Compositions and methods for non-viral delivery of therapeutic compounds

Abstract

The present disclosure provides compositions and methods for non-viral delivery of therapeutics, and methods of preparing said compositions.

Description

COMPOSITIONS AND METHODS FOR NON-VIRAL DELIVERY OF THERAPEUTICCOMPOUNDS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 610,979, filed December 15, 2023, which is incorporated herein by reference for all purposes.

[0002] Throughout this application various publications, patents, and / or patent applications are referenced. The disclosures of the publications, patents and / or patent applications are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art to which this disclosure pertains.TECHNICAL FIELD

[0003] The present disclosure provides compositions and methods for non-viral delivery of therapeutic agents, and methods of preparing said compositions.BACKGROUND

[0004] Delivery of therapeutic agents to a patient can be hindered by the limited ability of such compound to reach a target cell or tissue, or by restricted entry of the therapeutic agent into the cell or tissue. In general, the delivery of therapeutic agents is restricted by membranes of cells. Consequently, to overcome these restrictions and limitations to delivery, one approach is to use much higher concentrations of a therapeutic agent than necessary for treatment, which results in increased risk of adverse drug reactions and side effects.

[0005] Another strategy to address the delivery restrictions and limitations is to improve the transport of a compound into target cells or tissues using lipid molecules. Lipids can take advantage of mechanisms that exist for selective entry into a cell. For example, a cationic lipid may interact with a drug agent and provide contact with a cell membrane. Lipid molecules can also be organized into liposomes or particles as carriers for drug agents. Liposomal drug carriers can protect a drug molecule from degradation while improving its uptake by cells. Lipid- containing nanoparticle compositions, liposomes, and lipoplexes have proven effective as transport vehicles into cells and / or intracellular compartments for biologically active substances such as small molecule drugs, proteins, and nucleic acids.

[0006] Transfection is a method of delivering nucleic acids into cells. Transfection can be viral or non-viral. Viral vectors are usually very efficient at promoting gene transfer into eukaryoticcells, but they tend to be very immunogenic and not suitable for repeated dosing. Non-viral vectors tend to be much less efficient in terms of transfection efficiency but usually less likely to induce a strong inflammatory response than viral vectors. Among the non-viral vectors, cationic lipids and cationic polymers have been extensively studied and their transfection efficiency has been clearly demonstrated in vitro (Drean M. (2017) Biomacromolecules 18:440; Rose V. (2017) Polym. Chem. 8:353; Pitard B. (2002) Somat Cell Mol. Genet. 27:5-15). However, aggregation in tissue fluids, toxicity and low in vivo efficiency have hampered their clinical use.

[0007] Numerous compositions and methods exist in the art for delivery of plasmid DNA (therapeutics) into cells. One of the more efficient ways for transfection is via electroporation (Sardesai N. et al. (2011) Curr. Opin. Immunol. 23:421-429). However, electroporation requires a specialized and expensive instrument (e.g., Cellectra®) for injection and may cause permanent cell damage. Methods that rely on simple polymer-based formulations for transfection and delivery of therapeutics into cells (described for example in US7, 709,452 and US8,367,631), are significantly less efficient.

[0008] Yet other transfection methods rely on lipid-based formulations. Commonly used lipid-based transfection reagents are Lipofectamine™ and Invivofectamine™ sold by Thermo Fisher. These reagents are easy to use, without the need for user optimization, and Lipofectamine™ can be used for different payloads (DNA and RNA). However, these reagents have numerous drawbacks as well. For example, Lipofectamine™ can only be used for in vitro transfections, whereas Invivofectamine™ can only be used for in vivo transfections. Although Lipofectamine™ is effective in certain cells, it has low efficacy in primary cells. Invivofectamine™ can only be used to transfect siRNA and miRNA, and can only be administered intravenously. Moreover, both transfection reagents are also extremely expensive.

[0009] Despite numerous existing strategies to improve transfection in vivo, cost efficient, effective targeted delivery of biologically active substances such as small molecule drugs, proteins, and nucleic acids represents a continuing medical challenge.

[0010] Thus, there exists a need to develop compositions and methods to improve transfection and delivery of therapeutic agents such as nucleic acids, proteins and / or small molecule drugs into cells in vitro and in vivo. Preferably, such compositions can be universally used for transfection in vitro and in vivo, have high efficiency of transfection in any cell, and be relatively inexpensive.SUMMARY

[0011] The compositions described herein are simple to prepare. In embodiments, the same composition can be used for both in vitro and in vivo transfections, the compositions have very high efficacy in all types of cells (e.g., compared to Lipofectamine™) and are suitable to use with various nucleic acids, and the compositions can be less expensive than Lipofectamine™ and Invivofectamine™.

[0012] In an aspect, provided herein is a composition for transfecting a cell, in vitro or in vivo, comprising at least one nucleic acid contained in an EPS (lipid shell)~LPX (iipoplex) delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0013] In an aspect, provided herein is a method for transfecting a cell with one or more nucleic acids comprising: contacting the cell in vitro with a composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0014] In another aspect, provided herein is a method for treating or preventing a disease or alleviating a symptom of a disease comprising administering to a subject in need thereof a composition for transfecting a cell comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0015] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (f) preparing an EPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the EPS prepared in step (f), and optionallyadding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (h ) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell

[0016] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodi sparse or polydisperse LPX comprising one or more nucleic acids; (b) agitating the LPX prepared in step (a); (c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (d) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution;(e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (fi agitating the mixture prepared in step (e), thereby forming a composition for transfecting a cell.

[0017] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) w'ith the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a smallmolecule; and (i) agitating the mixture prepared in step (h), thereby forming & composition for transfecting a cell.

[0018] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanopanicles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX; (c) preparing an EPS by forming liposomes, micelles, or other self-assembled lipid nan op articles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution; (d) mixing the LPX prepared in step (b) with the EPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (e) agitating the mixture prepared in step (d), thereby forming a composition for transfecting a cell.DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 Two top scatter plots show transfection efficiency of LCF96 (transfecting 0.5 pg or 1.0 pg GFP mRNA) in HEK293 cells. Two bottom scatter plots are positive controls that show transfection efficiency of 0.5 pg or 1.0 pg GFP mRNA using Lipofectamine™ MessengerMAX™ (LCPOMAX). Vertical axis shows side scatter in arbitrary' units. Horizontal axis shows GFP fluorescence in relative fluorescence units.

[0020] FIG. 2 scatter plots show transfection efficiency of LCF96, LCF107, LCF108, and LCF109 (transfecting 1.0 pg or 3.0 pg GFP mRNA) in Jurkat cells. Two bottom scatter plots are positive controls that show transfection efficiency of 1.0 pg or 3.0 pg GFP mRNA using Lipofectamine™ MessengerMAX’™ (LIPOMAX). Vertical axis shows side scatter in arbitrary’ units. Horizontal axis shows GFP fluorescence in relative fluorescence units.

[0021] FIG. 3 scatter plots show transfection efficiency of LCF96 and LCF108 (transfecting 1.0 pg or 3.0 pg GFP mRNA) in human primary T cells. Two bottom scatter plots are positive controls that show transfection efficiency of 1.0 pg or 3.0 pg GFP mRNA usingLipofectamine™ MessengerMAX™ (LIPOMAX). Vertical axis shows side scatter in arbitrary units. Horizontal axis shows GFP fluorescence in relative fluorescence units.

[0022] FIG. 4 shows in vivo imaging of anaesthetized mice injected with rsF591, rsF616, or rsF592 (transfecting 1.0 pg Luc mRNA) followed by D-luciferin injection 3 hours later.

[0023] FIG. 5 Two top scatter plots show transfection efficiency of LCF155 and LCF157 (transfecting 1.0 pg pDNA) in HEK293 cells. Two bottom scatter plots are positive controls that show transfection efficiency of 1.0 pg pDNA using Lipofectamine™ 2000 and Lipofectamine™ LTX. Vertical axis shows side scatter in arbitrary units. Horizontal axis shows GFP fluorescence in relative fluorescence units.

[0024] FIG. 6 Two top scatter plots show transfection efficiency of LCF155 and LCF157 (transfecting 1.0 pg pDNA) in Jurkat cells. The bottom scatter plot is a positive control that shows transfection efficiency of 1.0 pg pDNA using Lipofectamine™ 2000. Vertical axis shows side scatter in arbitrary' units. Horizontal axis shows GFP fluorescence in relative fluorescence units.

[0025] FIG. 7 shows in vivo imaging of anaesthetized mice injected with rsF615 (transfecting 8.0 pg pDNA) followed by two D-luciferin injection 3 hours later and 6 hours later.

[0026] FIG. 8A shows bioluminescence images of mRNA expression in heart, lung, liver, spleen, and kidney, of mice that were injected with rsF598, rsF599, rsF600, or rsF601 (transfecting 1.0 pg Luc mRNA per mouse) followed by D-luciferin injection 6 hours later.

[0027] FIG. 8B shows relative biodistribution of the quantified IVIS images of heart, lung, liver, spleen, and kidney of mice described in FIG. 8A.

[0028] FIG. 9A shows bioluminescence images of mRNA expression in heart, lung, liver, spleen, and kidney, of mice that were injected with rsF627 or rsF628 (transfecting 1.0 pg Luc mRNA per mouse) followed by D-luciferin injection 3 hours later.

[0029] FIG. 9B shows relative biodistribution of the quantified IVIS images of heart, lung, liver, spleen, and kidney of mice described in FIG. 9A.

[0030] FIG. 9C shows luciferase expression level in the lung of mice described in FIG. 9A.

[0031] FIG. 10A shows bioluminescence images of mRNA expression in heart, lung, liver, spleen, and kidney, of mice that were injected with rsF620 or rsF621 (transfecting 1.0 pg Luc mRNA per mouse) followed by D-luciferin injection 3 hours later.

[0032] FIG. 10B shows relative biodistribution of the quantified IVIS images of heart, lung, liver, spleen, and kidney of mice described in FIG. 10A.

[0033] FIG. 10C shows luciferase expression level in the lung of mice described in FIG.10A

[0034] FIG. 11 shows fluidic / microfluidic processes for the preparation of LPS-LPX. FIG. 11A shows the process for preparation of LPS-LPX where the payload (nucleic acid(s)) is inside the LPX. FIG. 11A shows the process for preparation of LPS-LPX where the payload (nucleic acid(s)) is on the outside of LPX. FIG. 11C shows the process for preparation of LPS-LPX where there are two payloads (nucleic acid(s)), one is inside the LPX and another is on the outside of LPX.DESCRIPTIONDefinitions

[0035] Unless defined otherwise, technical and scientific terms used herein have meanings that are commonly understood by those of ordinary skill in the art unless defined otherwise. Generally, terminologies pertaining to techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, transgenic cell production, protein chemistry and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional procedures well known in the art and as described in various general and more specific references that are cited and discussed herein unless otherwise indicated. See, e g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). A number of basic texts describe standard antibody production processes, including, Borrebaeck (ed) Antibody Engineering, 2nd Edition Freeman and Company, NY, 1995; McCafferty et al. Antibody Engineering, A Practical Approach IRL at Oxford Press, Oxford, England, 1996; and Paul (1995) Antibody Engineering Protocols Humana Press, Towata, N.J., 1995; Paul (ed.), Fundamental Immunology, Raven Press, N.Y, 1993;Coligan (1991) Current Protocols in Immunology) Wiley / Greene, NY; Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, Calif, and references cited therein; Coding Monoclonal Antibodies: Principles and Practice (2nd ed.) Academic Press,New York, N.Y., 1986, and Kohler and Milstein Nature 256: 495-497, 1975. All of the references cited herein are incorporated herein by reference in their entireties. Enzymatic reactions and enrichment / purification techniques are also well known and are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The terminology used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0036] The headings provided herein are not limitations of the various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0037] Unless otherwise required by context herein, singular terms shall include pluralities and plural terms shall include the singular. Singular forms “a”, “an” and “the”, and singular use of any word, include plural referents unless expressly and unequivocally limited on one referent.

[0038] It is understood the use of the alternative (e.g., “or”) herein is taken to mean either one or both or any combination thereof of the alternatives.

[0039] The term “and / or” used herein is to be taken mean specific disclosure of each of the specified features or components with or without the other. For example, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0040] As used herein, terms “comprising”, “including”, “having” and “containing”, and their grammatical variants, as used herein are intended to be non-limiting so that one item or multiple items in a list do not exclude other items that can be substituted or added to the listed items. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0041] As used herein, the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured ordetermined, i.e., the limitations of the measurement system. For example, “about” or “approximately” can mean within one or more than one standard deviation per the practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 10% (i.e., ±10%) or more depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure, unless otherwise stated, the meaning of “about” or “approximately” should be assumed to be within an acceptable error range for that particular value or composition.

[0042] The term “coronavirus infection” refers to a human or animal that has cells that have been infected by a coronavirus. The infection can be established by performing a detection and / or viral titration from respiratory samples, or by assaying blood-circulating coronavirusspecific antibodies. The detection in the individuals infected with coronavirus is made by conventional diagnostic methods, such as molecular biology (e.g., PCR), which are known to those skilled in the art.

[0043] The term “subject” as used herein refers to human and non-human animals, including vertebrates, mammals and non-mammals. In one embodiment, the subject can be human, non- human primates, simian, ape, murine (e.g., mice and rats), bovine, porcine, equine, canine, feline, caprine, lupine, ranine or piscine.

[0044] The term “administering”, “administered” and grammatical variants refers to the physical introduction of a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, transdermal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation. In one embodiment, the formulation is administered via a non-parenteral route, e.g., orally. Other non-parenteral routes include atopical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0045] The terms “treatment” and “treating” refer to fighting a cancer or a viral infection in a human or animal subject. By virtue of the administration of at least one embodiment of the compositions described herein, the viral infection rate (infectious titer) in the subject will decrease, and the virus may completely disappear from the subject. The terms “treatment” and “treating” also refers to attenuating symptoms associated with the viral infection (e.g., respiratory syndrome, kidney failure, fever, and other symptoms relating to viral infections).

[0046] The terms "effective amount", “therapeutically effective amount” or “effective dose” or related terms may be used interchangeably and refer to an amount of the therapeutic agent that when administered to a subject, is sufficient to affect a measurable improvement or prevention of a disease or disorder associated with a cancer or a viral infection. For example, administering an effective dose sufficient to inhibit the proliferation and / or replication of the coronavirus, and / or the development of the viral infection within the subject. Therapeutically effective amounts of the therapeutic agents provided herein, when used alone or in combination with an antiviral agent, will vary depending upon the relative activity of the therapeutic agent, and depending upon the subject and disease condition being treated, the weight and age and sex of the subject, the severity of the disease condition in the subject, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art using known techniques.

[0047] The terms “therapeutic agent” and “therapeutics” as used herein refer to an agent (e.g., compound or composition described herein) that when administered to a subject will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or reoccurrence) of an injury, disease, pathology, or condition, or their symptoms or the intended therapeutic effect, e.g., treatment or amelioration of an injury, disease, pathology or condition, or their symptoms including any objective or subjective parameter of treatment such as abatement; remission; diminishing of symptoms or making the injury, pathology or condition more tolerable to the patient; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a patient’s physical or mental well-being.

[0048] The terms "peptide", "polypeptide" and "protein" and other related terms used herein are used interchangeably and refer to a polymer of amino acids and are not limited to any particular length. Polypeptides may comprise natural and non-natural amino acids. Polypeptides include recombinant or chemically synthesized forms. These terms encompass native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, chimeric proteins and fusion proteins) of a protein sequence as well as post-translationally, or otherwise covalently or non-covalently, modified proteins.

[0049] The terms “nucleic acid”, "polynucleotide" and "oligonucleotide" and other related terms used herein are used interchangeably and refer to polymers of nucleotides and are not limited to any particular length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids include DNA molecules (e.g., cDNA, pDNA or genomic DNA), RNA molecules (e.g., mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA), analogs of the DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. Nucleic acid molecule can be singlestranded or double-stranded. In one embodiment, nucleic acids comprise a one type of polynucleotides or a mixture of two or more different types of polynucleotides.

[0050] The term “plasmid DNA” is used to refer to a plasmid that includes a promoter (and enhancer) and either separate genes for a light chain and a heavy chain of a monoclonal antibody (or bispecific antibody or antigen-binding fragment of a monoclonal or bispecific antibody) or a protein-coding gene, e.g., encoding an antibody fragment such as, for example, Fab or scFv, or a single-chain antibody. In embodiments, the protein coding gene can encode a hormone, a cytokine, an enzyme, an immunogenic peptide, or the like.

[0051] In embodiments, the protein coding gene can encode an antibody fragment. In embodiments, the protein coding gene can encode a Fab. In embodiments, the protein coding gene can encode an scFv. In embodiments, the protein coding gene can encode a hormone, a cytokine, an enzyme, an immunogenic peptide, or the like. In embodiments, the protein coding gene can encode a hormone, the protein coding gene can encode a cytokine. In embodiments, the protein coding gene can encode an enzyme. In embodiments, the protein coding gene can encode an immunogenic peptide.

[0052] In embodiments, the nucleic acid may carry therapeutic genes, regulatory sequences for transcription or for replication, modified or unmodified antisense sequences, regions for binding to other cellular components, and the like.

[0053] The term “mutation”, “modification”, or “variation”, or related terms, refers to a change in a nucleic acid sequence or amino acid sequence that differs from a reference nucleic acid sequence or a reference amino acid sequence, respectively. Examples of mutations includes a point mutation, insertion, deletion, amino acid substitution, inversion, rearrangement, splice, sequence fusion (e.g., gene fusion or RNA fusion), truncation, transversion, translocation, nonsense mutation, sequence repeat, single nucleotide polymorphism (SNP), or other genetic rearrangement.

[0054] An "antibody" and “antibodies” and related terms used herein refers to an intact immunoglobulin or to an antigen binding portion thereof (or an antigen binding fragment thereof) that binds specifically to an antigen. Antigen binding portions (or the antigen binding fragment) may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions (or antigen binding fragments) include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

[0055] Antibodies include recombinantly produced antibodies and antigen binding portions. Antibodies include non-human, chimeric, humanized and fully human antibodies. Antibodies include monospecific, and multispecific (e.g., bispecific, trispecific and higher order specificities) antibodies. Antibodies include tetrameric antibodies, light chain monomers, heavy chain monomers, light chain dimers, heavy chain dimers. Antibodies include F(ab’)2 fragments, Fab’ fragments and Fab fragments. Antibodies include single domain antibodies, monovalent antibodies, single chain antibodies, single chain variable fragment (scFv), camelized antibodies, affibodies, disulfide-linked Fvs (sdFv), anti-idiotypic antibodies (anti-Id), minibodies.Antibodies include monoclonal and polyclonal populations.

