Targeted nanoparticles encapsulating water-soluble therapeutics, methods of production, and use

Abstract

Presented are targeted coated nanoparticles that have a desired in vitro or in vivo effect, compositions of the targeted and coated nanoparticles, methods of use of the coated nanoparticles to achieve therapeutic or prophylactic effects in cells, organs, or subjects, and processes for forming the targeted nanoparticle.

Description

[0001] OLSI-OOl-PCT

[0002] TARGETED NANOPARTICLES ENCAPSULATING WATER-SOLUBLE THERAPEUTICS, METHODS OF PRODUCTION, AND USE

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This present application claims priority to U. S. Provisional Patent Application No.

[0005] 63 / 719.864, filed November 13, 2024, the contents of which are incorporated by reference herein in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under National Institutes of Health Award 1R43AI177159-01A1. The Government has certain rights in the invention.

[0008] FIELD OF THE INVENTION

[0009] Embodiments of the invention provide hybrid polymer-lipid nanoparticle compositions encapsulating nucleic acids or proteins, a process of nanoparticle production, and methods for use of the compositions for therapeutic or prophylactic effect in cells, organs, tissues, or subjects.

[0010] BACKGROUND OF THE INVENTION

[0011] Nucleic acids including RNA and DNA have short half-lives and are rapidly degraded by RNases or DNases, respectively, when administered as soluble species. Therapeutic or clinical uses of RNA and DNA have required encapsulation into delivery vehicles that provide protection from clearance, enhance uptake into cells (usually by endocytosis), and facilitate entry of the nucleic acids into the cytosol. Encapsulation of the RNA or DNA relied on charge interactions (“complexation”) between the negatively charged nucleic acids and a positively charged lipid or polymer. This component also provides for endosomal escape of the nucleic acid species. Lipid-based delivery’ vehicles, called lipid nanoparticles (LNPs) are the most widely deployed, having been used in mRNA vaccines (e.g. Comimaty) and rare diseases (e.g. Onpattro).

[0012] Of particular interest in the field of nanoparticle delivery' is the ability’ to reach different tissues following systemic (e.g. intravenous) administration. Nanoparticles generally clear to the liver, taken up by Kupffer Cells or hepatocytes. Targeting to extra-hepatic tissues or cells requires reducing the non-specific clearance rate to the liver cells and providing a means to OLSI-OOl-PCT

[0013] enhance uptake in the cells of interest. The field remains limited by delivery challenges and is seeking low toxicity, redosable, and targetable solutions to deliver genes and protein cargo.

[0014] PCT / US2022 / 053948 (PCT’948) described a delivery vehicle that did not rely on charge interactions for encapsulation. This approach captured the RNA or DNA in an inert polymer core and then applied a coating containing one or more lipid species. The invention also described methods of producing these coated nanoparticles using inverse Flash NanoPrecipitation which employs rapid confined mixing in particular mixing geometries. Certain embodiments of this invention describe coatings that include a blend of lipids, including a cationic ionizable lipid, a helper lipid, and a cholesterol or sterol.

[0015] Embodiments of PCT’948 also describe different amphiphilic stabilizers for the outer surface of the coated nanoparticle (“second stabilizing agent”). These provide steric stabilization during handling to provide a controlled nanoparticle size. A common hydrophilic region of the stabilizer is poly(ethylene glycol) (PEG). One class of these stabilizers was PEG-lipids such as PEG-DMG or PEG-DSPE. Another class of surface stabilizers in PCT'948 used polymers instead of lipids for the hydrophobic portion: for example, poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA). or poly(caprolactone) (PCL). These hydrophobic polymers are linked, for example, to PEG to form amphiphilic stabilizers such as PLA-b-PEG, PLGA-b-PEG, or PCL-b-PEG. Herein, the nanoparticles described in PCT'948 will be termed Coated Inverse NanoCarriers (CINCs) or coated nanocarriers or coated nanoparticles.

[0016] SUMMARY OF THE INVENTION U S. Patents 10,231,937 and 11,554,101 and 11,103,461 and 11,737,981 and 11,731,099, the corresponding international applications, as well as International Application Publications W02020018890 and W02020227350 and W02021046078 and WO2023122331 are incorporated in their entirety herein and present methods and compositions for encapsulating hydrophilic compounds in nanoparticles without charge complexation that instead rely on solubility-driven precipitation of water-soluble therapeutics including nucleic acids. This approach is collectively termed “inverse Flash NanoPrecipitation” (iFNP) herein.

[0017] These patents describe compositions and a process where a hydrophilic active agent referred to as the encapsulated agent - such as ribonucleic acid (RNA), deoxyribonucleic acid (DNA), oligonucleotides, proteins, or peptides - and a stabilizing agent, such as a block copolymer, are dissolved in at least one polar solvent stream which is rapidly mixed with at least one non-polar antisolvent stream. Upon mixing, the encapsulated agent precipitates, forming the nanoparticle core. The hydrophilic region(s) of the stabilizing agent stick or adhere OLSI-OOl-PCT

[0018] to and / or are within the nanoparticle core, and the hydrophobic region(s) of the stabilizing agent face the external non-polar antisolvent phase, for example, the hydrophobic region(s) of the stabilizing agent form a shell around the core, to form an inverse nanoparticle.

[0019] Further, the inverse nanoparticle is transferred into a water-miscible reforming solvent, and a second stabilizing agent is dissolved at a desired ratio. PCT’ 948 provides for the incorporation of one or more additive agents such as lipids or lipid blends. An embodiment of these lipid blends includes a cationic ionizable lipid, a neutral helper lipid and a cholesterol or sterol.

[0020] In a second mixing step, the additive(s) and second stabilizing agent sticks to, adheres to, and / or is in contact with the collapsed hydrophobic region of the first stabilizer agent. This forms a coated nanoparticle or coated nanocarrier composition with a hydrophilic active, such as a nucleic acid, in a hydrophilic core, surrounded by a hydrophobic shell, with a hydrophilic surface coating to prevent aggregation in aqueous conditions.

[0021] The present invention provides at least two unexpected improvements over known techniques, enabling the unique capability to deliver therapeutic nucleic acids to specific tissues: (1) replacing the cholesterol / sterol component of lipid blends with a hydrophobic vitamin; and (2) including a targeting moiety linked to the terminal end of the second stabilizing agent or a portion of a mixture of second stabilizing agents. These improvements may be employed alone or in combination. In some aspects, the compositions utilize only the hydrophobic vitamin improvement. In some aspects, the compositions utilize only include the targeting moiety improvement. In some aspects, the compositions utilize both improvements.

[0022] The use of hydrophobic vitamins affords greater nanoparticle size stability during processing and improves the encapsulation efficiency of the cargo (the relative fraction remaining within the coated nanoparticle core after processing), which was not expected or predicted in the prior art.

[0023] This disclosure for the first time describes the use of targeting moieties on nanoparticles that contain a precipitated core of hydrophilic cargo. This disclosure provides approaches and compositions for targeted nanoparticles with a hydrophilic core of mRNA, DNA. or other biologies without charge complexation. This approach overcomes the very low loading and potency of liposome and polymersome approaches previously described.

[0024] The hydrophobic vitamin includes but is not limited to vitamin D, vitamin K, vitamin E, or vitamin A and their variants. This modification may modulate liver expression (such as reducing liver expression in instances where extra-hepatic delivery is preferred) and it may enhance physical stability and encapsulation efficiency of the nucleic acid when used with a OLSI-OOl-PCT

[0025] second stabilizing agent that is a PEG-polymer (e.g., PLA-b-PEG, PLGA-b-PEG, or PCL-b-PEG). Coated nanoparticles based on PCT’ 948 but with a hydrophobic vitamin substitution and PEG-polymer surface may be termed stealth CINCs (sCINCs) herein to provide clarity or may retain the “coated nanocarrier” terminology and can be recognized by context and description. Inclusion of a targeting moiety allows for cell-specific uptake of the coated nanoparticle and its cargo. Exemplary targeting moieties include but are not limited to peptides, proteins, antibodies, antibody fragments, oligonucleotides, small molecule ligands, polymers monosaccharides and polysaccharides.

[0026] This disclosure describes embodiments of these targeted coated nanoparticles, methods of production, and methods of use as a pharmaceutical agent.

[0027] As noted, the present disclosure provides at least two improvements upon known compositions include the lipid blend additive agents and the second stabilizing agents, that enable the creation of stealth Coated Inverse NanoCarriers (sCINCs) that are useful for targeted nanoparticles for delivery7to cells or tissues or organs of interest. First, the cholesterol / sterol component is replaced with a hydrophobic (fat-soluble) vitamin or derivative thereof. This may reduce liver accumulation and expression following systemic administration of the nanoparticle. In instances where the second stabilizing agent is poly(lactic acid)- block-poly(ethylene glycol) (PLA-PEG) or poly(caprolactone)-block-PEG (PCL-PEG) or similar polymer stabilizers, this modification provides physical stability (such as reduced size growth during coated nanoparticle handling) and encapsulation efficiency enhancements over formulations described in PCT’948. Second, the second stabilizing agent or fraction thereof is modified with one or more targeting moieties to provide for specific engagement with cell surface receptor(s) on a cell population in need of delivery7. This allows for targeting to tissues or cells or for the modification of internalization through engagement with surface receptor(s).

[0028] An embodiment of the present disclosure includes a coated nanoparticle composition including one or more encapsulated agents and a first stabilizing agent, with an exterior surface formed by a lipid blend comprising a cationic ionizable lipid, a hydrophobic vitamin or derivative, a neutral helper lipid and a second stabilizing agent or agents. In an embodiment of the present disclosure, all or a portion of the second stabilizing agent or agents are modified with a targeting moiety. Compositions include but are not limited to an encapsulated agent or agents selected from peptides, proteins, or nucleic acid classes including but not limited to RNA, DNA, mRNA (messenger RNA), siRNA (small interfering RNA), microRNA, circular RNA, antisense oligonucleotides, RNA / DNA blends. tRNA (transfer RNA), aptamers, DNA origami, or chemically-modified polynucleotides, including nucleic acid species with natural OLSI-OOl-PCT

[0029] (e.g., from methylation or acety lation) or artificial substitutions, and these can be in circular or linear form or in complex with other molecules. The composition may include a salt, for example, a magnesium salt, or a free acid form. The encapsulated agent may be more than one agent such as one or more of nucleic acids, peptides, proteins, or second or more nucleic acids. The encapsulated agent may be multiple nucleic acid sequences. In this text, the terms nanoparticle, nanocarrier, and NC are used interchangeably. Inverse nanoparticle or inverse nanocarrier refers to those nanoparticles formed by the first stabilizing agent and the cargo.

[0030] In an embodiment, a composition includes a first stabilizing agent that is a diblock, triblock, or comb copolymer. For example, the hydrophilic block(s) of the first stabilizing agent may be selected from dextran or other polysaccharides, poly(aspartic acid), or poly(glutamic acid). For example, the hydrophobic block(s) may be selected from poly(lactic acid), poly(lactic-co-gly colic acid), or poly(caprolactone).

[0031] In an embodiment, a composition includes neutral helper lipids selected from phospholipids such as POPC (l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DSPC (l,2-distearoyl-s-glycero-3-phosphocholine), hydrogenated soybean phosphatidylcholine (HSPC), DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3 -phosphoethanolamine), PLPC (l-Palmitoyl-2-lauroyl-sn-glycero-3-phosphocholine), or combinations.

[0032] In an embodiment, a composition includes a cationic lipid, or cationic ionizable lipid, or combinations of multiple cationic ionizable lipids.

[0033] In an embodiment, a composition includes a hydrophobic vitamin selected from the vitamin D group or related compounds (Vitamin Di, D2, Ds, D4, or Ds or ergocalciferol, cholecalciferol, 22-dihydroergocalciferol, sitocalciferol, calcitriol, alfacalcidol, doxercalciferol, 22-oxacalcitriol, falecalcitriol, or paricalcitol).

[0034] In an embodiment, a composition includes a hydrophobic vitamin selected from the vitamin E group or related compounds (alpha-, beta-, gamma-, delta- tocopherol or tocotrienol, tocopheryl acetate, and related compounds).

[0035] In an embodiment, a composition includes a hydrophobic vitamin selected from vitamin A or related compounds (retinoids, carotenoids, retinol, retinol esters, retinyl acetate, retinyl linoleate, retinyl palmitate, and retinyl propionate, alpha-, beta-, gamma-, or delta-carotene, and derivatives).

[0036] In an embodiment, a composition includes a hydrophobic vitamin selected from the vitamin K group or related compounds (Vitamin Ki, K2, or K3, or related compounds). OLSI-OOl-PCT

[0037] In an embodiment, compositions include a second amphiphilic stabilizing agent that is a diblock, triblock, or comb copolymer, or a lipid conjugate, or mixtures. The hydrophilic region of the polymeric or lipid conjugate second stabilizing agent can be selected from poly(ethylene glycol), poly(sarcosine), poly(aspartic acid), poly(glutamic acid), poly(lysine), or poly(arginine), or combinations. The hydrophobic polymer block can be selected from poly(lactic acid), poly(lactic-co-gly colic acid), poly(caprolactone). Compositions include PEG (polyethylene glycol, polyoxyethylene, poly (ethylene oxide)) lipids such as PEG-DMG (1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol) or PEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]), or PEG-b-PLGA (polyethylene glycol-block-poly(lactic-co-gly colic acid)) or PEG-b-PLA (polyethylene glycol-block-poly(lactic acid)), or PEG-b-PCL (poly(ethylene glycol)-block-poly(caprolactone)), or mixtures. A portion or all of the second stabilizing agent may be conjugated to one or more targeting moieties. For example, 0.1 %, 1%, 5%, 10%, 30%, 50%, 90%, or 99% of the stabilizing agent may be conjugated (linked) to the one or more targeting moieties.

[0038] The conjugation of the targeting moiety may be achieved through any known means for coupling two species. The conjugation may be carried out prior to nanoparticle coating (i.e.. between the stabilizing agent and the targeting moiety). The conjugation may alternatively be carried out after nanoparticle assembly. The coupling may be between a reactive group on the terminal end of the second stabilizing agent’s hydrophilic block or blocks (e.g. the end of the PEG chain that is located on the surface of the coated nanoparticle) and a reactive group on the targeting moiety. The reactive group on the second stabilizing agent may be conjugated anywhere on the molecule that is accessible to the aqueous solvent on the surface of the coated nanoparticle. Suitable combinations include but are not limited to N-hydroxysuccinimide esters reacting with amines, maleimides reacting with thiols, or azide-alkyne “click” chemistry. For example, a fraction of PEG-b-PLA stabilizing agent can be substituted with maleimide-PEG-b-PLA to provide a tunable density of functional groups on the surface for conjugation with a thiol located on the targeting moiety. In an embodiment, the targeting moiety is conjugated to the second stabilizing agent before assembly onto the coated nanocarrier surface.

[0039] Suitable targeting moieties include sugars, polysaccharides, oligonucleotides, peptides, proteins, recombinant proteins, antibodies, nanobodies, single-chain variable fragments (scFvs), antibody fragments, N-acetylgalactosamine, triantennary N-acetylgalactosamine. or small molecule compounds. Targeting moieties should have a site or sites suitable to conjugation to the second stabilizing agent that does not interfere with receptor interactions. In certain embodiments, the targeting moiety is an antibody or antibody fragment directed against OLSI-OOl-PCT

[0040] T cell surface markers CD3, CD4, CD5, CD7, CD8, CD25, or combinations. In certain embodiments, the targeting moiety is an antibody or antibody fragment directed against endothelial cell surface receptors such as PEC AM, transferrin, VC AM, or ICAM. In certain embodiments, the targeting moiety is an antibody or antibody fragment directed against hematopoietic stem cell markers CD 117 or CD 133 or cMPL. In some embodiments, a molecule or protein that binds to the targeting moiety is conjugated to the second stabilizing agent before or after coated nanoparticle assembly. The targeting moiety is then mixed with coated nanoparticle to allow binding onto the surface. In some embodiments, this molecule or protein that bind the targeting moiety is an immunoglobulin binding protein or peptide such as protein A, protein G, or fragments or derivatives thereof.

[0041] A process of the present disclosure is for preparing the coated nanocarrier composition involving forming an inverse nanocarrier using inverse Flash NanoPrecipitation, exchanging the inverse nanocarrier into a reforming solvent along with additive agent lipid blend and the second stabilizing agent or agents, and assembling the coating using Flash NanoPrecipitation. The coated nanocarrier may then be conjugated to a targeting moiety and / or further processed into a pharmaceutical form.

[0042] The inverse nanocarrier is produced by inverse Flash NanoPrecipitation. One or more solvent streams containing the dissolved encapsulated agent or agents and the first stabilizing agent is rapidly micromixed with an antisolvent stream or streams, optionally containing a divalent cation such as calcium. This mixing process drives precipitation of the encapsulated agent or agents which is then stabilized from further growth by the stabilizing agent. The micromixing process is carried out using mixers such as the confined impinging jet mixer or the multi-inlet vortex mixer. A representative solvent would be dimethylsulfoxide (DMSO) and a representative antisolvent would be dichloromethane (DCM).

[0043] The inverse nanocarrier is then combined with the additive agent lipid blend and the second stabilizing agent or agents at a specified or desired composition. A solvent exchange process, such as distillation, is employed to remove the antisolvent and introduce the reforming solvent, for example, acetonitrile. Some or all of the lipid blend may be added after the solvent exchange to the reforming solvent has been completed.

[0044] The inverse nanocarrier and additive agent lipid blend and second stabilizing agent(s) in the reforming solvent are then rapidly micromixed with an aqueous antisolvent using a mixer as described above to create the coated nanocarrier.

[0045] In some embodiments, the targeting moiety or moieties was pre-conjugated to the stabilizing agent before coating and unconjugated targeting moiety was already removed OLSI-OOl-PCT

[0046] (purified) before coating. Further processing steps common to the nanoparticle field, such as buffer exchange by ultrafiltration or diafiltration, can be employed to remove residual reforming solvent and prepare the pharmaceutical composition.

[0047] In some embodiments, the targeting moiety or moieties is conjugated onto the surface of the coated nanocarrier after assembly. The coated nanocarriers may be combined w ith the targeting moiety in a suitable reaction buffer under appropriate time and temperature conditions for the reaction to the proceed. Further processing steps may be employed to remove unconjugated targeting moiety and residual reforming solvent. These include tangential flow filtration, size exclusion chromatography, ultracentrifugation, and / or dialysis. Compositional adjustments may be made to prepare a pharmaceutical composition.

[0048] Methods of the present disclosure provide for delivering a therapeutic and / or prophylactic to a cell or organ. Delivery of a therapeutic and / or prophylactic to a cell involves administering a coated nanocarrier pharmaceutical composition including the encapsulated agent to a subject, where administration of the composition involves contacting the cell with the composition. Coated nanocarrier compositions and / or pharmaceutical compositions including one or more coated nanocarrier compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery of an encapsulated agent or agents to one or more particular cells, tissues, organs, or systems or groups thereof, such as the hepatic system.

[0049] The step of contacting a cell, such as a mammalian cell, with a coated nanocarrier may be performed in vivo, ex vivo, in culture, or in vitro. In certain embodiments, the mRNA or DNA encapsulated agent encodes a chimeric antigen receptor (CAR) to be expressed on a T cell to achieve T cell-mediated cell killing directed against the CAR target. In certain embodiments, additional mRNA or DNA sequences are co-encapsulated to provide further T cell functionality. In one embodiment, the additional sequence(s) encodes for the Foxp3 transcription factor. In another embodiment, the CAR and Foxp3 sequences are encoded on the same DNA sequence. In certain embodiments, an mRNA as the encapsulated agent in a coated nanocarrier may encode a recombinant polypeptide that may replace one or more polypeptides that may be reduced or substantially absent in a cell contacted with the nanoparticle composition. The one or more substantially absent polypeptides may be lacking due to a genetic mutation of the encoding gene or a regulatory pathway thereof. In certain embodiments, one or more nucleic acids including an interfering RNA sequence (e.g., siRNA) may be encapsulated in the coated nanocarrier to provide a method for treatment in vitro and in vivo of a disease or disorder in a mammal by downregulating or silencing the transcription and / or translation of OLSI-OOl-PCT

[0050] one or more target nucleic acid sequences or genes of interest. In certain embodiments, an mRNA sequence encoding an endonuclease and a small guide RNA may be the encapsulated agents in a coated nanocarrier to provide a method for treatment in vitro and in vivo by gene editing. In certain embodiments, the coated nanocarriers co-encapsulate an mRNA sequence encoding a transposase and a DNA sequence encoding a protein of interest.

[0051] BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figures 1A-1B are schematics showing various techniques, including: (1A) targeted coated nanocarriers produced by the presently disclosed techniques where the targeting moiety is conjugated after assembly, and (IB) targeted coated nanocarriers produced using second stabilizing agents that are already conjugated with a targeting moiety prior to use in the coating step.

[0053] Figures 2A-2D are plots showing results from in vitro cell incubation and in vivo dosing effects for Example 4. Coated nanocarriers with or without CD4 targeting were incubated with 3A9 cells or with expanded murine T cells (isolated from mouse splenocytes). An LNP control was included for comparison. “Serum incubated” samples were exposed to whole human serum for 3 hours at 37°C before dosing to the cells. In vivo tests were in naive BALB / cJ mice dosed with untargeted or CD4-targeted CINCs at 1 mg / kg. Splenocy tes were isolated at 24 hours post dose and stained for surface CD4 to determine whether binding and internalization had occurred.

[0054] Figure 3 shows whole animal images (gray scale) showing the locations of luminescence signal. Dark coloration corresponds to higher luminescence values. Formulations were prepared in example 5.

[0055] Figures 4A-4B show plots for an analysis of Jurkat T cell expression for coencapsulated and singly delivered genetic cargos for example 6, including GFP positivity (4A) and Luciferase activity' (4B). GFP positivity is the result for DNA cargo and luciferase was encoded by mRNA.

[0056] Figure 5 shows a plot of transfection efficiency for CD117-CINCs in EML stem cells after 24 hour incubation, dosed at 1.25 ug / mL mRNA basis and analyzed by flow cytometry, as prepared in example 7. MFI - median fluorescence intensity.

[0057] Figure 6 shows a plot of analytical SEC traces of coated nanocarriers before and after preparative scale SEC purification (example 8). Antibody was first labeled with Alexa Fluor 594 before coupling to the coated nanocarrier. The clear loss of the free antibody fraction after preparative scale purification confirms the efficiency of this purification. OLSI-OOl-PCT

[0058] Figure 7: Peripheral blood mononuclear cell population analyzed for GFP positivity 18 hours post dose of varied T cell targeted coated nanoparticles, prepared in example 9, encapsulating eGFP mRNA. CD4 and CD8 are T cell populations.

[0059] Figure 8: After dosing BALB / c mice according to example 9, CD3+ splenocytes (pan-T cell marker) analyzed for GFP positivity show specific delivery to spleen-resident T cells above background and untargeted nanocarriers.

[0060] Figures 9A-9B are plots showing (9A) bioluminescence of CINC and sCINC formulations as prepared in Example 10, where the highest luminescence flux is rendered darkest in this grey scale image, and in both formulations, signal is located in the liver, and (9B) quantification of the luminescence values in the liver as a function of composition changes resulting in the sCINC formulations described herein.

[0061] Figures 10A-10B are plots showing elution patterns of coated nanocarriers made using either PCL-b-PEG or PCL-b-PEG-AF647 as the second stabilizing agent. Elution behavior was similar for the absorbance of the unlabeled version (10A) and the AlexaFluor647 (AF647) labeled versions (10B).

[0062] Figure 11 is a plot showing GFP positivity measured by flow cytometry in splenocytes 24 hours after dosing (n=3).

[0063] Figure 12 is a plot showing Jurkat positivity for GFP expression, 2 days after a 0.625 ug / mL nanoparticle dose (DNA basis).

[0064] Figure 13 is a plot showing Dose responsive GFP expression in Jurkat T cells 2 days after dosing nanoparticles with DNA plasmid cargo.

[0065] Figures 14A-14B are plots showing transfection of primary T cells in vitro shows potency of different targeting antibodies (14A) and dose-responsive expression (14B). Significance of targeted vs null-CINC comparison: *P<0.05, **P<0.001.

[0066] Figures 15A-15B are plots showing flow analysis of example 16 OKT3-CINCs in primary T cells, including GFP positivity (15A) and live cell counts (15B).

[0067] Figure 16 is a plot showing GFP positivity in naive T cells.

[0068] Figures 17A-17B are plots showing Luciferase activity ratio between spleen and liver samples (17A, analyzed ex vivo by IVIS imaging) and neck lymph node and whole-body signal (17B, analyzed in-life by IVIS imaging). Dots represent the ratio for an individual animal receiving the indicated formulation. **p<0.006

[0069] Figure 18 is a plot showing TNF-alpha levels in sera 3 hours post dose, showing significantly higher immune activation for the LNP compared to CINC formulations as measured by Legendplex analysis. Individual dots represent the value for an individual mouse OLSI-OOl-PCT

[0070] (determined by the average of technical duplicates). CINC samples were not statistically significantly higher compared to buffer. The LNP was higher (****p<0.0001).

[0071] Figure 19 is a plot showing Interferon-alpha levels in sera 3 hours post dose, showing significantly higher immune activation for the LNP compared to CINC formulations as measured by Legendplex analysis. Individual dots represent the value for an individual mouse (determined by the average of technical duplicates). CINC samples were not statistically significantly higher compared to buffer. The LNP was higher (****p<0.0001).

[0072] Figure 20 is a plot showing GFP positivity at the indicated timepoint for each CINC formulation from Example 19, showing DNA delivery for all formulations except the untargeted control CM-227-F. All formulations except CM-227-A exhibited increasing DNA expression over time, regardless of how cells were treated.

[0073] Figure 21 is a plot showing Live cell percent in the flow cytometry analysis of Example 19 showing high viability for all treatments.

[0074] Figure 22 is a plot showing the effect of papain treatment on CINC physical stability. Figure 23 is a plot showing GFP expression from mRNA cargo in Jurkat T cells after 24 hours incubation measured by flow cytometry for Example 20.

[0075] DETAILED DESCRIPTION

[0076] Embodiments of the present disclosure are discussed in detail below. In describing embodiments, specific terminology is employed for the sake of clarity. However, the present disclosure is not intended to be limited to the specific terminology so selected. A person skilled in the relevant art will recognize that other equivalent parts can be employed and other methods developed without parting from the spirit and scope of the invention. All references cited herein are incorporated by reference as if each had been individually incorporated.

[0077] Definitions of several terms follow.

[0078] In this specification, solvent or solvent stream are terms used interchangeably to refer to an organic solvent, aqueous buffer, or mixture of the same, often with certain organic or inorganic compounds dissolved. " Stream" often, but not solely, is used in contexts where a continuous mixing or flow process is used or will be used. The properties of the solvent are dictating by the process as defined below.

[0079] In this specification, “antisolvent’' or “antisolvent stream” are terms used interchangeably to refer to an organic solvent, aqueous buffer, or mixture of the same, often with certain organic or inorganic compounds dissolved. “Stream” often, but not solely, is used in contexts where a continuous mixing or flow process is used or will be used. The properties OLSI-OOl-PCT

[0080] of the antisolvent are dictated by the process as defined below. In patents and manuscripts related to Flash NanoPrecipitation (FNP) or inverse Flash NanoPrecipitation (iFNP). the term “non-solvent’’ or “non-process solvent” may be used with the same intended meaning.

[0081] All solvents are miscible to some degree in each other. “Miscible” solvents as referred to herein are those that when mixed at the ratios used in the process would produce solutions that have no more than 20% of the volume of the minor phase not dissolved in the majority phase. “Immiscible” solvents as referred to herein are those that when mixed at the volume ratios used in the process produce a second phase with more than 20% of the minor phase not dissolved in the maj ority phase.

[0082] In this specification, the term “soluble” refers to the state of being molecularly dissolved within an indicated solvent or aqueous system. This requires that the solvent solubilize all regions, portions, or groups of the agent, species, polymer, or compound. Species that have precipitated, micellized, or formed collapsed polymer globules are not molecularly dissolved.

[0083] In this specification, the terms “species” and “agent” refer to compounds or molecules - organic or inorganic - that are included within the process.

[0084] In this specification, the terms “nanoparticles” (“NPs”), “particles”, and “nanocarriers” are used interchangeably, unless a distinction is indicated by the context. Particles in embodiments of the present disclosure that have hydrophilic or more polar cores are at times referred to as “inverse nanocarriers”, to contrast them with nanocarriers that have hydrophobic or less polar cores. However, for the sake of brevity, when the context indicates that particles having hydrophilic or more polar cores according to an embodiment of the present disclosure are being discussed, these may be simply referred to as “particles” or “nanoparticles”. “Coated nanocarriers” refers to “inverse nanocarriers” that have been further processed to have a surface permitting them to be dispersed in water without aggregation. Nanoparticles or nanocarriers typically have hydrodynamic mass average diameters as determined by dynamic light scattering to be between 10 nm and 800 nm. “Coated Inverse NanoCarriers” (CINCs) is at times used interchangeably with “coated nanocarrier.” Further, “Stealth CINCs” refer specifically to compositions where the additive agents were a blend of cationic ionizable lipid, a hydrophobic vitamin, and a helper lipid while the second stabilizing agent was PLA-PEG or PCL-PEG. “Targeted coated nanocarriers” describes coated nanocarriers that have a targeting moiety conjugated to the surface.

[0085] The term “water-dispersed” refers to the property of being colloidally stable in a water environment such as an aqueous buffer. “Colloidal stability” refers to nanoparticles or other OLSI-OOl-PCT

[0086] small particles that do not aggregate in the solvent. Colloidal stability may be imparted through a polymer brush that provides a steric barrier to nanoparticle aggregation, because it is solvated by the dispersing liquid, e.g. water.

[0087] The term '‘lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many- organic solvents. They are usually divided into at least three classes: (1) “simple lipids”, which include fats and oils as well as waxes; (2) “compound lipids”, which include phospholipids and glycolipids; and (3) “derived lipids” or “structural lipids” such as steroids or sterols. A “phospholipid” is a lipid that includes a phosphate moiety7and one or more carbon chains, such as unsaturated fatty acid carbon chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g.. one or more unsaturated bonds). A helper lipid describes neutral lipids, often phospholipids, that are incorporated into a lipid mixture with cationic ionizable lipids.

