, DMG-PEG2000 = approximately 0.75%; for example, MC3 or SM102 molar ratio is 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-Maleimide = 0.75%, DMG-PEG2000 = 0.75%.

[0121] In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: LNP-DSPE-PEG(2000)-MAL-S-RBC, wherein...

[0122] LNP-DSPE-PEG(2000)-MAL refers to any LNP as defined herein that contains DSPE-PEG(2000)-MAL, wherein optionally the amount of DSPE-PEG(2000)-MAL in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined herein (e.g., lipids suitable for lipid particles as described herein);

[0123] RBC refers to any red blood cell as defined herein, such as red blood cells treated with reducing agents, such as red blood cells treated with thiol reducing agents;

[0124] -S- refers to the group containing S formed by the reaction of the maleimide group with the thiol group of RBC, where the S atom comes from the thiol group of RBC.

[0125] Optionally, multiple lipid particles can be coupled to the RBC, wherein the lipid particles may be the same or different.

[0126] In some embodiments, the present invention provides a method for preparing a directly covalently coupled lipid particle-erythrocyte conjugate, comprising mixing lipid particles containing maleimide groups and erythrocytes treated with a reducing agent such that the two are covalently coupled via the maleimide groups and nucleophilic groups on erythrocyte membrane proteins, for example, comprising...

[0127] (1) Loading lipids containing maleimide groups into lipid particles;

[0128] (2) Surface chemical modification of adult natural erythrocytes to expose nucleophilic groups on erythrocyte membrane proteins; and collection and concentration of the modified erythrocytes;

[0129] (3) Mix the lipid particles in (1) and the red blood cells obtained in (2) such that the two are covalently coupled to nucleophilic groups on red blood cell membrane proteins via maleimide groups; in some embodiments, the ratio of the lipid particles to red blood cells is 0.1-1000 μg of lipid particles per 1 mL of red blood cells.

[0130] (4) Collect the lipid particle-erythrocyte conjugate obtained in (3).

[0131] In one embodiment, the nucleophilic group on the erythrocyte membrane protein is a side-chain thiol group.

[0132] In one embodiment, the side-chain thiol group is a thiol group resulting from the reduction of cysteine.

[0133] In one implementation, thiol reducing agents are used to chemically modify erythrocytes.

[0134] In one implementation, the thiol reducing agent is TCEP.

[0135] In some embodiments, the final concentration of TCEP is between 0.1 mM and 10 mM, for example, 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 2.5 mM.

[0136] III-2 Lipid particle-erythrocyte conjugate covalently coupled via a linker and its preparation method

[0137] In some embodiments, one or more lipid particles are covalently coupled to erythrocytes via a linker. In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: lipid particle-linker-erythrocyte.

[0138] In some embodiments, the connector can be used to attach one or more lipid particles to erythrocytes (e.g., to erythrocyte membrane proteins) to form a lipid particle-erythrocyte conjugate. In some embodiments, one or more lipid particles attached to one or more connectors can be coupled to erythrocytes. In some embodiments, the connector is a bivalent connector. In some embodiments, the lipid particle-erythrocyte conjugate can be prepared using a connector having reactive functional groups for covalently attaching to erythrocytes and lipid particles. In some embodiments, the connector reacts with nucleophilic groups on both the lipid particles and erythrocyte membrane proteins to form covalent bonds. For example, in some embodiments, the amino group on a lysine residue on an erythrocyte membrane protein can form a chemical bond with a reactive functional group of the connector, which then couples the connector to a thiol group on the lipid particle to prepare the lipid particle-erythrocyte conjugate.

[0139] Nucleophilic groups on erythrocyte membrane proteins include, but are not limited to: (i) N-terminal amino groups, (ii) side-chain amino groups, such as the amino group of lysine, (iii) side-chain thiol groups, such as the thiol group of cysteine, and (iv) glycosyl or amino groups (in the case of glycosylation), such as amino groups on the side chain. The amino, thiol, and hydroxyl groups are nucleophilic and capable of reacting with linker moieties and electrophilic groups on the linkers to form covalent bonds with: (i) active esters, such as NHS esters, HOBt esters, halocarbamates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehydes, ketones, carboxyl groups, and maleimide groups.

[0140] In some embodiments, the nucleophilic group of the erythrocyte is an N-terminal amino group or a side-chain amino group (e.g., the amino group of lysine), and / or the nucleophilic group on the lipid particle is a side-chain thiol group (e.g., the thiol group of free cysteine, or the thiol group exposed after disulfide bond reduction).

[0141] In some embodiments, lipid particles, such as LNP, comprise lipids containing thiol groups (SH), and optionally one or more other lipids suitable for lipid particles. In some embodiments, the amount of the thiol-containing lipids in the lipid particles is 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%). In another embodiment, the thiol-containing (SH) lipids suitable for the lipid particles of the present invention include, but are not limited to, one or more of the following: ionizable lipids containing thiol groups, auxiliary lipids containing thiol groups, steroids containing thiol groups, and / or PEG lipids containing thiol groups.

[0142] In another embodiment, the thiol-containing lipid suitable for the lipid particles of the present invention is a thiol-containing PEG lipid, preferably a thiol-containing DSPE-PEG lipid, and most preferably a thiol-containing DSPE-PEG(2000), such as DSPE-PEG(2000)-SH.

[0143] In another embodiment, the lipid particles of the present invention, such as LNP, comprise DSPE-PEG(2000)-SH, and optionally one or more other lipids suitable for lipid particles. In some embodiments, the DSPE-PEG(2000)-SH content in the lipid particles, such as LNP, is 0.1%-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%).

[0144] In one embodiment, the DSPE-PEG(2000)-SH of this disclosure has the following molecular structure:

[0145] In another embodiment, the lipid particles of the present invention, such as LNP, are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; DMG-PEG2000.

[0146] In another embodiment, the lipid particles of the present invention, such as LNP, are composed of the following five lipids: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; and DMG-PEG2000.

[0147] In another embodiment, the lipid particles of the present invention, such as LNP, are composed of the following five components: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-SH; and DMG-PEG2000, wherein the molar ratio of MC3 or SM102 is 40-60%, cholesterol is 30-45%, DSPC is 5-15%, DSPE-PEG2000-SH is 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%), and DMG-PEG2000 is 0. 0.1%-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%); for example, MC3 or SM102 molar ratio is approximately 50%, cholesterol = approximately 38.5%, DSPC = approximately 10%, DSPE-PEG2000-SH = approximately 0.75%, DMG-PEG2000 = approximately 0.75%; for example, MC3 or SM102 molar ratio is 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-SH = 0.75%, DMG-PEG2000 = 0.75%.

[0148] In one embodiment, the connector has functional groups capable of reacting with thiol groups present on the lipid particles to form covalent bonds to the lipid particles. Non-limiting exemplary examples of such reactive functional groups include maleimide, haloacetamide, α-haloacetyl, reactive esters such as succinimide ester, 4-nitrophenyl ester, pentafluorophenyl ester, tetrafluorophenyl ester, acid anhydride, acyl chloride, sulfonyl chloride, isocyanate, and isothiocyanate, preferably, maleimide (MAL).

[0149] In some embodiments, the connector has functional groups that can form covalent bonds with the amino group (-NH2) on lysine residues in erythrocytes to attach to the erythrocytes. Such reactive functional groups, without limitation or exemplarity, include, but are not limited to, reactive esters such as NHS esters, HOBt esters, halocarbamates, and acid halides, preferably, N-hydroxysuccinimide esters (NHS esters).

[0150] The linker may comprise one or more linker components. Exemplary linker components include 6-maleimide hexanoyl (“MC”), maleimide propionyl (“MP”), p-aminobenzyloxycarbonyl (“PAB”), and NHS esters such as N-succinimide 4-(2-pyridylthio)valerate (“SPP”) and 4-(N-maleimide methyl)cyclohexane-1-carboxylate (“MCC”). The linker may also comprise an amino acid, such as one or more glycines. Various linker components are known in the art.

[0151] In some embodiments, the linker can be attached to lipid particles by reacting with the thiol group of free cysteine ​​residues in the lipid particles, and to erythrocytes by reacting with the amino group (-NH2) on lysine residues in erythrocyte membrane proteins.

[0152] Exemplary connectors include, but are not limited to:

[0153] Bis-maleimide-trioxanediol (BMPEO), N-(β-maleimidepropoxy)-N-hydroxysuccinimide ester (BMPS), N-(ε-maleimidehexanoyloxy)succinimide ester (EMCS), N-[γ-maleimidebutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinyl sulfone (HBVS), succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), m-maleimidebenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidephenyl)butyrylhydrazine (MPBH), 3-(bromoacetamido)propionate succinimide ester (SB) AP), iodoacetyl succinimide (SIA), (4-iodoacetyl)aminobenzoic acid succinimide (SIAB), N-succinimide-3-(2-pyridyl dithio)propionate (SPDP), N-succinimide-4-(2-pyridyl thio)valerate (SPP), 4-(N-maleimide methyl)cyclohexane-1-carboxylic acid succinimide (SMCC), NHS-(PEG)n-MAL (where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more, such as NHS-P EG2-MAL, NHS-PEG3-MAL, NHS-PEG6-MAL, NHS-PEG8-MAL, NHS-PEG24-MAL), 4-(p-maleimide phenyl)butyrate succinimide (SMPB), succinimide-6-[(β-maleimide propamido)hexanoate] (SMPH), iminothione (IT), sulfonyl-EMCS, sulfonyl-GMBS, sulfonyl-KMUS, sulfonyl-MBS, sulfonyl-SIAB, sulfonyl-SMCC and sulfonyl-SMPB, and (4-vinyl sulfone)benzoate succinimide (SVSB), and including bis-maleimide reagents (such as dithiobismaleimide ethane (DTME), 1, 4-Bismaleimide butane (BMB), 1,4-bismaleimide-2,3-dihydroxybutane (BMDB), bismaleimide hexane (BMH), bismaleimide ethane (BMOE), BM(PEG)2 and BM(PEG)3), bifunctional derivatives of imino esters (such as dimethyl diimide adipate hydrochloride), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), bis-diazo derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).In some embodiments, the bismaleimide reagent allows the thiol group of a cysteine ​​residue in the antibody to be linked to a thiol-containing drug moiety, a linker, or a linker-drug intermediate. Other functional groups that react with the thiol group include, but are not limited to, iodoacetamide, bromoacetamide, vinylpyridine, disulfides, pyridyl disulfides, isocyanates, and isothiocyanates.

[0154] In some embodiments, the connector is 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester or its water-soluble analogue, such as sulfon-SMCC (Sulfo-SMCC), or SMCC-GGG, or succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), or NHS-(PEG)n-MAL (where n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more), such as NHS-PEG2-MAL or NHS-PEG. 24 -MAL.

[0155] In some embodiments, the connector is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), or sulfon-SMCC, preferably sulfon-SMCC.

[0156] In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: LNP-DSPE-PEG(2000)-S-SMCC-RBC, wherein...

[0157] LNP-DSPE-PEG(2000) refers to any LNP as defined herein that contains DSPE-PEG(2000)-SH, wherein optionally the amount of DSPE-PEG(2000)-SH in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined herein (e.g., lipids suitable for lipid particles as described herein);

[0158] RBC refers to any red blood cell as defined in this article;

[0159] SMCC refers to SMCC or its derivatives, such as LC-SMCC, SMCC, or sulfon-SMCC.

[0160] -S- refers to the group containing S formed by the reaction of the thiol group (SH) in LNP with the maleimide group of the linker SMCC, where the S atom comes from the thiol group of lipid DSPE-PEG(2000)-SH in LNP.

[0161] Optionally, multiple lipid particles can be coupled to the RBC, wherein the lipid particles may be the same or different.

[0162] In some embodiments, the present invention also provides a method for preparing lipid particle-erythrocyte conjugates covalently coupled via a linker, comprising mixing erythrocytes linked to the linker and lipid particles containing thiol groups (SH) such that the two are covalently coupled via the linker, for example comprising...

