Compositions and methods for delivery of agents

Abstract

The present disclosure provides lipid nanoparticle compositions and methods of use. Among other things the present disclosure provides lipid nanoparticle compositions which increased specificity for specific cells or tissues. The present disclosure provides methods of use of the disclosed lipid nanoparticles.

Description

Attorney Docket No.: 2013260-0047COMPOSITIONS AND METHODS FOR DELIVERY OF AGENTSBackground

[0001] The targeted delivery of nucleic acids represents a continuing medical challenge. Attempts have been made to use lipid nanoparticles as delivery vehicles for biologically active substances. There remains a poor understanding of which compositions and methods of lipid nanoparticles provide specific tissue targeted delivery.Summary

[0002] The present disclosure recognizes a need for compositions, preparations, and methods of use of lipid nanoparticles (LNPs) for the targeted delivery of nucleic acids. Among other things, the present disclosure recognizes that specific compositions, preparations, and methods of use of nanoparticles are useful for the targeted delivery of nucleic acids to particular tissues and cell types in vivo. In some embodiments, the present disclosure provides methods and compositions for delivering a nucleic acid to a tissue of a subject. In some embodiments, a tissue is extrahepatic. In some embodiments, a tissue is immune tissue. In some embodiments, the present disclosure provides compositions and methods of use of nanoparticles for the delivery of nucleic acids to immune cells.

[0003] Among other things, the present disclosure documents surprisingly efficient delivery of payload(s), including expression of nucleic acid payload(s) to immune cells. In some embodiments, such delivery is to T cells. In some embodiments, such delivery is to B cells.

[0004] In some embodiments delivery and expression of nucleic acid payloads by LNPs of the present disclosure is achieved by functionalization of the LNPs. In some embodiments functionalization is achieved by direct conjugation of a targeting entity to the surface of an LNP.

[0005] In some embodiments the present disclosure provides a functionalized nucleic acid lipid particle composition comprising: lipid components that encapsulate aAttorney Docket No.: 2013260-0047 payload to form a particle; and a targeting entity. In some embodiments, a payload is an RNA. In some embodiments, an RNA encodes a therapeutic agent. In some embodiments, an RNA encodes a chimeric antigen receptor.

[0006] In some embodiments, lipid components comprise an ionizable lipid, a sterol, a helper lipid, and a PEG-lipid. In some embodiments a PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, Maleimide-PEG-DSPE, Azide-PEG-DSPE and DBCO-PEG-DSPE. In some embodiments, a helper lipid is a phospholipid. In some embodiments, a helper lipid is DSPC.

[0007] In some embodiments, a targeting entity comprises an antibody agent. In some embodiments, an antibody agent is covalently linked to the surface of the nucleic acid lipid particle by DTT reduction, SATA thio conjugation or DBCO conjugation.

[0008] In some embodiments an antibody agent binds CD19. In some embodiments, an antibody agent comprises an anti-CD19 antibody or antibody fragment. In some embodiments, an antibody agent binds CD3. In some embodiments, an antibody agent comprises an anti-CD3 antibody or antibody fragment. In some embodiments, and antibody agent binds CD79b. In some embodiments, an antibody agent comprises an anti-CD79b antibody or antibody fragment.

[0009] In some embodiments, the present disclosure provides a method of delivering a nucleic acid to a tissue or a cell of a subject, the method comprising administering to the subject a functionalized nucleic acid lipid particle described herein. In some embodiments, a tissue is or comprises immune tissue. In some embodiments, a cell is or comprises an immune cell. In some embodiments, the immune tissue is spleen or thymus. In some embodiments, the immune cell is a B-cell or T-cell.

[0010] In some embodiments, the present disclosure provides a method of treating a subject suffering from a disease, the method comprising administering to the subject a functionalized nucleic acid lipid particle as described herein. In some embodiments, a disease is a cancer. In some embodiments, a disease is a disease effecting a T-cell or B-cell.Attorney Docket No.: 2013260-0047

[0011] In some embodiments, the present disclosure provides a method of manufacturing a nucleic acid lipid particle comprising combining a nucleic acid with lipid components including an ionizable lipid, a helper lipid, a sterol, a PEG-lipid. In some embodiments, the present disclosure provides a method of manufacturing a functionalized nucleic acid lipid particle comprising combining a payload with lipid components and directly conjugating a targeting moiety to the lipid nanoparticle encapsulating the pay load.Brief Description of the Drawing

[0012] Figures 1A-1C depict mAb reduction condition optimization and characterization of reduced mAb.

[0013] Figure 2 depicts mAb reduction condition optimization and characterization of reduced mAb.

[0014] Figure 3 depicts biodistribution of various LNP compositions in B6 Albino mice.

[0015] Figure 4 depicts biodistribution of T-mAb-LNPs with Lipid 5 versus LIP091 peripheral core compositions in B6 Albino mice.

[0016] Figures 5A-5C depict surface-functionalized T-MAB-LNPs with mAb 2- LNP demonstrates selective T cell targeting over off-target cells in human PBMCs.

[0017] Figures 6A and 6B depicts T-cell targeted CD19 CAR T-mAb 2-LNP uptake in cultured human PBMCs results in T-cell activation.

[0018] Figure 7 demonstrates B-cell depletion in mouse peripheral blood after CD19 CAR T-LNP in situ treatment.

[0019] Figures 8A-8F demonstrate Thy 1.1 RNA outperformed mCherry as the reporter RNA in vivo. A and B), Representative FACS histogram plots for mCherry vs Thy 1.1 signal in A20 cells. A20 cells were transfected with aCD19 and ISO-LNP loaded with mCherry or Thy 1.1 RNA at 0.2 mg / mL. After 24 h, cells were washed, and protein expression was measured through flow cytometry. C-F) Representative FACS histogramAttorney Docket No.: 2013260-0047 plots and quantification of mCherry vs Thyl.l signal in spleen B-cells in vivo. 8-week-old BALB / c mice were injected with mCherry or Thyl.l RNA LNP through tail vein injection at 0.5 mg / kg. After 18 hours, spleens were harvested, processed into single cells, and subsequently stained with surface markers. Dead cells and red blood cells were excluded by viability dye and TER-119. B-cells were identified as B220+CD3-. N=l-4 / group. Data represents mean ± SEM.

[0020] Figures 9A-9C depict screening of anti-CD19 antibodies in vitro and in vivo. 9A) In vitro transfection efficiency (mCherry MFI) of anti-CD19 antibodies of different clones from different vendors. A20 cells were transfected with aCD19-RG7-Peripheral and ISO-RG7-Peripheral LNPs loaded with mCherry at 0.2 mg / mL. After 24 h, cells were washed, and protein expression was measured through flow cytometry. 9B and 9C) In vivo cell binding (DiD+%) and protein expression (mCherry+%) of the top 3 candidates from in vitro screening versus the initial 1D3 antibody in spleen and bone marrow B-cells. 8-week- old BALB / c mice were injected with mCherry LNPs through tail vein injection at 0.5 mg / kg. After 18 hours, spleens and bone marrow were harvested, processed into single cells, and subsequently stained with surface marker. Dead cells and red blood cells were excluded by viability dye and TER- 119. B-cells were identified as B220+CD3-. N=3-4 / group. Two- way ANOVA with Dunnett’s multiple comparison test to aCD19 (1D3, BioXCell, aCD19 / RG7=0.1). Data represents mean + SEM.

[0021] Figures 10A-10L depict screening of targeting antibodies to improve B-cell delivery in vitro and in vivo. 10A to 10F) In vitro screening of B-cell targeting antibodies. Splenocytes were harvested from 8-week-old BALB / c mice and transfected with mAh functionalized LNP loaded with Thy 1. 1 RNA and DiD dye at 0.2 mg / mL. After 4h, cells were washed, stained with fluorescent labeled anti-Thyl. l mAb. The protein expression and cell uptake were measured by Thyl.l (A to C) and DiD (D to F) fluorescent signals in B- cells, myeloid cells and T-cells through flow cytometry. 10G-10L) In vivo validation of B cell targeting antibodies. Eight- week-old BALB / c mice were injected with Thyl.l RNA and DiD co-encapsulated LNP through tail vein injection at 0.5 mg / kg. After 18 hours, spleens were harvested, processed into single cells, and subsequently stained with surface markers. Dead cells and red blood cells were excluded by viability dye and TER-119. B-cells, T-cells and myeloid cells were identified as B220+CD3-, B220-CD3+, and B220-CD3-,Attorney Docket No.: 2013260-0047 respectively. The quantification was presented by % of positive cells (G to I) and mean fluorescent intensity (J to L). N=3-4 / group. Two-way ANOVA with Dunnett’s multiple comparison test to aCD79b-LNPs. Data represents mean ± SEM.

[0022] Figures 11A-11J demonstrate dose titration of aCD79b-RG7-Peripheral LNP in spleen and bone marrow B-cell subpopulations and non-B-cells. Eight-week-old BALB / c mice were injected with DiD dye and Thy 1.1 encoding RNA co-encapsulated LNPs through tail vein at 0.1 and 0.033 mg / kg. After 18 hours, spleens and bone marrows were harvested, processed into single cells, and subsequently stained with viability dye and surface markers. Dead cells and red blood cells were excluded by viability dye and TER- 119. B-cells, developing B-cells, mature B-cells, T-cells and myeloid cells were identified as B220+CD3- , B220i»CD3-, B220h,ghCD3-, B220-CD3+, and B220-CD3-, respectively. N=3-4 / group. Two-way ANOVA with Tukey’s multiple comparison. Data represents mean + SEM.

[0023] Figures 12A-12D demonstrate Optimization of core LNP compositions for potent and selective B-cell targeting in vivo. (A and B) Quantification of percentage of positive cells (A) and mean fluorescent intensity (B) for DiD and Thyl. 1 of standard LNP and aCD79b-standard LNP versus peripheral LNP and aCD79b-peripheral LNP in spleen B- cells in vivo. C and D) Quantification of cell uptake and protein expression by mean fluorescent intensity of DiD (C) and Thy 1. 1 (D) of LNP in B-cells, myeloid cells and T- cells. 10- week-old BALB / c mice were injected with Thy 1.1 RNA LNP through tail vein injection at 0.1 mg / kg. After 18 hours, spleens were harvested, processed into single cells, and subsequently stained with surface markers. Dead cells and red blood cells were excluded by viability dye and TER- 119. B-cells, myeloid cells and T-cells were identified as B220+CD3-, B220-CD3-, and B220-CD3+, respectively. N=2-3 / group. Two-way ANOVA with Dunnett’s multiple comparison test to aCD79b-Peripheral LNPs for A and B, and to B- cells for C and D). Data represents mean ± SEM.

[0024] Figures 13A- 13F demonstrate direct conjugation of aCD79b onto the peripheral LNP compared to the previous two-layer method in vivo. A and D) Thyl.l RNA expression after LNPs treatments in spleen mature and developing B-cells. (One-way ANOVA with Dunnett’s multiple comparison test to the aCD79b-Peripheral LNP). B and E) B-cell size change indicated by FSC after LNPs treatments in spleen mature and developing B-cells. C and F) IgM expression after LNPs treatments in spleen mature and developing B-Attorney Docket No.: 2013260-0047 cells. 10-week-old BABL / c mice were injected with Thyl.l RNA encapsulated aCD79b- LNPs through tail vein injection at 0.1 mg / kg. After 18 hours, spleens were harvested, processed into single cells, and subsequently stained with surface marker. B-cells, developing B-cells, mature B-cells, T-cells and myeloid cells were identified as CD45+B220+IgM+, CD45+B220io»lgM+, CD45+B220h>ghlgM+, CD45+CD3+, and CD45+CDllb+, respectively. N=4 / group. One-way ANOVA with Dunnett’s multiple comparison test to the control. Data represents mean ± SEM.

[0025] Figures 14A-14B demonstrate tissue level distribution of aCD79b-Peripheral LNP versus standard LNP core and peripheral LNP core. A) Representative IVIS bioluminescence images of all organs. 10-week-old B6- Albino mice were injected with fLuc RNA LNP through tail vein injection at 0.3 mg / kg. After 18 hours, D-luciferin was injected intraperitoneally at 150 mg / kg. 10 min later. The thymus, lungs, livers, spleens, and lymph nodes were then harvested for ex vivo imaging. N=5 / group. (b) Quantification of the mean radiance. Two-way ANOVA with Dunnett’s multiple comparison test comparing to aCD79b-peripheral LNP. Data represents mean ± SEM.