[0056] A “neutralizing antibody” and related terms refers to an antibody that is capable of specifically binding to the neutralizing epitope of its target antigen (e.g., coronavirus spike protein) and substantially inhibiting or eliminating the biological activity of the target antigen(e g., coronavirus spike protein). The neutralizing antibody can reduce the biological activity of the target antigen by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher levels of reduced biological activity.

[0057] An “antigen binding domain,” “antigen binding region,” or “antigen binding site” and other related terms used herein refer to a portion of an antigen binding protein that contains amino acid residues (or other moieties) that interact with an antigen and contribute to the antigen binding protein's specificity and affinity for the antigen. For an antibody that specifically binds to its antigen, this will include at least part of at least one of its CDR domains.

[0058] The terms "specific binding", "specifically binds" or "specifically binding" and other related terms, as used herein in the context of an antibody or antigen binding protein or antibody fragment, refer to non-covalent or covalent preferential binding to an antigen relative to other molecules or moieties (e g., an antibody specifically binds to a particular antigen relative to other available antigens). In one embodiment, an antibody specifically binds to a target antigen if it binds to the antigen with a dissociation constant KD of 10‘5M or less, or 10‘6M or less, or 10'7M or less, or 10'8M or less, or 10'9M or less, or 10'10M or less.

[0059] An "antibody fragment", "antibody portion", "antigen-binding fragment of an antibody", or "antigen-binding portion of an antibody" and other related terms used herein refer to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab1, Fab'-SH, F(ab')2; Fd; and Fv fragments, as well as dAb; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide. Antigen binding portions of an antibody may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), and complementarity determining region (CDR) fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer antigen binding properties to the antibody fragment.

[0060] The terms “Fab”, “Fab fragment” and other related terms refers to a monovalent fragment comprising a variable light chain region (VL), constant light chain region (CL), variable heavy chain region (VH), and first constant region (Cm). A Fab is capable of binding an antigen.An F(ab')2 fragment is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region. A F(Ab’)2 has antigen binding capability. An Fd fragment comprises VH and CHI regions. An Fv fragment comprises VL and VH regions. An Fv can bind an antigen. A dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain (U.S. Patents 6,846,634 and 6,696,245; U.S. published Application Nos. 2002 / 02512, 2004 / 0202995, 2004 / 0038291, 2004 / 0009507, 2003 / 0039958; and Ward et al., Nature 341:544- 546, 1989).

[0061] A single-chain antibody (scFv) is an antibody in which a VL and a VH region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain. Preferably the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., 1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

[0062] Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VH and VL domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2: 1121-23). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites. Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites. Similarly, tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.

[0063] The term “human antibody” refers to antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (e.g., a fully human antibody). These antibodies may be prepared in a variety of ways, examples of which are described below, including through recombinant methodologies or through immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and / or light chain-encoding genes.

[0064] As used herein, the term “variant” polypeptides and “variants” of polypeptides refers to a polypeptide comprising an amino acid sequence with one or more amino acid residuesinserted into, deleted from and / or substituted into the amino acid sequence relative to a reference polypeptide sequence. Polypeptide variants include fusion proteins. In the same manner, a variant polynucleotide comprises a nucleotide sequence with one or more nucleotides inserted into, deleted from and / or substituted into the nucleotide sequence relative to another polynucleotide sequence. Polynucleotide variants include fusion polynucleotides.

[0065] As used herein, the term “derivative” of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, e.g., via conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. Unless otherwise indicated, the term “antibody” includes, in addition to antibodies comprising two full-length heavy chains and two full-length light chains, derivatives, variants, fragments, and muteins thereof, examples of which are described below.

[0066] The term “hinge” refers to an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the overall construct and movement of one or both of the domains relative to one another. Structurally, a hinge region comprises from about 10 to about 100 amino acids, e.g., from about 15 to about 75 amino acids, from about 20 to about 50 amino acids, or from about 30 to about 60 amino acids. In embodiments, the hinge region is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids in length. The hinge region can be derived from a hinge region of a naturally-occurring protein, such as a CD8 hinge region or a fragment thereof, a CD8a hinge region, or a fragment thereof, a hinge region of an antibody (e.g., IgG, IgA, IgM, IgE, or IgD antibodies), or a hinge region that joins the constant domains CHI and CH2 of an antibody. The hinge region can be derived from an antibody and may or may not comprise one or more constant regions of the antibody, or the hinge region comprises the hinge region of an antibody and the CH3 constant region of the antibody, or the hinge region comprises the hinge region of an antibody and the CH2 and CH3 constant regions of the antibody, or the hinge region is a non-naturally occurring peptide, or the hinge region is disposed between the C- terminus of the scFv and the N-terminus of the transmembrane domain. In embodiments, the hinge region comprises any one or any combination of two or more regions comprising an upper, core or lower hinge sequences from an IgGl, IgG2, IgG3 or IgG4 immunoglobulin molecule. In embodiments, the hinge region comprises an IgGl upper hinge sequence EPKSCDKTHT. In embodiments, the hinge region comprises an IgGl core hinge sequence CPXCP, wherein X is P,R or S. In embodiments, the hinge region comprises a lower hinge sequence APELLGGP. In embodiments, the hinge is joined to an Fc region (CH2) having the amino acid sequence SVFLFPPKPKDT. In embodiments, the hinge region includes the amino acid sequence of an upper, core and lower hinge and comprises EPKSCDKTHTCPPCPAP-ELLGGP. In embodiments, the hinge region comprises one, two, three or more cysteines that can form at least one, two, three or more interchain disulfide bonds.

[0067] The term “Fc” or “Fc region” as used herein refers to the portion of an antibody heavy chain constant region beginning in or after the hinge region and ending at the C-terminus of the heavy chain. The Fc region comprises at least a portion of the CH2 and CH3 regions and may, or may not, include a portion of the hinge region. Two polypeptide chains each carrying a half Fc region can dimerize to form a full Fc domain. An Fc domain can bind Fc cell surface receptors and some proteins of the immune complement system. An Fc region can bind a complement component Clq. An Fc domain exhibits effector function, including any one or any combination of two or more activities including complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), opsonization and / or cell binding. An Fc domain can bind an Fc receptor, including FcyRI (e.g., CD64), FcyRII (e.g, CD32) and / or FcyRIII (e.g., CD16a). In embodiments, the Fc region can include a mutation or several mutations that increases or decreases any one or any combination of these functions. In embodiments, the Fc domain comprises LALA mutations (e.g., equivalent to L234A, L235A according to Kabat numbering) which reduces effector function (see, for example Hezareh et al. (2001) J Virol 12161-12168). In embodiments, the Fc domain comprises a LALA-PG mutation (e.g., equivalent to L234A, L235A, P329G according to Kabat numbering) which reduces effector function. In embodiments, the Fc domain mediates serum half-life of the protein complex, and a mutation in the Fc domain can increase or decrease the serum half-life of the protein complex. In embodiments, the Fc domain affects thermal stability of the protein complex, and mutation in the Fc domain can increase or decrease the thermal stability of the protein complex.

[0068] The term “labeled antibody” or related terms as used herein refers to antibodies and their antigen binding portions thereof that are unlabeled or joined to a detectable label or moiety for detection, wherein the detectable label or moiety is radioactive, colorimetric, antigenic, enzymatic, a detectable bead (such as a magnetic or electrodense (e.g., gold) bead), biotin,streptavidin or protein A. A variety of labels can be employed, including, but not limited to, radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).

[0069] The “percent identity” or “percent homology” and related terms used herein refers to a quantitative measurement of the similarity between two polypeptide or between two polynucleotide sequences. The percent identity between two polypeptide sequences is a function of the number of identical amino acids at aligned positions that are shared between the two polypeptide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polypeptide sequences. In a similar manner, the percent identity between two polynucleotide sequences is a function of the number of identical nucleotides at aligned positions that are shared between the two polynucleotide sequences, taking into account the number of gaps, and the length of each gap, which may need to be introduced to optimize alignment of the two polynucleotide sequences. A comparison of the sequences and determination of the percent identity between two polypeptide sequences, or between two polynucleotide sequences, may be accomplished using a mathematical algorithm. For example, the "percent identity" or "percent homology" of two polypeptide or two polynucleotide sequences may be determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters.

[0070] A "vector" and related terms used herein refers to a nucleic acid molecule (e.g., DNA or RNA) which can be operably linked to foreign genetic material (e.g., nucleic acid transgene). Vectors can be used as a vehicle to introduce foreign genetic material into a cell (e.g., host cell). Vectors can include at least one restriction endonuclease recognition sequence for insertion of the transgene into the vector. Vectors can include at least one gene sequence that confers antibiotic resistance or a selectable characteristic to aid in selection of host cells that harbor a vector-transgene construct. Vectors can be single-stranded or double-stranded nucleic acid molecules. Vectors can be linear or circular nucleic acid molecules. One type of vector is a "plasmid," which refers to a linear or circular double stranded extrachromosomal DNA molecule which can be linked to a transgene, and is capable of replicating in a host cell, and transcribing and / or translating the transgene. A viral vector typically contains viral RNA or DNA backbone sequences which can be linked to the transgene. The viral backbone sequences can be modifiedto disable infection but retain insertion of the viral backbone and the co-linked transgene into a host cell genome. Examples of viral vectors include retroviral, lentiviral, adenoviral, adeno- associated, baculoviral, papovaviral, vaccinia viral, herpes simplex viral and Epstein Barr viral vectors. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors comprising a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0071] An "expression vector" is a type of vector that can contain one or more regulatory sequences, such as inducible and / or constitutive promoters and enhancers. Expression vectors can include ribosomal binding sites and / or polyadenylation sites. Regulatory sequences direct transcription, or transcription and translation, of a transgene, such as a DNA or RNA transgene, linked to the expression vector which is transduced into a host cell. The regulatory sequence(s) can control the level, timing and / or location of expression of the transgene. The regulatory sequence can, for example, exert its effects directly on the transgene, or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Regulatory sequences can be part of a vector. Further examples of regulatory sequences are described in, for example, Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif, and Baron et al., 1995, Nucleic Acids Res. 23:3605-3606.

[0072] A transgene is “operably linked” to a vector when there is linkage between the transgene and the vector to permit functioning or expression of the transgene sequences contained in the vector. In one embodiment, a transgene is "operably linked" to a regulatory sequence when the regulatory sequence affects the expression (e.g., the level, timing, or location of expression) of the transgene.

[0073] The terms "transfected" or "transformed" or "transduced" or other related terms used herein refer to a process by which exogenous nucleic acid (e g., transgene) is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" host cell is one which has been transfected, transformed or transduced with exogenous nucleic acid (transgene). The host cell includes the primary subject cell and its progeny. Exogenous nucleic acids encoding at least a portion of any of the anti-spike protein antibodies described herein can beintroduced into a host cell. Expression vectors comprising at least a portion of any of the antispike protein antibodies described herein can be introduced into a host cell, and the host cell can express polypeptides comprising at least a portion of the anti-spike protein antibody.

[0074] 1 he terra “’transfection” is used herein generally to mean the method of delivery and introduction of biologically functional nucleic acid into a cell , e.g a eukaryotic cell, in such a way that the nucleic acid retains its function within the cell. The method may involve the use of poly(laciide-co-glycolide) (PLGA), ISCOMs, liposomes, niosomes. virosomes, block copolymers, Pluronic block copolymers, chitosan, and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nucleof ection, piezoelectric permeabilization, sonoporation, iontophoresis, ultrasound, SQZ high speed cell deformation mediated membrane disruption, corona plasma, plasma facilitated delivery, tissue tolerable plasma, laser microporation, shock wave energy, magnetic fields, contactless magneto- permeabilization, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high pressure tail vein injection, and the like, as known in the art.

[0075] Transfection methods of this invention may be applied to cells in vitro or in vivo. The term transfection includes the more specific meaning of delivery and introduction of expressible nucleic acid into a cell such that the cell is rendered capable of expressing that nucleic acid. The term expression means any manifestation of the functional presence of the nucleic acid within a cell, including both transient expression and stable expression. Nucleic acids include both DNA and R.NA without size limits from any source comprising natural and non-natural bases. Nucleic acids can have a variety of biological functions. They may carry- therapeutic genes, regulatory sequences for transcription or for replication, modified or unmodified antisense sequences, regions for binding to other cellular components, etc. They may direct the synthesis of a polypeptide specific for an infectious agent or may be capable of remedying a genetic or acquired deficiency. They can encode proteins, comprise regulatory regions, function as inhibitors of gene orRNA expression (e.g., antisense DNA or RNA), function as inhibitors of proteins, function to inhibit cell growth or kill cells, catalyze reactions or function in a diagnostic or other analytical assay.

[0076] The term “transfection agent” as used herein refers to compounds used herein for transfection. In embodiments, transfection agent is a polymer, a lipid, a surfactant, or any combinati on thereof

[0077] The term “therapeutic gene” is intended to mean in particular any gene encoding a protein product having a therapeutic effect. The protein product thus encoded may be a protein, a peptide, and the like. In embodiments, the nucleic acid may also comprise one or more genes encoding an antigenic peptide capable of generating an immune response in humans or animals. In embodiments, the nucleic acid can be used for cell therapy. In embodiments, the nucleic acid makes it possible to produce either vaccines or immuno-therapeutic treatments. In embodiments, vaccines or immuno-therapeutic treatments can be used for preventing or treating cancers. In embodiments, such vaccines or immuno-therapeutic treatments can be used to treat viral infections. In embodiments, the viral infection is a coronavirus.

[0078] A host cell can be a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell. The phrase “transgenic host cell” or "recombinant host cell" can be used to denote a host cell that has been introduced (e.g., transduced, transformed or transfected) with a nucleic acid either to be expressed or not to be expressed. A host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid. It is understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to, e.g., mutation or environmental influence, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0079] Thus the terms "host cell" or “or a population of host cells” or related terms as used herein may refer to a cell (or a population thereof or a plurality of host cells) to be used for production of the antibody or fragment thereof, is a cell or cells into which foreign (exogenous or transgene) nucleic acids have been introduced, for example, to direct production of the anti-spike protein antibody by the production host cell. The foreign nucleic acids can include an expression vector operably linked to a transgene, and the host cell can be used to express the nucleic acid and / or polypeptide encoded by the foreign nucleic acid (transgene). A host cell (or a population thereof) can be a cultured cell, can be extracted from a subject, or can be the cell of an organism,including a human subject. The host cell (or a population of host cells) includes the primary subject cell and its progeny without any regard for the number of generations or passages. The host cell (or a population thereof) includes immortalized cell lines. Progeny cells may or may not harbor identical genetic material compared to the parent cell. In one embodiment, a production host cell describes any cell (including its progeny) that has been modified, transfected, transduced, transformed, and / or manipulated in any way to express an antibody, as disclosed herein. In one example, the host cell (or population thereof) can be transfected or transduced with an expression vector operably linked to a nucleic acid encoding the desired antibody, or an antigen binding portion thereof, as described herein. Production host cells and populations thereof can harbor an expression vector that is stably integrated into the host’s genome or can harbor an extrachromosomal expression vector. In one embodiment, host cells and populations thereof can harbor an extrachromosomal vector that is present after several cell divisions or is present transiently and is lost after several cell divisions.

[0080] In other contexts, the disclosure may use the term “host cell” or “host cells” to refer to a cell or cells that are infected with a virus (such as a coronavirus), cells capable of being infected by a virus (e.g., lung cells of a subject), or cells used in assays or experiments testing their ability to be infected by a virus. Other terms for virally-infected cells, cells capable of being infected by a virus, or cells used in assays that include viral infection procedures, may include, as nonlimiting examples, “target cells”, “susceptible cells”, “test cells”, “virus propagating cells”, “infected cells”, and the like.

[0081] The terms “lipid” or “lipid moiety” are used in accordance with its ordinary meaning in chemistry and refer to a hydrophobic molecule which is typically characterized by an aliphatic hydrocarbon chain. In embodiments, the lipid moiety includes a carbon chain of 3 to 100 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 50 carbons. In embodiments, the lipid moiety includes a carbon chain of 5 to 25 carbons. In embodiments, the lipid moiety includes a carbon chain of 8 to 525 carbons. Lipid moieties may include saturated or unsaturated carbon chains, and may be optionally substituted. In embodiments, the lipid moiety is optionally substituted with a charged moiety at the terminal end. In embodiments, the lipid moiety is an alkyl or heteroalkyl optionally substituted with a carboxylic acid moiety at the terminal end. Lipids are also 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 manyorganic 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” such as steroids.

[0082] ‘LPS-LPX delivery system”, as used herein refers to transfection reagents described herein, comprising a lipoplex (LPX) encapsulated inside a lipid shell (LPS), and optionally with a targeting ligand, an imaging ligand, a digesting agent, or a small molecule on the lipid shell (LPS). A lipoplex is a complex of one or more lipids with a cargo, such as a nucleic acid; as such, the cargo (e.g., nucleic acid) may be contained in the LPS-LPX delivery system. In embodiments, the LPX comprises one or more ionizable cationic lipids and optionally one or more helper lipids. In embodiments, the LPS is a lipid micelle, a liposome, or a self-assembled lipid nanoparticle. In embodiments, the LPS comprises one or more ionizable cationic lipids and / or one or more helper lipids capable of forming a lipid micelle, a liposome, or a selfassembled lipid nanoparticle. In embodiments, the ionizable cationic lipid may be any one of the lipids in Table 1 . In embodiments, the helper lipid is a phospholipid, pegylated lipid, cholesterol, or cholesterol derivative. The nucleic acids s) can be incorporated in the LPX, as described herein, prior to its encapsulation inside the LPS. In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule may be in contact with the LPS. In embodiments, an imaging ligand, a digesting agent, or a small molecule may be inside the LPX. In embodiments, an imaging ligand, a digesting agent, or a small molecule may be between the surface of the LPS and the LPX.

[0083] As used herein, the expression - a targeting ligand “in contact with LPS” means a targeting ligand on the surface of the LPS; or a targeting ligand with one end in the micellar or liposomal bilayer and with its other end protruding outside of the lipid shell.

[0084] The terms “cationic lipid” or “ionizable cationic lipid” are used interchangeably herein and refer to lipids that are protonated (e.g., >50% protonated) at low pH (e.g., pH 4), which makes them positively charged, but they may remain neutral at physiological pH (e.g., pH 7.4). In embodiments, an ionizable cationic lipid is any lipid recited in Table 1.