[0163] (1) Reacting nucleophilic groups (e.g., NH2 on lysine residues) on erythrocyte membrane proteins with a divalent linker reagent to covalently form linker-connected erythrocytes. In some embodiments, the linker reagent, such as SMCC or sulfo-SMCC, has a final concentration between 0.1 mM and 10 mM, for example, 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 0.5 mM; preferably, the erythrocytes are collected and concentrated.

[0164] (2) Preload lipids containing thiol groups (SH) into lipid particles;

[0165] (3) Mix the red blood cells connected to the adapter in (1) and the lipid particles obtained in (2) so that the two are covalently coupled through the adapter; in some embodiments, the ratio of the lipid particles to red blood cells is 0.1-1000 μg of lipid particles per 1 mL of red blood cells.

[0166] (4) Collect the lipid particle-erythrocyte conjugate obtained in (3).

[0167] In another embodiment, the thiol-containing (SH) lipid is selected from one or more of the following: ionizable lipids containing thiol groups, auxiliary lipids containing thiol groups, steroids containing thiol groups, and / or PEG lipids containing thiol groups.

[0168] In another embodiment, the thiol-containing lipid is a thiol-containing PEG lipid, preferably a thiol-containing DSPE-PEG lipid, and most preferably a thiol-containing DSPE-PEG(2000), such as DSPE-PEG(2000)-SH.

[0169] In another embodiment, the thiol-containing lipid is coupled to a membrane protein on a red blood cell via a linker, such as a lysine-containing membrane protein on the red blood cell, preferably a mature red blood cell, such as an adult natural red blood cell. In another embodiment, the thiol-containing lipid is linked to an amino group on a red blood cell via a linker, preferably a mature red blood cell, such as an adult natural red blood cell.

[0170] In another embodiment, the connector has functional groups that can react with thiol groups on lipids containing thiol groups to form covalent bonds to the LNP, and its functional groups can react with nucleophilic groups on erythrocyte membrane proteins to connect to erythrocytes.

[0171] In another embodiment, the nucleophilic groups on erythrocyte membrane proteins include, but are not limited to: (i) N-terminal amino groups, (ii) side-chain amino groups, such as the amino group of lysine, (iii) side-chain thiol groups, such as the thiol group of cysteine, and (iv) glycosyl or amino groups (in the case of glycosylation), such as amino groups on the side chain. The amino, thiol, and hydroxyl groups are nucleophilic and capable of reacting with electrophilic groups on the linker moiety and linker reagent to form covalent bonds including: (i) active esters, such as NHS esters, HOBt esters, halocarbamates, and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups.

[0172] In another embodiment, the nucleophilic group on the erythrocyte membrane protein is -NH2 of the lysine side chain, and the functional group of the linker is capable of reacting with -NH2 of the lysine side chain on the erythrocyte membrane protein to attach to the erythrocyte.

[0173] In another embodiment, the connector is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), or sulfon-SMCC, preferably sulfon-SMCC.

[0174] In one embodiment, the final concentration of the connector, such as SMCC or sulfo-SMCC, is between 0.1 mM and 10 mM, for example, 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 0.5 mM.

[0175] In one embodiment, the nucleophilic group on the erythrocyte membrane protein is an N-terminal amine group or a side-chain amine group.

[0176] III-3. Lipid particle-erythrocyte conjugates covalently coupled to recognition motifs via sorting enzymes and their preparation methods.

[0177] In some embodiments, the erythrocytes of the present invention react with the sorting enzyme recognition motif in the lipid particles to form a covalent coupling. In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: lipid particle-connector-erythrocyte.

[0178] In some embodiments, RBCs are mature red blood cells (e.g., natural adult red blood cells) obtained by treatment with a reducing agent such as a thiol reducing agent as described above, which contain cysteine ​​residues containing a thiol group, for example, in the extracellular domain of at least one of its endogenous membrane proteins (e.g., at an internal site of the extracellular domain) where the disulfide bond is reduced to have a free thiol group.

[0179] In some embodiments, lipid particles, such as LNPs, are preloaded with or contain lipids containing sorting enzyme recognition motifs, and optionally one or more other lipids suitable for lipid particles. In some embodiments, the amount of the lipid containing the sorting enzyme recognition motif in the lipid particle is 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%).

[0180] In yet another embodiment, the lipids containing a sorting enzyme recognition motif suitable for the lipid particles of the present invention include, but are not limited to, one or more of the following: ionizable lipids containing a sorting enzyme recognition motif, auxiliary lipids containing a sorting enzyme recognition motif, steroids containing a sorting enzyme recognition motif, and / or PEG lipids containing a sorting enzyme recognition motif. In yet another embodiment, the lipids containing a sorting enzyme recognition motif suitable for the lipid particles of the present invention are PEG lipids containing a sorting enzyme recognition motif, preferably DSPE-PEG containing a sorting enzyme recognition motif, and most preferably DSPE-PEG (2000) containing a sorting enzyme recognition motif.

[0181] The term "sorting enzyme," also known as a transaminas, refers to an enzyme possessing transaminasal activity. Transaminases typically catalyze the formation of a peptide bond (amide bond) between an acyl donor and a nucleophilic acyl acceptor. Sorting enzymes recognize substrates containing a sorting enzyme recognition motif, such as the amino acid sequence LPSTG. The sorting enzyme cleaves the recognition motif between the residues threonine and glycine. Molecules recognized by a sorting enzyme (i.e., containing a sorting enzyme recognition motif) are sometimes referred to herein as "sorting enzyme substrates." Triglycine and even diglycine motifs at the N-terminus have been shown to be sufficient to support SrtA reactions (Clancy, KW et al., Peptide Science 94 (2010) 385-396). Suitable sorting enzymes will be apparent to those skilled in the art, including, but not limited to, sorting enzyme A, sorting enzyme B, sorting enzyme C, and sorting enzyme D. The amino acid sequences of the sorting enzymes and the nucleotide sequences encoding them are known to those skilled in the art. In one particular embodiment, the sorting enzyme is Staphylococcus aureus sorting enzyme A. In the reaction, firstly, sorting enzyme A recognizes a substrate containing an LPXTG amino acid sequence motif and cleaves the amide bond between Thr and Gly via the active site Cys to generate a sorting enzyme A-substrate thioester intermediate. Then, this thioester acyl-enzyme intermediate is decomposed by nucleophilic attack of the amino group of a second substrate containing oligoglycine, generating a covalently linked conjugate molecule and regenerating sorting enzyme A.

[0182] For enzymatic conjugation, soluble truncated sorting enzyme A lacking a transmembrane region can be used, such as the truncated SrtA containing 60-206 amino acid residues for Staphylococcus aureus. The sorting enzyme A-mediated reaction results in the linkage of a molecule containing a sorting enzyme recognition sequence (sorting motif) to a molecule containing a sorting enzyme acceptor sequence (e.g., one or more N-terminal glycine residues).

[0183] In some embodiments, the present invention contemplates the use of variants of naturally occurring sorting enzymes. A wealth of structural information is available regarding sorting enzymes such as sorting enzyme A, including NMR or crystal structures of SrtA alone or in combination with a sorting enzyme recognition sequence (see, for example, Zong Y et al., J. Biol Chem. 2004, 279, 31383-31389). The active site and substrate-binding pocket of Staphylococcus aureus SrtA have been determined. Functional variants can be generated by, for example, by deletion or substitution of the active site or substrate-binding pocket without disrupting or significantly altering the sorting enzyme. In some embodiments, directed evolution of SrtA can be performed using a FRET (fluorescence resonance energy transfer)-based selection assay as described by Chen et al., Sci. Rep. 2016, 6(1), 31899. In some embodiments, functional variants of Staphylococcus aureus SrtA may be those described in CN10619105A and CN109797194A. In some implementations, the Staphylococcus aureus SrtA variant can be a truncated variant, for example (compared to wild-type Staphylococcus aureus SrtA) removing 25-60 (e.g., 30, 35, 40, 45, 50, 55, 59 or 60) amino acids from the N-terminus.

[0184] In some embodiments, the functional variant of *Staphylococcus aureus* SrtA used in this invention may be a *Staphylococcus aureus* SrtA variant comprising one or more mutations of D124G, Y187L, E189R, and F200L at the amino acid positions of D124, Y187, E189, and F200, and optionally also comprising one or more mutations of P94S / R, D160N, D165A, K190E, and K196T. In some embodiments, the aforementioned mutated amino acid positions are numbered according to the wild-type *Staphylococcus aureus* SrtA number. In some embodiments, a variant of sortase A with higher transaminastic activity than naturally occurring sortase A may be used. In some embodiments, the activity of the sortase A variant is at least about 10, 15, 20, 40, 60, 80, 100, 120, 140, 160, 180, or 200 times that of the wild-type *Staphylococcus aureus* sortase. In some embodiments, this sorting enzyme variant is used in the compositions or methods of the present invention. In some embodiments, relative to wild-type Staphylococcus aureus SrtA, the sorting enzyme variant comprises any one or more of the following substitutions: P94S / R, E105K, E108A, E108Q, D124G, D160N, D165A, Y187L, E189R, K190E, K196T, and F200L mutations. In some embodiments, the SrtA variant may have 25-60 amino acids (e.g., 30, 35, 40, 45, 50, 55, 59, or 60) removed from the N-terminus.

[0185] In some embodiments, the sorting enzyme variant may also contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conserved amino acid mutations. Conserved amino acid mutations that do not significantly affect protein activity are well known in the art.

[0186] In one specific embodiment, the sorting enzyme is a variant of Staphylococcus aureus transpeptidase A (mgSrtA). In some embodiments, the sorting enzyme comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1, or is composed of said amino acid sequence. In some embodiments, the nucleic acid encoding the sorting enzyme comprises the nucleotide sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleotide sequence shown in SEQ ID NO:2, or is composed of said nucleotide sequence.

[0187] In another embodiment, the sorting enzyme recognizes a motif comprising or composed of LPXTG. Common recognition motifs include, for example, LPKTG, LPATG, and LPNTG. In some embodiments, LPETG is used. However, motifs falling outside this common sequence can also be recognized. For example, in some embodiments, the fourth position of the motif contains “A,” “S,” “L,” or “V” instead of “T,” such as LPXAG, LPXSG, LPLXLG, or LPXVG, such as LPNAG, LPESG, LPELG, or LPEG. In some embodiments, the fifth position of the motif contains “A” instead of “G,” such as LPXTA, such as LPNTA. In some embodiments, the second position of the motif contains “G” or “A” instead of “P,” such as LGXTG or LAXTG, such as LGATG or LAETG. In some embodiments, the first position of the motif contains “I” or “M” instead of “L,” such as MPXTG or IPXTG, such as MPKTG, IPKTG, IPNTG, or IPETG. Pishesha et al. (2018) described various recognition motifs for sorting enzyme A.

[0188] In some embodiments, the sorting enzyme recognition sequence is LPXTG, where X is a standard or non-standard amino acid. In some embodiments, X is selected from D, E, A, N, Q, K, or R. In some embodiments, the recognition sequence is selected from LPXTG, LPXAG, LPXSG, LPXLG, LPXVG, LGXTG, LAXTG, LSXTG, NPXTG, MPXTG, IPXTG, SPXTG, VPXTG, YPXRG, LPXTS, and LPXTA, where X can be any amino acid, for example, in some embodiments, an amino acid selected from D, E, A, N, Q, K, or R. In one specific embodiment, the sorting enzyme recognition motif provided in this application is LPETG. In one embodiment, the sorting enzyme recognition motif can be modified to improve its recognition efficiency; preferably, LPETG is modified to improve its affinity for the sorting enzyme, for example, by adding G to the C-terminus of the recognition sequence, for example, the modified sequence is LPETGG.

[0189] In one embodiment, the lipid particles of the present invention, such as LNP, further comprise DSPE-PEG(2000) containing a sorting enzyme recognition motif, wherein the sorting enzyme recognition motif is LPETG. In yet another embodiment, the sorting enzyme recognition motif may be modified to improve its affinity; preferably, the modification is the addition of G, such as LPETGG, to the C-terminus of the sorting enzyme recognition motif.