[0026] Figures 15 -15F demonstrate in vitro transfection of human PMBCs by anti-human B-cell targeting mAbs functionalized on peripheral LNPs. A) Percentage of Thyl.l+ cells in B-cell populations in PMBC post transfection. B) Viability of PMBCs post transfection. C) Mean fluorescent intensity (MFI) of cell activation marker CD69 in B-cell populations post-transfection. D to F) Mean fluorescent intensity (MFI) of Thyl.l in B-cells, myeloid cells and T-cells post-transfection. Human PBMCs were plated into U-shape 96- well plate in RPMI-1640 containing 10% FBS at 105 cells per well. Cells were treated with DiD dye and Thyl.l encoding RNA co-encapsulated LNPs at 2, 0.2 and 0.02 ug / mL. After 20 h, cells were then washed with IX DPBS three times and stained with cell markers, including viability dye, CD79b (receptor internalization), CD20 (B cells), CD 11b (myeloid cells), CD69 (B-cell activation), CD3 (T-cells) and analyzed by flow cytometry. N=3.Definitions

[0027] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art,Attorney Docket No.: 2013260-0047 familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0028] Administration: As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc.. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g. intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0029] Agent : As used herein, the term “agent”, may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polymer. In some embodiments, the term may refer to a compound or entity that comprises one or more polymeric moieties. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that is substantially free of a particular polymer or polymeric moiety. In some embodiments, the term may refer to a compound, molecule, or entity that lacks or is substantially free of any polymer or polymeric moiety.

[0030] Amelioration : as used herein, refers to the prevention, reduction or palliation of a state, or improvement of the state of a subject. Amelioration includes, but does notAttorney Docket No.: 2013260-0047 require complete recovery or complete prevention of a disease, disorder or condition (e.g., radiation injury).

[0031] Amino acid', in its broadest sense, as used herein, the term “amino acid” refers to a compound and / or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. “Standard amino acid” refers to any of the twenty standard L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.

[0032] Analog: As used herein, the term “analog” refers to a substance that shares one or more particular structural features, elements, components, or moieties with a reference substance. Typically, an “analog” shows significant structural similarity with the reference substance, for example sharing a core or consensus structure, but also differs in certain discrete ways. In some embodiments, an analog is a substance that can be generated from the reference substance, e.g., by chemical manipulation of the reference substance. InAttorney Docket No.: 2013260-0047 some embodiments, an analog is a substance that can be generated through performance of a synthetic process substantially similar to (e.g., sharing a plurality of steps with) one that generates the reference substance. In some embodiments, an analog is or can be generated through performance of a synthetic process different from that used to generate the reference substance.

[0033] Antagonist: Those skilled in the art will appreciate that the term“antagonist”, as used herein, may be used to refer to an agent, condition, or event whose presence, level, degree, type, or form correlates with decreased level or activity of another agent (i.e., the inhibited agent, or target). In general, an antagonist may be or include an agent of any chemical class including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity that shows the relevant inhibitory activity. In some embodiments, an antagonist may be direct (in which case it exerts its influence directly upon its target); in some embodiments, an antagonist may be indirect (in which case it exerts its influence by other than binding to its target; e.g., by interacting with a regulator of the target, so that level or activity of the target is altered).

[0034] Antibody’. As used herein, the term “antibody” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses a polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. For example, in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the referenceAttorney Docket No.: 2013260-0047CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent in or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art to correspond to CDRsl, 2, and 3 of an antibody variable domain; in some such embodiments, an antibody agent in or comprises a polypeptide or set of polypeptides whose amino acid sequence(s) together include structural elements recognized by those skilled in the art to correspond to both heavy chain and light chain variable region CDRs, e.g., heavy chain CDRs 1, 2, and / or 3 and light chain CDRs 1, 2, and / or 3. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent may be or comprise a polyclonal antibody preparation. In some embodiments, an antibody agent may be or comprise a monoclonal antibody preparation. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a particular organism, such as a camel, human, mouse, primate, rabbit, rat; in many embodiments, an antibody agent may include one or more constant region sequences that are characteristic of a human. In some embodiments, an antibody agent may include one or more sequence elements that would be recognized by one skilledAttorney Docket No.: 2013260-0047 in the art as a humanized sequence, a primatized sequence, a chimeric sequence, etc. In some embodiments, an antibody agent may be a canonical antibody (e.g., may comprise two heavy chains and two light chains). In some embodiments, an antibody agent may be in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multispecific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™ ); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®;Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc. ].

[0035] Approximately: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 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 stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0036] Associated: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of, stage of, etc the disease, disorder, or condition (e.g., across a relevantAttorney Docket No.: 2013260-0047 population). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0037] Biologically active: as used herein, refers to an observable biological effect or result achieved by an agent or entity of interest. For example, in some embodiments, a specific binding interaction is a biological activity. In some embodiments, modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity. In some embodiments, presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.

[0038] Cancer-. The terms "cancer", “malignancy”, "neoplasm", "tumor", and "carcinoma", are used herein to refer to cells that exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a tumor may be or comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. The present disclosure specifically identifies certain cancers to which its teachings may be particularly relevant. In some embodiments, a relevant cancer may be characterized by a solid tumor. In some embodiments, a relevant cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers,Attorney Docket No.: 2013260-0047 breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like.

[0039] Chemotherapeutic Agent-. The term “chemotherapeutic agent”, has used herein has its art-understood meaning referring to one or more pro-apoptotic, cytostatic and / or cytotoxic agents, for example specifically including agents utilized and / or recommended for use in treating one or more diseases, disorders or conditions associated with undesirable cell proliferation. In many embodiments, chemotherapeutic agents are useful in the treatment of cancer. In some embodiments, a chemotherapeutic agent may be or comprise one or more alkylating agents, one or more an thracy clines, one or more cytoskeletal disruptors (e.g. microtubule targeting agents such as taxanes, maytansine and analogs thereof, of), one or more epothilones, one or more histone deacetylase inhibitors HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., that share a relevant anti-proliferative activity). In some particular embodiments, a chemotherapeutic agent may be or comprise one or more of Actinomycin, All-trans retinoic acid, an Auiristatin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Curcumin, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Maytansine and / or analogs thereof (e.g. DM1) Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, a Maytansinoid, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, and combinations thereof. In some embodiments, a chemotherapeutic agent may be utilized in the context of an antibody-drug conjugate. In some embodiments, a chemotherapeutic agent is one found in an antibody-drug conjugate selected from the group consisting of: hLLl -doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLLl-SN-38, hRS7-Pro-2-P-Dox, hMN-14- Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLLl-Pro-2- P-Dox, P4 / D10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glembatumomab vedotin, SAR3419, SAR566658,Attorney Docket No.: 2013260-0047BIIB015, BT062, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG- 5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG- 7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, vorsetuzumab mafodotin, and lorvotuzumab mertansine.

[0040] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0041] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets ofAttorney Docket No.: 2013260-0047 circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0042] Composition: Those skilled in the art will appreciate that the term “composition” may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0043] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. To avoid prolixity, it is also understood that any composition or method described as "comprising" (or which "comprises") one or more named elements or steps also describes the corresponding, more limited composition or method "consisting essentially of" (or which "consists essentially of") the same named elements or steps, meaning that the composition or method includes the named essential elements or steps and may also include additional elements or steps that do not materially affect the basic and novel characteristic(s) of the composition or method. It is also understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes the corresponding, more limited, and closed-ended composition or method "consisting of" (or "consists of") the named elements or steps to the exclusion of any other unnamed element or step. In any composition or method disclosed herein, known or disclosed equivalents of any named essential element or step may be substituted for that element or step.

[0044] Designed: As used herein, the term “designed” refers to an agent (i) whose structure is or was selected by the hand of man; (ii) that is produced by a process requiring the hand of man; and / or (iii) that is distinct from natural substances and other known agents.

[0045] Encapsulated: The term “encapsulated” is used herein to refer to substances that are completely surrounded by another material.

[0046] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to beAttorney Docket No.: 2013260-0047“engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature. For example, in some embodiments of the present invention, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example by substitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0047] Excipient: as used herein, refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect. Suitable pharmaceutical excipients include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0048] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to the generation of any gene product from the nucleic acid sequence. InAttorney Docket No.: 2013260-0047 some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc); (3) translation of an RNA into a polypeptide or protein; and / or (4) post- translational modification of a polypeptide or protein.

[0049] Human : In some embodiments, a human is an embryo, a fetus, an infant, a child, a teenager, an adult, or a senior citizen.

[0050] “Improved, ” “increased” or “reduced”: As used herein, these terms, or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with an agent of interest may be “improved” relative to that obtained with a comparable reference agent. Alternatively or additionally, in some embodiments, an assessed value achieved in a subject or system of interest may be “improved” relative to that obtained in the same subject or system under different conditions (e.g., prior to or after an event such as administration of an agent of interest), or in a different, comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and / or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and / or prevalence of difference that is required or sufficient to achieve such statistical significance.

[0051] In vitro". The term “in vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0052] In vivo: as used herein refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).Attorney Docket No.: 2013260-0047

[0053] Nanoparticle: As used herein, the term “nanoparticle” refers to a discrete entity of small size, e.g., typically having a longest dimension that is shorter than about 1000 nanometers (nm) and often is shorter than 500 nm, or even 100 nm or less. In many embodiments, a nanoparticle may be characterized by a longest dimension between about 1 nm and about 100 nm, or between about 50 nm to 200nm, or between about 1 pm and about 500 nm, or between about 1 nm and 1000 nm. In many embodiments, a population of nanoparticles is characterized by an average size (e.g., longest dimension) that is below about 1000 nm, about 750 nm, about 500 nm, about 200 nm, about 100 nm, about 50 nm, about 40 nm, about 30 nm, about 20 nm, or about 10 nm and often above about 1 nm. In many embodiments, a nanoparticle may be substantially spherical (e.g., so that its longest dimension may be its diameter). In some embodiments, a nanoparticle has a diameter of less than 100 nm as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they comprise an enclosed compartment, separated from the bulk solution by a micellar membrane, typically comprised of amphiphilic entities which surround and enclose a space or compartment (e.g., to define a lumen). In some embodiments, a micellar membrane is comprised of at least one polymer, such as for example a biocompatible and / or biodegradable polymer.

[0054] Nucleic acid As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, "nucleic acid" refers to an individual nucleic acid residue (e.g., a nucleotide and / or nucleoside); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, a "nucleic acid" is or comprises RNA; in some embodiments, a "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively orAttorney Docket No.: 2013260-0047 additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl- uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5- propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or protein. In some embodiments, a nucleic acid includes one or more introns. In some embodiments, nucleic acids are prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template in vivo or in vitro), reproduction in a recombinant cell or system, and chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long. In some embodiments, a nucleic acid is partly or wholly single stranded; in some embodiments, a nucleic acid is partly or wholly double stranded. In some embodiments a nucleic acid has a nucleotide sequence comprising at least one element that encodes, or is the complement of a sequence that encodes, a polypeptide. In some embodiments, a nucleic acid has enzymatic activity.

[0055] Payload: In general, the term “payload”, as used herein, refers to an agent that may be delivered or transported by association with another entity. In some embodiments, such association may be or include a covalent linkage; in some embodiments such association may be or include non-covalent interaction(s). In some embodiments, association may be direct; in some embodiments, association may be indirect. The termAttorney Docket No.: 2013260-0047“payload” is not limited to a particular chemical identity or type; for example, in some embodiments, a payload may be or comprise, for example, an entity of any chemical class including, for example, a lipid, a metal, a nucleic acid, a polypeptide, a saccharide (e.g., a polysaccharide), small molecule, or a combination or complex thereof. In some embodiments, a payload may be or comprise a biological modifier, a detectable agent (e.g., a dye, a fluorophore, a radiolabel, etc.), a detecting agent, a nutrient, a therapeutic agent, etc., or a combination thereof. In some embodiments, a payload may be or comprise a cell or organism, or a fraction, extract, or component thereof. In some embodiments, a pay load may be or comprise a natural product in that it is found in and / or is obtained from nature; alternatively or additionally, in some embodiments, the term may be used to refer to one or more entities that is man-made in that it is designed, engineered, and / or produced through action of the hand of man and / or is not found in nature. In some embodiments, an pay load may be or comprise an agent in isolated or pure form; in some embodiments, such agent may be in crude form.

[0056] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or nonaqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.Attorney Docket No.: 2013260-0047

[0057] Reference: As used herein describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0058] Subject: As used herein, the term “subject” refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms). In some embodiments, a subject is suffering from a relevant disease, disorder or condition. In some embodiments, a subject is susceptible to a disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0059] Suffering from’. An individual who is “suffering from” a disease, disorder, and / or condition displays one or more symptoms of a disease, disorder, and / or condition and / or has been diagnosed with the disease, disorder, or condition.

