Compositions and methods for targeted lipid nanoparticles

Abstract

The present invention relates to lipid-based nanoparticles (e.g., lipid nanoparticles), formulations containing lipid-based nanoparticles, and methods of using the lipid-based nanoparticles and formulations thereof to treat diseases or conditions. The present invention provides a lipid-based nanoparticle comprising (a) an active agent, and (b) a plurality of capture binding domains presented on the outer surface of the nanoparticle, wherein each capture binding domain is attached to the lipid-based nanoparticle by a site-specific linkage such that each capture binding domain is presented in substantially the same orientation and is capable of capturing a targeting moiety in an orientation that allows the targeting moiety to interact with its target. The present invention also provides a lipid-based nanoparticle comprising (a) an active agent, and (b) a plurality of targeting molecules presented on the outer surface of the nanoparticle, wherein each targeting molecule is attached to the lipid-based nanoparticle by a site-specific linkage such that each targeting molecule is presented in substantially the same orientation and is capable of binding to a target on the surface of a cell.

Description

Technical Field

[0001] This invention relates to lipid-based nanoparticles (e.g., lipid nanoparticles), formulations containing lipid-based nanoparticles, and methods of treating diseases or conditions using said lipid-based nanoparticles and formulations thereof.

[0002] Cross-references to related applications

[0003] This application claims priority to Australian Provisional Application AU2023903447, filed on 27 October 2023, the entire contents of which are incorporated herein by reference. Background Technology

[0004] The successful application of two mRNA-lipid nanoparticle (LNP)-based vaccines in combating the COVID-19 pandemic demonstrates the potential of LNPs for efficient in vivo delivery of large synthetic mRNA payloads. However, the low delivery efficiency and low specificity of current LNP formulations limit their application beyond vaccines. Passive targeting of LNPs is typically achieved by modulating lipid formulations (e.g., different lipids or compositions) to enrich them in target organs or cells. Different charged lipids recruit different serum proteins to LNPs, leading to altered biodistribution in vivo. Library screening of hundreds of LNP formulations has enhanced the ability to modulate the biodistribution of different LNPs in vivo, but this approach remains time-consuming and labor-intensive, and lacks a clear understanding of the specific cells targeted.

[0005] There is a need to provide new compositions and methods to improve the delivery efficiency and / or specificity of lipid-based nanoparticles.

[0006] Any reference to prior art in this specification does not constitute an acknowledgment or implied acceptance that such prior art is part of common general knowledge in any jurisdiction, nor does it constitute an acknowledgment or implied acceptance that such prior art can be reasonably expected to be understood, regarded as relevant and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0007] The present invention provides a lipid-based nanoparticle comprising (a) an active agent and (b) a plurality of capture-binding domains present on the outer surface of the nanoparticle, wherein each capture-binding domain is connected to the lipid-based nanoparticle via site-specific linkage, such that each capture-binding domain is presented in substantially the same orientation and is capable of capturing the target portion in an orientation that allows the target molecule to interact with its target.

[0008] In any aspect or embodiment, the nanoparticles are liposomes, lipid nanoparticles, micelles, or other particles described herein. In one embodiment, the lipid-based nanoparticles are amorphous.

[0009] In any aspect or implementation, each of the multiple capture-binding domains is linked to the lipid-based nanoparticle via the same site. For example, the sites where site-specific connections are formed on each capture-binding domain are identical.

[0010] In any aspect or implementation, each capture-binding domain is linked to a lipid-based nanoparticle via a single-site-specific connection.

[0011] In any aspect or implementation, each capture-binding domain is attached to a lipid-based nanoparticle at a location remote from the site where it interacts with the target molecule. For example, when the capture-binding domain is an antibody or its antigen-binding fragment, the site where the capture-binding domain interacts with the target molecule is one or more complementarity-determining regions (CDRs), and the antibody or its antigen-binding fragment is attached to the lipid-based nanoparticle at a location remote from the one or more CDRs.

[0012] In any aspect or implementation, each of the multiple binding domains is linked to a lipid-based nanoparticle via a modified amino acid side chain.

[0013] In any aspect or implementation, each capture-binding domain is not linked to the lipid-based nanoparticle via its N-terminus or C-terminus.

[0014] In any aspect or implementation, each capture-binding domain is linked to a lipid-based nanoparticle at a location of a non-β-sheet or α-helical component.

[0015] In any aspect or implementation, the capture-binding domain is attached to the lipid-based nanoparticle at a location on the outer surface of the binding domain that is free from steric hindrance.

[0016] In any aspect or implementation, sites on the capture-binding domain for site-specific connections have been identified to allow the capture-binding domain to be presented in an optimized orientation when it is linked to a lipid-based nanoparticle. In one implementation, the location on the capture-binding domain for site-specific connections can be determined by any of the methods described herein (including examples) to allow the capture-binding domain to be presented in an optimized orientation.

[0017] In any aspect or implementation, the sites on the capture-binding domain for site-specific linkages have been identified such that when the capture-binding domain, which is linked to a lipid-based nanoparticle, binds to its target molecule, the target molecule is presented in an optimized orientation.

[0018] In any aspect or implementation, a targeting molecule is any molecule that binds to a molecule expressed on the surface of a target cell. Exemplary targeting molecules and the molecules expressed on the surface of target cells to which the targeting molecule binds are described herein.

[0019] In any aspect or implementation, site-specific linkage is formed between a first coupling group on the lipid-based nanoparticle and a second coupling group on the capture-binding domain.

[0020] In any aspect or embodiment, the first coupling group is located on a hydrophobic molecule, preferably on a lipid. Typically, the lipid is a phospholipid, a structured lipid, a PEGylated lipid, or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, such as DSPE-PEG.

[0021] In any aspect or embodiment, the first coupling group is an olefin or alkyne dipolarophile, a thiol or mercapto, maleimide, trans-cyclooctene, tetraazine, or thiolene. Exemplary dipolarophiles are strained cyclic olefins or cyclic alkynes. Preferred strained cyclic olefins or cyclic alkynes include cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazenecyclooctyne ketones.

[0022] In any aspect or embodiment, the second coupling group is a 1,3-dipolar, a thiol or mercapto, maleimide, trans-cyclooctene, tetraazine, or thiolene. Typically, the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate, preferably an azide.

[0023] In one embodiment, the azide is azide-phenylalanine (azPhe), wherein the capture-binding domain is modified to contain azPhe. In one embodiment, the thiol or mercapto group is provided by a cysteine ​​residue. Typically, a cysteine ​​residue is introduced into the capture-binding domain.

[0024] When the first and second coupling groups are thiols or mercapto groups, the site-specific connection is a disulfide bond.

[0025] When the first coupling group is maleimide and the second coupling group is thiol or mercapto, the site-specific connection is thiosuccinimide.

[0026] In any aspect or implementation, each capture-binding domain is an antibody or antibody fragment, such as a nanobody or single-chain variable fragment, affinity compound, aptamer, peptide, protein A, protein G, protein L, or spyCatcher. Preferably, the capture-binding domain is a nanobody.

[0027] In any aspect or implementation, when the capture-binding domain is an antibody, the site-specific linker may be located at or equivalent to amino acids 12 to 17 of the antibody's FR1; amino acids 84 to 91 of the antibody's FR3; or amino acids 117 to 119 of the antibody's FR4. Non-natural amino acids or cysteines may be introduced (e.g., by mutating existing residues or inserting additional residues) at or equivalent to amino acids 12 to 17 of the antibody's FR1, amino acids 84 to 91 of the antibody's FR3, or amino acids 117 to 119 of the antibody's FR4, unless the amino acid is proline.

[0028] In any aspect or implementation, each capture-binding domain binds to the Fc region of the antibody, preferably to the CH2 or CH3 domain.

[0029] In any aspect or implementation, each capture-binding domain binds to the framework region of an antibody, affinity, or fragment thereof (e.g., scFv or nanobody).

[0030] In any aspect or implementation, the capture-binding domain is any Fc-binding antibody described herein, including TP1107 or a variant thereof.

[0031] On one hand, the present invention provides a lipid nanoparticle comprising (a) an activator and (b) a plurality of nanobodies present on the outer surface of the nanoparticle, wherein each nanobodies is linked to the lipid nanoparticle via site-specific coupling, such that each nanobodies are presented in substantially the same orientation and are capable of capturing the target portion in an orientation that allows the target portion to interact with its target. The site-specific coupling is between a first coupling group (i.e., dibenzocyclooctylene) formed on the PEGylated lipid and a second coupling group (i.e., an azide located on the amino acid side chain at or equivalent to the 12th to 17th amino acids, preferably the 13th amino acid, in the FR1 of the nanobodies); preferably, the nanobodies are bound to the CH2 or CH3 domain of the Fc region of the antibody. Preferably, the nanobodies comprise CDR1 as shown in SEQ ID NO: 2, 9, or 15; CDR2 as shown in SEQ ID NO: 3, 10, or 16; and CDR3 as shown in SEQ ID NO: 4, 11, or 17. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. More preferably, the nanobody comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 1.

[0032] On one hand, the present invention provides a lipid nanoparticle comprising (a) an activator and (b) a plurality of nanobodies present on the outer surface of the nanoparticle, wherein each nanobodies is linked to the lipid nanoparticle via site-specific coupling, such that each nanobodies are presented in substantially the same orientation and are capable of capturing the target portion in an orientation that allows the target portion to interact with its target. The site-specific coupling is formed between a first coupling group (i.e., a thiol or thiol group) formed on the PEGylated lipid and a second coupling group (i.e., a thiol or thiol group on the side chain of an amino acid at or equivalent to the 12th to 17th amino acid position, preferably the 13th amino acid position, in the FR1 of the nanobodies); preferably, the nanobodies are bound to the CH2 or CH3 domain of the Fc region of the antibody. Preferably, the nanobodies comprise CDR1 as shown in SEQ ID NO: 2, 9, or 15; CDR2 as shown in SEQ ID NO: 3, 10, or 16; and CDR3 as shown in SEQ ID NO: 4, 11, or 17. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. More preferably, the nanobody comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 1.

[0033] On the other hand, the present invention provides lipid-based nanoparticles as described herein, which further comprise a plurality of targeting molecules, wherein the targeting molecules are bound by capture-binding domains. The targeting molecule is any molecule that binds to molecules expressed on the surface of a target cell. Exemplary targeting molecules and molecules expressed on the surface of target cells to which the targeting molecules bind are described herein.

[0034] On the other hand, the present invention provides a conjugate comprising a lipid capable of incorporating lipid-based nanoparticles and a capture-binding domain, wherein the capture-binding domain is conjugated to the lipid via site-specific linkage.

[0035] In this regard, the lipid is a phospholipid, a structured lipid, a PEGylated lipid, or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, such as DSPE-PEG.

[0036] In this respect, the conjugate is formed between a first coupling group on the lipid and a second coupling group on the trapping binding domain. The first coupling group can be an alkene or alkyne dipolarophile, or a thiol or mercapto. An exemplary dipolarophile is a strained cyclic alkene or cyclic alkyne. Preferred strained cyclic alkenes or cyclic alkynes include cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazenecyclooctyne ketone. The second coupling group can be a 1,3-dipolar, or a thiol or mercapto. Typically, the 1,3-dipolar is an azide, nitrile oxide, nitrone, or isocyanate, preferably an azide.

[0037] In one embodiment, the azide is azide-phenylalanine (azPhe), wherein the capture-binding domain is modified to contain azPhe.

[0038] When the first and second coupling groups are thiols or mercapto groups, the site-specific connection is a disulfide bond.

[0039] When the first coupling group is maleimide and the second coupling group is thiol or mercapto, the site-specific connection is thiosuccinimide.

[0040] In this regard, the capture-binding domain is an antibody or antibody fragment, such as a nanobody or a single-chain variable fragment, affinity compound, aptamer or peptide, preferably a nanobody.

[0041] In this regard, when the capture-binding domain is an antibody, the site-specific linker can be located at or equivalent to amino acids 12 to 17 of the antibody's FR1; amino acids 84 to 91 of the antibody's FR3; or amino acids 117 to 119 of the antibody's FR4. Non-natural amino acids or cysteines can be introduced at or equivalent to amino acids 12 to 17 of the antibody's FR1, amino acids 84 to 91 of the antibody's FR3, or amino acids 117 to 119 of the antibody's FR4, unless the amino acid is proline.

[0042] In this regard, the capture-binding domain binds to the Fc region of the antibody, preferably to the CH2 or CH3 domain. The capture-binding domain can be any Fc-binding antibody described herein, including TP1107 or a variant thereof.

[0043] On the other hand, the present invention provides lipid-based nanoparticles comprising the conjugates of the present invention as described herein.

[0044] In any aspect or embodiment, each lipid-based nanoparticle has at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180, 200, 300, 400, or 800 capture-binding domains. Preferably, each lipid-based nanoparticle has at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180, or 200 capture-binding domains.

[0045] In any aspect or embodiment, the lipid-based nanoparticles of the present invention exhibit higher cell-binding capacity compared to corresponding lipid-based nanoparticles in which the capture-binding domains are randomly oriented. Higher binding capacity can manifest as a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase in binding capacity as determined by any of the methods described herein (including examples). Furthermore, when the active agent encodes a protein, the lipid-based nanoparticles of the present invention exhibit higher protein expression levels compared to corresponding lipid-based nanoparticles in which the capture-binding domains are randomly oriented. Higher protein expression levels can manifest as a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase in expression as determined by any of the methods described herein (including examples). Lipid-based nanoparticles with randomly oriented capture-binding domains can be formed by conjugating the capture-binding domains to lipid-based nanoparticles through reaction with EDC / NHS molecules and random lysine residues.

[0046] In any aspect or embodiment, the lipid-based nanoparticles of the present invention exhibit higher cell-binding capacity compared to corresponding lipid-based nanoparticles lacking any capture-binding domains. Higher binding capacity can be manifested as a 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold increase in binding as determined by any of the methods described herein (including examples). Furthermore, when the active agent encodes a protein, the lipid-based nanoparticles of the present invention exhibit higher protein expression levels compared to corresponding lipid-based nanoparticles lacking any capture-binding domains. Higher protein expression levels can be manifested as a 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold increase in expression as determined by any of the methods described herein (including examples).

[0047] In any aspect or embodiment, the lipid-based nanoparticles of the present invention may be more stable and less aggregated than corresponding lipid-based nanoparticles in which the capture-binding domains are randomly oriented. Lipid-based nanoparticles with randomly oriented capture-binding domains can be formed by conjugating the capture-binding domains to lipid-based nanoparticles through reaction with EDC / NHS molecules and random lysine residues.

[0048] On the other hand, the present invention provides a method for preparing functionalized lipid-based nanoparticles, comprising contacting lipid-based nanoparticles with a conjugate as described herein, thereby forming functionalized lipid-based nanoparticles. The lipid-based nanoparticles are pre-formulated nanoparticles capable of encapsulating active agents, and may be referred to as basic lipid nanoparticles. Typically, basic lipid nanoparticles do not possess active targeting capabilities prior to incorporation with the conjugate as described herein.

[0049] In one embodiment, the present invention provides a method for preparing functionalized lipid nanoparticles, the method comprising: Solutions of cationic and / or ionizable lipids, phospholipids, structured lipids, and PEGylated lipids are mixed to form lipid nanoparticles, and The formed lipid nanoparticles are brought into contact with the conjugate of the present invention. Thus, functionalized lipid nanoparticles were prepared.

[0050] Preferably, the method further includes adding an active agent in the step of mixing lipids.

[0051] In one embodiment, the lipid nanoparticles are formed with a lipid component to an active agent weight ratio between about 5:1 and about 50:1. In another embodiment, the final lipid concentration in the solution is between about 5.5 mM and about 50 mM, preferably diluted with ethanol.

[0052] A lipid solution can be formed by mixing solutions of cationic and / or ionizable lipids, phospholipids, structured lipids, and PEGylated lipids to obtain the desired molar ratio. In one embodiment, the lipid solution is rapidly injected into a solution containing an active agent at a flow rate of about 0.5 mL / min to about 8 mL / min (preferably about 4 mL / min) using a microfluidic system (e.g., a Nano-Assembler microfluidic system) to prepare a suspension with a water to ethanol ratio of about 1:1 to about 4:1 (preferably 3:1).

[0053] Typically, the NP ratio (nitrogen-phosphorus ratio) is maintained between 4 and 7.

[0054] On the other hand, the present invention provides a method for preparing a conjugate, comprising mixing a lipid having a first coupling group as described herein with a capture domain having a second coupling group as described herein, thereby preparing the conjugate. Typically, the mixing is carried out at 37°C or about 37°C for 24 hours or about 24 hours. For example, when the first coupling group on the lipid is an alkene or alkyne dipolarophile and the second coupling group on the capture-binding domain is a 1,3-dipolar, the lipid and the capture-binding domain are mixed in a 2:1 molar ratio (DBCO: azide) and incubated at 37°C or about 37°C for 24 hours or about 24 hours. Alternatively, the lipid and the capture-binding domain are mixed in a 2:1 molar ratio (DBCO: azide) and incubated at a temperature below 37°C (e.g., 4°C or about 4°C) to prepare the conjugate.

[0055] On the other hand, the present invention provides a lipid-based nanoparticle comprising (a) an active agent and (b) a plurality of targeting molecules present on the outer surface of the nanoparticle, wherein each targeting molecule is linked to the lipid-based nanoparticle via a site-specific connection, such that each targeting molecule is presented in substantially the same orientation and is capable of binding to a target on the cell surface.

[0056] In any embodiment of this invention, the nanoparticles are liposomes, lipid nanoparticles, micelles, or other particles described herein. In one embodiment, the lipid-based nanoparticles are amorphous.

[0057] In any implementation thereof, each of the plurality of targeting molecules is linked to the lipid-based nanoparticle through the same site. For example, the sites on each targeting molecule that form site-specific connections are identical.

[0058] In any implementation thereof, each targeting molecule is linked to a lipid-based nanoparticle via a single-site specific linker.

[0059] In any implementation thereof, each targeting molecule is attached to a lipid-based particle at a location remote from the target binding site. For example, when the targeting molecule is an antibody or its antigen-binding fragment, the target binding site is one or more CDRs, and the antibody or its antigen-binding fragment is attached to a lipid-based nanoparticle at a location remote from the one or more CDRs.

[0060] In any implementation thereof, each of the plurality of target molecules is linked to a lipid-based nanoparticle via a modified amino acid side chain.

[0061] In any implementation thereof, each targeting molecule is not linked to a lipid-based particle via its N-terminus or C-terminus.

[0062] In any implementation thereof, each targeting molecule is linked to a lipid-based particle at a location of a non-β-sheet or α-helical component.

[0063] In any implementation thereof, each targeting molecule is attached to a lipid-based particle at a location on the outer surface of the binding domain in a sterically unobstructed manner.

[0064] In any embodiment of this aspect, sites on the target molecule for site-specific attachment have been identified to allow the target molecule to be presented in an optimized orientation when it is attached to lipid-based nanoparticles. In one embodiment, the sites on the target molecule for site-specific attachment can be determined by any of the methods described herein (including examples) to allow the target molecule to be presented in an optimized orientation.

[0065] In any implementation thereof, the targeting molecule is any molecule that binds to a molecule expressed on the surface of a target cell. Exemplary targeting molecules and molecules expressed on the surface of target cells to which the targeting molecule binds (e.g., “targets”) are described herein.

[0066] In any implementation thereof, site-specific linkage is formed between a first coupling group on the lipid-based nanoparticle and a second coupling group on the target molecule.

[0067] In any embodiment of this aspect, the first coupling group is located on a hydrophobic molecule, preferably on a lipid. Typically, the lipid is a phospholipid, a structured lipid, a PEGylated lipid, or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, such as DSPE-PEG.

[0068] In any embodiment of this aspect, the first coupling group is an olefin or alkyne dipolarophile, a thiol or mercapto, maleimide, trans-cyclooctene, tetraazine, or thiolene. Exemplary dipolarophiles are strained cyclic olefins or cyclic alkynes. Preferred strained cyclic olefins or cyclic alkynes include cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazoxide octyneone.

[0069] In any embodiment of this aspect, the second coupling group is a 1,3-dipolar, a thiol or mercapto, maleimide, trans-cyclooctene, tetraazine, or thiolene. Typically, the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate, preferably an azide. In one embodiment, the azide is azide-phenylalanine (azPhe), wherein the capture-binding domain is modified to include azPhe. In one embodiment, the thiol or mercapto is provided by a cysteine ​​residue. Typically, a cysteine ​​residue is introduced into the capture-binding domain.

[0070] When the first and second coupling groups are thiols or mercapto groups, the site-specific connection is a disulfide bond.

[0071] When the first coupling group is maleimide and the second coupling group is thiol or mercapto, the site-specific connection is thiosuccinimide.

[0072] In any embodiment of this aspect, when the target molecule is an antibody, the site-specific link may be located at or equivalent to amino acids 12 to 17 of the antibody's FR1; amino acids 84 to 91 of the antibody's FR3; or amino acids 117 to 119 of the antibody's FR4. Non-natural amino acids or cysteines may be introduced (e.g., by mutating existing residues or inserting additional residues) at or equivalent to amino acids 12 to 17 of the antibody's FR1, amino acids 84 to 91 of the antibody's FR3, or amino acids 117 to 119 of the antibody's FR4, unless the amino acid is proline.

[0073] In one embodiment of this invention, a lipid nanoparticle is provided comprising (a) an activator and (b) a plurality of nanobodies present on the outer surface of the nanoparticle, wherein each nanobodies is linked to the lipid nanoparticle via a site-specific connection such that each nanobodies are presented in substantially the same orientation and are capable of binding to a target on its cell surface, wherein the site-specific connection is formed between a first coupling group (i.e., dibenzocyclooctylene) and a second coupling group (i.e., an azide located on the side chain of an amino acid at or equivalent to the 12th to 17th amino acids, preferably the 13th amino acid, in FR1 of the nanobodies). Preferably, the nanobodies comprise CDR1 as shown in SEQ ID NO: 23, 30, 38 or 45; CDR2 as shown in SEQ ID NO: 24, 31, 39 or 46; and CDR3 as shown in SEQ ID NO: 25, 32, 40 or 47. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively. More preferably, the nanobody comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 22 or 37.

[0074] In one embodiment of this invention, a lipid nanoparticle is provided comprising (a) an activator and (b) a plurality of nanobodies present on the outer surface of the nanoparticle, wherein each nanobodies is linked to the lipid nanoparticle via a site-specific connection such that each nanobodies are presented in substantially the same orientation and are capable of binding to a target on its cell surface, wherein the site-specific connection is formed between a first coupling group (i.e., a thiol or thiol group) formed on the PEGylated lipid and a second coupling group (i.e., a thiol or thiol group on the side chain of an amino acid at or equivalent to the 12th to 17th amino acids, preferably the 13th amino acid, in the FR1 of the nanobodies). Preferably, the nanobodies comprise CDR1 as shown in SEQ ID NO: 2, 9, or 15; CDR2 as shown in SEQ ID NO: 3, 10, or 16; and CDR3 as shown in SEQ ID NO: 4, 11, or 17. In one embodiment, the nanobodies comprise CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. In one embodiment, the nanobody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively. More preferably, the nanobody comprises, or is composed of, the amino acid sequence shown in SEQ ID NO: 1.

[0075] As used herein, the position or equivalent position of the 13th amino acid can be determined with reference to SEQ ID NO: 1, 22 or 38.

[0076] In any embodiment of this aspect, the lipid-based nanoparticles of the present invention exhibit higher cell-binding capacity compared to corresponding lipid-based nanoparticles in which the target molecule is randomly oriented. Higher binding capacity may manifest as a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase in binding as determined by any of the methods described herein (including examples). Furthermore, when the active agent encodes a protein, the lipid-based nanoparticles of the present invention exhibit higher protein expression levels compared to corresponding lipid-based nanoparticles in which the target molecule is randomly oriented. Higher protein expression levels may manifest as a 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold increase in expression as determined by any of the methods described herein (including examples). Lipid-based nanoparticles with randomly oriented target molecules can be formed by conjugating the target molecule to the lipid-based nanoparticles through reaction with EDC / NHS molecules and random lysine residues.

[0077] In any embodiment of this aspect, the lipid-based nanoparticles of the present invention exhibit higher cell-binding capacity compared to corresponding lipid-based nanoparticles without any targeting molecules. Higher binding capacity can be manifested as a 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold increase in binding capacity as determined by any of the methods described herein (including examples). Furthermore, when the active agent encodes a protein, the lipid-based nanoparticles of the present invention exhibit higher protein expression levels compared to corresponding lipid-based nanoparticles without any targeting molecules. Higher protein expression levels can be manifested as a 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold increase in expression capacity as determined by any of the methods described herein (including examples).

[0078] In any embodiment of this invention, the lipid-based nanoparticles may be more stable and less aggregated than corresponding lipid-based nanoparticles in which the target molecules are randomly oriented. Lipid-based nanoparticles with randomly oriented target molecules can be formed by conjugating target molecules to lipid-based nanoparticles through reaction with EDC / NHS molecules and random lysine residues.

[0079] On the other hand, the present invention provides a conjugate comprising a lipid capable of incorporating lipid-based nanoparticles and a targeting molecule, wherein the targeting molecule is conjugated to the lipid via a site-specific link.

[0080] In this regard, the lipid is a phospholipid, a structured lipid, a PEGylated lipid, or a cationic or ionizable lipid. Preferably, the lipid is a PEGylated lipid, such as DSPE-PEG.

[0081] In this respect, the conjugate is formed between a first coupling group on the lipid and a second coupling group on the target molecule. The first coupling group can be an alkene or alkyne dipolarophile, or a thiol or mercapto. An exemplary dipolarophile is a strained cyclic alkene or cyclic alkyne. Preferred strained cyclic alkenes or cyclic alkynes include cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazenecyclooctyne ketone. The second coupling group can be a 1,3-dipolar, or a thiol or mercapto. Typically, the 1,3-dipolar is an azide, nitrile oxide, nitrone, or isocyanate, preferably an azide. In one embodiment, the azide is azoxyphenylalanine (azPhe), wherein the capture-binding domain is modified to include azPhe.

[0082] When the first and second coupling groups are thiols or mercapto groups, the site-specific connection is a disulfide bond.

[0083] When the first coupling group is maleimide and the second coupling group is thiol or mercapto, the site-specific connection is thiosuccinimide.

[0084] In this regard, the target molecule is an antibody or antibody fragment, such as a nanobody or a single-chain variable fragment, affinity compound, aptamer or peptide, preferably a nanobody.

[0085] In this regard, when the target molecule is an antibody, the site-specific linker can be located at or equivalent to amino acids 12 to 17 in the FR1 of the antibody; amino acids 84 to 91 in the FR3 of the antibody; or amino acids 117 to 119 in the FR4 of the antibody. Non-natural amino acids or cysteines can be introduced (e.g., by mutating existing residues or inserting additional residues) at or equivalent to amino acids 12 to 17 in the FR1 of the antibody, amino acids 84 to 91 in the FR3 of the antibody, or amino acids 117 to 119 in the FR4 of the antibody, unless the amino acid is proline.

[0086] In any aspect or embodiment, each lipid-based nanoparticle contains at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180, 200, 300, 400, or 800 targeting molecules. Preferably, each lipid-based nanoparticle contains at least about 10, 20, 40, 50, 80, 100, 120, 140, 160, 180, or 200 targeting molecules.

[0087] On the other hand, the present invention provides a pharmaceutical composition comprising lipid-based nanoparticles as described herein, and pharmaceutically acceptable carriers, diluents, or excipients.

