WO2016077639A2 - Nanovesicular therapies - Google Patents

Abstract

Compositions comprising nanomembrane delivery complexes, methods of generating nanomembrane delivery complexes, and methods of treating or preventing diseases, disorders or conditions therewith.

Description

NANO VESICULAR THERAPIES

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/078,967, "NANO VESICULAR THERAPIES", filed November 12, 2014, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The field of the invention is pharmaceutical compositions for the treatment of conditions, diseases and disorders and methods of making same.

BACKGROUND OF THE INVENTION

[0003] Intrinsic issues that are associated with free drugs particularly with small interfering RNA (siRNA) and other nucleic acids include poor solubility, poor stability, unwanted toxicity and/or an inability to cross cell membranes. These issues have propelled the development of new drug delivery systems. The in vivo pharmacokinetics and therapeutic activity of drugs generally needs to be improved, as drug costs are rising and drug pipelines are constricting. Fuelled by many advances in nanotechnology and biotechnology, the past decades have witnessed rapid growth in the research and development of drug delivery devices in the form of polymeric nano- and/ or microparticles, liposomes and micelles, among others. The success of these devices relies largely on the selection of appropriate design parameters to address the physicochemical limitations of free drugs (that is, solubility and stability) and to overcome biological hurdles in reaching the target (that is, the first-pass effect, immune clearance, cell entry and off-target deposition). There is a continued need for additional therapeutic modalities.

SUMMARY OF THE INVENTION

[0004] Provided herein are methods and compositions of nanomembrane delivery complexes that can be isolated from human cells (a parent or "producer" cell) and can confer therapeutic benefits by interacting with, signaling to, or delivering therapeutic molecules to a recipient or "target" cell. Nanomembrane delivery complexes are cell-like in protein, lipid, and/or nucleic acid composition. Producer cells may be selected based on the composition of secreted nanomembrane delivery complexes and optionally engineered to modify the secreted composition of nanomembrane delivery complexes. Nanomembrane delivery complexes may be extracted or isolated from the producer cells (and optionally modified) and formulated into a therapeutic composition. Nanomembrane delivery complexes are novel therapeutic modalities.

[0005] Nanomembrane delivery complexes can be extracted or isolated from the supernatant of producer cells and comprise a membrane and other functional entities, including, e.g., one or more of a protein, a lipid, and a nucleic acid that enable interaction with and delivery of a payload to a target cell. Provided herein are methods of modifying and isolating such producer cell-derived nanomembrane delivery complexes that exhibit various advantages in therapeutic delivery to target cells and tissues. Target cells and tissues may exhibit significant beneficial phenotypic modifications including altered morphology; altered expression of key metabolites, transcripts, or proteins; altered signaling states; altered proliferation; altered secretion and other desirable features.

[0006] The nanomembrane delivery complexes described herein can provide significant advantages over other nanoparticle therapeutic systems, including one or more of: limited or no undesired immunogenicity, e.g., when self-derived nanomembrane delivery complexes are used, greater stability in the blood due to evasion of complement and coagulation factors, efficient delivery into the cytosol of the target cell, and/or fewer off-target effects.

[0007] Aspects of the invention relate to preparations of isolated nanomembrane delivery complexes. In some embodiments, the nanomembrane delivery complexes have a diameter of 20 to 150 nanometers.

[0008] In some embodiments, the nanomembrane delivery complexes comprise a payload, such as, e.g., a therapeutic polypeptide, polynucleotide, polysaccharide, lipid, small molecule or toxin. The payload can be a polynucleotide, such as, e.g., m NA, miRNA, shRNA, dsDNA, IncRNA, and siRNA. In some embodiments, a mRNA payload encodes a therapeutic polypeptide that can have enzymatic activity.

[0009] In some embodiments, the nanomembrane delivery complexes comprise an imaging agent, such as, e.g., a radionuclide, an isotope, a fluorescent/fluorochrome molecule, a dye, and a chemiluminescent molecule.

[0010] In some embodiments, the nanomembrane delivery complexes comprise a receiver polypeptide that is capable of directing the nanomembrane delivery complex to a target. [0011] In some embodiments, the nanomembrane delivery complexes comprise and a surface molecule, such as, e.g., CD47, CD55, CD40, CD63, CD9, CD133 or CD59.