[0060] Therapeutic agent: As used herein, the phrase “therapeutic agent” refers to an agent that, when administered to a subject, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.Attorney Docket No.: 2013260-0047

[0061] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” means an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be appreciated by those of ordinary skill in this art, the effective amount of a substance may vary depending on such factors as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the effective amount of compound in a formulation to treat a disease, disorder, and / or condition is the amount that alleviates, ameliorates, relieves, inhibits, prevents, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0062] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, treatment may be phrophylactic; for example may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition and / or for delaying onset or decreasing rate of development or worsening of one or more features of a disease, disorder and / or condition.Detailed Description of Certain EmbodimentsLipid Nanoparticles

[0063] In some embodiments, the present disclosure provides lipid nanoparticles (LNP). The present disclosure provides, at least in part, the discovery that surface functionalization of a lipid nanoparticle enhances association of a LNP with particular tissues. In some embodiments, the present disclosure provides functionalized lipidAttorney Docket No.: 2013260-0047 nanoparticles which exhibit increased delivery of a payload to specific tissues relative to other tissues. In some embodiments, the present disclosure provides functionalized lipid nanoparticles which exhibit tropism for specific tissues relative to other tissues. In some embodiments, the present disclosure provides functionalized lipid nanoparticles which exhibit increased delivery of a payload to specific cells relative to other cells. In some embodiments, the present disclosure provides functionalized lipid nanoparticles which exhibit tropism for specific tissues relative to other tissues.

[0064] In some embodiments, the present disclosure provides functionalized LNPs comprising an ionizable lipid. In some embodiments, the present disclosure provides functionalized LNPs comprising at least one cationic lipid. In some embodiments, the present disclosure provides functionalized LNPs comprising at least one cationic ionizable lipid. In some embodiments, the term “cationic ionizable lipid” refers to lipid and lipid- like molecules with nitrogen atoms that can acquire charge (pKa). In some embodiments, a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of 5, 6, 7, 8, 9, 10, 11 at physiological pH. In some embodiments a cationic ionizable lipid comprises one or more groups which is protonated at physiological pH but deprotonates and has no charge at a pH above 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments a cationic ionizable lipid comprises one or more groups which are protonated and have a charge at a pH above 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the ionizable cationic group may contain one or more protonatable amines which are able to form a cationic group at physiological pH.

[0065] Specifically, in some embodiments, a cationic ionizable lipid has a high pKA. In some embodiments a high pKa is a pKa greater than 7. In some embodiments a high pKa is a pKa greater than 7.4. In some embodiments, a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of between 7 and 8, 7.5 and 8.5, 8 and 9, 8.5 and 9.5, 9 and 10. In some embodiments, a cationic ionizable lipid for use in accordance with the present disclosure has a pKa of between 7.2 and 8.2, 7.4 and 8.4, 7.6 and 8.6, 7.8 and 8.8, 8.0 to 9.0, 8.2 to 9.2, 8.4 to 9.4, 8.6 to 9.6, 8.8 to 9.8, 9.0 to 10.0. In some embodiments, a useful cationic ionizable lipid has a pKa of 7. 1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7,7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, or 10.0. . This use and efficacy of cationic lipids in lipid nanoparticles as described herein is particularly surprising in light of the understanding in the field that pKas >7 are notAttorney Docket No.: 2013260-0047 optimum for LNP payload delivery. See Jayaraman et al., Angew. Chem. Int. Ed. 2012, 51, 8529 -8533. Indeed, Jayaraman et al states that LNP potency rapidly decreases if the pKa is outside of the range of 6.2 to 6.5. Further, with respect to intramuscular administration of LNP Hassett et al., Molecular Therapy: Nucleic Acids Vol. 15 April 2019 states that a lipid pKa range of 6.6-6.9 is optimal.

[0066] In some embodiments, a cationic ionizable lipid with a high pKA (e.g., >7) is disclosed or described in WO2020219876; US20210162053; US20160317676; and WO2021113365 each of which is incorporated herein in their entirety. In some embodiments, a cationic ionizable lipid with a high pKA (e.g., >7 is one selected from those listed in Table 1.Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047

[0067] Among other things, the present disclosure demonstrates that use of a high pKa cationically ionizable lipid in functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to particular tissues or cells. In some embodiments, functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to tissues other than the liver (i.e., extrahepatic delivery). In some embodiments, functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to immune tissue. In some embodiments, functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to immune cells. In some embodiments, functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to B cells. In some embodiments, functionalized LNPs as described herein may be particularly useful to achieve delivery of nucleic acids to T cells.

[0068] In some embodiments the present disclosure provides functionalized LNPs comprising a at least first cationic ionizable lipid and a second cationic ionizable lipid. In some such embodiments, more than one (e.g., each) of such at least first and second cationic ionizable lipids has a high pKa (e.g., greater than , e.g., greater than 7.4) as described herein. Alternatively, in some such embodiments, only one (i.e., a first) cationic ionizable lipid has such a high pKa (e.g., greater than 7, e.g., greater than 7.4) as described herein. In some embodiments, a second cationic ionizable lipid is a non-high-pKa lipid, e.g., in that it has a pKa below 7, e.g., about 6.8, 6.6, 6.4, 6.2, 6.0, 5.8, 5.6, 5.4, 5.2, 5.0 or lower. In some embodiments, a non-high-pKa lipid (e.g., a second lipid) has a pKa of about 6.4 or lower. In some embodiments, a non-high-pKa lipid (e.g., a second lipid) has a pKa of about 6.4. In some embodiments, a non-high-pKa lipid (e.g., a second lipid) has a neutral pKa.

[0069] In some embodiments, a cationic ionizable lipid with a non-high pKa (e.g., <7) is disclosed or described in Finn et al., 2018 Cell Reports 22, 2227-2235; Jayaraman et al., Angew. Chem. Int. Ed. 2012, 51, 8529 -8533; Hassett et al., Molecular Therapy: Nucleic Acids Vol. 15 April 2019; W02015074085; W02020118041; W02020072605; WO2020252589; WO2021055849; WO2018232120; W0202103070I; W02020146805; W02019036000; W02018200943; and WO2018191657 each of which is incorporated herein in their entirety. In some embodiments, a cationic ionizable lipid with a non-high pKA (e.g., <7) is one selected from those listed in Table 4Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047PCT / US2024 / 036593, the entire contents of which is incorporated herein by reference.

[0071] In some embodiments, a functionalized LNP comprises a lipid selected fromTables 5, 6 and / or 7.Table 5. Exemplary Amino lipidsAttorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Table 6. Additional Exemplary Amino lipidsAttorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047Table 7. Additional Exemplary Amino lipidsAttorney Docket No.: 2013260-0047Attorney Docket No.: 2013260-0047

[0072] In some embodiments, a functionalized LNP comprises about 0-80 mol % of high pKa cationic ionizable lipid as described herein; in some embodiments, a functionalized LNP comprises two or more high pKa cationic ionizable lipids that, together make up such 0-80 mol % of the functionalized LNP. In some embodiments, a functionalized LNP comprises about 20-80 mol % of high pKa cationic ionizable lipid. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 7-, 75, 80 mol % of high pKa cationic ionizable lipid.

[0073] In some embodiments, a functionalized LNP comprises about 0-60 mol % of cationic ionizable lipid that is not high pKa (e.g., that is characterized by a pKa below aboutAttorney Docket No.: 2013260-00477, such as a pKa of about 6.4 or a neutral pKa); in some embodiments, a functionalized LNP comprises two or more non-high pKa cationic ionizable lipids that, together, make up such 0-60 mol % of the functionalized LNP. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mol % of a non-high-pKa cationic ionizable lipid.

[0074] In some embodiments, the present disclosure provides functionalized LNPs comprising at least one sterol. In some embodiments, a functionalized LNP comprises about 0-40 mol% of a sterol. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20, 25, 30, 35, 40 mol% of a sterol.

[0075] In some embodiments, a sterol is cholesterol, or a variant or derivative thereof. In some embodiments, a cholesterol is modified, for example oxidized. Unmodified cholesterol can be acted upon by enzymes to form variants that are side-chain or ring oxidized. In some embodiments, a cholesterol can be oxidized on the beta-ring structure or on the hydrocarbon tail structure. Exemplary cholesterols that are considered for use in the disclosed functionalized LNPs include but are not limited to 25 -hydroxy cholesterol (25- OH), 20a-hydroxycholesterol (20a-OH), 27 -hydroxy cholesterol, 6-keto-5a- hydroxycholesterol, 7-ketocholesterol, 7 |3-hydroxy cholesterol, 7 a-hydroxy cholesterol, 7[3- 25-dihydroxycholesterol, beta-sitosterol, stigmasterol, brassicasterol, campesterol, or combinations thereof. In some embodiments, side-chain oxidized cholesterol can enhance cargo delivery relative to other cholesterol variants. In some embodiments, a cholesterol is an unmodified cholesterol.

[0076] In some embodiments, the present disclosure provides functionalized LNPs comprising at least one helper lipid. In some embodiments a helper lipid is a phospholipid. In some embodiments, a functionalized LNP comprises about 0-20 mol% of a helper lipid. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20 mol% of a helper lipid.

[0077] Exemplary phospholipids include but are not limited to 1 ,2-distearoyL snglycero-3 -phosphocholine (DSPC), 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn- glycerophosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-Attorney Docket No.: 2013260-0047 dipalmitoyl-sn-glycero-3 -phosphocholine (DPPC) , 1 ,2-diundecanoyl-sn- glycerophosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-0-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoy l-sn-glycero-3 -phosphocholine (OChemsPC), 1-hexadecyl snglycero-3 -phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,1.2-diarachidonoyl-sn-glycero-3 -phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2- diarachidonoyl-sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3- phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), l-stearoyl-2-oleoyl-phosphatidy ethanolamine (SOPE), l-stearoyl-2 oleoylphosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), or combinations thereof. In some embodiments, a phospholipid is DSPC. In some embodiments, a phospholipid is DMPC.

[0078] In some embodiments, a phospholipid comprises l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-(succinyl) (succinyl PE), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), cholesterol, l,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) (succinyl-DPPE),1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or a combination thereof.

[0079] In some embodiments, the present disclosure provides functionalized LNPs comprising at least one PEGylated lipid. In some embodiments, a functionalized LNP comprises about 0-25 mol% of a PEGylated lipid. In some embodiments, a functionalized LNP comprises about 0, 5, 10, 15, 20, 25 mol% of a PEGylated lipid.Attorney Docket No.: 2013260-0047

[0080] In some embodiments, inclusion of a PEGylated lipid can be used to enhance lipid nanoparticle colloidal stability in vitro and circulation time in vivo. In some embodiments, PEGylation is reversible in that the PEG moiety is gradually released in blood circulation. Exemplary PEGylated- lipids include but are not limited to PEG conjugated to saturated or unsaturated alkyl chains having a length of C6-C20, PEG-modified-28- phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides (PEG-CER), PEG-modified dialkylamines, PEG-modified diacylglycerols (PEG-DAG), PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEGylated-lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DSG or a PEGDSPE lipid.

[0081] In some embodiments, the present disclosure provides functionalized LNPs comprising at least one PEG-ligand. In some embodiments, a functionalized LNP comprises about 0-2 mol% of a PEG-ligand. In some embodiments, a functionalized LNP comprises about 0, 0.01, 0.05, 0.1, 0.5, 0.75, 1, 1.5, 1.75, 2 mol% of a PEG-ligand. In some embodiments, a ligand (e.g., as included in a PEG-ligand) is or comprises: antibodies targeting cell surface proteins, hyaluronic acid, small molecules, peptides, and / or peptides that target integrins.Functionalized LNPs

[0082] The present disclosure provide functionalized LNPs directed to particular tissues. In some embodiments, functionalized LNPs of the present disclosure are directed to particular tissues by functionalization of the LNPs.

[0083] In some embodiments, LNPs of the present disclosure are functionalized by conjugation of a targeting entity directly to the surface of an LNP described herein.

[0084] In some embodiments, a targeting entity is an agent. In some embodiments, a targeting entity is an antibody agent. In some embodiments an antibody agent is directly conjugated to an LNP. In some embodiments, an antibody agent is or comprises an antibody or antibody fragment that binds or targets a specific antigen expressed by and / or present on a desired cell type or tissue.Attorney Docket No.: 2013260-0047

[0085] In some embodiments, a targeting entity comprises a primary antibody agent and a secondary antibody agent. In some embodiments a secondary antibody agent binds an Fc region. In some embodiments, a secondary antibody agent is or comprises an anti-human Fc, anti-mouse Fc, anti-rabbit Fc, or anti-hamster Fc antibody or antibody fragment. In some embodiments, a secondary antibody agent is or comprises, RG7 / 1.30 for Fc domain of Rat IgG2a, RG7 / 11.1 for Fc domain of Rat IgG2b. In some embodiments a primary antibody or fragment is an antibody or fragment that binds or targets a specific antigen of the desired cell type or tissue. In some embodiments, a primary antibody or fragment binds or targets a B cell surface receptor. In some embodiments, a primary antibody or fragment binds or targets CD19, CD79b, CD20, CD23, CD38, or CD138.