[0190] In yet another embodiment, the lipids containing sorting enzyme recognition motifs suitable for the lipid particles of the present invention, such as LNP, comprise or consist of DSPE-PEG(2000)-LPETG.

[0191] In yet another embodiment, the lipid particles of the present invention, such as LNPs, comprise DSPE-PEG(2000)-LPETG, and optionally one or more other lipids suitable for lipid particles. In some embodiments, the DSPE-PEG(2000)-LPETG content in the lipid particles, such as LNPs, is 0.1%-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%).

[0192] In one embodiment, the DSPE-PEG(2000)-LPETG of the present invention has the following molecular structure:

[0193] In yet another embodiment, the lipid particles of the present invention, such as LNP, are selected from MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; DMG-PEG2000.

[0194] In yet another embodiment, the lipid particles of the present invention, such as LNP, are composed of the following five lipids: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; and DMG-PEG2000.

[0195] In yet another embodiment, the lipid particles of the present invention, such as LNP, are composed of the following five components: MC3 or SM102; cholesterol; DSPC; DSPE-PEG2000-LPETG; and DMG-PEG2000, wherein the molar ratio of MC3 or SM102 is 40-60%, cholesterol is 30-45%, DSPC is 5-15%, DSPE-PEG2000-LPETG is 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%), and DMG-PEG2000 is 0. 0.1%-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%); for example, MC3 or SM102 molar ratio is approximately 50%, cholesterol = approximately 38.5%, DSPC = approximately 10%, DSPE-PEG2000-LPETG = approximately 0.75%, DMG-PEG2000 = approximately 0.75%; for example, MC3 or SM102 molar ratio is 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-LPETG = 0.75%, DMG-PEG2000 = 0.75%.

[0196] In yet another embodiment, the sorting enzyme recognition motif is conjugated to the extracellular portion of a erythrocyte membrane protein via a linker. In one embodiment, the linker comprises a G peptide and a maleimide alkyl chain (C2-8), preferably the maleimide alkyl chain (C2-8) is conjugated to the erythrocyte membrane protein, and / or the G peptide is conjugated to a lipid containing the sorting enzyme recognition motif via a sorting enzyme-mediated reaction.

[0197] In yet another embodiment, the G-peptide is a linear or branched peptide. In still another embodiment, the G-peptide is a branched peptide comprising two or more branching units, wherein one or more lipid particles are coupled to one or more branching units. In a specific embodiment, the branching units have the same structure.

[0198] In another embodiment, the branching unit consists of an amino acid sequence K (GGG), wherein glycine in parentheses is conjugated with the ε-amino group of the side chain of lysine to form a branch, and lysine forms a peptide bond with other amino acids through its α-amino group to form the backbone of the "G peptide". Optionally, an extension chain, such as COCH2CH2-PEG6-NH, may be added between K and G in the branching unit K (GGG).

[0199] In yet another embodiment, the G-peptide has the following structure: GGGSK, K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) or K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-NH2, wherein the glycine in parentheses is conjugated with the ε-amino group of the lysine side chain to form a branched chain. In yet another embodiment, the G-peptide has the following structure: K(GGG)-GGG-K(GGG)-GGG-K(GGG).

[0200] In yet another embodiment, the maleimide-alkyl chain (C 2-8 It is 6-maleimide hexanoic acid or 4-maleimide butyric acid, preferably 6-maleimide hexanoic acid.

[0201] In yet another embodiment, (PEG)n is present between the G peptide and 6-maleimide hexanoic acid, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more.

[0202] In a preferred embodiment, the small peptide is conjugated to a lipid particle containing a sorting enzyme recognition motif via an oligoglycine residue in its branched chain, mediated by a sorting enzyme. In a specific embodiment, multiple identical or different lipid particles are conjugated to a linker containing multiple branched units via the above reaction. In one embodiment, the linker is linked to a membrane protein of the erythrocyte via its maleimide alkyl chain (C2-8) and to a lipid particle containing a sorting enzyme recognition motif via a sorting enzyme-mediated reaction using its G-containing small peptide. In a specific embodiment, multiple identical or different lipid particles are simultaneously conjugated to the erythrocyte via this linker. Preferably, at least two lipid particles are simultaneously conjugated to the erythrocyte. In one embodiment, the lipid particles conjugated to the erythrocyte are identical.

[0203] Specifically, when the linker contains only one oligoglycine (e.g., GGG) that can react with lipid particles containing sorting enzyme recognition sequences, the G-containing peptide is called a G1 peptide; when the linker contains two oligoglycines that can react with lipid particles containing sorting enzyme recognition sequences, the G-containing peptide is called a G2 peptide; when the linker contains three oligoglycines that can react with lipid particles containing sorting enzyme recognition sequences, the G-containing peptide is called a G3 peptide; and so on, yielding G4, G5, and so on. Linkers containing the corresponding peptides can also be designated G1, G2, G3, G4, and G5. G6 is a G-peptide with extended branching (via PEG10) and containing three linkers. When two or more oligoglycines can react with lipid particles containing sorting enzyme recognition sequences, they each constitute a branched unit of the G-containing peptide, and the resulting linker is called a branched linker.

[0204] In a preferred embodiment, the connector has the structure shown below (G3):

[0205] In one embodiment, the sorting enzyme recognition motif is conjugated to the extracellular portion of a erythrocyte membrane protein via the aforementioned G3 structure.

[0206] In yet another implementation, multiple lipids containing sorting enzyme recognition motifs are conjugated to erythrocyte membrane proteins via branched connectors.

[0207] In yet another embodiment, the DSPE-PEG(2000) is modified to contain a sorting enzyme recognition motif.

[0208] In yet another embodiment, the DSPE-PEG(2000) can be linked to a sorting enzyme recognition motif via a flexible peptide (GS)n, where n = 1-10.

[0209] In some embodiments, the lipid particle-erythrocyte conjugate has the following structure: LNP-DSPE-PEG(2000)-LPETG-linker-RBC, preferably LNP-DSPE-PEG(2000)-LPETG-(G3)-RBC, wherein

[0210] LNP-DSPE-PEG(2000)-LPETG refers to any LNP as defined herein that contains DSPE-PEG(2000)-LPETG, wherein the amount of DSPE-PEG(2000)-LPETG in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined herein (e.g., lipids suitable for lipid particles as described herein);

[0211] RBC refers to any red blood cell as defined herein, such as red blood cells treated with reducing agents, such as red blood cells treated with thiol reducing agents;

[0212] The linker is any linker as defined herein that comprises a G peptide and a maleimide alkyl chain (C2-8) (e.g., 6-maleimide hexanoic acid or 4-maleimide butyric acid), preferably a linker having the structure as described above (G3).

[0213] The DSPE-PEG(2000)-LPETG is attached to the erythrocyte membrane (e.g., the extracellular domain of a membrane protein) via a linker.

[0214] In some embodiments, multiple lipid particles can be covalently linked to the RBC, wherein the lipid particles may be identical or different, for example, by attaching multiple lipid particles to a linker molecule having a branching unit, and / or by attaching multiple lipid particles to a red blood cell by a linker molecule having a branching unit. In some embodiments, the lipid particles attached to the RBC may also be attached to a red blood cell by multiple linker molecules having branching units.

[0215] The present invention also provides a method for preparing a lipid particle-erythrocyte conjugate covalently coupled to a sorting enzyme recognition motif, comprising mixing erythrocytes linked to a linker and lipid particles containing a sorting enzyme recognition motif in the presence of a sorting enzyme and under conditions suitable for the sorting enzyme to react, such that the sorting enzyme conjugates the lipid particles to the erythrocytes via the linker, for example including:

[0216] (1) Treating red blood cells with a reducing agent to allow adaptor molecules to attach to the extracellular domains of endogenous membrane proteins in red blood cells; and collecting and concentrating red blood cells;

[0217] (2) Treat the lipid particles so that they contain sorting enzyme recognition motifs;

[0218] (3) In the presence of a sorting enzyme, the red blood cells obtained in step 1) are brought into contact with the lipid particles obtained in step 2) under conditions suitable for the sorting enzyme to react, so that the sorting enzyme attaches the LNP to the endogenous membrane protein of the red blood cells through a second linker; in some embodiments, the ratio of the lipid particles to red blood cells is 0.1-1000 μg of lipid particles per 1 mL of red blood cells.

[0219] In one embodiment, red blood cells are treated with a reducing agent such that disulfide bonds in the extracellular domain of at least one endogenous membrane protein of the red blood cell (e.g., at an internal site of the extracellular domain) are reduced to have free thiol groups. In a specific embodiment, the adapter molecule is linked to the free thiol groups in the extracellular domain of the endogenous membrane protein of the red blood cell via its contained 6-maleimide hexanoic acid. In a specific embodiment, the lipid particles are linked to the G-containing peptide in the adapter molecule via a sorting enzyme recognition motif LPXTG, followed by a transamidation reaction of the sorting enzyme to form a lipid particle-LPXT-adaptor structure. In a specific embodiment, multiple lipid particles are conjugated to the adapter via adapter molecules having branched units. In a more specific embodiment, multiple lipid particles are conjugated to red blood cells via adapter molecules having branched units.

[0220] In some implementations, methods for processing red blood cells, adapters, and sorting enzymes can be found in WO2024 / 067295, which is incorporated herein by reference.

[0221] IV. Payload

[0222] In some embodiments, the lipid particles comprise one or more payloads. In some embodiments, the payload is a nucleic acid. In some embodiments, the payload is a nucleic acid, such as DNA and / or RNA.

[0223] In some embodiments, the lipid particles of the present invention, such as LNPs, contain one or more selected from miRNA, siRNA, dsDNA, or mRNA.

[0224] In some embodiments, the mass ratio of lipids to nucleic acids in the lipid particles of the present invention, such as LNP, can be any suitable ratio, for example, 5:1 to 50:1.

[0225] In some embodiments, the lipid particles of the present invention, such as LNP, have a lipid-to-nucleic acid mass ratio of 10:1 to 30:1.

[0226] In some embodiments, the lipid particles of the present invention, such as LNP, have a lipid-to-nucleic acid mass ratio of about 5:1, 10:1, 15:1, 20:1, 25:1, or 30:1.

[0227] In some implementations, the encapsulation efficiency of nucleic acids in lipid particles, such as LNPs, is greater than 50%, for example, the encapsulation efficiency can be about 55%, 60%, 65%, 70%, 75%, 79%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.

[0228] In some embodiments, the lipid particle-erythrocyte conjugate has 10-10000 nucleic acid molecules / RBC, for example, about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 or more nucleic acid molecules / RBC, with 9-270 nucleic acid molecules / RBC being the most preferred.

[0229] In one embodiment, the one or more payloads comprise or are RNA, wherein the RNA is selected from messenger RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antisense RNA, ribozymes, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA, circular RNA, tRNA, and / or combinations thereof.

[0230] In one embodiment, the payload is mRNA, preferably the mRNA encoding a therapeutic protein. In one embodiment, the RNA comprises a 5'-UTR, the mRNA of the target gene, and a 3'-UTR and Poly A linked in sequence. In one embodiment, the RNA is modified RNA.

[0231] In one embodiment, the one or more payloads comprise or are DNA, such as circular DNA, such as plasmids, or linear DNA, such as antisense DNA.

[0232] In some embodiments, the lipid particle-erythrocyte conjugate has a concentration greater than about 0.01 μg mRNA / mL RBC, for example, about 0.01-300 μg mRNA / mL RBC, 0.05-200 μg mRNA / mL RBC, 0.05-100 μg mRNA / mL RBC, 0.05-50 μg mRNA / mL RBC, or 0.08-10 μg mRNA / mL RBC, with a most preferred concentration of 0.1-3 μg mRNA / mL RBC.

[0233] In some implementations, nucleic acid molecules encapsulated in lipid particles express therapeutic proteins.

[0234] In some embodiments, the nucleic acid molecules encapsulated in the lipid particles can express therapeutic proteins in splenic immune cells. In some embodiments, the therapeutic proteins include proteins that effectively treat diseases, such as receptors, ligands, antibodies, or their antigen-binding fragments or chimeric antigen receptors. In some embodiments, the proteins may be immunomodulators, such as proteins that can be used to treat tumors, or proteins that can be used to treat autoimmune diseases.