[0088] As used herein, "expression" of a nucleic acid sequence refers to the transcription from DNA into mRNA. the translation of an mRNA into a polypeptide or protein, and / or post-translational modification of a polypeptide or protein.

[0089] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0090] As used herein, the term "in vivo" refers to events that occur within an organism. The term "ex vivo" refers to events that occur outside of an organism

[0091] As used herein, the term “peptide” or "polypeptide" or “protein” or "polypeptide of interest" refers to a polymer of amino acid residues typically joined by peptide bonds that can be produced naturally (e.g., isolated or purified) or synthetically. Polypeptides include proteins and enzymes while shorter polypeptides that lack higher-order structure are terms peptides.

[0092] As used herein, “nucleic acid”, “nucleotide”, or '‘oligonucleotide” refer to polymers of nucleotides or the individual nucleotide themselves. mRNA and DNA are longer polymers of nucleotides with particular structures and functions established in the field.

[0093] The term “antibody ” is used in the broadest sense and includes monoclonal antibodies (mAb), including full length or intact monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multi-specific antibodies (e.g., bi-specific antibodies), and antibody fragments long enough to exhibit the desired binding / recognizing activity. “Antibody fragments” comprise a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind an antigen. OLSI-OOl-PCT

[0094] The term “targeting moiety” is directed to any type of molecule capable of specifically recognizing and interacting / binding with cell surface receptors or antigens whose expression may be restricted to or enriched on specific cell. In some embodiments, the targeting moiety is selected from, but not limited to: antibodies, peptides, oligonucleotides ligands, ligand-mimic, small molecules, agonists and / or antagonists. In some embodiments, the targeting moiety may be any type of antibody, or a fragment thereof. In some embodiments, the targeting antibody is a monoclonal antibody.

[0095] As used herein, "targeted cells" refers to any one or more cells of interest. The cells may be found in vitro, in vivo, in situ, or in the tissue or organ of an organism. The organism may be an animal, such as a mammal or a human.

[0096] As used herein, the term "treating" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and / or reducing incidence of one or more symptoms or features of a particular infection, disease, disorder, and / or condition. For example, "treating" cancer may refer to inhibiting survival, grow th, and / or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology' associated with the disease, disorder, and / or condition.

[0097] As used herein, “functionalized”, “conjugated”, or “coupled” refers to the act of linking a moiety to the end of a polymer chain, often by means of a covalent bond.

[0098] “Surface receptor” refers to any biologic component found on the exterior surface or layer of a cell. Surface receptors may include, but are not limited to, proteins or polysaccharides that are present on the exterior of a cell.

[0099] “Chimeric antigen receptor (CAR)” is a surface receptor protein engineered to have an external domain that binds a target antigen or protein (usualty derived from the variable region of an antibody), linked to internal domains that exert a signaling function within the cell. These internal signaling domains for T cells may be those associated with the T cell receptor. These other domains may include but are not limited to: a hinge domain, a transmembrane domain, co-stimulatory domains such as CD3^, CD28, CD137 (4-1BB), 0X40, ICOS, or combinations.

[0100] The terms “maleimide” and “maleimide moiety” may interchangeably be used and are directed to a chemical compound having the formula H2C2(CO)2NH. In some embodiments, the maleimide may be bound / conjugated / linked to another compound or polymer. OLSI-OOl-PCT

[0101] “Isotonic” refers to a solution having osmotic pressure the same or nearly the same as bodily fluids, typically achieved through the dissolution of sodium chloride or potassium chloride salts to adjust the ionic strength as required.

[0102] Lipid nanoparticles are non-viral formulations for the intracellular delivery of RNA therapeutics such as siRNA, mRNA, and DNA (Cullis & Hope, Lipid Nanoparticle Systems for Enabling Gene Therapies. Molecular Therapy (2017) 25). Additional methods for RNA encapsulation include polyplexes and liposomes.

[0103] Targeted Nanoparticles

[0104] Nanoparticles are taken up by the reticuloendothelial system and hepatocytes upon systemic administration. Following intramuscular administration, nanoparticles are taken up by antigen presenting cells, dendritic cells, and other phagocytes in the lymphatic system. These distributions are suitable for certain applications, including certain infectious disease vaccines and certain gene replacement therapies. Targeted nanoparticles are of interest as a means to redirect the biodistribution of a systemically administered cargo or to direct the uptake into a cell of interest when exposed to the targeted nanoparticles in vitro or in vivo or ex vivo. The present disclosure provides compositions and processes that are suitable for targeted nanoparticles encapsulating nucleic acids or protein cargo.

[0105] Inverse Flash NanoPrecipitation (iFNP)

[0106] The inverse Flash NanoPrecipitation (iFNP) process can be used to create “inverse” nanocarriers with encapsulated hydrophilic agents, such as water-soluble peptides, proteins, and nucleic acids like RNA, DNA, antisense oligonucleotides, miRNA, siRNA, tRNA, plasmids, and nucleotides (see FIGS. 1A-1B). iFNP uses rapid micromixing in a confined geometry of miscible solvent and antisolvent streams to effect high supersaturation of components that are soluble in the solvent and insoluble in the antisolvent. The micromixing can be achieved in various geometries. Although not intended to be limiting, two such geometries have been previously described and analyzed: the Confined Impinging Jet mixer (CI J) (Johnson, B. K., Prud’homme. R. K. Chemical processing and micromixing in confined impinging jets. AIChE Journal 2003, 49, 2264-2282) and the multi-inlet vortex mixer (MIVM) (Liu, Y., Cheng, C., Liu, Y., Prud’homme, R. K., Fox, R. O. Mixing in a multi-inlet vortex mixer (MIVM) for flash nano-precipitation. Chemical Engineering Science 2008, 63. 2829- 2842). These examples are meant to be illustrative rather than limiting or exhaustive. OLSI-OOl-PCT

[0107] In iFNP high velocity inlet streams cause turbulent mixing that occurs in a central cavity. The time for solvent / antisolvent mixing is more rapid than the precipitation of the components. In iFNP the encapsulated agents are hydrophilic, and the antisolvent is more nonpolar than the solvent. The inverse Flash NanoPrecipitation process and compositions are described in: U S. Patents 10,231,937 and 11,554,101 and 11,103,461 and 11,737,981 and 11,731.099, the corresponding international applications, as well as International Application Publications W02020018890 and W02020227350 and WO2021046078 and WO2023122331: Pagels & Prud’homme, Polymeric nanoparticles and microparticles for the delivery of peptides, biologies, and soluble therapeutics. J Control Release (2015) vol. 219; Markwaiter et al., Polymeric Nanocarrier Formulations of Biologies Using Inverse Flash NanoPrecipitation. The AAPS Journal (2020) Vol. 22; Markwaiter et al., Sustained release of peptides and proteins from polymeric nanocarriers produced by inverse Flash NanoPrecipitation. Journal of Controlled Release (2021) Vol. 334. These documents are incorporated into this application in their entirety. In some cases the term “Flash NanoPrecipitation” is used to refer to inverse Flash Nanoprecipitation. However, it should be clear from the encapsulated material, process solvent, and antisolvent whether Flash NanoPrecipitation or inverse Flash NanoPrecipitation is being used.

[0108] The inverse Flash NanoPrecipitation (iFNP) process can be used to create “inverse” nanocarriers with encapsulated hydrophilic agents, such as water-soluble peptides, proteins, and nucleic acids like RNA, DNA. antisense oligonucleotides. miRNA. siRNA, tRNA, plasmids, and nucleotides. These inverse nanocarriers have a hydrophobic surface imparted by a first stabilizing agent. The iFNP process can also include additional processing steps to produce water-dispersible “coated nanocarriers” with a hydrophilic coating imparted by a second stabilizing agent and additional features imparted through the inclusions of one or more additive agents. The additional process steps may include a solvent exchange to remove the non-polar antisolvent and introduce a water-miscible reforming solvent (if the antisolvent is not already water-miscible). The inverse nanocarriers and additional components and stabilizing agents in the reforming solvent are then rapidly micromixed using a confined mixing geometry to produce a water-dispersible “coated” nanocarrier. Thus, iFNP can refer to both the process step of producing an inverse nanocarrier and the series of steps required to produce a coated nanocarrier. OLSI-OOl-PCT

[0109] Improvements

[0110] The present disclosure provides various improvements over known techniques, including at least the following two improvements: (1) replacing the cholesterol / sterol component of lipid blends with a hydrophobic vitamin; and (2) including a targeting moiety linked to the terminal end of the second stabilizing agent or a subset of a mixture of second stabilizing agents. These improvements may be applied individually or in combination to produce coated nanocarriers or targeted coated nanocarriers.

[0111] Encapsulated Agent

[0112] In some embodiments of the process, the encapsulated agent is, for example, a hydrophilic peptide, protein, or nucleic acid. In an embodiment of the disclosure the encapsulated agent is a polynucleic acid or nucleic acid such as an oligonucleotide, dinucleotide, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), plasmid DNA, mRNA, hybrids thereof, RNAi-inducing (RNA interference-inducing) agents, short interfering RNA (siRNA). microRNA (miRNA), small hairpin RNA (shRNA), dicer substrate RNA (dsRNA), transfer RNA (tRNA), long non-coding RNA (IncRNA), guide RNA, ribozymes, catalytic DNA, aptamers, vectors, or an antisense oligonucleotide. The nucleic acid can be linear, looped, circular, double stranded, or single stranded. The mRNA may be self-amplifying mRNA. The mRNA may possess native nucleotides or be a modified mRNA containing nonnatural nucleotides. An antisense oligonucleotide can possess a modified backbone, or include locked nucleic acids, or be a gapmer. Nucleic acids including different sequences can be coencapsulated in the process.

[0113] A DNA or an mRNA may encode any polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. A polypeptide encoded by an mRNA may be of any size and may have any secondary structure or activity. In some embodiments, a polypeptide encoded by a DNA or an mRNA may have a therapeutic effect when expressed in a cell. The DNA or mRNA may contain coding and non-coding flanking regions such as 5-UTR and 3-UTR (untranslated region).

[0114] An siRNA may be capable of selectively knocking down or down regulating expression of a gene of interest. For example, an siRNA could be selected to silence a gene associated with a particular disease, disorder, or condition upon administration to a subject in need thereof of a nanoparticle composition including the siRNA. An siRNA may include a sequence that is complementary’ to an mRNA sequence that encodes a gene or protein of interest. In some embodiments, the siRNA may be an immunomodulatory siRNA. OLSI-OOl-PCT

[0115] In other embodiments, a recombinant polypeptide produced by translation of mRNA may alter the DNA of gene expression in a cell through encoding various endonucleases and accessor}' factors. Polypeptides including CRISPR (clustered regularly interspaced short palindromic repeats) associated (Cas) proteins, transcription activator-like effector nucleases (TALENs), or other endonucleases may be desirable to change a subject’s DNA to treat a disease. For example, an sgRNA-Cas9 complex can affect mRNA translation of cellular genes. In certain embodiments, a therapeutic and / or prophylactic is an sgRNA and / or Cas9 or other Cas protein variant, fragment, or subunit encoded by mRNA.

[0116] Nucleic acids and polynucleotides may include one or more naturally occurring components (either as ribonucleic or deoxyribonucleic acid), including any of the canonical nucleotides A (adenosine). G (guanosine). C (cytosine), U (uridine), or T (thymidine). In some embodiments, all or substantially all of the nucleotides including (a) the 5’-UTR, (b) the open reading frame (ORF), (c) the 3’-UTR, (d) the poly A tail, and any combination of (a, b, c, or d above) include naturally occurring canonical nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). Polynucleotides and nucleic acids may include one or more modified (e.g., altered or alternative) nucleobases. nucleosides, nucleotides, or combinations thereof. The nucleic acids and polynucleotides useful in a nanoparticle composition can include any useful modification or alteration, such as to the nucleobase, the sugar, or the intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). In certain embodiments, alterations (e.g, one or more alterations) are present in each of the nucleobase, the sugar, and the intemucleoside linkage. Alterations according to the present disclosure may be alterations of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), e.g., the substitution of the 2'-OH of the ribofuranosyl ring to 2'-H, threose nucleic acids (TNAs), glycol nucleic acids (GNAs). peptide nucleic acids (PNAs), locked nucleic acids (LNAs), or hybrids thereof. Additional alterations are described herein.

[0117] The polynucleotide may contain from about 1% to about 100% alternative nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e.. any one or more of A, G. U or C) or any intervening percentage (e.g., from 1% to 20%. from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%. from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to OLSI-OOl-PCT

[0118] 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%. from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of a canonical nucleotide (e.g., A, G, U, or C).

[0119] Non-canonical or modified bases may include, for example, one or more substitutions or modifications including but not limited to alkyl, aryl, halo, oxo, hydroxyl, alkyloxy. and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction. In some embodiments, the nucleobase is an alternative uracil. Exemplary nucleobases and nucleosides having an alternative uracil include pseudouridine (y). pyridin-4-one ribonucleoside. 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U). 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m3U), 5 -methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchnfU). 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5 -aminomethy 1-2 -thio-uracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thio-uracil (mnm5s2U), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U). 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnnfU), 5-carboxymethylaminomethyl-2 -thio-uracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (xm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil(xm5s2U), l-taurinomethyl-4-thio-pseudo uridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxy thy mine), 1-methyl-pseudouridine (mV). 1-ethyl-pseudouridine (EtV), 5 -methy 1-2 -thio-uracil (m’s2U), l-methyl-4-thio-pseudouridine (m's4'|i).

[0120] 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2-thio-l-methyl-pseudouridine, 1 -methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil. 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uraciL 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5.2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um). 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcnfUm), 5-carbamoylmethyl-2'-O-methyl- OLSI-OOl-PCT

[0121] uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3.2-0-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5 -cy anomethyluracil, 5-methoxy -2 -thio-uracil, and 5-[3-(l-E-propenylamino)]uracil.

[0122] In some embodiments, the nucleobase is an alternative cytosine. Exemplary nucleobases and nucleosides having an alternative cytosine include 5-aza-cytosine. 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C). 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1-deaza-pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytosine, 2-methoxy-5- methyl-cytosine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine. lysidine (k2C). 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4, N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine.

[0123] In some embodiments, the nucleobase is an alternative adenine. Exemplary nucleobases and nucleosides having an alternative adenine include 2-amino-purine, 2,6- diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6- chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyl-adenine (mlA), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A). 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6, N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy -adenine, N6,2'-O-dimethyl-adenosine (m6Am). N6. N6,2'-O-trimethyl-adenosine (m62Am), l,2'-O-dimethyl-adenosine (ml Am), 2-amino-N6-methyl-purine, 1 -thio-adenine, 8- OLSI-OOl-PCT

[0124] azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2.8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0125] In some embodiments, the nucleobase is an alternative guanine. Exemplary nucleobases and nucleosides having an alternative guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-dcmcthyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*). 7-deaza-guanine, queuosine (Q). epoxyqueuosine (oQ). galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQi), archaeosine (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G). 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1 -methyl-guanine (mlG), N2- methyl-guanine (m2G), N2, N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2, N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio- guanine, N2-methyl-6-thio-guanine, N2, N2-dimethyl-6-thio-guanine, N2-methyl-2'-O-methyl- guanosine (m2Gm), N2. N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl- guanosine (mlGm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm).

[0126] 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (mllm), 1-thio-guanine, and O-6-methyl-guanine.

[0127] The alternative nucleobase of a nucleotide can be independently a purine, a pyrimidine, or a purine or pyrimidine analog. For example, the nucleobase can be an alternative to adenine, cytosine, guanine, uracil, or hypoxanthine. In some embodiments, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3.4-d]pyrimi dines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2 -thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines. 5-halo, e.g., 5-bromo. 5-trifluoromethyl and other 5-substituted uracils and cytosines. 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a] 1,3,5 triazinones, 9-deazapurines, imidazo [4,5 -d] pyrazines, thiazolo[4,5-d]pyrimidines. pyrazin-2-ones, 1,2,4-triazine, pyridazine; or 1,3,5-triazine. When the nucleotides are depicted using the shorthand OLSI-OOl-PCT

[0128] A, G, C, T, or U, each leter refers to the representative base and / or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine.

[0129] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting alternative nucleotides include replacement of the oxygen in ribose (e.g., with S (sulfur), Se (selenium), or alkylene, such as methylene or ethylene); addition of a double bond (e.g.. to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino (that also has a phosphoramidate backbone)); multicyclic forms (e.g., tricyclo and “unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units atached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L- threofuranosyl-(3' 2')), and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone).

[0130] Alternative nucleotides can be altered on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. Backbone phosphate groups can be altered by replacing one or more of the oxygen atoms with a different substituent. The alternative nucleotides can include the wholesale replacement of an unaltered phosphate moiety with another intemucleoside linkage as described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be altered by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0131] The replacement of one or more of the oxygen atoms at the alpha position of the phosphate moiety (e.g., a-thio phosphate) can be provided to confer stability (such as against exonucleases and endonucleases) to RNA and DNA through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.

[0132] Polynucleotides may contain an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. A polynucleotide containing more than one functional ribosome binding site may OLSI-OOl-PCT

[0133] encode several peptides or polypeptides that are translated independently by the ribosomes (e.g., multi cistronic mRNA). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picomaviruses (e.g., FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV). and / or cricket paralysis viruses (CrPV).

[0134] A polynucleotide (e.g., an mRNA) may include a 5'-cap structure. The 5'-cap structure of a polynucleotide is involved in nuclear export and increasing polynucleotide stability and binds the mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in the cell and translation competency through the association of CBP with poly-A binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5'-proximal introns removal during mRNA splicing.

[0135] A polynucleotide or nucleic acid (e.g., an mRNA) may include a polyA sequence and / or polyadenylation signal. A polyA sequence may be formed entirely or mostly of adenine nucleotides or analogs or derivatives thereof. A polyA sequence may be a tail located adjacent to a 3’ untranslated region of a nucleic acid.

[0136] The encapsulated agent may be an interfering RNA. The term “interfering RNA” or “RNAi” or “interfering RNA sequence” refers to single-stranded RNA (e.g.. mature miRNA) or double-stranded RNA (i.e., duplex RNA such as siRNA, aiRNA (asymmetric interfering RNA), or pre-miRNA) that is capable of reducing or inhibiting the expression of a target gene or sequence (e.g., by mediating the degradation or inhibiting the translation of mRNAs which are complementary to the interfering RNA sequence) when the interfering RNA is in the same cell as the target gene or sequence. Interfering RNA thus refers to the single-stranded RNA that is complementary to a target mRNA sequence or to the double-stranded RNA formed by two complementary' strands or by a single, self-complementary strand. Interfering RNA may have substantial or complete identity to the target gene or sequence, or may include a region of mismatch (i.e.. a mismatch motif). The sequence of the interfering RNA can correspond to the full-length target gene, or a subsequence thereof.

[0137] Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g., interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, for example, about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, for example, about 20-24. 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary sequence of the double-stranded siRNA is 15- OLSI-OOl-PCT

[0138] 60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides in length, for example, about 20-24, 21-22, or 21-23 nucleotides in length, and the double-stranded siRNA is about 15-60. 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, for example about 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes may include 3' overhangs of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and 5' phosphate termini. Examples of siRNA include, without limitation, a double-stranded polynucleotide molecule assembled from two separate stranded molecules, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single stranded molecule, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acidbased linker; a double-stranded polynucleotide molecule with a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0139] In one embodiment, the nucleic acid is an antisense oligonucleotide directed to a target gene or sequence of interest. The terms “antisense oligonucleotide7’ or “antisense” include oligonucleotides such as single strands of DNA or RNA that are complementary to a chosen polynucleotide sequence. Antisense RNA oligonucleotides prevent the translation of complementary RNA strands by binding to the RNA. Antisense DNA oligonucleotides can be used to target a specific, complementary (coding or non-coding) RNA. If binding occurs, this DNA / RNA hybrid can be degraded by the enzyme RNase H. In a particular embodiment, antisense oligonucleotides include from about 10 to about 60 nucleotides, for example, from about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, an embodiment can be utilized in instances where non-target specific-activities are found with antisense, or where an antisense sequence containing one or more mismatches with the target sequence is for a particular use.

[0140] As described above, the encapsulated agent can be a polynucleic acid or nucleic acid such as an oligonucleotide, dinucleotide, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), plasmid DNA, mRNA, hybrids thereof, RNAi-inducing agents, short interfering RNA (siRNA), microRNA (miRNA), small hairpin RNA (shRNA), dicer substrate RNA (dsRNA), transfer RNA (tRNA), ribozymes, catalytic DNA, aptamers, vectors, or an antisense oligonucleotide. The encapsulated agent can be a single species or combinations of multiple types of oligonucleotides or oligonucleotides with multiple sequences. An mRNA sequence OLSI-OOl-PCT

[0141] may be co-encapsulated with a DNA sequence, or with a shorter RNA sequence. A DNA sequence may be co-encapsulated with one or more other DNA sequences, or with a short RNA sequence or oligonucleotide. The DNA sequence may encode for multiple gene products in a polycistronic format.

[0142] The encapsulated agent or agents can include non-nucleic acid species including hydrophilic small molecules, peptides, recombinant proteins, or proteins. For example, a protein encapsulated agent could be a Cas endonuclease. Non-nucleic acid species that are coencapsulated with nucleotide-based encapsulated agents may be included because they have a therapeutic benefit, or they may aid in the encapsulation, delivery, or expression of the nucleotide-based encapsulated agent. Co-encapsulated species may include a peptide with a DNA or mRNA sequence.

[0143] Stabilizing Agent

[0144] The stabilizing agents suitable to iFNP have been described in U S. Patents 10,231,937 and 11,554,101 and 11,103,461 and 11,737.981 and 11,731,099, the corresponding international applications, as well as International Application Publications W02020018890 and W02020227350 and W02021046078 and WO2023122331. The stabilizing agent used in iFNP is also referred to here as a first stabilizing agent or first stabilizing polymer. The agent can be a copolymer of a more polar block coupled with a more nonpolar (or less polar) block. The term “block” may be interpreted as either a distinct domain with a single molecular composition, or it may mean a region of the polymer chain that has regions that are predominantly more polar and other regions that are less polar. The polarity may be imparted by the monomers forming the polymer backbone or grafted pendant groups or chains attached to the main polymer backbone. For example, the copolymer may be amphiphilic (the more nonpolar block is not water soluble), however, this is not a requirement and copolymers may be fully water soluble or fully non-w ater soluble, as long as solubilities of the blocks differ significantly enough in the antisolvent to enable surface stabilization driven by assembly of the stabilizing agent on the nanoparticle surface. The copolymer should self-assemble in the antisolvent, with the more polar blocks precipitating and the more nonpolar blocks remaining soluble. When used in the iFNP process to make particles, the more polar blocks go to the core of the particle, and the more nonpolar blocks form a sterically protective shell or brush. The sterically protective shell prevents particle aggregation and prevents release of encapsulated material during subsequent processing steps. OLSI-OOl-PCT

[0145] Nanoparticles formed by the disclosed process can be formed with graft, block, or random copolymers. For example, these copolymers can have a molecular weight between about 1000 g / mole and about 1,000,000 g / mole, or between about 3000 g / mole and about 25,000 g / mole, or at least about 2000 g / mole.

[0146] The copolymers are formed of repeat units or blocks that have different solubility characteristics. For example, these repeat units can be in groups of at least two forming a block of a given character. Depending on the method of synthesis, these blocks could be of all the same repeat unit or contain different repeat units dispersed throughout the block, but still yielding blocks of the copolymer with polar and more non-polar portions. These blocks can be arranged into a series of two blocks (diblock) or three blocks (triblock), or more (multiblock), forming the backbone of a block copolymer. In addition, the polymer chain can have chemical moieties covalently attached or grafted to the backbone. Such polymers are graft polymers. Block units making up the copolymer can occur in regular intervals or they can occur randomly making a random copolymer. In addition, grafted side chains can occur at regular intervals along the polymer backbone or randomly making a randomly grafted copolymer. In graft polymers, polar blocks may be grafted on anon-polar polymer. Alternatively, non-polar blocks may be grafted on a more polar polymer chain. In graft copolymers, the length of a grafted moiety can vary. For example, the grafted segments can be equivalent to 2 to 22 ethylene units in length. The grafted hydrophobic groups which create at least one less polar region of the copolymer may include tocopherol, tocopherol derivatives, lipids, alcohols with carbon numbers from 12 to 40, cholesterols, other sterols, unsaturated and / or hydrogenated fatty acids, salts, esters or amides thereof, fatty acids, mono-, di-or triglycerides, waxes, ceramides, cholesterol derivatives, or combinations. The grafting of the polymer backbone can be useful to enhance solvation or nanoparticle stabilization properties.

[0147] The copolymer used in the compositions and methods of an embodiment may be formed of blocks of at least two repeat units or with a minimum contour length equivalent to at least 25 ethylene units. Contour lengths are the linear sum of the polymer backbone, the molecular dimensions of which can be approximated using the Polymer Handbook, 4th Edition, eds. J. Brandrup, E. H. Immergut. and E. A. Grulke, assoc, ed. A. Abe. D. R. Bloch, 1999, New York. John Wiley & Sons, which is hereby incorporated by reference in its entirety.

[0148] Examples of suitable nonpolar (or hydrophobic or less polar) blocks in a copolymer include but are not limited to the following: acrylates including methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate (BA), isobuty l acrylate, 2-ethyl acrylate, and t-butyl acrylate; methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacrylate; OLSI-OOl-PCT

[0149] acrylonitriles; methacrylonitrile; vinyls including vinyl acetate, vinylversatate, vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, vinyl phenols, and vinyllimidazole; aminoalkyls including aminoalkylacrylates, aminoalkylsmethacrylates, and aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose phthalate, poly(D, L-lactide), poly(D, L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate). poly (alkylcarbonate) and poly(orthoesters), polyesters. poly(hydroxyvaleric acid), polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides, polyphosphazenes, poly(amino acids), lactic acid, poly(lactic acid) (PLA), caprolactone, poly(caprolactone) (PCL), glycolic acid, poly(glycolic acid), and their copolymers, e.g., poly(lactic-co-glycolic acid) (PLGA); hydrophobic peptide-based polymers and copolymers based on poly(L-amino acids), poly(ethylene-vinyl acetate) (" EVA") copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene, and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl methylether and other vinyl ethers, polyamides (nylon 6.6), polyurethane, poly(ester urethanes), poly(ether urethanes), and poly(esterurea). Hydrophobically modified sugar polymers, such as HPMC (hydroxypropyl methylcellulose), acetalated dextran, or acetylated dextran can be used. For example, polymeric blocks can include poly(ethylenevinyl acetate), poly(D, L-lactic acid) oligomers and polymers, poly(L-lactic acid) oligomers and polymers, poly(gly colic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone). poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid), or polypropylene sulfide). For example, for non-biologically related applications polymeric blocks can include, for example, polystyrene, polyacrylates, and butadienes.

[0150] Natural products with sufficient hydrophobicity to act as the non-polar portion of the polymer include the following: hydrophobic vitamins (for example vitamin E, vitamin K, and vitamin A), carotenoids, and retinols (for example, beta carotene, astaxanthin, trans and cis retinal, retinoic acid, folic acid, dihydrofolate, retinylacetate, retinyl palmintate), cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol, alpha-tocopherol, alphatocopherol acetate, alphatocopherol nicotinate, estradiol, lipids, alcohols with carbon numbers from 12 to 40, cholesterols, other sterols, unsaturated and / or hydrogenated fatty acids, salts, esters or amides thereof, fatty acids, mono-, di-or triglycerids, waxes, ceramides, cholesterol derivatives, or mixtures thereof. For example, a natural product is vitamin E which can be readily obtained as a vitamin E succinate, which facilitates functionalization to amines and OLSI-OOl-PCT

[0151] hydroxyls on the active species. As will be understood, these components, including the hydrophobic vitamins, are conjugated to a polymer.

[0152] Examples of suitable polar (or hydrophilic or more polar) blocks in an amphiphilic polymer that is a block copolymer include, but are not limited to the following: carboxylic acids including acry lic acid, methacry lic acid, itaconic acid, and maleic acid; polyoxyethylenes or polyethylene oxide; polyacrylamides and copolymers thereof with dimethyl-aminoethyl-methacrylate, diallyl-dimethyl-ammonium chloride, vinylbenzyl trimethylammonium chloride, acrylic acid, methacrylic acid, 2-acryamideo-2-methylpropane sulfonic acid and styrene sulfonate, polyvinyl pyrrolidone, starches and starch derivatives, dextran and dextran derivatives; polypeptides, such as polylysines, polyarginines, polyaspartic acids, polyglutamic acids; poly hyaluronic acids, alginic acids, polylactides, polyethyleneimines, polyionenes, polyacrylic acids, and polyiminocarboxylates, gelatin, and unsaturated ethylenic mono or dicarboxylic acids. To prepare anionic copolymers, acrylic acid, methacrylic acid, and / or poly aspartic acid or glutamic acid polymers can be used. To produce cationic copolymers, DMAEMA (dimethyl aminoethyl methacrylate), polyvinyl pyridine (PVP), and / or dimethyl aminoethyl acrylamide (DMAMAM) can be used. Polar blocks of the stabilizing polymer may be non-charged, cationic, cationizable, anionic, or anionizable, or combinations of these. A listing of suitable polar, water soluble, polymers can be found in Handbook of Water-Soluble Gums and Resins, R. Davidson, McGraw-Hill (1980), which is hereby incorporated by reference in its entirety.

[0153] The nonpolar polymers and polar polymers each may be selected to have a molecular weight or be within a range of molecular weights. For example, and not to be construed as limiting, dextran, poly(aspartic acid), and poly(glutamic acid) polymers (alone or as a component of a copolymer) may have molecular weights within a range of from about 500 to about 500,000 Da, from about 500 to about 50,000 Da, or from about 750 Da to about 20,000 Da. For example, and not to be construed as limiting, PLA, PLGA, and PCL polymers (alone or as a component of a copolymer) may have molecular w eights within a range of from about 500 to about 500,000 Da, from about 500 to about 50,000 Da, or from about 750 Da to about 20.000 Da.