[0086] In some embodiments, antibody agents are modified by DTT, TCEP, SATA ((N-succinimidyl S -acetylthioacetate), SATP (N-succinimidyl-S-acetylthiopropionate), NHS-PEG4-DBCO, NHS-PEG4-Azide, or NHS-PEG4-ester to expose functional groups such as sulfhydryl, DBCO, and Azide groups. In some embodiments, an antibody agent contains 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 functional groups.Target Tissues

[0087] In some embodiments, functionalized LNPs of the present disclosure target particular tissues or cells. In some embodiments, functionalized LNPs of the present disclosure target immune tissues and / or immune cells. In some embodiments, a functionalized LNP of the present disclosure targets a particular tissue or cell based on a targeting entity. In some embodiments a targeting entity binds a particular tissue or cell.

[0088] In some embodiments, an immune tissue is spleen, bone marrow, thymus. In some embodiments, an immune cell is a B-cell or a T-cell. In some embodiments, a targeting entity binds a B-cell. In some embodiments, a targeting entity binds a T-cell. In some embodiments a targeting entity binds or targets a B-cell surface receptor. In some embodiments a targeting entity binds or targets a T-cell surface receptor. In some embodiments, a targeting entity binds or targets CD19, CD79b CD20, CD23, CD38, or CD138. In some embodiments, a targeting entity binds or targets CD3, CD62L, CD197, CD45.Attorney Docket No.: 2013260-0047Payload

[0089] In some embodiments, functionalized LNPs of the present disclosure comprises a payload. In some embodiments, functionalized LNPs of the present disclosure encapuslate a payload. In some embodiments a payload is an agent

[0090] In some embodiments, a payload is a nucleic acid. Exemplary nucleic acids may be or include deoxyribonucleic acid (DNA), ribonucleic acid (RNA) RNA, analogs, and / or combinations thereof. For example, in some embodiments, a nucleic acid may be or comprise single-stranded RNA, single-stranded DNA, double-stranded RNA, double stranded DNA, triple-stranded DNA, siRNA, shRNA, sgRNA, mRNA, miRNA, and / or antisense DNA. In some embodiments, a nucleic acid may include one or more non-natural residues as is known in the art.

[0091] In some embodiments, a nucleic acid comprises a nucleic acid sequence that encodes a therapeutic. In some embodiments, a nucleic acid comprises a nucleic acid sequence that encodes a chimeric antigen receptor (CAR). Such CARs are known in the art (see, e.g., Gill et al., Immunol. Rev. 263:68-89 (2015); Stauss et al., Curr. Opin. Pharmacol. 24: 113-118 (2015)). In some embodiments, a nucleic acid comprises a nucleic acid sequence that encodes a CAR that binds CD19 (e.g., a CAR19). In some embodiments, a nucleic acid comprises a nucleic acid sequence that encodes a CAR that binds BCMA.

[0092] In some embodiments, a nucleic acid (e.g., an RNA) may be sequence engineered, for example to remove immunogenic sequence motifs. In some embodiments, a nucleic acid is sequence engineered to remove TLR7 or TLR8 stimulation motifs. In some embodiments, a nucleic acid is sequence engineered to remove motifs selected from the group consisting of KNUNDK motifs, UCW motifs, UNU motifs, UWN motifs, USU motifs, KWUNDK motifs, KNUWDK motifs, UNUNDK motifs, KNUNUK motifs, and combinations thereof. In some embodiments, a nucleic acid is sequence engineered as described in W02020 / 033720 the entirety of which is incorporated herein by reference.

[0093] In some embodiments a payload is a therapeutic or detection agent. In some embodiments, a detection agent is a visual marker (e.g., colormetric or fluorescent). In some embodiments, a detection agent is a surface expressed protein (e.g., Thy 1.1).Attorney Docket No.: 2013260-0047Uses

[0094] In some embodiments, functionalized LNPs of the present disclosure target or attach to specific tissues. In some embodiments, the present disclosure recognizes that inclusion of a targeting entity on the surface of an LNP results in more effective targeting of LNPs and thus delivery of a payload to specific tissues or cells. In some embodiments, functionalized LNPs as described herein preferentially target tissues other than liver tissues (i.e., extrahepatic). In some embodiments, functionalized LNPs as described herein preferentially target immune tissue or immune cells relative to other tissues. In some embodiments, functionalized LNPs as described herein preferentially target B cells or T cells, e.g., relative to liver tissue or cells.

[0095] In some embodiments, provided functionalized LNPs achieve extrahepatic delivery at a level that is significantly higher than that observed with an appropriate reference LNP. In some embodiments, provided functionalized LNPs achieve immune tissue and / or immune cell delivery at a level that is significantly higher than that observed with an appropriate reference LNP.

[0096] In some embodiments, provided functionalized LNPs achieve preferential extrahepatic, immune tissue and / or immune cell delivery relative to liver delivery to a degree greater than that observed with an appropriate reference LNP. For example, in some embodiments, an RNA delivered with a provided functionalized LNP is preferentially expressed in immune tissue and / or immune cells relative to liver at ratios that may be 5, 10, 20, 30, 40 , 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 or more greater than those observed with a reference LNP. In some embodiments, whole body:liver; tumorliver, spleemliver, B- celhliver, T-cell:liver, B-cell: T-cell, B-cell or T-cell / myeloid cell expression ratio is 1, 2, 3, or 4 orders of magnitude greater for a provided functionalized LNP than for a relevant reference LNP.

[0097] In some embodiments, provided functionalized LNPs achieve whole body, immune tissue, and / or immune cell delivery to a level that is as least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of its level of liver delivery. In some embodiments, provided functionalized LNPs achieve B-Attorney Docket No.: 2013260-0047 cell or T-cell delivery to a level that is as least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of its level of myeloid cell delivery. In some embodiments, provided functionalized LNPs achieve immune tissue, and / or immune cell delivery that is reasonably comparable to its liver delivery. In some embodiments, provided functionalized LNPs achieve preferential whole body, immune tissue, or immune cell delivery relative to liver delivery. In some embodiments, provided functionalized LNPs achieve preferential whole body, immune tissue, or immune cell delivery relative to myeloid cell delivery. In some embodiments, provided functionalized LNPs achieve whole body, immune tissue, or immune cell delivery at a level that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold or more that of its level of liver delivery. In some embodiments, provided functionalized LNPs achieve whole body, immune tissue, or immune cell delivery at a level that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 fold or more that of its level of myeloid cell delivery.

[0098] In some embodiments, provided functionalized LNPs achieve both material levels of whole immune tissue and / or immune cell expression and significant preference for delivery to immune tissue and / or immune cell e.g., relative to liver (i.e., extrahepatic). In some embodiments, functionalized LNPs as described herein delivering nucleic acids to extrahepatic tissues (e.g., immune tissues or immune cells) can be selected from one of the following compositions:Attorney Docket No.: 2013260-0047

[0099] In some embodiments, functionalized LNPs as described herein can be used to treat cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents for the treatment of cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids for the treatment of cancer. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to cancers of immune tissue and / or immune cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to cancers of immune tissue and / or immune cells.

[0100] In some embodiments, functionalized LNPs as described herein can be used to treat diseases of T-cells. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to T-cells cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to T-cells cells.

[0101] In some embodiments, functionalized LNPs as described herein can be used to treat diseases of B cells. In some embodiments, functionalized LNPs as described herein can be used to deliver therapeutic agents to B cells. In some embodiments, functionalized LNPs as described herein can be used to deliver nucleic acids to B cells.Attorney Docket No.: 2013260-0047

[0102] In some embodiments, disclosed functionalized LNPs can be used to treat a subject suffering from hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non- Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, breast cancer, gastro-intestinal cancers and nervous system cancers, benign lesions such as papillomas. In some embodiments, disclosed functionalized LNPs can be used to treat a subject suffering from autoimmune disorders, including but not limited to rheumatoid arthritis, multiple sclerosis, type I diabetes, Addison disease, celiac disease, dermatomyositis, Graves disease, Hashimoto thyroiditis, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, and systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis.

[0103] In some embodiments, functionalized LNPs used to treat a subject suffering from a disease can be administered as combination therapy. In some embodiments, a functionalized LNP used to treat a subject suffering from a disease can be administered to a subject in combination with other methods of treatment (e.g., standard of care treatment) for the disease.

[0104] In some embodiments, functionalized LNPs of the present disclosure are evaluated for potency, selectivity, and / or tolerability. In some embodiments, potency is measured by level of RNA expression in a specific targeted tissue (e.g., T-cell, B-cell). In some embodiments, selectivity is measured by ratio of RNA expression in a specific targeted tissue relative to another tissue or a control tissue (e.g., immune tissue or cells / liver). In some embodiments, potency and selectivity are evaluated through in vivo and / or ex vivo measurements of RNA expression. In some embodiments, potency and selectivity are evaluated through in vivo and / or ex vivo measurements of expression of the protein encoded by an RNA.

[0105] In some embodiments, tolerability is evaluated with respect to common markers of immunogenicity, complement activation, and liver toxicity. These markers mayAttorney Docket No.: 2013260-0047 include, but are not limited to, IFNg, IFNa, IL- lb, IL-6, MCP-1, TNFa, IP- 10, sC5b-9, C3a, ALT, and AST.Methods of Manufacture

[0106] Methods of manufacturing lipid nanoparticles are known in the art. In one embodiment, the disclosed lipid nanoparticles are manufactured using microfluidics. For exemplary methods of using microfluidics to form lipid nanoparticles, see Leung, A.K.K, et al., J Phys Chem, 116:18440-18450 (2012), Chen, D„ et al., J Am Chem Soc, 134:6947- 6951 (2012), and Belliveau, N.M., et al., Molecular Therapy- Nucleic Acids, 1: e37 (2012). Briefly, the payload, such as a nucleic acid, is prepared in one buffer. The other lipid nanoparticle components (e.g., an ionizable lipid; a sterol; a helper lipid; and a PEGylated lipid) are prepared in another (e.g., a second) buffer. A syringe pump introduces the two solutions into a microfluidic device. The two solutions come into contact within the microfluidic device to form lipid nanoparticles encapsulating the cargo.

[0107] In some embodiments, a functionalized LNP of the present disclosure is provided in a pharmaceutical composition. In some embodiments, the present disclosure provides a pharmaceutical composition comprising a functionalized LNP as described herein (e.g., a functionalized LNP comprising a nucleic acid) and excipients or accessory ingredients. Pharmaceutical compositions, LNP formulations and method of administration of LNPs are known in the art. Some formulations and methods of administration are described in, for example, WO2012135805 and WO201711286 each of which are incorporated herein in their entirety. In addition, techniques for formulation and administration of LNPs may be found in “ Remington ' s Pharmaceutical Sciences" Mack Publishing Co, Easton , Pa .latest edition .

[0108] In some embodiments, antibody agents are conjugated to an LNP (e.g., directly conjugated to an LNP toe make a functionalized LNP). In some embodiments, prior to interacting with an LNP, an antibody agent is modified to expose the functional groups in a buffer for 1, 2, 4, and 24 h hrs. In some embodiments, antibody agents are purified by desalting or dialysis. In some embodiments, LNPs are then incubated with antibody orAttorney Docket No.: 2013260-0047 antibody fragments in the buffer for 2, 4, or 24 h. In some embodiments, unconjugated antibody agents are removed by gel fdtration.

[0109] In some embodiments, a method for manufacturing functionalized LNPs is optimized to achieve the best targeting efficiency. In some embodiments, adjusted parameters include but are not limited to the ratio between the lipids in the lipid composition of the LNP core, the number of the functional groups per antibody agent, , the time length of various steps, the purification process, or any combination thereof.ExemplificationExample 1: Functionalized Lipid Nanoparticle Delivery of RNA to T-Cells

[0110] Blood cancer has an estimated global incidence of 6% amongst all cancer types, and approximately 1.24M people are diagnosed per year (2020 data). In the US, it is estimated that 186,000 new blood cancer diagnoses are made each year. CAR T therapies have shown remarkable efficacy for the treatment of B-cell malignancies, achieving remission rates ranging from 60 to 93%. A major current limitation is the high cost of therapy, estimated at $300K-$500K per patient, and the fact that only 2% of US hospitals are equipped to administer CAR therapy. CAR T manufacturing typically takes 9-14 days and the vein-to-vein time ranges from 3 to 5 weeks: an unacceptable timeframe for patients with rapidly progressing cancers. In addition, treatment with genetically modified and permanently CAR-expressing T cells results in the depletion of malignant and normal B cells and is associated with hypogammaglobulinemia. On-target, off- tumor toxicities present an increased risk of infection. Unfortunately, infections remain a major risk factor for mortality after CAR T-cell infusion. B-cell aplasia often persists for over 6 months, extending to years. Recent developments in RNA technology allow non-viral CAR therapies to benefit from the transitory expression of CARs in T cells and circumvent numerous potential risks of viral vector transduction, while also offering significant cost advantages over cell-based and ex vivo therapies. The shift to RNA CAR T cell therapy provides a major opportunity to reduce therapy cost as RNA manufacturing has significantly lower COGS than traditional CAR T cell therapies.Attorney Docket No.: 2013260-0047

[0111] While CAR T therapies are presently only approved for treatment of heme malignancies, this therapeutic modality has also demonstrated great value in the treatment of autoimmune disorders. Autoreactive B cells are intricately involved in the pathogenesis of many autoimmune diseases, hence CAR T therapy directed against circulating B cells can also address autoimmune diseases through rapid depletion of autoreactive B cells. Recent clinical evidence suggests that transient deep depletion of B-cells not only enables disease remission but can also allow for the recovery of B-cell numbers without reappearance of autoreactive B cells. CAR T therapy for autoimmune diseases has shown signs of safety and efficacy in early-stage clinical trials for systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis, myasthenia gravis (NCT04146051), and MS, with more clinical trials ongoing (NCT06428188, NCT06420154, NCT06056921, NCT06373081, NCT06350110, NCT05085431, NCT05859997). In these trials, CAR T cells are targeted toward CD 19 or BCMA cell surface markers on healthy and self-reactive B cells / plasma cells.