[0088] On the other hand, the present invention provides a method for introducing an active agent (e.g., nucleic acid) into a cell, preferably in vivo, the method comprising contacting the cell with lipid-based nanoparticles as described in the present invention, thereby introducing the active agent (e.g., nucleic acid) into the cell.

[0089] On the other hand, the present invention provides a method for in vivo delivery of an active agent, the method comprising administering the lipid-based nanoparticles of the present invention to a subject in need, thereby delivering the active agent to the subject.

[0090] On the other hand, the present invention provides a method for treating or preventing a disease or condition in a subject in need, the method comprising administering to the subject the lipid-based nanoparticles or pharmaceutical composition described herein, thereby treating or preventing the disease or condition in the subject in need.

[0091] On the other hand, the present invention provides the use of the lipid-based nanoparticles or pharmaceutical compositions described herein in the preparation of medicaments for treating or preventing diseases or conditions suffered by subjects in need.

[0092] On the other hand, the present invention provides lipid-based nanoparticles or pharmaceutical compositions for the treatment or prevention of diseases or conditions in subjects in need.

[0093] On the other hand, the present invention provides a method for generating a target polypeptide in cells (preferably mammalian cells), the method comprising contacting the cells with lipid-based nanoparticles as described in the present invention, wherein the activator is mRNA encoding the target polypeptide, and the mRNA is translatable in the cells to generate the target polypeptide.

[0094] On the other hand, the present invention provides a method for delivering mRNA into cells (preferably mammalian cells), the method comprising administering to a subject lipid-based nanoparticles as described herein, wherein the active agent is mRNA, thereby delivering mRNA into cells.

[0095] In any respect or implementation, the cell is a mammalian cell.

[0096] As used herein, unless the context otherwise requires, the term “comprise” and its variations (e.g., comprising / comprises / comprised) are not intended to exclude other additives, components, integers, or steps.

[0097] Other aspects of the invention, as well as other embodiments of the foregoing aspects, will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0098] Figure 1 Electron microscopy structural analysis enabled the rational engineering design of nanobodies for antibody capture. a) Two-dimensional projection (transmission electron microscopy image) of the nanobodies-antibody complex, scale bar 200 Å. b) Masked 3D refined image of the nanobodies (arrows indicated)-antibody structure (solid arrows indicated); and c) 3D reconstructed image of the two-dimensional projection. The Gln13 residue was determined as the optimal coupling site (hollow arrows indicated). d) Rendered model of the LNP modified with nanobodies. This model assumes an LNP diameter of 85 nm.

[0099] Figure 2LNP formulation capable of efficiently capturing antibodies with optimal orientation. Inserting TP1107 into an LNP forms stable particles and efficiently captures antibodies. a) Overview of the TP1107-LNP generation process. b) Protein Simple Jess Western blotting shows the lipid conjugate DSPE-PEG2000-TP1107. optimal and DSPE-PEG2000-TP1107 random c) Protein Simple Jess Western blotting revealed the presence of the lipid conjugate LNP-TP1107 containing the inserted LNP. optimal and LNP-TP1107 random d, e, f, and g) unmodified LNP, LNP-TP1107 optimal and LNP-TP1107 random The particle size distribution, mode particle size, zeta potential, and encapsulation efficiency. h) The TP1107 content of each LNP from four different batches of LNP. optimal Or TP1107 random The quantity of i and j)mAb TfR LNP-TP1107 marked optimal and LNP-TP1107 random The particle size distribution and mode particle size. All data are averages, and the error bars represent the mean ± standard deviation of independent repeated experiments (n = 3–5).

[0100] Figure 3 DSPE-PEG 2000 -LNPs maintain high antibody-mediated binding while limiting non-specific cell binding. a) Left panel: Mean fluorescence intensity (MFI) of Cy5 in Jurkat cells after incubation with DMG-LNP or DSPE-LNP; Right panel: eGFP expression level in Jurkat cells after incubation with DMG-LNP or DSPE-LNP. b) Cy5 MFI (left) and eGFP expression levels (right) in Jurkat cells after incubation with human TfR-targeted LNP, isotype control, and unmodified LNP. c) Calculated fold change in LNP binding and mRNA expression between targeted LNP and unmodified LNP (using DMG-LNP or DSPE-LNP). All data are averages, and error bars represent mean ± standard deviation (n = 3 independent wells).

[0101] Figure 4Optimally oriented antibodies exhibited significantly higher cell binding capacity and protein expression levels compared to randomly oriented antibodies. a) Mean fluorescence intensity (MFI) of Cy5; b) Percentage of eGFP-positive cells; c) Jurkat cells at a concentration of 0.5 ng / μL versus antibodies with different amounts of mAb. TfR (Each LNP contains 7 to 297 antibodies) Functionalized TP1107 optimal或random - Mean fluorescence intensity (MFI) of eGFP after 4 hours of LNP incubation. d) Cy5 MFI; e) Percentage of eGFP-positive cells; f) mAb of Jurkat cells reacting with lysine at a concentration of 1 ng / μL mRNA. TfR -LNP or excessive mAb load TfR mAb TfR -LNP-TP1107 optimal eGFP MFI after 24 hours of incubation. TP11007 functionalized with a set of mAbs (CD3, CD4, CD5, CD7 and isotype control). optimal - LNP was incubated with Jurkat cells for 24 hours. i) Cy5 MFI; j) percentage of eGFP-positive cells; j) eGFP MFI. All data are averages, and error bars represent mean ± standard deviation (n = 3). P-values ​​were calculated using one-way ANOVA combined with Tukey post-hoc tests. P < 0.0001. Data points represent independent wells. The experiment was repeated three times.

[0102] Figure 5 Screening of T-cell targeting antibodies revealed that different surface receptors exhibited varying mRNA delivery potential through targeting the LNP system. a) Schematic diagram showing the process of collecting human PBMCs and evaluating the specific targeting ability of the active LNP targeting system by flow cytometry. bg) Human PBMCs incubated with a group of mAb-LNPs (CD3, CD4, CD5, CD7, isotype control, and unmodified LNP) at a concentration of 2 ng / μL mRNA for 24 hours. b) CD4+ T cell population; c) CD8+ T cell population; d) CD56+ NK cell population; e) CD14-positive monocytes; f) CD19+ B cell population. The top figure represents LNP binding (Cy5 MFI), and the bottom figure represents mRNA delivery (eGFP MFI). g) The ratio of eGFP MFI to Cy5 in the group with significant LNP binding (Cy5 MFI > 3000 and positive). Data points represent three technical replicates from three donors. All data are donor means, and error bars represent standard deviations. P < 0.05, P < 0.01, P < 0.001, P < 0.0001; the results were obtained by two-way ANOVA combined with Tukey's post-hoc test (comparing row mean - main row effect).

[0103] Figure 6 LNP-TP1107 optimal and LNP-TP1107 random With a range of different proportions of antibodies and TP1107 optimal / random Joint incubation.

[0104] Figure 7 Even if the antibody to TP1107 ratio is 1:8, LNP-TP1107 optimal Compared to LNP-TP1107 random It still exhibits more effective targeted delivery. (via LNP-TP1107) optimal或random Percentage of transfected cells. All data are averages, and error bars represent mean ± standard deviation (n = 3).

[0105] Figure 8 In the short term, antibodies with optimal orientation outperformed conventional labeled antibodies on LNPs. a) Left panel: Mean fluorescence intensity (MFI) of Cy5; Middle panel: Percentage of GFP-positive cells; Right panel: Jurkat cells vs. TP1107 optimal -LNP or lysine-coupled mAb TfR - eGFP MFI after incubation with LNP for different times (0.5, 1, 2, 4 hours). All data are averages, and error bars represent the mean ± standard deviation (n = 3).

[0106] Figure 9 Expression levels of CD2, CD3, CD4, CD5, and CD7 in Jurkat cells. Jurkat cells were incubated with antibody at a 1:100 dilution at 4°C for 1 hour. After washing three times with cold PBS, cells were stained with Alexa Fluor 647-labeled goat anti-mouse antibody at a 1:1000 dilution at 4°C for 1 hour. The mean fluorescence intensity of Cy5 was collected and analyzed as the expression level of the surface marker. The simulated control sample represents cells incubated with secondary antibody only. All data are averages, and the error bars represent mean ± standard deviation (n = 3).

[0107] Figure 10The TP1107 capture system attaches antibodies to LNPs with optimal orientation. A schematic diagram comparing traditional antibody conjugation methods with the optimally oriented antibody capture system is shown. a) Typically, antibodies are conjugated to nanoparticles by reacting with EDC / NHS molecules and random lysine residues. However, the random orientation of the antibody on the nanoparticle surface leads to impaired antibody activity. b) Conjugating the anti-mouse / rat IgG1 nanobody TP1107 to lysine residues using NHS-azide enables high-throughput antibody screening, but the antibody remains randomly oriented. c) The optimally oriented TP1107 captures antibodies with optimal orientation, maximizing binding efficiency and enabling simple and rapid antibody screening.

[0108] Figure 11 Targeted LNP delivery to MDA-MB-231 cancer cells. These cells exhibit high levels of EGFR expression and low levels of Her2 expression. Left panel: Binding of LNP to MDA-MB-231 cells after 24 hours of incubation. Right panel: Protein expression levels (luciferase) after 24 hours of incubation.

[0109] Figure 12 Targeted LNP delivery to BT-474 cancer cells. These cells exhibit high levels of Her2 expression and low levels of EGFR expression. Left panel: LNP binding to BT474 cells after 24 hours of incubation. Right panel: Protein expression levels (luciferase) after 24 hours of incubation.

[0110] Figure 13 In vivo targeted distribution of different LNP formulations. Mice were administered a low-specificity LNP formulation, a CD3-targeting LNP formulation, an isotype control, and either comparative LNP formulation #1 or comparative LNP formulation #2. The distribution of LNPs in the spleen (A), bone marrow (B), liver (C), and lung (D) was determined. Detailed Implementation

[0111] Certain embodiments of the invention will now be described in detail. Although the invention will be described in conjunction with these embodiments, it should be understood that the purpose is not to limit the invention to these embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents, all of which may be included within the scope of the invention as defined in the claims.

[0112] Those skilled in the art will recognize that many methods and materials similar to or equivalent to those described and used herein can be used in the practice of this invention. This invention is by no means limited to the methods and materials described and used herein. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or obvious in the text or drawings. All these different combinations constitute various alternatives to the invention.

[0113] All patents and publications cited in this article are incorporated herein in their entirety through citation.

[0114] For the purposes of interpreting this specification, terms used in the singular will also include the plural forms, and vice versa.

[0115] Unless otherwise expressly stated, 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. Furthermore, any methods or materials similar to or equivalent to those described or made herein may be used in the practice of this invention. For the purposes of this invention, the following terms are defined as follows.

[0116] As used herein, the terms “an,” “a,” or “the” include not only aspects containing only a single member but also aspects containing multiple members. For example, unless the context clearly indicates otherwise, the singular forms of “an,” “a,” and “the” include plural referents. Thus, for example, reference to “a cell” includes multiple such cells, reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so on. For the purposes of interpreting this specification, terms used in the singular will also include the plural forms, and vice versa.

[0117] Efficient and more precise delivery of active agents (e.g., mRNA) is crucial for advancing mRNA therapy beyond its current use as an emergency vaccine. Lipid nanoparticle (LNP) formulations are an effective method for encapsulating and protecting mRNA, but non-specific cellular uptake leads to off-target delivery, and delivery efficiency to target cells is extremely low. Antibody-functionalized LNPs are an effective method for targeted mRNA delivery, but antibody modification requires complex conjugation and purification processes, resulting in loss of antibody affinity. This invention proposes a simple method for capturing antibodies at optimal orientation on the surface of LNPs without antibody modification or complex purification processes. This strategy results in protein expression levels more than 1000 times higher than untargeted LNPs and more than 10 times higher than traditional antibody functionalization techniques. This method enables rapid development of targeted LNPs and holds promise for broadening the application scope of mRNA therapy.

[0118] The antibody capture system described in this paper offers two main advantages over existing methods for preparing antibody-targeted LNPs. First, this highly efficient capture system enables the simple and rapid preparation of LNPs targeting a variety of different cell surface targets (e.g., receptors) without complex and inefficient purification processes. Second, the antibody is captured in an optimized or optimal orientation, thereby increasing the targeting efficiency of the LNP and subsequent protein expression levels by more than an order of magnitude. This simple antibody capture system allows for rapid screening of the most suitable receptors as targets. The method outlined in this paper shows broad application prospects in the delivery of various therapeutic agents (e.g., nucleic acids).

[0119] definition

[0120] As used herein, the terms “about” and “approximately” when applied to one or more target values ​​refer to values ​​similar to the reference value. In some embodiments, the terms “about” or “approximately” refer to a value falling within the range of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the reference value, unless otherwise stated or obvious from the context (unless the value exceeds 100% of the possible value). For example, when referring to a given amount of a compound in a lipid component of a nanoparticle composition, “about” may mean ±10% of the value. For example, a nanoparticle composition containing a lipid component having about 40% of a given compound may contain 30% to 50% of that compound.

[0121] As used herein, the term "compound" is intended to include all isomers and isotopes of the structures shown. "Isotope" refers to atoms having the same atomic number but different mass numbers due to differences in the number of neutrons in their nuclei. For example, isotopes of hydrogen include tritium and deuterium. Furthermore, the compounds, salts, or complexes of this disclosure can be prepared as solvates and hydrates by combining with solvents or water molecules using conventional methods.

[0122] As used herein, the term "delivery" refers to providing an entity to a destination. For example, delivering a therapeutic and / or preventative agent to a subject may include administering to the subject a nanoparticle composition comprising the therapeutic and / or preventative agent (e.g., via intravenous, intramuscular, intradermal, or subcutaneous route). Applying a nanoparticle composition to a mammal or mammalian cells may include contacting one or more cells with the nanoparticle composition.

[0123] As used herein, the term "enhanced delivery" refers to a nanoparticle delivering more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) a therapeutic and / or prophylactic agent to a target tissue (e.g., mammalian liver) compared to the level of delivery of a control nanoparticle (e.g., MC3, KC2, or DLinDMA) to the target tissue. The level of nanoparticle delivery to a specific tissue can be determined by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agent in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agent in the tissue to the total amount of therapeutic and / or prophylactic agent in the tissue. It should be understood that enhanced delivery of nanoparticles to a target tissue does not need to be determined in treated subjects, but can be determined in alternative models such as animal models (e.g., rat models).

[0124] As used herein, the term "specific delivery" refers to the delivery of more (e.g., at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times) therapeutic and / or prophylactic agents to a target tissue (e.g., mammalian liver) compared to off-target tissues (e.g., mammalian spleen). The delivery level of nanoparticles to a specific tissue can be determined by comparing the amount of protein produced in the tissue to the weight of the tissue, comparing the amount of therapeutic and / or prophylactic agents in the tissue to the weight of the tissue, comparing the amount of protein produced in the tissue to the total amount of protein in the tissue, or comparing the amount of therapeutic and / or prophylactic agents in the tissue to the total amount of therapeutic and / or prophylactic agents in the tissue.

[0125] As used herein, “encapsulation efficiency” refers to the amount of therapeutic and / or preventive agents that become part of the nanoparticle composition relative to the initial total amount of therapeutic and / or preventive agents used in preparing the nanoparticle composition. For example, if 97 mg of the total 100 mg of therapeutic and / or preventive agents initially provided to the composition are encapsulated in the nanoparticle composition, the encapsulation efficiency can be expressed as 97%. As used herein, “encapsulation” can mean complete, substantial, or partial closure, confinement, enclosure, or wrapping.

[0126] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or more closely related antigens via an antigen-binding domain contained within an Fv. The term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR transplanted antibodies, primate-derived antibodies, deimmunized antibodies, synthetic humanized antibodies, and haptens).

[0127] Antibodies typically contain constant domains, which can be arranged into constant regions, constant fragments, or crystallizable fragments (Fc). An exemplary form of antibody contains a four-chain structure as its basic unit. A full-length antibody consists of two covalently linked heavy chains (approximately 50 to 70 kDa) and two light chains (approximately 23 kDa each). The light chains typically contain a variable region (if present) and a constant domain; in mammals, the light chain is a κ light chain or a λ light chain. The heavy chain typically contains a variable region and one or two constant domains, which are linked to other constant domains via hinge regions. Mammalian heavy chains belong to one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains, as well as the heavy and light chains, are linked together by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds may vary in different antibody types. Each chain has an N-terminal variable region (V... H or V LEach of the light chain consists of approximately 110 amino acids and one or more constant domains located at the C-terminus. The constant domains of the light chain (C-terminus...) L (approximately 110 amino acids long) and the first constant domain of the heavy chain (C H1 The antibody heavy chain (330 to 440 amino acids long) is aligned and linked by disulfide bonds. The variable region of the light chain is aligned with the variable region of the heavy chain. The antibody heavy chain may contain two or more additional C164 ... H Structural domains (e.g., C) H2 C H3 (etc.), and can be found in C H1 With C H2 Hinge regions are contained between constant structural domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass. In one example, the antibody is a mouse (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is a humanized antibody, a synthetic humanized antibody, a chimeric antibody, a CDR-transplanted antibody, or a deimmunized antibody.

[0128] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably and refer to antibodies that essentially retain their complete form, as opposed to the antigen-binding fragment of an antibody. Specifically, whole antibodies include antibodies with both heavy and light chains containing an Fc region. The constant domain can be a wild-type sequence constant domain (e.g., a human wild-type sequence constant domain) or a variant of its amino acid sequence.

[0129] As used herein, a “variable region” refers to the portion of the antibody light chain and / or heavy chain that specifically binds to the antigen, as defined herein, including the amino acid sequence of the complementarity-determining regions (CDRs); namely CDR1, CDR2, and CDR3, and the frame regions (FRs). For example, a variable region may contain three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4) and three CDRs. H This refers to the variable region of the heavy chain. V L It refers to the variable region of the light chain.

[0130] As used herein, the term "complementarity-determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in the antibody variable region that play a major role in binding to specific antigens. Each variable region (V H or V L It typically has three CDRs, designated CDR1, CDR2, and CDR3. H The CDRs in this paper are also referred to as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to V. HCDR 1 and CDR H2 correspond to V H CDR 2 and CDR H3 correspond to V H CDR 3. Similarly, V L The CDRs in this paper are referred to as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to V. L CDR 1 and CDR L2 correspond to V L CDR 2 and CDRL3 correspond to V L CDR 3. In one example, the amino acid positions assigned to CDR and FR are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to CDR and FR are defined according to the enhanced Chothia numbering scheme (http: / / www.bioinfo.org.uk / mdex.html). This invention is not limited to FRs and CDRs as defined by the Kabat numbering system, but covers all numbering systems, including the canonical numbering system or the numbering system of Chothia and Lesk, J. Mol. Biol. 196: 901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273: 927-948, 1997; the numbering system of Honnegher and Plükthun, J. Mol. Biol. 309: 657-670, 2001; or the IMGT system discussed in Giudicelli et al., Nucleic Acids Res. 25: 206-211, 1997.

[0131] The "frame region" (FR) refers to the variable region residues excluding the CDR residues. In this paper, the FRs of VH are also referred to as FRH1, FRH2, FRH3, and FRH4, where FRH1 corresponds to VH. H FR1, FRH2 correspond to V H FR2, FRH3 correspond to V H FR3, FRH4 correspond to V H FR 4. Similarly, V LThe FRs in this paper are referred to as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to V. L FR1, FRL2 correspond to V L FR2, FRL3 correspond to V L FR3, FRL4 correspond to V L FR 4.

[0132] Lipid-based nanoparticles

[0133] As described herein, nanoparticles can be lipid-based. Lipid-based systems include oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0134] Imagine using lipid formulations to introduce at least one active agent into host cells (in vitro, ex vivo, or in vivo). Alternatively, at least one active agent may associate with lipids. This lipid-associated active agent may be encapsulated within the aqueous interior of lipid nanoparticles or liposomes, dispersed within a lipid bilayer of lipid nanoparticles or liposomes, attached to lipid nanoparticles or liposomes via linker molecules that simultaneously associate with both the lipid nanoparticles or liposomes and the active agent (e.g., oligonucleotides), embedded in lipid nanoparticles or liposomes, complexed with lipid nanoparticles or liposomes, dispersed in a lipid-containing solution, mixed with lipids, bound to lipids, contained in lipids in suspension, contained in or complexed with micelles, or otherwise associated with lipids. Compositions associated with lipids, lipid / nucleic acid, or lipid / expression vectors are not limited to any specific structure in solution. For example, these compositions may exist as bilayers, in micelle form, or exhibit a “collapsed” structure. These compositions may also simply be dispersed in solution, potentially forming aggregates of non-uniform size or shape.

[0135] In various embodiments, lipid-based nanoparticles may comprise lipids or derivatives thereof. Lipids are fatty substances and may be natural or synthetic lipids. For example, lipids include naturally occurring fat droplets in the cytoplasm, as well as compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, aldehydes, and polymers (e.g., PEGylated lipids).

[0136] In any aspect or implementation, lipid-based nanoparticles may have the composition or properties described herein (including examples). For example, lipid-based nanoparticles may be the low-specificity LNPs described in the examples.

[0137] The average particle size of the nanoparticles of the present invention can be greater than or equal to about 40 nm or 100 nm, for example, as determined by dynamic light scattering (DLS). For example, the average particle size can be greater than or equal to approximately 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230 nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275 nm, 280 nm, 285 nm, 290 nm, etc. nm, 295nm, 300 nm, 305 nm, 310 nm, 315 nm, 320 nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350nm, 355 nm, 360 nm, 365 nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405nm, 410nm, 415nm, 420nm, 425nm, 430nm, 435nm, 440nm, 445nm, 450nm, 455nm, 460nm, 465nm, 470nm, 475nm, 480nm, 485nm, 490nm or 500nm.

[0138] In some embodiments, the average particle size of the nanoparticles of the present invention may be about 40 nm to about 500 nm, about 45 nm to about 500 nm, about 50 nm to about 500 nm, about 55 nm to about 500 nm, about 60 nm to about 500 nm, about 65 nm to about 500 nm, about 70 nm to about 500 nm, about 75 nm to about 500 nm, about 80 nm to about 500 nm, about 90 nm to about 500 nm, about 100 nm to about 500 nm, about 110 nm to about 500 nm, about 120 nm to about 500 nm, about 130 nm to about 500 nm, about 140 nm to about 500 nm, about 150 nm to about 500 nm, about 160 nm to about 500 nm, about 170 nm to about 500 nm, about 180 nm to about 500 nm, about 190 nm to about 500 nm, about 200 nm to about 500 nm, about 210 nm, or about 210 nm. nm to 500 nm, approximately 220 nm to 500 nm, approximately 230 nm to 500 nm, approximately 240 nm to 500 nm, approximately 250 nm to 500 nm, approximately 260 nm to 500 nm, approximately 270 nm to 500 nm, approximately 280 nm to 500 nm, approximately 300 nm to 500 nm, approximately 310 nm to 500 nm, approximately 320 nm to 500 nm, approximately 330 nm to 500 nm, approximately 340 nm to 500 nm, approximately 350 nm to 500 nm, approximately 360 nm to 500 nm, approximately 370 nm to 500 nm, approximately 380 nm to 500 nm, approximately 390 nm to 500 nm, approximately 400 nm to 500 nm, approximately 410 nm to 500 nm, approximately 420 nm to 500 nm, approximately 430 nm to 500 nm, approximately 4 ... nm to about 500 nm, about 450 nm to about 500 nm, about 460 nm to about 500 nm, about 470 nm to about 500 nm, about 480 nm to about 500 nm, or about 490 nm to about 500 nm.

[0139] In some embodiments, the average particle size of the nanoparticles of the present invention may be about 40 nm to about 490 nm, about 40 nm to about 480 nm, about 40 nm to about 470 nm, about 40 nm to about 460 nm, about 40 nm to about 450 nm, about 40 nm to about 440 nm, about 40 nm to about 430 nm, about 40 nm to about 420 nm, about 40 nm to about 430 nm, about 40 nm to about 420 nm, about 40 nm to about 410 nm, about 40 nm to about 400 nm, about 40 nm to about 390 nm, about 40 nm to about 380 nm, about 40 nm to about 370 nm, about 40 nm to about 360 nm, about 40 nm to about 350 nm, about 400 nm to about 340 nm, about 40 nm to about 330 nm, about 40 nm to about 320 nm, about 40 nm to about 310 nm, about 40 nm to about 300 nm, about 40 nm to about 290 nm, about 40 nm to about 300 nm, about 40 nm to about 290 nm, about 40 nm to about 390 nm, about 40 nm to about 380 nm, about 40 nm to about 370 nm, about 40 nm to about 360 nm, about 40 nm to about 350 nm, about 400 nm to about 340 nm, about 40 nm to about 330 nm, about 40 nm to about 320 nm, about 40 nm to about 310 nm, about 40 nm to about 300 nm, about 40 nm to about 290 nm, about 40 nm to about 390 nm, about 40 nm to about nm to 280 nm, about 40 nm to 270 nm, about 40 nm to 260 nm, about 40 nm to 250 nm, about 40 nm to 240 nm, about 40 nm to 230 nm, about 40 nm to 220 nm, about 40 nm to 210 nm, about 40 nm to 200 nm, about 40 nm to 190 nm, about 40 nm to 180 nm, about 40 nm to 170 nm, about 40 nm to 160 nm, about 40 nm to 150 nm, about 40 nm to 140 nm, about 40 nm to 130 nm, about 40 nm to 120 nm, about 40 nm to 110 nm, about 40 nm to 100 nm, about 40 nm to 95 nm, about 40 nm to 90 nm, about 40 nm to 85 nm, about 40 nm to 80 nm, about 40 nm to 75 nm, about 40 nm to 70 ... nm to about 65 nm, about 40 nm to about 60 nm, about 40 nm to about 55 nm, about 40 nm to about 50 nm, or about 40 nm to about 45 nm.

[0140] Nanoparticles can be relatively homogeneous. A polydispersity index can be used to indicate the homogeneity of a nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can be from about 0 to about 0.25, for example, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be from about 0.10 to about 0.20.

[0141] As used in this article, “Zeta potential” refers to the electrokinetic potential of lipids (e.g., lipids in nanoparticles).

[0142] The zeta potential of nanoparticles can be used to indicate the electrokinetic potential of the particles. For example, the zeta potential can describe the surface charge of the nanoparticles. Typically, nanoparticles with low charge (whether positive or negative) are ideal because nanoparticles with high charge may interact with cells, tissues, and other components in the body in undesirable ways. However, the lipid nanoparticles of the present invention have an exceptionally high negative zeta potential. In some embodiments, the zeta potential of the nanoparticles can be from about -50 mV to about +10 mV, preferably from about -10 mV to about +5 mV. Furthermore, in some embodiments, the zeta potential of the nanoparticles can be from about -20 mV to about -5 mV, from about -15 mV to about -5 mV, from about -10 mV to about -5 mV, or from about -20 mV to about -10 mV. Furthermore, in some embodiments, the zeta potential of the nanoparticles can be -20 mV to -5 mV, -15 mV to -5 mV, -10 mV to -5 mV, or -20 mV to -10 mV. Furthermore, in some embodiments, the zeta potential of the nanoparticles can be about -5 mV, about -10 mV, about -15 mV, or about -20 mV. Furthermore, in some embodiments, the zeta potential of the nanoparticles can be -5 mV, -10 mV, -15 mV, or -20 mV.

[0143] Lipid-based nanoparticles may be any of those described herein, including those listed in the examples (e.g., Example 1).

[0144] Cationic and / or ionizable lipids

[0145] Any of a variety of cationic lipids can be used in the lipid-based nanoparticles described herein.