[0154] The lists above of nonpolar polymers and polar polymers should not be considered exclusive of one another. Copolymers of two polymers given in a single list may have sufficient differences in solubilities in a given antisolvent to be used in this process. As an illustrative example, polyethylene oxide) and poly(acrylic acid) are both given in the list of polar polymers. However, poly(ethylene oxide) is soluble in chloroform and acetone, while OLSI-OOl-PCT

[0155] poly(acrylic acid) is not. Therefore, copolymers of poly(ethylene oxide) and poly(acrylic acid) may be used in this process with chloroform or acetone as the antisolvent.

[0156] Stabilizing agent polymers can be linear polymers including one or more polar blocks and one or more nonpolar blocks. For example, the nonpolar block(s) can be poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), a polyester, a poly (ortho ester), poly [(carboxy phenoxy )propane-sebacic acid], a polyphosphoester, a polyester amide, a polyurethane, a polyvinyl acrylate, a poly(amino acid), or a hydrophobically modified polysaccharide. For example, the polar block(s) can be a poly(amino acid) such as poly(glutamic acid) (PGlu), poly(aspartic acid) (PAsp), poly(lysine), poly(arginine), poly(serine), poly (threonine), poly(glutamine), poly(asparagine). poly(cysteine), or combinations or modifications of these. For example, the polar block(s) can be a polysaccharide such as cellulose, dextran, maltodextrin, dextrin, dextran sulfate, dextrin sulfate, hyaluronic acid, pectins, amylopectin, amylose, pullulan, xylan, carrageenan, chitin, chitosan, starch, or combinations or modifications of these. For example, the stabilizing polymer can be PAsp-6-PLA. PAsp-h-PLGA, PAsp-6-PCL. PAsp-h-PLA-h-PAsp. PAsp-ft-PLGA-b-PAsp, poly(ethylene glycol)-6-PLA-h-PAsp. poly(ethylene glycol)- / >-PLGA-6-PAsp. poly(ethylene glycol)-h-PCL-h-PAsp, PGlu-6-PLA, PGlu-A-PLGA, PGlu-h-PCL, PGlu-6-PLA-h-PGIu. PGlu-6-PLGA-h-PGlu, poly(ethylene glycol)-h-PLA-h-PGlu, poly(ethylene glycol)- / :>-PLGA- / ?-PGIu. poly(ethylene glycol)- / i-PCL- / i-PGIu. or other combinations thereof. For example, the stabilizing polymer can be a polysaccharide-h-PLGA. a polysaccharide-h-PLA, a polysaccharide- / >-PCL, or a higher order block copolymer composed of polysaccharide and PLA, PCL, and / or PLGA blocks.

[0157] Stabilizing agents that are block copolymers include poly(styrene)- / ?-poly(acrylic acid), poly(lactic acid)- / >-poly(aspartic acid). poly(lactic acid-co-glycolic acid)-h-poly(aspartic acid), polyethylene glycol)-6-poly(lactic acid)-Z?-poly(aspartic acid), poly(aspartic acid)- / >-poly(lactic acid-co-glycolic acid)- -poly(aspartic acid), dextran- / >-poly(lactic acid), and dextran-d-poly(lactic-co-gly colic acid).

[0158] In an embodiment, the stabilizing polymer is a comb polymer of or including a hydrophilic backbone and hydrophobic branches or grafts. In an embodiment, the backbone of the comb polymer can be branched, like a dextran or a poly(aspartic acid) produced through condensation polymerization. In an embodiment, the backbone of the comb polymer can be linear, like cellulose. In an embodiment, the hydrophilic backbone of the comb polymer is a polysaccharide. For example, the backbone can be cellulose, dextran, maltodextrin, dextrin, dextran sulfate, dextrin sulfate, hyaluronic acid, pectins, amylopectin, amylose, pullulan, xylan, OLSI-OOl-PCT

[0159] carrageenan, chitin, chitosan, starch, or combinations or modifications of these. In an embodiment, the hydrophilic backbone of the comb polymer is a poly(amino acid) such as poly(glutamic acid), poly(aspartic acid), poly(lysine), poly(arginine), poly(serine), poly(threonine), poly(glutamine), poly(asparagine), poly(cysteine), or combinations or modifications of these. In an embodiment, the backbone is polyvinyl alcohol. In an embodiment, the nonpolar side chains are poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL). a polyester, a poly(ortho ester). poly[(carboxyphenoxy)propane-sebacic acid], a polyphosphoester, a polyester amide, a polyurethane, a polyvinyl acrylate, a poly(amino acid), or a hydrophobically modified polysaccharide. The comb polymer may have between 20, 30, 40, 50, 60, 70, or 80wt% and 70, 80, 90, 95wt% nonpolar side chains. The backbone of the comb polymer can be between 500 Da, 1000 Da, 2500 Da, 5000 Da, 10000 Da, or 20000 Da and 20000 Da, 50000 Da, 100000 Da, or 500000 Da. Each nonpolar side chain or graft can be between 100, 500, 1000, 5000 Da, 10000 Da, or 20000 Da and 20000 Da, 50000 Da, 100000 Da, or 500000 Da. The comb polymer may have between 20, 30, 40, 50, 60, 70, or 80wt% and 70, 80, 90, 95wt% nonpolar side chains. Someone skilled in the art will recognize that the number of nonpolar side chains per polar backbone that will result in the proper wt% of nonpolar groups will depend on the molecular weights of each species. For example, the stabilizing polymer can be a dextran-graft-PLGA, dextran-graft-PLA, dextran-graft-PCL, or another polysaccharide with PLA, PCL, or PLGA grafted on. For example, the stabilizing comb polymer can be PAsp-graft-PLA, PAsp-graft-PLGA, PAsp-graft-PCL or another poly(amino acid) with PLA, PLGA, or PCL grafted on.

[0160] Random copolymers include hydroxypropyl cellulose, methyl cellulose, ethyl methyl cellulose, hydroxypropylmethylcellulose, carboxymethyl cellulose, or a combination of these. A cellulosic polymer can include hydroxypropyl, hydroxyethyl, hydroxymethyl, succinate, and / or acetate substitution(s). In another embodiment, poly(meth)acrylate-based random copolymers are used as stabilizers. Eudragit polymers produced by Evonik Industries are one commercialized version of these. However, embodiments are not limited to the aforementioned random copolymers. Stabilizing random copolymers may have molecular weights ranging from about 0.1 kDa, 1 kDa, 10 kDa, 100 kDa, or 1000 kDato about 5 kDa, 50 kDa, 100 kDa, 1000 kDa or greater.

[0161] The first stabilizing agent may be a blend of different stabilizing agents.

[0162] Salts OLSI-OOl-PCT

[0163] Other non-liquid compounds that aid in the solvent quality of the streams may be added and are also considered part of the solvent or antisolvent. For example, a surfactant, a salt, or a cosolvent may be added to a solvent and considered part of the solvent. These excipient compounds may or may not be in the coated nanocarrier, depending on the requirements of the final product.

[0164] Patent application W02021046078 describes suitable salt forms of the nucleic acid or polynucleic acid. The protonated (free acid) form may be used, or a form of the nucleic acid that is partially (1%, 5%, 10%, 15%, 25%, 50%, 75%, 99% of acid groups neutralized) or fully neutralized with sodium hydroxide, lithium hydroxide, Tris (tris(hydroxymethyl)aminomethane), triethylamine, ammonia, cesium hydroxide, potassium hydroxide, choline, thiamine, pyroxidine, urea, guanidine, diphenhydramine, a primary amine, a secondary amine, a quaternary amine, or a tertiary amine may be used.

[0165] Any method known to the field may be used to modify the salt form of the nucleic acid. The nucleic acid may be neutralized in situ in the solvent stream. The nucleic acid may be neutralized in a salt exchange process and then purified and isolated prior to dissolution in the solvent. The nucleic acid salt form may be modified after in vitro transcription by buffer selection.

[0166] Crosslinking of acidic residues within a stabilizing copolymer, such as poly(aspartic acid) or poly(acrylic acid) or poly(glutamic acid), have been described (US Patents 10,231,937 and 11,103.461 and Pagels, R. F.; Prud’homme. R. K., Polymeric nanoparticles and microparticles for the delivery of peptides, biologies, and soluble therapeutics. J Control Release 2015, vol. 219, 519-535, these documents are hereby incorporated herein in their entirety). Suitable crosslinkers include but are not limited to salt forms of calcium, iron, zinc, manganese, divalent cations, and trivalent cations. Polyvalent amines, such as spermine, tetraethylene pentaamine, protamine, poly(lysine), or other organic amines may be used.

[0167] For example, the nanoparticle can be crosslinked during assembly of the nanoparticle. For example, the nanoparticle can be crosslinked after assembly of the nanoparticle. The crosslinking can be covalent crosslinking. For example, the crosslinking can be disulfide crosslinking. For example, the crosslinking can occur through "‘Click Chemistry’; The crosslinking can involve cleavable ester linkage of the types described in USP application 13 / 969,449, Particulate Constructs for Release of Active Agents, Law rence Mayer, et al. The crosslinking can be non-covalent. For example, the crosslinking can be ionic, chelation, acidbase, or hydrogen bonding crosslinking. OLSI-OOl-PCT

[0168] A crosslinking agent can be added to crosslink the copolymer. For example, the crosslinking agent can be added to crosslink groups of the copolymer having anionic functionality or character. For example, the crosslinking agent can be an alkaline earth halide, a magnesium halide, magnesium chloride, a calcium halide, calcium chloride, a transition metal halide, zinc halide, an iron halide, iron(III) chloride, spermine, or combinations. For example, the crosslinking agent can be a metal acetate, an alkaline earth acetate, a transition metal acetate, calcium acetate, or combinations. For example, the crosslinking agent can be chromium(III) acetate, or another chromium (III) salt. For example, the crosslinking agent can be a metal nitrate, an alkaline earth nitrate, a transition metal nitrate, calcium nitrate, zinc nitrate, iron nitrate, or combinations. Other bio-compatible multi-cationic water-soluble agents may be used as crosslinking agents, for example, to crosslink anionic sections of the copolymer. For example, the water-soluble agent can include tobramycin and the tobramycin can crosslink the copolymer. One example is tetraethylene pentamine. Ammonia or another chemical with basic character can be added to promote ionic interactions between the cationic crosslinker and the hydrophilic groups of the copolymer, if they have anionic functionality.

[0169] Alternatively, the shell of the nanoparticle may be crosslinked to enhance nanoparticle stability. For example, the end groups or other groups of the hydrophobic (non-polar) polymer chains or blocks can be crosslinked. For example, the crosslinking can be ionic or covalent. For example two or more hydrophobic polymer chains may be crosslinked directly or through the addition of a multifunctional crosslinker. For example, a hydrophobic polymer shell that contains groups with functionality “X” (on the chain end or throughout the chain) that is reactive to functionality “Y” may be crosslinked using a crosslinker that has tw o or more “Y” functionality groups. For example, the X and Y functional groups can be thiols and acrylates, thiols and maleimides, azides and alkynes, amines and acids, or vice versa. Functional groups X and Y can be groups that are reactive through '‘click chemistry.’’ Added catalysts or other agents may be needed or used to induce the crosslinking of the shell.

[0170] The crosslinking agent may also be used to neutralize charge on the nucleic acid. In the absence of an acidic residue on the stabilizing copolymer, the crosslinking agent may still be included in the process.

[0171] Solvent

[0172] Suitable antisolvents have been described in US Patents 10,231,937 and 11,103,461. Examples of polar process solvents include, but are not limited to, water, alcohols, acetone, acetonitrile, glycol ethers, dimethyl sulfoxide (DMSO), dimethylformamide, N-methyl-2- OLSI-OOl-PCT

[0173] pyrrolidone, dihydrolevoglucosenone, glycofurol, and mixtures thereof. In an embodiment, the encapsulated agent is dissolved in a process solvent or mixture. The stabilizing agent is dissolved in the same process solvent or mixture, or may be dissolved in a different process solvent. The solutions of encapsulated agent and stabilizing agent may be combined to form a single solution or may be maintained separately until the mixing process. The polar process solvent can be heated or pressurized or both to facilitate dissolution of the encapsulated agent and the stabilizing agent, depending on the dissolution characteristics. Selection of the polar process solvent is informed by the solubility characteristics of the encapsulated agent and the stabilizing agent. The polar process solvent containing the stabilizing agent is chosen such that the stabilizing agent is molecularly dissolved. This means that the process solvent solubilizes all parts of the stabilizing agent. The process solvent containing the encapsulated agent, if present, is also chosen such that material is molecularly dissolved. These process solvents may be, but are not required to be, the same. In some cases, both the stabilizing agent and the encapsulated agent may be dissolved in a single solution of the process solvent.

[0174] The concentration of the encapsulated agent may be within an order of magnitude of the concentration of the stabilizing agent. If the concentration of the encapsulated agent is much lower than the concentration of the stabilizing agent, then the final drug loading may be low (small). If the concentration of the encapsulated active is much higher than the concentration of the stabilizing agent, then the inverse nanoparticles may not be sufficiently stabilized to prevent aggregation. In an embodiment, encapsulated agents that are poorly soluble in the antisolvent are coated, encapsulated, or confined as a core component and sterically stabilized by stabilizing agent.

[0175] A stabilizing agent can be dissolved in a polar process solvent at a concentration of at least 0.01% by weight; the concentration of stabilizing agent can be at least 0.1% by weight to form a first process solution. In an embodiment, the stabilizing agent can be dissolved in the polar process solvent at a concentration in a range of from about 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, or 20 wt% to about 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, 20 wt%, or 40 wt%. A person of skill in the art will appreciate that a factor such as the economics of a process can constrain a lower bound of concentration, and that factors such as the viscosity of the process solution or the solubility limit of the copolymer in the polar process solvent can constrain an upper bound of concentration. For example, if the viscosity of the first process solution is much greater than that of the antisolvent, mixing of the first process solution with the antisolvent may be inhibited. A person of skill in the art will appreciate that factors such as the molecular weight of the copolymer and the composition of OLSI-OOl-PCT

[0176] the copolymer can affect the maximum concentration that can be attained in the polymer solution before the viscosity becomes too high (large).

[0177] In an embodiment, the encapsulated agent can be dissolved in the polar process solvent at a concentration in a range of from about 0.01 wt%, 0.1 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, or 20 wt% to about 0.2 wt%, 0.5 wt%, 1 wt%, 2 wt%, 5 wt%, 10 wt%, 20 wt%, or 40 wt%. A person of skill in the art will appreciate that a factor such as the economics of a process can constrain a lower bound of concentration, and that factors such as the viscosity of the process solution or the solubility limit in the polar process solvent can constrain an upper bound of concentration.

[0178] In an embodiment of the process, the solvent is DMSO (dimethylsulfoxide). In an alternative embodiment, the DMSO is in a mixture with water. For example, the water content in the DMSO and water mixture may be 0.5 volume percent (%vol), 1 %vol, 2.5 %vol, 5 %vol, 7.5 %vol, 10 %vol, 15 %vol, 20 %vol, or 25 %vol.

[0179] Antisolvent

[0180] In an embodiment, suitable antisolvents are more non-polar than the process solvent and have been described in US Patents 10,231,937 and 11,103,461. Examples include chloroform, dichloromethane, alcohols, alkanes such as hexane, tetrahydrofuran (THF), toluene, ethyl acetate, methyl acetate, acetonitrile, acetone, or combinations. Selection of the antisolvent is driven by the solubility characteristics of the stabilizing agent and the encapsulated agent. The encapsulated agent becomes supersaturated under the mixing conditions and precipitates from solution. Additionally, the antisolvent is selected such that the dissimilar solubility characteristics of regions or portions of the stabilizing agent are manifested, and the more polar portions of the copolymer can no longer exist in the soluble state, so that the stabilizing agent assembles onto the encapsulated agent and precipitates and produces a stable “inverse” nanocarrier. It is important to note that process solvents of one system may work well as the antisolvent in another system; thus, the listing above of an example as a process solvent should not be considered to exclude its use as an antisolvent, and the listing above of an example as an antisolvent should not be considered to exclude its use as a process solvent.

[0181] The antisolvent is chosen such that the more polar sections of the stabilizing agent rapidly precipitate while the more non-polar components of the stabilizing agent remain solubilized. Thus, the stabilizing agent can self-assemble into the desired nanoparticle form in the antisolvent. The antisolvent is chosen such that the encapsulated agent rapidly precipitates OLSI-OOl-PCT

[0182] in the final mixture. In some cases the process solvent and antisolvent can be fully miscible at the final composition. In some cases, no more than 20 volume percent of the process solvent may phase separate in the final composition.

[0183] In an embodiment, the crosslinking agent or salt can be incorporated into the antisolvent. In some embodiments, the crosslinking agent can be dissolved first in a secondary antisolvent. For example, a solution in methanol could be produced and then added to the dichloromethane antisolvent. The secondary antisolvent can be 1 vol%. 2 vol%, 5 vol%, 10 vol%, 20 vol% or higher (greater) within the antisolvent composition. The concentration of the crosslinking agent is selected to be a desired charge equivalent wi th respect to the total acid residue composition of the encapsulated agent and the stabilizing agent. The charge ratio is a molar ratio of the positive charge from the crosslinking agent to the negative charge from all acid residues. The crosslinking charge may be 0.1, or 0.2, or 0.25, or 0.3, or 0.5, or 0.6, or 0.7, or 0.75, or 0.9, or 1, or 1.2, or 1.5, or 2, or higher (greater) with respect to the acid charge.

[0184] Mixing

[0185] The intense micromixing of the process solution and the antisolvent can be effected in various geometries. The high velocity inlet streams cause turbulent flow and mixing that occurs in a central cavity7. The time for process solvent / antisolvent mixing is more rapid than the assembly time of the nanoparticles. While not meant to be limiting, two such geometries have been previously described and analyzed: The Confined Impinging Jet mixer (CIJ) (Johnson, B. K., Prud’homme, R. K. Chemical processing and micromixing in confined impinging jets. AIChE Journal 2003, 49, 2264-2282) and the multi-inlet vortex mixer (MIVM) (Liu, Y., Cheng, C., Liu, Y., Prud’homme. R. K., Fox, R. O. Mixing in a multi-inlet vortex mixer (MIVM) for flash nano-precipitation. Chemical Engineering Science 2008, 63. 2829- 2842). These examples are meant to be illustrative rather than limiting or exhaustive.

[0186] The fast mixing and high energy^ dissipation involved in the iFNP process provide mixing timescales that are shorter than the timescale for nucleation and grow th of particles, which leads to the formation of nanoparticles with active agent loading contents and size distributions not provided by other technologies. When forming the nanoparticles via inverse Flash NanoPrecipitation (iFNP), mixing occurs fast enough to allow high supersaturation levels, for example, as high as 10,000, of all components to be reached prior to the onset of aggregation. The supersaturation level is the ratio of the actual concentration of a material, for example, an encapsulated agent, in a solvent to the saturation concentration of that material in that solvent. For example, the supersaturation levels can be at least about 1, 3, 10, 30, 100, OLSI-OOl-PCT

[0187] 300, 1000, or 3000 and can be at most about 3, 10, 30, 100, 300, 1000, 3000, 10,000, 30,000, or 100.000. The timescales of aggregation of the encapsulated agent active material and copolymer self-assembly may be balanced. Therefore, the hydrophilic active material and polymers may precipitate simultaneously, and overcome the limitations of low encapsulated agent incorporations and aggregation found with other techniques based on slow solvent exchange (e.g., dialysis). The inverse Flash NanoPrecipitation process may be insensitive to the chemical specificity of the components, so that it is a broadly applicable nanoparticle formation technique.

[0188] In an embodiment, the encapsulated agent and stabilizing agent are dissolved in a single process solvent stream or solution. This stream is then rapidly mixed with an antisolvent in a mixer such that the time for solvent / antisolvent mixing is more rapid than the assembly time of the nanoparticles, for example, as described in WO 2017 / 112828 Al, WO 2015 / 200054, and US Patent 8,137,699. A stream is synonymous with a solution phase used as the input to the mixing process. The single process solvent stream may alternatively be mixed against more than one antisolvent stream. The flow rates of the solvent and antisolvent streams may be the same or different. The flow rates may be varied to further tune the solvent / antisolvent composition in the central cavity of a mixer. For example, the composition in the central cavity of the mixer can be 50 vol% solvent or less, or 40 vol% solvent or less, or 25 vol% solvent or less, or 10 vol% solvent or less. In an embodiment, the crosslinking agent, if included, may be dissolved in all antisolvent inlet streams. In an embodiment, the crosslinking agent, if included, may be dissolved in a single antisolvent stream or in multiple antisolvent streams.

[0189] In an embodiment, the encapsulated agent and stabilizing agent are dissolved in separate process solvent streams. The process solvent used to dissolve the stabilizing agent and the process solvent used to dissolve the encapsulated agent may be. but are not required to be, the same. For example, the encapsulated agent can be dissolved in a first polar process solvent to form an encapsulated agent solution, and the stabilizing agent can be dissolved in a second polar process solvent to form a stabilizing agent solution. The encapsulated agent and stabilizing agent solutions, are mixed, e.g., simultaneously mixed, with the antisolvent to form a mixed solution. The first polar process solvent and the second polar process solvent can be miscible, or they can be completely miscible (i.e., so that another phase is not formed) at the volumetric ratios at which they are mixed. The first polar process solvent and the antisolvent can be miscible, or they can be completely miscible (i.e., so that another phase is not formed) at the volumetric ratios at which they are mixed. The second polar process solvent and the OLSI-OOl-PCT

[0190] antisolvent can be miscible, or they can be completely miscible (i.e.. so that another phase is not formed) at the volumetric ratios at which they are mixed.

[0191] A person skilled in the art will recognize that all solvents are miscible to some degree in each other. Miscible solvents are used in the initial nanoparticle precipitation process. “Miscible” solvents as referred to herein are those that when mixed at the ratios used in the process would produce solutions that have no more than 20% of the volume of the minor phase (e.g.. a polar process solvent) not dissolved in the majority phase. Completely miscible solvents as referred to herein are those that when mixed at the ratios used in the nanoparticle formation process, the microparticle process, or another process would produce solutions with no phase separation. “Immiscible” solvents as referred to herein are those that when mixed at the ratios used in the process would produce solutions that have 20% or more of the volume of the minor phase not dissolved in the majority phase.

[0192] Nanoparticles can be produced from copolymers that are dissolved in a process solvent with no hydrophilic active material added.

[0193] Additional antisolvent may be added after the micromixing process to adjust the final composition of the solvent mixture as desired. The additional antisolvent may be added by inline mixing or it may already be present in the collection vessel for the outlet of the mixer. The final composition of the mixture may be adjusted so that the process solvent content is 25 vol% or less, or 20 vol% or less, or 15 vol% or less, or 10 vol% or less, or 5 vol% or less. This may be referred to as the quench or the collection bath or the dilution bath.

[0194] The size of the resulting nanoparticles from this process can be controlled by controlling the mixing velocity used to create them, the total mass concentration of the encapsulated agent and the stabilizing agent in the process solvent, the process solvent(s) and antisolvent(s), the ratio of the encapsulated agent and the stabilizing agent, and the supersaturation of the encapsulated agent and the stabilizing agent upon mixing with the antisolvent.

[0195] In a method, particles can be made that have sizes in the range of 15 nm to 10500 nm, sizes in the range of 20 nm to 6000 nm, sizes in the range of 20 nm to 1000 nm, sizes in the range of 35 nm to 400 nm. or sizes in the range of 40 nm to 300 nm. Sizes can be determined by dynamic light scattering. For example, particles can be made that have sizes of at least about 15 nm, 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, or 6000 nm, and have sizes of at most about 20 nm, 35 nm, 40 nm, 50 nm. 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, 6000 nm, or 10500 nm. Sizes reported and cited herein are the intensity average reported values as determined by the Malvern Nanosizer deconvolution program for particles smaller OLSI-OOl-PCT

[0196] than 2000 nm, and determined by scanning electron microscopy. Other intensity weighted deconvolution methods can be used to determine sizes of the nanoparticles.

[0197] Additive agents

[0198] In an embodiment, additive agents are added to the inverse nanocarrier and processed such that they assemble onto the surface of the inverse nanocarrier. Additive agents may be added before and / or after solvent exchange into the water-miscible reforming solvent. The exchange into a reforming solvent can be achieved by distillation or dialysis or other techniques known to the field. The exchange to an aqueous system can subsequently be achieved by rapidly mixing the organic stream containing components to be assembled with an aqueous antisolvent.

[0199] An embodiment employs additive agents that are a lipid blend of a cationic ionizable lipid, phospholipid (neutral helper lipid), and hydrophobic vitamin instead of cholesterol.

[0200] Phospholipids useful in the compositions and methods may be selected from the nonlimiting group consisting of l,2-distearoyl-s-glycero-3-phosphocholine (DSPC). 1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1.2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (Cl 6 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-gly cero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3- phosphocholine. l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamme, 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-oleoyl-phosphatidylcholine (SOPC), 1-Palmitoyl-2-lauroyl-sn-glycero-3-phosphocholine (PLPC), 1 -myristoyl-2-stearoyl-sn-glycero-3 -phosphocholine (MSPC), l-stearoyl-2-myristoyl-sn-glycero-3 -phosphocholine (SMPC), OLSI-OOl-PCT

[0201] sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol. phosphatidic acid. palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), and mixtures thereof. In some embodiments, a nanoparticle composition includes POPC. In certain embodiments, a nanoparticle composition includes DOPC. In some embodiments, a nanoparticle composition includes PLPC.

[0202] The phospholipid may be selected from anionic lipids. The term ‘"anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0203] The cationic ionizable lipid may be one known to the field. The cationic ionizable lipid may be permanently ionized, that is, it may be a quaternary amine bearing a permanent charge to form a cationic lipid. Such lipid blends have been widely described in publications and patents such as US Pat. 8,058,069, US Pat. 9,364,435, US Pat. 10,221,127, W02018170306A1, and W02020061367A1. This listing of patents and applications is not intended to be exhaustive, but rather to provide representative examples of suitable lipid blends.

[0204] A cationic lipid or cationic ionizable lipid may be selected from any list known in the field. See, for example, US Pat. 10,221,127, International Application Publications W02018170306A1 and W02020061367A1, U. S. Patent Publication Nos. 20060083780, 20060240554, and 20210101875, U. S. Pat. Nos. 5,208,036, 5,264,618. 5,279,833, 5,283,185, 5,753,613, and 5,785,992, WO2019246203A1, and US Pats. 10,562,849. US 10.888,626, and US 5,885,613. The term ‘‘cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). In some cases, the cationic lipids include a protonatable tertiary7amine (e.g., pH titratable) head group, alkyl chains, ether or ester linkages between the head group and alkyl chains, and 0 to 3 double bonds. In other cases, the cationic lipid contains branched lipid tails formed of saturated alkyl chains, such as those reported in W02020061367A1. Other cationic lipid variations include thiourea and squaramide moieties at the head group. Commonly used cationic lipids are summarized in the literature (Hou, Zaks. Langer, and Dong. Nature Reviews Materials, 6, 1078-1094, 2021. Kauffman, Webber, and Anderson. Journal of Controlled Release, 240. 227-234, 2016) and include Dlin-MC3-DMA, SM-102, ALC-0315, ePC, C12-200, cKK-E12, OF- OLSI-OOl-PCT

[0205] Deg-Lin, A2-iso5-2DC18, 3060iio, 503013, BAME-O16B, TT3, and FTT5. Cationic lipids can be multivalent, such as MVL5 and GL67.

[0206] Additional cationic lipids include: l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N, N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (Dlin-KC2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[l,3]-dioxolane (Dlin-KC3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[ 1.3]-dioxolane (Dlin-KC4-DMA). 2,2-dilinoleyl-5-dimethylaminomethyl-[l,3]-dioxane (Dlin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (Dlin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (Dlin-K-DMA), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (Dlin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (Dlin-DAC), l,2-dilinoleyoxy-3-morpholinopropane (Dlin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (Dlin-S-DMA), 1 -linoleoyl-2-linoleyloxy-3-dimethylaminopropane (Dlin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (Dlin-TMA), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (Dlin-TAP). 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (Dlin-MPZ). 3-(N, N-dilinoleylamino)-l,2-propanediol (DlinAP), 3-(N, N-dioleylamino)-l,2-propanedio (DOAP), l,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (Dlin-EG-DMA), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), l,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethylaminopropane (DSDMA). N-(l-(2,3-dioleyloxy)propyl)-N, N, N-tri methyl ammonium chloride (DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), 3-(N — (N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N, N-dimethyl-l-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA). N, N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N, N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), l,2-N, N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, Dlin-KC2-DMA (”XTC2"). 4-(dimethylamino)-butanoic acid. (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10, 13 -nonadecadi en-l-yl ester (Dlin-MC3-DMA), BP Lipid 323, 5 -(dimethylamino)- pentanoic acid, (6Z)-l,2-di-(4Z)-4-decen-l-yl-6-dodecen-l-yl ester (CL1), Dlin-MC4-DMA, BP Lipid 338, lipid 306-012B-3, BP lipid 399, BP Lipid 363, BP Lipid 337, BP Lipid 336, BP Lipid 418, BP Lipid 339, BP Lipid 368, BP Lipid 335, BP Lipid 377, BP Lipid 401, BP Lipid 338, or mixtures thereof. In addition to these, a cationic lipid may also be a lipid including a cyclic amine group.

[0207] Cationic ionizable lipid structures are as follows.

[0208] For BP Lipid 323:

[0209] For Dlin-MC4-DMA (CAS 1226909-66-5):

[0210] -......... For DLin-KC2-DMA (CAS 1190197-97-7):

[0211] For BP Lipid 338:

[0212] For 306-O12B-3:

[0213] For BP Lipid 399 (CAS 1799316-96-3):

[0214]

[0215] OLSI-OOl-PCT

[0216] For SM-102 (CAS 2089251-47-6):

[0217]

[0218] For ALC-0315 (CAS 2036272-55-4)

[0219] For BP Lipid 363:

[0220]

[0221] For BP Lipid 337: OLSI-OOl-PCT

[0222] Q N

[0223] For BP Lipid 336:

[0224]

[0225] For BP Lipid 418:

[0226] For BP Lipid 339:

[0227] O

[0228]

[0229] For BP Lipid 368:

[0230] For BP Lipid 335:

[0231]

[0232] OLSI-OOl-PCT

[0233] For BP Lipid 401 (CAS 2510781-15-2):

[0234] 0

[0235]

[0236] The hydrophobic vitamin may be selected from Vitamin D. Vitamin E. Vitamin A, or Vitamin K, their analogs, or their related compounds.