[0112] Despite existing limitations within the first five generations of CAR T therapy, the field is rapidly expanding, and the market shows a growth trend congruent with rapid scientific and clinical advancements (more than 900 clinical trials are underway globally), also driven by a healthy demand for this kind of immunotherapy. CAGR is estimated at 29.8% and it is projected that the global CAR T-cell therapy market will reach USD 88.5 billion by 2032. Great efforts are being made to fine-tune efficacy, attenuate CAR T-cell toxicity, and drive down cost of treatment.

[0113] The present example describes T cell-targeting lipid nanoparticles (T-mAb- LNPs). The present example demonstrates robust lipid nanoparticles with conjugated antibody agents that enable protein transfection (i.e., RNA encoding a protein delivery) to T cells in human PBMCs in vitro, avoiding both B and myeloid cells. Furthermore, in murine models, functional CAR expression, translated from a RNA payload, in splenic T cells show a substantial reduction of B cells present in the spleen after intravenous administration. T- mAb-LNPs have great potential towards lowering both the COGS and time to administration, while improving therapeutic success of CAR T therapy.

[0114] Materials. LIP003 (Lipid 5) was purchased from DC Chemicals (Shanghai, China). LIP091 was synthesized in house. Cholesterol was purchased from Millipore SigmaAttorney Docket No.: 2013260-0047(Burlington, USA). DSPC, PEG-DMG, Maleimide-PEG-DSPE were purchased from Avanti Polar lipids Inc. (Alabaster, USA). DTT and EDTA were purchased from Thermo Scientific (Waltham, USA). DiD was purchased from Invitrogen (Waltham, USA). Mouse IgGl (ISOtype Control, Clone MOPC-21), Mouse IgG2a (aCD3, Clone 0KT3) and Mouse IgGl (aCD4, Clone RPA-T4) were purchased from Bio X Cell (Lebanon, USA). Mouse lgG2A (aCD5, Clone 205919), Mouse IgG2b (aCD8 alpha, Clone 37006), and Mouse IgG2a (aCD8 alpha, Clone 1033123) were purchased from R&D Systems (Minneapolis, USA).

[0115] LNP Core Formulation. Lipids consisting of ionizable lipid, DSPC, cholesterol, PEG-lipid were dissolved in ethanol. RNA was dissolved in 50mM Citrate buffer pH 5.0. The ratio of cationic ionizable amines to RNA phosphates in the LNP was 6. In general, the lipid composition is 50.0 / 27.5 / 22.0 / 0.5 of ionizable lipid / cholesterol / helper lipid / PEG lipid. The organic and aqueous phases were combined at a flow rate of 12 mL / min on a Precision Nanosystems NanoAssemblr at a ratio of 3:1 (aqueous: organic). All LNP core for antibody functionalization also included a lipid dye DiD in the ethanol phase at 0.1 mol% to the total lipids. After mixing, the LNPs were immediately diluted to 16.5% ethanol using MilliQ water. The diluted product was dialyzed using Slide- A-Lyzer 10K MWCO cassettes (ThermoFisher) against a volume of IX PBS that was 300-fold in excess at 4 °C overnight. The dialyzed product was concentrated using Amicon Ultra lOOkDa centrifuge tubes and sterile filtered. Particle size and PDI were determined with Anton Paar Litesizer DLS 500 while RNA concentration and encapsulation were assessed by the Quant- IT Ribogreen assay (Invitrogen).

[0116] Representative Example of Reduction and Purification of Antibodies. Mouse anti-human CD3 (clone OKT3, isotype mouse IgG2a) was reduced in IX PBS containing various concentrations (0.2 mM, 1 mM and 5 mM) of dithiothreitol (DTT) and 5 mM EDTA for different durations (0.5 h, 1 h and 2 h) at room temperature. The reduced RG7 was then purified by buffer exchange to IX PBS with 5 mM EDTA using a 7K Zeba spin desalting column to remove excess DTT. The reduced antibody was quantified by Nanodrop.

[0117] Measurement of Thiols (-SH) per reduced antibody by Ellman 's assay. Two hundred microliters of reduced antibodies and Cysteine Hydrochloride Monohydrate standards in reaction buffer (IX PBS with 5 mM EDTA) were added into the microplate inAttorney Docket No.: 2013260-0047 duplicate. I. To each 200pL sample in the microplate, 20pL of working Ellman’s Reagent (0.5mg / mL in the reaction buffer) was added and mixed well. The absorbance of each well was measured at 405nm by a plate reader. The -SH concentrations of reduced antibodies was quantified against the various known concentrations of Cysteine Hydrochloride Monohydrate standards. The number of -SH per reduced antibody was calculated the ratio of the concentration of -SH to the concentration of reduced antibody.

[0118] Representative Example of SDS-PAGE Analysis of Reduced Antibodies. Rat anti-mouse CD3 (clone KT3, isotype rat IgG2a) was reduced in IX PBS containing various concentrations 1 mM DTT and 5 mM EDTA for Ih at room temperature. The reduced antibodies were heated at 65 °C for 5 min and loaded on a 10-well Mini Protean TGX precast gels 4-15% under non-reducing conditions. The gel was stained with SimplyBlue™ SafeStain.

[0119] Representative Example of ELISA Analysis of Antibody. The resDetect™ Anti-CD3 Antibody ELISA Kit (Aero Biosystems) was used to according to the manufacturer protocol. Briefly, the serial diluted (1.56ng / ml to 6.24 ng / ml) intact and reduced antibodies were added to Human CD3E & CD3G precoated microplate and incubated at 37°C for 1 hour. After washing, the secondary antibody HRP-Anti-Mouse IgG was added to the plate and incubated at 37°C for 1 hour. After washing, the substrate was loaded into the wells and monitor color development in proportion with the amount of antibody present. 'Hie reaction was stopped by the addition of a stop solution and the intensity of the absorbance can be measured at 450 nm and 630 nm. The OD Value was plotted against the antibody concentrations. The EC50 of intact and reduced antibodies was calculated using the Sigmoidal 4PL model in Prism.

[0120] Purifying T-mAb-LNPs From Non-conjugated Antibodies. Targeting antibodies conjugated to LNPs were separated from unconjugated antibodies using Sepharose CL4B beads on a gel filtration column with PBS serving as the mobile phase. Fractions containing pure LNPs were identified by the fluorescent signal of DiD-loaded LNPs and then were pooled together. RNA concentration and encapsulation were assessed by the Invitrogen Ribogreen assay. Antibody concentration on the LNP surface was assessed by the Pierce BCA protein assay kit.Attorney Docket No.: 2013260-0047

[0121] Cell culture and LNP transfection. Human primary blood mononuclear cells(PBMCs, ATCC PCS-800-01 1) were cultured in RPMI-1640 Medium (Gibco) supplemented with 1% Penicillin / Streptomycin (Gibco), and 10% HI-FBS (Gibco). Cells were maintained at 37°C and 5% CO2. For in vitro transfection, PBMCs were plated at a density of 100,000 cells per well in a 96-well U-bottom plate in RPMI-1640 medium. LNPs were then added to the cells at a final concentration of 2, 0.2, and 0.02 ug / mL. Eighteen hours after transfection, cells were washed with lx DPBS three times. Since Thy 1.1 RNA was used, cells were stained with PE-conjugated CD90.1 Antibody, anti-mouse / rat, REAfinity™ (Miltenyi Biotec) in 1:50 dilution in IX PEB (PBS / EDTA / BSA) buffer for 10 min in the dark at 4°C. Then, cells were washed with 1XPEB buffer three times and subjected to flow cytometry analysis.

[0122] General Animal Study Protocol. Protocols were approved by Charles River IACUC. BALB / c, C57BL / 6 and B6 Albino (female, 6-10 weeks) mice were purchased from Charles River Labs. Mice were housed in the Charles River Labs Facility.

[0123] Animal Study: T Cell-Targeting LNPs. Eight-week-old C57 / BL6 mice were injected with DiD dye and Thy 1.1 encoding RNA co-encapsulated T-mAb-LNPs through the tail vein at 0.3 mg / kg. After 18 hours, spleens were harvested, processed into single cells, and subsequently stained with viability dye and surface markers. Dead cells and red blood cells were excluded by viability dye and TER- 119. T-cells, B-cells and myeloid cells were identified as CD3+B220-, B220+CD3-, and CD3-B220-, respectively. N=3 / group.

[0124] Animal Study: Biodistribution of T Cell-Targeting LNPs. Eight- week-old B6-Albino mice were injected with fLuc RNA encapsulated LNPs through tail vein at 0.3 mg / kg. After 18 hours, D-luciferin was injected intraperitoneally at 150 mg / kg. Ten minutes later, the thymuses, lungs, livers, spleens, and lymph nodes were harvested for ex vivo imaging. N=5 / group.

[0125] Animal Study: CAR RNA POC. Eight-week-old C57 / BL6 mice were injected with CD19 CAR-encoding RNA T-mAb-LNPs through the tail vein at either 0.1 or 0.3 mg / kg on Day 0 and Day 2. On Day 6, blood was collected for CBC analysis (IDEXX), and spleens were harvested, processed into single cells, and subsequently stained with viabilityAttorney Docket No.: 2013260-0047 dye and surface markers. Flow cytometry was performed on a Attune CytPix Cytometer (ThermoFisher). N = 5 / group.

[0126] Cell isolation for flow cytometry staining. Spleens were harvested from mice and preserved in MACS® Tissue Storage Solution on ice. Spleens were mechanistically disrupted by pressing then through a 70 pm strainer. Cells were then spun down at 350g for 10 min and washed with 1XDPBS three times. Cells were then plated into a 96-well V- shape plate at 100,000 cells / well. After centrifuging at 350g for 10 min, cells were resuspended in 60 uL diluted Viogreen-Viobility Fixable Dye (Miltenyi Biotec) solution (1 : 100 in 1XPBS) and incubated for 8 minutes in the dark at room temperature. Cells were then stained with 50 uL of predetermined diluted surface markers solution (2 uL per antibody in total 50 uL 1XPEB buffer) for an additional 8 minutes in the dark at room temperature. Cells were then washed three times with 300 uL 1XPEB buffer and resuspended in 200 uL 1XPEB buffer for flow cytometry analysis on Attune CytPix Cytometer (Thermo Fisher).

[0127] Statistics. Means were compared with either one-way ANOVA or two-way ANOVA for comparison between multiple groups. Dunnett’s multiple comparison test was used when comparing to a one certain group. For comparison between two groups, unpaired two-tailed T-test was used. All statistics were performed with GraphPad Prism 10.

[0128] Figure 1 demonstrates mAb reduction condition optimization and characterization of reduced mAb. Figure 1A) Ellman’s assay demonstrates the current reaction condition (ImM DTT for 1 hr at r.t.) is optimal to expose ~ eight thiol groups on each antibody, likely attributed to the cleavage of four inter-chain disulfide bonds. Figure 1A B) SDS-PAGE shows the optimal reaction condition mostly generates bands at 75 kDa fitting to the heavy-light chain and reduction of 4 inter-chain disulfides. Individual heavy and light chains are also observed suggesting some break inter-HL disulfides. Figure 1A C) Binding affinities of intact versus reduced antibodies under optimal conditions are comparable, indicating functionality of the antibody is preserved after chemical manipulation.

[0129] Figure 2 demonstrates splenic T-cell targeting efficiency and selectivity of T mAb-LNPs in C57 / BL6 mice. Targeting T cells using T mAb-LNPs resulted in > 90%Attorney Docket No.: 2013260-0047 delivery and protein expression. In stark contrast to the peripheral LNP core, T mAb-LNP A shows significant (365 - fold, p < 0.0001) increase in mean fluorescence intensity (MFI) of protein marker expression in splenic T-cells while largely no difference in that of myeloid and B cells. Selectivity to T cells was found to be as high as 25-fold and 64-fold (p<0.0001) over myeloid and b cells respectively.