[0012] Aspects of the invention relate to pharmaceutical compositions comprising the nanomembrane delivery complex described herein and a pharmaceutically acceptable excipient or carrier. Optionally, the pharmaceutical compositions comprise a second therapeutic agent.

[0013] Aspects of the invention relate to dosage forms comprising the pharmaceutical compositions described herein. Optionally, the dosage forms are formulated as a sterile solution for intravenous injection.

[0014] Aspects of the invention relate to methods of treating a disease, disorder or condition described herein, such as, e.g. those listed in Table 3 and Table 4. The methods include administering to a subject in need thereof a pharmaceutical composition comprising nanomembrane delivery complex described herein, optionally in form of a suitable dosage form, in an amount effective to treat the disease, disorder or condition. In some embodiments, the nanomembrane delivery complex comprises a payload and delivers the payload to a target cell or tissue that is associated with the disease, disorder or condition in an amount effective to treat the disease, disorder or condition. A target cell can be an infected, impaired or dysregulated cell. A target tissue may include an infected, impaired or dysregulated cell.

[0015] In some embodiments, the nanomembrane delivery complex facilitates the contacting of an infected, impaired or dysregulated cell with the payload in sufficient proximity and for a sufficient duration to bring about a substantial killing of the infected, impaired or dysregulated cell, thereby treating the disease, disorder or condition.

[0016] In other embodiments, the nanomembrane delivery complex facilitates the contacting of the infected, impaired or dysregulated cell with the payload in sufficient proximity and for a sufficient duration to bring about a substantial restoration of the functionality of the infected, impaired or dysregulated cell, thereby treating the disease, disorder or condition. Optionally, an impaired enzyme function is restored or a dysregulated enzyme function regulated.

[0017] In some embodiments, the treatment regimen includes administering a second, standard-of-care therapy.

[0018] Aspects of the invention relate to method for producing a preparation of isolated nanomembrane delivery complexes. The methods include: providing a producer cell capable of generating a nanomembrane delivery complex; obtaining from the producer cell the nanomembrane delivery complex; modifying the isolated nanomembrane delivery complex with a payload; and isolating the modified nanomembrane delivery complex. In some embodiments, the nanomembrane delivery complexes are released by the producer cells into a culture medium. The nanomembrane delivery complexes may be modified by membrane perturbation. In some embodiments, the producer cell is a mammalian cell that is isolated or derived from a cell line.

[0019] In some embodiments, the producer cell comprises one or more surface molecules, such as, e.g., CD47, CD55, CD40, CD63, CD9, CD133 and CD59. In some embodiments, the producer cell naturally comprises the surface molecule. In some embodiments, the producer cell is modified to comprise the surface molecule.

[0020] In some embodiments, the isolated nanomembrane delivery complex comprises one or more surface molecules, such as, e.g., CD47, CD55, CD40, CD63, CD9, CD133 and CD59. In some embodiments, the isolated nanomembrane delivery complex naturally comprises the surface molecule. In some embodiments, the isolated nanomembrane delivery complex is modified to comprise the surface molecule.

[0021] Optionally, the preparation of isolated nanomembrane delivery complexes produced by the methods described herein is formulated into a pharmaceutical composition. Optionally, the pharmaceutical composition is tested or analyzed. The test or analysis may include one or more of: i) analyzing the presence or activity of the payload, ii) analyzing the potency of the nanomembrane delivery complex in a phenotypic or functional assay iii) detecting the presence or absence of one or more biomarkers from the producer cell, iv) analyzing the size distribution of the nanomembrane delivery complexes, and/or v) analyzing the membrane composition of the nanomembrane delivery complexes.

[0022] Aspects of the invention relate to pharmaceutical compositions comprising a purified population of nanomembrane delivery complexes operably associated with a therapeutic agent. Optionally, the nanomembrane delivery complex comprises a targeting receiver, which can be a protein or peptide. In some embodiments, the targeting receiver is at least partially exposed on an external surface of the nanomembrane delivery complex. The therapeutic agent may comprise a nucleic acid. The nucleic acid can be a ribonucleic acid. The ribonucleic acid can be a mRNA, miRNA, siRNA, or shRNA. In some embodiments, the purified population of nanomembrane delivery complexes comprise cellular membrane fragments.