[0237] The hydrophobic vitamin may be selected from the vitamin D group or related compounds (Vitamin Di, D2, D3, D4, or Ds or ergocalciferol, cholecalciferol. 22- dihydroergocalciferol, sitocalciferol. 25-hydroxycholecalciferol, calcitriol, alfacalcidol, doxercalciferol, 5,6-trans-Ergocalciferol, dihydrotachysterol, 24R,25-Dihydroxyvitamin D3, 22-oxacalcitriol, falecalcitriol, or paricalcitol or related compounds and derivatives).

[0238] The hydrophobic vitamin may be selected from the vitamin E group or related compounds (alpha-tocopherol, beta-tocopherol, gamma-tocopherol, delta-tocopherol. alpha- tocotrienol, beta- tocotrienol, gamma- tocotrienol, delta- tocotrienol, tocopheryl acetate (vitamin E acetate), tocopherol acetates, tocopherol esters, tocotrienol or related compounds and derivatives).

[0239] The hydrophobic vitamin may be selected from vitamin A or related compounds (retinoids, carotenoids, retinol, retinaldehydes, hydroxy-substituted retinoids, retinoid ketones, dimethyl retinoid species, 3-Dehydro Retinol; retinoid stereoisomers including but not limited OLSI-OOl-PCT

[0240] to 9-cis, 11 -cis, 13-cis, trans or certain combinations thereof, all of which may be found in Retinol, Retinaldehyde, Retinoic acid or various retinyl esters such as. retinyl acetate, retinyl linoleate, retinyl palmitate, retinyl stearate and retinyl propionate, alpha-carotene, betacarotene, gamma-carotene, or delta-carotene, lycopene, or related compounds and derivatives).

[0241] The hydrophobic vitamin may be selected from the vitamin K group or related compounds (Vitamin Ki, K2, or K3, menatetrenone, or related compounds and derivatives).

[0242] Second stabilizing agents

[0243] The second stabilizing agent may include PEG-modified lipids, PEG-containing block copolymers, PEG-ft-PLA (PLA-PEG), PEG-6-PLGA (PLGA-PEG), PEG-6-PCL (PCL-PEG), dextran-modified lipids, dextran-containing block copolymers. poly(sarcosine)-modified lipids, poly(sarcosine)-containing block copolymers, poly(sarcosine)-6-PLA, poly(sarcosine)-A-PLGA. and / or poly(sarcosine)- / )-PCL. The stabilizing agent has a water-soluble (or hydrophilic) region and a hydrophobic region.

[0244] The second stabilizing agent can be a copolymer of a hydrophilic block coupled with a hydrophobic block, for example, the second stabilizing agent can be a second stabilizing amphiphilic agent or second stabilizing amphiphilic copolymer. Inverse nanocarriers coated by the disclosed process can be coated with graft, block, or random amphiphilic copolymers. These amphiphilic polymers can have a molecular weight of between about 1000 g / mole and about 50.000 g / mole, between about 3000 g / mole and about 25,000 g / mole. or at least about 2000 g / mole.

[0245] Examples of suitable hydrophobic blocks in an amphiphilic polymer that is a block copolymer include, but are not limited to the following: acrylates including methyl acry late, ethyl acry late, propyl acrylate, n-butyl acrylate (BA), isobutyl acrylate, 2-ethyl acry late, and t-butyl acrylate; methacrylates including ethyl methacrylate, n-butyl methacrylate, and isobutyl methacry late; acrylonitriles; methacry lonitrile; vinyls including vinyl acetate, vinyl versatate, vinylpropionate, vinylformamide, vinylacetamide, vinylpyridines, vinyl phenols and vinylimidazole; aminoalkyls including aminoalkylacrylates, aminoalkydmethacrylates, and aminoalkyl(meth)acrylamides; styrenes; cellulose acetate phthalate, cellulose acetate succinate, hydroxypropy lmethylcellulose phthalate, poly(D, L lactide), poly (D, L-lactide-co-glycolide), poly(glycolide), poly(hydroxybutyrate). poly(alkylcarbonate) and poly(orthoesters), polyesters, poly(hydroxyvaleric acid), polydioxanone, poly(ethylene terephthalate), poly(malic acid), poly(tartronic acid), polyanhydrides, polyphosphazenes, poly(amino acids), and their copolymers; hydrophobic peptide-based polymers and copolymers OLSI-OOl-PCT

[0246] based on poly(L-amino acids), poly(ethylene-vinyl acetate) (“EVA”) copolymers, silicone rubber, polyethylene, polypropylene, polydienes (polybutadiene, polyisoprene, and hydrogenated forms of these polymers), maleic anhydride copolymers of vinyl methylether and other vinyl ethers, polyamides (nylon 6,6), polyurethane, poly(ester urethanes), poly(ether urethanes), and poly(esterurea). For example, polymeric blocks include poly(ethylenevinyl acetate), poly(D. L-lactic acid) oligomers and polymers, poly(L-lactic acid) oligomers and polymers, poly(glycolic acid), copolymers of lactic acid and glycolic acid, poly(caprolactone). poly(valerolactone), polyanhydrides, copolymers of poly(caprolactone) or poly(lactic acid).

[0247] Natural products with sufficient hydrophobicity to act as the hydrophobic portion of the amphiphilic polymer include, for example, hydrophobic vitamins (for example, vitamin E, vitamin K, and vitamin A), carotenoids, sterols, cholesterols, and retinols (for example beta carotene, astaxanthin, trans- and cis-retinal, retinoic acid, folic acid, dihydrofolate, retinylacetate, retinyl palmintate), cholecalciferol, calcitriol, hydroxycholecalciferol, ergocalciferol, alpha-tocopherol, alpha-tocopherol acetate, alpha-tocopherol nicotinate, and estradiol.

[0248] Examples of suitable hydrophilic blocks in an amphiphilic polymer include but are not limited to the following: carboxylic acids including acrylic acid, methacrylic acid, itaconic acid, and maleic acid; polyoxyethylenes or polyethylene oxide; polyacrylamides and copolymers thereof with dimethylaminoethylmethacrylate, diallyldimethylammonium chloride, vinylbenzyltrimethylammonium chloride, acrylic acid, methacrylic acid, 2-acrylamido-2-methylpropane sulfonic acid and styrene sulfonate, polyvinyl pyrrolidone, starches and starch derivatives, dextran and dextran derivatives; polypeptides, such as poly(lysine), poly(arginine), poly(glutamic acid), poly(sarcosine); polyhyaluronic acids, alginic acids, polylactides, polyethyleneimines, polyionenes, polyacrylic acids, and polyiminocarboxylates, poly(ethylene glycol), gelatin, and unsaturated ethylenic mono or dicarboxylic acids. For example, the hydrophilic blocks can be of poly(ethylene glycol). To produce cationic amphiphilic polymers DMAEMA (dimethylaminoethylmethacrylate), polyvinyl pyridine (PVP), chitosan, poly (lysine), poly(ethylenimine) or dimethylaminoethylacrylamide (DMAMAM) can be used. A representative list of suitable cationic polymers may be found in the literature, such as Kauffman, Webber, and Anderson. Journal of Controlled Release, 240, 227-234 (2016), and a further listing of suitable hydrophilic polymers can be found in Handbook of Water-Soluble Gums and Resins. R. Davidson, McGraw-Hill (1980); these documents are hereby incorporated by reference herein in their entirety. OLSI-OOl-PCT

[0249] The polymer blocks can be diblock, triblock, or multiblock repeats. For example, the amphiphilic polymer can be polystyrene-block-poly(ethylene glycol) (PS-ft-PEG), poly(lactic acid)-block-poly(ethylene glycol) (PLA-h-PEG). poly(caprolactone)-block-poly(ethylene glycol) (PCL-h-PEG), poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) (PLGA- / >-PEG), or tri-block forms of the diblock copolymers listed above. Furthermore, triblock copolymers such as poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (PEO-h-PPO-ft-PEO) or poly(ethylene oxide)-block-poly(butylene oxide)-block-poly(ethylene oxide) (PEO-ft-PBO-ft-PEO) may be used.

[0250] In an amphiphilic polymer that is a graft copolymer, the length of a grafted moiety can vary. For example, the grafted segments can be alkyl chains of 4 to 22 carbons or equivalent to 2 to 11 ethylene units in length. Grafted groups may also include lipids, phospholipids or cholesterol. The grafting of the polymer backbone can be useful to enhance solvation or nanoparticle stabilization properties. Suitable chemical moieties grafted to the block unit of the copolymer include alkyl chains containing species such as amides, imides, phenyl, carboxy, aldehyde, or alcohol groups.

[0251] In some embodiments, the second stabilizing agent is a conjugated lipid. Examples of suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, dextran-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), sarcosine-lipid conjugates, and mixtures thereof. In certain embodiments, the particles include either a PEG-lipid conjugate together with a CPL. In certain embodiments, the PEG lipid is selected from a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, and / or a PEG-modified dialkylglycerol. For example, some embodiments include a pegylated diacylglycerol (PEGDAG) such as 1 -(monomethoxy -poly ethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate, or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (PEG-DPSE).

[0252] The second stabilizing agent may be a blend of different stabilizing agents. For example, two block copolymers may be used at a desired ratio. For example, a PEG-lipid and a block copolymer may be used at a desired ratio. Two second stabilizing agents may be OLSI-OOl-PCT

[0253] blended, where the only difference between the two agents is that one is modified with a reactive group that is suitable for conjugation to a targeting moiety. For example, the second stabilizing agent may be a blend of PLA-b-PEG and PLA-b-PEG-maleimide. The ratio may be from 99% maleimide to 1% maleimide or 0.1% maleimide on a molar basis.

[0254] Reactive groups for conjugation to targeting moiety

[0255] A portion or all of the second stabilizing agent or agents may be conjugated to one or more targeting moieties. For example, 0.1 %. 1%, 5%. 10%. 30%, 50%, 90% or 99% of the stabilizing agent may be conjugated (linked) to the one or more targeting moieties. The conjugation may be completed before or after coated nanoparticle assembly.

[0256] The conjugation of the targeting moiety may be achieved through any known means for coupling two species. The conjugation may be made via covalent bond, electrostatic interactions, or hydrophobic (van der Waals) interactions. The conjugation may be earned out prior to nanoparticle coating (i.e., a reaction between the second stabilizing agent and the targeting moiety). The conjugation may alternatively be carried out after coated nanoparticle assembly.

[0257] The coupling may be between a first reactive group on the hydrophilic block, for example on the terminal end of the hydrophilic block (e.g. the end of the PEG chain that is located on the surface of the coated nanoparticle opposite the end conjugated to the hydrophobic block), and a second reactive group on the targeting moiety. In one embodiment, the first reactive group is bound to the hydrophobic portion of the second stabilizing agent. In another embodiment, the first reactive group is bound to the hydrophilic portion of the second stabilizing agent (for example, PEG). In one embodiment, the second reactive group is covalently attached to the targeting moiety. In another embodiment, the second reactive group is covalently attached to a intermediate molecule or biologic that bind to the targeting moiety after conjugation to the second stabilizing agent.

[0258] The first reactive group and second reactive group can be any functional groups known to those of skill in the art to together form a covalent bond, for example under mild reaction conditions or physiological conditions. In some embodiments, the first reactive group or second reactive group are selected from the group consisting of maleimides, N-hydroxysuccinimide (NHS) esters, carbodiimides, hydrazide, pentafluorophenyl (PFP) esters, phosphines, hydroxymethyl phosphines, psoralen, imidoesters, pyridyl disulfide, isocyanates, vinyl sulfones, alpha-haloacetyls, aryl azides, acyl azides, alkyl azides, diazirines, benzophenone, epoxides, carbonates, anhydrides, sulfonyl chlorides, alkynes, cyclooctyne, aldehydes, and sulfhydryl groups. In some embodiments, the first reactive group or second reactive group is OLSI-OOl-PCT

[0259] selected from the group consisting of free amines ( — NH2), free sulfhydryl groups ( — SH), free hydroxide groups ( — OH), carboxylates, hydrazides, azides, alkynes, and alkoxyamines. In some embodiments, the first reactive group is a functional group that is reactive toward sulfhydryl groups, such as maleimide, pyridyl disulfide, or a haloacetyl. In one embodiment, the first reactive group is a maleimide.

[0260] In one embodiment, the second reactive group is a sulfhydryl group. The sulfhydryl group can be installed on the targeting domain using any method known to those of skill in the art. In one embodiment, the sulfhydryl group is present on a free cysteine residue. In one embodiment, the sulfhydryl group is revealed via reduction of a disulfide on the targeting domain, such as through reaction with 2-mercaptoethylamine. In one embodiment, the free cysteine residue is present through engineering of the targeting moiety amino acid sequence. In one embodiment, the sulfhydryl group is installed via a chemical reaction, such as the reaction between a free amine and 2-iminothilane or N-succinimidyl S-acetylthioacetate (SATA).

[0261] In some embodiments, first or second reactive groups are those used in “click” chemistry. Bioorthogonal “click” chemistry comprises the reaction between a functional group such as an azide, a nitrile oxide, a nitrone, an isocyanide and similar compounds with an alkene or an alkyne. Exemplary alkenes and alkynes include any strained cycloalkenes and cycloalkynes known to those of skill in the art, including, but not limited to, cyclooctynes, dibenzocyclooctynes, monofluorinated cyclcooctynes. difluorinated cyclooctynes, and biarylazacyclooctynone

[0262] Suitable reactive group combinations include but are not limited to N-hydroxysuccinimide esters reacting with amines, maleimides reacting with thiols, or azidealkyne “click” chemistry.

[0263] The second stabilizing agent may a blend of agents with a first reactive group and agents with no reactive group at any ratio from 0% to 100% reactive groups. In one embodiment, a fraction of PEG-b-PLA stabilizing agent can be substituted with maleimide-PEG-b-PLA. The maleimide fraction may be 0.5 mol% or 1 mol% or 5 mol% or 10 mol% or 50 mol% or 90 mol%, to provide a tunable density of reactive groups on the surface for conjugation. The first reactive group may be present on a portion of or all of the second stabilizing agent before coated nanoparticle assembly, or it may be reacted to the terminal end of the stabilizer after coated nanoparticle assembly. The terminal end of the stabilizer refers to that region of the second stabilizing agent that is opposite that linked to the hydrophobic portion of the stabilizing agent. OLSI-OOl-PCT

[0264] In one embodiment, the second stabilizing agent is a blend of PEG-b-PLA and maleimide-PEG-DSPE which is conjugated to the targeting moiety before or after coated nanoparticle assembly.

[0265] Additive agent composition

[0266] The additive agent or agents is added at a desired mass or mole ratio with respect to the encapsulated agent mass (for example, mass or moles of mRNA). For the purposes of this text, the encapsulated agent mass is the amount of encapsulated agent in inverse nanocarrier after the iFNP assembly step. (This accounts for process hold-up in the mixer.) When multiple additive agents are added, they are also added at a defined mass or mole ratio to each other. Simple screening experiments may be employed to identify suitable compositions of the encapsulated agent, first stabilizing agent, additive agent(s), and second stabilizing agent(s). For a given inverse nanocarrier composition, the additive agent or agents may be added at a mass ratio selected from the range of 1:0.01 to 1:100 (encapsulated agent to additive agent or agents). The mass ratio may be selected from the range of 1:0.25 to 1:25. The mass ratio may be selected from the range of 1:0.5 to 1:15.

[0267] In an embodiment where the encapsulated agent is a nucleic acid species, an “N: P ratio”, which is the molar ratio of nitrogen groups on the cationic species to the phosphate groups on the nucleic acid species, can be described. In the disclosed process, the cationic species may be included at an N: P ratio of 0.1: 1 to 20:1. The N: P ratio may be selected from the range of 1:1 to 6: 1. The specific value may be selected on an empirical basis through activity-based screening assays suitable to the particular application. This ratio may be 1:1, or 2:1, or 3:1, or 6:1 or 10:1 or 20:1 or any similar ratio.

[0268] The ratio of the multiple additive agents to each other may be selected empirically through direct screening experiments. The ratio may be selected based on a known mechanism or synergistic ratio. In considering the molar composition of these three additive agents, the cationic ionizable lipid may be included at a mole ratio of 0% to 75%, the phospholipid may be included at a ratio of 0% to 50%, and the hydrophobic vitamin may be included at a ratio of 0% to 50%. The mole ratio of the second stabilizing agent may also be taken into account. For example, the cationic ionizable lipid may be included at a mole ratio of 0% to 75%, the phospholipid may be included at a ratio of 0% to 50%, the hydrophobic vitamin may be included at a ratio of 0% to 50%, and the second stabilizing agent may be included at a ratio of 0.5% to 20%. OLSI-OOl-PCT

[0269] The second stabilizing agent or agents is added at a desired mass or mole ratio with respect to the encapsulated agent mass and the additive agents. When multiple second stabilizing agents are added, they are also added at a defined mass or mole ratio to each other. For a given inverse nanocarrier composition, the second stabilizing agent or agents may be added at a mass ratio selected from the range of 1:0.01 to 1: 100 (encapsulated agent to second stabilizing agent(s)). When two stabilizing agents are added, they may be at a defined ratio with respect to one another. That ratio may be selected from the range of 1:0.01 to 1: 100. The ratio may be selected based on a known mechanism or synergistic ratio or based upon standard screening studies.

[0270] Additive agents and second stabilizing agents may be added at any point after inverse nanoparticle assembly but before aqueous mixing to form the coated nanocarrier. In some embodiments, the agents are added prior to solvent exchange. In some embodiments the agents are added after the solvent exchange. In some embodiments, a portion of the agents are added before solvent exchange and the remaining portion of the agents are added after the solvent exchange.

[0271] After addition of the agent or agents, the dispersion of the inverse nanocarrier and the agent or agents may be aged for a specified period of time. The age time may be 5 minutes or more. The age time may be 15 minutes or more. The age time may be 1 hour or more. The age time may be 24 hours or more. One skilled in the art will recognize that stability considerations, including degradation profiles of the nucleic acid, may dictate age conditions. The age process may be carried out under controlled temperature conditions. The dispersion of inverse nanocarrier and agent or agents may be aged at 37°C or higher. The dispersion of inverse nanocarrier and agent or agents may be aged at 20°C or higher. The dispersion of inverse nanocarrier and agent or agents may be aged at 2-8°C or higher. The dispersion of inverse nanocarrier and agent or agents may be aged at -20°C or higher. The dispersion of inverse nanocarrier and agent or agents may be stirred or agitated (using any technique known to the field, including magnetic stir bars, stirred tanks using impellers, overhead impellers of any suitable design including retreat curve impellers or chevron impellers) or may be stored static without agitation.

[0272] The provided examples further exemplify but should not be construed as to limit the ratios of the additive agent species. OLSI-OOl-PCT

[0273] Solvent exchange

[0274] The solvent exchange for iFNP can serve to transfer the inverse nanocarrier into a water-miscible organic solvent referred to as the reforming solvent, in preparation for the coating process. The reforming solvent should be selected such that the hydrophobic block or region of the first stabilizing agent remains soluble during solvent exchange. Representative but non-limiting reforming solvents include acetonitrile, propionitrile, acetone, or tetrahydrofuran.

[0275] The solvent exchange from antisolvent to reforming solvent can be achieved by any method known to the field. For example, distillation or dialysis can be employed. If used, the distillation may be carried out using standard techniques known to the field. If the solvent exchange is achieved through a distillation method, the reforming solvent can have a higher boiling point than the antisolvent, or an azeotrope composition that allows for removal of the antisolvent by evaporation. The distillation may be completed in a put-and-take fashion (concentrating and then adding additional reforming solvent before repeating the process). The distillation may be run as a constant volume distillation (concentrating to a desired volume and then continually adding reforming solvent at a rate approximately matching the evaporation rate of the solvent mixture).

[0276] In an embodiment, the dispersion of inverse nanocarriers, additive agent(s), and second stabilizing agent in the antisolvent is first concentrated to a desired concentration before adding a first portion of the reforming solvent as required by the selected distillation method. As a non-limiting example, given an approximately 1 mg RNA batch size in dichloromethane (DCM), the antisolvent, the volume was reduced from around 4.5 mL to around 2 mL by distillation. Then, 8 mL of acetonitrile, the reforming solvent, was added. The mixture was then concentrated to about 1.5 mL and 8 mL of acetonitrile was added again. This was repeated until acetonitrile had been added 3 times. The dispersion in acetonitrile was then concentrated to a target range. A practitioner of ordinary skill in the art will recognize that the volumes and associated concentration ranges listed here may be adjusted to accommodate process constraints such as minimum or maximum volume constraints.

[0277] The distillation should proceed until the residual antisolvent content has been reduced such that the solution can be homogeneously mixed with the aqueous antisolvent in the coating step. That is, the distillation should proceed, so that the residual antisolvent does not form a second liquid phase.

[0278] One skilled in the art will recognize that distillation unit operations can be conducted with a range of operating temperatures, pressures, and total mass concentrations of species in OLSI-OOl-PCT

[0279] the solvents. Considerations such as process throughput, economics, vessel volumes, time constraints, cooling and heating capabilities, and encapsulated agent stability may dictate these decisions. Generally speaking, the temperature and pressure should be selected to afford distillative conditions identifiable from vapor-liquid equilibrium data for the contemplated solvent mixture. Temperature may be selected in the range from 5°C to 50°C, or 15°C to 40°C or 25°C to 35°C. Pressure may be selected in the range from 10 torr to 400 torr, or 50 torr to 250 torr. As the distillation progresses and the solvent composition changes, the temperature and pressure may also be adjusted as required. The total mass concentration of the encapsulated agent, first stabilizing agent, additive agent(s) and second stabilizing agent can be in the range from 0.1 mg / mL to 200 mg / mL or from 0.5 mg / mL to 40 mg / mL.

[0280] Formation of coated nanocarriers - the coating step mixing process

[0281] In an embodiment, the inverse nanocarrier with additive agent and second stabilizing agent is then processed into an aqueous environment. This process is referred to as the “coating step’" because it drives assembly of the second stabilizing agent on the nanocarrier surface and ensures the formation of a hydrophobic layer around the encapsulated agent. The method of coating the nanocarriers is as previously described by Johnson et al., termed “Flash NanoPrecipitation” (FNP), Johnson, B. K., et al., AIChE Journal (2003) 49:2264-2282 and U.S. Pat. No. 8,137,699, which are incorporated herein by reference in their entirety. This mixing technique was also used in the formation of the inverse nanocarrier. Solvent quality is rapidly reduced by micromixing against water or an aqueous buffer or mixture wherein the time of mixing is faster than the aggregation of the nanoparticles and balances with the timescale of stabilizing agent self-assembly.

[0282] While not meant to be limiting, two such geometries are the Confined Impinging Jet mixer (CIJ) and the multi-inlet vortex mixer (MIVM). These examples are meant to be illustrative rather than limiting or exhaustive and are mentioned above.

[0283] The vortex mixer consists of a confined volume chamber where one solvent stream containing reforming solvent with inverse nanocarrier, additives, and stabilizing agent, is mixed at high velocity with one or more solvent streams containing water or an aqueous buffer. When coating the nanoparticles via Flash NanoPrecipitation, mixing occurs fast enough to allow high supersaturation levels of all components to be reached prior to the onset of inverse nanocarrier aggregation. The Flash NanoPrecipitation process may be insensitive to the chemical specificity of the components, so that it is a broadly applicable nanoparticle coating OLSI-OOl-PCT

[0284] technique. The confined impinging jet mixer is a similar mixing geometry to the vortex mixer but with only one inlet stream for the reforming solvent and one for the aqueous stream.

[0285] In an embodiment, the coating formed by the second stabilizing agent can have an inner region and an outer region. The inner region can include hydrophobic or less polar region(s) of the second stabilizing agent, and the outer region can include hydrophilic or more polar region(s) of the second stabilizing agent.

[0286] In an embodiment, the coated nanocarrier includes a nanoparticle with multiple regions. The core contains the encapsulated agent, such as a water soluble agent, for example, a nucleic acid, and hydrophilic or more polar region(s) of the first stabilizing agent, for example, a first stabilizing amphiphilic copolymer, such as region(s) including poly(glutamic acid). Around this core, i.e., surrounding the core, is a shell including hydrophobic or less polar region(s) of the first stabilizing agent, for example, a first stabilizing amphiphilic copolymer, such as region(s) including PLGA, additive agent(s) (such as a lipid, phospholipid, or lipid blends), and hydrophobic or more polar region(s) of a second stabilizing agent, for example, a second stabilizing amphiphilic agent, such as a second stabilizing amphiphilic copolymer (also termed a coating stabilizer or coating polymer). The shell can have an interior surface and an exterior surface, and the interior surface can be in contact with the core. The second stabilizing amphiphilic agent can include hydrophilic or more polar region(s) and hydrophobic or less polar region(s). The hydrophilic or more polar region(s) of the second stabilizing amphiphilic agent can be at the exterior surface of the shell, for example, as a corona on the exterior surface of the shell, which extends beyond the exterior surface of the shell, away from a center of the coated nanocarrier or nanoparticle, for example, towards an environment that surrounds the coated nanocarrier or nanoparticle. The corona can be around the shell, i.e., the corona can surround the shell. That is, the hydrophilic or more polar region(s) of the second stabilizing amphiphilic agent can be in contact with the environment that is outside of the shell and outside of the nanoparticle as a whole. The hydrophobic or less polar region(s) of the second stabilizing amphiphilic agent can be within the shell. For example, the hydrophobic or less polar region(s) of the first stabilizing amphiphilic copolymer and the hydrophobic or less polar region(s) of the second stabilizing amphiphilic agent can be in contact with each other. For example, the surface of the coated nanocarrier can include the hydrophilic or less polar region(s) of the second stabilizing agent, for example, poly(ethylene glycol) (PEG). The targeting moiety, if included, can be linked to the second stabilizing agent and be located at the surface of the coated nanoparticle or at the corona of the coated nanoparticle. OLSI-OOl-PCT

[0287] For example, the shell can include non-polar, less polar, or hydrophobic agents or regions of agents, including of the first stabilizing agent, the additive agent, and of the second stabilizing agent.

[0288] In a method, nanoparticles can be made that have sizes in the range of 15 nm to 10500 nm, sizes in the range of 20 nm to 6000 nm, sizes in the range of 20 nm to 1000 nm, sizes in the range of 35 nm to 400 nm, or sizes in the range of 40 nm to 300 nm. Sizes can be determined by dynamic light scattering. For example, particles can be made that have sizes of at least about 15 nm, 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, or 6000 nm and have sizes of at most about 20 nm, 35 nm, 40 nm, 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 600 nm, 900 nm, 1000 nm, 2000 nm, 4000 nm, 6000 nm, or 10500 nm. Sizes reported and cited herein are determined by dynamic light scattering using the manufacturer-installed deconvolution programs.

[0289] A nanoparticle composition may be relatively homogenous. A polydispersity index (PDI) may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index of from about 0 to about 0.3, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.29. or 0.30 or be within a range between two of these values. In some embodiments, the poly dispersity index of a nanoparticle composition may be from about 0.10 to about 0.20.

[0290] The zeta potential may describe the surface charge of a nanoparticle. Compositions can be selected to provide different nanocarrier zeta potentials. In some embodiments, the zeta potential of a nanoparticle composition may be from about -30 mV to about +30 mV, from about -20 mV to about +20 mV, from about -12 mV to about +10 mV, from about -12 mV to about +5 mV, from about -12 mV to about 0 mV, from about -12 mV to about -5 mV, from about -10 mV to about +20 mV, from about -5 mV to about +15 mV, from about -5 mV to about +10 mV, from about -5 mV to about +5 mV, from about -5 mV to about 0 mV, from about 0 mV to about +20 mV. from about 0 mV to about +15 mV. from about 0 mV to about + 10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about +10 mV.

[0291] Nanoparticles that are mixtures of polymers and lipids are reviewed by Hadinoto, Sundaresan, Cheow. European Journal of Pharmaceutics and Biopharmaceutics (2013) 85, 427-443, which is hereby incorporated by reference herein in its entirety. The existing methods OLSI-OOl-PCT

[0292] all rely on charge complexation to encapsulate the RNA, as with lipid nanoparticles, and employ different emulsification or nanoprecipitation techniques to form a polymer core. For example, the process discussed herein is distinct in that it employs sequential nanoprecipitation steps to apply sequential layers to the nanoparticle.

[0293] Formation of coated nanocarriers - stream composition

[0294] The concentrations, stabilizing agent, reforming solvent, and aqueous buffer (antisolvent) used in the coating process may be optimized such that individual inverse nanocarriers are coated. The number of mixer inlet streams may be governed by the mixer geometry. The multi-inlet vortex mixer generally contains four streams, whereas the confined impinging jet mixer generally contains two streams. The volumetric ratios of these streams are selected as described above for inverse Flash NanoPrecipitation (iFNP) and Flash NanoPrecipitation (FNP) to ensure suitable mixing speed and intensity. The aqueous content after mixing can be selected to be 50 vol% or more with the reforming solvent. The aqueous content after mixing can be 75 vol% or more, or 90 vol% or more. Further, an additional aqueous dilution after mixing can be employed to adjust the residual reforming solvent content to produce a stable nanocarrier dispersion.

[0295] The aqueous composition can be deionized water, or a suitable buffer such as phosphate-buffered saline, or 4-(2 -hydroxy ethyl)- 1 -piperazineethanesulfonic acid (HEPES) buffer, acetate buffer, tris buffer, citrate buffer, or another organic or inorganic buffer controlling the pH in the range of from 2 to 8, or from 4 to 7.5, such as those in the list of Good’s buffers. These representative buffers are not meant to be limiting. The aqueous composition in the mixer can be deionized water, but the additional aqueous dilution after mixing can be a suitable buffer. The aqueous stream may be unbuffered but contain species to tune the osmolarity of the aqueous system. Osmolytes can be chosen, such as sugars, PEG oligomers, salts, or amino acids.

[0296] When the targeting moiety is linked to the coated nanocarrier after assembly, the aqueous composition can be selected to be compatible with the reactive groups present in the system. For example, buffers can contain ethylenediaminetetraacetic acid. Buffers containing primary amines such as tris can be avoided and HEPES used instead.