[0146] The cationic lipids used in this invention can be any of a variety of lipids carrying a net positive charge at physiological pH. Such lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleoyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and N,N-distearyl-N,N-dimethylammonium bromide (DDAB). N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristoyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[2(speramido)ethyl]-N,N-dimethyl-1-propanediamine trifluoroacetate (DOSPA), octacosanoamidoglycylspermine (DOGS) ), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxaproxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamoyl-3-dimethylaminopropane Alkane (DOcarbDAP), 1,2-N,N'-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLinCDAP), ((4-hydroxybutyl)azadialkyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]octanoate-1-octylnonyl ester (SM-102) and mixtures thereof. The aforementioned lipids and their related analogues have been described in the following documents: U.S. Patent Publications 20060083780 and 20060240554; U.S. Patents 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992; and PCT Publication WO96 / 10390, the entire contents of which are incorporated herein by reference for all purposes.In addition, a variety of commercially available formulations of cationic lipids are available and can be used in this invention. These formulations include, for example, LIPOFECTIN® (commercially available cationic liposomes containing DOTMA and DOPE, purchased from GIBCO / BRL, Grand Island, NY, USA); LIPOFECTAMINE® (commercially available cationic liposomes containing DOSPA and DOPE, purchased from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic liposomes containing DOGS, purchased from PromegaCorp., Madison, WI, USA).

[0147] In addition, cationic lipids of formula (I) having the following structure can also be used in this invention.

[0148]

[0149] Where R 1 and R 2 Independently selected from H, OH or C1-C5 alkyl groups, R 3 and R 4 Independently selected from alkyl groups containing about 4 to about 20 carbon atoms, and R 3 and R 4 At least one of them contains at least two unsaturated sites. In some cases, R 3 and R 4 Same, i.e., R 3 and R 4 All are linoleic (C 18 ), etc. In other cases, R 3 and R 4 Different, i.e., R 3 It is a tetradecanetrienyl (C 14 R 4 It is an oil-based (C 18 In a preferred embodiment, the cationic lipid represented by formula (I) has a symmetrical structure, i.e., R 3 and R 4 Same. In another preferred embodiment, R 3 and R 4 All contain at least two unsaturated sites. In some implementations, R 3 and R 4 Independently selected from the group consisting of: dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and eicosadienyl. In a preferred embodiment, R 3 and R 4 All are linoleic acid-based. In some implementations, R 3 and R 4It contains at least three unsaturated sites, independently selected from, for example, dodecathoritrienyl, tetradecathoritrienyl, hexadecathoritrienyl, linolenic acid, and eicosoritrienyl. In a particularly preferred embodiment, the cationic lipid represented by formula (I) is 1,2-dilinoleoyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA).

[0150] In addition, cationic lipids of formula (II) having the following structure can also be used in this invention.

[0151]

[0152] Where R 1 and R 2 Independently selected from H or C1-C5 alkyl groups, R 3 and R 4 Independently selected from alkyl groups containing about 10 to about 20 carbon atoms, and R 3 and R 4 At least one of them contains at least two unsaturated sites. In some cases, R 3 and R 4 Same, i.e., R 3 and R 4 All are linoleic (C 18 ), etc. In other cases, R 3 and R 4 Different, i.e., R 3 It is a tetradecanetrienyl (C 14 R 4 It is an oil-based (C 18 In a preferred embodiment, the cationic lipid of the present invention has a symmetrical structure, i.e., R 3 and R 4 Same. In another preferred embodiment, R 3 and R 4 All contain at least two unsaturated sites. In some implementations, R 3 and R 4 Independently selected from the group consisting of: dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and eicosadienyl. In a preferred embodiment, R 3 and R 4 All are linoleic acid-based. In some implementations, R 3 and R 4 It contains at least three unsaturated sites, and is independently selected from, for example, dodecathoritrienyl, tetradecathoritrienyl, hexadecathoritrienyl, linalyl and eicosoritrienyl.

[0153] In addition, cationic lipids (or salts thereof) of formula (III) having the following structure can also be used in this invention.

[0154]

[0155] Where R 1 and R 2 The same or different, and independently, C is an optional substitution. 12 -C 24 Alkyl, optionally substituted C 12 -C 24 alkenyl, optionally substituted C 12 -C 24 alkynyl or optionally substituted C 12 -C 24 Acyl group; R 3 and R 4 The same or different, and independently of optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, or optionally substituted C1-C5 ynyl, or R 3 and R 4 They can be linked to form optionally substituted heterocycles containing 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; R 5 It is absent or is hydrogen or C1-C6 alkyl to form a quaternary ammonium salt; m, n and p are the same or different and are independently 0 or 1, provided that m, n and p are not simultaneously 0; q is 0, 1, 2, 3 or 4; Y and Z are the same or different and are independently O, S or NH.

[0156] In some embodiments, the cationic lipid represented by formula (III) is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2, 2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpiperazinyl-[1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino) Acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleothio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.C1) 1,2-Dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.C1), 1,2-dilinoleoyloxy-3-(N-methylpiperazinyl)propane (DLin-MPZ), 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleooxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof. In a preferred embodiment, the cationic lipid represented by formula (III) is DLin-K-C2-DMA (XTC2).

[0157] Preferably, the cationic lipid is DODAP, DLin-DMA, DLin-K-DMA, DLin-K2-DMA, or DLin-MC3-DMA.

[0158] Cationic lipids typically comprise about 40 mol% to about 60 mol%, about 40 mol% to about 55 mol%, about 40 mol% to about 50 mol%, about 40 mol% to about 45 mol%, about 45 mol% to about 60 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 60 mol% of the total lipids in the particles.

[0159] Cationic lipids typically account for approximately 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol of the total lipids in the particles.

[0160] Phospholipids

[0161] As used herein, "phospholipid" refers to a lipid comprising a phosphate moiety and one or more carbon chains (e.g., unsaturated fatty acid chains). Phospholipids may contain one or more multiple bonds (e.g., double or triple bonds) (e.g., one or more unsaturated bonds). Some phospholipids can facilitate fusion with membranes. For example, cationic phospholipids can interact with one or more negatively charged phospholipids in a membrane (e.g., a cell membrane or intracellular membrane). Fusion of phospholipids with membranes allows one or more components of a lipid-containing composition to cross the membrane, thereby allowing, for example, the delivery of one or more components to a cell.

[0162] The lipid component of lipid-based nanoparticles or compositions may comprise one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. Typically, phospholipids may comprise a phospholipid moiety and one or more fatty acid moieties. For example, phospholipids may be lipids represented by formula (IV):

[0163] Where R p R1 and R2 represent the phospholipid moieties, while R1 and R2 represent the fatty acid moieties. These moieties may or may not contain unsaturated bonds and may be the same or different.

[0164] The phospholipid portion may be selected from the non-restricted group consisting of: phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, 2-lysophosphatidylcholine, and sphingomyelin.

[0165] The fatty acid portion may be selected from the non-restricted group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0166] The study also envisions encompassing non-natural substances, including naturally occurring materials that have been modified and substituted (including branching, oxidation, cyclization, and alkynylation). For example, phospholipids can be functionalized or cross-linked via one or more alkynes (e.g., alkenyl groups where one or more double bonds are replaced by triple bonds). Under appropriate reaction conditions, alkynes can undergo copper-catalyzed cycloaddition reactions upon exposure to azides. Such reactions could be used to functionalize lipid bilayers of nanoparticle compositions to facilitate membrane permeation or cell recognition, or to conjugate nanoparticle compositions with useful components such as targeting or imaging modalities (e.g., dyes).

[0167] The envisioned phospholipids include lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lecithin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dihexadecanophosphate, distearate, dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), and palmitoylphosphatidylglycerol (DPPC). Phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), 4-(N-maleimidemethyl)-cyclohexane-1-carboxylic acid dioleoyl phosphatidylethanolamine (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearate phosphatidylethanolamine (DSPE), monomethyl phosphatidylethanolamine, dimethyl phosphatidylethanolamine, ditransoleoyl phosphatidylethanolamine (DEPE), stearoyl oleoyl phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoyl phosphatidylcholine, and mixtures thereof. Other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids may also be used. The acyl group in these lipids is preferably derived from a group having a C 10 -C 24 Acyl groups of fatty acids in the carbon chain, such as lauroyl, myristyl, palmitoyl, stearyl, or oleoyl.

[0168] In some embodiments, the nanoparticle composition comprises DSPC. In some embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0169] Phospholipids typically comprise about 5 mol% to about 20 mol%, about 5 mol% to about 15 mol%, about 5 mol% to about 10 mol%, about 10 mol% to about 20 mol%, or about 15 mol% to about 20 mol% of the total lipids in the particles.

[0170] Phospholipids typically account for 5 mol% to 20 mol%, 5 mol% to 15 mol%, 5 mol% to 10 mol%, 10 mol% to 20 mol%, or 15 mol% to 20 mol% of the total lipids in the particles.

[0171] Structural lipids

[0172] The lipid component of the nanoparticle composition may comprise one or more structural lipids. The structural lipids may be selected from, but are not limited to, cholesterol, coccosterol, sitosterol, ergosterol, campesterol, stigmasterol, phytosterol, tomatine, ursolic acid, α-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and corticosteroids (e.g., prednisolone, dexamethasone, prednisolone, and hydrocortisone) or combinations thereof. Furthermore, the structural lipid may be squalane, squalene, or combinations thereof.

[0173] Structural lipids may include lipids containing geraniol acetate, farnesyl acetate, or geraniol geraniol, or their ethers, esters, or other derivatives.

[0174] Structural lipids typically comprise about 30 mol% to about 50 mol%, about 30 mol% to about 45 mol%, about 30 mol% to about 40 mol%, about 30 mol% to about 35 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, or about 45 mol% to about 50 mol% of the total lipids in the particles.

[0175] Structural lipids typically account for 30 mol% to 50 mol%, 30 mol% to 45 mol%, 30 mol% to 40 mol%, 30 mol% to 35 mol%, 35 mol% to 50 mol%, 40 mol% to 50 mol%, or 45 mol% to 50 mol% of the total lipids in the particles.

[0176] PEGylated lipids

[0177] The lipid component of lipid-based nanoparticles or compositions may contain one or more PEG or PEG-modified lipids. Such substances may also be referred to as PEGylated lipids. As used herein, "PEG lipid" or "PEGylated lipid" refers to a lipid containing a polyethylene glycol component. PEG lipids may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, PEG lipids may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or PEG-DSPE lipids.

[0178] In another embodiment, the PEGylated lipid may be 1,2-dimyristic-rac-glycerol-3-methoxypolyethylene glycol-2000, also known as DMG-PEG.

[0179] In another embodiment, the PEGylated lipid may be 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide (ALC-0159).

[0180] PEGylated lipids may have a PEG component with a molecular weight of any practically desired value, including but not limited to approximately 100 Daltons (Da) to 10,000 Da or higher (including but not limited to 0.1 to 10 kDa in some cases). The molecular weight of PEG may have a wide range, including but not limited to approximately 100 Da to approximately 10,000 Da or higher. The molecular weight of PEG may be approximately 100 Da to approximately 100,000 Da, including but not limited to 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of PEG is about 100 Da to 10000 Da, about 1000 Da to 9000 Da, about 1000 Da to 8000 Da, about 1000 Da to 7000 Da, about 1000 Da to 6000 Da, about 1000 Da to 5000 Da, about 1000 Da to 4000 Da, about 1000 Da to 3000 Da, or about 1000 Da to 2000 Da. In some embodiments, the molecular weight of PEG is about 1000 Da to 5000 Da. In some embodiments, the molecular weight of PEG is about 2000 Da to 5000 Da.

[0181] PEGylated lipids typically comprise about 0.05 mol% to about 10 mol% of the total lipids in the particles, or about 0.06 mol% to about 10 mol%, about 0.07 mol% to about 10 mol%, about 0.08 mol% to about 10 mol%, about 0.09 mol% to about 10 mol%, about 0.1 mol% to about 10 mol%, about 0.15 mol% to about 10 mol%, about 0.2 mol% to about 10 mol%, about 0.25 mol% to about 10 mol%, about 0.3 mol% to about 10 mol%, about 0.3 mol% to about 10 mol%, about 0.35 mol% to about 10 mol%, about 0.4 mol% to about 10 mol%, about 0.45 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.55 mol% to about 10 mol%, about 0.6 mol% to about 10 mol%, and about 0.65 mol% of the total lipids in the particles. mol% to about 10 mol%, about 0.7 mol% to about 10 mol%, about 0.75 mol% to about 10 mol%, about 0.8 mol% to about 10 mol%, about 0.85 mol% to about 10 mol%, about 0.9 mol% to about 10 mol%, about 0.95 mol% to about 10 mol%, about 1.0 mol% to about 10 mol%, about 1.5 mol% to about 10 mol%, about 2.0 mol% to about 10 mol%, about 2.5 mol% to about 10 mol%, about 3.0 mol% to about 10 mol%, about 3.5 mol% to about 10 mol%, about 4.0 mol% to about 10 mol%, about 4.5 mol% to about 10 mol%, about 5.0 mol% to about 10 mol%, about 6.0 mol% to about 10 mol%, about 7.0 mol% to about 10 mol%, about 8.0 mol% to about 10 mol% or about 9.0 mol% to approximately 10 mol%.

[0182] Alternatively, PEGylated lipids typically comprise about 0.05 mol% to about 10 mol%, about 0.05 mol% to about 9 mol%, about 0.05 mol% to about 8 mol%, about 0.05 mol% to about 7 mol%, about 0.05 mol% to about 6 mol%, about 0.05 mol% to about 5 mol%, about 0.05 mol% to about 4.5 mol%, about 0.05 mol% to about 4 mol%, about 0.05 mol% to about 3.5 mol%, about 0.05 mol% to about 3 mol%, about 0.05 mol% to about 2.5 mol%, about 0.05 mol% to about 2 mol%, about 0.05 mol% to about 1.5 mol%, about 0.05 mol% to about 1 mol%, about 0.05 mol% to about 0.95 mol%, about 0.05 mol% to about 0.9 mol%, and about 0.05 mol% to about 0.85 mol% of the total lipids in the particles. mol%, about 0.05 mol% to about 0.8 mol%, about 0.05 mol% to about 0.75 mol%, about 0.05 mol% to about 0.7 mol%, about 0.05 mol% to about 0.65 mol%, about 0.05 mol% to about 0.6 mol%, about 0.05 mol% to about 0.55 mol%, about 0.05 mol% to about 0.5 mol%, about 0.05 mol% to about 0.45 mol%, about 0.05 mol% to about 0.4 mol%, about 0.05 mol% to about 0.35 mol%, about 0.05 mol% to about 0.3 mol%, about 0.05 mol% to about 0.25 mol%, about 0.05 mol% to about 0.2 mol%, about 0.05 mol% to about 0.15 mol%, about 0.05 mol% to about 0.1 mol%, about 0.05 mol% to about 0.09 mol%, about 0.05 mol% to about 0.08 mol%, about 0.05 mol% to about 0.07 mol%, or about 0.05 mol% to about 0.06 mol%.

[0183] PEGylated lipids typically comprise 0.05 mol% to 10 mol%, 0.06 mol% to 10 mol%, 0.07 mol% to 10 mol%, 0.08 mol% to 10 mol%, 0.09 mol% to 10 mol%, 0.1 mol% to 10 mol%, 0.15 mol% to 10 mol%, 0.2 mol% to 10 mol%, 0.25 mol% to 10 mol%, 0.3 mol% to 10 mol%, 0.3 mol% to 10 mol%, approximately 0.35 mol% to 10 mol%, 0.4 mol% to 10 mol%, 0.45 mol% to 10 mol%, 0.5 mol% to 10 mol%, 0.55 mol% to 10 mol%, 0.6 mol% to 10 mol%, 0.65 mol% to 10 mol%, and 0.7 mol% to 10 mol% of the total lipids in the particles. mol%, 0.75 mol% to 10 mol%, 0.8 mol% to 10 mol%, 0.85 mol% to 10 mol%, 0.9 mol% to 10 mol%, 0.95 mol% to 10 mol%, 1.0 mol% to 10 mol%, 1.5 mol% to 10 mol%, 2.0 mol% to 10 mol%, 2.5 mol% to 10 mol%, 3.0 mol% to 10 mol%, 3.5 mol% to 10 mol%, 4.0 mol% to 10 mol%, 4.5 mol% to 10 mol%, 5.0 mol% to 10 mol%, 6.0 mol% to 10 mol%, 7.0 mol% to 10 mol%, 8.0 mol% to 10 mol%, or 9.0 mol% to 10 mol%.

[0184] PEGylated lipids typically comprise 0.05 mol%, 0.06 mol%, 0.07 mol%, 0.08 mol%, 0.09 mol%, 0.1 mol%, 0.15 mol%, 0.2 mol%, 0.25 mol%, 0.3 mol%, 0.35 mol%, 0.4 mol%, or 0.45 mol% of the total lipids in the particles.

[0185] As used in this article, “molar percentage” and “mol%” are used interchangeably.

[0186] Capture combined domain

[0187] As used herein, the capture-binding domain can be any molecule capable of binding to another molecule. For example, the capture-binding domain can be an antibody or its antigen-binding fragment, such as a nanobody or a single-chain variable fragment, protein A, protein G, protein L, or a spyCatcher.

[0188] Specifically, the capture-binding domain used in this invention binds to or specifically binds to the target molecules described herein. Such target molecules are typically portions of cell surface targets (e.g., membrane receptors). The capture-binding domain can be any peptide or protein (e.g., antibody or antibody fragment, including nanobodies) capable of binding to the target molecule. Specific embodiments of suitable capture-binding domains include nanobodies, such as single-domain antibodies.

[0189] The capture-binding domain binds to or specifically binds to the target molecule, so that it does not substantially interfere with the binding activity of the target molecule.

[0190] The capture-binding domain can bind to a single or specific site on the target molecule, or it can bind to multiple sites.

[0191] By selecting the binding sites of the capture-binding domain on the target molecule, the target molecule is oriented to the target molecule, thereby exposing the binding domain of the target molecule so that it can bind to its target.

[0192] When bound to multiple capture-binding domains, each bound target molecule presents itself in a non-random orientation on the outer surface of the lipid-based nanoparticle, substantially exposing and / or providing accessibility of each target molecule's binding domain to its target, i.e., an "optimal" or "optimized" orientation. This non-random orientation of the target molecule is achieved by linking the capture-binding domains to the lipid-based nanoparticle via site-specific connections. These site-specific connections orient each capture-binding protein on the outer surface of the lipid-based nanoparticle, ensuring that each target molecule has a substantially identical non-random orientation, protruding outward from the lipid-based nanoparticle, when bound to the capture-binding domain. Compared to the case where the capture-binding domain is randomly oriented on the outer surface of the lipid-based nanoparticle, the orientation formed by each capture-binding domain and its bound target molecule enhances the delivery efficiency of the lipid-based nanoparticle to specific target cells or cell populations, for example... Figure 10 As shown.

[0193] Advantageously, in any method of the present invention, when the target molecule binds to the capture-binding domain, it facilitates the delivery of the capture-binding domain and any compounds (i.e., lipid-based nanoparticles) to the site where the ligand or target of the target molecule is located.

[0194] The capture-binding domain can bind to the Fc region of the antibody, preferably the CH2 or CH3 domain.

[0195] Alternatively, the capture-binding domain binds to the framework region of an antibody, affinity emulsion, or fragment thereof (e.g., scFv or nanobody).

[0196] In a preferred embodiment, the capture-binding domain is any Fc-binding antibody described herein, including TP1107 or a variant thereof.

[0197] Capture-binding domains include, but are not limited to, antibodies, antibody fragments (e.g., Fab2, Fab, scFV, VHH domains), and other proteins or peptides.

[0198] According to a specific embodiment of the present invention, the capture-binding domain includes a complementary determinant region that binds to the target molecule.

[0199] In any embodiment, the capture-binding domain comprises a sequence having any complementarity-determining region (CDR) having the amino acid sequences shown in Table 1. Preferably, the amino acid sequences of the framework region are also shown in Table 1 below.

[0200] In another preferred embodiment, the capture-binding domain comprises CDRH1, CDRH2, and / or CDRH3 of a single-domain antibody having a variable heavy chain as defined in SEQ ID NO: 1.

[0201] In another preferred embodiment, the capture-binding domain includes a heavy-chain variable region comprising: CDRH1 as shown in SEQ ID NO: 2; CDRH2 as shown in SEQ ID NO: 3; and CDRH3 as shown in SEQ ID NO: 4; CDRH1 as shown in SEQ ID NO: 9; CDRH2 as shown in SEQ ID NO: 10; and CDRH3 as shown in SEQ ID NO: 11; or CDRH1 as shown in SEQ ID NO: 15; CDRH2 as shown in SEQ ID NO: 16; and CDRH3 as shown in SEQ ID NO: 17.

[0202] In another preferred embodiment, the capture-binding domain includes a heavy chain variable region that contains or consists of the sequence of SEQ ID NO: 1.

[0203] Table 1: TP1107 sequence

[0204] Targeted molecules

[0205] As described herein, the capture-binding domain binds to a target molecule (or equivalently referred to as a "targeting domain," "targeting portion," or "targeting ligand"), which serves to target a delivery carrier (e.g., a LNP) to a specific cell or cell population. In this aspect of the invention, the target molecule binds to cell surface molecules on the target cell, while the capture-binding domain binds to the target molecule. In this aspect, the target molecule can bind to lipid-based nanoparticles in a non-covalent manner via the capture domain.

[0206] It is also envisioned that the targeting molecule will be directly (rather than through a trapping domain) linked to the lipid nanoparticle. In this respect, the targeting molecule is covalently linked to the lipid-based nanoparticle.

[0207] When directly attached to lipid-based nanoparticles, multiple target molecules exhibit a non-random orientation on the outer surface of the nanoparticles, substantially exposing and / or providing accessibility of each target molecule's binding domain to its target—i.e., an "optimal" or "optimized" orientation. This non-random orientation of the target molecules is achieved by attaching them to lipid-based nanoparticles via site-specific linkages. These site-specific linkages orient each target molecule on the outer surface of the lipid-based nanoparticle, resulting in each target molecule having a substantially identical non-random orientation protruding outward from the lipid-based nanoparticle. Compared to the case where target molecules are randomly oriented on the outer surface of lipid-based nanoparticles, the orientation formed by each target molecule enhances the delivery efficiency of the lipid-based nanoparticles to specific target cells or cell populations.

[0208] Targeting molecules may include any suitable binding agent capable of specifically interacting with and binding to target cell ligands on the surface of target cells or tissues. Targeting molecules may be naturally occurring or engineered. Targeting molecules may include, but are not limited to, proteins, peptides, antibodies or antibody fragments, immunoglobulins or immunoglobulin fragments, small molecules, aptamers, vitamins, nucleic acid molecules, etc. No limitation is imposed on the targeting molecules contemplated herein, provided that any particular targeting molecule (a) can bind (covalently or non-covalently) to a capture-binding domain, and / or (b) can induce or facilitate the localization or targeting of a delivery vector to a target cell or tissue through binding or other interactions between the targeting molecule (optionally bound to a capture-binding domain) and a target cell ligand on the target cell or tissue.

[0209] Target cell ligands may include endogenous ligands present on the cell surface or in the extracellular space, such as carbohydrates, lipids, polysaccharides, proteins, glycoproteins, glycolipids, peptides, cell membrane components (e.g., cholesterol), etc.

[0210] In some embodiments, the endogenous ligand on the target cells is specific to the target cells, meaning it is expressed and / or present only on the target cells, or at least present in very low amounts in non-target cells. For example, the endogenous ligand on the target cells may be a disease-related protein, such as a cancer cell surface protein, which is typically not expressed in healthy cells. In other embodiments, the target ligand on the target cells may be an engineered or otherwise non-naturally occurring ligand, such as a transgenic target cell expressing a non-natural cell surface protein. Suitable targeting ligands can be selected to leverage the unique properties of the target cells, enabling the composition to distinguish between target cells and non-target cells.

[0211] In some embodiments, the target cells are one or more of the following: T cells (e.g., CD4+ T cells, CD8+ T cells, and CD25+ T cells), NK cells, CD14+ monocytes, B cells (including CD25+ B cells), dendritic cells, NKT cells, tumor cells (e.g., breast cancer, colon cancer, pancreatic cancer, ovarian cancer, and lung cancer cells), bone marrow progenitor cells, renal interstitial cells, and macrophages. Preferably, the target cells are one or more of the following: CD4+ T cells, CD8+ T cells, NK cells, or CD14+ monocytes.

[0212] Exemplary target molecules are shown in Table 2a below.

[0213] Table 2a: Exemplary Target Molecules

[0214] In a specific implementation scheme, the target molecule is selected from: anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody or anti-CD7 antibody, or any combination thereof, and the antibody is preferably a monoclonal antibody.

[0215] In another preferred embodiment, the capture-binding domain comprises CDRH1, CDRH2, and / or CDRH3 of a single-domain antibody having a variable heavy chain as defined in SEQ ID NO: 22.

[0216] In another preferred embodiment, the targeting molecule includes a heavy chain variable region comprising: CDRH1 as shown in SEQ ID NO: 23; CDRH2 as shown in SEQ ID NO: 24; and CDRH3 as shown in SEQ ID NO: 25; or CDRH1 as shown in SEQ ID NO: 30; CDRH2 as shown in SEQ ID NO: 31; and CDRH3 as shown in SEQ ID NO: 32.

[0217] In another preferred embodiment, the capture-binding domain includes a heavy chain variable region that contains or consists of the sequence of SEQ ID NO: 22.

[0218] In another preferred embodiment, the capture-binding domain comprises CDRH1, CDRH2, and / or CDRH3 of a single-domain antibody having a variable heavy chain as defined in SEQ ID NO: 37.

[0219] In another preferred embodiment, the targeting molecule includes a heavy chain variable region comprising: CDRH1 as shown in SEQ ID NO: 38; CDRH2 as shown in SEQ ID NO: 39; and CDRH3 as shown in SEQ ID NO: 40; or CDRH1 as shown in SEQ ID NO: 45; CDRH2 as shown in SEQ ID NO: 46; and CDRH3 as shown in SEQ ID NO: 47.

[0220] In another preferred embodiment, the capture-binding domain includes a heavy chain variable region that contains or consists of the sequence of SEQ ID NO: 37.

[0221] Table 2b: 7D12 sequence

[0222] Table 2c: 2D3 Sequences

[0223] Adhesion

[0224] In various embodiments of the invention, the capture-binding domain is conjugated to the lipid via site-specific linkage. Exemplary conjugation methods may include forming covalent bonds. In one embodiment, the conjugation is reversible, such that the delivery vehicle can dissociate from the target domain upon exposure to specific conditions or chemical agents. In another embodiment, the conjugation is irreversible, such that the delivery vehicle does not dissociate from the target domain under normal conditions.

[0225] In some embodiments, the conjugation includes a covalent bond formed between the lipid and the capture-binding domain. The lipid is activated by functionalization via a first coupling group. In one embodiment, the lipid comprises a first coupling group capable of reacting with a second coupling group. In one embodiment, the activated lipid is an activated PEGylated lipid. In one embodiment, the first coupling group is bound to the polyethylene glycol portion of the PEGylated lipid. In one embodiment, the second functional group is covalently linked to or constitutes part of the capture-binding domain.