[0023] Aspects of the invention relate to pharmaceutical compositions comprising a purified population of nanomembrane delivery complexes operably associated with a therapeutic agent that is produced by the process of: providing a plurality of human cells isolated from a human subject; treating the plurality of isolated human cells under conditions effective to generate the population of nanomembrane delivery complexes; and purifying the generated population of nanomembrane delivery complexes from the plurality of human cells. Optionally, the process comprises the step of contacting the isolated human cells or the generated purified population with a therapeutic agent, under conditions such that the therapeutic agent is operably associated with the generated purified population. In some embodiments, the therapeutic agent is present within, on or in the membrane of the nanomembrane delivery complex.

[0024] Aspects of the invention relate to methods of preparing a pharmaceutical composition. The methods include: providing a plurality of donor cells (producer cells) from a human subject; treating the plurality of donor cells (producer cells) under conditions effective to generate a population of nanomembrane delivery complexes; and optionally purifying the nanomembrane delivery complexes from the donor cells (producer cells). Optionally, the methods may include the step of loading into the donor cells (producer cells) or the nanomembrane delivery complexes a therapeutic agent.

[0025] Aspects of the invention relate to methods delivering a therapeutic agent to a human subject in need thereof. The methods include the step of administering an effective amount of a nanomembrane delivery complex loaded with a payload to a human subject. In some embodiments, the loaded nanomembrane delivery complex localizes to a target cell, tissue or organ in the human subject. In some embodiments, the loaded nanomembrane delivery complex localizes to a target cell in the human subject. In some embodiments, the loaded nanomembrane delivery complex localizes to subcellular space within a target cell in the human subject, such as, e.g., the cytoplasm. The loaded nanomembrane delivery complex may comprise a biological molecule exogenous to the target cell, tissue or organ.

[0026] In some embodiments, the producer cells are the same cell-type as the target cell. In some embodiments, the producer cells are a different cell-type from the target cell. [0027] Aspects of the invention relate to a synthetic nanomembrane delivery complex comprising: a synthetic receiver capable of interacting with a target cell, and a membrane comprising a parent cell (producer cell)-derived protein. In some embodiments, the synthetic nanomembrane delivery complex is between 20nm and lOOOnm in size. Optionally, the nanomembrane delivery complex comprises a payload that may be exogenous or endogenous to the nanomembrane delivery complex. In some embodiments, the receiver is capable of increasing the concentration of the payload in the tissue of the subject. The payload can be a nucleic acid. The nucleic acid can be a mR A, a miR A, a siR A or a shRNA.

Alternatively or in addition, the payload can be a polypeptide or a nucleic acid encoding a polypeptide capable of being translated in a target cell. In some embodiments, the payload can be delivered to the cytoplasm of a target cell. In other embodiments, the payload is delivered to the membrane of the target cell. In some embodiments, the membrane of the nanomembrane delivery complex fuses with a membrane of a target cell.

[0028] In some embodiments, the synthetic nanomembrane delivery complex is formulated for intravenous administration to the circulatory system of a mammalian subject. The mammalian subject can be a human.

[0029] Further aspects of the invention relate to preparations of nanomembrane delivery complexes for use as a medicament.

[0030] Further aspects of the invention relate to preparations of nanomembrane delivery complexes for use in the treatment of any one of the diseases listed in Table 3 and Table 4.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Provided herein are nanomembrane delivery complexes and pharmaceutical compositions and dosage forms thereof, and methods of generating same.

Nanomembrane Delivery Complexes

[0032] In some embodiments, the nanomembrane delivery complex comprises a membrane that forms a particle that has a diameter of between about 10-100 nm, 50-150 nm, 30-100 nm, 40-100 nm, 20-150 nm, 20-200 nm, 80-125 nm, 40-250 nm, 20-500 nm, or between about 10-1000 nm. In some embodiments, the membrane comprises lipids and fatty acids. In some embodiments, the membrane comprises one or more of phospholipids, glycolipids, fatty acids, sphingolipids, phosphoglycerides, sterols, cholesterols, and phosphatidylserine. In some embodiments, the lipid or fatty acid can be one or more of those listed in Table 5. In addition, the membrane may comprise one or more polypeptides and one or more polysaccharides, such as glycans.