[0297] The concentration of species in the reforming solvent can be selected to achieve the desired outcome. One skilled in the art will recognize that a concentration may be selected that is uneconomical because it is too dilute. Alternatively, concentrations may be selected that are too high and can result in inverse nanocarrier aggregation, for example, as the concentration OLSI-OOl-PCT

[0298] approaches the regime where, during coating, inverse nanocarrier collisions happen faster than the second stabilizing agent can reach the surface or where the stabilizing agent is in a polymer overlap regime. Typical concentration ranges that are suitable for coating include from 0.5 mg / mL to 50 mg / mL, or from 1 mg / mL to 20 mg / mL, or from 5 mg / mL to 15 mg / mL.

[0299] When a vortex mixer that has 4 inlet streams is used there are additional inlet stream composition possibilities. For example, one stream can be the reforming solvent, two streams can be the aqueous streams, and one stream can include a second reforming solvent or solvent mixture. For example, the second reforming solvent can include the second stabilizing agent when that agent has not been added directly to the inverse nanocarrier dispersion at another point in the process. This method allows for the use of stabilizing agents that do not have suitable solubility profiles in the desired reforming solvent.

[0300] Targeting moiety composition

[0301] The targeting moiety may comprise a nucleic acid, peptide, protein, antibody, antibody fragment, small molecule, organic molecule, inorganic molecule, glycan, sugar, monosaccharide, polysaccharide, oligonucleotide, hormone, polymer and similar species that targets the nanoparticle to a site, cell, tissue, or organ in particular need of the therapeutic agent. In certain embodiments, the nanoparticle comprises multivalent targeting, wherein multiple targeting moieties are linked to the nanoparticle. In certain embodiments, the targeting moiety specifically binds to a target or cell surface receptor associated with a site in need of an agent comprised within the delivery vehicle. For example, the targeting moiety may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Such a target can be a protein, protein fragment, antigen, or other biomolecule that is associated with the targeted site. In some embodiments, the targeting moiety is a ligand which specifically binds to a target.

[0302] In one embodiment, the targeting moiety comprises a peptide. In certain embodiments, the peptide targeting moiety specifically binds to a target of interest. The peptide may be made using chemical methods. For example, peptides can be synthesized by solid phase technique. The peptides can be post-translationally modified. For example, post-translational modifications that fall within the scope of the present disclosure include signal peptide cleavage, glycosylation, acetylation, methylation, isoprenylation, myristoylation, peptide cyclization or covalent intramolecular bonding etc. The peptides may include unnatural amino acids formed by post-translational modification or by introducing unnatural amino acids during translation. OLSI-OOl-PCT

[0303] In one embodiment, the targeting moiety comprises an isolated nucleic acid, including for example a DNA oligonucleotide or a RNA oligonucleotide. In certain embodiments, the nucleic acid targeting moiety specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0304] In one embodiment, the targeting moiety comprises an antibody, or antibody fragment. In certain embodiments, the antibody targeting moiety specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies. Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0305] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule, or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0306] Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends on several factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species. Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmacokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity.

[0307] In one embodiment, the targeting moiety is N-acetylgalactosamine (GalNAc) or mono-di- or tri-antennary N-acetylgalactosamine (GalNAc). In another embodiment, the targeting moiety is an oligosaccharide or a glycan composed of one of more molecules of glucose, N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose, fucose or sialic acid.

[0308] In some embodiments, the targeting moiety is an antibody-like molecule, such as VNARs or nanobodies. OLSI-OOl-PCT

[0309] In some embodiments, the targeting moiety acts to "detarget" or minimize interactions with certain cell populations. In one embodiment, the targeting moiety interacts with SIRPa. In one embodiment, the targeting moiety is CD47 or a fragment of CD47. In one embodiment the targeting moiety has the peptide sequence GNYTCEVTELTREGETIIELK.

[0310] In some embodiments, there may be a mixture of targeting moieties on the surface. In one embodiment, a targeting moiety is combined with a CD47 fragment at a defined ratio on the coated nanoparticle surface. There may be two targeting moiety species coupled to the surface. One species may be pre-conjugated to the second stabilizing agent and the second species may be added after coated nanoparticle assembly for post-conjugation. In another embodiment, the two species may both be added after coated nanoparticle assembly for postconjugation.

[0311] Targeting moiety conjugation

[0312] The reactive group on the targeting moiety may be incorporated during synthesis or production of the targeting moiety. In one embodiment, a lysine or cysteine is engineered into the peptide or protein sequence as the reactive group on the targeting moiety. In one embodiment, reactive groups are incorporated into an oligonucleotide through inclusion of a reactive nucleotide substrate. Any means known to those skilled in the art may be employed for the addition of a reactive group onto a biological substrate.

[0313] The reactive group on the targeting moiety may be incorporated after production by any known chemical or biological means. In one embodiment, one or more free thiol groups are prepared through reaction of SATA (N-succinimidyl S-acetylthioacetate) with lysines on a protein or peptide targeting moiety and subsequent deprotection of the thiol.

[0314] The targeting moiety may be conjugated to the coated nanoparticle after the coating process is completed. In one embodiment, the targeting moiety’ associates via electrostatic or other physical attraction after addition to the aqueous dispersion containing the coating nanocarrier. In one embodiment, the targeting moiety’ is added to the aqueous dispersion of the coated nanocarriers and reacted under suitable time and temperature conditions for the pair of reactive groups employed. The coated nanocarrier dispersion may be prepared for the targeting moiety reaction prior to targeting moiety addition. For example, the dispersion may be degassed to remove dissolved oxygen, or dialyzed to remove residual reforming solvent or incompatible salts. In one embodiment, the first reactive group is a maleimide present on the surface of the coated nanoparticle and the second reactive group is a thiol present on the targeting moiety. The targeting moiety is added to the aqueous dispersion of the coated OLSI-OOl-PCT

[0315] nanocarrier in a neutral HEPES buffer and allowed to react for between 1 and 18 hours at room temperature. In one embodiment, the reaction is at 4 degrees Celsius.

[0316] The targeting moiety may be added at a defined mole ratio with respect to the first reactive group present on the coated nanoparticle surface. The targeting moiety may be added at a mole ratio of 1: 1 with respect to the moles of the first reactive group, or at a ratio between 150:1 to 1:150. In one embodiment, a ratio is 1:12 of the targeting moiety to first reactive group. In one embodiment, excess first reactive group species still present at the end of the reaction with the targeting moiety are quenched through the addition of an inert species suitable for reaction with the first reactive species. This quench agent, for example, may be glycine or tris or cysteine.

[0317] The targeting moiety may be conjugated to the second stabilizing agent prior to the coating. The conjugation of the second stabilizing agent and the targeting moiety can be carried out in a suitable reaction buffer and under suitable reaction conditions. The conjugated stabilizing agent may then be purified, as described in the subsequent section, characterized before use as a second stabilizing agent in the coating step. In one embodiment, the targeting moiety is compatible with (that is, soluble and stable in) the reforming solvent. In one embodiment, N-Acetylgalactos amine (GalNac) or triantennary GalNac is conjugated to PEG-DSPE, blended at a desired ratio with unconjugated PEG-lipid such as PEG-DMG and used in the coating step.

[0318] The targeting moiety may be conjugated to a PEG-lipid and associated with the coated nanoparticle through a post-insertion technique (Allen et al., Biochimica et Biophysica Acta, 1995). In one embodiment, the conjugated PEG-lipid is dispersed in water or aqueous buffer and added to the coated nanocarrier dispersion at a desired ratio. The mixture is heated at 30 to 60 degrees Celsius for 30 minutes to 2 hours to allow insertion of the PEG-lipid conjugates into the coated nanocarrier surface.

[0319] In one embodiment, a small molecule or biologic is first conjugated via a chemical bond to the second stabilizing agent. The targeting moiety is then bound by this small molecule or biologic. The small molecule or biologic "‘binder’ can be conjugated to the second stabilizing agent prior to coated nanocarrier assembly or after assembly. The binding of the targeting moiety may take place under any time, temperature, or buffer conditions suitable to the nature of the binding interaction. OLSI-OOl-PCT

[0320] Post-processing and preparation of pharmaceutical form - purification

[0321] In certain embodiments, it is desirable to remove targeting moiety that is unconjugated from the targeting moiety conjugated coated nanocarrier dispersion. Any technique known to the field may be used for this purpose. In one embodiment, the mixture is separated using size exclusion chromatography to isolate the nanoparticle population from the targeting moiety population. This may be conducted at any suitable scale and by the known chromatographic approaches including simulated moving beds. In one embodiment, unconjugated CTree”) targeting moiety is removed by tangential flow filtration. The molecular weight cut-off of the filter may be selected to permit the targeting moiety to enter the permeate while the coated nanocarriers are retained in the retentate. In one embodiment, residual reforming solvent is removed by dialysis prior to purification. In one embodiment, the concentration of coated nanocarriers is adjusted by ultrafiltration or ultracentrifugation prior to purification by, for example, size exclusion chromatography. These approaches may be optimized for process performance by techniques familiar to those skilled in the art.

[0322] Post-processing and preparation of pharmaceutical form - buffer exchange

[0323] The coated nanocarrier may be further processed to a pharmaceutical form useful for treating a patient or subject in need thereof. These methods of preparing a pharmaceutical form are known to the field. For example, the coated nanocarrier can be prepared with a pharmaceutically acceptable carrier (e.g., physiological saline or phosphate buffer) selected in accordance with the route of administration and standard pharmaceutical practice. Normal buffered saline (e.g., 135-150 mMNaCl) can be employed as the pharmaceutically-acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. These are examples and are not meant to limiting what can be used. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, and the like. Carrier is distinct from the term “nanocarrier”. For example, the phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an undesireable allergic or similar unacceptable reaction when administered to a human.

[0324] The pharmaceutically-acceptable carrier is generally added following coated nanocarrier formation. The residual reforming solvent can be removed to an acceptable level as determined according to ICH (International Council for Harmonisation of Technical OLSI-OOl-PCT

[0325] Requirements for Pharmaceuticals for Human Use) guidelines. Any suitable method known in the art may be used for reforming solvent removal and carrier introduction. For example, suitable methods include but are not limited to dialysis, ultrafiltration, tangential flow filtration, diafiltration, and centrifugal ultrafiltration.

[0326] Post-processing and preparation of pharmaceutical form - excipients

[0327] Coated nanocarriers may include any substance useful in pharmaceutical compositions. For example, the composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. A number of pharmaceutically acceptable excipients are known in the art.

[0328] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation- exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked polyvinyl pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0329] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl OLSI-OOl-PCT

[0330] monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan Patty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80]. sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0331] A binding agent may be starch (e g. cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; poly methacrylates: waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

[0332] Examples of preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Examples of antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butyl ated hydroxy toluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Examples of antimicrobial preservatives OLSI-OOl-PCT

[0333] include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide. cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxy ethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Examples of antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, benzyl alcohol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxy benzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A. vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BEIT), ethylenediamine, sodium lauryl sulfate (SLS), sodium laury l ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®. PHENONIP®, methylparaben, GERMALL® 115. GERMABEN®!!, NEOLONE™, KATHON™, and / or EUXYL®.

[0334] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levubnate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof. OLSI-OOl-PCT

[0335] Examples of oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropylmyristate, jojoba, kukui nut, lavandin, lavender, lemon, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, vetiver, walnut, and wheat germ oils as well as butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, simethicone, isopropyl myristate, mineral oil. octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0336] Excipients may be included as cryoprotectants or lyoprotectants. Examples of excipients are glucose, sucrose, trehalose, lactose, mannitol, sorbitol, aerosil, maltose, fructose, dextran, glycerol, poly(vinyl pyrrolidone), poly(vinyl alcohol), glycine, cyclodextrins, hydroxypropyl-beta-cyclodextrin, gelatine, poly(ethylene glycol), alanine, sodium chloride, citrate, histidine, and starch denvatives.

[0337] Post-processing and preparation of pharmaceutical form - freezing or lyophilization The pharmaceutical form may be prepared in a liquid or solid form. Liquid forms may be stored at a suitable temperature. For example, the liquid form may be stored at room temperature, or at 2-8°C. The dispersion may be stored as a frozen liquid. For example, the coated nanocarrier dispersion can be stored at -12°C or less, or at -20°C or less, or at -80°C or less. The coated nanocarrier pharmaceutical form may be prepared as a lyophilized or spray dried powder. Methods known to the field may be employed to lyophilize or spray dry the coated nanocarrier pharmaceutical form, including the addition of cryoprotectant or lyoprotectant excipients described above.

[0338] Compositions

[0339] Compositions may include an encapsulated agent or agents selected from nucleic acid classes including but not limited to RNA, DNA, mRNA, siRNA, microRNA, circular RNA, antisense oligonucleotides, tRNA, or plasmids. In an embodiment, the composition may include a salt, for example, a calcium salt. In an embodiment, the encapsulated agent or agents may be a nucleic acid and a peptide, protein, or second nucleic acid, or combinations thereof. The encapsulated agent may be multiple nucleic acid sequences. OLSI-OOl-PCT

[0340] Compositions may include a first stabilizing agent that is a diblock, triblock, or comb copolymer, that includes a hydrophilic block. The hydrophilic block can be selected from, e.g., dextran, poly(aspartic acid), and poly(glutamic acid). The hydrophobic block can be selected from poly(lactic acid), poly(lactic-co-gly colic acid), and poly(caprolactone).

[0341] Compositions may include additive agents selected from lipid blend of phospholipids, POPC, PLPC, DSPC, HSPC, DOPC, DOPE, cationic lipids or cationic ionizable lipids, hydrophobic vitamins, cholecalciferol, ergocalciferol, and combinations.

[0342] Compositions may include a second stabilizing agent or agents that is a diblock, triblock, or comb copolymer, or a lipid conjugate, or mixtures. The hydrophilic polymer block or the hydrophilic lipid conjugate can be selected from poly(ethylene glycol), poly(sarcosine), poly(aspartic acid). poly(glutamic acid), poly(lysine), or poly(arginine). and combinations. The hydrophobic polymer block can be selected from poly(lactic acid), poly(lactic-co-glycolic acid), ad poly(caprolactone). Compositions include PEG lipids such as PEG-DMG or PEG-DSPE, or PEG-b-PLGA or PEG-b-PLA, or mixtures.

[0343] Compositions may include a targeting moiety chemically conjugated to second stabilizing agent or associated with the nanoparticle via electrostatic or hydrophobic interactions. The targeting moiety may be selected from peptides, proteins, antibodies, antibody fragments, oligonucleotides, sugars, hormones, or polysaccharides that have a specific interaction or ligand binding affinity with a site on a cell, tissue, or organ of interest.

[0344] Methods of use

[0345] The present application provides compositions and methods of delivering a therapeutic and / or prophylactic agent to a cell, tissue, or organ by administering a coated nanocarrier or targeted coated nanocarrier or stealth CINC pharmaceutical composition of said agent to a subject in need thereof. Delivery of a therapeutic and / or prophylactic agent to a subject may involve administering a coated nanocarrier pharmaceutical composition including the encapsulated agent, where administration of the composition involves contacting a cell membrane with the composition. Coated nanocarrier compositions and / or pharmaceutical compositions including one or more coated nanocarrier compositions may be administered to any patient or subject, including those patients or subjects that may benefit from a therapeutic effect provided by the delivery' of a therapeutic and / or prophylactic to one or more particular cells, tissues, organs, or systems or groups thereof, such as the hepatic system. Although the descriptions provided herein include those of compositions that are suitable for administration to humans or materials derived from humans, it will be understood by the skilled artisan that OLSI-OOl-PCT

[0346] such compositions may be suitable for administration to another living system, including animals or animal-derived materials. Modification of compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals or animal-derived materials may be done, and an ordinarily skilled veterinary pharmacologist may be able to design and / or perform such modification with ordinary, if any, experimentation. Subjects to which administration of the compositions is contemplated include, but are not limited to, humans, other primates, and other animals, including commercially relevant animals such as cattle, pigs, horses, sheep, cats, dogs, rodents, mice, chickens, and / or rats. Subjects to which administration of the compositions is contemplated also include materials derived from humans or other animals, such as cells, tissues, and organs.

[0347] Pharmaceutical forms may be prepared for administration by routes that include, without limitation, oral, topical, transdermal, inhalation, parenteral, intraocular, subretinal, sublingual, mucosal, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular or other direct injections into tissues or organs, intradermal, intrathecal, intraperitoneal, intraarterial, intracistemal, intracerebral, or intratumoral injection or infusion techniques. Pharmaceutical compositions of an embodiment are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions for administration to a subject or patient may take the form of one or more dosage units, where for example, a tablet, or other dosage form, may be a single dosage unit, and a container of a compound of an embodiment in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms may be know n or apparent, to those skilled in this art.

[0348] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, w etting agents, and / or suspending agents. Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable nonirritating excipients such as cocoa butter, polyethylene glycol, and / or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient. Solid dosage forms for oral administration include capsules, tablets, pills, films, powders, and granules. Dosage forms for topical and / or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, OLSI-OOl-PCT

[0349] for example, by dissolving and / or dispensing the compound in the proper medium. Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U. S. Patents 4,886,499; 5, 190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5, 141,496; and 5,417,662. Pharmaceutical compositions formulated for pulmonary or intranasal delivery may provide an active ingredient in the form of droplets of a solution and / or suspension or as a dry powder.

[0350] In general, the step of contacting a cell with a coated nanocarrier may be performed in vivo, ex vivo, in culture, or in vitro. The amount of coated nanocarrier contacted with a cell, and / or the amount of encapsulated agent therein, may depend on the type of cell or tissue being contacted, the means of administration, the physiochemical characteristics, and other factors.

[0351] In certain embodiments, an mRNA included in a coated nanocarrier may encode a natural or recombinant polypeptide, such as a protein, that may replace one or more polypeptides that may be reduced or substantially absent in a cell contacted with the nanoparticle composition. The one or more substantially absent polypeptides may be lacking due to a genetic mutation of the encoding gene or a regulatory pathway thereof. The one or more substantially absent polypeptides may be enzymes with partially or wholly absent activity. Alternatively, a recombinant polypeptide produced by translation of the mRNA may antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. An antagonistic recombinant polypeptide may be desirable to combat deleterious effects caused by activities of the endogenous protein, such as altered activities or localization caused by mutation. In other embodiments, a recombinant polypeptide produced by translation of the mRNA may alter the DNA of gene expression in a cell through encoding various endonucleases and accessory factors. Polypeptides including Cas proteins, TALENs, and other endonucleases may be desirable to change a subject’s DNA to treat a disease. In other embodiments, a recombinant polypeptide produced by translation of the mRNA in a cell may encode for a polypeptide not normally found in that cell. Such polypeptides may include but are not limited to transcription factors, insulin, antibodies, hormones, cytokines, complement factors, clotting factors, and growth factors and may be used to treat various diseases. Indeed, in some implementations, the disclosed compositions may include one or more proteins that regulate gene expression. Such proteins may include, e.g., transcription factors, methyltransferases, and other enzy mes that regulate gene expression epigenetically, as known in the art.

[0352] In certain embodiments, the coated nanocarrier composition with an encapsulated agent including of one or more nucleic acids forming an interfering RNA sequence (e.g., siRNA) or OLSI-OOl-PCT

[0353] an antisense oligonucleotide sequence provides a method for treatment in vitro and / or in vivo of a disease or disorder in a mammal or animal subject by downregulating or silencing the transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0354] In certain embodiments, the coated nanocarrier composition with an encapsulated agent including one or more nucleic acids forming a non-coding ribnonucleic acid (RNA) sequence (e.g.. transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), Piwi-interacting RNA (piRNA), small nucleolar RNA (snoRNA), messenger RNA (mRNA), or long non-coding RNA acid (IncRN A)) provides a method for treatment in vitro and / or in vivo of a disease or disorder in an animal by regulating the transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0355] In certain embodiments, the coated nanocarrier composition with an encapsulated agent including one or more nucleic acids forming a DNA sequence (e.g. DNA aptamers or donor sequences for homologous repair) provides a method for treatment in vitro and / or in vivo of a disease or disorder in an animal by regulating the sequence, transcription and / or translation of one or more target nucleic acid sequences or genes of interest.

[0356] In certain embodiments, a DNA included in a coated nanocarrier may encode a natural or recombinant RNA sequence, such as an mRNA, an siRNA, or another RNA species, whether natural or unnatural. In some embodiments, the utility of DNA included in a coated nanocarrier is subsumed by the same utility as RNA. In other embodiments, DNA included in a coated nanocarrier may contain or encode for additional elements that promote retention within the cell as an episomal complex or may promote integration into the chromosomal DNA. In some embodiments, coated nanocarrier-DNA complexes may be used to treat various diseases through both acute and extended expression. In some embodiments, DNA included in a nanocarrier may be used as a non-viral gene therapy with applications to various diseases.

[0357] In certain embodiments, a mRNA or DNA sequence included in a coated nanocarrier encodes a recombinant protein useful for engineering the behavior of the target cell. In one embodiment, the target cell is a T cell subpopulation and the encoded protein is a chimeric antigen receptor (CAR) engineered to bind to a target antigen and includes intracellular costimulatory domains to for signaling T cell activity in response to antigen binding.

[0358] Method of use: Treating diseases and disorders

[0359] Coated nanocarrier compositions may be useful for treating a disease, disorder, or condition. For example, such compositions may be useful in treating a disease, disorder, or OLSI-OOl-PCT

[0360] condition characterized by missing or aberrant protein or polypeptide activity. For example, a nanoparticle composition including an mRNA encoding a missing or aberrant polypeptide may be administered or delivered to a cell. Subsequent translation of the mRNA may produce the polypeptide, thereby reducing or eliminating an issue caused by the absence of or aberrant activity caused by the polypeptide. Because translation may occur rapidly, the methods and compositions may also be useful in the treatment of acute diseases, disorders, or conditions.

[0361] A nanoparticle composition including a DNA or an mRNA may treat cancer or proliferative disorders by encoding for growth suppressing or apoptotic proteins. Delivery of natural or recombinant TP53, p21INK4a, Rb, APC, SMAD2, SMAD3, BAX or other DNAs / mRNAs may be therapeutic in various cancers or fibrotic diseases such as colorectal cancers or hepatocellular cancers.

[0362] A nanoparticle composition including a DNA or an mRNA may treat an autoimmune or immune-mediated inflammatory disorder by encoding for an autoantigen or other peptides or proteins involved generating an autoimmune or deleterious immune response. Such autoantigens include, but are not limited to, any that are known to play a role in any of various autoimmune or immune-mediated inflammatory disorders, for example multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosis, scleroderma, psoriasis, dermatomyositis, pemphigus vulgaris, inflammatory' bowel disease, intersitial lung disease. In another embodiment, the nanoparticle composition includes a DNA and / or an mRNA co-encapsulated with other compounds that are capable of modulating immune responses, including, but not limited to immunomodulatory drugs, biologies or other molecules.

[0363] In other examples, a nanoparticle composition including an mRNA encoding an endonuclease, DNA-binding, or RNA-binding factor and further including one or more small guide RNAs (sgRNAs) can be used for various diseases or conditions. For example, mRNA encoding Cas9 and a guide RNA may be used to treat various inherited diseases such as sickle cell anemia or beta-thalassemia. Alternatively, mRNA encoding Casl3d and a guide RNA may be useful in treating RNA repeat disorders such as Huntington’s Disease or ALS (amyotrophic lateral sclerosis).

[0364] A nanoparticle composition including an siRNA or miRNA encapsulated agent or agents can be used to downregulate or silence the translation (i.e., expression) of a gene of interest. Genes of interest include, but are not limited to, genes associated w ith viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g.. cancer), angiogenic genes, immunomodulator genes such as those associated with inflammatory and OLSI-OOl-PCT

[0365] autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders.

[0366] A nanoparticle composition including a DNA and / or an mRNA may treat a cancer, autoimmune, or immune-mediated disorder by encoding for a chimeric antigen receptor (CAR) and delivering DNA or mRNA to a T cell that, when expressed on a relevant T cell population, causes T cells to ease or resolve the disease. Such a DNA and or RNA cargo may be designed to be expressed on T cells in a transient or a permanent manner. Such a DNA and or RNA-encoded CAR may be directed against a tumor antigen and drive cytotoxic T cell behavior against tumor cells. For example, the CAR may be directed against B cells or B cell subpopulations producing autoantibodies and the cytotoxic activity of the CAR-T cells can drive clearance of an autoimmune response. For example, the CAR may be expressed in cytotoxic T cells to kill viral-infected cells.

[0367] The CAR may be expressed in regulatory T cells to direct the regulatory T cells against antigens to induce tolerance or suppress an undesired immune response. In one embodiment, the antigens of interest might include human leukocyte antigens (HLA) to induce tolerance of a graft or organ transplant. In one example. mRNA or DNA sequences encoding a CAR and Foxp3 transcription factor are delivered to T cells to generate a regulatory' T cell phenotype. In one example, the CAR and Foxp3 sequences are separate nucleic acid species and are coencapsulated in the targeted coated nanocarrier. In another example, the CAR and Foxp3 are encoded on a single nucleic acid sequence. Such a nucleic acid sequence might include, but is not limited to, a DNA sequence containing an internal ribosome entry site between the two sequences. In one embodiment, a targeted coated nanoparticle directed to a T cell population, for example using CD4 targeting, induces regulatory T cell function through delivery of a nucleic acid sequence. The targeted coated nanoparticle may be contacted with T cells in vivo or ex vivo. Exemplary nucleic acid sequences to induce regulatory T cell behavior include the protein FoxP3 encoded as an mRNA or DNA, or an siRNA directed against cyclin dependent kinase 8 or cyclin dependent kinase 19 (CDK8 / 19).

[0368] In one embodiment, chimeric antigen receptor regulatory T cells are induced through the targeted delivery of CD4-targeted coated nanoparticles co-encapsulating a CAR mRNA and a FoxP3 mRNA and / or siRNA to knockdown CDK8 or CDK19.

[0369] Exemplary diseases that may benefit from tolerance induction include graft-versus-host disease, type 1 diabetes, solid organ transplant rejection (liver, kidney, lung, skin, or other tissues), systemic lupus erythematosus, inflammatory bowel disease, and multiple sclerosis. OLSI-OOl-PCT

[0370] In one embodiment, the coated nanocarriers are targeted to T cell populations using antibodies against CD3. CD4, CD5, CD7. CD8, CD25, CD47, CD117 or CD147. In one embodiment, the CAR is a first, second, third, or fourth generation CAR. In one embodiment, the CAR consists of an extracellular antigen binding domain, a hinge region, a transmembrane domain, and intracellular signaling and / or co-stimulatory domain(s). Exemplary target antigens for the CAR include but are not limited to HLA, Her2, or B cell markers such as CD 19 or BCMA.

[0371] In one embodiment, the targeted coated nanoparticle encapsulates a protein sequence of interest encoded in DNA and a transposase encoded in mRNA to a target cell of interest. This system provides for integration of the delivered DNA into the genome. An exemplary transposase is the Sleeping Beauty transposase and associated transposon system. In one embodiment, the coated nanoparticle encapsulates a CAR sequence of interest encoded in DNA and a transposase encoded in mRNA to a T cell population of interest.

[0372] In one embodiment, the targeted coated nanoparticle encapsulates a protein sequence of interest encoded in DNA or in mRNA to a target cell of interest where the protein sequence modifies the identity of that cell. An exemplary protein is a known transcription factor that modifies T cell activity.

[0373] In one embodiment, the targeted coated nanoparticle encapsulates a DNA, a guide RNA, and an enzyme or an mRNA encoding for an enzyme. For example, the enzyme can be an endonuclease such as cas9. This system may be employed to permanently edit the target cell of interest.

[0374] In certain embodiments, the targeting moiety is an antibody or antibody fragment directed against endothelial cell surface receptors such as PEC AM, transferrin, VC AM, or ICAM. In certain embodiments, the targeting moiety is an antibody or antibody fragment directed against hematopoietic stem cell markers CD117 or CD 133 or cMPL.

[0375] In another embodiment, a coated nanocarrier composition can be used to induce an immune response directed against one or more tumor-associated antigens or cells, such as cancer cells, expressing one or more tumor-associated antigens. An embodiment envisions the use of coding or non-coding DNAs or RNAs that lead to expression of tumor-associated antigens (also termed '‘antigen” herein) as the encapsulated agent in the coated nanocarrier. These antigens may include a sequence essentially corresponding to or being identical to the sequence of a tumor-associated antigen or one or more fragments thereof. In a method, a coated nanocarrier composition encapsulating an mRNA encoding for antigen is capable of inducing an antigen-specific immune response in a subject including administering to the subject an OLSI-OOl-PCT

[0376] effective amount to produce an antigen-specific immune response. In some embodiments, the antigen specific immune response includes a T cell response. In some embodiments, the antigen specific immune response includes a B cell response. In some embodiments, the method of producing an antigen-specific immune response involves a single administration of the vaccine. In some embodiments, the method further includes administering one or more booster doses of the vaccine. In some embodiments, the vaccine is administered to the subject by intradermal or intramuscular injection.

[0377] In some embodiments, a disease-associated antigen is a tumor antigen. In this embodiment, the coated nanocarrier compositions described herein may be useful in treating cancer or cancer metastasis. Examples for tumor antigens that may be useful in an embodiment are p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, the cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT). LAGE, LDLR / FUT, MAGE-A, for example, MAGE-A1, MAGE-A2. MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10. MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART- 1 / Mel an- A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2. SAGE, SART-1 or SART-3. SCGB3A2, SCP1, SCP2. SCP3. SSX. SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE and WT, for example, WT-1.