[0130] Figure 3 demonstrates biodistribution of various LNP compositions in B6 Albino mice. Modification from a standard formulation to a peripheral composition resulted in a 14-fold decrease in liver expression. T-mAb-LNPs further reduced liver signal by 3-fold compared to the peripheral core. In stark contrast to the peripheral core, T-mAb-LNPs exhibit a 10-fold increase in spleen expression. An increase (2-fold) in thymus expression was also observed.

[0131] Figure 4 demonstrates biodistribution of T-mAb-LNPs with Lipid 5 versus LIP091 peripheral core compositions in B6 Albino mice. T-mAb-LNPs using lipid 5 or LIP091 as the ionizable lipid showed a comparable biodistribution pattern of shifting from livers to spleens compared to their untargeted counterparts.

[0132] Figure 5 demonstrates surface-functionalized T-mAB-LNPs with mAb 2 demonstrate selective T cell targeting over off-target cells in human PBMCs. Human PBMCs were transfected in vitro with 2, 0.2, or 0.02 ug / ml of Thy 1.1 RNA T-mAb-LNPs. T, B, and myeloid cells were analyzed for Thy 1.1 expression by flow cytometry. Geometric mean (Thy 1.1 MFI, A, B, C) for T, B, and myeloid cells are shown, respectively. mAb 2 T- MAB-LNP performed the best with a selectivity of D) 50-fold and E) ~ 130-fold for T cells over both B cells and myeloid cells respectively. By comparison, RNA delivery via Lipofectamine MessengerMAX transfection reagent achieved very low expression in B and T cells.

[0133] Figure 6 demonstrates T-cell targeted CD19 CAR T-mAb 2-LNP uptake in cultured human PBMCs results in T-cell activation. Human PBMCs were treated with 0.02- 2 ug / ml CD 19 CAR T-mAb 2-LNP, control, or CD 19 CAR RNA T-LNP surface- functionalized with antibody isotype control mAb. 24h later cells were collected, stained and analyzed by FACS. A) T-LNP uptake by T, myeloid and B cell subsets as determined by DiD fluorescence (percentage and MFI). B) Activation of CD4+ and CD8+ T cell subsetsAttorney Docket No.: 2013260-0047 based on CD69 staining (percentage and MFI). We observed an 81-94% increase in DiD fluorescence in CD3+ cells treated with 0.2 and 2 pg / ml of the T-LNP, respectively, compared to the isotype control. In comparison, treatment with 2 |ig / ml led to a 10% increase in DiD fluorescence in myeloid (CD1 lb+) cells and a 20% increase in B (CD20+) cells. Changes in DiD fluorescence in these cells after treatment with 0.2 pg / ml were insignificant. In parallel, we noted a 32-47% increase in CD69 expression in CD4+ cells after treatment with 0.2 and 2 pg / ml of the T-LNP, respectively, and a 27-37% increase in CD69 expression in CD8+ cells. As a result, 40-50% of CD4+ and CD8+ cells were activated. * - p < 0.05, **** - p < 0.0001 (Two-Way ANOVA with Tukey's multiple comparisons test).

[0134] Figure 7 demonstrates B cell depletion in mouse peripheral blood after CD 19 CAR T-LNP in situ treatment. C57BL / 6 mice were treated intravenously with 0.3 mg / kg CD 19 CAR T-LNP on day 0 and 2 (8 weeks old females, n=5 / group). Blood was collected on day 6 and analyzed by multi-parameter flow cytometry. CBC analysis was conducted at IDEXX to obtain total counts for CD45+ cells (WBC counts). CD19 CAR T-LNPaCD3-002 and T-LNPaCD3-155 achieved 83% and 81% depletion in B cell counts, respectively. The murine CD 19 CAR RNA sequence was designed based on protein sequence from Kochenderfer et al. (2010 Blood; 116(19):3875-86). Non-translated mouse beta-actin RNA encapsulated into T-LNPaCD3-002 was used as a control. * - p < 0.05 (Brown-Forsythe ANOVA test with Dunnett’s T3 multiple comparisons test), *** - p < 0.001, **** - p < 0.0001 (Ordinary One-Way ANOVA with Dunnett’s multiple comparisons test).References1) Siegel, R. L., Miller, K D., Fuchs, H. E. & Jemal, A. Cancer Statistics, 2021. CA: A Cancer Journal for Clinicians 71 , 7-33, doi:https: / / doi org / 10 3322 / caac 21654:https: / / doi org / 10 3322 / caac 21654 (2021)1) Khan, A N , Asija, S , Pendhari, J & Purwar, R CAR-T cell therapy in hematological malignancies: Where are we now and where are we heading for? European Journal of Haematology 112 6-18, doi:https: / / doi org / 10 1111 / ejh 14076 (2024) European Journal of Haematology 112, 6-18, doi:https: / / doi org / 10 1111 / ejh 14076 (2024)1) Wat, J & Barmettler, S Hypogammaglobulinemia After Chimeric Antigen Receptor (CAR) T-Cell Therapy: Characteristics, Management, and Future Directions. J Allergy Clin Immunol PractW, 460-466, doi:10.1016 / j.jaip.2O21 .10.037 (2022).1) Rurik, J. G. etal. CAR T cells produced in vivo to treat cardiac injury. Science 375, 91-96, doi:10.1126 / science.abm0594 (2022).1) Parayath, N. N. & Stephan, M. T. In Situ Programming of CAR T Cells. Annu Rev Biomed Eng 23, 385-405, doi:10.1146 / annurev-bioeng-070620-033348 (2021 ).1) Schett, G., Mackensen, A. & Mougiakakos, D. CAR T-cell therapy in autoimmune diseases. The Lancet 402, 2034-2044, doi : 10.1016 / S0140-6736(23)01126-1 (2023) .Attorney Docket No.: 2013260-00471) Muller, F. ef al. CD19 CAR T-Cell Therapy in Autoimmune Disease — A Case Series with Follow-up. New England Journal of Medicine 390, 687-700, doi:doi:10.1056 / NEJMoa2308917 (2024).1) Wang, W. ef al. BC A-CD19 compound CAR T cells for systemic lupus erythematosus: a phase 1 open-label clinical trial. Ann Rheum Dis, doi:10.1136 / ard-2024-225785 (2024).1) Granit, V. ef al. Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1 b / 2a study. The Lancet Neurology 22, 578-590, doi : 10.1016 / S 1474-4422(23)00194- 1 (2023) .1) Haghikia, A. ef al. Anti-CD19 CAR T cells for refractory myasthenia gravis. The Lancet Neurology 22, 1104-1105, doi:10.1016 / S1474-4422(23)00375-7 (2023).1) Fischbach, F. et al. CD19-targeted chimeric antigen receptor T cell therapy in two patients with multiple sclerosis. Med 5, 550-558 e552, doi:10 1016 / j.medj.2024.03002 (2024).1) BioSpace. CAR T-Cell Therapy Market is Rising Rapidly at CAGR 29.8% by 2032, <https: / / www.biospace.com / article / car-t- cell-therapy-market-is-rising-rapidly-at-cagr-29-8-percent-by- 2032 / #:~dext=The%20global%20CAR%20T%2Dcell,USD%201.75%20billion%20in%202023> (2024).12 BioSpace. CAR T- Cell Therapy Market is Rising Rapidly at CAGR 29.8% by 2032, <www.biospace.com / article / car-t-cell-therapy-market-is- rising-rapidly-at-cagr-29-8-percent-by- 2032 / #:~:text=The%20global%20CAR%20T%2Dcell,USD%201.75%20billion%20in%202023> (2024).Example 2: Functionalized Lipid Nanoparticle Delivery of RNA to B-Cells

[0135] B lymphocytes possess multifaceted roles that are fundamental to immune function in vivo. At the forefront of these functions is their capacity to produce antibodies and cytokines, a cornerstone in orchestrating immune responses. B-cells exhibit a remarkable ability to generate memory cells, thereby fostering immunological memory and enhancing the body's capacity to mount a rapid and specific defense upon re-exposure to a pathogen. Furthermore, B lymphocytes play a critical role in T cell activation by serving as adept antigen-presenting cells and instigate the cascade of immune responses. Modulating B cell function has recently emerged as a promising avenue for advancing targeted therapeutic strategies in diverse immune-related disorders, ranging from cancer and inflammation to autoimmune conditions.

[0136] It is noteworthy that non-myeloid phagocytic system (MPS) immune cells, including B, T, and natural killer (NK) cells, present a unique challenge in transfection endeavors. This is due in part to their inherently weak phagocytotic capabilities, underscoring the intricacies associated with manipulating these crucial cellular components for therapeutic purposes. Recently, various researchers have made notable efforts in the targeted delivery of therapeutic agents to B-cells, presenting a promising strategy for modulating B cell function. Anderson et al. introduced an ionizable lipidoid, OF-Deg-Lin, featuring ester linkages, achieving efficient transfection of B-cells in the spleen, with 7%Attorney Docket No.: 2013260-0047Cy5 positive splenic B-cells observed at 0.75 mg / kg. Harashima et al. utilized a DODAP / DOPE combination with specific lipid ratios, resulting in nanoparticles of 200 nm size and a zeta potential of <-20 mV, demonstrating a 60% DiD-i- uptake in B-cells. Siegwart and colleagues incorporated a negatively charged lipid (18PA) into their SORT lipid nanoparticles (LNPs), enabling spleen- specific delivery and detecting 12% tdTomato-i- splenic B-cells at 0.3 mg / kg of Cre RNA in Cre-loxP tdTomato reporter mice. Wang et al. developed PEG-b-PEG-based cationic lipid- as sis ted nanoparticles (CLAN) with optimized PEG densities and zeta potentials, revealing Cy5 positive cells in 50% splenic, 70% lymphatic, and 50% bone marrow B-cells at 2 OD (80 pg) Cy5-siRNA / mouse. Furthermore, Anu-Puri explored a CD22-scFv conjugated liposome, demonstrating significantly enhanced binding to BJAB-cells (CD22+) compared to control liposomes.

[0137] The present examples demonstrates surface functionalization of LNPs with the anti-CD79b antibody (mAb) to achieve targeted expression of B cell RNA and proteins in vivo. CD79b, also known as the B-cell antigen receptor complex-associated protein beta chain, is expressed on the surfaces of nearly all B-cells. As a vital component of the B cell receptor, CD79b, in conjunction with CD79a and surface immunoglobulin, forms a heterodimeric signal-transduction unit. CD79b can undergo internalization upon antibody binding, presenting a unique opportunity for selective delivery of molecules of interest to B- cells. The anti-CD79b mAb has previously found application in antibody-drug conjugates designed for the prolonged depletion of proliferating B-cells, offering therapeutic benefits in the treatment of non-Hodgkin's lymphoma (NHL). One notable success in this realm is Polivy®, an FDA-approved anti-CD79b-monomethyl auristatin E (MMAE) conjugate, sanctioned in 2019 for the treatment of diffuse large B-cell lymphoma.

[0138] Diverse formulation screening strategies focusing on the identification of an optimal conjugation chemistry and reporter RNA were evaluated. Lipid composition of core LNPs was then optimized to redirect biodistribution away from hepatic tissues towards other organs, especially the spleen and bone marrow. Meanwhile, an extensive screening process for targeted antibodies ensued, encompassing various targeting antigens, clones, and manufacturers. For each targeted antibody, antibody surface densities were fine tuned by varying the amount of antibody used during the conjugation. The dose-titration of the optimized mAb-LNPs was explored from 0.5 mg / kg to 0. 1 and 0.033 mg / kg to balance theAttorney Docket No.: 2013260-0047 transfection efficiency and selectivity. The lead antibody was then directly conjugated to the LNP core using its optimum antibody surface densities to eliminate the unnecessary secondary mAb (RG7) component in the system. The biodistribution of the B-cell targeted LNP was evaluated using IVIS imaging. Lastly, anti-human B-cell mAbs were conjugated on the LNP to validate the potential for translation. These efforts culminated in the targeted delivery of RNA to over 90% of B-cells in mice and human PMBC, underscoring the potential of RNA as a therapeutic modality for immune cell-related disorders.Results1. Selection of reporter RNA to enable sensitive formulation screening.

[0139] To screen the formulations for optimal B cell delivery in vitro and in vivo, RNA-encoded reporter protein were encapsulated to assess the functional RNA delivery to B-cells in a cell-specific and cost-effective manner. Two common reporter RNAs for indication of functional RNA expression in vitro and in vivo were evaluated. mCherry is a widely used fluorescent reporter protein. However, it is believed to be immunogenic, as its precursor dsRed has one D(b)-restricted peptide sequence (SSLQDGCFI) that acted as an epitope in C57BL / 6 mice. Also, the brightness of mCherry is mild, which is 47 % of eGFP. Thyl.l is a highly conserved glycoprotein that is anchored to the surface of cells. Most mouse strains don’t express Thyl.l except AKR / J and PL strains. Thyl.l protein is reported to not elicit an immune response when injected in mice. Moreover, it can be stained with antibodies with different fluorophores to maximize the signal.