[0033] In some embodiments, the nanomembrane delivery complex is generated by a producer cell (or parental cell), such as, e.g., a mammalian cell. In some embodiments, the membrane of the nanomembrane delivery complex comprises one or more molecules derived from the producer cell. The nanomembrane delivery complex may be generated in a cell culture system and isolated, e.g. by separating the nanomembrane delivery complex from the producer cell. Separation may be achieved by sedimentation. For example, the

nanomembrane delivery complex may have a specific density between 0.5-2.0, 0.75-1.5, and 0.9-1.1 kg/m³.

[0034] In some embodiments, the nanomembrane delivery complex is synthetic. For example, the nanomembrane delivery complex may comprise a payload, such as, e.g., a therapeutic polypeptide, nucleic acid (DNA or RNA) or other polynucleotide, polysaccharide or glycan, lipid or fatty acid, small molecule or toxin such that the nanomembrane delivery complex is not naturally occurring. In some embodiments, the nanomembrane delivery complex is modified, e.g. by introducing a payload or otherwise modifying the content of the complex, e.g. by changing the protein, lipid or glycan content of the membrane. For example, nanomembrane delivery complex are first isolated from a producer cell and then modified as desired, thereby generating synthetic nanomembrane delivery complexes. In some embodiments, the producer cell is modified. For example, an exogenous nucleic acid, an exogenous polypeptide or small molecule or toxin may be introduced into to the producer cell. Alternatively or in addition, the producer cell may otherwise be modified, e.g. by modifying the cellular or membrane content, such as by changing the lipid or glycan content of the cell membrane. Nanomembrane delivery complexes generated from the modified producer cells comprise one or more of the modification of the producer cell. The process produces synthetic nanomembrane delivery complexes. In some embodiments, both the producer cell and the nanomembrane delivery complex isolated from the producer cell are modified as described herein.

[0035] In some embodiments, the nanomembrane delivery complex delivers the payload (therapeutic agent) to a target. The payload is a therapeutic agent that acts on a target (e.g. a target cell) that is contacted with the nanomembrane delivery complex. Contacting may occur, e.g. in vitro or in a subject. Payloads that may be introduced into a nanomembrane delivery complex and/or a producer cell include therapeutic agents such as, nucleotides (e.g. nucleotides comprising a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g. DNA or mRNA molecules that encode a polypepetide such as an enzyme, or RNA molecules that have regulatory function such as miRNA, dsDNA, IncRNA, siRNA), amino acids (e.g. amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g. enzymes), lipids, carbohydrates, and small molecules (e.g. small molecule drugs and toxins). The payload may comprise nucleotides, e.g. nucleotides that are labeled with a detectable or cytotoxic moiety (e.g. a radiolabel).

[0036] In some embodiments, the nanomembrane delivery complex comprises nucleotides and/or polynucleotides (e.g. nucleic acids). For example, the nanomembrane delivery complex may comprise RNA, DNA, mRNA, miRNA, dsDNA, IncRNA, siRNA, or singular nucleotides. In some embodiments, the nanomembrane delivery complex comprises one or more of the miRNAs listed in Table 6. In some embodiments, the nucleotides and polynucleotides are synthetic. For example, an exogenous nucleic acid may be introduced into the nanomembrane delivery complex and/or the producer cell. In some embodiments, the nucleic acid is DNA that can be transcribed into an RNA (e.g. an siRNA or mRNA) and in the case of an mRNA may be translated into a desired polypeptide. In some embodiments, the nucleic acid is an RNA (e.g. an siRNA or mRNA) and in the case of an mRNA may be translated into a desired polypeptide.

[0037] In some embodiments, the nanomembrane delivery complex comprises a nucleic acid, such as a RNA or DNA. The nucleic acid is delivered to a target cell as a payload. The target cell may transcribe a DNA payload into an RNA such as a siRNA. In case a mRNA is transcribed by the target cell form the DNA payload, the cell may translate the mRNA into a polypeptide (e.g. therapeutic polypeptide). The target cell may also translate a delivered mRNA payload into a polypeptide.