[0378] Mammalian cells to be contacted by the composition may include, but are not limited to, hepatocytes, epithelial cells, hematopoietic cells, monocyte cells, endothelial cells, lung cells, alveolar cells, type I alveolar cells, type II alveolar cells, bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, lymphoid cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor or cancer cells. The target cell may be an antigen presenting cell, a dendritic cell, a macrophage, a spleen cell, a lung cell, a liver sinusoidal cell, or Claudius' cell. Hensen cell, Merkel cell, Muller cell, Paneth cell, Purkinje cell, Schwann cell, Sertoli cell, acidophil cell, acinar cell, adipoblast, adipocyte, brown or white alpha cell, amacrine cell, beta cell, capsular cell, cementocyte, chief cell, chondroblast, chondrocyte, chromaffin cell, chromophobic cell, corticotroph, delta cell, Langerhans cell, follicular dendritic cell, enterochromaffin cell, ependymocyte, epithelial cell, basal cell, squamous cell, endothelial cell, OLSI-OOl-PCT

[0379] transitional cell, erythroblast, erythrocyte, fibroblast, fibrocyte, follicular cell, germ cell, gamete, ovum, spermatozoon, oocyte, primary oocyte, secondary oocyte, spermatid, spermatocyte, primary spermatocyte, secondary spermatocyte, germinal epithelium, giant cell, glial cell, astroblast, astrocyte, oligodendroblast, oligodendrocyte, glioblast, goblet cell, gonadotroph, granulosa cell, haemocytoblast, hair cell, hepatoblast, hepatocyte, hyalocyte, interstitial cell, juxtaglomerular cell, keratinocyte, keratocyte, lemmal cell, leukocyte, granulocyte, basophil, eosinophil, neutrophil, lymphoblast, B-lymphoblast, T-lymphoblast. lymphocyte, B-lymphocyte, T-lymphocyte, helper induced T-lymphocyte, Thl T-lymphocyte, Th2 T-lymphocyte, natural killer cell, thymocyte, macrophage, Kupffer cell, alveolar macrophage, foam cell, histiocyte, luteal cell, lymphocytic stem cell, lymphoid cell, lymphoid stem cell, macroglial cell, mammotroph, mast cell, medulloblast, megakaryoblast, megakaryocyte, melanoblast, melanocyte, mesangial cell, mesothelial cell, metamyelocyte, monoblast, monocyte, mucous neck cell, muscle cell, cardiac muscle cell, skeletal muscle cell, smooth muscle cell, myelocyte, myeloid cell, myeloid stem cell, myoblast, myoepithelial cell, myofibrobast, neuroblast, neuroepithelial cell, neuron, odontoblast, osteoblast, osteoclast, osteocyte, oxyntic cell, parafollicular cell, paraluteal cell, peptic cell, pericyte, peripheral blood mononuclear cell, phaeochromocyte, phalangeal cell, pinealocyte, pituicyte, plasma cell, platelet, podocyte, proerythroblast, promonocyte, promyeloblast, promyelocyte, pronormoblast, reticulocyte, retinal pigment epithelial cell, retinoblast, small cell, somatotroph, stem cell, sustentacular cell, teloglial cell, zymogenic cell, kidney cell, or glandular cell.

[0380] A person of ordinary skill in the art may employ standard methods to determine the appropriate dosing range and dosage levels for a coated nanocarrier pharmaceutical composition. For example, coated nanocarrier compositions in accordance with the present disclosure may be administered in vivo at dosage levels sufficient to deliver from about 0.0001 mg / kg to about lOO mg / kg, from about 0.001 mg / kg to about 50 mg / kg, from about 0.005 mg / kg to about 10 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.05 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 10 mg / kg, from about 2 mg / kg to about 10 mg / kg, from about 5 mg / kg to about 10 mg / kg, from about 0.0001 mg / kg to about 5 mg / kg, from about 0.001 mg / kg to about 5 mg / kg, from about 0.005 mg / kg to about 5 mg / kg, from about 0.01 mg / kg to about 5 mg / kg, from about 0.05 mg / kg to about 5 mg / kg, from about 0.1 mg / kg to about 5 mg / kg, or from about 1 mg / kg to about 5 mg / kg, where a dose of 1 mg / kg (mpk) provides 1 mg of an encapsulated agent per 1 kg of subject body weight. OLSI-OOl-PCT

[0381] Coated nanocarrier compositions including one or more encapsulated agents may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. The phrasec’in combination with’’ is not intended to imply that the agents must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of this application. For example, one or more nanoparticle compositions including one or more different therapeutic and / or prophylactics may be administered in combination. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. For example, each agent can be administered at a dose and / or on a time schedule determined for that agent.

[0382] Cleavable Stabilizing Agents

[0383] In some embodiments, the second stabilizing agent contains one or more cleavable linkers within a copolymer stabilizing agent. For example, this second stabilizing agent could take the form of a diblock copolymer with the structure [hydrophobic block]-[linker]-[hydrophilic block]. For example, the structure could be PLA-linker-PEG. The linker could be any cleavable linker known in the art. Representative linkers include enzymatically cleavable linkers, disulfide linkers, light-cleavable, or acid-cleavable (pH sensitive) linkers. Enzy matically cleavable linkers include those that are susceptible to cathepsin cleavage, or sulftase, glycosidase, galactosidase, phosphatase, pyrophosphatase, or glucuronidase cleavage. Cathepsin-cleavable linkers like valine-citruline based linkers have been used clinically in antibody-drug conjugates as linkers that are cleaved in the endosome. Beta-Glucoronide-based linkers (beta-glucuronic acid with or without para-aminobezyl spacer) have also been used for these purposes. Hydrolytically acid-cleavable linkers include hydrazones, acylhydrazones, phosphoramidates. silyl ethers, acetals, ketals, vinyl ethers, cis-acotinyls, or thiopropionates.

[0384] The cleavable stabilizing agent may completely replace the second stabilizing agent or may be used in combination with a non-cleavable second stabilizing agent. Blends of cleavable and non-cleavable agents may be used at any ratio from 1 % cleavable to 99% cleavable. Blends may also be created with reactive secondary stabilizing agents to enable conjugation to targeting moieties. For example, a blend of maleimide-PEG-PLA and PEG-linker-PLA can be used. Ternary blends of secondary stabilizing agents can be used, such as maleimide-PEG-b-PLA, PEG-PLA, and PEG-linker-PLA.

[0385] Examples of enzymatically cleavable linkers include: valine-citruline dipeptide and variants such as valine-citruline-PABC (para-aminobenzyl carbamate), valine-alanine dipeptide and variants, valine-glycine and variants, glycine-glycine and variants such as OLSI-OOl-PCT

[0386] glycine-glycine-phenyl alanine-glycine, glutamine-glycine-citruline tripeptide and variants, alanine-alanine-asparagine and variants, Glycine-Phenylalanine-Leucine-Glycine, Alanine-Leucine-Alanine-Leucine, phenylalaine-arginine dipeptide and variants, or phenyl-lysine dipeptide and variants. Peptide linkers, described using the three-letter amino acid code, may include: Val-Cit, Vai-Ala, Phe-Lys, Gly-Phe, Gly-Gly-Phe, Pro-Leu-Gly-Leu-Ala-Gly, Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, GPLGIAGQ, Asp-Glu-Val-Asp, Ile-Glu-Thr-Asp, Val-Asp-Glu-Val, Arg-X-(Lys / Arg)-Arg (e.g., RRKR), Arg-Val-Arg-Arg. Leu-Val-Pro-Arg, Pro-Arg-Ser, Gly-Gly-Arg-Arg, Ala- Arg- Arg, Ala-Ala-Pro-Val, MeOSuc-AAPV (protected N-term), Lys-Arg, Arg- Arg, or Gly-Arg.

[0387] The PAB spacer may be included with any appropriate peptide sequence. Suitable linkers are surveyed in Bargh et al. Chem. Soc. Rev., 2019. 48. 4361.

[0388] The cleavable PEG polymer may be formed using any suitable chemistry. For example, maleimide, NHS-ester, azide-alkyne, or any other coupling chemistry may be employed as suited to the particular polymer chemistry requirements.

[0389] Nitro-oleic acid inclusion

[0390] An alternative or additional helper lipid additive may be a nitrated form of unsaturated fatty acids, exemplified by nitro-oleic acid (NOA) or nitro-linoleic acid (NLA). Nitrated fatty acids are STING inhibitors and have been reported to reduce mortality and immunogenicity of DNA cargo in lipid nanoparticles (Patel et al. Nature Biotechnology, 2025). This lipid may be included with other additive lipids at a specified molar ratio. For example, the nitrated lipid may be added as an additional component at a 20% molar ratio to the other additive components, or at a ratio from 1% to 30%.

[0391] Assembly of lipid-polymer blends in a single mixing step

[0392] Nanoparticles comprising an amphiphilic polymeric (not lipid anchored) stabilizing agent (e.g. PLA-PEG, PLGA-PEG, PCL-PEG, and variants) and one or more lipid blends can be created in a single assembly step. The polymeric stabilizer provides two functional roles: it can provide a stable anchor for a targeting moiety (that is. it will not partition off the nanoparticle surface) and it also limits off-target cell uptake (that is, non-specific delivery). Such properties reduce non-specific clearance to the liver following IV administration while enhancing delivery to the target cell of interest. The lipid blends may contain cationic ionizable lipids, phospholipids, lipids, triglycerides, sterols, hydrophobic vitamins, and combinations at different ratios that afford colloidally stable nanoparticles with other desired functions. For example, the inclusion OLSI-OOl-PCT

[0393] of a cationic ionizable lipid or cationic lipid can enable intracellular delivery of nucleic acid cargo after internalization. The targeting functionalization approaches described herein may be used to created targeted nanoparticles. Cleavable stabilizing polymers may also be used according to the description above. For example, the composition could include a cationic ionizable lipid at molar ratio of 10 - 80%, a phospholipid and molar ratio from 0 - 50%, a cholesterol at molar ratio from 0 to 50%, a hydrophobic vitamin at molar ratio from 0 to 50%, and a polyester-PEG diblock copolymer at a molar ratio from 1 to 25%. The amphiphilic polymeric stabilizing agent may be a polyester-PEG diblock that is a mixture of unmodified polymer, functionalized polymer for targeting moiety conjugation, and cleavable polymer, combined at any desired ratio.

[0394] Nanoparticles may be formed that encapsulate nucleic acid cargo by any means known to the field, including rapid mixing of solvent and antisolvent streams in a confined impinging jet mixers and multi-inlet vortex mixers. Nucleic acid encapsulation can be achieved by including the nucleic acid cargo in an acidified aqueous antisolvent (e.g. acetate or citrate buffer at pH 4) during assembly. The relative mass loading of the nucleic acid can be modified to achieve sufficiently high encapsulation efficiency. The nanoparticles may then be processed using the methods described herein to form a pharmaceutical product.

[0395] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0396] EXAMPLES

[0397] Example 1

[0398] Coated nanocarriers were produced using the following approach. First, inverse nanocarriers were produced using dimethyl sulfoxide (DMSO) as the solvent and dichloromethane (DCM) as the antisolvent. The DMSO input contained mRNA at 1 mg / mL, a polyglutamic acid-comb-poly(lactic acid-co-glycolic acid) first stabilizing polymer was included at 3 mg / mL, and nuclease-free deionized water at 10 volume percent (vol%) with respect to DMSO. The mRNA was in the sodium salt form and encoded Green Fluorescent Protein (GFP). The DCM antisolvent contained either calcium chloride or magnesium chloride at charge ratio (with respect to the total negative charge on the mRNA and the stabilizing polymer) of 0.5 equivalents. Inverse nanocarrier assembly was carried out in a multi-inlet vortex mixer at a 1:3 DMSO: DCM volume ratio and the resulting nanoparticle dispersion was OLSI-OOl-PCT

[0399] collected in a vial containing additional DCM such that the final DMSO content was approximately 10 vol%. The volume of the input streams passed through the mixer was selected based on material needs for the subsequent analyses.

[0400] Lipids were added before the solvent exchange and second stabilizing polymers were added after the solvent exchange. The cationic ionizable lipid was ALC-0315 (6-((2-

[0401] hexyldecanoyl)oxy)-N-(6-((2-hexyldecanoyl)oxy)hexyl)-N-(4-hydroxybutyl)hexan-l-aminium) and was added at 2 charge equivalents with respect to the mRNA negative charge, after accounting for mixer hold-up losses. The neutral helper lipid was POPC (1-palmitoyl-2-

[0402] oleoyl-glycero-3-phosphocholine) and was added 10 mol% with respect to the other additive

[0403] agents and surface stabilizers. Either cholesterol, ethyl oleate, cholesteryl oleate, or a hydrophobic vitamin was added at a specific mol% as indicated in Table 1. The second stabilizing agent was PCL5kDa-b-PEG5kDa or PLA5kDa-b-PEG5kDa and was added according to Table 1.

[0404] ii Hl ldeperp

[0405] (l%)mo

[0406] Table 1: Summary of formulations prepared in example 1. Note that “CIL ” refers to the cationic ionizable lipid, "helper lipid” refers to POPC, and " Pi HdhbropocyEG” refers to the second t componen

[0407] stabilizing agent. ()l%mo

[0408] G () PEl%mo

[0409] 187-1 Ethyle Oleate PCL-b-PEG 47.3 10 36.3 6.4

[0410] 187-2 Cholesterol PCL-b-PEG 47.3 10 36.3 6.4

[0411] 187-3 Ethyle Oleate PLA-b-PEG 47.3 10 36.3 6.4

[0412] 187-4 - PCL-b-PEG 47.3 10 36.3 6.4

[0413] 190-1 Ethyle Oleate PCL-b-PEG 47.3 10 36.3 6.4

[0414] 190-2 Cholesteryl oleate PCL-b-PEG 47.3 10 36.3 6.4

[0415] 190-3 Ethyle Oleate PCL-b-PEG 45 9 33 13

[0416] 190-4 Ethyle Oleate PLA-b-PEG 45 9 33 13

[0417] MA-91-A Cholecalciferol PCL-b-PEG 48 15 25 12

[0418] MA-91-C Vitamin E acetate PCL-b-PEG 48 15 25 12

[0419]

[0420] MA-91-E Vitamin KI PCL-b-PEG 48 15 25 12

[0421] The solvent exchange was carried out with a put-and-take approach, as follows. The volumes used were defined relative to the volumes anticipated for the nanoparticle stream in the coating step. First, DCM was removed at 200 Torr (35°C) until the volume was 3x the nanoparticle coating step input volume. That is, a target of 1.5 ml for a 0.5 ml coating volume. OLSI-OOl-PCT

[0422] Then, 8x acetonitrile was added with mixing. After concentrating to 2x-3x final volume, 8x acetonitrile was added again and concentrated to slightly less than the final lx target. The second stabilizing polymer was then added (in acetonitrile) and the volume was diluted to the lx volume target.

[0423] The coated nanoparticles were creating by rapid mixing of the nanoparticle stream in a confined impinging jet mixer against a deionized water antisolvent stream. The resultant coated nanoparticles (nanocarriers) were collected in a vial containing neutral tris-buffered saline (TBS) at such a volume to afford a final acetonitrile concentration of 10 vol% or less. Samples were dialyzed in a large excess of TBS at 4°C overnight before concentration on an ultrafilter with a 100 kDa molecular weight cut-off to approximately 100 ug / mL (mRNA basis).

[0424] Samples were then analyzed by dynamic light scattering (DLS) for size and zeta potential. Ribogreen analysis was used to determine encapsulation efficiency by measuring the concentration of free mRNA and total mRNA (with and without triton X-100 lysis, respectively).

[0425] Table 2 summarizes the findings of this analysis. While ethyl oleate, cholesterol, and cholesteryl oleate all made nanoparticles of suitable size, the zeta potential (surface charge) was more negative and encapsulation efficiencies (EEs) were generally lower than would be desired. Crucially, all formulations resulted in size increases during the post-coating processing steps. The average size increase was 21%, and ranged from 12% (ethyl oleate) to 39% (cholesterol). When the present disclosure was implemented by substituting hydrophobic vitamins for cholesterol or ethyl oleate, all three vitamins tested - cholecalciferol (vitamin D), D, L-a-tocopheryl acetate (vitamin E acetate), and vitamin KI - resulted in stable nanoparticle size (average change, 0.1%, and range was -2% to 2%), neutral zeta potentials (between -5 mV and 5 mV), and EE values that were 14 percentage points higher on average than seen with ethyl oleate and cholesterol. These results highlight the notable process improvements that accrue from the use of hydrophobic vitamins instead of ethyl oleate or cholesterol for coated nanoparticle formulations using PCL-b-PEG and PLA-b-PEG as the second stabilizing agent.

[0426] Table 2: Summary of formulation results for example 1. Sizes are z-average size determined by DLS. “Final size” is after dialysis and concentration steps while “initial size ” is immediately after coating. PDI is the polydispersity index, where values less than 0.1 are considered highly uniform and values higher than 0.25 are considered polydisperse. “EE” is encapsulation OLSI-OOl-PCT

[0427] efficiency, where values of 100% indicate that all mRNA was located completely within the nanoparticles.

[0428] Formulation Hydrophobic Initial Final Size PDI Zeta EE Reference Component Size (nm) (nm) (mV) (%) 187-1 Ethyle Oleate 97 114 0.13 -9.4 63 187-2 Cholesterol 104 144 0.04 -6.4 76 187-3 Ethyle Oleate 98 111 0.06 -8.8 60 187-4 none 107 123 0.07 -8.1 57 190-1 Ethyle Oleate - 109 0.08 - 64 190-2 Cholesteryl oleate - 114 0.15 - 66 190-3 Ethyle Oleate - 97 0.11 - 57 190-4 Ethyle Oleate - 86 0.16 - 68 MA-91-A Cholecalciferol 105 103 0.16 -4.3 81 MA-91-C Vitamin E acetate 102 102 0.12 -4.8 81

[0429]

[0430] MA-91-E Vitamin KI 103 105 0.12 -3.3 73

[0431] Example 2

[0432] Additional formulation variations were prepared according to the methods of example 1 with modifications as noted here. Either magnesium or calcium was included at 0.5 charge equivalents as noted in Table 3 during the inverse nanocarrier formation. For the coating step, the POPC helper lipid and the hydrophobic vitamin were both added before solvent exchange. The hydrophobic vitamin was varied as noted in Table 3. After the solvent exchange into acetonitrile, the cationic ionizable lipid (CIL) was added at the specified charge equivalents relative to the mRNA content. Then, the second stabilizing agent was added. As before, either PCL5kDa-b-PEG5kDa or PLA5kDa-b-PEG5kDa was used, according to Table 3. These coating components were added at specific ratios relative to each other, as defined in Table 4. As in example 1, the coating step was carried out in a confined impinging jet mixer with a deionized water antisolvent of equal volume to the nanoparticles in acetonitrile. The coated nanocarriers were collected in additional aqueous buffer - either isotonic tris-buffered saline or isotonic HEPES-buffered saline with 2 mM EDTA - such that the final composition was less than 10 vol% acetonitrile.

[0433] Table 3: Summary of formulation components for Example 2. CIL is the cationic ionizable lipid, which were sourced from BroadPharm catalog.

[0434] Ref Metal CIL Hydrophobic Stabilizing Vitamin Agent

[0435]

[0436] CM-209-A Magnesium BP LIPID 323 Cholecalciferol PCL-b-PEG OLSI-OOl-PCT

[0437] CM-209-B Magnesium DLin-MC4-DMA Cholecalciferol PCL-b-PEG CM-209-C Magnesium BP LIPID 323 Cholecalciferol PLA-b-PEG CM-209-D Magnesium DLin-MC4-DMA Cholecalciferol PLA-b-PEG CM-209-E Magnesium BP LIPID 323 a-Tocopheryl acetate PCL-b-PEG CM-209-F Magnesium DLin-MC4-DMA a-Tocopheryl acetate PCL-b-PEG CM-210-A Magnesium DLin-MC4-DMA Cholecalciferol PLA-b-PEG CM-210-B Magnesium DLin-MC4-DMA Cholecalciferol PLA-b-PEG CM-210-C Magnesium DLin-MC4-DMA Cholecalciferol PLA-b-PEG CM-210-D Magnesium BP LIPID 323 Cholecalciferol PLA-b-PEG RH-42-A Magnesium DLin-MC4-DMA Cholecalciferol PLA-b-PEG PLA-b-PEG RH-42-B Magnesium DLin-MC4-DMA Cholecalciferol

[0438] (alternate lot) RH-49-A1 Magnesium BP LIPID 323 Cholecalciferol PLA-b-PEG RH-49-A2 Magnesium BP LIPID 323 Ergocalciferol PLA-b-PEG RH-49-A3 Magnesium BP LIPID 323 Retinol PLA-b-PEG RH-49-B1 Calcium BP LIPID 323 Cholecalciferol PLA-b-PEG RH-49-B2 Calcium BP LIPID 323 Ergocalciferol PLA-b-PEG

[0439]

[0440] RH-49-B3 Calcium BP LIPID 323 Retinol PLA-b-PEG

[0441] Table 4: Formulation summary for Example 2. CIL is the cationic ionizable lipid. Aqueous buffer is the dilution used after coated nanocarrier formation. Mole percentages refer to the relative content in the coating layer.

[0442] CIL Charge CIL POPC Vitamin PEG Aqueous Ref

[0443] Equivalents mol% mol% mol% mol% Buffer CM-209-A 2 48 15 25 12 TBS CM-209-B 2 48 15 25 12 TBS CM-209-C 2 48 15 25 12 TBS CM-209-D 2 48 15 25 12 TBS CM-209-E 2 48 15 25 12 TBS CM-209-F 2 48 15 25 12 TBS CM-210-A 3 48 15 25 12 TBS CM-210-B 3 50 15.6 26 8.4 TBS CM-210-C 3 49 15.3 25.5 10.2 HEPES / EDTA CM-210-D 3 50 10 30 10 HEPES / EDTA RH-42-A 2 48 15 25 12 HEPES / EDTA RH-42-B 2 48 15 25 12 HEPES-EDTA RH-49-A1 2 48 15 25 12 HEPES / EDTA RH-49-A2 2 48 15 25 12 HEPES / EDTA RH-49-A3 2 48 15 25 12 HEPES / EDTA RH-49-B1 2 48 15 25 12 HEPES / EDTA RH-49-B2 2 48 15 25 12 HEPES / EDTA

[0444]

[0445] RH-49-B3 2 48 15 25 12 HEPES / EDTA

[0446] Samples were analyzed by dynamic light scattering for size, polydispersity (PDI), and OLSI-OOl-PCT

[0447] surface charge (zeta potential, mV). Encapsulation efficiency was measured by Ribogreen quantification with and without triton X-100 lysis of the nanoparticles. Table 5 summarizes these findings. Sizes ranged from 113 nm to 167 nm with narrow poly dispersity values and neutral surface charges. EE values were high and are suitable for a commercially viable process. These results confirm the broad suitability7of hydrophobic vitamins from the vitamin A, vitamin E, and vitamin D classes for use in coated nanocarriers. They were implemented with a variety7of ionizable lipids and second stabilizing agents with acceptable process metrics. Table 5: Formulation analysis results for example 2.

[0448] Zeta

[0449] Ref Size (nm) PDI EE (%)

[0450] (mV)

[0451] CM-209-A 142 0.00 -0.2 81

[0452] CM-209-B 140 0.05 0.6 81

[0453] CM-209-C 128 0.04 -1.1 87

[0454] CM-209-D 132 0.02 -0.6 91

[0455] CM-209-E 140 0.05 -0.1 83

[0456] CM-209-F 139 0.07 0.0 71

[0457] CM-210-A 113 0.04 -0.7 95

[0458] CM-210-B 132 0.03 -1.3 98

[0459] CM-210-C 123 0.05 -1.6 99

[0460] CM-210-D 121 0.02 -1.4 93

[0461] RH-42-A 151 0.07 -2.9 - RH-42-B 167 0.11 -3.5 - RH-49-A1 128 0.01 0.0 92

[0462] RH-49-A2 141 0.04 -1.9 91

[0463] RH-49-A3 138 0.01 -2.2 91

[0464] RH-49-B1 132 0.04 -2.1 91

[0465] RH-49-B2 139 0.07 -3.2 84

[0466]

[0467] RH-49-B3 141 0.03 -4.4 83

[0468] Example 3

[0469] Coated nanoparticles were produced using the methods of Example 1 and Example 2 with modifications as noted here. The cargo was a 90 nucleotide single-stranded DNA sequence. After the inverse nanocarrier was produced, the CIL as indicated in Table 6 was added before solvent swap (195-5, 195-6, 195-7) or after solvent swap (214- A, 214-B, 214-C). The charge ratio and lipid ratios were varied as noted in Table 6. The hydrophobic vitamin was cholecalciferol (vitamin D) in all instances. Coated nanoparticles were collected in either isotonic Tris-buffered saline (195-5. 195-6, 195-7) or isotonic HEPES-buffered saline with 2 mM EDTA (214- A, 214-B, 214-C). The samples were then dialyzed in additional tris-buffered saline (195-5, 195-6, 195-7) or lOx more concentrated tris-buffered saline (214-A, 214-B, 214- OLSI-OOl-PCT

[0470] C).

[0471] Samples were analyzed as described in Example 1. These results (Table 6) indicate that hydrophobic vitamins used in lieu of cholesterol or ethyl oleate are suitable for the encapsulation single-stranded DNA. These data further highlight that the nanoparticles are stable to high salt concentrations, with only minor loss of encapsulation efficiency observed.

[0472] Table 6: Formulation summar Siiidbltecon sangzy for example 3 capturing single-stranded DNA encapsulation performance with cholecalciferolt agen. “CIL” refers to the cationic ionizable lipid, “helper lipid” refers to POPC, and “PEG ” refers to the second stabilizing agent. Sizes are z-average size determined by DLS. “Final size ” is after C CILh Eargeq dialysis and concentration steps while “initial size ” is immediately after coating. PDI is the polyd C)IL (l%imospersity index, where values less than 0.1 are considered highly uniform and values higher than 0.25 are considered polydisperse. “EE” is ii Hl ldeperp

[0473] encapsulation efficiency, where values of 100% indicate) (l%mo that all mRNA was located completely within the nanoparticles.

[0474] i Hdhbropocy

[0475] ) (l%t componenmo

[0476] G () PEl%mo

[0477] Ref. CIL

[0478] Si ()enmz

[0479] PDI

[0480] 195-5 ALC-0315 PLA-b- 2 48 10 35 7 10 0.0 85

[0481] PEG 4 9 EE (%) 195-6 ALC-0315 PCL-b- 2 48 10 35 7 11 0.0 82

[0482] PEG 3 9 195-7 ALC-0315 PCL-b- 2 53 20 20 7 14 0.1 89

[0483] PEG 7 0 214- BP LIPID PLA-b- 2 48 15 25 12 11 0.0 70 A 323 PEG 5 3 214-B BP LIPID PLA-b- 3 50 10 30 10 11 0.0 70

[0484] 323 PEG 5 4 214-C 306-012B-3 PLA-b- 2 48 15 25 12 16 0.0 70

[0485]

[0486] PEG 0 1

[0487] Example 4

[0488] Coated nanocarriers targeted to different cell ty pes may be prepared using antibodies coupled to the surface of coated nanoparticles through PEG stabilizers terminated with thiolreactive maleimide. This maleimide coupling process is amenable to any molecule containing a free thiol or lysines that can be reacted with protected thiols. A generalized protocol for OLSI-OOl-PCT

[0489] creating targeted coated nanocarriers follows. To prepare an antibody for coupling that lacks a free thiol, the antibody was first reacted with N-succinimidyl S-acetylthioacetate (SATA) which contains an N-hydroxy-succinimide (NHS) ester that is reactive to lysines on the antibody. This converts some of the surface lysines to protected thiols linked via a stable amide bond. The conditions for this first reaction can be any that are suitable for NHS-ester chemistry. In this example, the antibody (or antibody fragment) was buffer-exchanged into PBS at pH8. At small scales, this was typically done with desalting columns, though tangential flow filtration and dialysis are also techniques that could achieve the same end. Typically, IgG, or Fab”, or Fab fragments were used, though scFV and nanobodies are also suitable.

[0490] After buffer exchange, 9 mole equivalents of N-succinimidyl S-acetylthioacetate (SATA) in DMSO was added with gentle mixing. After reacting for 30 minutes to 3 hours, the excess SATA was removed using desalting columns with a 7 kDa molecular weight cut-off. Prior to coupling of the antibody to the coated nanocarrier, the SATA was cleaved using the manufacturer recommended conditions: a two-hour incubation at room temperature with hydroxylamine and EDTA. After cleavage to expose free thiols was complete, the antibody was desalted to remove excess reagents and used for coupling to the coated nanoparticle within 2 hours.

[0491] Coated nanocarriers were prepared according to Examples 1 and 2 with modifications as noted a here. A fraction of the second stabilizing agent was replaced with a maleimide-terminated PEG block copolymer (such as PLA-b-PEG-mal or PCL-b-PEG-mal). Typically, 1 % to 10 mole % (mol%) of the second stabilizing agent was replaced with the corresponding maleimide-terminated version. The coated nanocarriers were collected in a degassed isotonic HEPES-buffered saline containing 2 mM EDTA. Other buffers that do not contain maleimide-reactive components and maintain stability of the coated nanocarrier may also be used.

[0492] After the coating step, the cleaved antibody was added to the coated nanoparticles such that there was 7.5x molar excess of maleimide relative to the antibody. After 3 hours of incubation, a 5x molar excess of cysteine relative to maleimide may be added to quench residual maleimides. The samples were then stored at 4°C for approximately 18 hours.

[0493] Unconjugated antibody was removed from the coated nanoparticles using a Sepharose-packed size exclusion column validated to resolve the free (unconjugated) antibody fraction from the nanoparticle fraction. The appropriate elution fractions were concentrated to about 100 ug / mL, analyzed for EE and size, and flash frozen with 5 wt% sucrose added as a cryoprotectant.

[0494] In one example of this protocol, inverse nanocarriers were prepared with Cy5-labeled OLSI-OOl-PCT

[0495] mRNA in the guanidine salt form and using 2 equivalents of calcium chloride. The coating composition included SM-102 cationic lipid at 3 charge equivalents along with POPC, ethyl oleate, and PCL-b-PEG and PCL-b-PEG-mal (10% ratio with respect to the unmodified PEG). The coated nanocarriers were collected in isotonic degassed HEPES-buffered saline with 2 mM EDTA. Three controls were prepared: an untargeted control (null-CINC) as well as CD4- and null-targeted LNPs prepared using the same ionizable lipid used for the coated nanocarrier C'CINC”). The antibody was a Fab7’ version of the anti-mouse CD4 antibody clone GK.1.5. The antibody was coupled to LNPs using the technique described above but using a DSP-PEG-maleimide included at 25 mol% relative to the total PEG content of the LNP instead of the PCL-b-PEG stabilizers used for coated nanocarriers.