[0140] Isotype control-RG7-Peripheral LNP or aCD19-RG7-Peripheral LNP were tested in the A20 cell line, and protein expression was assessed with flow cytometry 24 h post-transfection. aCD19-RG7-Peripheral LNP showed very high levels of protein expression (>90%) in vitro in both mCherry and Thyl.l settings, while LNPs conjugated with isotype control were not effectively transfecting cells (Figure 8A and B). However, when mAb-LNP were intravenously injected into BALB / c mice, mCherry expression from aCD19-RG7-Peripheral LNP could barely be differentiated from the control (Figure 8C) with 20-30% percentage of positive cells and <100 MFI (Figure 8E). Interestingly, Thyl. l RNA encapsulated aCD19-RG7-Peripheral LNP maintained >60% transfection efficiencyAttorney Docket No.: 2013260-0047 and MFI levels at 10A3. Therefore, Thy 1.1 was selected as the reporter RNA for the following screening efforts.2. Screening targeting mAb to improve the B-cells delivery in vivo

[0141] Besides the initial targeting antibody, anti-CD19 (clone 1D3), we also investigated other targeting antibodies for in vivo B cell delivery. We started from a panel of other CD19 antibodies of different clones (1D3, 6D5, and 6OMP31) from different vendors. Among the aCD19 screened, all three 6D5 antibodies showed higher transfection efficiency in A20 cells than our initial antibody (1D3 from BioXCell at aCD19 / RG7=0.1:l, in circle) while peaking at different aCD19 / RG7 molar ratios (Figure 9A). The top three 6D5 LNPs (Arrows in Figure 3A) with their respective optimal antibody densities were proceeded into the in vivo test, in particular, 6D5 from Biolegend at aCD19a / RG7 =0.1: 1 and 0.01:1, and 6D5 from Southern at aCD19a / RG7=0.01:l. Unexpectedly, they didn’t exceed the initial aCD19 in spleen and bone marrow B-cells in vivo (Figure 9B and C). The discrepancy between in vitro and in vivo mAb targeting efficiency is likely due to the variance of antibody / receptor interactions in a dynamic blood flow system versus a static and overwhelmed cell culture system. Then, we expanded to antibodies targeting other receptors on B-cells such as CD79b, CD38, and CD20 using a similar conjugation and optimization methodology to CD19. Since A20 is a mature B cell line and doesn’t express CD38, which is more prevalent on B cell precursors, germinal center B-cells, and plasma cells, we tested these antibodies in splenocytes ex vivo to cover all receptors targeted by antibodies. Among the second panel screened, only the aCD79b excelled the aCD19 in the transfection efficiency with >70% Thyl.l+ and >90% DiD+ in splenic B-cells in vitro (Figure 10 A and D). All antibody conjugated LNPs showed low levels of protein expression in off-target cells, such as myeloid cells and T-cells (Figure 10 B, C, E and F). Other antibodies might not be suitable for targeted delivery to B-cells due to factors such as the clone of the antibody used or receptor-dependent factors such as expression level, internalization rate, or off-target tissue expression levels that could serve as antigen sinks. Therefore, the aCD79b- RG7 -Peripheral LNP and aCD19-RG7-Peripheral LNP at their optimal targeting antibody density of 1 : 1 and 0.1: 1 of mAb / RG7 molar ratios were selected for in vivo study.Attorney Docket No.: 2013260-0047

[0142] Consistent with the in vitro splenocytes transfection results, aCD79b-LNPs outperformed aCD19-LNPs in transfecting splenic B-cells in vivo (Figure 10 G and J), showing >80% Thy 1.1+ at 0.5 mg / kg. Thus, aCD79b was selected as the lead targeting mAb for the future development. It’ s worth mentioning that, in Figure 10 H, despite a 6-fold lower of MFI than B-cells, all three mAb-conjugated LNPs expressed Thyl. 1 in around 70% splenic myeloid cells, suggesting the compromised selectivity at this dose. In the next study, we tested whether lowering the dose could improve selectivity.3. Lowering the dose of antibody conjugated LNP to improve the selectivity

[0143] To improve the selectivity, we lower the dose 5 -fold and 15 -fold to see whether it will decrease the off-target expression in myeloid cells while keeping the high expression in B-cells in vivo. Meanwhile, in the experiments above ISO-RG7-Peripheral LNP also showed >50% Thy 1.1+ in B-cells. To elucidate the cause for the non-specificity, we added the peripheral LNP core to the comparison to see whether the core itself or ISO mAb caused the unspecific expression in B-cells.

[0144] As shown in Figure 11, aCD79b-RG7-Peripheral LNPs at 0.1 mg / kg still maintained the 70% transfection efficiency in spleen B-cells, which was significantly higher than the core and ISO counterparts at the same concentration (Figure 11A), while reduced the myeloid cell expression to around 50% (Figure 11G). Further decreasing the dose to 0.033 mg / kg reduced the splenic B cell protein expression to < 40%, which might be too low for effective targeting. Thus, 0.1 mg / kg was selected for future formulation screening and development.

[0145] Regarding the cause of unspecific expression of ISO-RG7-Peripheral LNP in B-cells, as shown in Figure 11A, the peripheral LNP core itself was potent in transfecting spleen cells at lower doses of 0.1 mg / kg (~50% Thyl.l+) and 0.033 mg / kg (—15% Thyl.1+%). Moreover, ISO-LNPs showed a similar transfection efficiency as the peripheral LNP core, suggesting the unspecific expression of ISO-LNPs in B-cells was likely due to the peripheral core composition other than the unspecific binding of ISO mAb to B cell receptors.

[0146] In the B cell subpopulations, aCD79b mAb-Peripheral LNP exhibited a higher expression in mature B-cells than developing B-cells, as CD79b has a more prevalentAttorney Docket No.: 2013260-0047 expression on the mature B-cells (Figure 11C and HE). As the bone marrow is composed of 90% immature or developing B-cells and 10% mature B-cells, the overall B cell targeting efficiency of aCD79b-RG7-Peripheral LNPs was lower in the bone marrow than in the spleens (Figure 11 B, D and F).4. Evaluating different LNP core compositions to balance the transfection efficiency and selectivity

[0147] Building upon a sophisticated screening platform, we advanced into combining LNP core composition optimization with targeting antibody screening. The standard LNP composition (ionizable lipid / cholesterol / DSPC / DMG-PEG2K=50 / 38.5 / 10 / 1.5) has been extensively proven to effectively deliver RNA to the liver upon systemic administration, but they showed inferior or no efficacy in extrahepatic tissues and cells. Furthermore, B-cells originate in the bone marrow and activate in the secondary lymphoid organs such as the spleen and lymph nodes, which might not be reached by the standard LNP formulation and therefore require optimized LNP formulation that targets away from the liver. We applied the design of experiments (DOE) methodology on lipid compositions and developed the next generation of LNP that redistributed the protein expression from the liver to spleen and bone marrow, coined here as the “peripheral LNP. We hypothesized that peripheral LNPs functionalized with B cell-targeting mAbs would outperform the standard liver-targeting LNPs.

[0148] In Figure 12A, the aCD79b functionalized on the standard LNP reached 60% and functionally expressed in 50% spleen B-cells, while the untargeted standard LNP core had a low capability of transfecting B-cells (<5%). The unconjugated peripheral LNP core itself showed about 25% Thy 1.1 expression, which was higher than its standard LNP core counterpart. More importantly, the level of B cell delivery was greatly enhanced by the incorporation of aCD79b, aCD79b-RG7-Peripheral LNP achieved delivery and protein expression in 70-80% of spleen B-cells in vivo at only 0.1 mg / kg, outperforming any other nanoparticle systems reported up to date. Regarding the mean fluorescent intensity (MFI), As shown in Figure 12B, aCD79b-RG7-Peripheral LNP exhibited around 100-fold and 15- fold protein expression over the unconjugated peripheral LNP core and aCD79b-RG7- Standard LNP, respectively. This suggested our DOE-optimized LNP core formulation is aAttorney Docket No.: 2013260-0047 good foundation for targeting antibody surface functionalization, and achieving high levels of cell uptake and protein translation.

[0149] Interestingly, aCD79b-RG7 -Peripheral LNP only improved the transfection efficiency in B-cells, however not myeloid cells and T-cells, thus presented a high selectivity toward non-B-cells (Figure 12C and D). In particular, the protein expression was 6-fold over myeloid cells and 44-fold over T-cells. In contrast, aCD79b-RG7-Standard LNP could only bring the B-cells expression to the same level as the myeloid cells, an indication of suboptimal selectivity. Here, we have developed a B-cell targeted LNP platform with high efficiency and selectivity.5. Direct conjugation of targeting mAbs to LNP to simplify the composition and streamline the process

[0150] After having established a highly efficient and selective B cell-targeting LNP system, we next considered removing unnecessary components in the final LNP formulation. The secondary antibody RG7 was initially incorporated into the system for high-throughput screening of antibodies. Choosing to adopt this strategy as our lead formulation will compromise future clinical translation due to the origin of different species and the complications of the purification process during scale-up. Therefore, we conjugated DTT modified aCD79b directly with maleimide-functionalized peripheral LNPs and applied the optimal targeting antibody density (aCD79b / Maleimide=0.32) from the screening (RG7 / Maleimide=0.32, aCD79b / RG7=l). In addition, due to the binding affinity limitation of RG7, all targeting antibodies previously screened were Rat IgG2a. In the scenario of direct conjugation, we were allowed to screen more aCD79b with different backgrounds and two more aCD79b were tested (Rat IgGl, Clone AT107-2, and Hamster IgG, Clone HM79- 11) and compared to the current aCD79b (Rat IgG2a, Clone 735451). As shown in Figure 13A, B-targeted LNPs generated by the direct conjugation method (aCD79b-Peripheral LNP) showed a higher transfection efficiency than the previous two-layer method (aCD79b- RG7-Peripheral LNP) in spleen mature B-cells. Such a result further emphasizing the benefits of switching to the direct conjugation method. We hypothesize the advantage in B cell delivery using a direct conjugation method might be due to the smaller size of a direct one-layer conjugation (~ 110 nm), which was equivalent to RG7 conjugated-LNP, than that of the two-layer LNPs (125 nm), potentially assisting tissue penetration and cell uptake.Attorney Docket No.: 2013260-0047Two new aCD79b antibodies showed significantly lower protein expression than the current aCD79b used in the system, so we continued using our original rat IgG2a aCD79b clone 735451.

[0151] Interestingly, we also observed the size (FSC) increase in mature B-cells after all four aCD79b-LNP treatments, which may indicate the activation and proliferation of B- cells (Figure 13B). Coincidently, the MFI of IgM was found to be decreased after aCD79b- LNP treatments, suggesting the internalization of IgM on the B cell surface (Figure 13C). This is likely due to the unique structure and function of CD79b, which along with CD79a and a surface immunoglobulin (IgM) comprise a heterodimeric signal-transduction component of the B cell receptor. Multiple aCD79b’s on the LNP surface may crosslink several CD79b receptors on B-cells, causing the co-internalization with adjacent IgM and subsequent signal advance for the functional consequences, activating B-cells and triggering B cell proliferation. As shown in Figure 13D to E, the tendency in spleen developing B- cells was consistent with that of the mature B-cells, with a relatively lower protein expression level and a more moderate B cell size increase, which again was related to the less expression of CD79b on the developing B cell populations.6. Biodistribution of B-cell targeting mAb conjugated-LNP

[0152] To analyze the overall tissue distribution of aCD79b-LNPs, we encapsulated firefly luciferase RNA into LNPs that were directly conjugated to aCD79b and compared it to unconjugated peripheral LNP core and standard LNP core. In Figure 14, 18h after injection, the luminescence signal for standard LNP core was primary detected in the livers. In contrast, the peripheral LNP core had nearly equal accumulation in livers and spleens with a 10-fold decrease in liver expression than the standard LNP core. aCD79b conjugation on the peripheral LNP core maintained the low liver expression while significantly boosting the signals in spleens, where B-cells were developed and activated.7. Anti-human B-cell targeted LNP in transfecting PMBCs

[0153] In the studies above, we have demonstrated 70-80% B-cell transfection efficiency and 6-fold selectivity over myeloid cells in mice using anti-mouse aCD79b-LNPs. To accelerate the translation into drug products for human use, we switched the targetingAttorney Docket No.: 2013260-0047 mAb to anti-human clones and tested them in human PMBCs. Besides anti-human aCD79b, we also incorporated anti-human aCD79a and aCD20 for B-cell targeting.