[0038] In some embodiments, the producer cell comprises a nucleic acid that may be transcribed (e.g. a DNA may be transcribed into a siRNA or mRNA) and in case a mRNA is made the mRNA may be translated by the producer cell into a polypeptide. The producer cell may also be modified with a non-translatable RNA (e.g. siRNA) or mRNA. In case a mRNA is transferred the producer cell may translate the mRNA into a polypeptide. Nanomembrane delivery complexes derived from the producer cell may then carry the non-translatable RNA, the transcribed RNA or the translated polypeptide as a payload. [0039] The nanomembrane delivery complex may interact with the target cell via membrane fusion and deliver payloads (e.g., therapeutic agents) in a nanomembrane delivery complex composition to the surface or cytoplasm of a target cell. In some embodiments, membrane fusion occurs between the nanomembrane delivery complex and the plasma membrane of a target cell. In other embodiments, membrane fusion occurs between the nanomembrane delivery complex and an endosomal membrane of a target cell.

[0040] In some embodiments, the nanomembrane delivery complex comprises polypeptides on its surface selected from CD47, CD55, CD40, CD63, CD9, CD133 and CD59. In some embodiments, the nanomembrane delivery complex is modified to contain the one or more polypeptides. In some embodiments, the producer cell is modified to contain the one or more polypeptides. In some embodiments, the producer cell naturally contains the one or more polypeptides and nanomembrane delivery complexes derived therefrom also contain the polypeptides. The levels of any desired surface marker may be modified directly on the nanomembrane delivery complex, e.g. by contacting the complex with recombinantly produced polypeptides to bring about insertion in or conjugation to the membrane of the complex. Alternatively or in addition, the levels of any desired surface marker may be modified directly on the producer cell, e.g. by contacting the complex with recombinantly produced polypeptides to bring about insertion in or conjugation to the membrane of the cell. Alternatively, the producer cell may be modified by transducing an exogenous nucleic acid into the producer cell to express a desired surface marker. The surface marker may already be naturally present on the producer cell, in which case the exogenous construct may lead to overexpression of the marker and increased concentration of the marker in or on the producer cell. Alternatively, a naturally expressed surface marker may be removed from the producer cell, e.g. by inducing gene silencing in the producer cell. The polypeptides may confer different functionalities to the nanomembrane delivery complex, e.g. specific targeting capabilities, delivery functions (e.g. fusion molecules), enzymatic functions, increased or decreased half-life in vivo, etc. In some embodiments, the polypeptides include, but are not limited to CD47, CD55, CD49, CD40, CD133, CD59, glypican-1, CD9, CD63, CD81, integrins, selectins, lectins, and cadherins.

[0041] In some embodiments, the nanomembrane delivery complex comprises a receiver polypeptide. The receiver polypeptide is synthetic. In some embodiments, the receiver polypeptide is introduced into the producer cell (e.g. an exogenous nucleic acid that encodes the receiver polypeptide is introduced into the producer cell or a recombinant receiver polypeptide that is made outside the producer cell (e.g. synthesized by a protein expression system)). In some embodiments, the receiver polypeptide (e.g. a recombinantly produced polypeptide) is introduced into the nanomembrane delivery complex directly, e.g. after the nanomembrane delivery complex is isolated from the producer cell. In some embodiments, the receiver polypeptide can be on the surface of the nanomembrane delivery complexes. In some embodiments, the receiver polypeptide is capable of targeting the nanomembrane delivery complex to a specific target, e.g. a target (such as a pathogen, a metabolite, a polypeptide complex or a cell, e.g. a non-functional cell or cancer cell) that circulates in the circulatory system of the subject, such as the blood, or a target that resides in a tissue (such as a diseased tissue).