[0496] The coated nanoparticles that were produced had very low EE values: 46% for the null-CINC and 33% for the CD4-CINC. This further highlights the limitations of ethyl oleate as a hydrophobic additive. In vitro tests compared the relative uptake of LNPs and CINCs with and without targeting, based on the Cy5 signal of the cells. In both 3A9 T cell hybridomas (Figure 2A) and in murine primary T calls (Figure 2B). targeted CINCs had significantly higher Cy5 MFI compared to null-CINCs. LNPs with and without targeting to CD4 were taken up at high levels by the 3A9 cells, indicating elevated non-specific binding and internalization. This lack of specificity7was not seen in the primary7T cells. Serum pre-incubation for 3 hours at 37°C had no effect on this finding. Binding and depletion of the CD4 surface signal was observed based on staining with a non-competing anti-CD4 antibody.

[0497] Null-CINCs and CD4-CINCs were dosed at 1 mg / kg (mRNA basis) to BALB / c mice to assess whether they could bind and deplete CD4 in vivo. After 24 hours, splenocytes were isolated and analyzed for CD4 signal. CD4-CINCs blocked approximately 50% of the CD4 whereas null-CINCs saw approximately 20% decrease relative to buffer-only Figure 2C. To further confirm successful conjugation of the CD4 antibody on the CINC surface, biolayer interferometry was used to confirm specific binding to mouse CD4. Figure 2D shows the binding curves for the unconjugated antibody as well as the null-CINC and the CD4-CINC. These results confirm successful conjugation due to the opposite directions of the free antibody binding curve and the CD4-CINC. Above about 70 nm. the interference signal causes a negative curve shift. As these CINCs are about 150 nm, this confirms that the antibody is in fact bound to the nanoparticle.

[0498] These findings together highlight the limitations of ethyl oleate as a hydrophobic additive for CINCs and also the potential of targeted CINCs to achieve high specificity through their use of second stabilizing agents like PCL-b-PEG and PLA-b-PEG. These results OLSI-OOl-PCT

[0499] demonstrate the CD4-specific nature of deliver}' in vitro and in vivo using orthogonal assays and metrics.

[0500] f Re

[0501] Example 5

[0502] To demonstra Sdtecone functional delivery of cargo in vivo using targeted coated nanocarriers, we prepared formulatioiiiblnt sangzs according to the generalized method described in example 4 with t agen

[0503] modifications as noted here. The nanocarriers encapsulated 5-methoxy-uridine modified Firefly Luciferase mRNA and u CILl%s moed 0.5 equivalents of magnesium chloride in the inverse nanocarrier step. Formulations used BP LIPID 323 from BroadPharm as the cationic ionizable OC PP

[0504] lipid included at 3 equivalents and addedl% mo after the solvent exchange. The helper lipid POPC and the hydrophobic vitamin cholecalciferol were added before the solvent exchange to i VDl%t mo

[0505] acetonitrile. Table 7 summarizes the second stabilizing agent (either PCLskDa-b-PEGskDa or PLAskDa-b-PEGskDa) and the molar ratio of the compon Siiibltangzents used in the coating step. For each l%t agen mo

[0506] formulation 10% of the stabilizing agent was replaced with the corresponding mal eimide functionalized version. Both agents were added after the solvent exchange. The coating step was run in a confined impinging jet mixer and the aqueous buffer dilution used isotonic, degassed HEPES-buffered saline with 2 mM EDTA. Aibdtnoy

[0507] Coated nanocarriers were targeted to desired tissues through the selection of antibodies or antibody fragments. Fab fragments were generated using the Pierce Fab Preparation Kit A Mlba:m according to the manufacturer protocol. In each case, the antibody was reacted with SATA and it rao deprotected according to the method described in example 4. The deprotected antibody was added to the coated nanocarrier at the mal eimide: antibody ratio described in Table 7. As described in example 4, unconjugated antibody was removed from the nanocarrier using preparative size exclusion chromatography. Formulations were then adjusted to the desired concentration, 5 wt% sucrose was dissolved, and aliquots were flash frozen until dosing.

[0508] Table 7: Formulation summary for example 7. MaT.mAb ratio refers to the relative molar excess of maleimide to the amount of antibody or antibody fragment added.

[0509] CM-206-C PCL-b-PEG 50 15 25 10 None 0

[0510]

[0511] CM-206-D PCL-b-PEG 50 15 25 10 GK1.5 IgG 10 OLSI-OOl-PCT

[0512] CM-206-E PCL-b-PEG 50 15 25 10 Isotype IgG 10 CM-211-N PLA-b-PEG 50 10 30 10 GKI.5 Fab 15 CM-211-P PLA-b-PEG 50 10 30 10 MEC13.3 IgG 15

[0513]

[0514] CM-211-R PLA-b-PEG 50 10 30 10 Isotype Fab 15

[0515] Formulations were analyzed by DLS for size, poly dispersity (PDI) and surface charge (zeta potential, mV). Ribogreen analysis afforded the encapsulation efficiency. These results are summarized in Table 8.

[0516] Table 8: Formulation analysis results for example 5.

[0517] Ref Size (nm) PDI Zeta (mV) EE (%)

[0518] CM-206-C 141 0.04 -0.7 69.4 CM-206-D 170 0.07 -1.3 65.7 CM-206-E 169 0.08 -2.1 66.5 CM-211-N 127 0.02 -3.5 90.6 CM-211-P 130 0.06 -2.9 91.4

[0519]

[0520] CM-211-R 130 0.02 -1.8 92.6

[0521] Formulations were then dosed to 8-12 week old BALB / c mice at 0.5 mg / kg (CM-211 samples) or 1 mg / kg (CM-206 samples) on an mRNA basis by intravenous tail vein injection. After 5 hours, bioluminescence was measured using the Spectral Instruments Ami HT. D-luciferin was injected at 150 mg / kg prior to whole animal and ex vivo organ imaging analysis. Table 9 summarizes these analysis results for the CM-206 series of samples. These results highlight the CD4-mediated enhancement in spleen delivery. The isolype antibody had some spleen enhancement, which may have been driven by Fc-mediated interactions. Whole animal imaging noted cervical lymph node expression only for CD4-targeted formulations.

[0522] Table 9: Ex vivo organ luminescence quantification for 206 samples dosed at 1 mgEg.

[0523] Values are photons / sec and are the average of n=2 animals.

[0524] Spleen signal Lung Liver Spleen Liver: Spleen

[0525] enhancement (p / s) (p / s) (p / s) ratio

[0526] versus 206-C CM- 4.9E+06 2.4E+07 3.0E+06 7.9 - 206- C

[0527] CM- 7.7E+06 3.0E+07 1.6E+07 1.9 5.3x 206-D

[0528] CM- 1.0E+07 4.0E+07 9.5E+06 4.2 3. lx

[0529]

[0530] 206-E

[0531] Table 10 summarizes the analysis results for the CM-211 series of samples. These results indicate that targeting of coated nanocarriers can drive changing patterns of tissue OLSI-OOl-PCT

[0532] expression (tropism). Significant changes in lung signal were seen with anti-CD31 (PECAM) targeting. CD4-mediated delivery to the spleen was also achieved with the Fab format of the antibody.

[0533] Table 10: Ex vivo organ luminescence quantification for 211 samples, dosed at 0.5 mg / kg. Values are photons / sec and are the average of n=2 animals.

[0534] Target organ Lung Liver Spleen Liver: Spleen Liver: Lung enhancement (ph / s) (ph / s) (ph / s) Ratio ratio

[0535] vs isotype CM- 1.18E+06 6.47E+06 2.26E+06 2.9 5.5 9.0 (spleen) 211-N

[0536] CM- 3.39E+08 7.26E+06 1.63E+06 4.5 0.02 204.2 (lung) 211-P

[0537] CM- 1.66E+06 4.42E+06 2.52E+05 17.5 2.7 -

[0538]

[0539] 211-R

[0540] FIG. 3 visually depicts the changes in expression biodistribution with different targeting antibodies for the CM-211 series of targeted coated nanoparticles. For CD4 targeting, tropism to the spleen and cervical lymph nodes can be seen through the gray splotches of signal. With CD31 (PECAM) targeting, the lung signal can be seen as two nodes of dark splotches. As expected, isotype targeting resulted in primarily non-specific liver clearance over time.

[0541] Example 6

[0542] Formulations encapsulating firefly luciferase mRNA, eGFP plasmid, or both were prepared using the methods of example 5 for samples CM-211 with modifications as noted here. Formulations used different ionizable lipids and targeting antibodies, as described in table 11. Anti -human CD3 clone 0KT3 (InVivoMab, Bio X Cell) and isotype controls (as noted in table 11) were conjugated using the methods described in example 4 and 5.

[0543] Formulations were characterized by DLS and Ribogreen as summarized in table 11. Formulations were then dosed at 1.25 ug / mL to Jurkat cells cultured in RPMI media supplemented with 10% fetal bovine serum. Luciferase samples were analyzed at 24 hrs post dose and GFP samples were analyzed at 48 hours post dose. FIGS. 4A-4B illustrate the successful deliver}' of DNA, mRNA, and co-encapsulated DNA and mRNA to Jurkat T cells using a variety7of cationic ionizable lipids in the coated nanocarrier formulations.

[0544] Table 11: Formulation summary for example 6. CIL is the cationic ionizable lipid used in the formulation.

[0545] Ref Core CIL mAh Size (nm) PDI Zeta EE

[0546]

[0547] (mV) (%) OLSI-OOl-PCT

[0548] RH-53-A DNA DLin-MC4- OKT3 176 0.13 -1.2 89

[0549] DMA RH-53-B DNA DLin-KC2- OKT3 173 0.14 -4.4 81

[0550] DMA RH-53-C DNA BP lipid 323 OKT3 184 0.18 -0.9 89 RH-53-D DNA BP lipid 338 OKT3 195 0.13 1 95 RH-53-E DNA BP Lipid 399 OKT3 172 0.15 -8 39 RH-53-F DNA BP lipid 320 OKT3 143 0.14 -14.2 9 RH-53-G DNA BP lipid 323 NONE 150 0.03 -1.2 84 CM-218- mRNA DLin-MC4- OKT3 162 0.12 -1.8 90 A DMA CM-218- mRNA DLin-KC2- OKT3 145 0.11 -7.2 69 B DMA CM-218- mRNA BP lipid 323 OKT3 147 0.10 -1.5 88 C CM-218- mRNA BP lipid 338 OKT3 185 0.15 0.1 95 D CM-218- mRNA BP Lipid 399 OKT3 157 0.13 -8.4 46 E CM-218- mRNA BP lipid 320 OKT3 124 0.12 -8.0 12 F CM-218- mRNA+DNA BP lipid 323 isotype 142 0.08 -2.0 90 G CM-218- mRNA+DNA BP lipid 323 OKT3 148 0.12 -2.6 92 H CM-218- mRNA+DNA DLin-MC4- OKT3 154 0.11 -3.7 94

[0551]

[0552] J DMA

[0553] Example 7

[0554] Hematopoietic stem and progenitor cell targeting was evaluated using CD117 targeted CINCs. Coated nanocarriers were prepared as described in Example 4, with slight modifications as noted here. 5-methoxy uridine modified eGFP mRNA (Trilink) was encapsulated in the sodium salt form. Magnesium chloride was used at 0.5 charge equivalents. POPC and cholecalciferol were added before solvent swap while the cationic ionizable lipid (CIL) and PLA-b-PEG / PLA-b-PEG-mal (second stabilizing agents, Nanosoft Polymers) were added after the solvent swap. The CIL identity and charge equivalents relative to the mRNA are noted in Table 12, where the relative ratios of the additive components are also defined on a mole basis. The mal eimide functionalized PLA-b-PEG replaced 10 mol% of the total amount of second stabilizing agent. CILs were sourced from Broadpharm. OLSI-OOl-PCT

[0555] Table 12: Formulation summary for Example 7. See earlier examples for abbreviation definitions.

[0556] Ref. CIL CIL CIL POPC VitD PEG Antibody Eq mol% mol% mol% mol%

[0557] 216-A DLin-MC4- 3 50 10 30 10 None DMA

[0558] 216-B DLin-MC4- 3 50 10 30 10 CD117

[0559] DMA

[0560] 216-C BP LIPID 3 50 10 30 10 CD117

[0561] 323

[0562] 216-D 306-O12B- 2 40 12 36 12 CD117

[0563]

[0564] 3

[0565] The antibody, InVivoMAb anti-mouse c-Kit CD117 (BE0280 from BioXCell), was conjugated to the coated nanocarriers and purified as described in Example 4. Analysis of the purified coated nanocarriers revealed a slight increase in size on conjugating the antibody, as would be expected for adding additional material to the surface of the nanoparticle. As in earlier examples, sizes, polydispersity, surface charges, and encapsulation efficiencies (EE) all met suitable quality criteria (Table 13).

[0566] Table 13: Formulation analysis for Example 7. See earlier examples for abbreviation definitions.

[0567] Ref. Size PDI Zeta EE

[0568] (nm) (mV) (%)

[0569] 216-A 14.3 0.09 -4.1 81.3

[0570] 216-B 152 0 13 -4.1 90.9

[0571] 216-C 167 0.13 ■5 1 89.7

[0572]

[0573] 216-D 156 0.10 -0.2 87.6

[0574] Formulations were dosed to EML cells, a multipotent hematopoietic precursor cell line, at 1.25 ug / mL and incubated for 24 hours by flow cytometry analysis for GFP signal. Transfection was confirmed to be CD117-mediated as the untargeted control and cell-only controls had only minor differences (4%) in GFP median fluorescence intensity (MFI). By contrast, two of the three targeted CINCs had elevated GFP signals up to 175% above background. See FIG. 5. These results highlight that targeted CINC performance is not restricted to T cell populations only and CD 117-mediated delivery to hematopoietic cells lines can be achieved. OLSI-OOl-PCT

[0575] Example 8

[0576] Coated nanocarriers encapsulating single-stranded DNA were prepared according to the methods used to make sample 216-B from Example 7. Instead of the CD117 antibody, an Alexa Fluor 594-labeled anti-CD4 antibody was used instead. Antibody was added at four different levels relative to the moles of PLA-b-PEG-maleimide. These were maleimide:antibody ratios of 12:1. 22:1, 150:1, or 0:1. After the coupling reaction was complete, a sample of the 12:1 condition was taken prior to SEC purification. Then, all nanocarrier samples were purified by the preparative-scale SEC.

[0577] The samples were analyzed by DLS and Ribogreen analysis. Fluorescence in the coated nanocarrier fraction was quantified to determine the approximate antibody content relative to the total nanoparticle mass. These results are summarized in Table 14. Additionally, samples were injected on an analytical SEC column (Agilent Bio SEC -5, 500 angstroms) to confirm that the preparative-scale SEC had successfully removed unconjugated antibody.

[0578] Table 14: Formulation summary for Example 8. “Mai: Ab ” is the maleimide:antibody ratio on a molar basis. The “Ab fraction” is the relative content of targeting antibody quantified in the nanoparticle fraction after size purification by SEC to remove unconjugated antibody.

[0579] Mal: Ab Size (nm) PDI Zeta EE (%) Ab ratio (mV) Fraction (% mass) 212-Al 12 119 0.12 -2.5 80 9.4 212- A2 22.5 127 0.08 -0.9 85 6.2 212-A3 150 125 0.04 -1.0 86 0.3

[0580]

[0581] 212-A4 0 125 0.04 -1.7 86 -

[0582] As expected, higher maleimide to protein ratios resulted in lower antibody loadings on the coated nanocarrier surface. EE, size, and zeta potential were not adversely impacted by the changing antibody content. These results confirm that single-stranded DNA can be encapsulated using the techniques described here. They also confirm that the coupling reaction follows expected trends - lower reaction ratios lead to lower antibody content in the final coated nanocarrier.

[0583] The analytical SEC analysis confirmed that the preparative scale SEC column successfully removed unconjugated antibody (FIG. 6). The analysis of 212- Al before purification also allows for an estimate of the reaction efficiency: approximately 42% conjugated after 3 hours. The reaction efficiency can be further optimized through changing conditions using techniques known in the field for maleimide-thiol reactions. OLSI-OOl-PCT

[0584] Example 9

[0585] Cell-specific delivery to T cell subpopulations was assessed using targeted coated nanocarriers prepared according to example 5 using slight modifications such as different antibodies conjugated to the surface. The nanoparticles encapsulated Nl-methylpseudouridine- modified eGFP mRNA (Trilink). All formulations used POPC and cholecalciferol in the coating step as in example 5. PLAskDa-b-PEGskDa was the second stabilizing agent in all cases and 10 mol% was replaced with a maleimide-modified version of the polymer. The cationic ionizable lipid (CIL) was varied as noted in Table 15 but all coating component ratios matched those used in CM-211 samples from example 5. Antibody coupling and purification was performed as described in example 5. The results of the DLS and Ribogreen analysis are also summarized in Table 15.

[0586] Table 15: Summary of formulation composition and analysis for example 9. See example 5 for definition summary.

[0587] Ref CIL Antibody target MakmAb Size PDI Zeta EE (clone) ratio (nm) (mV) (%) CM-211 -A BP LIPID 323 CD5 (53-7.3) 15 147 0.06 -3.6 85.9 CM-211-B Dlin-MC4- CD5 (53-7.3) 15 144 0.11 -3.6 86.1

[0588] DMA CM-211-C BP LIPID 323 CD3 (145-2C11) 15 162 0.11 -1.7 89.0 CM-211-D Dlin-MC4- CD3 (145-2C11) 15 148 0.12 -4.2 88.6

[0589] DMA CM-211-E BP LIPID 323 CD3 (500A2) 15 165 0.13 -2.1 87.3 CM-211-F Dlin-MC4- none - 142 0.07 -2.9 86.4

[0590] DMA CM-211-G BP LIPID 323 Isotype IgG 15 149 0.03 -0.5 90.4 CM-211-H BP LIPID 323 CD4 (YTS177) 15 148 0.07 -1.3 94.8 CM-211 -J BP LIPID 323 CD4 (RM4-4) 15 131 0.05 -0.9 83.8 CM-211-K BP LIPID 323 CD4 Fab (GK1.5) 15 145 0.04 -0.6 93.2 RH-56-A BP LIPID 323 Isotype Fab 15 155 0.13 -1.3 79.0 RH-56-B BP LIPID 323 CD4 Fab (GK1.5) 15 163 0.15 -1.7 82.0 CM-219-B BP LIPID 323 CD4 Fab (GK1.5) 40 146 0.03 -3.1 86.1 CM-219-C BP LIPID 323 CD4 Fab (GK1.5) 10 153 0.07 -1.3 84.0 CM-219-D BP lipid 338 CD4 Fab (GK1.5) 15 260 0.22 -0.6 87.9

[0591]

[0592] CM-219-E BP LIPID 323 CD31 (MEC13.3) 15 148 0.06 -1.1 85.2

[0593] Formulations were relatively monodisperse, with suitable size, surface charge and encapsulation efficiency. Formulations were dosed to 8-12 week old BALB / c mice at 0.5 mg / kg. 18 hours post dose, peripheral blood mononuclear cells (PBMCs) were isolated using SepMate kits according to manufacturer protocols. Spleens were collected and splenocytes OLSI-OOl-PCT

[0594] were isolated according to standard protocols. Cells were stained for viability', CD3, CD45, CD4 and CD8 before flow cytometry analysis for GFP positivity. FIG. 7 shows the GFP positivity for the CD4 and CD8 T cell populations in PBMCs. Targeted coated nanocarriers resulted in GFP positivity above background as well as the untargeted controls. Formulation changes led to different selectivity for T cell subpopulations. FIG. 8 depicts the GFP positivity in CD3+ splenocytes (that is, spleen-resident T cells) for the same formulations. These results demonstrate the antibody -mediated delivery of a genetic cargo to T cells in vivo.

[0595] Example 10

[0596] Coated nanoparticles were produced using the methods of Example 1 with slight modifications, encapsulating a 5-methoxy-Uridine modified Firefly Luciferase mRNA. Three formulations were prepared, each using 3 charge equivalents of BP LIPID 323. The CINC formulation used cholesterol and had PEG-DMG as the second stabilizing agent. The stealth CINC (sCINC) used cholecalciferol and had PCL-b-PEG as the second stabilizing agent. The CINC-Achol formulation replaced cholesterol with cholecalciferol. Samples were analyzed as described in Example 1 and then dosed to 8-12 week female BALB / c mice at 1 mg / kg by tail vein injection. Five hours after injection, luciferase activity was measured as described in example 5.

[0597] These findings demonstrate that the dual modifications of hydrophobic vitamin substitution and PEG-DMG substitution contribute to the reduced liver expression seen with stealth CINCs. These findings also demonstrate that hydrophobic vitamins can be substituted into the CINC compositions described in PCT’948. FIGS. 9A-9B demonstrate these changes in hepatic luminescence.

[0598] Example 11

[0599] As a demonstration of pre-coupling a targeting moiety to the second stabilizing agent by an alternative chemistry' to thiol-maleimide reactions, AlexaFluor647-azide (A10277 from ThermoFisher Scientific) yvas reacted with PCL-b-PEG-alkyne prior to coating of the inverse nanocarriers. Though this dye does not function for targeting, it demonstrates the approach that may be employed for a variety of small molecule, peptide and protein moieties that may be of interest for pre-coupling. This approach demonstrates that “click’’ chemistries may be used for coupling molecules to the surface of the coated nanoparticle.

[0600] PCL-b-PEG-alkyne was dispersed as micelles in 100 mM phosphate buffer (pH 7). This was achieved by dissolving the block copolymer in acetonitrile and then rapidly mixing this OLSI-OOl-PCT

[0601] solution with the phosphate buffer in confined impinging jet mixer. This was collected in a vial with additional buffer such that acetonitrile was less than 10 vol% of the final composition. The resulting micelles were concentrated to 80 uM by ultrafiltration with a 50 kDa molecular weight cut-off Amicon filter. The AlexaFluor647-azide (AF647) was dissolved in DMSO at 5 mg / ml. Copper sulfate was dissolved in water at 20 mM and tris-hydroxypropyltriazolylmethylamine (THPTA) was dissolved at 50 mM in water. Sodium ascorbate was dissolved in water at 20 mg / mL. The copper and THPTA were mixed at a 5:1 THPTA: Copper ratio (2.5 parts copper and 5 parts THPTA at the above concentrations). Then, all reactants and reagents were mixed as described in Table 16 and incubated at room temperature for 2 hours, protected from light.

[0602] Table 16: Reaction buffer composition for copper -catalyzed azide-alkyne reaction

[0603] vol (uL) final cone

[0604] PCL-b-PEG-alkyne (micelle) 900 75 uM

[0605] AF647 27 122.73 uM Copper / THPTA 15 0.1 mM

[0606] Sodium Ascorbate 50 5 mM

[0607] Phosphate buffer 8 -

[0608]

[0609] TOTAL 1000 -

[0610] The micelles were then dialyzed against a lOOx volume excess of lOmM EDTA in water for 16 hours followed by 8 hours of dialysis against deionized water to remove free dye and reactants. The Alexa Fluor-labeled polymer (PCL-b-PEG-AF647) was then lyophilized to a powder before use as a second stabilizing agent. This coupling was achieved using copper-mediated “click” chemistry’. Variations on this coupling approach, such as alternative reagents or buffers or conditions, can be employed to achieve the same effect. Variations may include alternative click-compatible reactive groups or amine-reactive groups such as N-hydroxysuccinimide. The azide-functionalized dye may be replaced with a targeting moiety such as a peptide, ligand, or other species of interest.

[0611] The PCL-b-PEG-AF647 was dissolved in acetonitrile and utilized as a second coating agent according to the methods described above. The coated nanocarriers were concentrated using 100 kDa Amicon ultrafilters and then filtered using a 0.45 pm filter. The dispersion was then injected onto a HiPrep Sephacry l S-500 size exclusion column (GE Healthcare, Chicago, IL). The mobile phase was 150 mM NaCl in endotoxin-free water, eluting at room temperature. The fractions (4 mL each) were collected and analyzed for fluorescence to track when the OLSI-OOl-PCT

[0612] AlexaFluor647 signal was eluting. FIGS. 10A-10B demonstrate that the PCL-b-PEG-AF647 elution pattern matches the elution profile of similarly sized nanocarriers with PCL-b-PEG as the second stabilizing agent, indicating that the second stabilizing agent was incorporated similarly in both cases. The elution pattern with PCL-b-PEG- AF 647 also confirms that the dye was not unbound, as this would have eluted at 90 minutes or later.

[0613] Example 12

[0614] Cell-specific delivery to T cell subpopulations was assessed using targeted coated nanocarriers prepared according to example 5 using slight modifications as noted here. The nanoparticles encapsulated Nl-methylpseudouridine-modified eGFP mRNA (Trilink). All formulations used POPC and cholecalciferol in the coating step as in example 5. PLAskDa-b-PEGskO or PCLskDa-b-PEGskDa was the second stabilizing agent as noted in Table 17. 10 mol% of each stabilizing agent was replaced with a maleimide-modified version of the polymer. The cationic ionizable lipid (CIL) was varied as noted in Table 17 but all coating component ratios matched those used in CM-211 samples from example 5. Formation of Fab formats, antibody coupling, and purification was performed as described in example 5. The results of the DLS and Ribogreen analysis are also summarized in Table 17.

[0615] Table 17: Formulation details and analysis results for example 12. CIL is the cationic ionizable lipid. Mal.mAb ratio is the maleimide ratio relative to the antibody on a molar basis. EE is the encapsulation efficiency.

[0616] Ref CIL Stabilizin Antibody Mal:m Size PDI Zet EE g clone Ab (nm) a (% Agent (format) ratio (mV )

[0617] ) RH-56-A BP Lipid PLA-b- Isotype 15 155 0.13 -1.3 78.

[0618] 323 PEG (Fab) 8 RH-56-B BP Lipid PLA-b- GK1.5 (Fab) 15 163 0.15 -1.7 81.

[0619] 323 PEG 8 CM-219- BP Lipid PLA-b- GK1.5 (Fab) 40 146 0.03 -3.1 86.

[0620] B 323 PEG 1 CM-219- BP Lipid PLA-b- GK1.5 (Fab) 10 153 0.07 -1.3 84.

[0621] C 323 PEG 0 CM-219- BP lipid PCL-b- GK1.5 (Fab) 15 260 0.22 -0.6 87.

[0622] D 338 PEG 9 CM-219- BP Lipid PLA-b- PECAM 15 148 0.06 -1.1 85.

[0623]

[0624] E 323 PEG (IgG) 2 OLSI-OOl-PCT

[0625] Example 13:

[0626] T cell coated nanocarriers are prepared using targeting antibodies against human CD3 or human CD4 or human CD5 according to the methods of example 6 and selecting from the composition ranges in Table 18. The genetic cargo is selected from a DNA plasmid or mRNA or combinations. A cationic ionizable lipid is selected from: BP Lipid 323, BP Lipid 363, BP Lipid 337, BP Lipid 338, BP Lipid 336, BP Lipid 418, BP Lipid 339, BP Lipid 368, BP Lipid 335, BP Lipid 377. or BP Lipid 401 or similar structures. They hydrophobic vitamin is selected from the vitamin D or vitamin E classes of compounds and the second stabilizing agent is selected from PCL-b-PEG or PLA-b-PEG or PLGA-b-PEG. The resulting formulations are tested in vitro on Jurkat cells, on resting or activated human peripheral blood mononuclear cells (PBMCs), or on activated human T cells. The resulting formulations are dosed at 0.1 to 3 mg / kg to PBMC -humanized NCG mice. Splenocytes are isolated after 24 hours and analyzed for positivity for the mRNA- and / or DNA-encoded gene(s).

[0627] Table 18: Composition ranges of formulations for Example 13. By definition, the mol% values must total to 100%. The maleimide content is a percentage of the total stabilizing agent amount.

[0628] Maleimide

[0629] Stabilizi

[0630] CIL POPC Vitamin (% of

[0631] ng agent

[0632] mol% mol% mol% stabilizing

[0633] mol%

[0634] agent)

[0635]

[0636] 45-55 10-20 20-35 5-15 1-10

[0637] Two CD3-targeted CINCs (Table 19) produced according to these compositions resulted in enhanced GFP positivity in the targeted human T cells while resulting in no change to positivity in the off-target mouse cells (FIG. 11).

[0638] Table 19: Composition description and characterization from example 13 mouse study.

[0639] CD3 CIL Other components Size PDI Zeta EE Antibody (nm) (mV) (%) RH-123- Teplizumab BP Lipid POPC / Vitamin D / PLA- 157 0.11 0 0.96 A2 338 PEG

[0640] RH-123-B OKT3 BP Lipid POPC / Vitamin D / PLA- 135 0.13 -0.3 0.97

[0641]

[0642] F(ab')2 363 PEG

[0643] Example 14:

[0644] T cell coated nanocarriers are prepared using a targeting antibody against human CD3 (clone OKT3) according to the methods of examples 4-6 and selecting from the composition ranges OLSI-OOl-PCT

[0645] in Table 18. The cargo was a GFP-encoding DNA plasmid. The ionizable lipid was varied according to Table 20 and the second stabilizing agent was PLA-PEG. Formulations were analyzed by DLS and Ribogreen, then dosed to Jurkat T cells at 0.625 mg / ml and analyzed for GFP positivity after 2 days in culture. The DLS results are summarized in Table 20.

[0646] Table 20: Formulation summary of OKT3-targeted coated nanocarriers and their potency in Jurkat culture

[0647] i i Sample Name | Cil Size (ran) PDi Zeta (mV)i EE | KH-57-A 1 37-A BP lipid 3381 185 0.18 2.7 | 90%

[0648] RH-57-8 | 8P Lipid 363 | 216 0.33 5.8! 98% j RH-57-C BP Lipid 418 i 215 0.20 4.7 92% j RH-57-D | BP lipid 336 { 200 0.23 3.7 93% j RH-574. j BP Lipid 335 i 374 0.28 3.1 i 96% [ RH-57-F j BP lipid 339 | 191 0.21. 1 ™.8. | 95%

[0649]

[0650] The GFP expression signal in Jurkats confirmed the potency of multiple different ionizable lipids in the targeted CINC formulation for DNA delivery (FIGS. 12-13). Further, the untargeted control (57-G) exhibited minimal background signal, confirming the expression is mediated by the targeting moiety. This is further corroborated by the dose response analysis.