[0226] The first and second coupling groups can be any functional groups known to those skilled in the art that co-form a covalent bond (e.g., under mild reaction conditions or physiological conditions). In some embodiments, the first or second coupling group is selected from the group consisting of: maleimide, phosphine, hydroxymethylphosphine, psoralen, imine ester, pyridyl disulfide, isocyanate, vinyl sulfone, α-haloacetyl, aryl azide, acyl azide, alkyl azide, tetrazine, methyltetrazine, trans-cyclooctene, methylcyclopropene, norbornene, diaziridine, benzophenone, epoxide, carbonate, acid anhydride, sulfonyl chloride, cyclooctyne, aldehyde, and mercapto. In some embodiments, the first or second coupling group is selected from the group consisting of: free mercapto (-SH), azide, strained alkyne, acylhydrazine, and alkoxyamine. In some embodiments, the first coupling group is a functional group that can react with a thiol group, such as maleimide, pyridyl disulfide, or haloacetyl. In one embodiment, the first coupling group is maleimide. In some embodiments, the first coupling group is a functional group that can react with an azide group, such as dibenzocyclooctyne, 4-dibenzocyclooctyne alcohol, or an alkyne. In one embodiment, the first coupling group is dibenzocyclooctyne.

[0227] In one embodiment, the second coupling group is a thiol group. The thiol group can be introduced into the capture-binding domain by any method known to those skilled in the art. In one embodiment, the thiol group is present on a free cysteine ​​residue.

[0228] In one embodiment, the second coupling group is an azide group. The azide group can be introduced into the trap-binding domain by any method known to those skilled in the art. In one embodiment, the azide group is present on an azide-phenylalanine residue.

[0229] In some embodiments, the lipid and capture-binding domains are functionalized via groups for "click" chemistry. Bioorthogonal "click" chemistry involves the reaction of a functional group containing a 1,3-dipolar (e.g., azide, nitrile oxide, nitrone, isonitrile) with an alkene or alkyne dipolarophile. Exemplary dipolarophiles include any strained cyclic alkenes and cyclic alkynes known to those skilled in the art, including but not limited to cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, and biarylazazenecyclooctyne ketones.

[0230] Surfactant

[0231] Lipid-based nanoparticles may contain one or more therapeutic and / or preventative agents. This disclosure provides methods for delivering therapeutic and / or preventative agents to mammalian cells or organs, optionally generating target peptides in mammalian cells, and treating diseases or conditions in mammals in need, the methods comprising administering to and / or contacting mammalian cells with lipid-based nanoparticles containing therapeutic and / or preventative agents.

[0232] Therapeutic agents and / or preventative agents include bioactive substances, also known as "active agents." A therapeutic agent and / or preventative agent may be a substance that, once delivered to a cell or organ, produces desired changes in the cell, organ, or other body tissue or system. Such agents can be used to treat one or more diseases, conditions, or symptoms. In some embodiments, the therapeutic agent and / or preventative agent is a small molecule drug that can be used to treat a specific disease, condition, or symptom. Examples of pharmaceuticals that can be used in nanoparticle compositions include, but are not limited to: antitumor agents (e.g., vincristine, doxorubicin, mitoxantrone, camptothecin, cisplatin, bleomycin, cyclophosphamide, methotrexate, and streptozotocin), anticancer agents (e.g., actinomycin D, vincristine, cytarabine, anthracyclines, alkylating agents, platinum compounds, antimetabolites, and nucleoside analogs, such as methotrexate, purine analogs, and pyrimidine analogs), antiinfective agents, local anesthetics (e.g., debucaine and chlorpromazine), and β-adrenergic blockers (e.g., propranolol, timolol, and labetalol). Antihypertensive agents (e.g., clonidine and hydralazine), antidepressants (e.g., imipramine, amitriptyline, and doxepin), anticonvulsants (e.g., phenytoin), antihistamines (e.g., diphenhydramine, chlorpheniramine, and promethazine), antibiotics / antibacterial agents (e.g., gentamicin, ciprofloxacin, and cefoxitin), antifungal agents (e.g., miconazole, terconazole, econazole, isoconazole, butonazole, clotrimazole, itraconazole, nystatin, naftifine, and amphotericin B), antiparasitic agents, hormones, hormone antagonists, immunomodulators, neurotransmitter antagonists, antiglaucoma agents, vitamins, anesthetics, and imaging agents.

[0233] The present invention provides novel lipid-based nanoparticles comprising one or more active agents, methods for preparing lipid-based nanoparticles, and methods for delivering and / or applying lipid-based nanoparticles (e.g. for treating diseases or conditions).

[0234] In some embodiments, the active agent or therapeutic agent is completely encapsulated within the lipid portion of the lipid particle, enabling the active agent in the lipid-based nanoparticles to resist enzymatic degradation, such as nuclease or protease degradation, in aqueous solution. In other embodiments, the lipid-based nanoparticles are substantially non-toxic to mammals, such as humans.

[0235] In some embodiments, the active agent or therapeutic agent comprises nucleic acid. In some cases, the nucleic acid comprises interfering RNA molecules, such as siRNA, aiRNA, miRNA, or mixtures thereof. In other cases, the nucleic acid comprises single-stranded or double-stranded DNA, RNA, or DNA / RNA hybrids, such as antisense oligonucleotides, ribozymes, plasmids, immunostimulatory oligonucleotides, or mixtures thereof.

[0236] The mRNA content in lipid-based nanoparticles may depend on the size, sequence, and other characteristics of the mRNA. The mRNA content in lipid-based nanoparticles may also depend on the size, composition, desired target, and other characteristics of the lipid-based nanoparticles. The relative content of mRNA with other components, such as lipids, may also vary. In some embodiments, the weight ratio (wt / wt) of the lipid component to mRNA in the nanoparticle composition may be from about 5:1 to about 50:1, for example, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, and 50:1. For example, the weight ratio of the lipid component to mRNA may be from about 10:1 to about 40:1. The mRNA content in the nanoparticle composition can be determined, for example, by absorption spectroscopy (e.g., UV-Vis absorption spectroscopy).

[0237] In some embodiments, the weight ratio of lipid component to mRNA in the nanoparticle composition is from about 5:1 to about 50:1. In some embodiments, the weight ratio is from about 10:1 to about 40:1.

[0238] In some embodiments, one or more mRNAs, lipids, and their amounts may be selected to provide a specific N:P ratio. The N:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to phosphate groups in the mRNA. Generally, a lower N:P ratio is preferred. One or more mRNAs, lipids, and their amounts may be selected to provide an N:P ratio of about 2:1 to about 8:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, and 8:1. In some embodiments, the N:P ratio may be about 2:1 to about 5:1. In a preferred embodiment, the N:P ratio may be about 4:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0239] In some embodiments, the N:P ratio of the nanoparticle composition is from about 2:1 to about 8:1. In specific embodiments, the N:P ratio is from about 2:1 to about 5:1. In preferred embodiments, the N:P ratio is about 4:1. In some embodiments, the N:P ratio is from about 5:1 to about 8:1. For example, the N:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

[0240] In other embodiments, the active agent or therapeutic agent comprises a peptide or polypeptide. In some cases, the peptide or polypeptide comprises an antibody, such as a polyclonal antibody, a monoclonal antibody, an antibody fragment; a humanized antibody, a recombinant antibody, a recombinant human antibody, a Primatized™ antibody, or a mixture thereof. In other cases, the peptide or polypeptide comprises a cytokine, a growth factor, an apoptosis factor, a differentiation-inducing factor, a cell surface receptor, a ligand, a hormone, a small molecule (e.g., a small organic molecule or compound), or a mixture thereof.

[0241] In some embodiments, the active agent is a therapeutic agent, or a salt or derivative thereof. The therapeutic agent derivative may itself possess therapeutic activity, or it may be a prodrug that becomes active after further modification. Thus, in one embodiment, the therapeutic agent derivative retains some or all of the therapeutic activity compared to an unmodified agent, while in another embodiment, the therapeutic agent derivative is a prodrug lacking therapeutic activity but becomes active after further modification.

[0242] A. Nucleic acid

[0243] In some embodiments, the lipid-based nanoparticles of the present invention are associated with nucleic acids to form nucleic acid-lipid-based nanoparticles (e.g., NALP). In some embodiments, the nucleic acid is completely encapsulated within the lipid particles. As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, wherein fragments containing no more than 60 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides.

[0244] In specific embodiments, the oligonucleotides of the present invention are about 15 to about 60 nucleotides in length. Nucleic acids can be administered alone in the lipid particles of the present invention, or in combination with (e.g., co-administered) the lipid-based nanoparticles of the present invention comprising peptides, polypeptides, or small molecules (e.g., conventional pharmaceuticals).

[0245] In the context of this invention, the terms "polynucleotide" and "oligonucleotide" refer to polymers or oligomers of nucleotide or nucleoside monomers composed of naturally occurring bases, sugars, and inter-sugar (backbone) bonds. The terms "polynucleotide" and "oligonucleotide" also include polymers or oligomers composed of non-naturally occurring monomers or portions thereof, and which function similarly. Such modified or substituted oligonucleotides are generally superior to their natural forms due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0246] Oligonucleotides are generally classified as deoxyribonucleotides or ribonucleotides. Deoxyribonucleotides consist of a 5-carbon sugar called deoxyribose, which is covalently linked to phosphate groups at its 5' and 3' carbon positions, forming an alternating, unbranched polymer. Ribonucleotides consist of a similar repeating structure, where the 5-carbon sugar is ribose.

[0247] According to the present invention, the nucleic acids present in nucleic acid-lipid-based nanoparticles include any known form of nucleic acid. The nucleic acids used herein may be single-stranded DNA or RNA (e.g., precursor mRNA, mature mRNA, mRNA), or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples of double-stranded DNA are described herein, including, for example, structural genes, genes containing regulatory and termination regions, and self-replicating systems such as viral DNA or plasmid DNA. Examples of double-stranded RNA are described herein, including, for example, siRNA and other RNAi agents, such as aiRNA and precursor miRNA. Single-stranded nucleic acids include, for example, antisense oligonucleotides, ribozymes, mature miRNAs, and triple-stranded oligonucleotides.

[0248] The lipid-based nanoparticles of the present invention contain or encapsulate mRNA that encodes a target polypeptide. Preferably, the mRNA is translatable in cells to produce the target polypeptide. The target polypeptide can be any antigenic or immunogenic polypeptide, such as polypeptides used to stimulate the humoral immune system (e.g., B cells or T cells). The target polypeptide can be used for therapeutic or prophylactic immunization in mammals (preferably humans). The target polypeptide can be used to produce a therapeutic or prophylactic effect on a disease or condition (preferably an infection). The infection can be caused by any microorganism, such as bacteria, viruses, fungi, or protozoa. The virus can be any virus, including but not limited to coronaviruses, preferably SARS-CoV or SARS-CoV-2.

[0249] The nucleic acids of this invention can have different lengths, generally depending on the specific form of the nucleic acid. For example, in a specific embodiment, the length of a plasmid or gene can be from about 100 to about 100,000 nucleotide residues. In a specific embodiment, the length of an oligonucleotide can be from about 10 to about 100 nucleotides. In various related embodiments, the lengths of single-stranded, double-stranded, and triple-stranded oligonucleotides can be from about 10 to about 60 nucleotides, from about 15 to about 60 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, or from about 20 to about 30 nucleotides.

[0250] In specific embodiments, the oligonucleotides (or their chains) of the present invention specifically hybridize or complement a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" indicate a sufficient degree of complementarity to allow stable and specific binding between the DNA or RNA target and the oligonucleotide. It should be understood that the oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence to achieve specific hybridization. In a preferred embodiment, the oligonucleotide is considered specifically hybridizable when binding to the target sequence interferes with the normal function of the target sequence, resulting in loss of its efficacy or expression, and when there is sufficient complementarity to avoid non-specific binding to non-target sequences under conditions requiring specific binding (i.e., under physiological conditions for in vivo assays or therapeutic treatments; under conditions under which the assay is performed for in vitro assays). Therefore, the oligonucleotide may contain one, two, three, or more base substitutions relative to the gene region or mRNA sequence it targets or specifically hybridizes with.

[0251] 1. siRNA

[0252] The siRNA component in the nucleic acid-lipid particles of the present invention can silence the expression of a target gene. Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides. In some embodiments, the siRNA contains at least one modified nucleotide. Modified siRNA is generally less immunostimulatory than the corresponding unmodified siRNA sequence and retains RNAi activity against the target gene. In some embodiments, the modified siRNA contains at least one 2'OMe purine or pyrimidine nucleotide, such as 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and / or 2'OMe-cytidine nucleotide. In a preferred embodiment, one or more uridine and / or guanosine nucleotides are modified. The modified nucleotide may be present in one strand (i.e., the sense strand or the antisense strand) or both strands of the siRNA. The siRNA sequence may have overhangs (e.g., Elbashir et al., Genes Dev. , 15: 188 (2001) or Nyilnen et al., CellThe 3' or 5' protruding end described in 107: 309 (2001) may also not have a protruding end (i.e., have a flat end).

[0253] In the double-stranded region of the siRNA double helix, the modified siRNA typically contains about 1% to about 100% (e.g., about 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%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%) of modified nucleotides. In some embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more nucleotides in the siRNA double helix contain modified nucleotides.

[0254] In some implementations, less than about 25% (e.g., less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of the siRNA double-stranded region contains modified nucleotides.

[0255] In other embodiments, the siRNA double-stranded region contains approximately 1% to approximately 25% (e.g., approximately 1% to 25%, 2% to 25%, 3% to 25%, 4% to 25%, 5% to 25%, 6% to 25%, 7% to 25%, 8% to 25%, 9% to 25%, 10% to 25%, 11% to 25%, 12% to 25%, 13% to 25%, 14% to 25%, 15% to 25%, 16% to 25%, 17% to 25%, 18% to 25%, 19% to 25%, 20% to 25%, 21% to 25%, 22% to 25%, 23% to 25%, 24% to 25%, etc.) or approximately 1% to approximately 20% (e.g., approximately 1% to 20%, 2% to 20%, 3% to 25%). 0%, 4% to 20%, 5% to 20%, 6% to 20%, 7% to 20%, 8% to 20%, 9% to 20%, 10% to 20%, 11% to 20%, 12% to 20%, 13% to 20%, 14% to 20%, 15% to 20%, 16% to 20%, 17% to 20%, 18% to 20%, 19% to 20%, 1% to 19%, 2% to 19%, 3% to 19%, 4% to 19%, 5% to 19%, 6% to 19%, 7% to 19%, 8% to 19%, 9% to 19%, 10% to 19%, 11% to 19%, 12% to 19%, 13% to 19%, 14% to 19%, 15% to 19%, 16% to 19%, 1 7% to 19%, 18% to 19%, 1% to 18%, 2% to 18%, 3% to 18%, 4% to 18%, 5% to 18%, 6% to 18%, 7% to 18%, 8% to 18%, 9% to 18%, 10% to 18%, 11% to 18%, 12% to 18%, 13% to 18%, 14% to 18%, 15% to 18%, 16% to 18%, 17% to 18%, 1% to 17%, 2% to 17%, 3% to 17%, 4% to 17%, 5% to 17%, 6% to 17%, 7% to 17%, 8% to 17%, 9% to 17%, 10% to 17%, 11% to 17%, 12% to 17%, 13% to 17%, 14% to 17%. Nucleotides of 15% to 17%, 16% to 17%, 1% to 16%, 2% to 16%, 3% to 16%, 4% to 16%, 5% to 16%, 6% to 16%, 7% to 16%, 8% to 16%, 9% to 16%, 10% to 16%, 11% to 16%, 12% to 16%, 13% to 16%, 14% to 16%, 15% to 16%, 1% to 15%, 2% to 15%, 3% to 15%, 4% to 15%, 5% to 15%, 6% to 15%, 7% to 15%, 8% to 15%, 9% to 15%, 10% to 15%, 11% to 15%, 12% to 15%, 13% to 15%, 14% to 15%, etc., contain modified nucleotides.

[0256] In a further embodiment, for example, when one or both strands of the siRNA are selectively modified at uridine and / or guanosine nucleotides, the resulting modified siRNA may contain less than about 30% of the modified nucleotides (e.g., less than about 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the modified nucleotides) or about 1% to about 30% of the modified nucleotides (e.g., about 1% to 30%). 2% to 30%, 3% to 30%, 4% to 30%, 5% to 30%, 6% to 30%, 7% to 30%, 8% to 30%, 9% to 30%, 10% to 30%, 11% to 30%, 12% to 30%, 13% to 30%, 14% to 30%, 15% to 30%, 16% to 30%, 17% to 30%, 18% to 30%, 19% to 30%, 20% to 30%, 21% to 30%, 22% to 30%, 23% to 30%, 24% to 30%, 25% to 30%, 26% to 30%, 27% to 30%, 28% to 30%, or 29% to 30% of modified nucleotides.

[0257] a. Screening of siRNA sequences

[0258] Applicable siRNA sequences can be identified using any method known in the art. Typically, Elbashir et al., Nature , 411: 494-498 (2001) and Elbashir et al, EMBO J. The method described in 20: 6877-6888 (2001) is similar to that described by Reynolds et al. Nature Biotech. This is combined with the rational design rules proposed in , 22(3): 326-330 (2004).

[0259] Typically, the nucleotide sequence at the 3' end of the AUG start codon in the transcript of the target gene is scanned to look for dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, where N is C, G, or U) (see, for example, Elbashir et al.). EMBO J.,20: 6877-6888 (2001)). Nucleotides immediately adjacent to the 3' end of a dinucleotide sequence are identified as potential siRNA sequences (i.e., target sequences or sense strand sequences). Typically, 19, 21, 23, 25, 27, 29, 31, 33, 35 or more nucleotides immediately adjacent to the 3' end of a dinucleotide sequence are identified as potential siRNA sequences. In some embodiments, the dinucleotide sequence is an AA or NA sequence, and 19 nucleotides immediately adjacent to the 3' end of an AA or NA dinucleotide are identified as potential siRNA sequences. siRNA sequences are typically distributed at different locations along the length of the target gene. To further improve the silencing efficiency of siRNA sequences, potential siRNA sequences can be analyzed to identify sites that do not contain homologous regions to other coding sequences (e.g., coding sequences in target cells or organisms). For example, a suitable siRNA sequence of about 21 base pairs typically has no more than 16 to 17 consecutive base pairs homologous to coding sequences in target cells or organisms. If the siRNA sequence needs to be expressed by the RNA Pol III promoter, then choose an siRNA sequence that does not contain more than 4 consecutive A or T.

[0260] Once a potential siRNA sequence is identified, its complementary sequence (i.e., antisense sequence) can be designed. Potential siRNA sequences can also be analyzed according to various criteria known in the art. For example, to improve silencing efficiency, siRNA sequences can be analyzed using rational design algorithms to identify sequences with one or more of the following characteristics: (1) a G / C content of approximately 25% to approximately 60%; (2) at least three A / U sequences at positions 15 to 19 of the positive strand; (3) no internal repeat sequences; (4) an A sequence at position 19 of the positive strand; (5) an A sequence at position 3 of the positive strand; (6) a U sequence at position 10 of the positive strand; (7) no G / C sequence at position 19 of the positive strand; and (8) no G sequence at position 13 of the positive strand. siRNA design tools that integrate algorithms for assigning appropriate values ​​to the above characteristics and facilitate siRNA screening are available, for example, at http: / / boz094.ust.hk / RNAi / siRNA. Those skilled in the art will understand that sequences with one or more of the above characteristics can be selected as potential siRNA sequences for further analysis and testing.

[0261] In addition, potential siRNA sequences that meet one or more of the following criteria are generally excluded: (1) sequences containing four or more consecutive identical bases; (2) sequences containing G homopolymers (i.e., to reduce non-specific effects that may be caused by the structural characteristics of such polymers); (3) sequences containing triplet motifs (e.g., GGG, CCC, AAA, or ITT); (4) sequences containing seven or more consecutive G / C sequences; and (5) candidate sequences containing four or more direct repeats of bases, resulting in an internal foldback structure. However, those skilled in the art will understand that sequences with one or more of the above characteristics can still be selected as potential siRNA sequences for further analysis and testing.

[0262] In some implementations, the potential siRNA sequence can be further analyzed based on the asymmetry of the siRNA duplex, as described by Khvorova et al. Cell , 115: 209-216 (2003) and Schwarz et al, Cell As described in , 115:199-208 (2003). In other embodiments, the potential siRNA sequence can be further analyzed based on the secondary structure of the target site, for example, as described by Luo et al. Biophys. Res. Commun. As described in , 318: 303-310 (2004). For example, the secondary structure of the target site can be modeled using the Mfold algorithm (available at http: / / www.bioinfo.rpi.edu / applications / mfold / rna / forml.cgi) to screen siRNA sequences that are more accessible to the target site, where the site has fewer base pairing and stem-loop secondary structures.

[0263] Once a potential siRNA sequence is identified, its immunostimulatory properties can be analyzed, for example, using in vitro cytokine assays or in vivo animal models. Motifs in the sense and / or antisense strands of the siRNA sequence, such as GU-rich motifs (e.g., 5'-GU-3', 5'-UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.), can also indicate the potential immunostimulatory nature of the sequence. Once an siRNA molecule is found to be immunostimulatory, it can be modified according to the methods described herein to reduce its immunostimulatory properties. As a non-limiting example, the siRNA sequence can be exposed to mammalian responder cells under conditions that induce a detectable immune response in cells to determine whether the siRNA is an immunostimulatory or non-immunostimulatory siRNA. Mammal responder cells can be derived from unsensitized mammals (i.e., mammals that have not previously been exposed to the gene product corresponding to the siRNA sequence). Mammal responder cells can be, for example, peripheral blood mononuclear cells (PBMCs), macrophages, etc. Detectable immune responses may include the production of cytokines or growth factors, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-12, or combinations thereof. The immunostimulatory properties of siRNA molecules identified as immunostimulatory can then be reduced by replacing at least one nucleotide on the sense and / or antisense strands with a modified nucleotide. For example, less than about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in the double-stranded region of the siRNA duplex can be replaced with a modified nucleotide (e.g., a 2'OMe nucleotide). The modified siRNA can then be exposed to mammalian responding cells as described above to confirm that its immunostimulatory properties have been reduced or eliminated.

[0264] In vitro analytical methods suitable for detecting immune responses include, but are not limited to: the biclonal antibody sandwich immunoassay described by David et al. (US Patent No. 4,376,110); the monoclonal-polyclonal antibody sandwich assay (Wide et al., published in Kirkham and Hunter, eds.). Radioimmunoassay Methods E. and S. Livingstone, Edinburgh (1970); Western blotting as described by Gordon et al. (US Patent No. 4,452,901); labeled ligand immunoprecipitation (Brown et al., J. Biol. Chem. , 255: 4980-4983 (1980)); Enzyme-linked immunosorbent assay (ELISA) (Raines et al., 255: 4980-4983 (1980)); J. Biol. Chem. , 257: 5154-5160 (1982)); Immunocytochemistry techniques, including the use of fluorescent dyes (Brooks et al., 257: 5154-5160 (1982)); Clin. Exp. Immunol., 39: 477 (1980)); and the active neutralization method (Bowen-Pope et al., , Proc. Natl. Acad. Sci. USA , 81: 2396-2400 (1984)). In addition to the immunoassays described above, other immunoassays as described in U.S. Patent Nos. 3,817,827, 3,850,752, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, and 4,098,876 may also be used. The entire contents of the above references are incorporated herein by reference for all purposes.

[0265] Non-limiting examples of in vivo models for detecting immune responses include Judge et al. Mol. Ther. The in vivo mouse cytokine induction assay described in ,13: 494-505 (2006). In some embodiments, the assay may be performed as follows: (1) siRNA is administered via standard intravenous injection via tail vein; (2) blood is collected via cardiac puncture approximately 6 hours after administration and processed into plasma for cytokine analysis; (3) cytokines (e.g., mouse and human IFN-α (PBL Biomedical; Piscataway, NJ); human IL-6 and TNF-α (eBioscience; San Diego, Calif.); and mouse IL-6, TNF-α and IFN-γ (BDBiosciences; San Diego, Calif.)) are quantitatively detected using a sandwich ELISA kit according to the manufacturer's instructions.

[0266] Monoclonal antibodies that specifically bind to cytokines and growth factors are commercially available from multiple sources and can also be prepared using methods known in the art (see, for example, Kohler et al.). Nature , 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)). Methods for preparing monoclonal antibodies have been previously described and can be achieved by any method known in the art (Buhring et al., in Hybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, the monoclonal antibody is labeled (e.g., any composition detectable by spectroscopic, photochemical, biochemical, electrical, optical, or chemical means) for easy detection.

[0267] b. Preparation of siRNA molecules

[0268] siRNA can be provided in various forms, including, for example, one or more isolated small interfering RNA (siRNA) duplexes, longer double-stranded RNA (dsRNA), or siRNA or dsRNA transcribed from a transcription cassette in a DNA plasmid. siRNA sequences may have overhangs (e.g., Elbashir et al.). Genes Dev. , 15: 188 (2001) or Nykänen et al., Cell The 3' or 5' protruding end described in ,107: 309 (2001) may also not have a protruding end (i.e., have a flat end).

[0269] Long precursor RNAs can be provided using a population of RNAs, or siRNAs can be prepared using long precursor RNAs that are substantially or completely identical to the selected target sequence. These RNAs can be isolated, synthesized, and / or cloned from cells or tissues using methods well known to those skilled in the art. The RNAs can be a mixed population (derived from cells or tissues, transcribed from cDNA, obtained through subtraction, screening, etc.) or represent a single target sequence. The RNAs can be naturally occurring (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerases and PCR products or cloned cDNA), or chemically synthesized.

[0270] To form long dsRNA, for synthetic RNA, its complementary strand must be transcribed in vitro and hybridized to form dsRNA. If a naturally occurring RNA population is used, a complementary RNA strand must also be provided (e.g., for forming dsRNA for digestion by *E. coli* RNAse III or Dicer), for example, by transcribing cDNA corresponding to the RNA population or using RNA polymerase. The precursor RNA is then hybridized to form double-stranded RNA for digestion. dsRNA can be administered directly to the subject or pre-digested in vitro before administration.

[0271] Methods for RNA isolation, RNA synthesis, nucleic acid hybridization, cDNA library construction and screening, and PCR are all well-known in the field (see, for example, Gubler and Hoffman). Gene , 25: 263-269 (1983); Sambrook et al., ibid.; Ausubel et al., ibid.), PCR methods are also well known in the art (see U.S. Patent Nos. 4,683,195 and 4,683,202). PCR Protocols: A Guide to Methods and Applications (Innis et al., eds., 1990). Expression libraries are also well known to those skilled in the art. Other foundational literature disclosing the general methods used in this invention includes: Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd edition, 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al., 1994). The entire contents of the above references are incorporated herein by reference for all purposes.

[0272] Preferably, the siRNA is chemically synthesized. The oligonucleotides constituting the siRNA molecule of the present invention can be synthesized using various techniques known in the art, such as those employed by Usman et al. J. Am. Chem. Soc. , 109: 7845 (1987); Scaringe et al, Nucl. Acids Res. , 18: 5433 (1990); Wincott et al, Nucl. Acids Res. ,23: 2677-2684 (1995); and Wincott et al., Methods Mol. Bio. The technique described in 74:59 (1997) is applicable to the synthesis of oligonucleotides. Common nucleic acid protecting groups and coupling groups are utilized, such as a 5'-terminal dimethoxytriphenylmethyl group and a 3'-terminal phosphoramide group. As a non-limiting example, small-scale synthesis can be performed using an Applied Biosystems synthesizer at a scale of 0.2 μmol. Alternatively, a 0.2 μmol-scale synthesis can be performed using a 96-well plate synthesizer manufactured by Protogene (Palo Alto, Calif.). However, larger or smaller-scale synthesis is also within the scope of this invention. Reagents suitable for oligonucleotide synthesis, RNA deprotection methods, and RNA purification methods are known to those skilled in the art.