Payloads

[0042] Nanomembrane delivery complexes may comprise payloads such as peptides, proteins, DNA, RNA, siRNA, and other macromolecules and small therapeutic molecules. In some embodiments, the payload is transferred to a producer cell by applying controlled injury to the cell for a predetermined amount of time in order to cause perturbations in the cell membrane such that the payload can be delivered to the inside of the cell (e.g., cytoplasm). In some embodiments the payload is transferred to a nanomembrane delivery complex isolated from a producer cell by applying controlled injury to the nanomembrane delivery complex for a predetermined amount of time in order to cause perturbations in the complex membrane such that the payload can be delivered to the inside of the nanomembrane delivery complex. In some embodiments the payload of the nanomembrane delivery complex may be loaded within the membrane or interior portion of the nanomembrane delivery complex.

[0043] The payload may be a therapeutic agent selected from a variety of known small molecule pharmaceuticals. Alternatively, the payload may be a therapeutic agent selected from a variety of macromolecules, such as, e.g., an inactivating peptide nucleic acid (PNA), an RNA or DNA oligonucleotide aptamer, an interfering RNA (iRNA), a peptide, or a protein.

[0044] In some embodiments, the payload that may be delivered to a target by a nanomembrane delivery complex includes, but is not limited to, RNA, DNA, siRNA, mRNA, IncRNA, iRNA, polypeptides, enzymes, cyotkines, antibodies, antibody fragments, small molecules, chemotherapeutics, metals, viral particles, imaging agents, and plasmids. [0045] In some embodiment, the nanomembrane delivery complex comprises a payload of siR A capable of interfering with the expression of an oncogene or other dysregulating polypeptide. In some embodiments, the siRNA is capable of interfering with the expression of BCR-ABL, clusterin, survivin, B-catenin, CXCR4, BRCA-1, or BRCA-2.

[0046] In another embodiment, the nanomembrane delivery complex comprises a payload of antibodies, scFv, or nanobody that have intracellular targets including, but not limited to, tau, amyloid beta, WT1, LMP2, HPV E6 E7, MAGE A3, p53, NY-ESO-1, MelanA/MARTl, Ras, gplOO, proteinase 3, bcr-abl, tyrosinase, surviving, hTERT, and ML- IAP.

[0047] In another embodiment, the nanomembrane delivery complex comprises a payload of proteins, antibodies, polypeptides, or mRNAs encoding a polypeptides that include IL-1, IL-2, insulin, erythropoietin, anti-TNF alpha, glucocerebrosidase, interferon beta la, interferon beta lb, agalsidase beta, velaglucerase alfa, dornase alfa, alpha galactosidase A, idursulfase, adalimumab, etancercept, rituximab, infliximab, trastuzumab, bevacizumab, filgrastim, and ranibizumab.

[0048] In another embodiment, the nanomembrane delivery complex comprises a payload of miRNA, including, but not limited to, let-7a, let- 7b, let-7c, mir-34, miR-101, miR- 215, or miR-16.

[0049] In another embodiment, the nanomembrane delivery complex comprises a payload of small molecules, including, but is not limited to, doxorubicin, daunorubicin, docetaxel, irinotecan, taxanes, topoisomerase inhibitors, cyclophosphamide, vinca alkaloids, cisplatin, retinoids, nucleotide analogs, and kinase inhibitors.

[0050] In some embodiments the payload of the nanomembrane delivery complex is a nucleic acid molecule, e.g. mRNA or DNA, and the nanomembrane delivery complex targets the payload to the cytoplasm of the recipient or target cell, such that the nucleic acid molecule can be translated (if mRNA) or transcribed and translated (if DNA) and thus produce the polypeptide encoded by the payload nucleic acid molecule within the target cell. In one embodiment the polypeptide encoded by the payload nucleic acid molecule is secreted by the target cell, thus modulating the systemic concentration or amount of the polypeptide encoded by the payload nucleic acid molecule in the subject. In one embodiment the polypeptide encoded by the payload nucleic acid molecule is not secreted by the target cell, thus modulating the intracellular concentration or amount of the polypeptide encoded by the payload nucleic acid molecule in the subject. In one embodiment the polypeptide encoded by the payload nucleic acid molecule is toxic to the target cell or to other cell or tissue in the subject, e.g. toxic to a cancer cell. In one embodiment, the polypeptide encoded by the payload nucleic acid molecule is not toxic to the target cell or other cell or tissue in the subject, e.g. is therapeutically beneficial or corrects a disease phenotype.