[0235] In some embodiments, the therapeutic protein is a chimeric antigen receptor. In one embodiment, the chimeric antigen receptor includes an extracellular binding region that specifically binds to an antigen, such as a tumor-associated antigen.

[0236] V. Pharmaceutical compositions or combination products

[0237] In one aspect, the present invention provides pharmaceutical compositions or formulations comprising lipid particle conjugates. These compositions or formulations may also optionally comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0238] In one aspect, the present invention provides blood preparations, such as human blood preparations, comprising the lipid particle conjugates of the present invention. In some embodiments, the human blood preparations of the present invention comprise 10-1000 μg / mL of lipid particle conjugates, for example 10-500 μg / mL, for example at 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or more, or any range of said values.

[0239] In some embodiments, the blood product of the present invention is a leukocyte-reducing blood product, i.e., a blood product from which leukocytes have been filtered out. In some embodiments, the blood product of the present invention is a human blood leukocyte-reducing blood product.

[0240] In some embodiments, the blood product of the present invention may be an allogeneic blood product, such as an allogeneic human blood product. In some embodiments, the red blood cells in the blood product are derived from healthy subjects. In some embodiments, the red blood cells in the blood product may be mature red blood cells differentiated from stem cells (e.g., obtained by culturing stem cells to differentiate into mature red blood cells).

[0241] In some embodiments, the blood product of the present invention may be an autologous blood product, such as an autologous human blood product. In some embodiments, the red blood cells in the blood product are derived from the subject to be treated.

[0242] In one aspect, the present invention also provides combination products (e.g., pharmaceutical combination products) comprising the lipid particle conjugate of the present invention, and one or more other therapeutic agents. The combination products of the present invention can be used in the treatment methods of the present invention.

[0243] The present invention also provides a complete set of medicine boxes comprising the combined products, for example, the complete set of medicine boxes comprising, within the same package:

[0244] - A first container containing the lipid particle conjugate of the present invention or a pharmaceutical composition or formulation comprising the present invention;

[0245] - A second container containing one or more other therapeutic agents or pharmaceutical compositions or preparations containing them.

[0246] In some embodiments, the therapeutic agent is selected from any substance effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants or immune agonists). Preferably, the therapeutic agent is selected from tumor vaccines, immune checkpoint inhibitor antibodies, or immune agonist antibodies.

[0247] VII. Uses and Therapies

[0248] The present invention also provides methods for treating diseases, activating the immune system, or modifying immune cells in the spleen, such as myeloid cells like CD11b+ myeloid cells, using the lipid particle-erythrocyte conjugate of the present invention, including administering the lipid particle-erythrocyte conjugate of the present invention, or a drug or human blood preparation containing the conjugate, to a subject.

[0249] In some implementations, the disease will benefit from immune system activation or from the modification of CD11b+ myeloid cells.

[0250] In some implementations, the modification of CD11b+ myeloid cells refers to the introduction of nucleic acid drugs contained in lipid particles into CD11b+ myeloid cells by red blood cells, such as the expression of mRNA in CD11b+ myeloid cells.

[0251] In some implementations, the disease is, for example, a tumor or an immune system disease.

[0252] In some embodiments, the tumor is cancer. In some embodiments, the tumor is a tumor that is positive for tumor antigen (TA) or tumor-associated antigen (TAA). In one embodiment, the TA-positive tumor refers to the presence of TA expression or TA nucleic acid in an individual's tumor tissue or tumor cells (e.g., cancerous tissue or cancer cells). In one embodiment, the TA or TAA-positive tumor refers to the presence of TAA expression or TAA nucleic acid in an individual's tumor tissue or tumor cells (e.g., cancerous tissue or cancer cells), for example, compared to an individual's adjacent normal tissue or normal cells (e.g., normal cells in tissue) or the same tissue or cells in a healthy individual, or compared to TAA protein levels (e.g., expression), or TAA nucleic acid levels; or compared to TAA activity.

[0253] In some implementations, the disease is an immune system-related disease, such as an autoimmune disease.

[0254] In some embodiments, the lipid particle-erythrocyte conjugate of the present invention can be used to modify immune cells in the spleen, such as CD11b+ myeloid cells, for example, by administering the conjugate to an individual. In some embodiments, immune cells in the spleen containing the lipid particle-erythrocyte conjugate of the present invention, such as myeloid cells like CD11b+ myeloid cells, migrate to lesions such as tumor lesions and are able to specifically recognize and phagocytose tumor cells (e.g., TAA-positive tumor cells) targeted by the nucleic acids contained in the lipid particles, thereby achieving tumor killing.

[0255] In some embodiments, immune cells in the spleen containing the lipid particle-erythrocyte conjugate of the present invention, such as myeloid cells like CD11b+ myeloid cells, can also activate immune cells, such as T cells, in lesions, such as tumor lesions, to achieve tumor killing.

[0256] In some embodiments, the lipid particle-erythrocyte conjugate of the present invention can be used to stimulate the host's immune system, such as enhancing cellular immune responses. "Stimulating the immune system" may include any one or more of the following: overall increased immune function, increased T cell function, increased B cell function, restored lymphocyte function, increased IL-2 receptor expression, increased T cell responsiveness, increased T cell activity or natural killer cell activity or lymphokine-activated killer (LAK) cell activity, increased T cell or natural killer cell survival, increased expression of cytotoxic effector proteins, etc. The lipid particle-erythrocyte conjugate of the present invention (and compositions, pharmaceutical compositions, formulations, combination products, etc., including blood products comprising it) can be administered by any suitable method, preferably by infusion or injection, such as parenteral administration. Parenteral administration includes intravenous or intra-arterial administration. Preferably, the lipid particle-erythrocyte conjugate of the present invention is administered by intravenous or intra-arterial infusion.

[0257] For the prevention or treatment of disease, the appropriate dosage of the lipid particle-erythrocyte conjugate of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the lipid particle-erythrocyte conjugate, the severity and progression of the disease, whether it is administered for preventative or therapeutic purposes, previous treatments, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The lipid particle-erythrocyte conjugate is appropriately administered to the patient as a single treatment or after a series of treatments.

[0258] In some embodiments, the lipid particle-erythrocyte conjugate of the present invention, or a drug or formulation comprising it, is administered in combination with one or more therapeutic agents or other therapeutic agents. In some embodiments, the therapeutic agent is radiotherapy or surgical treatment. In some embodiments, the therapeutic agent is selected from any substance effective in cancer, including chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants or immune agonists, such as immunomodulatory proteins). Preferably, the therapeutic agent is selected from tumor vaccines, immune checkpoint inhibitor antibodies, or immune agonist antibodies.

[0259] In other respects, the present invention provides the use of the lipid particle-erythrocyte conjugate of the present invention, or a pharmaceutical or human blood preparation comprising the present invention, in the production or preparation of a pharmaceutical for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0260] In other respects, the present invention provides the lipid particle-erythrocyte conjugate of the present invention, or a pharmaceutical or human blood preparation comprising the present invention, for use as described herein, such as for a therapy, for example for treating the related diseases or conditions mentioned herein.

[0261] In some implementations, the red blood cells in the lipid particle-red blood cell conjugate are derived from the subject to be treated or a healthy subject. Example:

[0262] Example 1: Process Study of mRNA-LNP-RBC

[0263] 1.1 mRNA-LNP-RBC process route selection and formulation screening

[0264] Experimental objective: To evaluate the feasibility of conjugating mRNA-LNP to erythrocytes using different process pathways (chemical coupling or enzyme catalysis).

[0265] Experimental methods:

[0266] mRNA in vitro transcription and purification

[0267] In vitro amplification

[0268] Add a certain amount of DNA template (the amino acid sequence and nucleic acid sequence of each protein are shown in the sequence listing), T7 reaction solution, ATP, GTP, CTP, N1-Me-pseudoUTP, cap analog and T7 polymerase to a clean 1.5mL centrifuge tube according to the kit requirements. Mix well and react at 37℃ for 2 hours.

[0269] Purification and identification

[0270] After the reaction, an appropriate amount of DNase I was added and mixed thoroughly. The reaction was then continued at 37°C for 15 minutes. The resulting mixture was then mixed with an appropriate amount of enzyme-free water and 7.5M LiCl solution and frozen at -20°C for 30 minutes. After 30 minutes, the reaction mixture was removed and centrifuged at 15,000 rpm for 15 minutes at 4°C to obtain mRNA precipitate. The supernatant was discarded, and the precipitate was washed with 500 mL of ice-cold ethanol and centrifuged again at 15,000 rpm for 10 minutes at 4°C to obtain mRNA precipitate once more. The supernatant was discarded, and the mixture was dried at room temperature. An appropriate amount of TE solution was added to dissolve the precipitate to obtain the mRNA product, and the concentration of the mRNA product was measured using a micro-UV-Vis spectrophotometer (DS-11+, Denovix).

[0271] The mRNA product from the previous step can be further purified using NanoGeldT20 packing material according to the manufacturer's requirements to obtain a higher purity mRNA product to meet the requirements of in vivo experiments. The final product is mRNA containing CapG(OMe)AG and N1-Me-pseudoUTP modifications and polyA100.

[0272] The reagents used in preparation routes 1-3 are shown in Table 1.

[0273] Table 1

[0274] Pathway 1: Preparation of mRNA-LNP-RBC-1:

[0275] (a) Preparation and purification of mRNA-LNP

[0276] Weigh appropriate amounts of different lipid powders and dissolve them in anhydrous ethanol at room temperature with shaking, so that the mass concentrations of the lipids are as shown in Table 2.

[0277] Table 2

[0278] Preparation of mRNA-LNP

[0279] In clean centrifuge tubes, the five lipids were mixed according to the specific molar ratios in the formula (MC3 or SM102 = 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-Maleimide = 0.75%, DMG-PEG2000 = 0.75%) and diluted with anhydrous ethanol to the specified concentration. A certain amount of purified mRNA was taken into a clean centrifuge tube and diluted with 10mM, pH=4 citrate-sodium citrate buffer, so that the final volume of the aqueous phase solution containing mRNA was 3 times the volume of the anhydrous ethanol phase containing lipids (V). mRNA :V lipid =3:1). Transfer the ethanol phase and aqueous phase solutions to two clean syringes respectively. Place the syringes in the corresponding positions on the microfluidic device (NWDPSII120 model from Nanomicro Instruments Technology). Attach the sample tube and waste liquid tube to the microfluidic device. Adjust the flow rate to 4 mL / min, the sample volume to 0.4-12 mL, and the initial waste liquid volume to be no less than 0.15 mL. Then start the program and monitor the sample preparation process (the mixing volume ratio of the aqueous phase to the ethanol phase is 3:1). When the program is complete, remove the sample tube. At this point, the solvent in the prepared mRNA-LNP is citrate-sodium citrate buffer containing 25% ethanol.

[0280] Purification and identification of mRNA-LNP

[0281] The prepared mRNA-LNP solution was quickly transferred to a clean centrifuge tube, and 100 times its volume of PBS buffer (pH = 7.4) was added. The solution was then transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000 x g at room temperature. Once the solution volume was reduced to its original volume, another 100 times its volume of PBS buffer was added, and ultrafiltration was performed to the desired volume. At this point, the solvent in the purified mRNA-LNP was PBS buffer at pH = 7.4. An appropriate amount of the mRNA-LNP solution was used for particle size and particle size distribution index (PDI) testing (Zetasizer Lab, Malvern), as well as mRNA concentration and encapsulation efficiency testing (SpectraMaxiD5, Molecular Devices). The results are shown in Table 3.