[0273] siRNA molecules can also be synthesized using tandem synthesis, where two strands are synthesized as a single, continuous oligonucleotide fragment or strand, separated by a cleavable linker. This linker is then cleaved to obtain independent fragments or strands that can hybridize to form a siRNA duplex. The linker can be a polynucleotide linker or a non-nucleotide linker. Tandem synthesis of siRNA is readily applicable to porous / plate synthesis platforms as well as large-scale synthesis platforms using batch reactors, synthesis columns, etc. Alternatively, siRNA molecules can be assembled from two distinct oligonucleotides, one containing the sense strand of the siRNA and the other containing the antisense strand. For example, each strand can be synthesized separately and assembled together via hybridization or ligation after synthesis and / or deprotection. In other cases, siRNA molecules can be synthesized as a single, continuous oligonucleotide fragment, where self-complementary sense and antisense regions hybridize to form a siRNA duplex with a hairpin-like secondary structure.

[0274] c. Modification of siRNA sequences

[0275] In some respects, the siRNA molecule comprises a double-stranded structure with two strands, and the double-stranded region contains at least one modified nucleotide, wherein each strand is about 15 to about 60 nucleotides in length. Advantageously, the modified siRNA is less immunostimulatory than the corresponding unmodified siRNA sequence, but retains the ability to silence the expression of the target sequence. In a preferred embodiment, the degree of chemical modification introduced into the siRNA molecule achieves a balance between reducing or eliminating the immunostimulatory properties of the siRNA and preserving RNAi activity. As a non-limiting example, the siRNA molecule targeting the target gene may be minimally modified (e.g., the modification ratio is less than about 30%, 25%, 20%, 15%, 10%, or 5%) at selective uridine and / or guanosine nucleotides within the siRNA double-stranded structure, thereby eliminating the immune response induced by the siRNA while retaining its ability to silence the expression of the target gene.

[0276] Examples of modified nucleotides suitable for this invention include, but are not limited to, ribonucleotides having the following groups: 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl. Modified nucleotides having a Northern conformation (e.g., Saenger, Principles of Nucleic Acid Structure The modifications described in Springer-Verlag Ed. (1984) also apply to siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-furanose) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thioethyl nucleotides, 2'-deoxy-2'-fluoro(2'F) nucleotides, 2'-deoxy-2'-chloro(2'Cl) nucleotides, and 2'-azidonucleotides. In some cases, the siRNA molecules described herein contain one or more G-clamp nucleotides. G-clamp nucleotides refer to modified cytosine analogs whose modifications confer their ability to form hydrogen bonds simultaneously with both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides within the duplex (see, for example, Lin et al.). J Am. Chem. Soc. ,120: 8531-8532 (1998)). Furthermore, nucleotides having nucleotide base analogs (e.g., C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitrazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole) (see, for example, Loakes, 120: 8531-8532 (1998)) can be used. Nucl. Acids Res. , 29: 2437-2447(2001)) incorporate siRNA molecules.

[0277] In some implementations, the siRNA molecule may also contain one or more chemical modifications, such as a terminal cap portion, phosphate backbone modifications, etc. Examples of terminal cap portions include, but are not limited to: inverted deoxygenated baseless residues, glycerol modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-dehydrated hexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentafuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide portions, 3'-3'-inverted baseless portions, 3'-2'-inverted nucleotide portions, 3'-2'-inverted baseless portions, 5'-5'-inverted nucleotide portions, 5' -5'-inverted baseless moiety, 3'-5'-inverted deoxygenated baseless moiety, 5'-aminoalkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-aminophosphate, 5'-aminophosphate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-thiophosphate, 5'-thiophosphate, dithiophosphate, and bridged or non-bridged methylphosphonates or 5'-mercaptomeric moiety (see, for example, U.S. Patent No. 5,998,203; Beaucage et al., Tetrahedron 49: 1925 (1993)). Non-limiting examples of phosphate backbone modifications (i.e., the formation of modified internucleotide links) include thiophosphates, dithiophosphates, methylphosphonates, triphosphates, morpholinophosphates, amide esters, carbamates, carboxymethyl esters, acetamide esters, polyamides, sulfonates, sulfonamides, aminosulfonates, methyl acetals, thiomethyl acetals, and alkylsilyl substitutions (see, for example, Hunziker et al.). Nucleic Acid Analogues: Synthesis and Properties , published in Modern Synthetic Methods VCH, 331-417 (1995); Mesmaeker et al. Novel Backbone Replacements for Oligonucleotides , published in Carbohydrate Modifications in Antisense Research (ACS, 24-39 (1994)). Such chemical modifications can occur at the 5' and / or 3' ends of the sense strand, antisense strand, or both strands of the siRNA. The entire contents of the above references are incorporated herein by reference for all purposes.

[0278] In some embodiments, the sense and / or antisense strands of the siRNA molecule may also include a 3' end overhang having about one to four (e.g., one, two, three, or four) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Other examples of modified nucleotides and types of chemical modifications that may be introduced into the siRNA molecule are described, for example, in British Patent No. GB ​​2,397,818B and U.S. Patent Publications Nos. 20040192626, 20050282188, and 20070135372, the entire contents of which are incorporated herein by reference for all purposes.

[0279] The siRNA molecules described herein may optionally contain one or more nonnucleotides in one or both strands. As used herein, the term "nonnucleotide" refers to any group or compound that can replace one or more nucleotide units incorporated into a nucleic acid chain (including sugar and / or phosphate substitutions) and allow the remaining bases to exert their activity. This group or compound is baseless, meaning it does not contain commonly recognized nucleotide bases such as adenine, guanine, cytosine, uracil, or thymine, and therefore lacks a base at the 1' position.

[0280] In other embodiments, chemical modification of siRNA includes attaching a conjugate to the siRNA molecule. The conjugate can be attached to the 5' and / or 3' ends of the sense and / or antisense strands of the siRNA via covalent bonds (e.g., biodegradable linkers). The conjugate can also be attached to the siRNA via, for example, a carbamate group or other linking group (see, for example, U.S. Patent Publications 20050074771, 20050043219, and 20050158727). In some cases, the conjugate is a molecule that facilitates siRNA delivery to cells. Examples of conjugated molecules suitable for attachment to siRNA include, but are not limited to: steroids (e.g., cholesterol), glycols (e.g., polyethylene glycol (PEG)), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folic acids (e.g., folic acid and its analogues and derivatives), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, cell receptor ligands capable of mediating cellular uptake, and combinations thereof (see, for example, U.S. Patent Publications 20030130186, 20040110296, and 20040249178; U.S. Patent No. 6,753,423). Other examples include the following conjugated molecules as described in U.S. Patent Publications 20050119470 and 20050107325: lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleavage agents, and crosslinking agents. Other examples also include the following conjugated molecules as described in U.S. Patent Publication 20050153337: 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cation-modified pyrimidines, cationic peptides, guanidino groups, amidine groups, and cationic amino acids. Other examples also include the following conjugated molecules as described in U.S. Patent Publication 20040167090: hydrophobic groups, membrane-active compounds, cell-penetrating compounds, cell-targeting signaling agents, interaction modifiers, and steric stabilizers. Other examples also include the conjugated molecules as described in U.S. Patent Publication 20050239739. The type of conjugate used and its degree of conjugation to the siRNA molecule can be evaluated to improve the pharmacokinetic characteristics, bioavailability, and / or stability of the siRNA while preserving RNAi activity. Therefore, those skilled in the art can use various well-known in vitro cell culture or in vivo animal models to screen siRNA molecules with different conjugates to identify siRNA molecules with modified properties and intact RNAi activity. The entire contents of the aforementioned patent literature are incorporated herein by reference for all purposes.

[0281] d. Target genes

[0282] The siRNA component in the nucleic acid-lipid particles described herein can be used to downregulate or silence the translation (i.e., expression) of target genes. Target genes include, but are not limited to: genes associated with viral infection and survival, genes associated with metabolic diseases and conditions (e.g., liver diseases and conditions), genes associated with tumorigenesis and cell transformation (e.g., cancer), angiogenesis genes, and immune regulatory genes (e.g., genes associated with inflammation and autoimmune responses).

[0283] Genes associated with metabolic diseases and conditions (e.g., liver-targeting conditions and liver diseases and conditions) include, but are not limited to: genes involved in dyslipidemia (e.g., liver X receptors such as LXRα and LXRβ (Genbank accession number NM_007121), farnesol X receptor (FXR) (Genbank accession number NM_005123), sterol regulatory element binding protein (SREBP), site 1 protease (SIP), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), apolipoprotein B (ApoB) (Genbank accession number NM_000384), apolipoprotein CIII (ApoC3) (Genbank accession numbers NM_000040 and NG_008949, region: 5001.8164), and apolipoprotein E (ApoE) (Genbank accession numbers NM_000041 and NG_007084, region: 5001.8164). 5001.8612); and genes associated with diabetes (e.g., glucose-6-phosphatase) (see, for example, Forman et al., Cell , 81: 687 (1995); Seol et al, Mol. Endocrinol. , 9: 72 (1995); Zavacki et al, Proc. Natl. Acad. Sci. USA , 94:7909 (1997); Sakai et al., Cell , 85: 1037-1046 (1996); Duncan et al, J. Biol. Chem. , 272: 12778-12785 (1997); Willy et al., Genes Dev. ,9: 1033-1045 (1995); Lehmann et al, J. Biol. Chem. , 272: 3137-3140 (1997); Janowski et al., Nature , 383: 728-731 (1996); and Peet et al., Cell, 93: 693-704 (1998). Those skilled in the art will understand that genes associated with metabolic diseases and conditions (e.g., liver-targeting conditions and liver diseases and conditions) include genes expressed by the liver itself as well as genes expressed by other organs and tissues. Silencing sequences encoding genes associated with metabolic diseases and conditions can be conveniently combined with conventional pharmaceutical agents used to treat the disease or condition. Non-limiting examples of siRNA molecules targeting the ApoB gene include those described in U.S. Patent Publication No. 20060134189, the entire contents of which are incorporated herein by reference for all purposes. Non-limiting examples of siRNA molecules targeting the ApoC3 gene include those described in U.S. Provisional Application No. 61 / 147,235, filed January 26, 2009, the entire contents of which are incorporated herein by reference for all purposes.

[0284] Examples of gene sequences associated with tumorigenesis and cellular transformation (e.g., cancer or other tumors) include: mitotic kinases such as Eg5 (KSP, KIF11; Genbank accession number NM_004523); serine / threonine kinases such as Polo-like kinase 1 (PLK-1) (Genbank accession number NM_005030; Barr et al., Nat. Rev. Mol. Cell Biol.,5: 429-440 (2004)); tyrosine kinases, such as WEE1 (Genbank accession numbers NM_003390 and NM_001143976); apoptosis inhibitory proteins, such as XIAP (Genbank accession number NM_001167); COP9 signaling subunits, such as CSN1, CSN2, CSN3, CSN4, CSN5 (JAB1; Genbank accession number NM_006837), CSN6, CSN7A, CSN7B, and CSN8; ubiquitin ligases, such as COP1 (RFWD2; Genbank accession numbers NM_022457 and NM_001001740); and histone deacetylases, such as HDAC1, HDAC2 (Genbank accession number NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, etc. Non-limiting examples of siRNA molecules targeting the Eg5 and XIAP genes include those described in U.S. Patent Application No. 11 / 807,872, filed May 29, 2007, the entire contents of which are incorporated herein by reference for all purposes. Non-limiting examples of siRNA molecules targeting the PLK-1 gene include those described in U.S. Patent Publications Nos. 20050107316 and 20070265438; and those described in U.S. Patent Application No. 12 / 343,342, filed December 23, 2008, the entire contents of which are incorporated herein by reference for all purposes. Non-limiting examples of siRNA molecules targeting the CSN5 gene include those described in U.S. Provisional Application No. 61 / 045,251, filed April 15, 2008, the entire contents of which are incorporated herein by reference for all purposes.

[0285] Other examples of gene sequences associated with tumorigenesis and cell transformation include translocation sequences, such as MLL fusion genes and BCR-ABL (Wilda et al.). Oncogene , 21: 5716 (2002); Scherr et al, Blood , 101: 1566(2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO and AML1-MTG8 (Heidenreich et al., ... Blood , 101: 3157 (2003)); overexpressed sequences, such as multidrug resistance genes (Nieth et al., , 101: 3157 (2003)); FEBS Lett. , 545: 144 (2003); Wu et al., Cancer Res. , 63: 1515 (2003)), cyclins (Li et al, , Cancer Res., 63: 3593 (2003); Zou et al., Genes Dev. , 16: 2923 (2002)), β-catenin (Verma et al, , Clin Cancer Res. , 9: 1291 (2003)), telomerase gene (Kosciolek et al, , 9: 1291 (2003)), Mol Cancer Ther. , 2: 209 (2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1 (Genbank accession numbers NM_005228, NM_201282, NM_201283 and NM_201284; see also Nagy et al., Exp. Cell Res. , 285: 39-49 (2003)), ErbB2 / HER-2 (Genbank accession numbers NM_004448 and NM_001005862), ErbB3 (Genbank accession numbers NM_001982 and NM_001005915) and ErbB4 (Genbank accession numbers NM_005235 and NM_001042599)); and mutant sequences, such as RAS (reviewed in Tuschl and Borkhardt, 285: 39-49 (2003)). Mol. Interventions , 2: 158 (2002)). Non-limiting examples of siRNA molecules targeting the EGFR gene include those described in U.S. Patent Application No. 11 / 807,872, filed May 29, 2007, the entire contents of which are incorporated herein by reference for all purposes.

[0286] The sequence encoding the DNA repair enzyme can be silenced and used in combination with chemotherapeutic agents (Collis et al.). Cancer Res. (63:1550 (2003)). Genes encoding proteins associated with tumor migration are also target sequences, such as integrins, selectins, and metalloproteinases. The above examples are not exclusive. Those skilled in the art will understand that any complete or partial gene sequence that promotes or induces tumorigenesis or cell transformation, tumor growth, or tumor migration can be used as a template sequence.

[0287] Angiogenesis genes can promote new blood vessel formation. Among them, vascular endothelial growth factor (VEGF) (Reich et al.) is particularly important. Mol. Vis. VEGFR is of most interest. siRNA sequences targeting VEGFR are described, for example, in GB2396864; U.S. Patent Publication No. 20040142895; and CA2456444, the entire contents of which are incorporated herein by reference for all purposes.

[0288] Anti-angiogenic genes can inhibit the formation of new blood vessels. These genes are particularly useful for treating cancers in which angiogenesis is involved in the pathological process. Examples of anti-angiogenic genes include, but are not limited to: endostatin (see, for example, U.S. Patent No. 6,174,861), angiostatin (see, for example, U.S. Patent No. 5,639,725), and VEGFR2 (see, for example, Decaussin et al.). J. Pathol. , 188: 369-377 (1999)), the entire contents of which are incorporated herein by reference for all purposes.

[0289] Immunomodulatory genes are genes that regulate one or more immune responses. Examples of immunomodulatory genes include, but are not limited to: cytokines, such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.); interleukins (e.g., IL-2, IL-4, IL-12 (Hill et al.)). J. Immunol. , 171: 691 (2003)), IL-15, IL-18, IL-20, etc.; interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.); and TNF. Fas and Fas ligand genes are also immune regulatory target sequences (Song et al., 171: 691 (2003)). Nat. Med. , 9: 347 (2003)). This invention also covers genes encoding secondary signaling molecules in hematopoietic and lymphocyte cells, such as Tec family kinases, such as Bruton's tyrosine kinase (BTK) (Heinonen et al., F , 9: 347 (2003)). EBS Lett. , 527: 274 (2002)).

[0290] Cellular receptor ligands include ligands capable of binding to cell surface receptors (e.g., insulin receptor, EPO receptor, G protein-coupled receptor, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) to regulate (e.g., inhibit, activate, etc.) the physiological pathways involved by that receptor (e.g., blood glucose regulation, blood cell development, mitosis, etc.). Examples of cellular receptor ligands include, but are not limited to: cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, G protein-coupled receptor ligands, etc. Templates encoding trinucleotide repeat sequences (e.g., CAG repeat sequences) can be used to silence pathogenic sequences in neurodegenerative diseases caused by trinucleotide repeat sequence amplification (e.g., spinal bulbar muscular atrophy and Huntington's disease) (Caplen et al., ...). Hum. Mol. Genet. , 11: 175(2002)).

[0291] In addition to therapeutic silencing of the expression of any of the aforementioned genes, the siRNAs described herein can also be used for research and development applications, as well as diagnostic, preventative, prognostic, clinical, and other healthcare applications. As a non-limiting example, siRNAs can be used in target validation studies to determine whether a target gene has the potential to be a therapeutic target. siRNAs can also be used in target identification studies to discover genes that may serve as potential therapeutic targets.

[0292] 2. aiRNA

[0293] Similar to siRNA, asymmetric interfering RNA (aiRNA) can recruit the RNA-induced silencing complex (RISC) and effectively silence multiple genes in mammalian cells by mediating a sequence-specific cleavage of the target sequence between the 10th and 11th nucleotides relative to the 5' end of the antisense strand (Sun et al.). Nat. Biotech. , 26: 1379-1382 (2008)). Typically, aiRNA molecules comprise a short RNA duplex with a sense strand and an antisense strand, wherein the duplex contains overhangs at both the 3' and 5' ends of the antisense strand. aiRNAs are usually asymmetric in structure because the sense strand is shorter at both ends compared to the complementary antisense strand. In some respects, the design, synthesis, and annealing conditions of aiRNA molecules can be similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences can be selected and prepared using the screening methods described above for siRNA sequences.

[0294] In another embodiment, aiRNA duplexes of various lengths (e.g., about 10 to 25, 12 to 20, 12 to 19, 12 to 18, 13 to 17, or 14 to 17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) can be designed with overhangs at the 3' and 5' ends of the antisense strand to target the target mRNA. In some cases, the length of the sense strand of the aiRNA molecule is about 10 to 25, 12 to 20, 12 to 19, 12 to 18, 13 to 17, or 14 to 17 nucleotides, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In other cases, the antisense strand of the aiRNA molecule is about 15 to 60, 15 to 50, or 15 to 40 nucleotides in length, more commonly about 15 to 30, 15 to 25, or 19 to 25 nucleotides, and preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides.

[0295] In some embodiments, the 5' antisense overhang contains one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3' antisense overhang contains one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In some aspects, the aiRNA molecule described herein may contain one or more modified nucleotides, such as those located in the double-stranded (double-stranded) region and / or the antisense overhang. As a non-limiting example, the aiRNA sequence may contain one or more modified nucleotides as described above for the siRNA sequence. In a preferred embodiment, the aiRNA molecule contains a 2'OMe nucleotide, such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, or a mixture thereof.

[0296] In some embodiments, the aiRNA molecule may contain an antisense strand corresponding to the antisense strand of the siRNA molecule (such as one of the siRNA molecules described herein). In other embodiments, the aiRNA molecule may be used to silence the expression of any of the target genes described above, such as genes associated with viral infection and survival, genes associated with metabolic diseases and conditions, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immune regulatory genes (such as genes associated with inflammation and autoimmune responses), ligand receptor genes, and genes associated with neurodegenerative diseases.

[0297] 3. miRNA

[0298] Typically, microRNAs (miRNAs) are single-stranded RNA molecules approximately 21 to 23 nucleotides in length that regulate gene expression. miRNAs are encoded by genes and transcribed from the DNA of these genes, but they themselves are not translated into proteins (non-coding RNAs). Instead, each primary transcript (pri-miRNA) is processed to form a short stem-loop structure called pre-miRNA, which eventually forms a functional mature miRNA. Mature miRNA molecules are partially or completely complementary to one or more messenger RNA (mRNA) molecules, and their primary function is to downregulate gene expression. For methods of identifying miRNA molecules, see, for example, Lagos-Quintana et al. Science , 294: 853-858; Lau et al., Science , 294: 858-862; and Lee et al., Science , 294:862-864.

[0299] The genes encoding miRNAs are much longer than the processed mature miRNA molecules. miRNAs are first transcribed into primary transcripts (pri-miRNAs) with a cap and a polyA tail, which are then processed in the cell nucleus into short stem-loop structures of about 70 nucleotides, called pre-miRNAs. In animals, this processing is carried out by a protein complex called the microprocessor complex, which consists of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al.). Nature These pre-miRNAs are then processed into mature miRNAs in the cytoplasm through interaction with the endonuclease Dicer, simultaneously initiating the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., 432: 231-235 (2004)). Nature , 409:363-366 (2001)). Both the sense and antisense strands of DNA can serve as templates for the generation of miRNAs.

[0300] When Dicer cleaves the stem-loop structure of pre-miRNA, two complementary short RNA molecules are formed, but only one integrates into the RISC complex. This strand, called the guide strand, is selected by the Argonaute protein (a catalytically active RNase in the RISC complex) based on the stability of its 5' end (Preall et al.). Curr. Biol. , 16: 530-535 (2006)). Another chain, called the anti-guide chain or transit chain, is degraded as a substrate of the RISC complex (Gregory et al., 16: 530-535 (2006)). Cell , 123: 631-640 (2005)). After integration into the active RISC complex, the miRNA pairs with its complementary mRNA molecule and induces the degradation of the target mRNA and / or translational silencing.

[0301] Mammalian miRNA molecules are typically complementary to sites in the 3' UTR of target mRNA sequences. In some cases, annealing of miRNA with target mRNA blocks the protein translation mechanism, thereby inhibiting protein translation. In other cases, annealing of miRNA with target mRNA promotes the cleavage and degradation of target mRNA, a mechanism similar to RNA interference (RNAi). miRNAs can also target methylation at genomic sites corresponding to their target mRNAs. Typically, miRNAs function synergistically with a group of proteins collectively known as miRNPs.

[0302] In some aspects, the miRNA molecules described herein are about 15 to 100, 15 to 90, 15 to 80, 15 to 75, 15 to 70, 15 to 60, 15 to 50, or 15 to 40 nucleotides in length, more typically about 15 to 30, 15 to 25, or 19 to 25 nucleotides, and preferably about 20 to 24, 21 to 22, or 21 to 23 nucleotides. In other aspects, the miRNA molecule may contain one or more modified nucleotides. As a non-limiting example, the miRNA sequence may contain one or more modified nucleotides as described above for the siRNA sequence. In a preferred embodiment, the miRNA molecule contains 2'OMe nucleotides, such as 2'OMe-guanosine nucleotide, 2'OMe-uridine nucleotide, or mixtures thereof.

[0303] In some implementations, miRNA molecules can be used to silence the expression of any of the target genes mentioned above, such as genes associated with viral infection and survival, genes associated with metabolic diseases and conditions, genes associated with tumorigenesis and cell transformation, angiogenesis genes, immune regulation genes (e.g., genes associated with inflammation and autoimmune responses), ligand receptor genes, and genes associated with neurodegenerative diseases.

[0304] In other embodiments, the lipid particles of the present invention (e.g., nucleic acid-lipid particles) are used to administer one or more agents that block the activity of miRNAs targeting a specific mRNA. Examples of blocking agents include, but are not limited to, sterically hindered oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides. Such blocking agents may bind directly to miRNAs or to miRNA-binding sites on the target mRNA.

[0305] 4. Antisense oligonucleotides

[0306] In one embodiment, the nucleic acid is an antisense oligonucleotide targeting a target gene or sequence. The term "antisense oligonucleotide" or "antisense" includes an oligonucleotide complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA complementary to a selected sequence. Antisense RNA oligonucleotides prevent the translation of the complementary RNA strand by binding to RNA. Antisense DNA oligonucleotides can be used to target specific complementary (coding or non-coding) RNA. If binding occurs, the DNA / RNA hybrid can be degraded by the RNase H enzyme. In one specific embodiment, the antisense oligonucleotide comprises about 10 to about 60 nucleotides, more preferably about 15 to about 30 nucleotides. The term also covers antisense oligonucleotides that may not be perfectly complementary to the desired target gene. Therefore, the present invention can be used when antisense oligonucleotides have non-target-specific activity, or when one or more mismatched antisense sequences with the target sequence are most preferred for a particular use.

[0307] Antisense oligonucleotides have been demonstrated as effective and targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit protein synthesis of target genes. The efficacy of antisense oligonucleotides in inhibiting protein synthesis has been well-established. For example, the synthesis of polygalacturonase and muscarinic acetylcholine receptor M2 has been inhibited by antisense oligonucleotides targeting their respective mRNA sequences (see U.S. Patents 5,739,119 and 5,759,829). Furthermore, examples of antisense inhibition against nucleoprotein cyclins, multidrug resistance genes (MDR1), ICAM-1, E-selectin, STK-1, striatal GABAA receptors, and human EGF have been validated (see Jaskulski et al.). Science , 240: 1544-6 (1988); Vasanthakumar et al., Cancer Commun. , 1: 225-32 (1989); Penis et al, Brain Res Mal Brain Res. , 15; 57:310-20 (1998); and U.S. Patent Nos. 5,801,154, 5,789,573, 5,718,709, and 5,610,288. Furthermore, antisense constructs that can inhibit and be used to treat various abnormal cell proliferations (e.g., cancer) are described (see U.S. Patent Nos. 5,747,470, 5,591,317, and 5,783,683). The entire contents of the foregoing references are incorporated herein by reference for all purposes.

[0308] Methods for preparing antisense oligonucleotides are known in the art and can be readily applied to prepare antisense oligonucleotides targeting any polynucleotide sequence. The selection of antisense oligonucleotide sequences specific to a given target sequence is based on analysis of the selected target sequence and its secondary structure, T... m Binding energy and relative stability are determined. The selection of antisense oligonucleotides can be based on their relative inability to form dimers, hairpin structures, or other secondary structures that reduce or hinder their specific binding to target mRNA in host cells. Highly preferred mRNA target regions include regions at or near the AUG translation start codon, and sequences highly complementary to the 5' end region of the mRNA. These secondary structure analyses and target site selection considerations can be performed, for example, using software such as OLIGO primer analysis software version 4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al.). Nucleic Acids Res. , 25: 3389-402 (1997)).

[0309] 5. Ribozymes

[0310] According to another embodiment of the invention, nucleic acid-lipid particles are bound to a ribozyme. The ribozyme is an RNA-protein complex with a specific catalytic domain and endonuclease activity (see Kim et al.). Proc. Natl. Acad Sci. USA. , 84: 8788-92 (1987); and Forster et al., Cell , 49: 211-20 (1987)). For example, many ribozymes accelerate phosphoester transfer reactions in a highly specific manner, typically cleaving only one of several phosphoester bonds in an oligonucleotide substrate (see Cech et al., 49: 211-20 (1987)). Cell , 27: 487-96 (1981); Michel et al, J. Mol. Biol. , 216:585-610 (1990); Reinhold-Hurek et al., Nature , 357: 173-6 (1992)). This specificity is attributed to the fact that the substrate must bind to the internal guide sequence (“IGS”) of the ribozyme through specific base pairing interactions before a chemical reaction can occur.

[0311] At least six basic types of naturally occurring enzymatic RNA molecules are currently known. These RNA molecules can all catalyze the hydrolysis of RNA phosphodiester bonds in a trans-regulatory manner under physiological conditions (thus cleaving other RNA molecules). Typically, the mechanism of action of enzymatic nucleic acids involves first binding to a target RNA. This binding occurs through the target-binding moiety of the enzymatic nucleic acid, which is closely adjacent to the enzymatically cleaving moiety responsible for cleaving the target RNA. Therefore, the enzymatic nucleic acid first recognizes and binds to the target RNA through complementary base pairing; once bound to the correct site, it exerts its enzymatic action to cleave the target RNA. Strategic cleavage of this target RNA disrupts its ability to guide the synthesis of encoded proteins. After binding to and cleaving the RNA target, the enzymatic nucleic acid is released from the RNA to seek the next target and can repeatedly bind to and cleave new targets.