[0085] The term “helper lipid” refers to lipids that improve nanoparticle stability, fluidity, blood compatibility, oligonucleotide delivery efficiency, and transfection activity. In embodiments, the helper lipids include, but is not limited to, some phospholipids, DOPE, DOPC,ALC-0159, DEPE, DLOPE, POPE, DSPC, cholesterol, cholesterol-based lipids, and PEGylated lipids. In embodiments, the helper lipid can be any lipid recited in Table 2.

[0086] The term “organic phase” as used herein is used in accordance with its ordinary meaning in chemistry and refers to a solution comprising a solvent which includes carbon (an organic solvent). Non-limiting examples of organic solvents include acetic acid, acetone, acetonitrile, benzene, 1 -butanol, 2-butanol, 2-butanone, t-butyl alcohol, carbon tetrachloride, chlorobenzene, chloroform, cyclohexane, 1,2-di chloroethane, di ethylene glycol, diethyl ether, diglyme (di ethylene glycol , dimethyl ether), 1,2-dimethoxy ethane (glyme, DME), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,4-di oxane, ethanol, ethyl acetate, ethylene glycol, glycerin, heptane, hexamethylphosphoramide (HMPA), hexamethylphosphorous, triamide (HMPT), hexane, methanol, methyl t-butyl ether (MTBE), methylene chloride, N-methyl-2-pyrrolidinone (NMP), nitromethane, pentane, petroleum ether (ligroine), 1 -propanol, 2-propanol, pyridine, tetrahydrofuran (THF), toluene, tri ethyl amine, o- xylene, m-xylene, or p-xylene. In embodiments, the organic solvent is or includes chloroform, dichloromethane, methanol, ethanol, tetrahydrofuran, or dioxane.

[0087] The term “aqueous phase” as used herein is used in accordance with its ordinary meaning in chemistry and refers to a solution where the solvent is water.

[0088] The term “agitation” as used herein refers to any type of agitation or mixing. Some non-limiting examples of agitation include vortexing, sonication, pipetting, stirring, shaking, and the like.

[0089] The present disclosure provides methods for treating a subject testing positive for an infection or a subject suffering from cancer. The present disclosure also provides methods for treating a subject suspected of being infected or at risk of being infected with a virus.Compositions for Transfection

[0090] In an aspect provided herein is a composition for transfecting a cell, m vitro or m vivo, comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the composition for transfecting a cell comprises a DNA contained in an LPS- LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the composition for transfecting a cell comprises an RNA contained in an LPS-LPX delivers- system, and optionally a targeting ligand, an imaging ligand.a digesting agent, or a small molecule. In embodiments, the composition for transfecting a cell comprises a DNA and an RNA each contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0091] In embodiments, the targeting ligand is in contact with the LPS. In embodiments, the imaging ligand is in contact with the I., PS, inside the LPX, or between the surface of the LPS and the LPX. In embodiments, the imaging ligand is in contact with the LPS. In embodiments, the imaging ligand is inside the LPX. In embodiments, the imaging ligand is between the surface of the LPS and the LPX. In embodiments, the digesting agent is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In embodiments, the digesting agent is in contact with the LPS. In embodiments, the digesting agent is inside the LPX. In embodiments, the digesting agent is between the surface of the LPS and the LPX. In embodiments, the small molecule is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In embodiments, the small molecule is in contact with the LPS. In embodiments, the small molecule is inside the LPX. In embodiments, the small molecule is between the surface of the LPS and the LPX.

[0092] In embodiments, provided herein is a composition for transfecting a cell comprising one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, provided herein is a composition for transfecting a cell comprising two nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, provided herein is a composition for transfecting a cell comprising three nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, provided herein is a composition for transfecting a cell comprising four nucleic acids contained in an LPS-LPX delivery' system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. Where multiple nucleic acids are present, they may or may not be present in the same individual LPS-LPX structures (e.g., one LPS-LPX may contain a first nucleic acid, and another LPS-LPX may contain a second nucleic acid; or one LPS-LPX may contain both first and second nucleic acids).

[0093] In embodiments, the composition for transfecting a cell comprises at least one nucleic acid contained in an LPX-LPS delivery system. In embodiments, the nucleic acid is DNA, RNAor DNA and RNA. In embodiments, the nucleic acid is DNA. In embodiments, the nucleic acid is RNA. In embodiments, the nucleic acid is a DNA and an RNA. In embodiments, the nucleic acid comprises a therapeutic gene.

[0094] In embodiments, the RNA is mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA In embodiments, the RNA is mRNA In embodiments, the RNA is siRNA. In embodiments, the RNA is miRNA. In embodiments, the RNA is RN Ai. In embodiments, the RNA is saRNA. In embodiments, the RNA is taRNA. In embodiments, the RNA is shRNA.

[0095] In embodiments, the DNA is a plasmid DNA. In embodiments, the plasmid DNA is a nanoplasmid DNA. In embodiments, the nanoplasmid DNA encodes a monoclonal antibody.

[0096] In embodiments, the protein coding gene can encode an antibody fragment. In embodiments, the protein coding gene can encode a Fab. In embodiments, the protein coding gene can encode an scFv. In embodiments, the protein coding gene can encode a hormone, a cytokine, an enzyme, an immunogenic peptide, or the like. In embodiments, the protein coding gene can encode a hormone. In embodiments, the protein coding gene can encode a cytokine. In embodiments, the protein coding gene can encode an enzyme. In embodiments, the protein coding gene can encode an immunogenic peptide.

[0097] In embodiments, the nucleic acid may comprise one or more therapeutic genes, regulatory sequences for transcription or for replication, modified or unmodified antisense sequences, regions for binding to other cellular components, and the like.

[0098] In embodiments, the nucleic acid may comprise therapeutic genes. In embodiments, the nucleic acid comprises a therapeutic gene. In embodiments, the nucleic acid may comprise regulatory sequences for transcription. In embodiments, the nucleic acid comprises a regulatory sequence for transcription. In embodiments, the nucleic acid may comprise regulatory sequences for replication. In embodiments, the nucleic acid comprises a regulatory sequence for replication. In embodiments, the nucleic acid may comprise modified antisense sequences. In embodiments, the nucleic acid comprises a modified antisense sequence. In embodiments, the nucleic acid may comprise unmodified antisense sequences. In embodiments, the nucleic acid comprises an unmodified antisense sequence. In embodiments, the nucleic acid may comprise regions for binding to other cellular components. In embodiments, the nucleic acid comprises a region for binding to other cellular components.

[0099] The term “therapeutic gene” is intended to mean any gene encoding a protein product (e g., peptide) having a therapeutic effect. In embodiments, the nucleic acid may also comprise one or more genes encoding an antigenic peptide capable of generating an immune response in humans or animals. In embodiments, the nucleic acid can be used for cell therapy. In embodiments, the nucleic acid makes it possible to produce either vaccines or immunotherapeutic treatments. In embodiments, vaccines or immuno-therapeutic treatments can be used for preventing or treating cancers. In embodiments, such vaccines or immuno-therapeutic treatments can be used to treat viral infections. In embodiments, the viral infection is a coronavirus infection.

[0100] In embodiments, the LPS-LPX delivery system comprises an EPS encapsulating an LPX.

[0101] In embodiments, the LPX comprises one or more ionizable cationic lipids and optionally one or more helper lipids. In embodiments, the LPX comprises one ionizable cationic lipid. In embodiments, the LPX comprises two ionizable cationic lipids. In embodiments, the LPX comprises three ionizable cationic lipids In embodiments, the LPX comprises four ionizable cationic lipids. In embodiments, the LPX comprises five ionizable cationic lipids.

[0102] In embodiments, the LPX comprises one ionizable cationic lipid and one helper lipid. In embodiments, the LPX comprises one ionizable cationic lipid and two helper lipids In embodiments, the LPX comprises one ionizable canonic lipid and three helper Lipids. In embodiments, the LPX comprises one ionizable cationic lipid and four helper lipids. In embodiments, the LPX comprises one ionizable cationic lipid and five helper lipids.

[0103] In embodiments, the LPX comprises tW'O ionizable cationic lipids and one helper lipid. In embodiments, the LPX comprises two ionizable cationic lipids and two helper lipids. In embodiments, the LPX comprises two ionizable cationic lipids and three helper lipids. In embodiments, the LPX comprises two ionizable cationic lipids and four helper lipids. In embodiments, the LPX comprises two ionizable cationic lipids and five helper lipids.

[0104] In embodiments, the LPX comprises three ionizable cationic lipids and one helper lipid. In embodiments, the LPX comprises three ionizable cationic lipids and two helper lipids. In embodiments, the LPX comprises three ionizable cationic lipids and three helper lipids. In embodiments, the LPX comprises three ionizable cationic lipids and four helper lipids. In embodiments, the LPX comprises three ionizable cationic lipids and five helper lipids

[0105] In embodiments, the LPX comprises four ionizable cationic lipids and one helper lipid. In embodiments, the LPX comprises four ionizable cationic lipids and two helper lipids. In embodiments, the LPX comprises four ionizable cationic lipids and three helper lipids. In embodiments, the LPX comprises four ionizable cationic lipids and four helper lipids. In embodiments, the LPX comprises four ionizable cationic lipids and five helper lipids.

[0106] In embodiments, the LPX comprises five ionizable cationic lipids and one helper lipid In embodiments, the LPX comprises five ionizable cationic lipids and two helper lipids In embodiments, the LPX comprises five ionizable cationic lipids and three helper lipids In embodiments, the LPX comprises five ionizable cationic lipids and four helper lipids. In embodiments, the LPX comprises five ionizable cationic lipids and five helper lipids.

[0107] In embodiments, the LPS is a lipid micelle, a liposome, or a self-assembled lipid nanoparticle. In embodiments, the LPS is a lipid micelle In embodiments, the LPS is a liposome In embodiments, the LPS is a self-assembled lipid nanoparticle.

[0108] In embodiments, the LPS comprises one or more ionizable cationic lipids and / or one or more helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid. In embodiments, the LPS comprises two ionizable cationic lipids. In embodiments, the LPS comprises three ionizable cationic lipids In embodiments, the LPS comprises four ionizable cationic lipids. In embodiments, the LPS comprises five ionizable cationic lipids. In embodiments, the LPS comprises one helper lipid. In embodiments, the LPS comprises two helper lipids. In embodiments, the LPS comprises three helper lipids In embodiments, the LPS comprises four helper lipids. In embodiments, the LPS comprises five helper lipids

[0109] In embodiments, the LPS comprises one ionizable cationic lipid and one helper lipid. In embodiments, the LPS comprises one ionizable cationic lipid and two helper lipids In embodiments, the LPS comprises one ionizable cationic lipid and three helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid and four helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid and five helper lipids In embodiments, the LPS comprises one ionizable cationic lipid and two helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid and three helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid and four helper lipids. In embodiments, the LPS comprises one ionizable cationic lipid and five helper lipids

[0110] In embodiments, the LPS comprises two ionizable cationic lipids and one helper lipid In embodiments, the LPS comprises two ionizable cationic lipids and two helper lipids. In embodiments, the LPS comprises two ionizable cationic Lipids and three helper lipids. In embodiments, the LPS comprises two ionizable cationic lipids and four helper lipids. In embodiments, the LPS comprises two ionizable cationic lipids and five helper lipids.

[0111] In embodiments, the LPS comprises three ionizable cationic lipids and one helper lipid In embodiments, the LPS comprises three ionizable cationic lipids and two helper lipids In embodiments, the LPS comprises three ionizable cationic lipids and three helper lipids. In embodiments, the LPS comprises three ionizable cationic lipids and four helper lipids. In embodiments, the LPS comprises three ionizable cationic lipids and five helper lipids.

[0112] In embodiments, the LPS comprises four ionizable cationic lipids and one helper lipid In embodiments, the LPS comprises four ionizable cationic lipids and two helper lipids In embodiments, the LPS comprises four ionizable cationic lipids and three helper lipids. In embodiments, the LPS comprises four ionizable cationic lipids and four helper lipids In embodiments, the LPS comprises four ionizable cationic lipids and five helper lipids.

[0113] In embodiments, the LPS comprises five ionizable cationic lipids and one helper lipid In embodiments, the LPS comprises five ionizable cationic lipids and two helper lipids In embodiments, the LPS comprises five ionizable cationic lipids and three helper lipids. In embodiments, the LPS comprises five ionizable cationic lipids and four helper Lipids. In embodiments, the LPS comprises five ionizable cationic lipids and five helper lipids.

[0114] Some non-limiting examples of ionizable cationic lipids (e.g., of the LPS or LPX) include lipids provided in Table 1 below.Table 1:'ll* Where no information is provided as to specific vendor, the lipids are readily available from numerous vendors[00115J In embodiments, an ionizable cationic lipid (e.g., of the LPS or LPX) is selected from any of those disclosed in the following US Patents or US Patent Publications Nos.US20150376115A1, US20160376224A1, US20170119904A1, US20180185516A1,US20190022247A1, US20200046838A1, US20200172472A1, US20200283372A1,US20210122703A1, US20210128488A1, US20210395188A1, US20220081392A1,US20220106257A1, US20220218622A1, US20220218622A1, US10077232B2,US20190240339A1, US10561732B2, US10653780B2, US20200282060A1,US20220133636A1, US20150141678A1, US20150239926A1, US20170190661 Al,US20180170866A1, US20180222863 Al, US8034376B2, US20090163705A1, andUS20110097720A1, all of which are incorporated by reference herein in their entirety .

[0116] In embodiments, the ionizable cationic lipid (e.g., of the LPS or LPX) is SM-102, ALC-0315, DODMA, DOTMA, DOTAP, DC-cholesterol, JK-0315-CA, C 12-200, JK-102-CA, ALC-BAE-0315, Me2N+HexDecA, KT-001, TU-C3-HAD, TU-B3-HAD, TU-D3-HAD, or SSEC-ALC-0315.

[0117] In embodiments, the ionizable cationic lipid (e.g., of the LPS or LPX) is DOTMA, DO TAP, DC-cholesterol, or KT-001 .

[0118] In embodiments, the helper lipid (e.g., of the LPS or LPX) is a phospholipid, pegylated lipid, cholesterol, or cholesterol derivative.

[0119] In embodiments, the helper lipid (e.g., of the LPS or LPX) is a phospholipid. In embodiments, the helper lipid (e.g., of the LPS or LPX) is a pegylated lipid. In embodiments, the helper lipid (e.g., of the LPS or LPX) is cholesterol. In embodiments, the helper lipid (e.g., of the LPS or LPX) is a cholesterol derivative.

[0120] Some non-limiting examples of helper lipid (e.g., of the LPS or LPX) include lipids provided in Table 2 below. All the lipids in Table 2 can be purchased from Avanti Polar Lipids.Table 2:c

[0121] In embodiments, the helper lipid (e.g., of the LPS or LPX) is DSPC, cholesterol, DMG-PEG2000, or DOPE. In embodiments, the helper lipid (e.g., of the LPS or LPX) is DSPC. In embodiments, the helper lipid (e.g., of the LPS or LPX) is cholesterol. In embodiments, the helper lipid (e.g., of the LPS or LPX) is DMG-PEG2000. In embodiments, the helper lipid (e.g., of the LPS or LPX) is DOPE.

[0122] In embodiments, the LPX comprises KT-001. DSPC, cholesterol, DO I'M A, DC- cholesterol. DOPE, or any combinations thereof.

[0123] In embodiments, the LPX comprises KT-001 and DSPC. In embodiments, the LPX comprises KT-001, DSPC, and cholesterol. In embodiments, the LPX comprises KT-001, DOTMA, DSPC, and cholesterol. In embodiments, the LPX comprises KT-001, DSPC, cholesterol and DMG-PEG2000. In embodiments, the LPX comprises DC-cholesterol and DOPE

[0124] In embodiments, the LPS comprises KT-001 , DSPC, cholesterol, DMG-PEG2000, DOTMA, DOTAP, or any combinations thereof.

[0125] In embodiments, LPS comprises DC-cholesterol and DOTAP. In embodiments, LPS comprises DC-cholesterol and DOPE. In embodiments, LPS comprises KT-001 , DSPC, cholesterol, and DMG-PEG2000. In embodiments, LPS comprises KT-001, DOTMA, DSPC, cholesterol and DMG-PEG2000

[0126] In embodiments, the molar ratio of KT-001 to DSPC (e.g., in the LPX) is in the range of about 1 :3 to about 10: 1, e.g., about 1 :3, about 1 :2, about 1: 1, about 2: 1, about 3:1, about 4: 1, about 5:1, about 10: 1, or about 15: 1. In embodiments, the ratio of KT-001 :DSPC is 4.5: 1 to 5.5:1, 4.7: 1 to 5.3: 1, 4.8:1 to 5.2: 1, or 4.9: 1 to 5.1: 1. In embodiments, the ratio of KT-001 to DSPC is about 5: 1. In embodiments, the ratio of KT-001 to DSPC is 5: 1.

[0127] In embodiments, the molar ratio of KT-001 to DSPC to cholesterol (e.g., in the LPX) may be between about 20-80:2-30: 10-60. In embodiments, the molar ratio of KT-001 :DSPC:cholesterol is 40: 10:40 to 60: 10:40, 40:20:80 to 60:20:50, 45: 10:45 to 55: 10:45, or 45: 10:30 to 55:10:45. In embodiments, the molar ratio of KT-001 :DSPC:cholesterol is about 50: 10:38.5. In embodiments, the molar ratio of KT-001 :DSPC:cholesterol is 50:10:38.5.

[0128] In embodiments, the molar ratio of KT-001 to DOTMA to DSPC to cholesterol (e g., in the LPX) may be between about 20-80: 1-20:2-30: 10-60. In embodiments, the molar ratio of KT-001 :DOTMA:DSPC:cholesterol is 35:5:10:40 to 55:5: 10:40, 35: 10:20:80 to 55: 10:20:50, 30:5: 10:45 to 50:5: 10:45, 40:2:4:30 to 50: 10:20:45, or 40:5: 10:30 to 50:5: 10:45. In embodiments, the molar ratio of KT-001:DOTMA:DSPC:cholesterol is about 45:5: 10:38.5. In embodiments, the molar ratio of KT-001 :DOTMA:DSPC:cholesterol is 45:5: 10:38.5.