[0282] Table 3. Detection results of mRNA-LNP under process route 1

[0283] (b) Preparation and purification of mRNA-LNP-RBC-1

[0284] Packed red blood cells (RBCs) after TCEP treatment

[0285] Take an appropriate amount of EDTA-anticoagulated whole blood (mouse or human) into a centrifuge tube, add 10 times the volume of PBS buffer (pH=7.4), mix well, and centrifuge at 900xg for 4 minutes at room temperature. Remove the supernatant, add another 10 times the volume of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Take one aliquot of 0.5M TCEP stock solution, thaw it, and dilute it to 5mM with PBS buffer to obtain a 2X TCEP PBS solution. Add an equal volume of 2X TCEP PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of TCEP 2.5mM. Place the centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Repeat the above washing steps once to obtain TCEP-treated concentrated red blood cells.

[0286] Preparation of mRNA-LNP-RBC-1

[0287] Take an appropriate amount of TCEP-treated concentrated red blood cells obtained in the previous step and add them to a centrifuge tube. Add the corresponding volume of PBS buffer and the purified and characterized mRNA-LNP as described in step (2)(a) above, so that the ratio of mRNA-LNP to concentrated red blood cells is 100 μg mRNA-LNP per 1 mL of concentrated RBC, and the concentration of RBC in the system is 2.5E9 / mL. Place the well-mixed centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix. Centrifuge at 900xg for 4 minutes at room temperature and discard the supernatant. Repeat the above washing steps once to obtain the conjugated mRNA-LNP-RBC-1, i.e., mRNA-LNP-Mal.

[0288] Pathway 2: Preparation of mRNA-LNP-RBC-2:

[0289] (a) Preparation and purification of mRNA-LNP

[0290] Weigh appropriate amounts of different lipid powders and dissolve them in anhydrous ethanol with shaking at room temperature to achieve the following lipid mass concentrations:

[0291] Preparation of mRNA-LNP

[0292] In clean centrifuge tubes, the five lipids were mixed according to the specific molar ratios in the formula (MC3 or SM102 = 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-SH = 0.75%, DMG-PEG2000 = 0.75%) and diluted with anhydrous ethanol to the specified concentration. A certain amount of purified mRNA was placed in a clean centrifuge tube and diluted with 10mM, pH=4 citrate-sodium citrate buffer, so that the final volume of the aqueous phase solution containing mRNA was three times the volume of the anhydrous ethanol phase containing lipids (V). mRNA :V lipid =3:1). Transfer the ethanol phase and aqueous phase solutions to two clean syringes respectively, place the syringes in the corresponding positions on the microfluidic device, and load the sample tube and waste liquid tube into the microfluidic device; adjust the corresponding parameters of the program (flow rate, sample volume, waste liquid volume, etc.) and start the program, monitor the sample preparation process (the mixing volume ratio of aqueous phase to ethanol phase is 3:1), and remove the sample tube when the program is completed. At this time, the solvent in the prepared mRNA-LNP is citrate-sodium citrate buffer containing 25% ethanol.

[0293] Purification and identification of mRNA-LNP

[0294] The prepared mRNA-LNP solution was quickly transferred to a clean centrifuge tube, and 100 times its volume of PBS buffer (pH = 7.4) was added. The solution was then transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000 x g at room temperature. Once the solution volume had reduced to its original volume, another 100 times its volume of PBS buffer was added, and ultrafiltration was performed to the desired volume. At this point, the solvent in the purified mRNA-LNP was PBS buffer at pH = 7.4. An appropriate amount of the mRNA-LNP solution was used for particle size and particle size distribution index (PDI) testing (Zetasizer Lab, Malvern), as well as mRNA concentration and encapsulation efficiency testing (SpectraMaxiD5, Molecular Devices). The results are shown in Table 4.

[0295] Table 4. Detection results of mRNA-LNP under process route 2

[0296] (b) Preparation and purification of mRNA-LNP-RBC-2

[0297] SMCC-treated packed red blood cells

[0298] Take an appropriate amount of EDTA-anticoagulated whole blood (mouse or human) into a centrifuge tube, add 10 volumes of PBS buffer (pH=7.4), mix well, and centrifuge at 900xg for 4 minutes at room temperature. Remove the supernatant, add another 10 volumes of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Take one aliquot of 2 mg sulfo-SMCC powder, dissolve it completely in 1.8 mL of ultrapure water, add 0.2 mL of 10xPBS buffer, mix well to obtain a 1 mg / mL (2.29 mM) SMCC PBS solution. Add an appropriate volume of SMCC PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of SMCC 0.5 mM. Place the centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 volumes of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Repeat the above washing steps once to obtain SMCC-treated concentrated red blood cells.

[0299] Preparation of mRNA-LNP-RBC-2

[0300] Take an appropriate amount of concentrated red blood cells obtained from the previous step after SMCC treatment and add them to a centrifuge tube. Add the corresponding volume of PBS buffer and the purified and characterized mRNA-LNP as described in step (3)(a) above, so that the ratio of mRNA-LNP to concentrated red blood cells is 100 μg mRNA-LNP per 1 mL of concentrated RBC, and the concentration of RBC in the system is 2.5E9 / mL. Place the well-mixed centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix. Centrifuge at 900xg for 4 minutes at room temperature and discard the supernatant. Repeat the above washing steps once to obtain the conjugated mRNA-LNP-RBC-2, i.e., mRNA-LNP-SH.

[0301] Pathway 3: Preparation of mRNA-LNP-RBC-3

[0302] (a) Preparation and purification of mRNA-LNP

[0303] Weigh appropriate amounts of different lipid powders and dissolve them in anhydrous ethanol with shaking at room temperature to achieve the following lipid mass concentrations:

[0304] Preparation of mRNA-LNP

[0305] In clean centrifuge tubes, the five lipids were mixed according to the specific molar ratios in the formula (MC3 or SM102 = 50%, cholesterol = 38.5%, DSPC = 10%, DSPE-PEG2000-LPETG = 0.75%, DMG-PEG2000 = 0.75%) and diluted with anhydrous ethanol to the specified concentration. A certain amount of purified mRNA was taken into a clean centrifuge tube and diluted with 10mM, pH=4 citrate-sodium citrate buffer, so that the final volume of the aqueous phase solution containing mRNA was 3 times the volume of the anhydrous ethanol phase containing lipids (V). mRNA :V lipid =3:1). Transfer the ethanol phase and aqueous phase solutions to two clean syringes respectively, place the syringes in the corresponding positions on the microfluidic device, and load the sample tube and waste liquid tube into the microfluidic device; adjust the corresponding parameters of the program (flow rate, sample volume, waste liquid volume, etc.) and start the program, monitor the sample preparation process (the mixing volume ratio of aqueous phase to ethanol phase is 3:1), and remove the sample tube when the program is completed. At this time, the solvent in the prepared mRNA-LNP is citrate-sodium citrate buffer containing 25% ethanol.

[0306] Purification and identification of mRNA-LNP

[0307] The prepared mRNA-LNP solution was quickly transferred to a clean centrifuge tube, and 100 times its volume of PBS buffer (pH = 7.4) was added. The solution was then transferred to a 100 kDa ultrafiltration tube and centrifuged at 2000 x g at room temperature. Once the solution volume had reduced to its original volume, another 100 times its volume of PBS buffer was added, and ultrafiltration was performed to the desired volume. At this point, the solvent in the purified mRNA-LNP was PBS buffer at pH = 7.4. An appropriate amount of the mRNA-LNP solution was used for particle size and particle size distribution index (PDI) testing (Zetasizer Lab, Malvern), as well as mRNA concentration and encapsulation efficiency testing (SpectraMaxiD5, Molecular Devices). The results are shown in Table 5.

[0308] Table 5. Detection results of mRNA-LNP under process route 3

[0309] (b) Preparation and purification of mRNA-LNP-RBC-3

[0310] TCEP-treated packed red blood cells

[0311] Take an appropriate amount of EDTA-anticoagulated whole blood (mouse or human) into a centrifuge tube, add 10 times the volume of PBS buffer (pH=7.4), mix well, and centrifuge at 900xg for 4 minutes at room temperature. Remove the supernatant, add another 10 times the volume of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Take one aliquot of 0.5M TCEP stock solution, thaw it, and dilute it to 5mM with PBS buffer to obtain a 2X TCEP PBS solution. Add an equal volume of 2X TCEP PBS solution to the concentrated red blood cells obtained in the previous step to make the final concentration of TCEP 2.5mM. Place the centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer, mix, centrifuge at 900xg for 4 minutes at room temperature, and discard the supernatant. Repeat the above washing steps once to obtain TCEP-treated concentrated red blood cells.

[0312] G-peptide-conjugated thrombocytopenic erythrocytes

[0313] Take an appropriate amount of TCEP-treated concentrated red blood cells obtained in the previous step and add them to a centrifuge tube. Add an equal volume of PBS buffer and an appropriate amount of 10 mg / mL (6.90 mM) G peptide solution to make the final concentration of G peptide 0.3125 mM. Place the centrifuge tube on a rotary mixer and react at 37°C for 15 minutes. After the reaction, add 10 times the volume of PBS buffer and mix. Centrifuge at 900 x g for 4 minutes at room temperature and discard the supernatant. Repeat the above washing steps once to obtain concentrated red blood cells conjugated with G peptide.

[0314] Preparation of mRNA-LNP-RBC-3

[0315] Take an appropriate amount of concentrated red blood cells conjugated with the G peptide obtained in the previous step and add the corresponding volume of PBS buffer, purified and characterized mRNA-LNP as described in step (3)(a) above, and mgSortase transpeptidase (SEQ ID No: 1) to a centrifuge tube, so that the ratio of mRNA-LNP to concentrated red blood cells is 100 μg mRNA-LNP per 1 mL of concentrated RBC, the concentration of mgSortase transpeptidase is 10 μM, and the concentration of RBC in the system is 2.5E9 / mL. Place the well-mixed centrifuge tube on a rotary mixer and react at 37°C for 1 hour. After the reaction, add 10 times the volume of PBS buffer and mix. Centrifuge at 900xg for 4 minutes at room temperature and discard the supernatant. Repeat the above washing steps once to obtain the conjugated mRNA-LNP-RBC-3, i.e., mRNA-LNP-LPETG.

[0316] 1.2 Preparation of linkerless mRNA-LNP

[0317] Weigh appropriate amounts of different lipid powders and dissolve them in anhydrous ethanol with shaking at room temperature to achieve the following lipid mass concentrations:

[0318] Preparation of mRNA-LNP

[0319] In clean centrifuge tubes, the four lipids were mixed in the specified molar ratios (MC3 = 50%, cholesterol = 38.5%, DSPC = 10%, DMG-PEG2000 = 1.5%) and diluted with anhydrous ethanol to the specified concentration. A certain amount of purified mRNA was then placed in a clean centrifuge tube and diluted with 10 mM, pH 4 citrate-sodium citrate buffer, so that the final volume of the aqueous phase containing mRNA was three times the volume of the anhydrous ethanol phase containing lipids (V). mRNA :V lipid =3:1). Transfer the ethanol phase and aqueous phase solutions to two clean syringes respectively, place the syringes in the corresponding positions on the microfluidic device, and load the sample tube and waste liquid tube into the microfluidic device; adjust the corresponding parameters of the program (flow rate, sample volume, waste liquid volume, etc.) and start the program, monitor the sample preparation process (the mixing volume ratio of aqueous phase to ethanol phase is 3:1), and remove the sample tube when the program is completed. At this time, the solvent in the prepared mRNA-LNP is citrate-sodium citrate buffer containing 25% ethanol.

[0320] Table 6. Detection results of mRNA-LNP without linkers

[0321] Example 2: In vitro characterization of mRNA-LNP-RBC

[0322] Experimental objective: To investigate the distribution of mRNA-LNP-RBC-1 on the surface of erythrocytes.

[0323] Experimental Methods: Luc-mRNA (encoding Luciferase mRNA, Luciferase amino acid sequence: SEQ ID NO:3; nucleotide sequence: SEQ ID NO:4) was labeled using the same HiSynthT7 Co-transcription RNA synthesis kit, replacing 20% ​​of N1-Me-pseudo UTP with Cy5-UTP (APExBIO, B8333) to obtain fluorescent Luc-mRNA, i.e., Cy5-Luc-mRNA. Cy5-Luc-mRNA was then microfluidically mixed with a lipid formulation in the appropriate proportion to obtain Cy5-Luc-mRNA-LNP (preparation method as described in process route 1 of Example 1); red blood cells were labeled with CFSE dye (ThermoFisher, C34554) to obtain fluorescently labeled red blood cells.