[0312] Enzymatic nucleic acid molecules can form, for example, hammerhead structures, hairpin structures, hepatitis D virus, group I introns, or RNase P RNA (associated with an RNA guide sequence) or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described, for example, by Rossi et al. Nucleic Acids Res. , 20: 4559-65 (1992). Examples of hairpin structure motifs are described, for example, in EP0360257; Hampel et al., Biochemistry , 28: 4929-33 (1989); Hampel et al, Nucleic Acids Res., 18: 299-304 (1990); and U.S. Patent No. 5,631,359. Examples of hepatitis D virus motifs are described, for example, by Perrotta et al. Biochemistry , 31: 11843-52 (1992). Examples of RNaseP motifs are described, for example, by Guerrier-Takada et al., Cell , 35: 849-57 (1983). Examples of Neurospora vs. RNA ribozyme motifs are described, for example, by Saville et al., Cell , 61: 685-96 (1990); Saville et al, Proc. Natl. Acad. Sci. USA , 88: 8826-30 (1991); Collins et al, Biochemistry , 32: 2795-9 (1993). Examples of Group I introns are described, for example, in U.S. Patent No. 4,987,071. An important feature of the enzymatic nucleic acid molecule used according to the invention is that it has a specific substrate binding site complementary to one or more target gene DNA or RNA regions, and has a nucleotide sequence conferring RNA cleavage activity to the molecule within or around the substrate binding site. Therefore, the ribozyme construct is not necessarily limited to the specific motifs mentioned herein. The entire contents of the foregoing references are incorporated herein by reference for all purposes.

[0313] Methods for preparing ribozymes targeting any polynucleotide sequence are known in the art. Ribozymes can be designed and synthesized according to, for example, the methods described in PCT publications WO93 / 23569 and WO94 / 02595, for in vitro and / or in vivo testing as described therein. The entire contents of the aforementioned PCT publications are incorporated herein by reference for all purposes.

[0314] Ribozyme activity can be optimized by altering the length of the ribozyme-binding arm or by chemically synthesizing ribozymes modified to: prevent degradation of the ribozyme by serum ribonuclease (see, for example, PCT Publications WO92 / 07065, WO93 / 15187, WO91 / 03162 and WO94 / 13688; EP92110298.4; and U.S. Patent No. 5,334,711, which describe various chemical modifications that can be made to the sugar moiety of enzymatic RNA molecules, the entire contents of which are incorporated herein by reference for all purposes), enhance the efficacy of the ribozyme in the cell, and remove stem II bases to shorten RNA synthesis time and reduce the need for chemical agents.

[0315] 6. Immunostimulatory oligonucleotides

[0316] The nucleic acids associated with the lipid particles of the present invention may be immunostimulatory nucleic acids, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) capable of inducing an immune response when administered to a subject (which may be a mammal such as a human). ISSs include, for example, certain palindromic sequences capable of forming hairpin-like secondary structures (see Yamamoto et al., J. Immunol. ,148: 4072-6 (1992)), or CpG motifs, as well as other known ISS features (e.g., multiG domains; see PCT Publication No. WO96 / 11266, the entire contents of which are incorporated herein by reference for all purposes).

[0317] When an immunostimulatory nucleic acid can induce an immune response without specifically binding to and reducing the expression of a target sequence, it is considered non-sequence-specific. Therefore, although some immunostimulatory nucleic acids may contain sequences corresponding to naturally occurring gene or mRNA regions, they can still be considered non-sequence-specific immunostimulatory nucleic acids.

[0318] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide having a methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide, wherein the cytosine in the CpG dinucleotide is methylated cytosine. In another embodiment, the nucleic acid comprises at least two CpG dinucleotides, wherein at least one of the CpG dinucleotides has a methylated cytosine. In another embodiment, each cytosine in the CpG dinucleotides present in the sequence is methylated cytosine. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, wherein at least one CpG dinucleotide contains a methylated cytosine. Examples of immunostimulatory oligonucleotides suitable for the compositions and methods of the present invention are described in PCT application No. PCT / US08 / 88676, filed December 31, 2008; PCT Publications Nos. WO02 / 069369 and WO01 / 15726; U.S. Patent No. 6,406,705; and Raney et al. J. Pharm. Exper. Ther. , 298: 1185-92 (2001), the entire contents of which are incorporated herein by reference for all purposes. In some embodiments, the oligonucleotides used in the compositions and methods of the present invention have a phosphodiester (“PO”) backbone or a phosphate thioester (“PS”) backbone, and / or contain at least one methylated cytosine residue in the CpG motif.

[0319] B. Other surfactants

[0320] In some embodiments, the active agent associated with the lipid-based nanoparticles of the present invention may comprise one or more therapeutic proteins, peptides, or small organic molecules or compounds. Non-limiting examples of such therapeutically effective active agents or drugs include antitumor drugs (e.g., chemotherapy drugs, hormone therapy agents, immunotherapy agents, radiotherapy agents, etc.), lipid-lowering agents, antiviral drugs, anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cell angiogenesis agents, signal transduction inhibitors, cardiovascular drugs (e.g., antiarrhythmic drugs), hormones, vasoconstrictors, and steroids. These active agents may be administered alone in the lipid particles of the present invention or in combination with (e.g., co-administered) the lipid particles of the present invention containing nucleic acids (e.g., interfering RNA).

[0321] Non-restrictive examples of chemotherapy drugs include platinum-based drugs (e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, isopropylplatin, saplatin, etc.), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, busulfan, melphalan, nitrogen mustard, uramustine, thiotepa, nitrosoureas, etc.), antimetabolites (e.g., 5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin, capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, pemetrexed, raltitrexed, etc.), phytoalkaloids (e.g., vincristine, vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel, docetaxel, etc.), and topoisomers. Enzyme inhibitors (e.g., irinotecan (CPT-11; Camptosar), topotecan, acridine, etoposide (VP16), etoposide phosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin, doxorubicin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin, mitoxantrone, procainoxicin, etc.), tyrosine kinase inhibitors (e.g., gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®; OSI-1774), lapatinib (GW572016; GW2016), canenatinib (CI) 1033), smasani (SU5416), vatalani (PTK787 / ZK222584), sorafenib (BAY 43-9006), imatinib (Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101), vandetanib (Zactima™; ZD6474), etc.), their pharmaceutically acceptable salts, their stereoisomers, their derivatives, their analogs, and combinations thereof.

[0322] Examples of conventional hormone therapy agents include, but are not limited to, steroids (such as dexamethasone), finasteride, aromatase inhibitors, tamoxifen and goserelin, and other gonadotropin-releasing hormone agonists (GnRH).

[0323] Examples of conventional immunotherapeutic agents include, but are not limited to, immunostimulants (such as BCG, levamisole, interleukin-2, alpha-interferon, etc.), monoclonal antibodies (such as anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (such as anti-CD33 monoclonal antibody-kazidoxomil conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy agents (such as those used in combination with...). 111 In、 90 Y or 131 I-conjugated anti-CD20 monoclonal antibodies, etc.

[0324] Examples of conventional radiotherapy agents include, but are not limited to, radionuclides, such as 47 Sc、 64 Cu、 67 Cu、 37 Sr、 86 Y、 37 Y、 90 Y、 105 Rh、 111 Ag、 111 In、 117m Sn、 149 Pm, 153 Sm、 166 Ho、 177 Lu、 186 Re、 188 Re、 211 At and 212 Bi, these radionuclides are optionally conjugated with antibodies against tumor antigens.

[0325] Other antitumor drugs that can be used according to the present invention include, but are not limited to, Alkeran, Allopurinol, Altretamine, Amifostine, Anastrozole, AraC, Arsenic Trioxide, Bexarotene, biCNU, Carmustine, CCNU, Celecoxib, Cladribine, Cyclosporine A, Cytarabine, Cytoxamide, Dexrazoxane, DTIC, Estramustine, Exemestane, FK506, Gelatinumab-Ozomicin, Hydroxyurea, and Idarubicin. n), interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol, L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate, pegylated adenosine deaminase, pentostatin, porphyrin sodium, prednisone, rituximab, streptozotocin, STI-571, taxotere, temozolomide, VM-26, toremifene, retinoic acid, ATRA, valrubicin, and vinblastine. Other examples of antitumor drugs that can be used according to the present invention include roserine and its analogues or derivatives, epochyam, intracellular kinase inhibitors, and camptothecin.

[0326] Non-limiting examples of lipid-lowering agents used to treat lipid disorders or conditions associated with elevated triglycerides, cholesterol, and / or glucose include statins, fibrates, ezetimibe, thiazolidinediones, niacin, beta-blockers, nitroglycerin, calcium channel blockers, fish oils, and mixtures thereof.

[0327] Examples of antiviral drugs include, but are not limited to, abacavir, acyclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol, atazanavir, atripla, cidofovir, Combivir, darunavir, delavirdine, didanosine, docosanol, edoxudine, efavirenz, and emtricitabine. Tricitabine, Enfuvirtide, Entecavir, Virus entry inhibitors, Famciclovir, Fixed-dose combination drugs, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Fusion inhibitors, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir, Inosine, Integrase inhibitors, Type III interferons (e.g., IFN-λ molecules)Examples include IFN-λ1, IFN-λ2, and IFN-λ3; type II interferons (e.g., IFN-γ); type I interferons (e.g., IFN-α (e.g., PEGylated IFN-α), IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ); interferons; lamivudine; lopinavir; loviride; MK-0518; maraviroc; moroxydine; nelfinavir; nevirapine; and sorafenib. Nexavir, nucleoside analogs, oseltamivir, penciclovir, peramivir, pleconaril, podophyllotoxin, protease inhibitors, reverse transcriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir, stavudine, potentiators, tenofovir, tenofovir disoproxil fumarate The following are listed: disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valaciclovir, valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir, zidovudine, their pharmaceutically acceptable salts, their stereoisomers, their derivatives, their analogues, and mixtures thereof.

[0328] Other drugs include Zotarolimus, Sirolimus, Rapamycin, Everolimus, Biolimus, Myolimus, Novolimus, Temsirolimus, Defoolimus, Merilimus, Tacrolimus, Pimecrolimus, Ridaforolimus, their pharmaceutically acceptable salts, their stereoisomers, their derivatives, their analogues, and mixtures thereof.

[0329] Pharmaceutical Composition

[0330] Lipid-based nanoparticles can be formulated wholly or partially into pharmaceutical compositions. Pharmaceutical compositions may contain one or more nanoparticles. For example, a pharmaceutical composition may contain one or more nanoparticles that comprise one or more different therapeutic and / or preventative agents. Pharmaceutical compositions may also contain one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidance on the formulation and preparation of pharmaceutical compositions and agents can be found in, for example... Remington: The Science and Practice of Pharmacy , twenty one st Edition, ARGennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition unless any conventional excipient or adjuvant may be incompatible with one or more components of the lipid-based nanoparticles. An excipient or adjuvant may be incompatible with a component if the combination of such an excipient or adjuvant with a component of the lipid-based nanoparticles could result in any adverse biological effects or other harmful effects.

[0331] In some embodiments, one or more excipients or adjuvants may constitute more than 50% by mass or volume of the total pharmaceutical composition comprising lipid-based nanoparticles. For example, one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90%, or more of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable purity of the excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient conforms to the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0332] In the pharmaceutical compositions according to this disclosure, the relative amounts of one or more lipid-based nanoparticles, one or more pharmaceutically acceptable excipients, and / or any other ingredients will vary depending on the identity, body size, and / or condition of the treated subject and the route of administration of the composition. For example, the pharmaceutical composition may comprise 0.1% to 100% (by weight) of one or more lipid-based nanoparticles.

[0333] In some embodiments, the lipid-based nanoparticles and / or pharmaceutical compositions of the present invention are refrigerated or frozen for storage and / or transport (e.g., stored at 4°C or lower, such as about -150°C to about 0°C or about -80°C to about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C or -150°C)).

[0334] In some embodiments, the pharmaceutical compositions of the present invention comprise the lipid-based nanoparticles described herein, and one or more pharmaceutically acceptable carriers selected from Tris, acetates (e.g., sodium acetate), citrates (e.g., sodium citrate), physiological saline, PBS, and sucrose. In some embodiments, the pharmaceutical compositions of the present disclosure have a pH of about 7 to 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or 7.5 to 8, or 7 to 7.8). For example, the pharmaceutical compositions of the present disclosure comprise the nanoparticle composition described herein, Tris, physiological saline, and sucrose, with a pH of about 7.5 to 8, suitable for storage and / or transport at, for example, about -20°C. For example, the pharmaceutical compositions of the present disclosure comprise the lipid-based nanoparticles described herein and PBS, with a pH of about 7 to 7.8, suitable for storage and / or transport at, for example, about 4°C or lower. In the context of this disclosure, “stability,” “stabilization,” and “stable” refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions described herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, encapsulation changes, etc.) under given conditions of manufacture, preparation, transport, storage, and / or use (e.g., when stresses such as shear force, freeze-thaw stress, etc. are applied).

[0335] In a preferred embodiment, the lipid-based nanoparticles of the present invention are formulated with one or more disaccharides or disaccharide-containing molecules (e.g., sucrose, lactose, maltose, trehalose, maltitol, or lactitol) in any one or more buffer solutions described herein (including Tris HCl, Tris acetate, TT / AA). As shown in the examples below, the lipid-based nanoparticles formulated with sucrose can be reconstructed after thawing without aggregation.

[0336] Lipid-based nanoparticles and / or pharmaceutical compositions comprising one or more lipid-based nanoparticles may be administered to any patient or subject, including those who may benefit from the therapeutic effects resulting from the delivery of therapeutic and / or prophylactic agents to one or more specific cells, tissues, organs, or systems or groups thereof (e.g., the renal system). While the lipid-based nanoparticles and pharmaceutical compositions comprising lipid-based nanoparticles described herein are primarily intended for human administration, those skilled in the art will understand that such compositions are generally suitable for administration to any other mammal. Modifications to compositions intended for human administration to suit a variety of animal administration are well known, and such modifications can be designed and / or implemented by a generally skilled veterinary pharmacologist with routine experimental procedures, if applicable. Subjects envisioned to administer the compositions include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0337] Pharmaceutical compositions comprising one or more lipid-based nanoparticles may be prepared by any method known in or to be developed in the field of pharmacology. Typically, such preparation methods involve mixing the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dispensing, shaping, and / or packaging the product into the desired single- or multi-dose units.

[0338] The pharmaceutical compositions described in this disclosure may be prepared, packaged, and / or sold in bulk, single-dose, and / or multiple single-dose forms. As used herein, "unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., lipid-based nanoparticles). The amount of active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or an appropriate fraction of that dose, such as half or one-third of the dose.

[0339] Pharmaceutical compositions can be formulated in a variety of forms to suit a variety of routes and methods of administration. For example, pharmaceutical compositions can be formulated as liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable dosage forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal application (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and patches), suspensions, powders, and other dosage forms.

[0340] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters and mixtures thereof. In addition to inert diluents, oral compositions may also contain other therapeutic and / or preventative agents, other pharmaceutical agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or flavoring agents. In some embodiments for parenteral administration, the composition is mixed with a solubilizer (e.g., Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof).

[0341] Injectable formulations (e.g., sterile aqueous or oily suspensions for injection) can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic, parenteral-acceptable diluents and / or solvents, such as solutions using 1,3-butanediol as a solvent. Acceptable carriers and solvents include water, Ringer's solution (United States Pharmacopeia), and isotonic sodium chloride solution. Sterile fixative oils are commonly used as solvents or suspension media. For this purpose, any mild fixative oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0342] Injectable formulations can be sterilized in a variety of ways, such as by filtration through a bacterial trap filter, and / or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved or dispersed in sterile water or other sterile injection media before use.

[0343] Compositions for rectal or vaginal administration are typically suppositories, which are prepared by mixing the composition with suitable non-irritating excipients (such as cocoa butter, polyethylene glycol, or suppository waxes) that are solid at room temperature but liquid at body temperature, thus melting and releasing the active ingredient within the rectal or vaginal cavity.

[0344] Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient (e.g., sodium citrate or calcium hydrogen phosphate) and / or fillers or expanders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicate), binders (e.g., carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic), humectants (e.g., glycerin), disintegrants (e.g., agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate), solution blockers (e.g., paraffin), absorption enhancers (e.g., quaternary ammonium compounds), wetting agents (e.g., cetyl alcohol and glyceryl monostearate), absorbents (e.g., kaolin and bentonite, silicates), and lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate), and mixtures thereof. For capsules, tablets, and pills, the dosage form may contain a buffer.

[0345] Such solid compositions can be used as fillers in soft and hard-filled gelatin capsules, with excipients including lactose, high molecular weight polyethylene glycol, etc. Solid dosage forms such as tablets, sugar-coated pills, capsules, pellets, and granules can be prepared using coatings and shells (e.g., enteric coatings and other coatings well known in the pharmaceutical formulation field). They may optionally contain light-blocking agents and can release the active ingredient in compositional form only or preferentially in specific portions of the intestine, optionally employing a sustained-release mechanism. Examples of usable encapsulation compositions include lipid-based substances and waxes. Such solid compositions can be used as fillers in soft and hard-filled gelatin capsules, with excipients including lactose, high molecular weight polyethylene glycol, etc.

[0346] Dosage forms for topical and / or transdermal application may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and / or patches. Typically, the active ingredient is mixed under sterile conditions with pharmaceutically acceptable excipients and / or any desired preservatives and / or buffers (if necessary). Furthermore, this disclosure relates to the use of transdermal patches, which generally have the added advantage of delivering the compound into the body in a controlled manner. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the compound in a suitable medium. Additionally, the release rate can be controlled by providing a rate-controlled membrane and / or dispersing the compound in a polymer matrix and / or gel.

[0347] Devices suitable for delivering the intradermal pharmaceutical compositions described herein include short needle devices, such as those described in U.S. Patents 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662. The intradermal composition can be administered via devices that limit the effective depth of needle penetration into the skin, such as those described in PCT Publication WO99 / 34850 and its functional equivalents. Suitable jet injection devices deliver the liquid composition to the dermis via a liquid jet injector and / or deliver the liquid composition by generating a jet that reaches the dermis via a needle that pierces the stratum corneum. Such jet injection devices are described, for example, in U.S. Patent Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, and 5,466,220. Applicable patents include: Nos. 5,339,163; 5,312,335; 5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880; 4,940,460; and PCT Publications WO97 / 37705 and WO97 / 13537. Applicable ballistic powder / particle delivery devices utilize compressed gas to accelerate the delivery of powdered vaccines through the outer layer of skin to the dermis. Alternatively, intradermal administration can be performed using a conventional syringe according to the classic Mantoux method.

[0348] Formulations suitable for topical application include, but are not limited to, liquid and / or semi-liquid formulations, such as liniments, lotions, oil-in-water and / or water-in-oil emulsions (e.g., creams, ointments, and / or pastes), and solutions and / or suspensions. Topically applicable formulations may, for example, contain from about 1% to about 10% (by weight) of the active ingredient, although the concentration of the active ingredient may be up to its solubility limit in a solvent. Formulations for topical application may also contain one or more other ingredients described herein.

[0349] Pharmaceutical compositions may be prepared, packaged, and / or marketed as formulations suitable for oral administration to the lungs. Such formulations may comprise dry granules containing an active ingredient. These compositions are typically in dry powder form and are administered via a device comprising a dry powder reservoir (in which a propellant gas stream is directed to disperse the powder) and / or a self-propelled solvent / powder dispensing container (e.g., a device for dissolving and / or suspending the active ingredient in a low-boiling-point propellant and sealing it within the container). Dry powder compositions may contain a solid, finely powdered diluent (e.g., sugars) and are typically provided in unit doses.

[0350] Low-boiling-point propellants typically include liquid propellants with a boiling point below 65°F at atmospheric pressure. Typically, the propellant comprises 50% to 99.9% (by weight) of the composition, and the active ingredient comprises 0.1% to 20% (by weight). The propellant may also contain other components, such as liquid nonionic surfactants and / or solid anionic surfactants and / or solid diluents (whose particle size may be on the same order of magnitude as the particles containing the active ingredient).

[0351] Pharmaceutical compositions for pulmonary delivery may provide the active ingredient in the form of solution and / or suspension droplets. Such formulations may be prepared, packaged, and / or sold as aqueous and / or dilute alcoholic solutions and / or suspensions (optionally sterile) containing the active ingredient and readily applicable via any spray and / or nebulizer. Such formulations may also contain one or more other ingredients, including but not limited to flavoring agents (e.g., sodium saccharin), volatile oils, buffers, surfactants, and / or preservatives (e.g., methylparaben). The average diameter of the droplets provided via this route of administration may be from about 1 nm to about 200 nm.

[0352] The formulations described herein for pulmonary delivery are also suitable for intranasal delivery of the pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 μm to 500 μm. Such formulations are administered by nasal inhalation, that is, by bringing the container containing the powder close to the nose and rapidly inhaling it through the nasal cavity.

[0353] Formulations intended for nasal administration may, for example, comprise 0.1% (by weight) to 100% (by weight) of the active ingredient, and may include one or more other ingredients described herein. Pharmaceutical compositions may be prepared, packaged, and / or marketed as formulations intended for oral administration. Such formulations may be, for example, tablets and / or lozenges prepared using conventional methods, wherein the active ingredient may comprise, for example, 0.1% to 20% (by weight), the remainder comprising a composition that can dissolve and / or degrade in the oral cavity, and optionally one or more other ingredients described herein. Alternatively, formulations intended for oral administration may comprise powders and / or aerosols and / or nebulized solutions and / or suspensions containing the active ingredient. Such powders, aerosols, and / or nebulized formulations, after dispersion, may have an average particle size and / or droplet size of about 0.1 nm to about 200 nm, and may also include any one or more other ingredients described herein.

[0354] Pharmaceutical compositions may be prepared, packaged, and / or marketed as formulations suitable for ocular application. Such formulations may be, for example, eye drops, comprising, for example, solutions and / or suspensions of the active ingredient at 0.1 / 1.0% (by weight) of an aqueous or oily liquid excipient. Such drops may also contain buffers, salts, and / or one or more other ingredients described herein. Other useful ocularly applicable formulations include those containing the active ingredient in microcrystalline and / or liposomal formulations. The scope of this disclosure covers ear drops and / or eye drops.

[0355] Treatment methods for diseases and symptoms

[0356] The lipid-based nanoparticles of the present invention can be used to treat diseases, conditions, or symptoms. In particular, such compositions can be used to treat diseases, conditions, or symptoms characterized by the absence or abnormality of protein or peptide activity. For example, lipid-based nanoparticles containing an active agent (e.g., mRNA encoding a missing or abnormal peptide) can be administered or delivered to cells. Subsequent translation of the mRNA can produce the corresponding peptide, thereby reducing or eliminating the problems caused by the absence or abnormality of peptide activity. Because mRNA translation can occur rapidly, these methods and compositions can be used to treat acute diseases, conditions, or symptoms such as sepsis, stroke, and myocardial infarction. The therapeutic and / or preventative agents contained in the nanoparticle compositions can also alter the transcription rate of specific genes, thereby affecting gene expression.

[0357] Diseases, conditions, and / or symptoms characterized by abnormal protein or peptide function or activity that can be treated with the applicable composition include, but are not limited to, rare diseases, infectious diseases (as both vaccines and therapeutics), cancers and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular and renal vascular diseases, and metabolic diseases. Many diseases, conditions, and / or symptoms may be characterized by a lack of protein activity (or a significant reduction to the point of being unable to function normally). Such proteins may be absent or substantially nonfunctional. A specific example of a dysfunctional protein is a missense mutation in the cystic fibrosis transmembrane conduction regulator (CFTR) gene, which produces a dysfunctional CFTR protein variant that causes cystic fibrosis.

[0358] This disclosure provides a method for treating such diseases, conditions and / or symptoms, comprising administering to a subject lipid-based nanoparticles of the present invention containing RNA, wherein the RNA may be mRNA encoding a polypeptide that antagonizes or otherwise overcomes abnormal protein activity present in the subject's cells.

[0359] This disclosure provides methods relating to the administration of lipid-based nanoparticles comprising one or more therapeutic and / or preventive active agents, and pharmaceutical compositions comprising the same. For the purposes of the features and embodiments of this disclosure, the terms “therapeutic” and “preventive” are used interchangeably herein. Therapeutic compositions or compositions thereof for imaging, diagnostic, or preventive purposes may be administered to a subject at any reasonable dose and via any effective route of administration to achieve the prevention, treatment, diagnosis, or imaging of a disease, condition, and / or symptom and / or any other purpose. The specific dose administered to a particular subject may vary depending on factors such as the subject’s species, age, and general health condition; the purpose of administration; the specific composition used; the method of administration; and other relevant factors. Compositions according to this disclosure may be formulated in unit dose form to facilitate administration and ensure dose uniformity. However, it should be understood that the total daily dosage of the lipid-based nanoparticles or pharmaceutical compositions of this disclosure should be determined by an attending physician within the bounds of reasonable medical judgment. For any given patient, the specific therapeutic effective dose, prophylactic effective dose, or other appropriate dose level (e.g. for imaging) will depend on a number of factors, including the severity and nature of the condition being treated (if any); one or more therapeutic and / or prophylactic agents used; the specific composition used; the patient's age, weight, general health status, sex, and diet; the timing, route of administration, and excretion rate of the specific pharmaceutical composition used; the duration of treatment; the drugs used in combination with or concurrently with the specific pharmaceutical composition used; and similar factors well known in the medical field.

[0360] Lipid-based nanoparticles containing one or more therapeutic and / or preventative active agents can be administered via any route. In some embodiments, compositions containing one or more lipid-based nanoparticles described herein (including compositions for preventative, diagnostic, or imaging purposes) are administered via one or more of the following routes: oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, subcutaneous, intraventricular, percutaneous or intradermal, intradermal, rectal, vaginal, intraperitoneal, intraocular, subretinal, vitreous, topical (e.g., via powder, ointment, cream, gel, lotion, and / or drops), mucosa, nasal cavity, oral cavity, enterum, vitreous body, intratumoral, sublingual, intranasal; via endotracheal instillation, bronchial instillation, and / or inhalation; as oral sprays and / or powders, nasal sprays and / or aerosols, and / or via portal vein catheters. In some embodiments, the compositions can be administered via intravenous, intramuscular, intradermal, intraarterial, intratumoral, subcutaneous, intraocular, subretinal, vitreous, or inhalation. Typically, the most appropriate route of administration will depend on a number of factors, including: the properties of the nanoparticle composition containing one or more therapeutic and / or preventative agents (e.g., its stability in various bodily environments, such as the circulatory system and the gastrointestinal tract), the patient's condition (e.g., whether the patient can tolerate a particular route of administration), etc.