[0129] In embodiments, the molar ratio of KT-001 to DSPC to cholesterol to DMG- PEG2000 (e.g., in the LPX or the LPS) may be between about 20-80:2-30: 10-60:0.5-8. In embodiments, the molar ratio of KT-001 :DSPC:cholesterol:DMG-PEG2000 is 40: 10:40:2 to 60: 10:40:2, 40:20:80: 1.5 to 60:20:50: 1.5, 45: 10:45:2 to 55:10:45:2, 45: 10:30: 1 to 55: 10:45:1, or 45:5:30:0.5 to 55:25:45:2.5. In embodiments, the molar ratio of KT- 001 :DSPC:cholesterol:DMG-PEG2000 is about 50: 10:38.5: 1.5. In embodiments, the molar ratio of KT-00 EDSPC: cholesterol :DMG-PEG2000 is 50: 10:38.5:1.5.

[0130] In embodiments, the molar ratio of DC-cholesterol to DOPE (e.g., in the LPX or the LPS) is in the range of about 1 :3 to about 10: 1, e.g., about 1 :3, about 1 :2, about 1 :1, about 2: 1,about 3:1, about 4:1, about 5 : 1 , or about 10:1. In embodiments, the ratio of DC- cholesterokDOPE is 1.5:1 to 2.5:1, 1.7:1 to 2.3:1, 1.8:1 to 2.2:1, or 1.9:1 to 2.1:1. In embodiments, the ratio of DC-cholesterol to DOPE is about 2:1. In embodiments, the ratio of DC-cholesterol to DOPE is 2:1.

[0131] In embodiments, the molar ratio of DC-cholesterol to DOPE (e.g., in the LPS)is in the range of about 3 : 1 to about 10:1, e.g., about 3:1, about 2:1, about 1:1, about 1 :2, about 1 :3, about 1:4, or about 1:5. In embodiments, the ratio of DC-cholesterol :DOPE is 1:1.5 to 1:2.5, 1:1.7 to 1:2.3, 1:1.8 to 1:2.2, or 1:1.9 to 1:2.1. In embodiments, the ratio of DC-cholesterol to DOPE is about 1 :2. In embodiments, the ratio of DC-cholesterol to DOPE is 1 :2.

[0132] In embodiments, the molar ratio of DC-cholesterol to DOTAP (e.g., in the LPS) is in the range of about 1 :3 to about 10:1, e.g., about 1 :3, about 1 :2, about 1 : 1, about 2:1, about 3:1, about 4:1, about 5:1, or about 10:1. In embodiments, the ratio of DC-cholesterol :DOTAP is 1.5:1 to 2.5:1, 1.7:1 to 2.3:1, 1.8:1 to 2.2:1, or 1.9:1 to 2.1:1. In embodiments, the ratio of DC- cholesterol to DOTAP is about 2:1. In embodiments, the ratio of DC-cholesterol to DOTAP is 2:1.

[0133] In embodiments, the molar ratio of DC-cholesterol to DOTAP (e.g., in the LPS) is in the range of about 3:1 to about 10:1, e.g., about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, or about 1:5. In embodiments, the ratio of DC-cholesterol :DOTAP is 1:1.5 to 1:2.5, 1:1.7 to 1:2.3, 1:1.8 to 1:2.2, or 1:1.9 to 1:2.1. In embodiments, the ratio of DC-cholesterol to DOTAP is about 1:2. In embodiments, the ratio of DC-cholesterol to DOTAP is 1:2.

[0134] In embodiments, the molar ratio of KT-001 to DOTMA to DSPC to cholesterol to DMG-PEG2000 (e.g., in the LPS) may be between about 20-80:1-15:2-30:10-60:0.5-8. In embodiments, the molar ratio of KT-001 :DOTMA:DSPC:cholesterol:DMG-PEG2000 is 40:5:10:40:2 to 60:5:10:40:2, 35:10:20:80:1.5 to 55:10:20:50:1.5, 40:5:10:45:2 to 50:5:10:45:2, 40:5:10:30:1 to 50:5:10:45:1, or 40:1:5:30:0.5 to 50:15:25:45:2.5. In embodiments, the molar ratio of KT-001 :DOTMA:DSPC cholesterol :DMG-PEG2000 is about 45 / 5 / 10 / 38.5 / 1.5. In embodiments, the molar ratio of KT-001:DOTMA:DSPC:cholesterol:DMG-PEG2000 is 45 / 5 / 10 / 38.5 / 1.5.

[0135] In embodiments, the composition for transfecting a cell comprises a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the composition for transfecting a cell comprises a targeting ligand In embodiments, the composition for transfectinga cell comprises an imaging ligand. In embodiments, the composition for transfecting a ceil comprises a digesting agent. In embodiments, the composition for transfecting a cell comprises a small molecule.

[0136] In embodiments, a targeting ligand is in contact with the LPS. As used herein, the expression - a targeting ligand “in contact with LPS” means a targeting ligand on the surface of the LPS; or a targeting ligand with one end in the micellar or liposomal bilayer and with its other end protruding outside of the lipid shell.

[0137] In embodiments, the targeting ligand is DIJPA, folate, a peptide, or an antibody or an antigen binding fragment thereof. In embodiments, the targeting ligand is DUPA. In embodiments, the targeting ligand is folate. In embodiments, the targeting ligand is a peptide. In embodiments, the targeting ligand is an antibody or an antigen binding fragment thereof. In embodiments, the targeting ligand is an antibody. In embodiments, the targeting ligand is a Fab. In embodiments, the targeting ligand is a scFv.

[0138] Non-limiting examples of antibodies that can be used as targeting ligands include anti -FAP antibody, anti-CD2 antibody, anti-CD25 antibody, anti-CD3 antibody, anti-CD33 antibody, anti-CD30 antibody, anti-HER2 antibody, anti-CD22 antibody, anti-PSMA antibody, anti-TM4SF antibody, and anti-ICAMl antibody. In embodiments, some non-limiting examples of targeting ligands can be Fabs of the antibodies listed above.

[0139] In embodiments, the targeting ligand is anti-FAP antibody, anti-CD2 antibody, anti- CD25 antibody, anti-CD3 antibody, anti-CD33 antibody, anti-CD30 antibody, anti-HER2 antibody, anti-CD22 antibody, anti-PSMA antibody, anti-TM4SF antibody, or anti-ICAMl antibody.

[0140] In embodiments, the targeting ligand is anti-FAP Fab, anti-CD2 Fab, anti-CD25 Fab, anti-CD3 Fab, anti-CD33 Fab, anti-CD30 Fab, anti-HER2 Fab, anti-CD22 Fab, anti-PSMA Fab, anti-TM4SF Fab, or anti-ICAMl Fab.

[0141] Non-limiting examples of peptides that can be used as targeting ligands include RGD4C, PL1, angiopep-1, PEN-221, Ty3-octreotide, and the like. In embodiments, the targeting ligand is RGD4C, PL1, angiopep-1, PEN-221, or Ty3-octreotide.

[0142] In embodiments, an imaging ligand is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In embodiments, an imaging ligand is in contactwith the LPS. In embodiments, an imaging ligand is inside the LPX. in embodiments, an imaging ligand is between the surface of the LPS and the LPX.

[0143] Non-limiting examples of imaging ligands that can be used in the composition for transfecting a cell include l,2-Distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol-IR825 (DSPE-PEG-IR825) and diethylenetriaminepentaacetic acid (DTPA)-Gd. In embodiments, the imaging ligand is l,2-Distearoyl-sn-glycero-3-phosphoethanolamine- polyethylene glycol-IR825 (DSPE-PEG-IR825). In embodiments, the imaging ligand is diethylenetriaminepentaacetic acid (DTPA)-Gd.

[0144] In embodiments, a digesting agent is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX. In embodiments, a digesting agent is in contact with the LPS. In embodiments, a digesting agent is inside the LPX. In embodiments, a digesting agent is between the surface of the LPS and the LPX.

[0145] Non-limiting examples of digesting agents that can be used in the composition for transfecting a cell include hyaluronidase and collagenase. In embodiments, the digesting agent is hyaluronidase. In embodiments, the digesting agent is collagenase. In embodiments, the digesting agent is a degradative enzyme. In embodiments, the digesting agent is an enzyme that cleaves an oligomeric or polymeric substrate.

[0146] In embodiments, a small molecule is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX In embodiments, a small molecule is in contact with the LPS In embodiments, a small molecule is inside the LPX In embodiments, a small molecule is between the surface of the I., PS and the LPX.

[0147] Non-limiting examples of small molecules that can be used in the composition for transfecting a cell include molecules in Table 3.Table 3.

[0148] Other non-limiting examples of small molecules that can be used in the composition for transfecting a cell include anti-inflammatory small molecules and toxins. In embodiments, the small molecule is an NSAID or a toxin. In embodiments, the small molecule is a vitamin, saccharide, steroid, or chemotherapy drug. Yet other non-limiting examples of small molecules that can be used in the composition for transfecting a cell include folic acid, glucose, galactose, N-acetylgalac-tosaraine (GalNAc). In embodiments, the small molecule is folic acid, glucose, galactose, N-acetylgalactosamine (GalNAc), dexamethasone, paclitaxel, or doxorubicin.Pharmaceutical Compositions

[0149] In an aspect, provided herein are pharmaceutical compositions comprising the composition for transfecting a cell, in vitro and in vivo, as described herein, including in embodiments, and a pharmaceutically acceptable excipient.

[0150] In embodiments, the compositions for transfecting a cell described herein, may be formulated as a pharmaceutical composition. The pharmaceutical composition may include one or more of LPS-LPX delivery systems which comprises at least one nucleic acid contained in each LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a. small molecule. Pharmaceutical compositions may further include one or more pharmaceutically acceptable excipients or accessory ingredients such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents are available, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006.

[0151] Relative amounts of the one or more compositions for transfecting a cell, the one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending uponthe identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, a pharmaceutical composition may comprise between 0.1% and 100% (wt / wt) of one or more compositions for transfecting a cell.

[0152] Non-limiting examples of pharmaceutically acceptable excipients include water for injection (WFI), NaCl, PBS, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors, salt solutions (such as Ringer's solution), alcohols, fixed oils, polyethylene glycols, glycerine, propylene glycol, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, and the like.

[0153] The pharmaceutically acceptable excipient is usually added following the formation of composition for transfecting a cell. Thus, after the composition for transfecting a cell is formed, it can be diluted into pharmaceutically acceptable excipients such as normal buffered saline. Pharmaceutical compositions typically include a conventional pharmaceutical excipient and may additionally include other medicinal agents, carriers, adjuvants, additives and the like.

[0154] Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions of the invention may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms. Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying andsuspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0155] Pharmaceutical formulations containing the composition for transfecting a cell may take the form of liquid, solid, semi-solid or lyophilized powder forms, such as, for example, solutions, suspensions, emulsions, sustained-release formulations, tablets, capsules, powders, suppositories, creams, ointments, lotions, aerosols, patches or the like, e.g., in unit dosage forms suitable for simple administration of precise dosages.

[0156] For in vivo administration, administration can be in any manner known in the art, e.g., by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application (topical), transmucosal, or rectal administration.

[0157] In embodiments, the pharmaceutical compositions can be administered parenterally, e.g., intraarticularly, intravenously, intradermally, intrathecally, intraperitoneally, subcutaneously, or intramuscularly. In embodiments, the pharmaceutical compositions are administered intravenously or intraperitoneally by a bolus injection. In embodiments, parenteral preparation is enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0158] An injectable composition for parenteral administration (e.g., intravenous, intramuscular or intrathecal) will typically contain the composition in a suitable i.v. solution, such as sterile physiological salt solution. The composition may also be formulated as a suspension in an aqueous emulsion.Methods of Preparing Compositions for Transfection

[0159] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or m vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (f) preparing an LPS by forming liposomes,micelles, or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the EPS prepared in step (f), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, and (h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell

[0160] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising, (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (b) agitating the LPX prepared in step (a); (c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (d) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanopartides from a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution; (e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging Ligand, a digesting agent, or a small molecule; and (f) agitating the mixture prepared in step (e), thereby forming a composition for transfecting a cell.

[0161] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (f) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the third solution prepared in step (e); (g) mixing the LPX after step (d) with the LPSprepared in step (I); and (h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell.

[0162] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids: (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids, (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase and one or more lipids; (f) preparing an LPS by forming liposomes, micelles, or other self- assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution prepared in step (e); (g) mixing the LPX after step (cl) with the LPS prepared in step (f); and (h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell.

[0163] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vivo or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an organic phase and one or more lipids; (t) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the third solution prepared in step (e), (g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell.

[0164] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing athird solution com prising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX: (f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell

[0165] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or bi vivo comprising: (a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX; (c) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution; (d) mixing the LPX prepared in step (b) with the LPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (e) agitating the mixture prepared in step (d), thereby forming a composition for transfecting a cell.

[0166] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing athird solution com prising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX: (f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the fourth solution prepared in step (f), (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell.

[0167] in another aspect, provided herein is a method for preparing a composition for transfecting a cell In vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles, (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX: (f) preparing a fourth solution comprising an organic phase and one or more lipids; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g); and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell.

[0168] In another aspect, provided herein is a method for preparing a composition for transfecting a cell hi vitro or in vivo comprising, (a) preparing a first solution comprising an aqueous phase; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) wtth the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with thethird solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase and one or more lipids; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell

[0169] In another aspect, provided herein is a method for preparing a composition for transfecting a ceil in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids, (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids, (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (gl and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell.

[0170] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising, (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles; (b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX; (c) preparing an LPS by formingliposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution; (d) mixing the LPX prepared in step (b) with the LPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, and (e) agitating the mixture prepared in step (d), thereby forming a composition for transfecting a cell

[0171] In another aspect, provided herein is a method for preparing a composition for transfecting a cell vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles, (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g); and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell

[0172] In another aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles: (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase and one ormore lipids; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g); and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell.

[0173] In another aspect, provided herein is a method for preparing a composition for transfecting a cell ifi vitro or in vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids, (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparti des; (d) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX; (f) preparing a fourth solution comprising an organic phase and one or more lipids; (g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the fourth solution prepared in step (f); (h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell,

[0174] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or w vivo comprising: (a ) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the LPX prepared in step (d) with the third solution prepared in step (e); (g) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (h) preparing an LPS by forming liposomes, micelles, orother self-assembled lipid nanoparticles from the fourth solution prepared in step (g), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (g), (i) mixing the LPX after step (f) with the LPS prepared in step (h), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (j) agitating the mixture prepared in step (i), thereby forming a composition for transfecting a ceil.

[0175] A method for preparing a composition for transfecting a ceil in vitro or in vivo comprising: (a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids , thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids, (b) agitating the LPX prepared in step (a); (c) mixing the LPX prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids; (d) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution; (e) mixing the LPX prepared in step (c) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and (f) agitating the mixture prepared in step (e), thereby forming a composition for transfecting a cell.

[0176] In an aspect, provided herein is a method for preparing a composition for uansfecting a cell in vitro or z>? vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the LPX prepared in step (d) with the third solution prepared in step (e); (g) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; (h) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed w7ith thefourth solution prepared in step (g); (i ) mixing the LPX after step (f) with the LPS prepared in step (h); and (j) agitating the mixture prepared in step (i), thereby forming a composition for transfecting a ceil.

[0177] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or io vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids, (f) mixing the LPX prepared in step (d) with the third solution prepared in step (e); (g) preparing a fourth solution comprising an organic phase and one or more lipids; (b) preparing an I., PS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (g), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (g); (i) mixing the LPX after step (f) with the LPS prepared in step (h), and (j) agitating the mixture prepared in step (i), thereby forming a composition for transfecting a cell.

[0178] In an aspect, provided herein is a method for preparing a composition for transfecting a cell in vitro or io vivo comprising: (a) preparing a first solution comprising an aqueous phase and one or more nucleic acids; (b) preparing a second solution comprising an organic phase and one or more lipids; (c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids; (d) agitating the LPX prepared in step (c); (e) preparing a third solution comprising an aqueous phase and one or more nucleic acids; (f) mixing the LPX prepared in step (d) with the third solution prepared in step (e); (g) preparing a fourth solution comprising an organic phase and one or more lipids; (h) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from an aqueous phase which is then mixed with the fourth solution prepared in step (g); (i) mixing the LPX after step (f) with the i .PS prepared in step (h), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, and (j) agitating the mixture prepared in step (i), thereby forming a composition for transfecting a cell

[0179] In embodiments, the method for preparing a composition for transfecting a cell in vitro or in vivo, can incorporate the use of fluidic instruments. In embodiments, the method for preparing a composition for transfecting a cell in vitro or in vivo, can incorporate the use of microfluidic instruments. Schematic representations of such processes are depicted in FIG. 11A, FIG. 11B, and FIG. 11C

[0180] In embodiments, all steps involving mixing of aqueous phase with organic phase can incorporate the use of fluidic instruments. In embodiments, all steps involving mixing of aqueous phase with organic phase can incorporate the use of microfluidic instruments.

[0181] In embodiments, the method for preparing a composition for transfecting a cell in vitro or in vivo comprises a first process of preparing an LPX, a second process of preparing an EPS, and a third process of mixing the LPX with the EPS. In embodiments, the LPX encapsulates one or more nucleic acids. In embodiments, one or more nucleic acids are on the surface of the LPX (e.g., in contact with LPX ). In embodiments, the LPX encapsulates one or more nucleic acids and additionally one or more nucleic acids are on the surface of LPX. In embodiments, a targeting ligand, an imaging ligand, a digesting; agent, or a small molecule is in contact with the EPS. In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is inside the LPX. In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is between the surface of the LPS and the LPX.

[0182] In embodiments, the first process involves a first step of preparing a first solution comprising an aqueous phase. In embodiments, the first process involves a first step of preparing a first solution comprising an aqueous phase and optionally one or more nucleic acids. In embodiments, the first process involves a first step of preparing a first solution comprising an aqueous phase and one or more nucleic acids. In embodiments, the aqueous phase is an aqueous buffer. In embodiments, the aqueous buffer is an acetate buffer, citrate buffer, Tyrode’s buffer, TBT buffer, Tris buffer, TBS buffer, or Tris-Sucrose Buffer. In embodiments, the aqueous buffer is an acetate buffer. In embodiments, the aqueous buffer is a citrate buffer. In embodiments, the aqueous buffer is a Tyrode’s buffer. In embodiments, the aqueous buffer is a TBT buffer. In embodiments, the aqueous buffer is a Tris buffer. In embodiments, the aqueous buffer is a TBS buffer. In embodiments, the aqueous buffer is a Tris-sucrose buffer.