[0154] As shown in Figure 15A, all three aCD79b-peripheral LNPs tested achieved >90% Thy 1.1 expression in B-cell populations of PBMC and outperformed aCD79a and aCD20 functionalized LNPs, which were consistent with our previous mice in vitro / in vivo transfection results. Regarding the ranking of anti-human aCD79b, the B-cell expression of LNP conjugated with the clone CB3-1 was 1.2 and 3-fold higher than that of the clone 683023 and SN-8, respectively. However, SN-8 achieved 1500-fold selectivity towards myeloid cells, which was superior to the 200-fold and 10-fold attained by 683023 and CD3- 1. Therefore, SN-8 or its Fab and scFv will be selected if we will test the anti-human aCD79b-peripheral LNPs in humanized mouse model. In Figure 15B, viability of PMBC was maintained >80% across all tested groups, except MassengerMax at 2 ug / mL, which indicated our B-cell targeted LNP platform was more tolerable than the golden- standard of in vitro transfection. Moreover, in Figure 15C, SN-8 and CB3-1 were more prone to drive B -cells activation (CD69+) than 683023, which equipped us with the options of activating T-cells or not depending on the applicable indications.Materials

[0155] LIP003 (Lipid 5) was purchased from DC Chemicals (Shanghai, China). Cholesterol was purchased from Millipore Sigma (Burlington, USA). DSPC, PEG-DMG, Maleimide-PEG-DSPE were purchased from Avanti Polar lipids Inc. (Alabaster, USA). Mouse anti-rat IgG2a (clone RG7 / 1.30), rat IgG2a isotype control (clone 2A3), Rat antimouse CD19 mAb (clone 1D3), and Rat anti-mouse CD38 (NIMR-5) were purchased from Bio X Cell (Lebanon, USA). Rat anti-mouse CD 19 mAb (clone 1D3) was purchased from Novus Biologies (Centennial, USA). Rat anti-mouse CD19 mAb (clone 1D3), Rat antimouse CD19 mAb (clone 6D5), Rat anti-mouse CD19 mAb (clone 6OMP31), and Rat antimouse CD20 mAb (AISB12) were purchased from Invitrogen (Waltham, USA). Rat antimouse CD 19 mAb (clone 6D5), Rat anti-mouse CD38 (NIMR-5) and Rat anti-mouse CD38 mAb (clone 90) were purchased from SouthernBiotech (Birmingham, USA). Rat anti-mouse CD 19 mAb (clone 6D5) and Rat anti-mouse CD38 mAb (clone 90) were purchased from Biolegend (San Diego, USA). Rat anti-mouse CD79b mAb (clone 90) was purchased from R&D Systems (Minneapolis, USA). Rat anti-mouse CD79b mAb (clone AT107-2) wasAttorney Docket No.: 2013260-0047 purchased from Bio-Rad (Hercules, USA). Hamster anti-mouse CD79b mAb (clone HM79- 11) was purchased from Thermo Scientific (Waltham, USA). DTT and EDTA were purchased from Thermo Scientific (Waltham, USA). DiD was purchased from Invitrogen (Waltham, USA).Methods

[0156] Formulation for LNP core-. Thyl.l, mCherry or Luciferase RNA was formulated in LNPs containing a cationic ionizable LIP003 (Lipid 5), cholesterol, DS PC, PEG2K-DMG and DSPE-PEG2K-Maleimimde at a ratio of 50 / 38.5 / 10 / 1 / 0.5 for the standard LNP core and 50 / 27.5 / 22 / 0.5 for the peripheral LNP core. The ratio of cationic ionizable amines to RNA phosphates in the LNP was 6. Lipids were dissolved in ethanol and RNA was dissolved in 50mM Citrate buffer pH 5.0. The organic and aqueous phases were combined at a flow rate of 12 mL / min on a Precision Nanosystems NanoAssemblr at a ratio of 3:1 (aqueous: organic). The product was immediately diluted to 16.5% ethanol using MilliQ water. The diluted product was dialyzed against a volume of IX PBS that was 300- fold in excess. The dialyzed product was concentrated using Amicon Ultra lOOkDa centrifuge tubes and sterile filtered. RNA concentration and encapsulation were assessed by the Invitrogen Ribogreen assay.

[0157] Using DTT to introduce thio to antibody and maleimide / thio chemistry to conjugate antibody to LNPs: Anti-IgG secondary antibody (RG7) or targeting antibodies was reduced in IX PBS containing 1 mM dithiothreitol (DTT) and 5 mM EDTA for 1 hour at room temperature. The reduced antibodies were then purified by buffer exchange to IX PBS with 5 mM EDTA using a 7K Zeba spin desalting column (Thermo Scientific) to remove excess DTT. The reduced antibody was quantified by Nanodrop. The reduced antibody was added to LNP at various rations of antibody / maleimide ratios and incubated for 2 hours at room temperature.

[0158] Purifying unconjugated mAb'. Antibody conjugated-LNPs were separated from unconjugated antibodies using Sepharose CL4B beads (G-Biosciences) on a gel filtration column with PBS as the mobile phase. Fractions containing pure LNPs were identified by the fluorescent signal of DiD-loaded LNPs and then were pooled together. RNA concentration and encapsulation were assessed by the Invitrogen Ribogreen assay.Attorney Docket No.: 2013260-0047RG7 concentration was assessed by the Pierce BCA protein assay kit (Thermo Fisher Scientific). RNA concentration and encapsulation were assessed by the Ribogreen assay (Invitrogen).

[0159] Attaching targeting mAb (if the two-layer method was used): The targeting (primary) antibody was added to RG7-LNPs at various primary / secondary weight or molar ratios and incubated for 30 min at room temperature.

[0160] A20 cell culture and LNP transfection: A20 cells (ATCC, TIB-208) were cultured in RPMI-1640 Medium (Gibco) supplemented with 10% HI-FBS (Gibco). Cells were maintained at 37°C and 5% CO . For in vitro transfection, A20 cells were plated at a density of 100,000 cells per well in a 96-well U-bottom plate in Opti-MEM (Gibco). LNPs were then added to the cells at a final concentration of 0.2 ug / mL. Eighteen hours after transfection, cells were washed with lx DPBS three times. If Thy 1.1 RNA was used, cells were stained with PE-conjugated CD90.1 Antibody, anti-mouse / rat, REAfinity™ (Miltenyi Biotec) in 1:50 dilution in IX PEB (PBS / EDTA / BSA) buffer for 10 min in the dark at 4°C. Then, cells were washed with 1XPEB buffer three times and subjected to flow cytometry analysis.

[0161] PMBC culture and LNP transfection: Human primary blood mononuclear cells (PBMCs, ATCC PCS -800 -01 1) were cultured in RPMI-1640 Medium (Gibco) supplemented with 1% Penicillin / Streptomycin (Gibco), and 10% HI-FBS (Gibco). Cells were maintained at 37 °C and 5% COr. For in vitro transfection, PBMCs were plated at a density of 100,000 cells per well in a 96-well U-bottom plate in RPMI-1640 medium. LNPs were then added to the cells at a final concentration of 2, 0.2, and 0.02 ug / mL. Eighteen hours after transfection, cells were washed with lx DPBS three times. Cells were stained with viability dye in 1: 100 dilution in 1XDPBS for 8 min followed by a mixture of cell markers including anti-CD79b (receptor internalization), anti-CD20 (B cells), anti-CDl lb (myeloid cells), anti-CD69 (B-cell activation), anti-CD3 (T-cells) and anti-CD90.1 (REAfinity™ , Miltenyi Biotec) in 1:50 dilution in IX DPBS for 10 min. Then, cells were washed with 1XPEB (PBS / EDTA / BSA) buffer three times and subjected to flow cytometry analysis.Attorney Docket No.: 2013260-0047

[0162] General Animal Study Protocol: Protocols were approved by Charles River IACUC. BALB / c, C57BL / 6 and B6 Albino (female, 6-10 weeks) mice were purchased from Charles River Labs. Mice were housed in the Charles River Labs Facility.

[0163] Biodistribution: Eight-week-old B6-Albino mice were injected with Luciferase RNA encapsulated LNPs through tail vein at 0.3 mg / kg. After 18 hours, D- luciferin was injected intraperitoneally at 150 mg / kg. Ten minutes later, the thymuses, lungs, livers, spleens, and lymph nodes were harvested for ex vivo imaging. N=5 / group.

[0164] In vivo transfection efficiency: Eight-week-old C57 / BL6 mice were injected with DiD dye and mCherry or Thy 1.1 encoding RNA co-encapsulated LNPs through the tail vein. After 18 hours, spleens were harvested from mice and preserved in MACS® Tissue Storage Solution on ice. Spleens were mechanistically disrupted by pressing then through a 70 pm strainer. Cells were then spun down at 350g for 10 min and washed with 1XDPBS three times. Cells were then plated into a 96-well V-shape plate at 100,000 cells / well. After centrifuging at 350g for 10 min, cells were resuspended in 60 uL diluted Viogreen-Viobility Fixable Dye (Miltenyi Biotec) solution (1: 100 in 1XPBS) and incubated for 8 minutes in the dark at room temperature. Cells were then stained with 50 uL of predetermined diluted surface markers solution (2 uL per antibody in total 50 uL 1XPEB buffer) for an additional 8 minutes in the dark at room temperature. Cells were then washed three times with 300 uL 1XPEB buffer and resuspended in 200 uL 1XPEB buffer for flow cytometry analysis on Attune CytPix Cytometer (ThermoFisher). Dead cells and red blood cells were excluded by viability dye and TER-119. T-cells, B-cells and myeloid cells were identified as CD3+B220- , B220+CD3-, and CD3-B220-, respectively. N=3-4 / group.Equivalents

[0165] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the following claims:

Claims

Attorney Docket No.: 2013260-0047ClaimsWe claim:

1. A functionalized nucleic acid lipid particle composition comprising: lipid components that encapsulate a payload to form a particle; and a targeting entity.

2. The functionalized nucleic acid lipid particle of claim 1, wherein the payload is an RNA.

3. The functionalized nucleic acid lipid particle of claim 2, wherein the payload is an mRNA.

4. The functionalized nucleic acid lipid particle of claim 2, wherein the RNA encodes a therapeutic agent.

5. The functionalized nucleic acid lipid particle of claim 2, wherein the RNA encodes a chimeric antigen receptor (CAR).

6. The functionalized nucleic acid lipid particle of claim 1, wherein the lipid components comprise an ionizable lipid, a sterol, a helper lipid, and a PEG-lipid.

7. The functionalized nucleic acid lipid particle of claim 6, where in the PEG-lipid is selected from the group consisting of PEG-DMG, PEG-DSPE, Maleimide-PEG-DSPE, Azide-PEG-DSPE and DBCO-PEG-DSPE.

8. The functionalized nucleic acid lipid particle of claim 6, where in the helper lipid is a phospholipid.

9. The functionalized nucleic acid lipid particle of claim 8, where in the helper lipid is a DSPC.

10. The functionalized nucleic acid lipid particle of claim 1, wherein the targeting entity comprises an antibody agent.

11. The functionalized nucleic acid lipid particle of claim 10, wherein the antibody agent is covalently linked to the surface of the nucleic acid lipid particle by DTT reduction, SATA thio conjugation or DBCO conjugation.Attorney Docket No.: 2013260-004712. The functionalized nucleic acid lipid particle of claim 10, wherein the antibody agent binds CD 19.

13. The functionalized nucleic acid lipid particle of claim 12, wherein the antibody agent comprises an anti-CD19 antibody or antibody fragment.

14. The functionalized nucleic acid lipid particle of claim 10, wherein the antibody agent binds CD3.

15. The functionalized nucleic acid lipid particle of claim 14, wherein the antibody agent comprises an anti-CD3 antibody or antibody fragment.

16. The functionalized nucleic acid lipid particle of claim 10, wherein the antibody agent binds CD79b.

17. The functionalized nucleic acid lipid particle of claim 16, wherein the antibody agent comprises an anti-CD79b antibody or antibody fragment.

18. A method of delivering a nucleic acid to a tissue or a cell of a subject, the method comprising: administering to the subject functionalized nucleic acid lipid particle of any one of claims 1-16.

19. The method of claim 18, wherein the tissue is or comprises immune tissue.

20. The method of claim 18, where the cell is or comprises an immune cell.

21. The method of claim 19, wherein the immune tissue is spleen or thymus.

22. The method of claim 20, wherein the immune cell is a B-cell or T-cell.

23. A method of treating a subject suffering from a disease, the method comprising: administering to the subject functionalized nucleic acid lipid particle of any one of claims 1-16.

24. The method of claim 23, wherein the disease is a cancer.Attorney Docket No.: 2013260-004725. The method of claim 23, wherein the disease is a disease effecting a T-cell or B-Cell.

26. A method of manufacturing a nucleic acid lipid particle comprising combining a nucleic acid with lipid components including an ionizable lipid, a helper lipid, a sterol, and a PEG- lipid.

27. A method of manufacturing a functionalized nucleic acid lipid particle comprising combining a payload with lipid components and directly conjugating a targeting moiety to the lipid nanoparticle encapsulating the payload.

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