[0361] In some embodiments, the compositions according to this disclosure may be administered at dose levels sufficient to deliver the following therapeutic and / or prophylactic agents (e.g., mRNA): about 0.0001 mg / kg to about 10 mg / kg, about 0.001 mg / kg to about 10 mg / kg, about 0.005 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.05 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 0.0001 mg / kg to about 5 mg / kg, about 0.001 mg / kg to about 5 mg / kg, about 0.005 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 5 mg / kg, about 0.05 mg / kg to about 5 mg / kg, about 0.1 mg / kg to about 5 mg / kg. mg / kg, about 1 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.0001 mg / kg to about 2.5 mg / kg, about 0.001 mg / kg to about 2.5 mg / kg, about 0.005 mg / kg to about 2.5 mg / kg, about 0.01 mg / kg to about 2.5 mg / kg, about 0.05 mg / kg to about 2.5 mg / kg, about 0.1 mg / kg to about 2.5 mg / kg, about 1 mg / kg to about 2.5 mg / kg, about 2 mg / kg to about 2.5 mg / kg, about 0.0001 mg / kg to about 1 mg / kg, about 0.001 mg / kg to about 1 mg / kg, about 0.005 mg / kg to about 1 mg / kg, about 0.01 mg / kg to about 1 mg / kg, about 0.05 mg / kg to about 1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 0.0 ... The dosage is approximately 0.001 mg / kg to about 0.25 mg / kg, about 0.005 mg / kg to about 0.25 mg / kg, about 0.01 mg / kg to about 0.25 mg / kg, about 0.05 mg / kg to about 0.25 mg / kg, or about 0.1 mg / kg to about 0.25 mg / kg, wherein a dose of 1 mg / kg (mpk) represents 1 mg of therapeutic and / or prophylactic agent per 1 kg of subject body weight. In some embodiments, a therapeutic and / or prophylactic agent (e.g., mRNA) of the nanoparticle composition may be administered at a dose of about 0.001 mg / kg to about 10 mg / kg.In other embodiments, a therapeutic and / or prophylactic agent may be administered at a dose of about 0.005 mg / kg to about 2.5 mg / kg. In some embodiments, an administered dose may be administered at a dose of about 0.1 mg / kg to about 1 mg / kg. In other embodiments, an administered dose may be administered at a dose of about 0.05 mg / kg to about 0.25 mg / kg. Doses may be administered once or multiple times daily, with each dose being the same or different, to obtain the desired mRNA expression level and / or therapeutic, diagnostic, preventative, or imaging effect. The desired dose may be delivered, for example, at the following frequencies: three times daily, twice daily, once daily, every other day, every three days, once weekly, once every two weeks, once every three weeks, or once every four weeks. In some embodiments, the desired dose may be delivered by multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more). In some embodiments, a single dose may be administered, for example, before or after surgery, or when an acute illness, condition, or symptom occurs.

[0362] The lipid-based nanoparticles of the present invention, comprising one or more therapeutic and / or preventative active agents, can be used in combination with one or more other therapeutic, preventative, diagnostic, or imaging agents. The term "combined use" does not mean that these agents must be administered simultaneously and / or formulated together for delivery, although such delivery methods are all within the scope of this disclosure. For example, one or more lipid-based nanoparticles comprising one or more different therapeutic and / or preventative agents can be administered in combination. The lipid-based nanoparticles can be administered simultaneously, before, or after one or more other desired pharmaceutical or therapeutic measures. Typically, each agent will be administered at a dosage and / or timing regimen determined for that agent. In some embodiments, this disclosure covers the combined delivery of lipid-based nanoparticles, compositions, or compositions thereof for imaging, diagnostic, or preventative purposes with agents that improve their bioavailability, reduce and / or alter their metabolism, inhibit their excretion, and / or alter their distribution in vivo.

[0363] It should also be understood that active agents used in combination for therapeutic, preventative, diagnostic, or imaging purposes may be co-present in a single composition or administered separately in different compositions. Generally, the dose level of each agent is not expected to exceed its dose level when used in combination. In some embodiments, the dose level when used in combination may be lower than the dose level when used alone.

[0364] The specific combination of therapies (medications or treatments) used in a combination therapy regimen will take into account the compatibility between the required drugs and / or treatments and the desired therapeutic effect. It should also be understood that the therapies used may achieve the desired effect for the same condition (e.g., a composition for treating cancer may be administered concurrently with a chemotherapy agent) or may achieve different effects (e.g., controlling any adverse effects, such as infusion-related reactions).

[0365] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more individual features mentioned or obvious in the text or drawings. All these different combinations constitute various alternatives to the invention.

[0366] Example

[0367] As illustrated in the following embodiments, the inventors have developed a simple antibody capture system that requires no antibody modification and ensures that the antibody attaches to LNPs in an optimized or optimal orientation. The inventors achieved this by modifying LNPs with a nanobody, TP1107, which possesses highly specific antibody capture capabilities. The inventors used single-particle transmission electron microscopy to determine the position and orientation of TP1107 when binding to mouse IgG1 and identified the optimal position of TP1107 on the LNP surface. To control the orientation of TP1107 on the LNP surface, the inventors utilized codon recoding technology with amber stop codons (UAG) to specifically incorporate the synthetic amino acid p-azidophenylalanine (azPhe) with an azide group. TP1107azPhe was modified with DSPE-PEG2000-DBCO to achieve its insertion into the LNP. Due to the high affinity between TP1107 and the Fc domain of IgG1, only the targeting antibody needs to be added to the TP1107-functionalized LNP, without further purification. The inventors demonstrated that, compared to unmodified LNPs, optimally targeted antibodies improved LNP binding affinity and mRNA delivery efficiency by over 1000-fold, and by more than an order of magnitude compared to traditional antibody modification methods. The inventors also verified that this technology can be used for rapid screening of multiple antibodies to obtain optimal specificity and expression efficiency. Using these actively targeted LNPs, the inventors successfully transfected specific cell populations in primary human peripheral blood mononuclear cell (PBMC) culture systems with minimal off-target expression. This method facilitates the rapid development of actively targeted LNPs and holds promise for expanding the application scope of mRNA therapies.

[0368] Example 1 - Materials and Methods

[0369] mRNA synthesis

[0370] All IVT mRNAs were synthesized from PCR templates containing an upstream T7 promoter and a subsequent codon-optimized ORF. All constructs included a 5' UTR, a 3' UTR, and a 125-polyA tail. All mRNAs were transcribed using the HiScribe T7 High-Yield RNA Synthesis Kit (New England Biolabs). Co-transcriptional capping was performed using CleanCap Reagent AG (TriLink Biotechnologies). All uridines were replaced with N1-methylpseudouridine (TriLink Biotechnologies). IVT reactions were DNase-treated to eliminate the template, and dsRNA was removed by cellulose purification (Baiersdörfer et al.). Molecular Therapy-Nucleic Acids (2019; 15: 26-35). The final product was purified by sodium acetate precipitation.

[0371] LNP preparation

[0372] The preparation method for LNP is based on previous reports and modified (Veiga et al., Nat Commun, 2018, 9(1):4493). A lipid mixture consisting of Dlin-MDA-MC3, DSPC (Avanti Polar Lipids), cholesterol (Sigma), and 14:0 PEG2000PE (DMG-PEG2000) or 18:0 PEG2000 PE (DSPE-PEG2000) (Avanti Polar Lipids) was prepared as a 20 mM stock solution in ethanol. The molar composition used was 50:10:38.5:1.5. The lipid solution was mixed with an aqueous mRNA solution in 10 mM citrate buffer (pH 4) using a microfluidic mixing device Nanoassemblr® (Precision Nanosystems, Vancouver BC) at a volume ratio of organic to aqueous phase of 1:3 and a total flow rate of 4 mL / min. The resulting LNP was then immediately diluted twice with PBS (pH 7.4) and dialyzed overnight. The next day, the LNP was filtered through a 0.22-micron filter.

[0373] LNP characterization

[0374] The particle size distribution, number of particles per mL, and mode size of LNPs were determined using a NanoSight NS300 (Malvern Panalytical) by diluting LNPs 250 to 1000 times with PBS. The zeta potential of LNPs was measured using a Zetasizer Nano ZS (Malvern Panalytical) dynamic light scattering instrument. Total mRNA content and encapsulation efficiency were determined using the standard Ribogreen (ThermoFisher) assay.

[0375] Molecular cloning of sdAb

[0376] The gene encoding the TP1107 sdAb sequence was synthesized as a gene fragment (Integrated DNA Technologies) and used for cloning into the pET His6 TEV LIC cloning vector (Pleiner et al.). J Cell Biol ,2018; 217(3): 1143-1154). Based on sequence alignment with three other sdAbs, the Q13 site was determined to be a conserved site and modified to a UAG codon for incorporation into azPhe (Chin et al., 2018; 217(3): 1143-1154). J of the Am Chem Soc, 2002; 124(31):9026-9027).

[0377] sdAb expression and purification

[0378] pET-TP1107 was combined with pEVOl-pAzF (this plasmid is described by Young et al.), J Mol Biol (2010; 395(2): 361-74) was co-transformed into B-95.ΔA Escherichia coli. This strain expresses an orthogonal mechanism that can recognize the UAG codon and incorporate azPhe during protein translation. B-95.ΔA Escherichia coli strain (described in Mukai et al., 2010; 395(2): 361-74) Sci Rep (2015, 5, 9699) is a unique expression vector in which 95 original UAG codons have been replaced and release factor 1 (RF-1) has been eliminated to improve the incorporation efficiency of azPhe.

[0379] Overnight cultures were inoculated into fresh TB medium containing appropriate antibiotics and cultured at 37°C with shaking until the optical density (OD600) reached 0.7 to 1.0. sdAb expression was induced by the addition of IPTG (2 mM), L-arabinose (0.02%), and azPhe amino acids (2 mM). Protein expression was continued at 30°C for 12 to 14 hours, after which bacteria were collected by centrifugation. The bacterial pellet was collected by centrifugation (4000 × g, 20 min), resuspended in Ni-NTA wash buffer, and then lysed using an autoclave (Avestin Emulsiflex C5).

[0380] After lysis, cell debris was centrifuged (12000 × g, 30 min), and the supernatant was collected and purified using an immobilized metal affinity chromatography (IMAC) column. Size exclusion chromatography (SEC) was also employed to remove non-specifically bound proteins using a Superdex 75 10 / 300 GL gel filter column (GE Healthcare). The concentration of sdAb was determined at 280 nm using a Nanodrop (Thermo) spectrophotometer.

[0381] Conjugation of sdAb with DSPE-PEG2000-DBCO

[0382] sdAb (TP1107) doped with azide optimal The sdAb can be directly conjugated to DBCO-PEG2000-DSPE via the SPAAC chemical method. To illustrate the effect of randomly oriented sdAbs, a 2-molar excess of NHS-azide (198 Da, Thermo) was first conjugated to a TP1107 sdAb. Unconjugated excess NHS-azide linkers were removed using a 7K MWCO Zeba desalting column (Thermo). The azide-modified / incorporated sdAb was then conjugated to DSPE-PEG with a 0.5-molar excess. 2000 -DBCO mixture, incubated at 37°C for 24 hours, forms TP1107. random No further purification is required.

[0383] Calculation of the number of TP1107 per LNP

[0384] The number of sdAbs for each LNP is calculated as follows.

[0385]

[0386] The concentration of sdAb was calculated using JESS Simple Western™ (Bio-Techne) via Western blotting, while the number of LNPs was determined using NanoSight NS300 (Malvern Panalytical).

[0387] Table 4

[0388] Binding affinity of sdAb-Ab

[0389] The binding affinity (Kb) between TP1107 sdAb and CD71 was determined using the BLItz (Pall ForteBio) biolayer interferometry system. D Initially, the streptavidin biosensor was hydrated with 10 mM PBS under oscillating conditions. First, a baseline reading was recorded in PBS for 30 seconds, followed by loading TP1107-biotin (300 nM) for 120 seconds, and then an additional 120 seconds of washing / baseline reading. CD71 (300 nM) was added to the sample holder, and the binding process was recorded for 120 seconds, followed by a 120-second dissociation step. D The values ​​were determined using BLItz curve fitting software.

[0390] The conjugation of mAbTfR with DSPE-PEG2000-DBCO

[0391] First, a 5-fold molar excess of NHS-azide was conjugated to purified mAbTfR (OKT9), an antibody kindly provided by Justine Mintern's team. Unconjugated excess NHS-azide was removed using a 7K MWCO Zeba desalting column (ThermoFisher). The azide-mAbTfR was then incubated overnight at 37°C with DSPE-PEG2000-DBCO at a DBCO:azide ratio of 2:1. No further purification was required.

[0392] The active targeting module was inserted into the LNP and functionalized mAb-TP1107 was prepared. optimal / random LNP

[0393] The DSPE-PEG2000-TP1107 mixture was added to the prepared LNP at a weight ratio of 0.5%. The reaction system was incubated at 4°C for 48 hours. Free TP1107 or unreacted DSPE-PEG2000-DBCO was removed by ultrafiltration using an Amicon 100 kDa MWCO (Merck) filter. A total of 5 washes were performed (3000 rpm, 5 min). The antibody was then mixed with TP1107. optimal / randomLNPs were mixed in a selected ratio and incubated overnight at 4°C to prepare functionalized LNPs. Pre-conjugated mAbTfR-PEG2000-DSPE (0.5% w / w lipid mass) was mixed with LNPs and incubated overnight to prepare mAbTfR-lysine-responsive LNPs.

[0394] Cell culture maintenance

[0395] Jurkat cells were maintained in RMPI medium (Gibco) containing 10% fetal bovine serum and penicillin-streptomycin (100 U / mL). Cells were cultured in a humidified incubator at 37°C and 5% CO2, and routine or mycoplasma contamination tests were performed.

[0396] Collection and purification of human PBMCs

[0397] Blood was collected from healthy individuals according to the experimental protocol. 10 to 30 mL of blood was collected and diluted with PBS, then carefully layered at a 1:1 volume ratio onto Ficoll-Paque PLUS density gradient medium. After centrifugation at 400 g for 40 minutes, the PBMC layer was collected and washed twice with pre-warmed RPMI medium. PBMCs can be used directly in experiments or stored in cryopreservation medium at -80°C for later use.

[0398] Cell binding and transfection experiments using functionalized LNPs

[0399] To determine the binding and transfection efficiency of functionalized LNPs to Jurkat cells, approximately 100,000 cells were added to each well of a 96-well plate. mRNA was added to the cells at a final concentration of 1 ng / μL, and the cells were incubated at 37°C for different times. The cells were then centrifuged at 400 × g for 5 min and washed three times with 2% FBS-PBS. The cells were resuspended in 80 μL of 2% FBS-PBS, and the mean fluorescence intensity was measured using a Straedigm S1000EXi flow cytometer. eGFP and Cy5 fluorescence were excited at 488 nm and 642 nm, respectively, and fluorescence emission signals were collected at 520 / 20 nm and 676 / 29 nm, respectively.

[0400] Experiments on human PBMC binding and transfection using functionalized LNPs

[0401] To evaluate the binding and transfection efficiency of functionalized LNPs, approximately 500,000 PBMCs were added to each well of a 96-well plate, along with functionalized LNPs at a final concentration of 1 ng / μL. The cells were then incubated at 37°C for 24 hours. The PBMCs were subsequently centrifuged at 400 × g for 5 minutes and washed three times with 2% FBS-PBS. To identify the phenotype of each cell subpopulation, cells were stained with the following antibodies: αCD3 mAb (clone OKT3, Biolegend), αCD4 mAb (clone OKT4, Biolegend), αCD8 mAb (clone SK1, Biolegend), αCD19 mAb (clone HIB19, Biolegend), αCD14 mAb (clone HCD14, Biolegend), and αCD56 mAb (clone 5.1H11, Biolegend), along with cell viability dye (eBioscience™ Fixable Viability Dye eFluor™ 780, ThermoFisher), and incubated on ice for 30 minutes. All antibodies were used in conjunction with Human TruStain FcX™ (Biolegend) according to the manufacturer's instructions and diluted 1:200. After washing away excess antibody, cells were resuspended in 100 μL of 2% FBS-PBS for flow cytometry analysis (Stratedigm S1000EXi). Cells were identified using the following combination of surface markers: CD4+ T cells (CD3+ and CD4+), CD8+ T cells (CD3+ and CD8+), monocytes (CD3-, CD19-, CD56-, and CD14+), NK cells (CD3-, CD19-, CD14-, and CD56+), and B cells (CD3- and CD19+). eGFP and Cy5 fluorescence were excited at 488 nm and 642 nm, respectively, and fluorescence emission signals were collected at 520 / 20 nm and 676 / 29 nm, respectively.

[0402] Statistical analysis

[0403] Data are expressed as mean ± standard deviation, based on results from at least n = 3 independent experiments. Statistical significance was analyzed using GraphPad Prism 9.0 software via one-way ANOVA combined with post-hoc tests.

[0404] Example 2 - Determination of the structure of nanobody / antibody-binding complex using electron microscopy

[0405] To design nanobodies that capture antibodies with optimal orientation, it is crucial to define the binding sites between the nanobodies and antibodies. Previous studies have shown that the TP1107 nanobodies can bind to the Fc region of mouse and rat IgG1. To determine the precise binding sites of the nanobodies, the inventors used single-particle negative staining transmission electron microscopy (TEM) to image and analyze the protein complex. TP1107 was mixed with a mouse anti-human transferrin receptor (clone OKT9) monoclonal antibody (mAbTfR) at a 2:1 ratio, and negatively stained with uranyl formate on a carbon membrane, followed by imaging at 120 kV. Single particles were selected and extracted using RELION software, and two-dimensional classification analysis was performed. Figure 1 (A) (Zivanov et al., elife , 2018; 7: e42166). Two-dimensional classification average images of the complex show a Y-shaped IgG structure, with two additional densities observed at the Fc region terminus, corresponding to two antibody-bound nanobodies ( Figure 1 A).

[0406] These particles were then reconstructed in 3D, and a de novo model was built using cryoSPARC. This model was then imported into RELION as a reference model for 3D refinement (Punjani et al.). Nature methods , 2017; 14(3): 290-296). The generated three-dimensional model shows two distinct foliate densities in the Fc region of IgG ( Figure 1 (B) By rigid-body fitting of the nanobody structural model, the inventors identified these foliated structures as binding sites for the nanobodies. This allowed them to determine the possible orientations of TP1107 when it binds to the antibody structure. Due to the structural similarities and conserved regions among different nanobodies, the structural model of TP1107 was constructed using MODELLER software, with templates derived from known high-resolution crystal structures of the anti-EGFR nanobody 7D12 and the anti-GFP nanobody Nanotrap (Mitchell et al.). Proteins: Structure, Function, and Bioinformatics, 2018; 86(7): 697-706; Schmitz et al., Structure , 2013; 21(7): 1214-1224). The resolution of the three-dimensional reconstructed surface is sufficient to fit the nanobody and antibody structure model, thereby showing the orientation of TP1107 when binding to the Fc region of the IgG structure ( Figure 1 (B and C). The reconstruction results show that the nanobody binds laterally to the Fc region of the antibody, thus enabling the assessment of the final orientation of the nanobody after binding with the antibody.

[0407] Example 3 - Rational Design of Nanobodies for Optimally Oriented Antibody Capture

[0408] To determine the optimal site for coupling TP1107 to the nanoparticle surface, the inventors utilized the three-dimensional structure of the TP1107 / mAbTfR complex to screen for residues located opposite the mAbOKT9 binding site. The "lateral" binding pattern of the TP1107 nanobody relative to the IgG structure indicates that the Gln13 site ( Figure 1 (In B and C, marked in red) are the ideal sites for conjugating TP1107 to the surface of nanoparticles to achieve optimal antibody capture.

[0409] Example 4 - TP1107 expression with site-specific azide incorporation

[0410] Using Methanococcus japonicus ( Methnocaldococcus jannaschii Orthogonal tRNA / synthesizer pairs (Chin et al.) J of the Am Chem Soc, 2002; 124(31): 9026-9027) and genome recoding of E. coli host B-95.ΔA (Mukai et al., ... Scientific Reports , 2015; 5(1): 9699), prepared TP1107 (TP1107) with azidophenylalanine (AzPhe) introduced at the Gln13 site. optimal The inventors also expressed TP1107 without the introduction of AzPhe and randomly modified lysine using NHS-azides (TP1107). random The incorporation efficiency of azide in these nanobodies was determined using the click-reaction fluorescent probe DBCO-Cy5. Each batch of TP1107 optimal and TP1107 random The DoL (Dominance Level) of TP1107 remained stable at approximately 0.5, but due to the randomness of NHS modification, some TP1107... random Nanobodies carry multiple azide groups.

[0411] Example 5 - LNP Antibody Capture System

[0412] Subsequently, TP1107 optimal and TP1107 random With DSPE-PEG respectively 2000 -DBCO lipids were modified. The lipids were conjugated to TP1107 at a 2:1 molar ratio (DBCO: azide). Capillary Western blotting analysis showed 14% TP1107. optimal Coupled with a single DSPE-PEG2000, a single band of 22 kDa was observed. Figure 2 (B). In comparison, 7.9% of TP1107 randomConjugation with a single DSPE-PEG2000 resulted in three additional bands at 26 kDa (5%), 32 kDa (2.7%), and 36 kDa (2.5%), indicating conjugation with 2, 3, and 4 lipid molecules, respectively. Figure 2 B).

[0413] DLin-MC3-DMA was used as the ionizable lipid, and LNPs (“MC3-LNP”) were formulated at a molar ratio of 50:10:38.5:1.5 (ionizable lipid:DSPC:cholesterol:DMG-PEG2000). The average hydrodynamic particle size of MC3-LNP, as determined by NanoSight, was 83 nm. Figure 2 (See Tables D and S1). To allow nanobody insertion, a DSPE-PEG2000-TP1107 mixture was added to LNP at a weight ratio of 0.5% (DSPE-PEG2000-TP1107 to total lipids) and incubated at 4°C for 48 hours. Unincorporated DSPE-PEG2000-TP1107 was removed by 100 kDa MWCO ultrafiltration. Western blotting results showed that no unmodified nanobodies were detected after purification. Figure 2 (C). TP1107 optimal A single band is observed, corresponding to a nanobody modified with a single PEG2000-DSPE, while TP1107... random Four bands were observed, corresponding to 1 (57%), 2 (28%), 3 (15%), and 4 or more (8%) PEG2000-DSPE modifications, respectively. This band distribution is similar to that of DSPE-PEG2000-TP1107. random The observed patterns are consistent ( Figure 2 B).

[0414] Subsequently, the inventors evaluated the basic physical properties of the TP1107-modified LNP. The particle size of the LNP was determined using NanoSight. optimal The average particle size is 85 nm, LNP-TP1107 random The average particle size was 92 nm, both of which were similar to the particle size of unmodified LNP. Figure 2 (See E and Table 3). Because the small size of the nanobodies (2 × 2 × 4 nm) is within the NanoSight measurement error range, the inventors did not expect a significant increase in the particle size of the functionalized LNPs. LNP-TP1107 random A second particle size peak of 132 nm also appeared. This indicates that randomly labeled TP1107 induced a certain degree of LNP aggregation. LNP-TP1107 optimalThe particle size distribution was the same as that of the unmodified LNP, and no particle aggregation was detected. The attachment of TP1107 did not change the surface charge or encapsulation efficiency of the LNP. Figure 2 F and Figure 2 (G). To determine the number of nanobodies attached to each particle, particle concentration was measured using NanoSight (Table 3), and nanobodily concentration was obtained by interpolation of calibrated Western Blot analysis results (Table 3). The average number of nanobodies on each LNP was calculated based on the ratio of the two: LNP-TP1107 optimal Approximately 200, LNP-TP1107 random 400 to 600 ( Figure 2 (H). Overall, these results indicate that TP1107 with optimized orientation can be efficiently incorporated into LNPs without affecting particle size; while the random concatenation method leads to TP1107 connecting multiple DSPE-PEG2000 particles and causes a small amount of particle aggregation (H). Figure 1 B).

[0415] Table 3: Summary of hydrodynamic particle sizes of different LNPs determined by NTA. All data are average ± standard. Standard deviation. N represents the number of independent experiments repeated.

[0416]

[0417] Functionalization of LNPs with a targeted antibody can be achieved by simply adding the antibody to TP1107-functionalized particles at a 2:1 excess ratio of TP1107 to the antibody. Due to the high affinity of TP1107 for the Fc domain of mouse or rat IgG1, all antibodies can capture it under these conditions. TP1107 was modified with an anti-transferrin receptor mouse IgG1 antibody (mAbTfR). optimal After LNP, the resulting LNPs exhibited a single particle size distribution (87 ± 3 nm, n = 5). Meanwhile, TP1107... random The resulting particles were larger (95 ± 8 nm, n = 5). This further indicates that the randomly oriented TP1107 particles exhibited poor stability and a degree of aggregation. These results confirm that the inventors were able to engineer TP1107 nanobodies with antibody capture capabilities onto the LNP surface without significantly altering particle size, surface charge, or efficient mRNA encapsulation capability.

[0418] Example 6 - Optimizing Specific Delivery to Cells

[0419] To design the most efficient active targeting LNP system, the base LNP formulation should minimize non-specific binding to cells. To this end, the inventors investigated two commonly used PEG lipids for LNP formulation: DMG-PEG2000 and DSPE-PEG2000. Unmodified DMG-PEG2000-LNP or DSPE-PEG2000-LNP were formulated using eGFP-encoding mRNA (incorporated with Cy5-labeled oligonucleotides) to evaluate their induced protein expression and LNP binding levels to cells. Both formulations were co-incubated with Jurkat cells at a total mRNA concentration of 1 ng / μL for 24 hours. Compared to the untargeted DSPE-PEG2000-LNP, the untargeted DMG-PEG2000-LNP showed more than 5-fold non-specific binding after 24 hours. Figure 3 (A). Untargeted DSPE-PEG2000-LNP produced only limited eGFP expression (MFI = 59 ± 8, n = 3), while untargeted DMG-PEG2000-LNP induced strong eGFP expression (MFI = 1680 ± 213, n = 3). Figure 3 (B). Subsequently, the inventors functionalized these LNPs using mAbTfR, which binds to human TfR on the surface of Jurkat cells. Simultaneously, a mouse IgG1 isotype control antibody (mAbiso) was used to assess the non-specific binding that might be induced by protein modification of the PEGylated LNP surface. Compared to unmodified LNPs, the binding levels of mAbTfR-DMG-PEG2000-LNP and DSPE-PEG2000-LNP to cells were significantly increased. The two targeting agents had similar binding levels to cells, but due to the lower non-specific binding of DSPE-PEG2000-LNP, its specific binding increased by 61-fold, while that of DMG-PEG2000-LNP increased by only 11-fold. Figure 3 The expression of eGFP generated from mRNA delivered by LNP showed a similar trend. Both mAbTfR-DMG-PEG2000-LNP and DSPE-PEG2000-LNP showed high levels of eGFP expression. Figure 3Similarly, unmodified DSPE-PEG2000-LNP and mAb control-DSPE-PEG2000-LNP consistently maintained low nonspecificity, with extremely low eGFP expression levels. Compared to unmodified DSPE-PEG2000-LNP, mAbTfR-DSPE-PEG2000-LNP showed an eGFP expression level that increased more than 1880-fold. Although mAbTfR-DMG-PEG2000-LNP and mAbTfR-DSPE-PEG2000-LNP had similar eGFP expression levels, DMG-LNP showed a higher nonspecific eGFP expression level, resulting in only a 73-fold increase compared to unmodified LNP. Figure 3 Therefore, the inventors subsequently used DSPE as the PEG component in all LNP formulations (referred to herein as "low-specificity LNP").