[0183] In embodiments, the aqueous buffer is in the pH range of about pH 2 to about pH 6. In embodiments, the aqueous buffer is in the pH range of about pH 2 to about pH 5.5. Inembodiments, the aqueous buffer is in the pH range of about pH 2 to about pH 5. In embodiments, the aqueous buffer is in the pH range of about pH 2 to about pH 4.5. In embodiments, the aqueous buffer is in the pH range of about pH 2.5 to about pH 5.5. In embodiments, the aqueous buffer is in the pH range of about pH 2.5 to about pH 5. In embodiments, the aqueous buffer is in the pH range of about pH 3 to about pH 5.5. In embodiments, the aqueous buffer is in the pH range of about pH 3 to about pH 5.

[0184] In embodiments, the aqueous buffer is about pH 2. In embodiments, the aqueous buffer is about pH 2.5. In embodiments, the aqueous buffer is about pH 3. In embodiments, the aqueous buffer is about pH 3.5. In embodiments, the aqueous buffer is about pH 4. In embodiments, the aqueous buffer is about pH 4.5. In embodiments, the aqueous buffer is about pH 5. In embodiments, the aqueous buffer is about pH 5.5. In embodiments, the aqueous buffer is about pH 6. In embodiments, the aqueous buffer is about pH 6.5. In embodiments, the aqueous buffer is about pH 7. In embodiments, the aqueous buffer is about pH 7.5. In embodiments, the aqueous buffer is about pH 8.

[0185] In embodiments, the aqueous buffer is in the pH range of about pH 7.0 to about pH 8.0. In embodiments, the aqueous buffer is about pH 7.0. In embodiments, the aqueous buffer is about pH 7.2. In embodiments, the aqueous buffer is about pH 7.4. In embodiments, the aqueous buffer is about pH 7.6. In embodiments, the aqueous buffer is about pH 7.8. In embodiments, the aqueous buffer is about pH 8.0.

[0186] In embodiments, the aqueous buffer is an acetate buffer. In embodiments, the aqueous buffer is a citrate buffer. In embodiments, the aqueous buffer is an acetate buffer adjusted to about pH 5. In embodiments, the aqueous buffer is an acetate buffer adjusted to about pH 4. In embodiments, the aqueous buffer is a citrate buffer adjusted to about pH 3. In embodiments, the aqueous buffer is a Tris-sucrose buffer. In embodiments, the aqueous buffer is a Tris-sucrose buffer adjusted to about pH 7.4.

[0187] In embodiments, the first solution is prepared by dissolving one or more nucleic acids in an aqueous phase. In embodiments, the first solution is prepared by dissolving one nucleic acid in an aqueous phase. In embodiments, the first solution is prepared by dissolving two nucleic acids in an aqueous phase. In embodiments, the first solution is prepared by dissolving three nucleic acids in an aqueous phase.

[0188] In embodiments, the first solution is prepared by dissolving one nucleic acid in an aqueous buffer. In embodiments, the first solution is prepared by dissolving one nucleic acid in an acetate buffer. In embodiments, the first solution is prepared by dissolving one nucleic acid in a citrate buffer. In embodiments, the first solution is prepared by dissolving one nucleic acid in a Tris-sucrose buffer.

[0189] In embodiments, the first solution is prepared by dissolving two nucleic acids in an aqueous buffer. In embodiments, the first solution is prepared by dissolving two nucleic acids in an acetate buffer. In embodiments, the first solution is prepared by dissolving two nucleic acids in a citrate buffer. In embodiments, the first solution is prepared by dissolving two nucleic acids in a Tris-sucrose buffer.

[0190] In embodiments the first process involves a second step of preparing a second solution comprising an organic phase and one or more lipids. In embodiments, the organic phase comprises a water miscible organic solvent. In embodiments, the water miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, THF, ethylamine, glycerol, or dioxane. In embodiments, the water miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, or THF. In embodiments, the water miscible organic solvent is ethanol.

[0191] In embodiments, the second solution is prepared by dissolving one or more lipids in an organic phase. In embodiments, the second solution is prepared by dissolving one or more lipids in a water miscible organic solvent. In embodiments, the second solution is prepared by dissolving one lipid in a water miscible organic solvent. In embodiments, the second solution is prepared by dissolving two lipids in a water miscible organic solvent. In embodiments, the second solution is prepared by dissolving three lipids in a water miscible organic solvent. In embodiments, the second solution is prepared by dissolving four lipids in a water miscible organic solvent. In embodiments, the second solution is prepared by dissolving five lipids in a water miscible organic solvent.

[0192] In embodiments, the second solution is prepared by dissolving one or more lipids in ethanol. In embodiments, the second solution is prepared by dissolving one lipid in ethanol. In embodiments, the second solution is prepared by dissolving two lipids in ethanol. In embodiments, the second solution is prepared by dissolving three lipids in ethanol. Inembodiments, the second solution is prepared by dissolving four lipids in ethanol. In embodiments, the second solution is prepared by dissolving five lipids in ethanol.

[0193] In embodiments, where the first solution does not comprise nucleic acid(s), the first solution and the second solution are combined together to form liposomes, micelles, or other self-assembled lipid nanoparticles, by any method known in the an. In embodiments, liposomes, micelles, or other self-assembled lipid nanoparticles can be formed by pipetting, vortexing, or sonicating the first solution and the second solution together In embodiments, the first solution and the second solution can be mixed using fluidics or microfluidics. In embodiments, the second solution can be dried and then rehydrated with the first solution. In embodiments, the second solution (organic phase) can be injected into the first solution. In embodiments, the fist and the second solution can be emulsified i.e., the first solution can be added into the second solution.

[0194] In embodiments, the first solution and the second solution are mixed, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids. In embodiments, the first solution and the second solution are mixed by pipetting, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids.

[0195] In embodiments, the first solution and the second solution are mixed. In embodiments, the first solution and the second solution are mixed by pipetting. In embodiments, the first solution and the second solution are mixed followed by agitating. In embodiments, the first solution and the second solution are mixed followed by vortexing or sonicating. In embodiments, the first solution and the second solution are mixed by pipetting followed by vortexing or sonicating. In embodiments, the first solution and the second solution are mixed followed by vortexing. In embodiments, the first solution and the second solution are mixed by pipetting followed by vortexing. In embodiments, the first solution and the second solution are mixed followed by sonicating. In embodiments, the first solution and the second solution are mixed by pipetting followed by sonicating. In embodiments, the first solution and the second solution are mixed using fluidics or microfluidics, as depicted in FIGS. 11A-C.

[0196] In embodiments, where the first solution does not include nucleic acids the first process involves a third step of preparing a third solution comprising an aqueous phase and one or more nucleic acids.

[0197] In embodiments, the third solution is prepared by dissolving one or more nucleic acids in an aqueous phase. In embodiments, the third solution is prepared by dissolving one nucleic acid in an aqueous phase. In embodiments, the third solution is prepared by dissolving two nucleic acids in an aqueous phase. In embodiments, the third solution is prepared by dissolving three nucleic acids in an aqueous phase.

[0198] In embodiments, the third solution is prepared by dissolving one nucleic acid in an aqueous buffer. In embodiments, the third solution is prepared by dissolving one nucleic acid in an acetate buffer. In embodiments, the third solution is prepared by dissolving one nucleic acid in a citrate buffer. In embodiments, the third solution is prepared by dissolving one nucleic acid in a Tris-sucrose buffer.

[0199] In embodiments, the third solution is prepared by dissolving two nucleic acids in an aqueous buffer. In embodiments, the third solution is prepared by dissolving two nucleic acids in an acetate buffer. In embodiments, the third solution is prepared by dissolving two nucleic acids in a citrate buffer. In embodiments, the third solution is prepared by dissolving two nucleic acids in a Tris-sucrose buffer.

[0200] In embodiments, where the first solution does not comprise nucleic acid(s), the first solution and the second solution are combined together to form liposomes, micelles, or other self-assembled lipid nanoparticles. In embodiments, said liposomes, micelles, or other selfassembled lipid nanoparticles are mixed with the aqueous third solution comprising one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX.

[0201] In embodiments, the second process involves a first step of preparing a third solution comprising an organic phase and one or more lipids. In embodiments the second process involves a first step of preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0202] In embodiments, the organic phase comprises a water miscible organic solvent. In embodiments, the water miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, THF, ethylamine, glycerol, or dioxane. In embodiments, the water miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, or THF. In embodiments, the water miscible organic solvent is ethanol.

[0203] In embodiments, the third solution is prepared by dissolving one or more lipids in an organic phase. In embodiments, the third solution is prepared by dissolving one or more lipids in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving one lipid in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving two lipids in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving three lipids in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving four lipids in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving five lipids in a water miscible organic solvent.

[0204] In embodiments, the third solution is prepared by dissolving one or more lipids in ethanol. In embodiments, the third solution is prepared by dissolving one lipid in ethanol. In embodiments, the third solution is prepared by dissolving two lipids in ethanol. In embodiments, the third solution is prepared by dissolving three lipids in ethanol. In embodiments, the third solution is prepared by dissolving four lipids in ethanol. In embodiments, the third solution is prepared by dissolving five lipids in ethanol.

[0205] In embodiments, the third solution is prepared by dissolving one or more lipids in an organic phase and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving one or more lipids in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving one lipid in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving two lipids in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving three lipids in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving four lipids in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the third solution is prepared by dissolving five lipids in a water miscible organic solvent and a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0206] In embodiments, the third solution is prepared by dissolving one or more lipids and a targeting ligand in an organic phase. In embodiments, the third solution is prepared by dissolving one or more lipids and a targeting ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving one lipid and a targeting ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving two lipids and a targeting ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving three lipids and a targeting ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving four lipids and a targeting ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving five lipids and a targeting ligand in a water miscible organic solvent.

[0207] In embodiments, the third solution is prepared by dissolving one or more lipids an imaging ligand in an organic phase. In embodiments, the third solution is prepared by dissolving one or more lipids and an imaging ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving one lipid and an imaging ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving two lipids and an imaging ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving three lipids and an imaging ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving four lipids and an imaging ligand in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving five lipids and an imaging ligand in a water miscible organic solvent.

[0208] In embodiments, the third solution is prepared by dissolving one or more lipids and a digesting agent in an organic phase. In embodiments, the third solution is prepared by dissolving one or more lipids and a digesting agent in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving one lipid and a digesting agent in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving two lipids and a digesting agent in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving three lipids and a digesting agent in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving four lipids and a digesting agent in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving five lipids and a digesting agent in a water miscible organic solvent.

[0209] In embodiments, the third solution is prepared by dissolving one or more lipids and a small molecule in an organic phase. In embodiments, the third solution is prepared by dissolving one or more lipids and a small molecule in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving one lipid and a small molecule in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving two lipids and a small molecule in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving three lipids and a small molecule in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving four lipids and a small molecule in a water miscible organic solvent. In embodiments, the third solution is prepared by dissolving five lipids and a small molecule in a water miscible organic solvent.

[0210] In embodiments, where the first solution does not include nucleic acids, a fourth solution is prepared comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. The fourth solution comprising art organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is prepared in the same manner as the third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0211] In embodiments, the second process involves a second step of preparing an LPS using the third solution and the aqueous phase for the preparation of liposomes, micelles, or other selfassembled lipid nanoparticles by any method known in the art. In embodiments, the second process involves a second step of preparing an LPS using the third solution and the aqueous phase, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule was added to the aqueous phase, for the preparation of liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art.

[0212] In embodiments, where the first solution does not include nucleic acids, the second process involves a second step of preparing an LPS using a fourth solution and the aqueous phase for the preparation of liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art. In embodiments, the second process involves a second step of preparing an LPS using the fourth solution and the aqueous phase, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule was added to the aqueousphase, for the preparation of liposomes, micelles, or other self-assembled lipid nanoparticles by any method known in the art.

[0213] In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by pipetting. In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by agitating. In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by vortexing. In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by sonicating. In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by using a microfluidics system. In embodiments, second process involves a second step of preparing an LPS using the third solution drying it and then rehydrating with the aqueous phase (thin film method). In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by injecting the third solution (organic phase) into the aqueous phase. In embodiments, second process involves a second step of preparing an LPS using the third solution and mixing it with the aqueous phase by emulsification i.e., slowly adding aqueous phase into the third solution (organic phase).

[0214] In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is dissolved in the aqueous phase prior to mixing with the third solution. In embodiments, a targeting ligand is dissolved in the aqueous phase prior to mixing with the third solution. In embodiments, an imaging ligand is dissolved in the aqueous phase prior to mixing with the third solution. In embodiments, a digesting agent is dissolved in the aqueous phase prior to mixing with the third solution Tn embodiments, a small molecule is dissolved in die aqueous phase prior to mixing with the third solution.

[0215] In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by pipetting. In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by agitating. In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by vortexing. In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by sonicating. In embodiments, second processinvolves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by using a microfluidics system. In embodiments, second process involves a second step of preparing an LPS using the fourth solution drying it and then rehydrating with the aqueous phase (thin film method). In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by injecting the fourth solution (organic phase) into the aqueous phase. In embodiments, second process involves a second step of preparing an LPS using the fourth solution and mixing it with the aqueous phase by emulsification i.e., slowly adding aqueous phase into the fourth solution (organic phase).

[0216] In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is dissolved in the aqueous phase prior to mixing with the fourth solution. In embodiments, a targeting ligand is dissolved in the aqueous phase prior to mixing with the fourth solution. In embodiments, an imaging ligand is dissolved in the aqueous phase prior to mixing with the fourth solution. In embodiments, a digesting agent is dissolved in the aqueous phase prior to mixing with the fourth solution. In embodiments, a small molecule is dissolved in the aqueous phase prior to mixing with the fourth solution.

[0217] In embodiments, LPX is mixed with LPS. In embodiments, a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added when LPX is mixed with LPS. In embodiments, a targeting ligand is added when LPX is mixed with LPS. In embodiments, an imaging ligand is added when LPX is mixed with LPS. In embodiments, a digesting agent is added when LPX is mixed with LPS. In embodiments, a small molecule is added when LPX is mixed with LPS

[0218] In embodiments, both the first and the third solution comprise an aqueous phase and one or more nucleic acids.Methods for Treating or Preventing a disease and for Transfecting In Vitro

[0219] Transfection methods of this invention may be applied to cells in vitro or in vivo. The present disclosure provides methods for transfecting a cell in vitro. In an aspect provided herein is a method for transfecting a cell with one or more nucleic acids comprising: contacting the cell in vitro with a composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the method provided herein for transfecting a cell with one nucleic acid comprises: contacting the cell in vitro with a composition comprising one nucleicacid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the method provided herein for transfecting a cell with two nucleic acids comprises: contacting the cell in vitro with a composition comprising two nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule. In embodiments, the method provided herein for transfecting a cell with three nucleic acids comprises: contacting the cell in vitro with a composition comprising three nucleic acids contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

[0220] In embodiments, the cell is a eukaryotic cell.

[0221] The present disclosure provides methods for prophylactically treating a subject who has not yet been infected with a viral infection, or who may be in the early stages of infection but is not yet exhibiting symptoms of the viral infection. The method comprising administering to a subject a composition for cell transfection as described herein including in embodiments.

[0222] The present disclosure provides methods for prophylactically treating a subject who has not yet been infected with a coronavirus infection, or who may be in the early stages of infection but is not yet exhibiting symptoms of a coronavirus infection.

[0223] In one embodiment, the prophylactic treatment can be administered to the asymptomatic subject at about 1-24 hours, about 24-48 hours, or about 48 hours to 3 days following exposure or possible exposure or suspected exposure to a viral infection. In one embodiment, the prophylactic treatment can be administered to the asymptomatic subject at about 3-5 days, or about 5-10 days, or about 10-14 days, or about 14-21 days, or about 21-30 days or longer time ranges following exposure to the viral infection.

[0224] In one embodiment, the prophylactic treatment can be administered to the asymptomatic subject at about 1-24 hours, about 24-48 hours, or about 48 hours to 3 days following exposure or possible exposure or suspected exposure to the coronavirus. In one embodiment, the prophylactic treatment can be administered to the asymptomatic subject at about 3-5 days, or about 5-10 days, or about 10-14 days, or about 14-21 days, or about 21-30 days or longer time ranges following exposure to the coronavirus.

[0225] The present disclosure provides methods for treating a subject comprising administering to a subject in need thereof a composition for cell transfection as described herein including in embodiments.

[0226] The present disclosure provides methods for alleviating a symptom of a disease in a subject comprising administering to a subject in need thereof a composition for cell transfection as described herein including in embodiments. In embodiments, the present disclosure provides methods for treating a subject in need of cell therapy.

[0227] In embodiments, the disease is a viral infection, diabetes, autoimmune disorder, or cancer. In embodiments, the disease is any disease that can be treated with cell therapy. In embodiments, the disease is a cancer. In embodiments, the disease is diabetes. In embodiments, the disease is an autoimmune disorder. In embodiments, the disease is a viral infection. In embodiments, the viral infection is a coronavirus.

[0228] T he methods of this disclosure can be used to generate transfected cells which express useful gene products. The methods of this disclosure are useful as a step in any therapeutic method requiring introduction of nucleic acids into cells including methods of gene therapy and viral inhibition and for introduction of antisense or antigene nucleic acids or ribozymes or RNA regulatory sequences or related inhibitory or regulatory nucleic acids into cells. In particular, these methods are useful in cancer treatment, in treatment of infectious diseases, in ri? vivo and ex vivo gene therapy, and in diagnostic methods.

[0229] The dosage of a composition for cell transfection administered to a patient will depend on a number of factors including the method and site of administration, patient age, weight and condition. Those of ordinary skill in the art can readily adjust dosages for a given type of administration, a given patient and for a given therapeutic application.

[0230] In embodiments, the subject is a mammal. In embodiments, the subject is human.Methods of Administration

[0231] T he compositions described herein may be formulated as solutions or suspensionsIn embodiments, the compositions described herein may be formulated as solutions. In embodiments, the compositions described herein may be formulated as suspensions.

[0232] In embodiments, the compositions described herein and formulated as a solution or a suspension, may be administered to a subject by injection. In embodiments, the compositionsdescribed herein and formulated as a solution or a suspension, may be administered to a subject by intramuscular, subcutaneous, or intradermal injection.[002331 In embodiments, the compositions described herein and formulated as a suspension, may be administered to a subject by intramuscular injection. In embodiments, the compositions described herein and formulated as a suspension, may be administered to a subject by subcutaneous injection. In embodiments, the compositions described herein and formulated as a suspension, may be administered to a subject by intradermal injection.