[0051] The mRNA may be naked or modified, as desired. mRNA modification that improve mRNA stability and/or decrease immunogenicity include, e.g., ARCA: anti-reverse cap analog (m₂ ^{73 ~}°GP₃G), GP₃G (Unmethylated Cap Analog), m⁷GP₃G (Monomethylated Cap Analog), m₃ ^{2 2 7}GP₃G (Trimethylated Cap Analog), m5CTP (5'-methyl-cytidine triphosphate), m6ATP (N6-methyl-adenosine-5'-triphosphate), s2UTP (2-thio-uridine triphosphate), and Ψ (pseudouridine triphosphate).

[0052] In some embodiments, the payload of the nanomembrane delivery complex is a miRNA or pre-miRNA molecule, and the nanomembrane delivery complex targets the payload to the cytoplasm of the target cell, such that the miRNA molecule can silence a native mRNA in the target cell.

[0053] In one embodiment, the nanomembrane delivery complex comprises as a receiver synaptobrevin, as a payload an mRNA molecule encoding ricin toxin, and is useful for targeting the payload mRNA to tumor cells such that the mRNA is translated and the cells are killed.

[0054] In one embodiment, the nanomembrane delivery complex comprises as a receiver mannose, as a payload an mRNA molecule encoding glucocerebrosidase, and is useful for targeting the payload mRNA to macrophages in a subject with Gaucher' s disease such that the mRNA is translated and the restorative enzyme is expressed, thus rescuing the recipient macrophage.

[0055] In some embodiments, the payload or receiver may be engineered for specific trafficking from the producer cell into the nanomembrane delivery complex. In some embodiments, the payload or receiver may be directed for trafficking by an addition of a molecule to the payload or receiver (e.g. conjugation or fusion of another molecule). In certain embodiments, the additional molecule may be appended via a linker. In other embodiments, the payload or receiver may be directed for trafficking by modifying the payload or receiver sequence (e.g., a nucleotide change for nucleic acid or an amino acid change for polypeptide). In some embodiments, a payload or receiver may be directed for trafficking by modifying the payload or receiver composition to share increased similarity with part or all of a lipid listed in Table 5, or a nucleic acid listed in Table 6.

[0056] In some embodiments, a nucleic acid payload may be engineered for specific trafficking from the producer cell into the nanomembrane delivery complex. In certain embodiments, a nucleic acid payload (e.g., mRNA or miRNA) may comprise a sequence in the coding or noncoding region that targets the nucleic acid to the nanomembrane delivery complex. In certain embodiments, the noncoding region may include a 3' UTR or 5' UTR.

[0057] In some embodiments the payload of the nanomembrane delivery complex may be a membrane protein delivered to the plasma membrane or endosomal membrane of the recipient cell.

[0058] Nanomembrane delivery complexes may comprise two or more payloads, including mixtures, fusions, combinations and conjugates, of atoms, molecules, etc. as disclosed herein, for example including but not limited to, a nucleic acid combined with a polypeptide; two or more polypeptides conjugated to each other; a protein conjugated to a biologically active molecule (which may be a small molecule such as a prodrug); and the like.

[0059] Suitable payloads include, without limitation, pharmacologically active drugs and genetically active molecules, including antineoplastic agents, anti-inflammatory agents, hormones or hormone antagonists, ion channel modifiers, and neuroactive agents. Examples of suitable payloads of therapeutic agents include those described in, "The Pharmacological Basis of Therapeutics," Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism;

Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases;

Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Suitable payloads further include toxins, and biological and chemical warfare agents, for example see Somani, S. M. (ed.), Chemical Warfare Agents, Academic Press, New York (1992)).

[0060] In some embodiments, the payload is a therapeutic agent, such as a small molecule drug or a large molecule biologic. Large molecule biologies include, but are not limited to, a protein, polypeptide, or peptide, including, but not limited to, a structural protein, an enzyme, a cytokine (such as an interferon and/or an interleukin), a polyclonal or monoclonal antibody, or an effective part thereof, such as an Fv fragment, which antibody or part thereof, may be natural, synthetic or humanized, a peptide hormone, a receptor, or a signaling molecule.