[0420] To further optimize antibody capture on LNPs, the inventors investigated how the antibody-to-TP1107 ratio affected cell binding levels and protein expression efficiency in delivering mRNA. To determine the antibody capture efficiency of LNPs, the inventors used natural PAGE and silver staining to detect the amount of free antibody in the functionalized solution. 100 ng of mAbTfR TP1107 containing different antibody:TP1107 ratios (1:8, 1:4, 1:2, 1:1, and 2:1) was used. optimal Or TP1107 random LNPs were loaded onto the gel. Free antibodies could enter the gel, while the LNPs and the antibodies captured on their surface remained in the wells. No free antibodies were detected at antibody:TP1107 ratios of 1:8, 1:4, or 1:2. Figure 6 This indicates that antibody capture is quantitative, and these LNPs can be used directly without further purification. A gradual increase in free antibody was observed at 1:1 and 2:1 ratios. To test cell binding and protein expression induced by LNPs with different antibody functionalization levels, the inventors targeted TP1107. random -LNP or TP1107 optimal - LNPs were co-incubated with Jurkat cells for 4 hours without removal of unbound antibodies. As expected, cell binding (Cy5 fluorescence) and eGFP expression levels increased with increasing number of targeting antibodies on the LNP surface, with maximum cell binding and eGFP expression levels observed at a mAbTfR to TP1107 ratio of 1:1. At the highest antibody to TP1107 ratio (2:1), cell binding and eGFP expression levels significantly decreased. Figure 4(A, B). This is likely due to the free antibody binding to the TfR receptor on the cell surface, thereby blocking further binding of the target LNP. Although the target LNP can be purified from the unbound antibody, this increases the complexity of the functionalization process and may lead to a decrease in LNP yield. Based on these data, the inventors determined that a 1:2 antibody:TP1107 ratio was the optimal functionalization ratio because no further purification of LNP was required at this ratio, and cell binding was increased by 135-fold and protein expression by 648-fold compared to the unmodified control. Although a 1:1 antibody:TP1107 ratio showed an approximately 21% increase in cell binding and an approximately 51% increase in protein expression, the inventors believe that the additional purification steps required are insufficient to justify this activity enhancement.

[0421] Subsequently, the inventors studied the number of matched antibodies in each LNP (LNP-TP1107). optimal Under the condition of ), the optimized orientation of TP1107 (TP1107) optimal Is it comparable to TP1107? random It exhibited superior cell binding and eGFP expression levels. Compared to LNP-TP1107 random Compared to LNP-TP1107 optimal The cell binding level increased by approximately 2-fold, and the eGFP expression level increased by more than 4-fold. Figure 4 (A to C).

[0422] The inventors also studied targeted delivery and protein expression over shorter incubation periods (30 minutes to 4 hours). Figure 4 (C to E). In the 4-hour experiment, for TP1107 optimal -LNP and TP1107 random -LNP (TP1107 to mAbTfR ratio of 2:1), cell binding levels continued to increase. eGFP expression was observed as early as 2 hours later, indicating that eGFP release and subsequent expression occur rapidly after initial interaction with cells.

[0423] The most common strategy for synthesizing actively targeted nanoparticles is to directly conjugate linkers to antibodies by reacting with lysine or cysteine ​​residues. The inventors intentionally compared this method with the antibody capture system of this invention. mAbTfR reacted with NHS-PEG4-azide, which reacted randomly with lysine residues (DoL 0.47). Subsequently, DBCO-PEG2000-DSPE lipids were conjugated with azid-modified antibodies using a similar approach to TP1107 conjugation. The inventors attempted to purify mAbTfR-lysine-LNP by ultrafiltration to remove excess mAbTfR-PEG2000-DSPE, but the yield was low, and sufficient material could not be obtained for subsequent experiments. To compare crude mAbTfR-lysine-LNP with mAbTfR-TP1107... optimal -LNP binding ability, inventor Xiang Jing TP1107 optimal -LNP-treated cells were treated with the same excess of free mAbTfR to mimic the receptor blocking effect that occurs in the presence of free mAbTfR. Similarly, mAbTfR-TP1107 optimal -LNP showed significant cell-binding levels ( Figure 4 The binding levels of mAbTfR-lysine-LNP to cells were similar, but the percentage of transfected cells (2% vs. 75%) and eGFP expression levels were significantly lower. Figure 4 The same trend was observed with shorter incubation times (F). Figure 8 (A to C). These results confirm that even in the presence of a large excess of free mAbTfR in the solution, mAbTfR-TP1107 optimal -LNP also outperformed mAbTfR-lysine-LNP.

[0424] Example 7 - Screening a group of targeted antibodies

[0425] To demonstrate the feasibility of the antibody capture system of the present invention in rapidly screening a group of different antibodies, the inventors prepared TP1107 antibodies carrying antibodies against CD3, CD4, CD5, and CD7, respectively. optimal -LNPs. These receptors are typically expressed on the surface of T lymphocytes, which are targets for drug delivery or chimeric antigen receptor T-cell (CAR-T) therapy.

[0426] First, the inventors determined the expression levels of these biomarkers on Jurkat cells. The results showed that CD3 expression was limited, but CD4, CD5, and CD7 were all significantly expressed on the cell surface. Figure 9 ).

[0427] Subsequently, the inventors investigated the binding affinity of LNPs to cells. The binding levels of mAbCD5 and mAbCD7-LNPs to Jurkat cells were higher than those of mAbCD3 and mAbCD4-LNPs. Figure 4 The nonspecific binding levels of mAb control and unmodified LNP were similar. The binding trend of mAb-LNP to Jurkat cells was similar to that observed with free mAb, indicating that the antibody capture method does not interfere with antibody-receptor binding. Interestingly, consistent with the results observed with mAbTfR LNP, the increase in eGFP expression level was even greater than the increase in cell binding level for targeted LNP. Both mAbCD5 and mAbCD7-LNP showed high expression levels (MFI 21633 ± x and 14207 ± y, respectively), and approximately 100% of cells expressed eGFP after 24 hours. Figure 4 (G to I). Although both unmodified LNP and isotype control LNP could bind nonspecifically to Jurkat cells, eGFP expression was not detected.

[0428] Example 8 - Maximizing protein expression in human PBMCs by targeting specific T cell receptors

[0429] To further demonstrate the efficacy of this LNP-targeting system, the inventors investigated its ability to target specific subsets of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were obtained from healthy blood donors and isolated using the Ficoll gradient purification method. Figure 5 (A). Human CD3, CD4, CD5, and CD7-targeting LNPs carrying eGFP mRNA and Cy5-labeled oligonucleotides were co-incubated with PBMCs for 24 hours. Subsequently, cell binding (Cy5 signaling) and eGFP expression levels of the LNPs were analyzed by flow cytometry, and the following cell populations were identified using antibody phenotype grouping: CD4+ T cells (CD3+, CD4+); CD8+ T cells (CD3+, CD8+); NK cells (CD3-, CD19-, CD56+); monocytes (CD3-, CD19-, CD14+); and CD19+ B cells (CD3-, CD19+). As expected, CD3, CD4, CD5, and CD7-targeting LNPs showed strong binding to CD4-positive T cells because these receptors are present on the T cell surface. Interestingly, mAbCD3 LNPs bound to CD4+ T cells at a lower level than mAbCD7 LNPs, but their eGFP expression levels were significantly higher. Figure 5(B). Furthermore, although the binding levels of mAbCD4 and mAbCD5 LNPs to CD4+ T cells decreased only slightly (binding levels decreased by 10% to 50%), their eGFP expression levels were significantly reduced (by 90%). A similar phenomenon was observed in CD8+ T cells. mAbCD7 LNP had the highest binding level to CD8+ T cells, but mAbCD3 LNP had a significantly higher eGFP expression level (B). Figure 5 This illustrates the role of receptors in mediating mRNA uptake and delivery, and highlights the importance of screening for optimal receptors. Only mAbCD7 LNP showed significant binding ability to NK cells, resulting in a corresponding high eGFP signal (C). Figure 5 D).

[0430] Neither the unmodified LNP nor the mAbiso LNP was detected to bind to T cells, B cells, or NK cells, but both bound to monocytes. Figure 5 Although unmodified LNPs showed only low binding levels, mAbiso LNPs showed high binding levels, comparable to the binding levels of mAbCD4 LNPs to CD4-positive T cells. mAbCD3, mAbCD4, mAbCD5, and mAbCD7 LNPs all showed high levels of binding ability to monocytes. Figure 5 Interestingly, although the binding level of mAbCD4 LNP to monocytes was similar to that of other targeted LNPs, it induced a significantly higher level of eGFP expression (E). Figure 5 As expected, for CD19-positive B cells, due to the lack of corresponding receptors on their surface, neither modified LNPs nor targeting LNPs showed binding or expression (E). Figure 5 (F).

[0431] To further understand the relationship between LNP binding and eGFP expression, the inventors calculated the ratio of eGFP expression to Cy5 binding signal in all samples where the Cy5 signal was significantly higher than the background signal. Figure 5 These results confirm that LNPs targeting CD3 are significantly more efficient in inducing protein expression.

[0432] Example 9 - Nanobodies directly target cell surface receptors

[0433] To demonstrate that nanobodies directly targeting cell surface receptors achieve cell binding and efficient transfection, LNPs modified with anti-Her2 nanobodies (2D3) or anti-EGFR nanobodies (7D12) were respectively used to bind to MDA-MB231 cells. Figure 11 ) or BT474 cells ( Figure 12 Co-incubation.

[0434] Approximately 30,000 cells were added to each well of a 96-well plate. mRNA was added to the cells at a final concentration of 1 ng / μL, and the plates were incubated at 37°C for 24 hours. The cells were then detached from the plate, centrifuged at 400 × g for 5 min, and washed three times with 2% FBS-PBS. The cells were resuspended in 80 μL of 2% FBS-PBS, and the mean fluorescence intensity was measured using a Straedigm S1000EXi flow cytometer. Cy5 fluorescence was excited at 642 nm, and the fluorescence emission signal was collected at 676 / 29 nm. To determine transfection efficiency, luciferase activity was measured using the Promega Nano-Glo® luciferase detection system according to the manufacturer's instructions.

[0435] In MDA-MB-231 cells (where EGFR expression was higher than Her2), EGFR-targeted LNPs exhibited higher cell binding levels and induced higher protein expression compared to Her2-targeted LNPs. Conversely, in BT474 cells (where Her2 expression was higher than EGFR), Her2-targeted LNPs exhibited higher cell binding levels and induced higher protein expression compared to EGFR-targeted LNPs. This indicates that directed nanobodies directly attached to the LNP surface can efficiently recognize target receptors on the cell surface and induce efficient protein translation.

[0436] Example 10 - In vivo targeted distribution of different LNP formulations

[0437] To demonstrate that LNPs can precisely target specific cells in vivo, mice were administered targeted LNPs, isotype control LNPs, or unmodified LNPs, all of which contained Cre-mRNA (100 µg / kg). Figure 13Twenty-four hours later, the mice were euthanized, perfused with cold PBS, and the liver, spleen, lungs, lymph nodes, and femur and tibia of the right hind limb were collected. The organs were then enzymatically digested to isolate tissue-resident immune cells. The enriched immune cells were washed and resuspended in PBS containing 2% FBS for staining analysis. To identify the phenotypes of different cell subpopulations, cells were stained with the following antibodies: αCD3e mAb (clone 145-2C11, BD Biosciences), αCD4 mAb (clone GK1.5, Biolegend), αCD8 mAb (clone 53-6.7, BD Biosciences), αCD19 mAb (clone 1D3, BD Biosciences), αCD11b mAb (clone M1 / 70, Biolegend), αCD11c mAb (clone N418, Biolegend), αI-A / IE mAb (clone M5 / 114.15.2, Biolegend), αNK1.1 mAb (clone PK136, Biolegend), αLy-6C mAb (clone HK1.4.rMAb, BD Biosciences), αLy-6G mAb (clone 1A8, BD Biosciences), αF4 / 80... mAb (clone BM8, Biolegend), αCD45 mAb (clone S18009F, Biolegend), and αCD64 (FcγRI) mAb (clone X54-5 / 7.1, Biolegend) were added along with Mouse BD Fc Block™ and cell viability dye (LIVE / DEAD™ fixative blue dead cell staining kit for UV excitation, ThermoFisher), and incubated on ice for 30 minutes. Excess antibody was then removed. Flow cytometry analysis was performed using a Cytek Aurora 5 laser flow cytometer, and data were analyzed using Flowjo software (BDBiosciences).

[0438] These results confirm that antibody-targeted LNPs can precisely deliver active mRNA payloads to target cells (T cells in this example), involving multiple organs, including the spleen, liver, bone marrow, blood, and lymph nodes. Non-targeted particles have limited delivery efficiency to target cells (less than 1%).

Claims

1. A lipid-based nanoparticle comprising (a) an active agent and (b) a plurality of capture-binding domains present on the outer surface of the nanoparticle, wherein each capture-binding domain is connected to the lipid-based nanoparticle via site-specific linkage, such that each capture-binding domain is presented in substantially the same orientation and is capable of capturing the target portion in an orientation that allows the target portion to interact with its target.

2. The lipid-based nanoparticles according to claim 1, wherein the nanoparticles are liposomes, lipid nanoparticles, or micelles.

3. The lipid-based nanoparticles according to claim 1 or 2, wherein each of the plurality of capture-binding domains is connected to the lipid-based nanoparticle through the same site.

4. The lipid-based nanoparticles according to any one of claims 1 to 3, wherein each capture-binding domain is specifically linked to the lipid-based nanoparticles via a single site.

5. The lipid-based nanoparticles according to any one of claims 1 to 4, wherein each of the plurality of binding domains is linked to the lipid-based nanoparticles via a modified amino acid side chain.

6. The lipid-based nanoparticles according to any one of claims 1 to 5, wherein each capture-binding domain is not connected to the lipid-based nanoparticle via its N-terminus or C-terminus.

7. The lipid-based nanoparticles according to any one of claims 1 to 6, wherein each capture-binding domain is attached to the lipid-based particle at a location remote from the antigen-binding site, preferably remote from the complementarity-determining region (CDR).

8. The lipid-based nanoparticles according to any one of claims 1 to 7, wherein each capture-binding domain is connected to the lipid-based particle at a position of a non-β-sheet or α-helical component.

9. The lipid-based nanoparticles according to any one of claims 1 to 8, wherein each capture-binding domain is connected to the lipid-based nanoparticle at a location on the outer surface of the binding domain and without steric hindrance.

10. The lipid-based nanoparticle according to any one of claims 1 to 9, wherein the sites on the capture-binding domain for site-specific linkage have been determined to allow the capture-binding domain to be presented in an optimized orientation when the capture-binding domain is linked to the lipid-based nanoparticle.

11. The lipid-based nanoparticle according to any one of claims 1 to 10, wherein the sites on the capture-binding domain for site-specific linkages have been determined to allow the target molecule to be presented in an optimized orientation when the capture-binding domain connected to the lipid-based nanoparticle binds to its target molecule.

12. The lipid-based nanoparticle according to any one of claims 1 to 11, wherein the site-specific connection is between a first coupling group formed on the lipid-based nanoparticle and a second coupling group on the capture-binding domain.

13. The lipid-based nanoparticles according to claim 12, wherein the first coupling group is located on a hydrophobic molecule, preferably on a lipid.

14. The lipid-based nanoparticles according to claim 13, wherein the hydrophobic molecule is a lipid, and wherein the lipid is a phospholipid, a structural lipid, a PEGylated lipid, or a cationic or ionizable lipid.

15. The lipid-based nanoparticles of claim 14, wherein the lipid is a PEGylated lipid.

16. The lipid-based nanoparticles of claim 15, wherein the PEGylated lipid is DMG-PEG or DSPE-PEG.

17. The lipid-based nanoparticles according to any one of claims 12 to 16, wherein the first coupling group is an olefin or an alkyne dipoleophile.

18. The lipid-based nanoparticles of claim 17, wherein the dipoleophile is a strained cyclic olefin or a cyclic alkyne.

19. The lipid-based nanoparticles according to claim 18, wherein the strained cyclic olefin or cyclic alkyne is cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazene octyne ketone.

20. The lipid-based nanoparticles according to any one of claims 12 to 19, wherein the second coupling group is a 1,3-dipolar.

21. The lipid-based nanoparticles of claim 20, wherein the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate.

22. The lipid-based nanoparticles of claim 21, wherein the 1,3-dipolar is an azide.

23. The lipid-based nanoparticles according to claim 22, wherein the azide is azidophenylalanine.

24. The lipid-based nanoparticles according to any one of claims 12 to 16, wherein the first coupling group is a thiol or a mercapto group.

25. The lipid-based nanoparticles according to any one of claims 12 to 16, wherein the second coupling group is a thiol or a mercapto group.

26. The lipid-based nanoparticles according to any one of claims 12 to 16, wherein the site-specific linkage is a disulfide bond.

27. The lipid-based nanoparticles according to any one of claims 1 to 26, wherein each capture-binding domain is an antibody or antibody fragment, such as a nanobody or a single-chain variable fragment, affinity compound, aptamer, or peptide.

28. The lipid-based nanoparticles according to any one of claims 1 to 27, wherein each capture-binding domain is a nanobody.

29. The lipid-based nanoparticles according to any one of claims 1 to 27, wherein the site-specific linker is located at amino acid positions 10 to 16 of FR1 in the antibody.

30. The lipid-based nanoparticles according to any one of claims 1 to 27, wherein the site-specific linker is located at the 10th to 16th amino acids, which are not proline, in the FR1 of the antibody.

31. The lipid-based nanoparticles according to any one of claims 1 to 30, wherein each capture-binding domain binds to the Fc region of an antibody.

32. The lipid-based nanoparticles according to claim 31, wherein the Fc region is a CH2 or CH3 domain.

33. The lipid-based nanoparticles of claim 31, wherein the trapping binding domain is TP1107 or a variant thereof.

34. The lipid-based nanoparticles according to any one of claims 1 to 33, further comprising a plurality of targeting molecules, wherein the targeting molecules are bound by the capture-binding domain.

35. A conjugate comprising a lipid capable of incorporating lipid-based nanoparticles and a capture-binding domain, wherein the capture-binding domain is conjugated to the lipid via site-specific linkage.

36. The conjugate according to claim 35, wherein the lipid is selected from phospholipids, structured lipids, PEGylated lipids, or cationic or ionizable lipids.

37. The conjugate according to claim 36, wherein the lipid is a PEGylated lipid.

38. The conjugate according to claim 37, wherein the PEGylated lipid is DMG-PEG or DSPE-PEG.

39. The conjugate according to any one of claims 35 to 38, wherein the conjugate is formed between a first coupling group on the lipid and a second coupling group on the capture-binding domain.

40. The conjugate according to claim 39, wherein the second coupling group is a 1,3-dipolar.

41. The conjugate according to claim 40, wherein the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate.

42. The conjugate according to claim 41, wherein the 1,3-dipolar is an azide.

43. The conjugate according to claim 42, wherein the azide is azidophenylalanine.

44. The conjugate according to any one of claims 35 to 43, wherein the first coupling group is an olefin or an alkyne dipoleophile.

45. The conjugate according to claim 44, wherein the dipoleophile is a strained cyclic olefin or a cyclic alkyne.

46. ​​The conjugate according to claim 45, wherein the strained cyclic olefin or cyclic alkyne is cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazene octyne ketone.

47. The conjugate according to any one of claims 35 to 46, wherein the capture-binding domain is an antibody or antibody fragment, such as a nanobody or a single-chain variable fragment, affinity, aptamer or peptide.

48. The conjugate according to claim 47, wherein the capture-binding domain is a nanobody.

49. The conjugate according to claim 48, wherein the nanobody is TP1107 or a variant thereof.

50. A lipid-based nanoparticle comprising (a) an active agent and (b) a plurality of targeting molecules present on the outer surface of the nanoparticle, wherein each targeting molecule is linked to the lipid-based nanoparticle via a site-specific connection such that each targeting molecule is presented in substantially the same orientation and is capable of binding to a target on the cell surface.

51. The lipid-based nanoparticles according to claim 50, wherein the nanoparticles are liposomes, lipid nanoparticles, or micelles.

52. The lipid-based nanoparticles according to claim 50 or 51, wherein each of the plurality of targeting molecules is linked to the lipid-based nanoparticles through the same site.

53. The lipid-based nanoparticles according to any one of claims 50 to 52, wherein each targeting molecule is specifically linked to the lipid-based nanoparticles via a single site.

54. The lipid-based nanoparticles according to any one of claims 50 to 53, wherein each of the plurality of binding domains is linked to the lipid-based nanoparticles via a modified amino acid side chain.

55. The lipid-based nanoparticles according to any one of claims 50 to 54, wherein each targeting molecule is not linked to the lipid-based nanoparticle via its N-terminus or C-terminus.

56. The lipid-based nanoparticles according to any one of claims 50 to 55, wherein each targeting molecule is attached to the lipid-based nanoparticle at a location remote from the antigen binding site, preferably remote from the complementarity-determining region (CDR).

57. The lipid-based nanoparticles according to any one of claims 50 to 56, wherein each targeting molecule is attached to the lipid-based nanoparticle at a position of a non-β-sheet or α-helical component.

58. The lipid-based nanoparticles according to any one of claims 50 to 57, wherein each targeting molecule is attached to the lipid-based nanoparticle at a sterically unobstructed location on the outer surface of the binding domain.

59. The lipid-based nanoparticle according to any one of claims 50 to 58, wherein the sites on the targeting molecule for site-specific attachment have been determined to allow the targeting molecule to be presented in an optimized orientation when the targeting molecule is attached to the lipid-based nanoparticle.

60. The lipid-based nanoparticle according to any one of claims 50 to 59, wherein the site-specific connection is between a first coupling group formed on the lipid-based nanoparticle and a second coupling group on the target molecule.

61. The lipid-based nanoparticles of claim 60, wherein the first coupling group is located on a hydrophobic molecule, preferably on a lipid.

62. The lipid-based nanoparticles according to claim 61, wherein the hydrophobic molecule is a lipid, and wherein the lipid is a phospholipid, a structural lipid, a PEGylated lipid, or a cationic or ionizable lipid.

63. The lipid-based nanoparticles according to claim 62, wherein the lipid is a PEGylated lipid.

64. The lipid-based nanoparticles according to claim 63, wherein the PEGylated lipid is DMG-PEG or DSPE-PEG.

65. The lipid-based nanoparticles according to any one of claims 60 to 64, wherein the first coupling group is an olefin or an alkyne dipoleophile.

66. The lipid-based nanoparticles according to claim 65, wherein the dipoleophile is a strained cyclic olefin or a cyclic alkyne.

67. The lipid-based nanoparticles according to claim 66, wherein the strained cyclic olefin or cyclic alkyne is cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazene octyne ketone.

68. The lipid-based nanoparticles according to any one of claims 60 to 67, wherein the second coupling group is a 1,3-dipolar.

69. The lipid-based nanoparticles according to claim 68, wherein the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate.

70. The lipid-based nanoparticles of claim 69, wherein the 1,3-dipolar is an azide.

71. The lipid-based nanoparticles of claim 70, wherein the azide is azidophenylalanine.

72. The lipid-based nanoparticles according to any one of claims 60 to 64, wherein the first coupling group is a thiol or a mercapto group.

73. The lipid-based nanoparticles according to any one of claims 60 to 64, wherein the second coupling group is a thiol or a mercapto group.

74. The lipid-based nanoparticles according to any one of claims 60 to 64, wherein the site-specific linkage is a disulfide bond.

75. The lipid-based nanoparticles according to any one of claims 50 to 74, wherein each targeting molecule is an antibody or antibody fragment, such as a nanobody or a single-chain variable fragment, affinity compound, aptamer, or peptide.

76. The lipid-based nanoparticles according to any one of claims 50 to 75, wherein each targeting molecule is a nanobody.

77. The lipid-based nanoparticles according to claim 75 or 76, wherein the site-specific linker is located at amino acid positions 10 to 16 of FR1 in the antibody.

78. The lipid-based nanoparticles according to claim 75 or 76, wherein the site-specific linker is located at the 10th to 16th amino acids, which are not proline, in the FR1 of the antibody.

79. A conjugate comprising a lipid capable of incorporating lipid-based nanoparticles and a targeting molecule, wherein the targeting molecule is conjugated to the lipid via a site-specific link.

80. The conjugate according to claim 79, wherein the lipid is selected from phospholipids, structured lipids, PEGylated lipids, or cationic or ionizable lipids.

81. The conjugate according to claim 80, wherein the lipid is a PEGylated lipid.

82. The conjugate according to claim 81, wherein the PEGylated lipid is DMG-PEG or DSPE-PEG.

83. The conjugate according to any one of claims 79 to 82, wherein the conjugate is formed between a first coupling group on the lipid and a second coupling group on the capture-binding domain.

84. The conjugate according to claim 83, wherein the second coupling group is a 1,3-dipolar.

85. The conjugate according to claim 84, wherein the 1,3-dipolar is an azide, a nitrile oxide, a nitrone, or an isocyanate.

86. The conjugate according to claim 85, wherein the 1,3-dipolar is an azide.

87. The conjugate according to claim 86, wherein the azide is azidophenylalanine.

88. The conjugate according to any one of claims 79 to 86, wherein the first coupling group is an olefin or an alkyne dipoleophile.

89. The conjugate according to claim 88, wherein the dipoleophile is a strained cyclic olefin or a cyclic alkyne.

90. The conjugate according to claim 89, wherein the strained cyclic olefin or cyclic alkyne is cyclooctyne, dibenzocyclooctyne, monofluorocyclooctyne, difluorocyclooctyne, or biarylazazene octyne ketone.

91. A lipid-based nanoparticle comprising the conjugate according to any one of claims 35 to 49.

92. A lipid-based nanoparticle comprising a conjugate according to any one of claims 79 to 90.

93. A method for preparing functionalized lipid-based nanoparticles, comprising contacting the lipid-based nanoparticles with a conjugate according to any one of claims 35 to 49 to form functionalized lipid-based nanoparticles.

94. A method for preparing functionalized lipid-based nanoparticles, comprising contacting the lipid-based nanoparticles with a conjugate according to any one of claims 79 to 90 to form functionalized lipid-based nanoparticles.

95. The method of claim 93, wherein the method further comprises the step of contacting the functionalized lipid-based nanoparticles with the target molecule.

96. The method according to any one of claims 93 to 95, wherein the lipid-based nanoparticles have an active agent that is encapsulated inside the nanoparticles, adhered to the surface of the nanoparticles, or integrated into the structure of the nanoparticles.

97. A method for preparing functionalized lipid nanoparticles, the method comprising: Solutions of cationic and / or ionizable lipids, phospholipids, structured lipids, and PEGylated lipids are mixed to form lipid nanoparticles, and The formed lipid nanoparticles are contacted with the conjugate according to any one of claims 35 to 49 or 79 to 90. Thus, functionalized lipid nanoparticles were prepared.

98. The method for preparing functionalized lipid nanoparticles according to claim 97, the method further comprising adding an active agent in the step of mixing lipids.

99. A functionalized lipid-based nanoparticle, which can be obtained by or has been obtained by the method according to any one of claims 93 to 98.

100. A pharmaceutical composition comprising lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, and a pharmaceutically acceptable carrier, diluent or excipient.

101. A method for introducing an active agent into cells, preferably cells present in vivo, the method comprising contacting the cells with lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, thereby introducing the active agent into the cells.

102. A method for in vivo delivery of an active agent, the method comprising administering lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99 to a subject in need, thereby delivering the active agent to the subject.

103. A method for treating or preventing a disease or condition in a subject of need, the method comprising administering to the subject a lipid-based nanoparticle according to any one of claims 1 to 34, 50 to 78 or 99, or a pharmaceutical composition according to claim 100, thereby treating or preventing a disease or condition in a subject of need.

104. Use of the lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, or the pharmaceutical composition according to claim 100, in the preparation of a medicament for treating or preventing a disease or condition suffered by a subject in need.

105. The lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, or the pharmaceutical composition according to claim 100, for the treatment or prevention of a disease or condition suffered by a subject in need.

106. A method for producing a target polypeptide in cells, preferably mammalian cells, the method comprising contacting the cells with lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, wherein the active agent is mRNA encoding the target polypeptide, wherein the mRNA is translatable in the cells to produce the target polypeptide.

107. A method for delivering mRNA to cells, preferably mammalian cells, the method comprising administering to a subject lipid-based nanoparticles according to any one of claims 1 to 34, 50 to 78 or 99, wherein the active agent is mRNA, thereby delivering mRNA to the cells.

Images