[0234] T he compositions described herein can be administered in a single dose or in multiple doses. In embodiments, the compositions are administered in a single dose. In embodiments, the compositions are administered in two doses. In embodiments, the compositions are administered in three doses. In embodiments, the compositions are administered in four doses. In embodiments, the compositions are administered in five doses

[0235] The compositions described herein may be used to treat viral infection, diabetes, autoimmune disorders, or cancer. The compositions described herein may be used to prevent a viral infection.

[0236] In embodiments, where the subject is treated for a viral infection, the injection may be delivered intramuscularly, subcutaneously, or intradermally. In embodiments, where the subject is treated for a viral infection, the injection may be delivered intramuscularly. In embodiments, where the subject is treated for a viral infection, the injection may be delivered subcutaneously. In embodiments, where the subject is treated for a viral infection, the injection may be delivered intradermally.

[0237] In embodiments, where the subject is vaccinated to prevent a viral infection, the injection may be delivered intramuscularly, subcutaneously, or intradermally. In embodiments, where the subject is vaccinated to prevent a viral infection, the injection may be delivered intramuscularly. In embodiments, where the subject is vaccinated to prevent a viral infection, the injection may be delivered subcutaneously. In embodiments, where the subject vaccinated to prevent a viral infection, the injection may be delivered intradermally.

[0238] In embodiments, where the subject is treated for cancer, the injection may be delivered intratumorally.

[0239] In embodiments, the vaccine or treatment is delivered directly to the lymphatic system. In embodiments, the vaccine or treatment is administered to the lymphatic system sub-dermally. In embodiments, the vaccine or treatment is administered to the lymphatic system intramuscularly. In embodiments, the vaccine or treatment is administered to the lymphatic system intranodally. In embodiments, the vaccine or treatment is administered to the lymphatic system transdermally.

[0240] In embodiments, the vaccine or treatment is administered into the lymphatic system via a patch. In embodiments, the patch comprises a polymer. In embodiments, the polymer is an absorbable polymer.

[0241] In embodiments, the vaccine or treatment is administered to the lymphatic system by a medical device comprising an array of microneedles.

[0242] Medical devices that comprise an array of microneedles suitable for use herein are known in the art. Particular exemplary structures and devices comprising a means for controllably delivering the vaccine or treatment to a subject are described in International Patent Application Publication Nos. WO 2014 / 188343, WO 2014 / 132239, WO 2014 / 132240, WO 2013 / 061208, WO 2012 / 046149, WO 2011 / 135531, WO 2011 / 135530, WO 2011 / 135533, WO 2014 / 132240, WO 2015 / 16821, and International Patent Applications PCT / US2015 / 028154 (published as WO 2015 / 168214 Al), PCT7US2015 / 028150 (published as WO 2015 / 168210 Al), PCT / US2015 / 028158 (published as WO 2015 / 168215 Al), PCT / US2015 / 028162 (published as WO 2015 / 168217 Al), PCT / US2015 / 028164 (published as WO 2015 / 168219 Al), PCT / US2015 / 038231 (published as WO 2016 / 003856 Al), PCT / US2015 / 038232 (published as WO 2016 / 003857 Al), PCT / US2016 / 043623 (published as WO 2017 / 019526 Al), PCT / US2016 / 043656 (published as WO 2017 / 019535 Al), PCT / US2017 / 027879 (published as WO 2017 / 189258 Al), PCT / US2017 / 027891 (published as WO 2017 / 189259 Al), PCT / US2017 / 064604 (published as WO 2018 / 111607 Al), PCT / US2017 / 064609 (published as WO 2018 / 111609 Al), PCT / US2017 / 064614 (published as WO 2018 / 111611 Al), PCI7US2017 / 064642 (published as WO 2018 / 111616 Al), PCT / US2017 / 064657 (published as WO 2018 / 111620 Al), and PCT / US2017 / 064668 (published as WO 2018 / 111621 Al), all of which are incorporated by reference herein in their entirety.

[0243] In embodiments, the vaccine or treatment is administered by applying one or more medical devices to one or more sites of the skin of a subject. In embodiments, the vaccine or treatment is delivered directly to the lymphatic system. One nonlimiting example of a medical device comprising a plurality of microneedles that is suitable for use with all of the methodsdisclosed herein is the Sofusa™ drug delivery platform available from Sorrento Therapeutics, Inc. see e.g., US Patent No: 10,737,082; International Patent Application PCT / US2019 / 034736 (published as WO 2019 / 232265) which are incorporated by reference herein in their entirety.

[0244] In embodiments, the medical device is placed in direct contact with the skin of the subject. In embodiments, an intervening layer or structure will be between the skin of the subject and the medical device. For example, surgical tape or gauze may be used to reduce possible skin irritation between the medical device and the skin of the subject. When the microneedles extend from the apparatus, they will contact and, in some instances, penetrate the epidermis or dermis of the subject in order to deliver the vaccine or treatment to the subject. The delivery of the vaccine or treatment can be to the circulatory system, the lymphatic system, the interstitium, subcutaneous, intramuscular, intradermal or a combination thereof. In embodiments, the vaccine or treatment is delivered directly to the lymphatic system of the subject. In embodiments, the vaccine or treatment is delivered to the superficial vessels of the lymphatic system.

[0245] In embodiments, the vaccination or treatment target is a lymph node, a lymph vessel, an organ that is part of the lymphatic system or a combination thereof. In embodiments, the vaccination or treatment target is a lymph node. In embodiments, the vaccination or treatment target is a specific lymph node as described elsewhere herein.

[0246] In embodiments, the medical device may comprise a needle array in the form of a patch. In embodiments, the array of needles is able to penetrate a most superficial layer of the stratum comeum and deliver the vaccine or treatment as described herein to at least a portion or all of the non-viable epidermis, at least a portion of or all of the viable epidermis, and / or at least a portion of the viable dermis of a subject and subsequently to the lymphatic system of the subject. These needles may further comprise nanotopography on the surface of the needle in a random or organized pattern. In embodiments, the nanotopography pattern may demonstrate fractal geometry.ABBREVIATIONSEtOH ethanolGFP green fluorescent proteinLuc luciferaseDMSO dimethyl sulfoxideDMF dimethylformamideTHF tetrahydrofuran pDNA plasmid DNAGal N Ac N-acetylgalactosamineEXAMPLESExample 1. Transfection of mRNA Using LPS-LPX Delivery System Compared to Lipofectamine™ MessengerMAX™ in HEK293 Cells.

[0247] In all formulations comprising mRNA, the mRNA is mainly found on the inside of the lipoplex (LPX).

[0248] Formulation LCF96 was prepared as follows: 1) The core (lipoplex, LPX) - Green fluorescent protein (GFP) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH) and cholesterol (5.68 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DSPC / cholesterol=50 / 10 / 38.5) were added to a 2 mL serum vial to make 23.28 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0249] Sodium acetate buffer 25mM, pH5 was prepared as follows: Commercial IM pH 5.0 acetate buffer (Thermo Scientific, Cat# J60964-AK) was diluted 40 times with water for injection.

[0250] 2) The lipid shell (LPS) - KT-001 (729.8 μL, 20 mg / mL in EtOH), DSPC (150.5 μL,20 mg / mL in EtOH), cholesterol (283.8 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1238.4 μL lipid solution (molar ratio of lipids: KT-001 / DSPC / Cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3715.11 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 toproduce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0251] Tris-Sucrose Buffer pH ~7.4 was prepared as follows: Tris base (9.688 g, 0.08 mol), sucrose (320.00 g), and water for injection (WFI 3.8 L) were added to a 6L beaker. The mixture was allowed to stir at room temperature for 2 hours or until all materials dissolved. The pH of the solution was adjusted with HC1 (IN) to pH 7.3 to 7.4. The overall volume of the solution was brought to 4 L, and the solution was sterile fdtered using 0.22 pM filter.

[0252] 3 ) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0253] Transfection in HEK293 Cells: Day 1 - Cells were gently pipetted to mix well. 10 μL aliquot of the cell suspension was used to perform Trypan Blue exclusion cell count using an automatic cell counter to detect the cell concentration and viability. Total cell number was calculated. Cells were transferred to 50 ml centrifuge tube, spun at 1200rpm or 250g for 5 minutes, old medium was discarded, and the cells were resuspended in fresh HEK293 completed medium (DMEM with 10% FBS) to adjust cells to concentration of 0.4X106 / ml. 24 well plate was then loaded with 0.5 ml of cell suspension per well (0.2xl06 / well) and the plate was incubated overnight at 37°C and 5% CO2. Day 2 - the medium was changed to fresh completed cell medium before transfection. 0.5 pg or 1 pg of mRNA-LPS-LPX (prepared above) was added to each well, and the plate was incubated for 24 hours at 37°C and 5% CO2. 0.5 pg or 1 pg / well of mRNA+Lipofectamine™ MessengerMAX™ (from Thermo Fisher) was used as positive control. HEK293 cells were used as negative control.

[0254] After 24 hours, transfection efficiency was determined via flow cytometry and transfection cell viability was determined by performing Trypan Blue exclusion cell count using an automatic cell counter.

[0255] FIG. 1 scatter plots show transfection efficiency ofLCF96 and Lipofectamine™ MessengerMAX™ (0.5 pg and 1 .0 pg GFP mRNA) in HEK293 cells. In easy to transfect cells, such as HEK293, the LPS-LPX delivery system improves transfection efficiency compared to Lipofectamine™ MessengerMAX™ from 90.4% to 99.4% (for 0.5 pg) and from 96.6% to 99.3% (for 1.0 pg). Cell viability is also improved compared to Lipofectamine™ MessengerMAX™, from 82% to 94%, as shown in Table 4.

[0256] Table 4:Example 2. Transfection of mRNA Using LPS-LPX Delivery System Compared to Lipofectamine™ MessengerMAX™ in Jurkat Cells.

[0257] Formulation LCF96 was prepared as described in Example 1.

[0258] Formulation LCF107 was prepared as follows: 1) The core (lipoplex, LPX) - Luciferase (Luc) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (13.14 μL, 20 mg / mL in EtOH), DOTMA (1.28 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH) and cholesterol (5.68 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT- 001 / DOTMA / DSPC / cholesterol=45 / 5 / 10 / 38.5) were added to a 2 mL serum vial to make 23.1 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0259] 2) The lipid shell (LPS) - KT-001 (729.8 μL, 20 mg / mL in EtOH), DSPC (150.5 μL,20 mg / mL in EtOH), cholesterol (283.8 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1238.4 μL lipid solution (molar ratio of lipids: KT-001 / DSPC / Cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3715.11 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 toproduce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0260] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.1 μL lipoplex (LPX), and then pipetted a few times up and down to mix.

[0261] Formulation LCF108 was prepared as follows: 1) The core (lipoplex, LPX) - Green fluorescent protein (GFP) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH) and cholesterol (5.68 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DSPC / cholesterol=50 / 10 / 38.5) were added to a 2 mL serum vial to make 23.28 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0262] 2) The lipid shell (LPS) - KT-001 (656.8 μL, 20 mg / mL in EtOH), DOTMA (63.9 μL, 20 mg / mL in EtOH), DSPC (150.5 μL, 20 mg / mL in EtOH), cholesterol (283.8 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1229.3 μL lipid solution (molar ratio of lipids: KT- 001 / DOTMA / DSPC / Cholesterol / DMG-PEG2000=45 / 5 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3687.9 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0263] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0264] Formulation LCF109 was prepared as follows: 1) The core (lipoplex, LPX) - Luciferase (Luc) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (13.14 μL, 20 mg / mL in EtOH), DOTMA (1.28 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH) and cholesterol (5.68 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DOTMA / DSPC / cholesterol=45 / 5 / 10 / 38.5) were added to a 2 mL serum vial to make 23.1 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0265] 2) The lipid shell (LPS) - KT-001 (656.8 μL, 20 mg / mL in EtOH), DOTMA (63.9 μL, 20 mg / mL in EtOH), DSPC (150.5 μL, 20 mg / mL in EtOH), cholesterol (283.8 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1229.3 μL lipid solution (molar ratio of lipids: KT- 001 / DOTMA:DSPC / Cholesterol / DMG-PEG2000=45 / 5 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3687.9 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0266] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.1 μL lipoplex (LPX), and then pipetted a few times up and down to mix.

[0267] Transfection in Jurkat Cells: Cells were gently pipetted to mix well. 10 μL aliquot of the cell suspension was used to perform Trypan Blue exclusion cell count using an automatic cell counter to detect the cell concentration and viability. Total cell number was calculated. Cells were transferred to 50 ml centrifuge tube, spun at 1200rpm or 250g for 5 minutes, old medium was discarded, and the cells were resuspended in fresh RPMI Serum Reduced Medium (SRM) to adjust cells to concentration of 1.0xl06 / ml. 24 well plate was then loaded with 0.5 ml of cell suspension per well (0.5xl06 / well). 1.0 pg or 3.0 pg of mRNA-LPS-LPX (prepared in in Examples 1 and 2) was added to each well, and the plate was incubated for 4 hours at 37°C and 5% CO2. 1 pg / well or 3 pg / well of mRNA+Lipof ectamine™ MessengerMAX™ was used as positive control. Jurkat cells were used as negative control.

[0268] After 4 hours, the plate was centrifuged at 250g for 5 minutes, and old medium was discarded. 500 μL of Jurkat completed medium (RPMI1640 with 10% FBS) was added into each well and the plate was incubated for 24 hours at 37°C and 5% CO2.

[0269] After 24 hours, transfection efficiency was determined via flow cytometry and transfection cell viability was determined by performing Trypan Blue exclusion cell count using an automatic cell counter.

[0270] FIG. 2 scatter plots show transfection efficiency of LCF96, LCF107, LCF108, LCF109, and Lipofectamine™ MessengerMAX™ (1.0 [tg and 3.0(itg) in Jurkat cells. In hard to transfect cells, such as Jurkat, the LPS-LPX delivery system (LCF107, LCF108, and LCF109) significantly improves transfection efficiency compared to Lipofectamine™ MessengerMAX™ from 14.1% to 99.8% (for 1 .0 pg) and from 23,4% to 99.9% (for 3.0 pg). Cell viability is slightly lower than that of Lipofectamine™ MessengerMAX™, as shown in Table 5.

[0271] Table 5:Example 3. Transfection of mRNA Using LPS-LPX Delivery System Compared to Lipofectamine™ MessengerMAX™ in Human Primary T Celis.

[0272] Formulation LCF96 was prepared as described in Example 1. Formulation LCF108 was prepared as described in Example 2.

[0273] Transfection in human primary T cells: Day 1 - A frozen vial of 3 day activated T cells was quickly thawed in a bead bath. The outside of the vial was wiped with 70% ethanol. The cell suspension was transferred to a 50 mL tube containing 10 ml of complete T cell media.The cells were centrifuged at 200g for 10 minutes, followed by resuspension of the cell pellet in 10 ml of complete T cell media. 10 μL aliquot of the cell suspension was used to perform Trypan Blue exclusion cell count using an automatic cell counter to detect the cell concentration and viability. T cell concentration was adjusted with complete T cell media in T75 flask to cell concentration of 1x106 / ml. The flask was incubated overnight at 37°C and 5% CO2. Day 2 - the medium was changed to fresh culture medium before transfection. Trypan Blue exclusion cell count was performed using an automatic cell counter to get cell concentration and viability. Cell concentration was adjusted with complete T cell medium to 1x106 / ml. 1 ml of cells were added to 24 well plate at IxlO6cells / ml. 1.0 pg or 3.0 pg of mRNA-LPS-LPX (prepared in Examples 1 and 2) was added to each well, and the plate was incubated for 24 hours at 37°C and 5% CO2. 1.0 pg / well or 3.0 pg / well of mRNA+Lipof ectamine™ MessengerMAX™ (from Thermo Fisher) was used as positive control. Human primary T cells were used as negative control. [00274J After 24 hours, transfection efficiency was determined via flow cytometry and transfection cell viability was determined by performing Trypan Blue exclusion cell count using an automatic cell counter.

[0275] FIG. 3 scatter plots show transfection efficiency of LCF96, LCF108, and Lipofectamine™ MessengerMAX™ (1 .0 pg and 3.0 pg) in human primary T cells. In hard to transfect cells, such as human primary T cells, the LPS-LPX delivery system (LCF108) significantly improves transfection efficiency compared to Lipofectamine™ MessengerMAX™ from 3.6% to 93.1% (for 1.0 pg) and from 9.1% to 99.5% (for .3 0 pg). Cell viability is slightly lower than that of Lipofectamine™ MessengerMAX™, as shown in Table 6

[0276] Table 6:Example 4. Transfection of mRNA Using LPS-LPX Delivery System Compared to Lipofectamine™ MessengerMAX™ in vivo.

[0277] Formulation rsF591 was prepared as described above in Example 1 for Formulation LCF96, the only difference was that in Formulation rsF591 the mRNA used was Luciferase mRNA instead of Green fluorescent protein (GFP) mRNA used in Formulation LCF96. Formulation rsF616 was prepared as described above in Example 2 for Formulation LCF108, the only difference was that in Formulation rsF616 the mRNA used was Luciferase mRNA instead of Green fluorescent protein (GFP) mRNA used in Formulation LCF108.

[0278] Formulation rsF592 was used as a control. Formulation rsF592 has the same LPX (core) as that of Formulations rsF591 and rsF616 (the cores were prepared in identical manner), but it does not have a lipid shell (LPS).

[0279] In vivo transfection: Formulations rsF591, rsF592, or rsF616 were reconstituted in sterile PBS and each formulation was administered intravenously into four healthy mice at a dose of 1 pg / mouse. Three hours post injection, the mice were administered intra-peritoneally with D- luciferin at a dose of 150 mg / kg body weight. The mice were anesthetized and placed on the imaging stage of the IVIS imaging system in the ventral position. Images were obtained 10 minutes after D-luciferin injection using the IVIS Imaging System (Xenogen, Alameda, CA). Photons emitted from the whole body were quantified using Living Image Software (Xenogen).

[0280] Mice following administration of rsF591 or rsF616 exhibited high levels of mRNA expression (i.e., luciferase activity), including regions of luminescence at or above 1 x 107p / sec / cm2 / sr that spanned most or all of the width of the torso. Mice following administration of rsF592 exhibited significantly less mRNA expression (i.e., luciferase activity), with no luminescence exceeding 0.3 x 107p / sec / cm2 / sr, showing that LPX alone is insufficient for transfection (shown in FIG. 4).Example 5. Transfection of pDNA Using LPS-LPX Delivery System Compared to Lipofectamine™ LTX or Lipofeetamine™ 2000 in HEK293 Cells.