[0324] The formulation CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was prepared by combining CFSE+RBCs with Cy5-Luc-mRNA (see process route 1 in Example 1 for preparation method). Labeling efficiency was evaluated using flow cytometry (Beckman-Coulter CytoFLEX), and CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was imaged using a Zessis 980 confocal microscope.

[0325] Experimental Results: As shown in Figure 3, flow cytometry results indicated that 99.96% of erythrocytes were coupled with Cy5-Luc-mRNA-LNP. Confocal imaging further confirmed that all erythrocytes in the field of view were coupled with Cy5-Luc-mRNA-LNP, and that Cy5-Luc-mRNA was uniformly distributed on the erythrocyte surface. These results suggest that mRNA-LNP can be successfully and efficiently coupled to the erythrocyte surface.

[0326] Example 3: Study on mRNA-LNP-RBC gene delivery efficiency

[0327] 3.1 Gene delivery / expression ratio of mRNA-LNP-RBC

[0328] Experimental Objective: Unlike mRNA-LNP, which enters cells via endocytosis, erythrocytes carry mRNA-LNP into cells via phagocytosis. Therefore, these two methods may result in different mRNA escape efficiencies. This study evaluated the mRNA-LNP-RBC gene delivery efficiency and compared it with the traditional cationic liposome transfection method.

[0329] Experimental Methods: Bone marrow-derived macrophages (BMDM) were isolated from the bone marrow of C57BL / 6 mice (Vitalliwa). The cells were cultured for 7 days under M-CSF cytokine (Peprotech, 315-02) induction, followed by 8 hours of induction with LPS (MCE, HY-D1056). After induction, Luc-LNP or GFP-LNP (preparation method as described in Example 1.2, 500 ng / well, where GFP amino acid sequence: SEQ ID NO:5; nucleotide sequence: SEQ ID NO:6) or Luc-LNP-RBC-1 (i.e., Luciferase mRNA-LNP-RBC-1) or GFP-LNP-RBC-1 (i.e., GFP mRNA-LNP-RBC-1) prepared according to process route 1 of Example 1 (10 μL / well, equivalent to 50 ng / well mRNA) were added to the BMDM cells. 24 hours later, the mRNA transfection level (Delivery of mRNA) and protein expression level (Production of proteins) were detected by flow cytometry. The escape efficiency of the delivered mRNA was evaluated based on the amount of mRNA entering and the efficiency of protein expression.

[0330] Experimental Results: As shown in Figure 4, compared to mRNA-LNP-RBC-1, although mRNA-LNP delivers more mRNA, the expression level of the target protein is far lower. This means that mRNA-LNP-RBC-1 can achieve more efficient protein expression with a lower amount of mRNA, and this was validated at both target sites. These results indicate that mRNA-LNP-RBC-1 has higher lysosomal escape compared to the mRNA-LNP gene delivery method, resulting in more efficient mRNA expression.

[0331] Therefore, the mRNA-LNP carried by red blood cells enters the cell through phagocytosis, resulting in higher mRNA delivery efficiency.

[0332] 3.2 Study on the gene delivery pathway of mRNA-LNP-RBC

[0333] Experimental Objective: This study compares the differences between mRNA-LNP and erythrocyte-mediated mRNA-LNP delivery pathways using fluorescence colocalization assays, exploring whether erythrocytes can improve mRNA delivery efficiency and protein expression levels by bypassing the traditional endosome-lysosome pathway and reducing endosome trapping.

[0334] Experimental methods: (1) Colocalization experiment: Bone marrow-derived macrophages (BMDM) were isolated from the bone marrow of C57BL / 6 mice (Vitolliwa) and cultured for 7 days under M-CSF (Peprotech, 315-02) induction, followed by stimulation with LPS (MCE, HY-D1056) for 8 hours. After induction, Cy5-Luc-LNP (fluorescently labeled Luciferase mRNA, 500 ng / well) or Cy5-Luc-LNP-RBC-1 (10 μL / well, mRNA content equivalent to 50 ng / well) prepared by process route 1 in Example 1 was added to BMDM and incubated for 6 hours. Subsequently, the colocalization of mRNA with early endosome marker EEA1 (Abcam, ab2900, 1:50) and late endosome / lysosome marker LAMP1 (Cell Signaling Technology, 99437S, 1:50) was detected by immunofluorescence. (2) Phagocytosis inhibition assay: BMDM from the same source was pretreated with 10 μM Cytochalasin D (Thermo Fisher, PHZ1063) for 1 hour to inhibit phagocytosis. After washing, Cy5-Luc-LNP (500 ng / well) or Cy5-Luc-LNP-RBC-1 (10 μL / well, equivalent to 50 ng / well of mRNA) was added, and the mixture was incubated for 6 hours. Flow cytometry was used to detect the uptake of Cy5-mRNA in BMDM.

[0335] Experimental results: Immunofluorescence results (Figures 5A-B) showed that 83% of the mRNA delivered by LNP co-localized with EEA1 and 50% with LAMP1; while the co-localization rate of mRNA delivered by erythrocytes was less than 5%, indicating that it does not depend on the classical endosome-lysosome pathway. Phagocytosis inhibition experiments showed that Cytochalasin D treatment reduced the uptake level of erythrocyte-delivered mRNA by 11.1-fold, confirming that phagocytosis is the main mechanism of erythrocyte-mediated mRNA uptake (Figure 5C).

[0336] This study demonstrates that erythrocyte-mediated mRNA delivery can bypass the endosome-lysosome pathway, efficiently enter myeloid cells through phagocytosis, and release mRNA in the cytoplasm, thereby enhancing protein expression efficiency.

[0337] Example 4: Tissue distribution and pharmacokinetic study of mRNA-LNP-RBC

[0338] 4.1 In vivo tissue distribution and gene expression of mRNA-LNP-RBC-1 and mRNA-LNP-RBC-2

[0339] Objective: To evaluate the distribution and expression of mRNA-LNP-RBC in mice and compare it with LNP. Luciferase (Luc, luciferase) and GFP (green fluorescent protein) were used for evaluation in this experiment.

[0340] Experimental methods: Luc-LNP (preparation method as described in Example 1.3, containing 500 ng / mouse mRNA, of which mRNA is the mRNA encoding Luciferase), Luc-LNP-RBC-1 (2e9 / mouse, containing 500 ng / mouse mRNA, of which mRNA is the mRNA encoding Luciferase), Luc-LNP-RBC-2 (2e9 / mouse, containing 500 ng / mouse mRNA), and Luc-LNP-RBC-3 (2e9 / mouse, containing 500 ng / mouse mRNA, of which mRNA is the mRNA encoding Luciferase) were prepared and injected into C57BL / 6 mice (Vitaliva) via tail vein. The expression of Luciferase in various tissues and organs was detected 20 hours after administration; n=3 in each group. The specific in vivo expression detection method is as follows: Prepare an aqueous solution of 15 mg / mL D-luciferin potassium salt (Beyotime, ST198), and inject 200 uL / mouse into the mice via intraperitoneal injection. 10 min later, the heart, liver, spleen, lung and kidney of the mice are taken for imaging analysis using a PerkinElmer IVI Slimina Series III in vivo imaging system.

[0341] GFP-LNP (preparation method as described in Example 1.3, containing 500 ng / mouse mRNA, where the mRNA is the mRNA encoding GFP) and GFP-LNP-RBC-1 (2e9 / mouse, containing 500 ng / mouse mRNA, where the mRNA is the mRNA encoding GFP) were prepared and injected into C57BL / 6 mice (Vitalliwa) via tail vein. Twenty hours after administration, the expression of GFP in the spleen was detected by flow cytometry (Beckman Coulter CytoFLEX), with n=2 mice in each group.

[0342] Experimental Results: As shown in Figure 6, Luc-LNP-RBC-1, Luc-LNP-RBC-2, and Luc-LNP-RBC-3 can all achieve in vivo gene delivery and expression, with expression mainly concentrated in the spleen. The left figure shows the distribution of each Luc-LNP carried by erythrocytes in various organs, while the right figure shows the distribution ratio of Luc-LNPs carried by erythrocytes in the liver and spleen.

[0343] As shown in Figure 7, consistent with previous reports, Luc-LNP, after tail vein infusion, was mainly distributed in the liver, where Luciferase expression was observed, with a liver expression rate as high as 97%. In contrast, Luc-LNP-RBC-1 was mainly distributed in the spleen, where Luciferase expression was observed, with a spleen expression rate as high as 90%. These results suggest that Luc-LNP-RBCs can alter the in vivo distribution of Luc-LNP, achieving effective gene delivery and expression within the spleen.

[0344] As shown in Figure 8, compared to GFP-LNP, GFP-LNP-RBC-1 is more effectively delivered to the spleen, and its expression of GFP protein is mainly concentrated in CD11b+ myeloid cells (CD11b+F4 / 80+ cells and CD11b+F4 / 80- cells). Other immune cells (i.e., cells other than CD11b+F4 / 80+ cells and CD11b+F4 / 80- cells, such as lymphocytes) do not express GFP. This demonstrates that erythrocytes deliver mRNA-LNP to the spleen, particularly the myeloid cells, through phagocytosis by CD11b+ myeloid cells in the spleen.

[0345] The above experimental results show that the target distribution of Luc-LNP-RBC is similar to that of RBC distribution (JANDL JH et al., Clinical determination of the sites of red cell sequestration in hemolytic anemias. J Clin Invest. 1956 Aug; 35(8):842-67. doi:10.1172 / JCI103338.PMID:13345887;PMCID:PMC441656;McArdel,SL, et al.(2021). Anti-tumor effects of RTX-240: an engineered red blood cell expressing 4-1BB ligand and interleukin-15. Cancer immunology, immunotherapy: CII The distribution of Luc-LNP-RBC differs significantly from that of mRNA-LNP (70, 2701-2719). This is because red blood cells transport the mRNA they carry to their target organs (spleen) and immune cells (CD11b+ myeloid cells).

[0346] 4.2 In vivo pharmacokinetic studies of mRNA-LNP-RBC

[0347] Experimental Objective: To evaluate the pharmacokinetics of Luc-mRNA-LNP-RBC-1 in C57BL / 6 mice.

[0348] Experimental Methods: As described above, CFSE+Cy5-Luc-mRNA-LNP-RBC-1 was prepared. CFSE+Cy5-Luc-mRNA-LNP-RBC-1 (2e9 / mouse, where the mRNA is encoding Luciferase) was injected into C57BL / 6 mice (Vitalliru) via tail vein injection. Blood samples were collected at 0.5 hours (Day 0, D0), 24 hours (D1), 48 hours (D2), 72 hours (D3), 96 hours (D4), and 120 hours (D5) after administration for flow cytometry analysis. The number of animals in each group was n=3.

[0349] Experimental Results: As shown in Figure 9, approximately 70% of Luc-mRNA-LNP-RBC-1 was cleared from mice within 5 days, and the Cy5-Luc-mRNA-LNP signal on the surface decreased with the clearance of erythrocytes. Since mRNA-LNP drugs are usually completely cleared within 24 hours (Lei Ci, et al., Biodistribution of Lipid 5, mRNA, and Its Translated Protein Following Intravenous Administration of mRNA-Encapsulated Lipid Nanoparticles in Rats. Drug Metab Dispos. 2023 Jul; 51(7):813-823; Kimberly J Hassett et al., mRNAvaccine trafficking and resulting protein expression after intramuscular administration. Mol Ther Nucleic Acids. 2023 Nov 24; 35(1):102083.), this result suggests that mRNA-LNP-RBC can effectively enhance the in vivo stability of mRNA-LNP.