[0651] Example 15:

[0652] Targeted CINCs encapsulating eGFP mRNA (N1 -methylpseudouridine modified) were prepared using the ionizable lipid BP lipid 363. The molar composition of the coating was 50 / 10 / 30 / 10 ionizable lipid / POPC / cholecalciferol / PLA-PEG. PLA-PEG-maleimide made up 10% of the PEG content and the respective antibodies (OK. T3, teplizumab, anti-CD5, anti-CD4) were included at 15: 1 mal eimide: antibody ratio. CD3-CINC (a) used an OKT3 targeting antibody and CD3-CINC (b) is teplizumab targeted. Processing was as in Example 4 and Table 21 documents the resulting CINC properties.

[0653] CINCs were then tested in activated and resting T cells. Peripheral blood mononuclear cells were monocyte-depleted and then T cells were activated and expanded using DynaBeads. After 6 days, cells were treated either with fresh CD3 / CD28 DynaBeads, IL-2, and IL-7 (“activated”) or just IL-2 and IL-7 (“rested”). After 24 hours, targeted nanoparticles with different targeting antibodies were dosed at 1 ug / ml (GFP mRNA basis). GFP positivity was measured by flow after 24 hours (FIGS. 14A-14B). Dose response data used the same conditions but with CINC OLSI-OOl-PCT

[0654] levels dosed as described. All CINC formulations had live cell counts that were 85% or higher relative to the untreated controls, with the exception of CD4-CINCs which were at 75% of the control level. These results indicate that targeted CINCs achieve expression in primary T cells in a targeted manner without toxicity.

[0655] Table 21: Summary of characterization of CINC formulations with different targeting antibodies.

[0656] Size (nm) Zeta (mV) EE

[0657] CD3-CINC (A) 215 1.6 94%

[0658] CD3-CINC (B) 182 0.75 98%

[0659] CD4-CINC 252 6.5 80%

[0660] CD5-CINC 200 1.5 95%

[0661]

[0662] null-CINC 164 0.5 96%

[0663] Example 16:

[0664] Additional ionizable lipids were screened in primary human T cells according to the methods of Example 15. Targeting was achieved using OKT3 antibody against human CD3, except for RH-72-H, which was a null-CINC without any targeting. The genetic cargo was GFP mRNA with no uridine modifications. The formulation results are summarized in Table 22.

[0665] Table 22: Characterization of OKT3-CINC formulations with different ionizable lipid component substitutions

[0666] Formulatian Manw Lipid i Site (nm) POi Zeta (mV) EE RH-72-A 88 Lipid 363 | 170 a.24 2.8 30211 RH-72-8 BP lipid 365 s 162 0.28 6,8 88% RH72-C BP Lipid 384 | 183 0,09 2.8 82%. RHAND. ip Ppid 333 ' ]. W.. 042...

[0667] J 184 aio 47 93% RH-72-F BP Lipid 401 ] 180 0,14 •3.4 84%

[0668] Lipid 388 | 70S 0.31 3.2 103%

[0669]

[0670] 8H72-H OP upsd 3O3? 181 047 3.9 99%

[0671] Targeted CINC formulations transfected primary T cells under both resting and activated conditions (FIGS. 15A-15B). No formulations caused a significant drop in live cell levels except for RH-72-G. These results show that targeted CINCs function with multiple different ionizable lipids. OLSI-OOl-PCT

[0672] Example 17:

[0673] A selection of formulations from example 16 were tested in naive primary T cells isolated by negative selection kits. CD3-CINCs resulted in GFP positivity while the null-CINC was no different than background signal (FIG. 16).

[0674] Example 18:

[0675] Targeted CINCs were prepared using three different anti-mouse antigen antibodies and 2 different ionizable lipids for evaluation in vivo using the methods of example 4 (see Table 23). The CINCs encapsulated Firefly Luciferase with a 5-methoxy uridine modification. Magnesium was included as the salt and the secondary stabilizing agent was PLA-PEG with 6.9% PLA-PEG-maleimide. The lipid molar composition was as noted in Table 23 and the cationic ionizable lipid was added at a 3x charge excess relative to the negative charges on the mRNA. The helper lipid was POPC and the hydrophobic vitamin was cholecalciferol. After CINC assembly, the targeting antibodies were added at a 12.5: 1 mal eimide: antibody ratio. A targeted LNP was created with anti-CD4 antibody and isotype antibody for comparison. Table 23: Formulation compositions for example 18.

[0676] CIL CIL % POPC % Vitamin % PEG % Antibody RH-89-A1 BP Lipid 338 47.8 9.6 28.7 13.9 anti-CD3 RH-89-B1 BP Lipid 363 47.8 9.6 28.7 13.9 anti-CD3 RH-89-A2 BP Lipid 338 47.8 9.6 28.7 13.9 anti-CD4 RH-89-B2 BP Lipid 363 47.8 9.6 28.7 13.9 anti-CD4 RH-89-A3 BP Lipid 338 47.8 9.6 28.7 13.9 Isotype

[0677]

[0678] RH-89-B3 BP Lipid 363 47.8 9.6 28.7 13.9 Isotype

[0679] CINCs were characterized by dynamic light scattering (DLS) and Ribogreen for encapsulation efficiency as summarized in Table 24. The CINCs were then dosed to female C57B16 mice at about 8-10 weeks of age at 0.5 mg / kg intravenous dose. After 3 hours, sera were collected and analyzed for cytokine levels. After 6 hours. IVIS imaging of the mice and harvested organs was completed to identify the biodistribution of expression.

[0680] Table 24: Formulation characterization results for Example 18 by DLS and Ribogreen encapsulation efficiency (EE) analysis after processing.

[0681] Pre-freeze Post-freeze

[0682] Size (nm) PDI Zeta (mV) Size (nm) PDI Zeta (mV) EE RH-89-A1 133 0.09 0.9 148 0.17 -0.5 95% RH-89-B1 130 0.1 1.1 133 0.12 -0.3 98% RH-89-A2 132 0.09 -0.1 145 0.16 -1.2 96%

[0683]

[0684] RH-89-B2 128 0.11 0.5 134 0.15 0.1 98% OLSI-OOl-PCT

[0685] RH-89-A3 134 0.08 -0.5 143 0.14 -0.5 96%

[0686]

[0687] RH-89-B3 127 0.14 0.7 137 0.22 -1.3 98%

[0688] The luciferase analysis at 6 hours revealed enhancement in the spleen with CD4 targeting. Similarly, the lymph nodes in the neck exhibited significant signal only with CD4 but not CD3 targeting (FIGS. 17A-17B). This may reflect the performance of a particular antibody clone rather than a broader CD3 effect. RH-89-B2 exhibited a 55% reduction in liver expression with targeting vs isotype, compared to a 25% increase with the LNP formulation. The liver signal reduction was 10% to 67% among the different CINCs tested. These results demonstrate that the biodistribution of protein expression can be tuned through the addition of targeting antibodies.

[0689] Cytokine analysis revealed statistically significant elevated cytokine levels with LNP administration but not with CINC administration (Figures 18 and 19). The results were similar for chemokines and all other cytokines tested in the mouse antiviral LegendPlex assay. These findings indicate that CINCs may provide an immune silent platform for delivery.

[0690] Example 19

[0691] Targeted CINCs were prepared with either DNA cargo or a 1:1 mixture of DNA and mRNA cargo, and then evaluated for DNA expression in Jurkat T cells. The DNA cargo was a GFP plasmid and the mRNA w-as a firefly luciferase. All CINCs used anti-CD3 antibody clone OKT3 and cationic ionizable lipid BP Lipid 338 (except for CM-227-F. the untargeted control, which had no antibody and used BP Lipid 363). Table 25 summarizes the variations and the characterization of the resulting CINCs.

[0692] The CINCs w ere then dosed to Jurkat T cells at 1 ug / ml (with 40,000 cells / well) and monitored at 48 hours and 96 hours for DNA expression by flow. As shown in FIG. 20, CINCs resulted in sustained DNA expression across 48 and 96 hours. All CINCs except CM-227-A resulted in increasing DNA expression between the time points. Only the untargeted control, CM-227-F, showed no expression. Process variations impacted the relative expression levels as well as the relative cell health (FIG. 21). Only RH-94-G and RH-94-H showed notable drops though they remained impressively high viability values.

[0693] Table 25: Formulation summary for Example 19

[0694] Sample Size Zeta Variation PDI EE Name (nm) (mV)

[0695] CM-227-A DNA / mRNA co-encapsulation, OKT3 167 0.13 0.7 96%

[0696]

[0697] OLSI-OOl-PCT

[0698] DNA / mRNA co-encapsulation, null- CM-227-F 141 0.03 0.4 98% CINC RH-94-A - 140 0.09 -0.1 96% RH-94-B 10: 1 maleimide: antibody ratio 154 0.16 3.4 97% RH-94-C 7.5:1 maleimide: antibody ratio 162 0.18 0.1 97% Nitro-oleic acid added at 20 % of total

[0699] RH-94-D 162 0.10 5.1 84% additive moles

[0700] RH-94-E PCL-PEG instead of PLA-PEG 194 0.21 -0.6 86% RH-94-F POPC to 4.8%, Vitamin D to 33.5% 159 0.22 6.8 97% RH-94-G POPC to 14.4%, Vitamin D to 23.9% 167 0.20 2.6 98% RH-94-H POPC to 19.1%, Vitamin D to 19.1% 180 0.11 2.2 97%

[0701]

[0702] Example 20

[0703] Targeted CINCs were prepared using a cleavable PEG secondary stabilizing agent according to the following process. The cleavable PEG was a 5 kDa PLA linked via thiol-maleimide chemistry to valine-citruline-PAB which was linked to a 5 kDa PEG via amide bond formed using a para-nitrophenyl (PNP) leaving group to generate PLA-VC-PEG. The cleavable PEG polymer may be formed using any suitable chemistry.

[0704] To synthesize the PLA-VC-PEG, a 3x molar excess of PEG-NEE was dissolved in dry DMSO at 50 mg / ml. This was added to the Mal-VC-PAB-PNP (CAS: 159857-81-5) powder and stirred at room temperature for 1 - 3 hours. Optionally, 1.1 equivalent of dry triethylamine may be added. Yellow color develops rapidly, indicating liberation of the PNP leaving group. The PEG-VC-mal product with excess PEG-NH3 may be isolated by dialysis or precipitation.

[0705] The PEG mixture, after drying, was then added to a solution of thiol-terminated PLA (PLA-SH) in DMF with 1 mg / ml Tris(hydroxypropyl)phosphine (THPP) or other suitable reducing agent. The PEG-VC-mal was added at 5x molar excess relative to the PLA to drive complete conversion of the PLA. The reaction was stirred at room temperature for 12 - 48 hours and then the PLA-VC-PEG was micellized by rapid precipitation in water and excess PEG-NH3 removed by dialysis or ultrafiltration. The final product was then isolated by lyophilization to afford a soluble white to yellow-white powder. OLSI-OOl-PCT

[0706] CINCs were prepared according to the methods of Example 2, using a Dasher GFP mRNA cargo and BP Lipid 323 as the cationic ionizable lipid. CINCs made with PLA-PEG were compared to CINCs made with different amounts of PLA-VC-PEG. The resulting CINCs were all stable to ultrafiltration and concentration, indicating stable PEG surfaces. The PLA-VC-PEG formulations were bigger than PLA-PEG formulations, possibly due to PLA homopolymer contamination that could be addressed through chemistry optimization (Table 26).

[0707] Table 26: Formulation summary for Example 20.

[0708] Name PEG Size (nm) PDI

[0709] CM-239-A PLA-PEG 118 0.201

[0710] CM-239-B PLA-VC-PEG

[0711] (1.4 mg) 225 0.194

[0712] CM-239-C PLA-VC-PEG

[0713] (1.8 mg) 198 0.156

[0714] CM-239-D PLA-VC-PEG

[0715]

[0716] (2.2 mg) 187.2 0.186

[0717] CM-239-A and CM-239-D were then incubated at 37 °C with or without papain, an enzyme that cleaves the VC linker. Cleavage of the PEG would be expected to result in aggregation and changes to zeta potential over time. FIG. 22 demonstrates the dramatic size increases upon papain treatment seen only with PLA-VC-PEG formulations. The change was observable visually as well within less than one minute. No effect was seen for non-cleavable PLA-PEG formulations. This demonstrates the potential for responsive CINC formulations.

[0718] To confirm whether this responsiveness correlated with improved potency of genetic cargo expression, CINCs were prepared according to the compositions of Example 18. The cargo was Dasher GFP mRNA (Aldevron) and the ionizable lipid was BP Lipid 338. Three formulations were prepared: CM-241-C used PLA-PEG and no targeting antibody; CM-241-D used PLA-PEG with 6.9% PLA-PEG-mal and anti-CD3 targeting antibody (0KT3); CM-241 -E used PLA-VC-PEG with 6.9% PLA-PEG-mal and anti-CD3 targeting antibody (0KT3). Table 27 summaries the physical properties of the resulting CINCs.

[0719] Table 27: Summary of formulation properties for Example 20 evaluation of mRNA delivery Size (nm) PDI Zeta (mV) EE CM-241-C Null-CINC 125 0.02 -1.0 99.2 CM-241-D OKT3-CINC (PLA-PEG) 154 0.15 -0.5 99.1

[0720]

[0721] CM-241-E OKT3-CINC (PLA-VC-PEG) 246 0.18 -0.8 95.7 OLSI-OOl-PCT

[0722] The CINCs were then tested in Jurkat T cells for GFP expression after 24 hours (FIG.

[0723] 23). The cleavable PEG (CM-241-E) was significantly more potent than the baseline formulation (CM-241-D), with the mean fluorescence intensity of the cleavable formulation CIL

[0724] being 10 times higher than CM-241-D across all doses tested. These results demonstrate the utility of a cleavable secondary stabilizing agent for modulating the potency of targeted CINCs.

[0725] Hleper

[0726] Example 21

[0727] Lipid-polymer nanoparticle compositions were assembled in the absence of an inverse iiddt aev

[0728] nanocarrier, relying on charge interactions to complex a nucleic acid entering the mixer via an acidified antisolvent, pH 4 acetate buffer. The solvent stream contained lipids in an acetonitrile / ethanol blend and the antisolvent contained the nucleic acid (such as mRNA) at a specified charge ratio relative to the cationic ionizable lipid amount in the solvent stream.

[0729] Samples (Table 28) were prepared at a 3:1 charge ratio without targeting ligands. The acetate buffer was removed by dialysis and samples were characterized by DLS and dosed to Huh7 cells to identify the effect of compositions on potency in liver cells

[0730] Table 28: Untargeted formulation details for example 21 CIL %

[0731] Hl %eper

[0732] Add %.

[0733] 249-1 ALC-0315 POPC Cholesterol 5k-5k PLA 3.000 47.8 14.4 23.9 13.9 249-2 ALC-0315 POPC Vit D* 5k-5k PLA 3.000 47.8 14.4 23.9 13.9 249-3 ALC-0315 POPC Cholesterol PEG-DMG 3.000 50.0 10.0 37.0 3.0 249-4 ALC-0315 POPC Vit D PEG-DMG 3.000 50.0 10.0 37.0 3.0 249-5 ALC-0315 POPC Cholesterol 5k-5k PLA 3.000 50.0 10.0 37.0 3.0

[0734]

[0735] 249-6 ALC-0315 POPC Vit D 5k-5k PLA 3.000 50.0 10.0 37.0 3.0 *Vit D: cholecalciferol was used

[0736] These formulations demonstrated significant compositions effects on hepatocyte expression, where the inclusion of both vitamin D and PLA-PEG reduced the expression from 98% to 1% (Table 29).

[0737] Table 29: Untargeted formulation results for example 21

[0738] Sample Name Size PDI Zeta Positivity in Huh7 (%) stdev

[0739] 249-1 178.9 0.1 -0.8 17.1 2.7

[0740] 249-2 192.9 0.02 2.3 1.4 0.4

[0741] 249-3 94.46 0.13 -3.7 98.6 0.4

[0742]

[0743] 249-4 77.52 0.17 0.3 50.8 1.1 OLSI-OOl-PCT

[0744] 249-5 263 0.09 -0.7 37.5 1.4

[0745]

[0746] 249-6 208.4 0.08 -2.9 1.5 0.3

[0747] To extend these findings to targeted formulations, samples were prepared at a 6:1 CIL

[0748] charge ratio and 6.9% of the PEG was replaced with maleimide-functionalized PEG (Table 30). Nanoparticles were dialyzed into PBS, and half of the batch was conjugated to OKT3 targeting ligands using techniques described herein (A samples) while the other half of the batch was unconjugated to produce a null target formulation (B samples).

[0749] iiddt aev

[0750] Table 30: Targeted formulation details for example 21

[0751] G PE

[0752] CIL Eq

[0753] 250-1 ALC-0315 POPC Choi 5k-5k PLA 6.000 47.8 14.4 23.9 13.9 250-2 ALC-0315 POPC Vit D 5k-5k PLA 6.000 47.8 14.4 23.9 13.9

[0754]

[0755] 250-3 BP Lipid 338 POPC Vit D 5k-5k PLA 6.000 47.8 14.4 23.9 13.9

[0756] Hl %eper

[0757] Formulations were tested in primary T cells at multiple doses after characterization by DLS and ribogreen analysis for encapsulation efficiency (EE). The results (Table 31) demonstrate that the inclusion of vitamin D does not hamper expression in T cells (samples 2 & 3). The low expression of untargeted “nulE nanoparticles demonstrates that the delivery’ is OKT3G PE% antibody mediated.

[0758] Table 31: Formulation characterization for example 21, part 2 including results of expression in primary T cells Sample Name Size (nm) PDI Zeta EE % Positivity (1 ug / ml)

[0759] CM-250-1A 150.3 0.11 -1.6 84.2 9.47

[0760] CM-250-1B 154 0.09 -0.2 89.4 0.15

[0761] CM-250-2A 161.6 0.09 -0.6 80.9 13.24

[0762] CM-250-2B 481.5 0.45 -8 69.2 0.01

[0763] CM-250-3A 136.5 0.11 0.8 91.3 69.67

[0764]

[0765] CM-250-3B 134.3 0.13 0.2 93.9 17.85

[0766] Pharmaceutical Compositions and Administration

[0767] In an embodiment of the present disclosure, nanoparticles are useful in pharmaceutical compositions prepared with a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier or diluent.

[0768] Nanoparticles of an embodiment of the present disclosure can be formulated as pharmaceutical compositions and administered to a subject in need of treatment, for example OLSI-OOl-PCT

[0769] a mammal, such as a human patient, in a variety of forms adapted to the chosen route of administration, for example, orally, nasally, intraperitoneally, or parenterally, by intravenous, intramuscular, topical or subcutaneous routes, or by injection into tissue.

[0770] Thus, nanoparticles of an embodiment of the present disclosure may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier, or by inhalation or insufflation. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the nanoparticles may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The nanoparticles may be combined with a fine inert powdered carrier and inhaled by the subject or insufflated. Such compositions and preparations should contain at least 0.1% nanoparticles. The percentage of the compositions and preparations may, of course, be varied and may conveniently be betw een about 2% to about 60% of the weight of a given unit dosage form. The amount of nanoparticles in such therapeutically useful compositions is such that an effective dosage level may be obtained.

[0771] The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, com starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as com starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above ty pe, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the nanoparticles may be incorporated into sustained-release preparations and devices. For example, the nanoparticles may be incorporated into time release capsules, time release tablets, and time release pills.

[0772] The nanoparticles may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the nanoparticles can be prepared in water, optionally mixed OLSI-OOl-PCT

[0773] with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary’ conditions of storage and use, these preparations can contain a preservative to prevent the grow th of microorganisms.

[0774] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders including the nanoparticles which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium including, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal. and the like. Isotonic agents, for example, sugars, buffers or sodium chloride can be included. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0775] Sterile injectable solutions are prepared by incorporating the nanoparticles in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze dry ing techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0776] For topical administration, the nanoparticles may be applied in pure form. How ever, it may be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0777] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Other solid carriers include nontoxic polymeric nanoparticles or microparticles. Useful liquid carriers include water, alcohols or glycols or water / alcohol / glycol blends, in which the nanoparticles can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate OLSI-OOl-PCT

[0778] bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0779] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0780] Examples of useful dermatological compositions which can be used to deliver the nanoparticles to the skin are known to the art; for example, see Jacquet et al. (U. S. Pat. No.

[0781] 4,608,392), Geria (U. S. Pat No. 4,992,478), Smith et al. (U. S. Pat. No. 4,559,157) and Wortzman (U. S. Pat. No. 4,820,508), all of which are hereby incorporated by reference.

[0782] Useful dosages of the compounds of formula I can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U. S. Pat. No. 4,938,949, which is hereby incorporated by reference.

[0783] For example, the concentration of the nanoparticles in a liquid composition, such as a lotion, can be from about 0.1-25% by weight, or from about 0.5-10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder can be about 0.1-5% by weight, or about 0.5-2.5% by weight.

[0784] The amount of the nanoparticles required for use in treatment may vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

[0785] Effective dosages and routes of administration of agents of an embodiment of the present disclosure can be conventional. The exact amount (effective dose) of the agent may vary from subject to subject, depending on, for example, the species, age, weight and general or clinical condition of the subject, the severity or mechanism of any disorder being treated, the particular agent or vehicle used, the method and scheduling of administration, and the like. A therapeutically effective dose can be determined empirically, by conventional procedures known to those of skill in the art. See. e.g., The Pharmacological Basis of Therapeutics. Goodman and Gilman, eds., Macmillan Publishing Co., New York. For example, an effective dose can be estimated initially either in cell culture assays or in suitable animal models. The animal model may also be used to determine the appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. A therapeutic dose can also be selected by analogy to dosages for OLSI-OOl-PCT

[0786] comparable therapeutic agents.

[0787] The particular mode of administration and the dosage regimen may be selected by the attending clinician, taking into account the particulars of the case (e.g., the subject, the disease, the disease state involved, and whether the treatment is prophylactic). Treatment may involve daily or multi-daily doses of compound(s) over a period of a few days to months, or even years.

[0788] In general, however, a suitable dose can be in the range of from about 0.001 to about 100 mg / kg, e.g., from about 0.01 to about 100 mg / kg of body weight per day. such as above about 0.1 mg per kilogram, or in a range of from about 1 to about 10 mg per kilogram body weight of the recipient per day. For example, a suitable dose may be about 1 mg / kg, 10 mg / kg, or 50 mg / kg of body weight per day.

[0789] The nanoparticles are conveniently administered in unit dosage form; for example, containing 0.05 to 10000 mg, 0.5 to 10000 mg, 5 to 1000 mg, or about 100 mg of active ingredient per unit dosage form.

[0790] The nanoparticles can be administered to achieve peak plasma concentrations of, for example, from about 0.5 to about 75 pM, about 1 to 50 pM, about 2 to about 30 pM, or about 5 to about 25 pM. Exemplary desirable plasma concentrations include at least or no more than 0.25, 0.5, 1, 5, 10, 25, 50, 75, 100 or 200 pM. For example, plasma levels may be from about 1 to 100 micromolar or from about 10 to about 25 micromolar. This may be achieved, for example, by the intravenous injection of a 0.05 to 5% solution of the nanoparticles, optionally in saline, or orally administered as a bolus containing about 1-100 mg of the nanoparticles. Desirable blood levels may be maintained by continuous infusion to provide about 0.00005 - 5 mg per kg body weight per hour, for example at least or no more than 0.00005, 0.0005, 0.005, 0.05, 0.5, or 5 mg / kg / hr. Alternatively, such levels can be obtained by intermittent infusions containing about 0.0002 - 20 mg per kg body weight, for example, at least or no more than 0.0002, 0.002, 0.02, 0.2, 2, 20, or 50 mg of the nanoparticles per kg of body weight.

[0791] The nanoparticles may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator.

[0792] The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Nothing in this specification should be considered as limiting the scope of the invention. All examples presented are representative and non-limiting. The above-described embodiments of the invention may be modified or varied, without departing from the invention, as appreciated OLSI-OOl-PCT

[0793] by those skilled in the art in light of the above teachings. It is therefore to be understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.

Claims

What is claimed is:

1. A composition of a coated nanocarrier comprising a hydrophilic encapsulated agent, a first stabilizing polymer, one or more additive agents, and one or more second stabilizing agents, wherein the one or more additive agents includes a hydrophobic vitamin and / or wherein a targeting moiety is conjugated to the one or more second stabilizing agents and is a ligand directed to a surface receptor or protein on a cell of interest.

2. The composition of claim 1, wherein the targeting moiety is conjugated to at least one of the one or more second stabilizing agents and is a ligand directed to a surface receptor or protein on a cell of interest.

3. The composition of claim 2. where the one or more additive agents include a hydrophobic vitamin.

4. The composition of claim 2 or 3, wherein the targeting moiety is an anti-CD3, anti-CD4, anti-CD5, anti-CD7, anti-CD8, anti-CD25, anti-CD117, or anti-CD147 antibody for targeting T cells.

5. The composition of claim 2 or 3, wherein the targeting moiety is an anti-CD 117 or antiCD 133 or anti-cMPL for targeting hematopoietic stem cells.

6. The composition of claim 2 or 3, wherein the targeting moiety is an antibody against an endothelial cell marker.

7. The composition of claim 6. wherein the antibody against an endothelial cell marker is an anti-PECAM antibody.

8. The composition of any one of claims 2-7, wherein the one or more second stabilizing agents is a PLA-PEG, PLGA-PEG, or PCL-PEG.

9. The composition of any one of claim 2-8. wherein the one or more second stabilizing agents comprise a cleavable stabilizing agent.

10. The composition of claim 9, w herein the cleavable stabilizing agent is a cleavable PEG polymer.

11. The composition of any one of claims 1-10, further comprising a cationic ionizable lipid.

12. The composition of any one of claims 1-11, wherein the hydrophilic encapsulated agent includes an RNA, a DNA, or a combination thereof.

13. The composition of claim 12, where the RNA or the DNA encodes for a chimeric antigen receptor.

14. The composition of any one of claims 1-13 which co-encapsulates a chimeric antigen receptor and a protein that regulates gene expression, encoded on mRNA or DNA, or mixtures of mRNA and DNA.

15. The composition of claim 14, wherein the protein that regulates gene expression is a transcription factor or methyltransferase.

16. A method of treatment, comprising administering a composition of any one of claims 1- 15. where the composition delivers an RNA and / or a DNA to treat a patient with cancer or immune disease.

17. A method of treatment, comprising administering a composition of any one of claims 1-15, where the composition delivers one or more RNA sequences and / or DNA sequences expressing a chimeric antigen receptor and a protein that regulates gene expression to treat a patient with cancer or immune disease.

18. The composition of claim 17, wherein the protein that regulates gene expression is a transcription factor or methyltransferase.

19. A composition for treating a cancer or immune disease, comprising a composition of any one of claims 1-15, where the composition delivers an RNA and / or a DNA to treat a patient with cancer or immune disease.

20. A composition for treating a cancer or immune disease, comprising a composition of any one of claims 1-15, where the composition delivers one or more RNA sequences and / or DNA sequences expressing a chimeric antigen receptor and a protein that regulates gene expression to treat a patient with cancer or immune disease.

21. The composition of claim 20, wherein the protein that regulates gene expression is a transcription factor or methyltransferase.

22. A pharmaceutical composition, comprising a composition of any one of claims 1-15, and a pharmaceutically acceptable earner.

23. A composition of a targeted encapsulated particle, comprising:lipid components;a hydrophilic encapsulated agent;a polymeric amphiphilic stabilizing agent; anda targeting moiety conjugated to the polymeric amphiphilic stabilizing agent, where the targeting moiety is a ligand directed to a surface receptor or protein on a cell of interest.

24. The composition of claim 23, wherein the polymeric amphiphilic stabilizing agent comprises a PLA-PEG, a PLGA-PEG. a PCL-PEG, or a combination thereof.Ill25. The composition of any one of claim 23 or 24, wherein the polymeric amphiphilic stabilizing agent comprises a cleavable stabilizing agent.

26. The composition of claim 25, wherein the cleavable stabilizing agent is a cleavable PEG polymer.

27. The composition of any one of claims 23-6, wherein the lipid components comprise a cationic ionizable lipid.

28. The composition of any one of claims 23-27, wherein the lipid components further comprise a helper or structural lipid.

29. The composition of any one of claims 23-28, wherein the lipid components further comprise a hydrophobic vitamin.

30. The composition of any one of claims 23-29, wherein the targeting moiety is an anti-CD3, anti-CD4, anti-CD5, anti-CD7, anti-CD8, anti-CD25, anti-CD117, or anti-CD147 antibody for targeting T cells.

31. The composition of any one of claims 23-29, wherein the targeting moiety7is an anti-CD117 or anti-CD133 or anti-cMPL for targeting hematopoietic stem cells.

32. The composition of any one of claims 23-29, wherein the targeting moiety is an antibody against an endothelial cell marker.

33. The composition of claim 32, wherein the antibody against an endothelial cell marker is an anti-PECAM antibody.

34. The composition of any one of claims 23-33, wherein the hydrophilic encapsulated agent includes an RNA, a DNA, or a combination thereof.

35. The composition of claim 34, where the RNA or the DNA encodes for a chimeric antigen receptor.

36. The composition of any one of claim 23-35, wherein a hydrophobic portion of the polymeric amphiphilic stabilizing agent is a hydrophobic block copolymer.

37. A method of treatment, comprising administering a composition of any one of claims 23- 36, where the composition delivers an RNA and / or a DNA to treat a patient with cancer or immune disease.

38. A method of treatment, comprising administering a composition of any one of claims 23-36, where the composition delivers one or more RNA sequences and / or DNA sequences expressing a chimeric antigen receptor and a protein that regulates gene expression to treat a patient with cancer or immune disease.

39. The composition of claim 38, wherein the protein that regulates gene expression is a transcription factor or methyltransferase.

40. A composition for treating a cancer or immune disease, comprising a composition of any one of claims 23-36, where the composition delivers an RNA and / or a DNA to treat a patient with cancer or immune disease.

41. A composition for treating a cancer or immune disease, comprising a composition of any one of claims 23-36, where the composition delivers one or more RNA sequences and / or DNA sequences expressing a chimeric antigen receptor and a protein that regulates gene expression to treat a patient with cancer or immune disease.

42. The composition of claim 41, wherein the protein that regulates gene expression is a transcription factor or methyltransferase.

43. A pharmaceutical composition, comprising a composition of any one of claims 23-36, and a pharmaceutically acceptable carrier.

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