[0281] In formulations comprising pDNA, the pDNA is mainly found on the outside of the lipoplex (LPX).

[0282] Formulation LCF155 was prepared as follows: 1) Lipid shell - DC-cholesterol (500 μL, 20 mg / mL in EtOH) and DOPE (346 μL, 20 mg / mL in EtOH) were added to a 5 mLEppendorf tube to make 846 μL lipid solution (molar ratio of lipids: DC- Cholesterol / DOPE=2 / 1). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4230 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0283] 2) The core (lipoplex, LPX) - Green fluorescent protein (GFP) pDNA (5 μL, 4 mg / mL in water) was quickly pipetted into DC-Cholesterol / DOPE lipid shell (8.47 μL) to yield 13.47 μL of lipoplex (pDNA / DC-cholesterol=l / l weight ratio). This solution was completed to 100 μL with Tris-Sucrose buffer pH 7.4.

[0284] 3) The lipid shell (LPS) - KT-001 (656.8 μL, 20 mg / mL in EtOH), DOTMA (63.9 μL, 20 mg / mL in EtOH), DSPC (150.5 μL, 20 mg / mL in EtOH), cholesterol (283.8 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1229.3 μL lipid solution (molar ratio of lipids: KT- 001 / DOTMA / DSPC / Cholesterol / DMG-PEG2000=45 / 5 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3687.9 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0285] 4) pDNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 100 μL lipoplex (LPX), and then pipetted a few times up and down to mix.

[0286] Formulation LCF157 was prepared was prepared as follows: 1) Lipid shell - DC- cholesterol (500 μL, 20 mg / mL in EtOH) and DOPE (346 μL, 20 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 846 μL lipid solution (molar ratio of lipids: DC- Cholesterol / DOPE=2 / 1). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles wereremoved by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4230 μL of Tris-Sucrose Buffer was drawn into a 10 mb BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0287] 2) The core (lipoplex, LPX) - Green fluorescent protein (GFP) pDNA (5 μL, 4 mg / mL in water) was quickly pipetted into DC-Cholesterol / DOPE lipid shell (8.47 μL) to yield 13.47 μL of lipoplex (pDNA / DC-cholesterol=l / l weight ratio). This solution was completed to 100 μL with Tris-Sucrose buffer pH 7.4.

[0288] 3) The lipid shell (LPS) was prepared as described above for LCF155.

[0289] 4) pDNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 100 μL lipoplex (LPX), and then pipetted a few times up and down to mix.

[0290] Transfection in HEK293 Cells: Day 1 - Cells were gently pipetted to mix well. 10 μL aliquot of the cell suspension was used to perform Trypan Blue exclusion cell count using an automatic cell counter to detect the cell concentration and viability. Total cell number was calculated. Cells were transferred to 50 ml centrifuge tube, spun at 1200rpm or 250g for 5 minutes, old medium was discarded, and the cells were resuspended in fresh HEK293 completed medium (DMEM with 10% FBS) to adjust cells to concentration of 0.4X106 / ml. 24 well plate was then loaded with 0.5 ml of cell suspension per well (0.2x106 / well) and the plate was incubated overnight at 37°C and 5% CO2. Day 2 - the medium was changed to fresh completed cell medium before transfection. 1 pg of pDNA-LPS-LPX (prepared in above) was added to each well, and the plate was incubated for 24 hours at 37°C and 5% CO2. 1 pg / well of pDNA+Lipofectamine™ LTX or 1 pg / well of pDNA+Lipofectamine™ 2.000 (from Thermo Fisher) were used as positive controls. HEK293 cells were used as negative control.

[0291] After 24 hours, transfection efficiency was determined via flow cytometry and transfection cell viability was determined by performing Trypan Blue exclusion cell count using an automatic cell counter.

[0292] FIG. 5 scatter plots show transfection efficiency of LCF155, LCF157, Lipofectamine™ 2000, and Lipofectamine™ LTX (1 .0 pg pDNA) in HEK293 cells. In easy totransfect cells, such as HEK293, the LPS-LPX delivery system significantly improves transfection efficiency compared to Lipofectamine™ 2000 or Lipofectamine™ LTX from about 67% to about 89%. Cell viability is comparable to that of the controls Lipofectamine™ 2000 or Lipofectamine™ LTX, as shown in Table 7.

[0293] Table 7:Example 6. Transfection of pDNA Using LPS-LPX Delivery System Compared to Lipofectamine™ 2000 in Jurkat Cells.

[0294] Formulations LCF155 and LCF157 were prepared as described in Example 5.

[0295] Transfection in Jurkat Cells: Cells were gently pipetted to mix well. 10 μL aliquot of the cell suspension was used to perform Trypan Blue exclusion cell count using an automatic cell counter to detect the cell concentration and viability. Total cell number was calculated. Cells were transferred to 50 ml centrifuge tube, spun at 1200rpm or 250g for 5 minutes, old medium was discarded, and the cells were resuspended in fresh RPMI Serum Reduced Medium (SRM) to adjust cells to concentration of 1.0xl06 / ml. 24 well plate was then loaded with 0.5 ml of cell suspension per well (0.5x106 / well). 1.0 pg pDNA-LPS-LPX (prepared in Example 5) was added to each well, and the plate was incubated for 4 hours at 37°C and 5% CO2. 1 pg / well of mRNA+Lipofectamine™ 2000 was used as positive control. Jurkat cells were used as negative control.

[0296] After 4 hours, the plate was centrifuged at 250g for 5 minutes, and old medium was discarded. 500 μL of Jurkat completed medium (RPMU640 with 10% FBS) was added into each well and the plate was incubated for 24 hours at 37°C and 5% CO2.

[0297] After 24 hours, transfection efficiency was determined via flow cytometry and transfection cell viability was determined by performing Trypan Blue exclusion cell count using an automatic cell counter.

[0298] FIG. 6 scatter plots show transfection efficiency of LCF155, LCF157, and Lipofectamine™ 2000 (1 .0 gg pDNA) in Jurkat cells. In hard to transfect cells, such as Jurkat, the LPS-LPX delivery system (LCF155 and LCF157) significantly improves transfection efficiency compared to Lipofectamine™ 2000 from about 2.5% to about 36% (for 1.0 tig pDNA). Cell viability is comparable to that of the control Lipofectamine2000, as shown in Table 8.Example 7. Transfection of pDNA Using LPS-LPX Delivery System m vivo.

[0299] Formulation rsF615 was prepared as described above in Example 1 for Formulation LCF96, the only difference was that in Formulation rsF615 luciferase pDNA (20 μL, 1 mg / mL in water) was used instead of Green fluorescent protein (GFP) mRNA used in Formulation LCF96 (the pDNA is mainly found inside the LPX).

[0300] In vivo transfection: Formulation rsF615 was reconstituted in sterile PBS and administered intravenously into two healthy mice at a dose of 8 pg / mouse. Three hours post injection and six hours post injection the mice were administered intra-peritoneally with D- luciferin at a dose of 150 mg / kg body weight. The mice were anesthetized and placed on the imaging stage of the IVIS imaging system in the ventral position. Images were obtained 10 minutes after D-luciferin injection using the IVIS Imaging System (Xenogen, Alameda, CA). Photons emitted from the whole body were quantified using Living Image Software (Xenogen).

[0301] Bioluminescence images of mice, following administration of rsF615, exhibited high levels of pDNA expression (i.e., Luc expression) (shown in FIG. 7), with regions of luminescence at or above 2 x 106p / sec / cm2 / sr that spanned most or all of the width of the torso.Example 8. Manipulating Organ Specificity of LPS-LPX Delivery System by Controlling Lipid Composition of LPS.[003021 In this experiment the LPX composition remained constant, while the composition of LPS is modified.

[0303] For all four formulations (rsF598, rsF599, rsF600, and rsF601) the core (LPX) was prepared in identical manner. 1) The core (lipoplex, LPX) - Luciferase mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH) and cholesterol (5.68 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT- 001 / DSPC / cholesterol=50 / 10 / 38.5) were added to a 2 mL serum vial to make 23.28 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0304] rsF598 2) The lipid shell (LPS) - DC-Cholesterol (500 μL, 20 mg / mL in EtOH) and DOPE (346 μL, 20 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 846 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOPE=2 / l). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4.23 mL of Tris- Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0305] rsF599 2) DC-Cholesterol (250 μL, 20 mg / mL in EtOH) and DOPE (692 μL, 20 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 942 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOPE=l / 2). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4.71 mL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were thencollected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.[003061 rsF600 2) DC-Cholesterol (500 μL, 20 mg / mL in EtOH) and DOTAP (325 μL, 20 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 825 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOTAP=2 / l). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4.125 mL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0307] rsF601 2) DC-Cholesterol (250 μL, 20 mg / mL in EtOH) and DOTAP (650 μL, 20 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 900 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOTAP=l / 2). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4.5 mL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0308] 3) For all four formulations (rsF598, rsF599, rsF600, and rsF601) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX), and then pipetted a few times up and down to mix.

[0309] In vivo transfection: Formulation rsF598, rsF599, rsF600, or rsF601 was reconstituted in sterile PBS and each formulation was administered intravenously into two healthy mice at a dose of Ipg / mouse. At 6 hours post injection the mice were administered intraperitoneally with D-luciferin at a dose of 150 mg / kg body weight. Major organs such as heart, lung, liver, spleen, and kidney were immediately excised and placed on the imaging stage of the IVIS imaging system for imaging. Images were obtained 10 minutes after D-luciferin injectionusing the IVIS Imaging System (Xenogen, Alameda, CA). Photons emitted from the organs and tissues were quantified using Living Image Software (Xenogen).

[0310] The composition of the lipoplex (LPX) was identical for the four formulations tested(rsF598, rsF599, rsF600, and rsF601). The composition of LPS contained one common component (DC-cholesterol) and one component that was changed. Formulations rsF598 and rsF599 contained a neutral lipid (DOPE) in different amounts. Formulations rsF600 and rsF601 contained a cationic lipid (DOTAP) in different amounts.

[0311] Bioluminescence images (FIG. 8A) of organs (heart, lung, liver, spleen, and kidney) of mice following administration of rsF598, rsF599, rsF600, or rsF601, show that rsF598 has more lung accumulation (>1 x 106p / sec / cm2 / sr in one specimen and about 0.6 x 106p / sec / cm2 / sr in the other) than rsF599 (both < 0.4 x 106p / sec / cm2 / sr), whereas rsF599 has more spleen accumulation (both >1 x 106p / sec / cm2 / sr) than rsF598 (both <0.4 x 106p / sec / cm2 / sr). Having higher DC-CHOL / DOPE ratio (rsF598) correlated with higher lung and lower spleen accumulation compared to lower DC-CHOL / DOPE ratio (rsF599). rsF600 had the highest liver accumulation (>1 x 106p / sec / cm2 / sr) with rsF598 giving about 0.6-1 x 106p / sec / cm2 / sr and the others being in the range of about 0.1-0.6 x 106p / sec / cm2 / sr. Values given for luminescence here and elsewhere in the examples correspond to the peak luminescence observed anywhere on the surface of the analyzed object.

[0312] Replacing neutral lipid (DOPE) with cationic lipid (DOTAP) increased liver accumulation and lowered lung accumulation.

[0313] FIG. 8B shows relative biodistribution of the quantified IVIS images of heart, lung, liver, spleen, and kidney. rsF598 has more lung distribution 30.7% (higher DC-CHOL / DOPE ratio) than 7.0% in rsF599. rsF599 has more spleen distribution 48.1% (lower DC-CHOL / DOPE ratio) than 6.6% in rsF598. Both rsF600 and rsF601 have strong liver accumulation, 80.2% and 74.6% respectively. These results indicate that the distribution of the LPS-LPX delivery system can be manipulated and passive targeting to specific organs can be achieved by changing LPS composition.Example 9. Enhancing Organ Specificity of LPS-LPX Delivery System with anti-ICAM Fab.

[0314] Formulation rsF627 was prepared as follows: 1) The core (lipoplex, LPX) -Luciferase (Luc) mRNA (10 μL, 1 mg / mL in water) and sodium acetate buffer (90 μL, 25 mMpH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 10 mg / mL in EtOH), DSPC (3.01 μL, 10 mg / mL in EtOH) and cholesterol (5.68 μL, 10 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DSPC / cholesterol=50 / 10 / 38.5) were added to a 2 mL serum vial to make 23.28 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0315] 2) The lipid shell (LPS) - KT-001 (73 μL, 10 mg / mL in EtOH), DSPC (15.1 μL, 10 mg / mL in EtOH), cholesterol (28.4 μL, 10 mg / mL in EtOH) and DMG-PEG2000 (7.4 μL, 10 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 123.9 μL lipid solution (molar ratio of lipids: KT-001 / DSPC / Cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. anti-ICAM Fab (139.9 μL, 664 μg / mL) and 231.8 μL of Tris- Sucrose Buffer was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0316] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0317] Formulation rsF628 was prepared as follows: 1) The core (lipoplex, LPX) - prepared as described above for rsF627.

[0318] 2) The lipid shell (LPS) - KT-001 (729.8 μL, 10 mg / mL in EtOH), DSPC (150.5 μL,10 mg / mL in EtOH), cholesterol (283.8 μL, 10 mg / mL in EtOH) and DMG-PEG2000 (74.3 μL, 10 mg / mL in EtOH) were added to a 5 mL Eppendorf tube to make 1238.4 μL lipid solution (molar ratio of lipids: KT-001 / DSPC / Cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5). The lipid solution was drawn into a 3 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 3715.2 μL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 3 / 1 toproduce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0319] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0320] Four mice were intravenously injected with lipopolysaccharide (from Escherichia coli O111 :B4, Sigma-Aldrich) at a dose of 7.5 mg / kg to establish endotoxemia induced acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS). One hour following the injection in vivo transfection was initiated using rsF627 or rsF628.

[0321] In vivo transfection: Formulation rsF627 or rsF628 was reconstituted in sterile PBS, and each formulation was intravenously administered into two ARDS mice at a dose of Ipg / mouse. At 3 hours post injection the mice were administered intra-peritoneally with D- luciferin at a dose of 150 mg / kg body weight. Major organs such as heart, lung, liver, spleen, and kidney were immediately excised and placed on the imaging stage of the IVIS imaging system for imaging. Images were obtained 10 minutes after D-luciferin injection using the IVIS Imaging System (Xenogen, Alameda, CA). Photons emitted from the organs and tissues were quantified using Living Image Software (Xenogen).

[0322] The composition of the lipoplex (LPX) was identical for the two formulations tested (rsF627 and rsF628). The lipid composition and lipid ratios of the LPS for the two formulations were identical, but rsF627 additionally included anti-ICAM Fab on the LPS.

[0323] Bioluminescence images (FIG. 9A) of organs (heart, lung, liver, spleen, and kidney) of ARDS mice following administration of rsF627 or rsF628, show that rsF627 has more lung accumulation (luminescence of about 0.1-0.3 x 107p / sec / cm2 / sr) than rsF628 (below limit of detection, i.e., < 0.05 x 107p / sec / cm2 / sr).

[0324] rsF628 is a liver targeting formulation, with more than 95% of overall luciferase expression being in the liver. Modifying LPS of rsF628 with anti-ICAM Fab resulted in Formulation rsF627, which exhibited 14% lung distribution, as shown in FIG. 9B.

[0325] FIG. 9C shows that overall luciferase expression level in the lung with Formulation rsF627 is 8 times higher than when using control Formulation rsF628.

[0326] These results indicate that active targeting with a targeting ligand (e.g., an antibody or an antibody fragment) can enhance organ specificity. The distribution of the LPS-LPX deliverysystem can be manipulated and active targeting to specific organs can be achieved by having a targeting ligand (e.g., an antibody or an antibody fragment) on LPS.Example 10. Enhancing Organ Specificity of LPS-LPX Delivery System by Controlling LPS lipid Composition and with anti-ICAM Fab.

[0327] Formulation rsF620 was prepared as follows: 1) The core (lipoplex, LPX) - prepared as described above for rsF627 in Example 9.

[0328] 2) The lipid shell (LPS) - DC-Cholesterol (500 μL, 10 mg / mL in EtOH) and DOPE(346 μL, 10 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 846 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOPE=2 / l). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. 4.23 mL of Tris-Sucrose Buffer was drawn into a 10 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag (100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0329] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0330] Formulation rsF621 was prepared as follows: 1) The core (lipoplex, LPX) - prepared as described above for rsF627 in Example 9.

[0331] 2) The lipid shell (LPS) - DC-Cholesterol (41 μL, 10 mg / mL in EtOH) and DOPE(56.6 μL, 10 mg / mL in EtOH) were added to a 1 mL Eppendorf tube to make 97.6 μL lipid solution (molar ratio of lipids: DC-Cholesterol / DOPE=2 / l). The lipid solution was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then, the syringe was loaded onto the Ignite NanoAssemblr cartridge. anti-ICAM Fab (183.72 μL, 664 pg / mL) and 304.28 μL of Tris-Sucrose Buffer was drawn into a 1 mL BD syringe. Air bubbles were removed by gently tapping the syringe. Then the syringe was loaded onto the Ignite NanoAssemblr cartridge. Flowrate was set at 12 mL / min with ratio of: Tris-Sucrose Buffer / Lipid solution = 5 / 1 to produce liposomes. The liposomes were then collected into a dialysis bag(100 KD) and dialyzed with Tris-Sucrose Buffer. The buffer was changed 3 times every 2 hours for a total of 6 hours.

[0332] 3) mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 123.28 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0333] Four mice were intravenously injected with lipopolysaccharide (from Escherichia coli O111 :B4, Sigma-Aldrich) at a dose of 7.5 mg / kg to establish endotoxemia induced acute lung injury (ALI) and its most severe form, acute respiratory distress syndrome (ARDS). One hour following the injection in vivo transfection was initiated using rsF620 or rsF621.

[0334] In vivo transfection: Formulation rsF620 or rsF621 was reconstituted in sterile PBS, and each formulation was intravenously administered into two ARDS mice at a dose of Ipg / mouse. At 3 hours post injection the mice were administered intra-peritoneally with D- luciferin at a dose of 150 mg / kg body weight. Major organs such as heart, lung, liver, spleen, and kidney were immediately excised and placed on the imaging stage of the IVIS imaging system for imaging. Images were obtained 10 minutes after D-luciferin injection using the IVIS Imaging System (Xenogen, Alameda, CA). Photons emitted from the organs and tissues were quantified using Living Image Software (Xenogen).