[0350] 4.3 Detection of drug loading in mRNA-LNP-RBC

[0351] Take 10 μL of the concentrated mRNA-LNP-RBC preparations prepared in routes 1, 2, and 3 above and place them in a 1.5 mL centrifuge tube. The mRNA in these mRNA-LNP-RBCs is the mRNA encoding Luciferase (amino acid sequence: SEQ ID NO:3; nucleotide sequence: SEQ ID NO:4). Add an appropriate amount of internal control mRNA (GFP (SEQ ID NO:5; SEQ ID NO:6) mRNA) to the cell lysis buffer (QIAGEN RNeasy Plus Mini Kit, 74136) and mix thoroughly to obtain the lysis buffer with the internal control added. Each 10 μL of concentrated RBC requires 600 μL of cell lysis buffer and 50 ng of internal control mRNA. To lyse N 10 μL of concentrated RBC samples, prepare (N+1) lysis buffers with the internal control mRNA added, i.e., take (N+1)*600 μL of lysis buffer and add (N+1)*50 ng of internal control mRNA. Centrifuge at 15000 rpm for 3 minutes and collect the supernatant. Perform gDNA removal, mRNA reverse transcription, and RealTime PCR (Light Cycler 96, Roche) according to the kit instructions.

[0352] Experimental Results: The modified mRNA-LNPs (including mRNA-LNP-Mal, mRNA-LNP-SH, and mRNA-LNP-LPETG) showed no significant abnormalities in physicochemical properties compared to unmodified mRNA-LNPs, and all could successfully undergo erythrocyte conjugation to obtain mRNA-LNP-RBCs. Drug loading assays of mRNA-LNP-RBCs (Figure 2) showed that the surface mRNA-LNP loading of engineered erythrocytes (mRNA-LNP-RBC-1, mRNA-LNP-RBC-2, and mRNA-LNP-RBC-3) prepared by the three methods was 0.1-3 μg mRNA / mL RBCs, meaning that approximately 9-270 mRNA molecules could be conjugated to each erythrocyte surface. However, when mRNA-LNPs without linkers were mixed with RBCs, the drug loading was below the detection limit (<10 pg / μL), indicating that the amount of mRNA-LNPs that could be conjugated to RBCs via adsorption was very low. This result demonstrates that the covalent coupling method used in this study significantly improved the LNP drug loading on the RBC surface.

[0353] sequence list

[0354] References

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Claims

1. A lipid particle-erythrocyte conjugate comprising lipid particles containing nucleic acids and erythrocytes, wherein the lipid particles are covalently coupled to the membrane surface of the erythrocytes, optionally the covalent coupling is performed via a linker.

2. The lipid particle-erythrocyte conjugate of claim 1, wherein the lipid particles are lipid nanoparticles (LNPs).

3. The lipid particle-erythrocyte conjugate of any of the preceding claims, wherein the nucleic acid comprises DNA and / or RNA.

4. The lipid particle-erythrocyte conjugate of claim 3, wherein the RNA is selected from spontaneous RNA (mRNA), short interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), RNA interference (RNAi) molecules, microRNA (miRNA), antisense RNA, ribozymes, Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), aguide RNA, a circular RNA, a tRNA, and / or combinations thereof; and / or The DNA mentioned therein is circular DNA, such as plasmids, or linear DNA, such as antisense DNA.

5. The lipid nanoparticles as claimed in any of the preceding claims, wherein the lipid nanoparticles comprise one or more lipids selected from the group consisting of ionizable lipids, cofactor lipids, steroids, and PEG lipids. For example, among them The ionizable lipid is preferably selected from: 3-(bis(dodecylamino)-N1,N1,4-tridecyl-1-piperazine ethylamine (KL10), N1-[2-(bis(dodecylamino)ethyl]-N1,N4,N4-tridecyl-1,4-piperazine diethylamine (KL22), 14,25-bis(tridecyl)-15,18,21,24-tetraaza-octacosane (KL25), 1,2-dilinolenicooxy-N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinolenico-4- Dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), heptadecano-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate (DLin-MC3-DMA) (abbreviated as MC3), 2,2-diolenoyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-diolenoyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[-cholest-5-en-3-yloxy) (2R)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA), (2S)-2-({8-[-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2R)), (2S)-2-({8-[-cholest-5-en-3-yloxy]octyl} (Oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecane-9,12-dien-1-yloxy]prop-1-amine (octyl-CLinDMA(2S)), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butyryl)oxy)heptadecanoic acid ester (L319), 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}9-heptadecyl octanoic acid ester (SM102) and / or combinations thereof, preferably ionizable lipids selected from MC3 or SM102; The auxiliary lipid is preferably selected from: 1,2-dilinoleoyl-sn-glycerol-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), 1,2-distearateoyl-sn-glycerol-3-phosphocholine (DSPC), 1,2-di-undecanoyl-sn-glycerol-3-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycerol -3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycerol-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterolylhemisuccino-sn-glycerol-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycerol-3-phosphocholine (C16LysoPC), 1,2-dilinolenoyl-sn-glycerol-3-phosphocholine, 1,2-disarachidonicoyl-sn-glycerol-3-phosphocholine, 1,2-bis(docohexanoyl-sn-glycerol-3-phosphocholine), 1 2-Dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-diphydanoyl-sn-glycerol-3-phosphate ethanolamine (ME16.0PE), 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinoleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dilinolenoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-diarachidonicoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-bis(docohexanoyl)-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, Sodium oleo-3-phosphate-rac-(1-glycerol) (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearate-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), 1-stearoyl-2-oleoylphosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), sphingomyelin and / or combinations thereof, more preferably, the auxiliary lipid is selected from DSPC; The steroids are preferably selected from: cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassosterol and / or combinations thereof, preferably selected from cholesterol; and / or The PEG lipid is preferably selected from: 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol (DMG-PEG), 1,2-distearyl-sn-glycerol-3-phosphate ethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), PEG-distearylglycerol (PEG-DSG), PEG-dispalmitoyl, PEG-dioleoyl, PEG-distearyl, PEG-diacylglycineamide (PEG-DAG), PEG-dispalmitoylphosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and / or combinations thereof, more preferably selected from DMG-PEG, such as DMG-PEG2000.

6. The lipid particle-erythrocyte conjugate of any of the preceding claims, wherein the lipid particle comprises one or more lipids containing maleimide groups, preferably wherein the amount of the lipid in the lipid particle is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%).

7. The lipid particle-erythrocyte conjugate of claim 6, wherein the lipid containing a maleimide group is selected from one or more of the following lipids: ionizable lipids containing a maleimide group, auxiliary lipids containing a maleimide group, steroids containing a maleimide group, and / or PEG lipids containing a maleimide group, preferably, the ionizable lipid, auxiliary lipid, steroid, or PEG lipid is selected from the lipids defined in claim 5.

8. The lipid particle-erythrocyte conjugate of any of the preceding claims, wherein the lipid containing a maleimide group is a PEG lipid containing a maleimide group, preferably a DSPE-PEG lipid containing a maleimide group, and most preferably a DSPE-PEG(2000) containing a maleimide group, such as DSPE-PEG(2000)-MAL.

9. The lipid particle-erythrocyte conjugate of any of the preceding claims, wherein the erythrocytes are erythrocytes treated with a reducing agent, such as erythrocytes treated with a thiol reducing agent. Optionally, the red blood cells treated with the reducing agent are obtained by a method comprising: (1) Red blood cells are separated and concentrated from human whole blood, optionally by filtration of leukocytes; or red blood cells are obtained from stem cells such as pluripotent stem cells such as induced pluripotent stem cells; (2) Treat red blood cells with reducing agents such as thiol reducing agents to chemically modify the surface of red blood cells; (3) Collect and concentrate the modified red blood cells.

10. The lipid particle-erythrocyte conjugate of claim 9, wherein the thiol reducing agent is TCEP.

11. The lipid particle-erythrocyte conjugate of any of the preceding claims, having the following structure: LNP-DSPE-PEG(2000)-MAL-S-RBC, wherein... LNP-DSPE-PEG(2000)-MAL refers to lipid nanoparticles as defined in claim 8 containing DSPE-PEG(2000)-MAL, optionally in an amount of 0.1-10% (e.g., 0.1%-5%, 0.1%-1%, or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined in claim 5; RBCs are red blood cells as defined in claim 9; -S- refers to the group containing S formed by the reaction of maleimide group with thiol group of red blood cell, where the S atom comes from thiol group of RBC.

12. A method for preparing a lipid particle-erythrocyte conjugate as claimed in any of the preceding claims, comprising mixing the lipid particles containing maleimide groups with erythrocytes treated with a reducing agent, such that the two are covalently coupled via maleimide groups and nucleophilic groups on erythrocyte membrane proteins, for example, the method comprising... (1) Loading the lipid containing the maleimide group into lipid particles. (2) Surface chemical modification of adult natural erythrocytes to expose nucleophilic groups on erythrocyte membrane proteins; and collection and concentration of the modified erythrocytes; (3) Mix the lipid particles in (1) and the red blood cells obtained in (2) (e.g., 0.1-1000 μg lipid particles per 1 ml of red blood cells) so that the two are covalently coupled by maleimide groups and nucleophilic groups on red blood cell membrane proteins; (4) Collect the lipid particle-erythrocyte conjugate obtained in (3).

13. The method of claim 12, wherein the nucleophilic group on the erythrocyte membrane protein is a side-chain thiol group.

14. The method of claim 13, wherein the side chain thiol group is a thiol group resulting from the reduction of cysteine.

15. The method of any one of claims 12-14, wherein a thiol reducing agent is used to chemically modify the surface of the erythrocytes.

16. The method of claim 15, wherein the thiol reducing agent is TCEP.

17. The method of claim 16, wherein the final concentration of TCEP is between 0.1 mM and 10 mM, for example 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 2.5 mM.

18. The lipid particle-erythrocyte conjugate of any one of claims 1-5, wherein the lipid particle comprises a lipid containing a thiol group (SH), preferably the amount of the thiol group (SH)-containing lipid in the lipid particle is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%).

19. The lipid particle-erythrocyte conjugate of claim 18, wherein the thiol-containing (SH) lipid is selected from one or more of the following: ionizable lipids containing thiol groups, accessory lipids containing thiol groups, steroids containing thiol groups, and / or PEG lipids containing thiol groups, preferably, the ionizable lipid, accessory lipid, steroid, or PEG lipid is selected from the lipids defined in claim 5; for example, the thiol-containing lipid is a PEG lipid containing thiol groups, preferably a DSPE-PEG lipid containing thiol groups, and most preferably a DSPE-PEG(2000) containing thiol groups, such as DSPE-PEG(2000)-SH.

20. The lipid particle-erythrocyte conjugate of claim 18 or 19, wherein the thiol-containing lipid is linked to an amino group on the erythrocyte via a linker, preferably the erythrocyte is a mature erythrocyte, such as an adult natural erythrocyte.

21. The lipid particle-erythrocyte conjugate of claim 20, wherein the functional group of the connector is capable of reacting with the thiol group on the lipid containing the thiol group to form a covalent bond to the lipid particle, and wherein the functional group is capable of reacting with the nucleophilic group on the erythrocyte membrane protein to connect to the erythrocyte.

22. The lipid particle-erythrocyte conjugate of claim 21, wherein the nucleophilic groups on the erythrocyte membrane proteins include, but are not limited to: (i) an N-terminal amino group, (ii) a side-chain amino group, such as an amino group of lysine, (iii) a side-chain thiol group, such as an thiol group of cysteine, and (iv) a glycosyl hydroxyl or amino group (in the case of glycosylation), such as an amino group on the side chain, and the connector has electrophilic groups: (i) active esters, such as NHS esters, HOBt esters, halocarboxylate esters and acid halides; (ii) alkyl and benzyl halides, such as haloacetamides; and (iii) aldehyde, ketone, carboxyl and maleimide groups, wherein the nucleophilic groups react with the electrophilic groups to form covalent bonds.

23. The lipid particle-erythrocyte conjugate of claim 21, wherein the nucleophilic group on the erythrocyte membrane protein is a -NH2 of the lysine side chain, and the functional group of the linker is capable of forming a covalent bond with the -NH2 of the lysine side chain on the erythrocyte membrane protein to connect to the erythrocyte.

24. The lipid particle-erythrocyte conjugate of any one of claims 20-23, wherein the linker is succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxy-(6-aminohexanoate) (LC-SMCC), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), or sulfonate-SMCC, preferably sulfonate-SMCC.

25. The lipid particle-erythrocyte conjugate according to any one of claims 18-24, having the following structure: LNP-DSPE-PEG(2000)-S-SMCC-RBC, wherein... LNP-DSPE-PEG(2000) refers to lipid nanoparticles as defined in claim 19 containing DSPE-PEG(2000)-SH, wherein optionally the amount of DSPE-PEG(2000)-SH in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined in claim 5; RBC stands for red blood cells; SMCC is the linker LC-SMCC, SMCC or sulfo-SMCC as defined in claim 24, where -S- refers to the group containing S formed by the reaction of the thiol group (SH) in LNP with the maleimide group of the linker SMCC, wherein the S atom comes from the thiol group of lipid DSPE-PEG(2000)-SH in LNP. Optionally, multiple lipid particles can be coupled to the RBC, wherein the lipid particles may be the same or different.

26. A method for preparing a lipid particle-erythrocyte conjugate as claimed in any one of claims 18-25, comprising mixing lipid particles containing erythrocytes linked to a linker and lipids containing thiol groups (SH) such that the two are covalently coupled via the linker, for example... (1) Reacting nucleophilic groups (e.g., NH2 on lysine) on red blood cell membrane proteins with a divalent linker reagent to form red blood cells connected to the linker via covalent bonds, preferably collecting and concentrating the red blood cells; (2) Preload lipids containing thiol groups (SH) into lipid particles; (3) Mix the red blood cells connected to the adapter in (1) and the lipid particles obtained in (2) (e.g., 0.1-1000 μg of lipid particles per 1 mL of red blood cells) so that the two are covalently coupled through the adapter; (4) Collect the lipid particle-erythrocyte conjugate obtained in (3).

27. The method of claim 26, wherein the nucleophilic group on the erythrocyte membrane protein is an N-terminal amine group or a side-chain amine group.

28. The method of claim 26 or 27, wherein the divalent connector is LC-SMCC, SMCC or sulfon-SMCC, preferably SMCC, for example, the final concentration of SMCC is between 0.1 mM and 10 mM, for example 0.5 mM to 10.0 mM, 0.5 mM to 5.0 mM, preferably about 0.5 mM.

29. The lipid particle-erythrocyte conjugate of any one of claims 1-5, wherein the lipid particles are preloaded or contain lipids containing sorting enzyme recognition motifs.

30. The lipid particle-erythrocyte conjugate of claim 29, wherein the lipid containing the sorting enzyme recognition motif is selected from one or more of the following: ionizable lipids containing the sorting enzyme recognition motif, auxiliary lipids containing the sorting enzyme recognition motif, steroids containing the sorting enzyme recognition motif, and / or PEG lipids containing the sorting enzyme recognition motif.

31. The lipid particle-erythrocyte conjugate of claim 29 or 30, wherein the lipid containing the sorting enzyme recognition motif is a PEG lipid containing the sorting enzyme recognition motif, preferably a DSPE-PEG containing the sorting enzyme recognition motif, most preferably a DSPE-PEG (2000) containing the sorting enzyme recognition motif, optionally, said DSPE-PEG (2000) is modified to contain the sorting enzyme recognition motif; for example, said DSPE-PEG (2000) is derived via flexible peptide (GS) n It is linked to the sorting enzyme recognition motif, where n = 1-10.

32. The lipid particle-erythrocyte conjugate of any one of claims 29-31, wherein the sorting enzyme recognition motif comprises or is composed of LPXTG, preferably, the sorting enzyme recognition motif is LPETG.

33. The lipid particle-erythrocyte conjugate of any one of claims 29-32, wherein the sorting enzyme recognition motif may be modified to enhance its affinity; preferably, the modification is the addition of G, such as LPETGG, to the C-terminus of the sorting enzyme recognition motif.

34. The lipid particle-erythrocyte conjugate of any one of claims 29-33, wherein the lipid containing the sorting enzyme recognition motif comprises or is composed of DSPE-PEG(2000)-LPETG.

35. The lipid particle-erythrocyte conjugate of any one of claims 29-34, wherein the sorting enzyme recognition motif is conjugated to the extracellular portion of an erythrocyte membrane protein via a linker, for example, the linker comprising a G peptide and a maleimide alkyl chain (C... 2-8 ), the maleimide alkyl chain (C 2-8 The G peptide conjugates with membrane proteins of erythrocytes, and the G peptide conjugates with lipids containing sorting enzyme recognition motifs via a sorting enzyme-mediated reaction.

36. The lipid particle-erythrocyte conjugate of any one of claims 35, wherein the G peptide is a linear peptide or a branched peptide.

37. The lipid particle-erythrocyte conjugate of claim 35 or 36, wherein the G peptide is a branched-chain peptide comprising two or more branching units, wherein one or more lipid particles are coupled to one or more branching units.

38. The lipid particle-erythrocyte conjugate of claim 37, wherein the branching unit is composed of the amino acid sequence K (GGG), wherein the glycine in parentheses is conjugated with the ε-amino group of the side chain of lysine to form a branch, and lysine forms a peptide bond with other amino acids through its α-amino group to form the backbone of the "G peptide", optionally, an extended chain, such as COCH2CH2-PEG6-NH, may be added between K and G in the branching unit K (GGG).

39. The lipid particle-erythrocyte conjugate of any one of claims 34-37, wherein the G peptide has the following structure: GGGSK, K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG), K(GGG)-GGG-K(GGG)-GGG-K(GGG)-GGG-K(GGG) GG)-GGG-K(GGG)-GGG-K(GGG) or K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-GGG-K[(COCH2CH2-PEG6-NH)-GGG]-NH2, preferably the G-peptide has the following structure: K(GGG)-GGG-K(GGG)-GGG-K(GGG).

40. The lipid particle-erythrocyte conjugate of any one of claims 35-39, wherein the maleimide alkyl chain (C 2-8 It is 6-maleimide hexanoic acid or 4-maleimide butyric acid, preferably 6-maleimide hexanoic acid.

41. The lipid particle-erythrocyte conjugate of any one of claims 35-40, wherein (PEG)n is contained between the G peptide and 6-maleimide hexanoic acid, wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more.

42. The lipid particle-erythrocyte conjugate according to any one of claims 35-41, wherein the linker has the structure shown below (G3):

43. The lipid particle-erythrocyte conjugate of any one of claims 35-42, wherein a plurality of lipids containing sorting enzyme recognition motifs are conjugated to membrane proteins (e.g., extracellular domains of membrane proteins) of erythrocytes via branched connectors.

44. The lipid particle-erythrocyte conjugate of any one of claims 29-43, wherein the erythrocytes are erythrocytes treated with a reducing agent, such as erythrocytes treated with a thiol reducing agent, obtained by a method comprising: (1) Red blood cells are separated and concentrated from human whole blood, optionally by filtration of leukocytes; or red blood cells are obtained from stem cells such as pluripotent stem cells such as induced pluripotent stem cells; (2) Treat red blood cells with thiol reducing agents to chemically modify the surface of red blood cells; (3) Collect and concentrate the modified red blood cells.

45. The lipid particle-erythrocyte conjugate of claim 44, wherein the thiol reducing agent is TCEP.

46. ​​The lipid particle-erythrocyte conjugate according to any one of claims 29-45, having the following structure: LNP-DSPE-PEG(2000)-LPETG-linker-RBC, preferably LNP-DSPE-PEG(2000)-LPETG-(G3)-RBC; wherein LNP-DSPE-PEG(2000)-LPETG refers to lipid nanoparticles as defined in claim 34 that contain DSPE-PEG(2000)-LPETG, wherein the amount of DSPE-PEG(2000)-LPETG in the LNP is 0.1-10% (e.g., 0.1%-5% or 0.1%-1% or 0.5%-1%, e.g., about 0.75%), and optionally the LNP may also contain one or more other lipids as defined in claim 5; Red blood cells are those defined as in claim 44; The connector is the connector as defined in any one of claims 35-42; The DSPE-PEG(2000)-LPETG is attached to the extracellular domain of the erythrocyte membrane protein via a linker.

47. A method for preparing a lipid particle-erythrocyte conjugate as claimed in any one of claims 29-46, comprising mixing erythrocytes linked to a linker and lipid particles containing a linker recognition motif in the presence of a sorting enzyme and under conditions suitable for the sorting enzyme to react, such that the sorting enzyme conjugates the lipid particles to the erythrocytes via the linker, for example comprising: (1) Treating red blood cells with a reducing agent such that the adapter molecule of any one of claims 35-42 is attached to the extracellular domain of an endogenous membrane protein of the red blood cells; and collecting and concentrating the red blood cells; and / or, (2) Treat the lipid particles so that they contain sorting enzyme recognition motifs; and / or, (3) In the presence of a sorting enzyme, the red blood cells obtained in step 1) are brought into contact with the lipid particles obtained in step 2) under conditions suitable for the sorting enzyme to react (e.g., 0.1-1000 μg of lipid particles per 1 mL of red blood cells) so that the sorting enzyme attaches the lipid particles to the endogenous membrane proteins of the red blood cells through a second linker.

48. The method of claim 47, wherein the reducing agent is a thiol reducing agent, such as TCEP.

49. A pharmaceutical composition or formulation comprising a lipid particle-erythrocyte conjugate as claimed in any one of claims 1-11, 18-25 and 29-46, or a lipid particle-erythrocyte conjugate prepared by the method of any one of claims 12-17, 26-28 and 47-48, and a pharmaceutical excipient.

50. The pharmaceutical composition or formulation of any one of claims 49, wherein the formulation is a human blood formulation, such as an autologous or allogeneic human blood formulation.

51. The pharmaceutical composition or formulation of any one of claims 49 or 50, wherein the human blood formulation is a human blood leukocyte preparation.

52. The pharmaceutical composition or formulation of any one of claims 49-51, wherein the human blood formulation comprises 10-1000 μg / mL of lipid particle-erythrocyte conjugate, for example, at concentrations of 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL, 250 μg / mL, 300 μg / mL, 350 μg / mL, 400 μg / mL, 450 μg / mL, or 500 μg / mL or higher; or The human blood preparation described herein comprises a lipid particle-erythrocyte conjugate having 10-10000 nucleic acid molecules / RBC, for example, about 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, or 9000 or more nucleic acid molecules / RBC, most preferably 9-270 nucleic acid molecules / RBC; or The human blood preparation described herein comprises a lipid particle-erythrocyte conjugate having a concentration greater than approximately 0.01 μg mRNA / 10 10 RBCs, for example, have approximately 0.01-300 μg mRNA / 10 10 RBCs, 0.05-200 μg mRNA / 10 10 RBCs, 0.05-100μg mRNA / 10 10 RBCs, 0.05-50 μg mRNA / 10 10 RBCs or 0.08-10 μg mRNA / 10 10 RBCs, with an optimal concentration of 0.1-3 μg mRNA / 10 10 RBCs.

53. The use of a lipid particle-erythrocyte conjugate as claimed in any one of claims 1-11, 18-25 and 29-46, or a lipid particle-erythrocyte conjugate prepared by any one of claims 12-17, 26-28 and 47-48, in the preparation of a medicament or human blood preparation for modifying CD11b+ myeloid cells in the spleen, preferably for modulating the host's immune system (e.g., enhancing or weakening cellular immune responses) or for treating cancer or autoimmune diseases in a subject.

54. The use of claim 53, wherein the red blood cells in the lipid particle-red blood cell conjugate are derived from a subject to be treated or a healthy subject.

55. The use as claimed in claim 53 or 54, wherein the drug or human blood preparation is administered in combination with one or more treatment methods or other therapeutic agents.

56. The use as claimed in any one of claims 53-55, wherein the treatment is radiotherapy or surgical treatment, and / or the therapeutic agent is selected from chemotherapeutic agents, other antibodies, cytotoxic agents, vaccines, etc.

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