[0335] The composition of the lipoplex (LPX) was identical for the two formulations tested (rsF620 and rsF621). The lipid composition and lipid ratios of the LPS for the two formulations were identical, but rsF621 additionally included anti-ICAM Fab on the LPS. Lipid composition of the LPS was chosen to target lungs.

[0336] Bioluminescence images (FIG. 10A) of organs (heart, lung, liver, spleen, and kidney) of ARDS mice following administration of rsF620 or rsF621, show that rsF621 has more lung accumulation (>5 x 105p / sec / cm2 / sr) than rsF620 (< 3.5 x 105p / sec / cm2 / sr).

[0337] rsF620 is a lung targeting formulation, with about 48% of overall luciferase expression in the lung. Modifying LPS of rsF620 with anti-ICAM Fab resulted in Formulation rsF621, which exhibited about 78% lung distribution, as shown in FIG. 10B.

[0338] FIG. 10C shows that overall luciferase expression level in the lung with Formulation rsF621 is 9 times higher than overall lung expression level with control Formulation rsF620.

[0339] These results indicate that active targeting with a targeting ligand (e.g., an antibody or an antibody fragment) can enhance organ specificity. The distribution of the LPS-LPX deliverysystem can be manipulated and active targeting to specific organs can be achieved by having a targeting ligand (e.g., an antibody or an antibody fragment) on LPS.Example 11. Characterization of LPS-LPX Delivery System.

[0340] Various LPS-LPX delivery systems were prepared, and their size, surface charge, and encapsulation efficiency were determined.

[0341] Size, surface charge, and poly dispersity index (PDI) of various LPS were determined using dynamic light scattering analyzer. The findings are summarized in Tables 9 and 10 below. PDI values indicate that LPS are monodispersed.

[0342] Table 9: Positive shell00343] Table 10: Neutral Shell

[0344] Formulations rsF598, rsF599, rsF600, and rsF601, were prepared as described above in Example 8. The size, surface charge, and encapsulation efficiency of the formulations were measured. The results of these measurements are summarized in Table 11.

[0345] Table 11:

[0346] Formulations LCF102, LCF96, LCF104, LCF105, and LCF106, were prepared as follows: The outside (LPS) for all the formulations was identical and was prepared as described in Example 1 above for LCF96.

[0347] LCF102: The inside (LPX) was prepared as follows: Green fluorescent protein (GFP) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH) and DSPC (4.3 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT-001 / DSPC =5 / 1) were added to a 2 mL serum vial to make 18.9 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5- 10 seconds of vortex at highest speed setting.

[0348] mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 118.9 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0349] LCF104: The inside (LPX) was prepared as follows: Green fluorescent protein (GFP) mRNA (20 μL, 1 mg / mL in water) and sodium citrate buffer (80 μL, 10 mM, pH 3) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH), cholesterol (5.68 μL, 20 mg / mL in EtOH), and DMG-PEG2000 (1 .49 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT- 001 / DSPC / cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to make 24.77 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0350] Sodium citrate buffer 10 mM, pH3: Commercial 0.5 M, pH 3.0 citrate buffer (Thermo Scientific, Cat No. J61391-AK) was diluted 50 times with water for injection.

[0351] mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 124.77 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0352] LCF105: The inside (LPX) was prepared as follows: Green fluorescent protein (GFP) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM, pH 4) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH), cholesterol (5.68 μL, 20 mg / mL in EtOH), and DMG-PEG2000 (1.49 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT- 001 / DSPC / cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to make 24.77 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0353] Sodium acetate buffer 25mM, pH4: Commercial IM pH 4.0 acetate buffer (Thermo Scientific, Cat. No. J60104-AK) was diluted 40 times with water for injection.

[0354] mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 124.77 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0355] LCF106: The inside (LPX) was prepared as follows: Green fluorescent protein(GFP) mRNA (20 μL, 1 mg / mL in water) and sodium acetate buffer (80 μL, 25 mM, pH 5) were added to a 1 mL Eppendorf tube to make 100 μL mRNA solution. KT-001 (14.6 μL, 20 mg / mL in EtOH), DSPC (3.01 μL, 20 mg / mL in EtOH), cholesterol (5.68 μL, 20 mg / mL in EtOH) and DMG-PEG2000 (1.49 μL, 20 mg / mL in EtOH) (molar ratio of lipids: KT- 001 / DSPC / cholesterol / DMG-PEG2000=50 / 10 / 38.5 / 1.5) were added to a 2 mL serum vial to make 24.77 μL lipid solution. The lipid solution was then quickly pipetted into the mRNA solution (and pipetted a few times up and down to mix), followed by 5-10 seconds of vortex at highest speed setting.

[0356] mRNA-LPS-LPX was prepared as follows: 100 μL lipid shell (LPS) was pipetted into 124.77 μL lipoplex (LPX) and then pipetted a few times up and down to mix.

[0357] The size, surface charge, and encapsulation efficiency of the formulations were measured. The results of these measurements are summarized in Table 12.

[0358] Table 12:

[0359] LPS determines the final surface charge / composition of the LPS-LPX delivery system. When the LPS is neutral, the LPS-LPX surface is always neutral regardless of LPX charges. When LPS is positively charged, the LPS-LPX surface is always positive.

[0360] The complete disclosures of all publications cited herein are incorporated herein by reference in their entireties as if each were individually set forth in full herein and incorporated.

[0361] Various modifications and alterations to the embodiments disclosed herein will become apparent to those skilled in the art without departing from the scope and spirit of this disclosure. Illustrative embodiments and examples are provided as examples only and are not intended to limit the scope of the present invention.

Claims

What is claimed:

1. A composition for transfecting a ceil in vitro or in vivo, comprising at least one nucleic acid contained in an LPS (lipid shell)-LPX (lipoplex) delivery' system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

2. A method for transfecting a cell with one or more nucleic acids comprising: contacting the cell in vitro with a composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule.

3. A method for treating or preventing a disease or alleviating a symptom of a disease comprising administering to a subject in need thereof a composition for transfecting a cell, the composition comprising at least one nucleic acid contained in an LPS-LPX delivery system, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule4. The composition for transfecting a cell or method of any one of claims 1-3, wherein the LPS-LPX delivery system comprises an LPS encapsulating an LPX.

5. A method for preparing a composition for transfecting a cell in vitro or in vivo comprising:(a) preparing a first solution comprising an aqueous phase and one or more nucleic acids;(b) preparing a second solution comprising an organic phase and one or more lipids;(c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids:(d) agitating the LPX prepared in step (c);(e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(t) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targetingligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution prepared in step (e),(g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell.

6. A method for preparing a composition for transfecting a cel I bi vitro or in vivo comprising:(a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids;(b) agitating the LPX prepared in step (a);(c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(d) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution;(e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(f) agitating the mixture prepared in step (e), thereby forming a composition for transfecting a cell.

7. A composition for transfecting a ceil hi vitro or in vivo., prepared by a method comprising:(a) preparing a first solution comprising an aqueous phase and one or more nucleic acids;(b) preparing a second solution comprising an organic phase and one or more lipids;(c) mixing the first solution prepared in step (a) with the second solution prepared in step(b) thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids;(d) agitating the LPX prepared in step (c);(e) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(t) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the third solution prepared in step (e), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution prepared in step (e);(g) mixing the LPX after step (d) with the LPS prepared in step (f), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(h) agitating the mixture prepared in step (g), thereby forming a composition for transfecting a cell.

8. A composition for transfecting a cell in vitro or in vivo, prepared by a method comprising:(a) mixing a first solution comprising an aqueous phase and one or more nucleic acids with a second solution comprising an organic phase and one or more lipids, thereby forming a monodisperse or polydisperse LPX comprising one or more nucleic acids;(b) agitating the LPX prepared in step (a),(c) preparing a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(d) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a third solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the third solution,(e) mixing the LPX after step (b) with the LPS prepared in step (d), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(f) agitating the mixture prepared in step (e), thereby forming a composition for transfecting a cell.

9. A method for preparing a composition for transfecting a cell zri vitro or in vivo compri sing:(a) preparing a first solution comprising an aqueous phase;(b) preparing a second solution comprising an organic phase and one or more lipids;( c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles;(d) preparing a third solution comprising an aqueous phase and one or more nucleic acids:(e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or poly disperse I..PX;(f) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f);(h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell.

10. A method for preparing a composition for transfecting a cel I bi vitro or in vivo comprising:(a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles;(b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX;(c) preparing an EPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution,(d) mixing the LPX prepared in step (b) with the EPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(e) agitating the mixture prepared in step (d), thereby forming a composition for transfecting a cell11. A composition for transfecting a cell in vitro or in vivo, prepared by a method cornpri sing’(a) preparing a first solution comprising an aqueous phase;(b) preparing a second solution comprising an organic phase and one or more lipids;(c) mixing the first solution prepared in step (a) with the second solution prepared in step (b) to produce liposomes, micelles, or other self-assembled lipid nanoparticles;(d) preparing a third solution comprising an aqueous phase and one or more nucleic acids;(e) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (c) with the third solution prepared in step (d) thereby forming a monodisperse or polydisperse LPX;(t) preparing a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule;(g) preparing an LPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from the fourth solution prepared in step (f), wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution prepared in step (f);(h) mixing the LPX prepared in step (e) with the LPS prepared in step (g), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(i) agitating the mixture prepared in step (h), thereby forming a composition for transfecting a cell12. A composition for transfecting a cell in vitro or bi vivo, prepared by a method comprising:(a) mixing a first solution comprising an aqueous phase with a second solution comprising an organic phase and one or more lipids to produce liposomes, micelles, or other self-assembled lipid nanoparticles;(b) mixing the liposomes, micelles, or other self-assembled lipid nanoparticles prepared in step (a) with a third solution comprising an aqueous phase and one or more nucleic acids, thereby forming a monodisperse or polydisperse LPX,(c) preparing an EPS by forming liposomes, micelles, or other self-assembled lipid nanoparticles from a fourth solution comprising an organic phase, one or more lipids, and optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule, wherein optionally a targeting ligand, an imaging ligand, a digesting agent, or a small molecule is added to an aqueous phase which is then mixed with the fourth solution,(d) mixing the LPX prepared in step (b) with the EPS prepared in step (c), and optionally adding a targeting ligand, an imaging ligand, a digesting agent, or a small molecule; and(e) agitating the mixture prepared in step (d), thereby forming a composition for transfecting a cell.

13. The composition for transfecting a cell or method of any one of claims 5- 12, wherein the aqueous phase of the first and / or the third solution is an aqueous buffer.

14. The composition for transfecting a cell or method of claim 13, wherein the aqueous buffer is a citrate buffer or an acetate buffer.

15. The composition for transfecting a cell or method of claim 13 or 14, wherein the aqueous buffer is in the pH range of about pH 2 to about pH 6.

16. The composition for transfecting a cell or method of claim 13, wherein the aqueous buffer is a Tris-sucrose buffer.

17. The composition for transfecting a cell or method of claim 13 or 16, wherein the aqueous buffer is in the pH range of about pH 7 to about pH 8.

18. The composition for transfecting a cell or method of any one of claims 5-17, wherein the organic phase comprises a water miscible organic solvent.

19. The method or composition of claim 18 wherein the water miscible organic solvent is ethanol, methanol, isopropanol, DMSO, acetone, acetonitrile, DMF, or THF.

20. The composition for transfecting a cell or method of any one of claims 1-19, wherein the nucleic acid is DNA, RNA, or DNA and RNA.

21. The composition for transfecting a cell or method of claim 20, wherein the RNA is or comprises mRNA, siRNA, miRNA, RNAi, saRNA, taRNA, or shRNA22. The composition for transfecting a cell or method of claim 21, wherein the RNA is or comprises mRNA.

23. The composition for transfecting a cell or method of claim 20, wherein the DNA is a plasmid DNA.

24. The composition for transfecting a cell or method of claim 23, wherein the plasmid DNA is nanoplasmid DNA25. The composition for transfecting a cell or method of any one of claims 20-24, wherein the nucleic acid comprises a therapeutic gene26. The composition for transfecting a cell or method of any one of claims 1-25, wherein the EPS is a lipid micelle, a liposome, or a self-assembled lipid nanopartide.

27. The composition for transfecting a cell or method of claim 26, wherein the LPS comprises one or more ionizable cationic lipids and / or one or more helper lipids28. The composition for transfecting a cell or method of any one of claims 1 -27, wherein the LPX comprises one or more ionizable cationic lipids and optionally one or more helper lipids29. The composition for transfecting a cell or method of claim 27 or 28, wherein the ionizable cationic lipid is selected from the lipids shown in Table I .

30. The composition for transfecting a cell or method of any one of claims 27-29, wherein the helper lipid is a phospholipid, pegylated lipid, cholesterol, or cholesterol derivative.

31. The composition for transfecting a cel I or method of claim 30, wherein the helper Lipid is a phospholipid.

32. The composition for transfecting a cell or method of claim 30, wherein the helper lipid is a pegylated lipid.

33. The composition for transfecting a cell or method of claim 30, wherein the helper lipid is a cholesterol34. The composition for transfecting a cell or method of claim 30, wherein the helper lipid is a cholesterol derivative.

35. The method of any one of claims 3-34, wherein the disease is cancer.

36. The composition for transfecting a cell or the method of any one of claims 1-35, wherein the LPX comprises KT-001, DSPC, cholesterol, DOTMA. DC-cholesterol, DOPE, or any combinations thereof.

37. The composition for transfecting a cell or the method of any one of claims 1 -35, wherein the LPS comprises KT-()()I. DSPC, cholesterol, DMG-PEG2000, DOTMA, DO TAP, or any combinations thereof.

38. The composition for transfecting a cell or the method of claim 36 or 37, wherein the LPX comprises KT-001 and DSPC.

39. The composition for transfecting a cell or the method of claim 38, wherein the molar ratio of KT-001 :DSPC in the LPX is 5:1.

40. The composition for transfecting a cell or the method of claim 36 or 37, wherein the LPX comprises KT-001, DSPC, and cholesterol.

41. The composition for transfecting a cell or the method of claim 40, wherein the molar ratio of KT-001 'DSPC .cholesterol in the LPX is 50: 10:38.5.

42. The composition for transfecting a cell or the method of claim 36 or 37, wherein the LPX comprises KT-001, DOTMA, DSPC, and cholesterol.

43. The composition for transfecting a cell or the method of claim 42, wherein the molar ratio of KT-001 :DOTMA;DSPC:cholesterol in die LPX is 45:5:10:38.5.

44. The composition for transfecting a cell or the method of claim 36 or 37, wherein the LPX comprises KT-001, DSPC, cholesterol and DMG-PEG200045. The composition for transfecting a cell or the method of claim 44, wherein the molar ratio of KT-001 :DSPC:cholesterol:DMG-PEG2000 m the LPX is 50: 10:38.5: 1.5.

46. The composition for transfecting a cell or the method of claim 36 or 37, wherein the LPX comprises DC-cholesterol and DOPE.

47. The composition for transfecting a cell or the method of claim 46, wherein the molar ratio of DC-cholesterol:DOPE in the LPX is 2: 1 .

48. The composition for transfecting a cell or the method of any one of claims 36-47, wherein the LPS comprises KT-001, DSPC, cholesterol, and DMG-PEG2000.49 The composition for transfecting a cell or the method of claim 48, wherein the molar ratio of KT-001 :DSPC:choiesterol:DMG-PEG2000 in the LPS is 50 / 10 / 38.5 / 1.5.

50. The composition for transfecting a cel I or the method of any one of claims 36-47, wherein the LPS comprises KT-001, DOTMA, DSPC, cholesterol, and DMG-PEG2000.

51. The composition for transfecting a cell or the method of claim 50, wherein the molar ratio of KT-001 :DOTMA:DSPC:cholesterol:DMG-PEG2000 in the LPS is 45 / 5 / 10 / 38.5 / 1.5.

52. The composition for transfecting a cell or the method of any one of claims 36-47, wherein the LPS comprises DC / -cholesterol and DOPE53. The composition for transfecting a cell or the method of claim 52, wherein the molar ratio of DC -cholesterol DOPE in the LPS is 2: 1 or 1 :2.

54. The composition for transfecting a cell or the method of any one of claims 36-47, wherein the I, PS comprises DC~cholesterol and DOTAP.

55. The composition for transfecting a cell or the method of claim 54, wherein the molar ratio of DC-cholesterol: DOTAP in the LPS is 2: 1 or 1 '256. The composition for transfecting a cell or the method of any one of claims 1-55, wherein the composition comprises a targeting ligand.

57. The composition for transfecting a cell or the method of claim 56, wherein the targeting ligand is in contact with the LPS.

58. The composition for transfecting a cell or the method of claim 56 or 57, wherein the targeting ligand is DUPA, folate, a peptide, or an antibody or an antigen binding fragment thereof.

59. The composition for transfecting a cell or the method of any one of claims 1-58, wherein the composition comprises an imaging ligand.

60. The composition for transfecting a cell or the method of claim 59, wherein the imaging ligand is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX.61 . The composition for transfecting a cell or the method of claim 59 or 60, wherein the imaging ligand is l,2-Distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-IR825 (DSPE-PEG-IR825) or diethylenetriaminepentaacetic acid (DTPA)-Gd.

62. The composition for transfecting a cell or the method of any one of claims 1-61, wherein the composition comprises a digesting agent.

63. The composition for transfecting a cell or the method of claim 62, wherein the digesting agent is in contact with the LPS, inside the LPX, or between the surface of the EPS and the LPX.

64. The composition for transfecting a cell or the method of claim 62 or 63, wherein the digesting agent is hyaluronidase or collagenase.

65. The composition for transfecting a cell or the method of any one of claims 1-64, wherein the composition comprises a small molecule.

66. The composition for transfecting a cell or the method of claim 65, wherein the small molecule is in contact with the LPS, inside the LPX, or between the surface of the LPS and the LPX.

67. The composition for transfecting a cell or the method of claim 65 or 66, wherein the small molecule is an NS AID or a toxin.

68. The composition for transfecting a cell or the method of claim 65 or 66, wherein the small molecule is a vitamin, saccharide, steroid, or chemotherapy' drug.

69. The composition for transfecting a cell or the method of claim 65 or 66, wherein the small molecule is a folic acid, glucose, galactose, N-acetylgalactosamine (GalNAc), dexamethasone, paclitaxel, or doxorubicin.

Images