[0061] Large molecule biologies are immunoglobulins, antibodies, Fv fragments, etc., that are capable of binding to antigens in an intracellular environment. These types of molecules are known as "intrabodies" or "intracellular antibodies." An "intracellular antibody" or an "intrabody" includes an antibody that is capable of binding to its target or cognate antigen within the environment of a cell, or in an environment that mimics an environment within the cell. Selection methods for directly identifying such "intrabodies" include the use of an in vivo two-hybrid system for selecting antibodies with the ability to bind to antigens inside mammalian cells. Such methods are described in PCT/GBOO/00876, incorporated herein by reference. Techniques for producing intracellular antibodies, such as anti-P-galactosidase scFvs, have also been described in Martineau et al., J Mol Biol 280: 117- 127 (1998) and Visintin et al, Proc. Natl. Acad. Sci. USA 96: 11723-1728 (1999).

[0062] Large molecule biologies include but is not limited to, at least one of a protein, a polypeptide, a peptide, a nucleic acid, a virus, a virus-like particle, an amino acid, an amino acid analogue, a modified amino acid, a modified amino acid analogue, a steroid, a proteoglycan, a lipid and a carbohydrate or a combination thereof (e.g., chromosomal material comprising both protein and DNA components or a pair or set of effectors, wherein one or more convert another to active form, for example catalytically).

[0063] A large molecule biologic may include a nucleic acid, including, but not limited to, an oligonucleotide or modified oligonucleotide, an antisense oligonucleotide or modified antisense oligonucleotide, an aptamer, a cDNA, genomic DNA, an artificial or natural chromosome (e.g., a yeast artificial chromosome) or a part thereof, RNA, including an siRNA, a shRNA, mRNA, tRNA, rRNA or a ribozyme, or a peptide nucleic acid (PNA); a virus or virus-like particles; a nucleotide or ribonucleotide or synthetic analogue thereof, which may be modified or unmodified.

[0064] The large molecule biologic can also be an amino acid or analogue thereof, which may be modified or unmodified or a non-peptide (e.g., steroid) hormone; a proteoglycan; a lipid; or a carbohydrate. If the large molecule biologic is a polypeptide, it can be loaded directly into a producer cell according to the methods described herein. Alternatively, an exogenous nucleic acid encoding a polypeptide, which sequence is operatively linked to transcriptional and translational regulatory elements active in a producer cell at a target site, may be loaded.

[0065] Small molecules, including inorganic and organic chemicals, may also be used as payloads of the nanomembrane delivery complexes described herein.

[0066] In some embodiments, the small molecule is a pharmaceutically active agent. Useful classes of pharmaceutically active agents include, but are not limited to, antibiotics, anti-inflammatory drugs, angiogenic or vasoactive agents, growth factors and

chemotherapeutic (anti-neoplastic) agents (e.g., tumour suppressers).

[0067] If a prodrug is loaded into the nanomembrane delivery complex in an inactive form it is often useful that the nanomembrane delivery complex further comprises an activating polypeptide which converts the inactive prodrug to active drug form. In an embodiment, activating polypeptides include, but are not limited to, viral thymidine kinase (encoded by Genbank Accession No. J02224), carboxypeptidase A (encoded by Genbank Accession No. M27717), a-galactosidase (encoded by Genbank Accession No. M13571), β- glucuronidase (encoded by Genbank Accession No. M15182), alkaline phosphatase (encoded by Genbank Accession No. J03252 J03512), or cytochrome P-450 (encoded by Genbank Accession No. D00003 N00003), plasmin, carboxypeptidase G2, cytosine deaminase, glucose oxidase, xanthine oxidase, β-glucosidase, azoreductase, t-gutamyl transferase, β-lactamase, and penicillin amidase.

[0068] Either the activating polypeptide or the exogenous gene encoding it may be transduced into a producer cell to generate a nanomembrane delivery complex. Both the prodrug and the activating polypeptide may be encoded by genes on the same exogenous nucleic acid. Furthermore, either the prodrug or the activating polypeptide of the prodrug may be transgenically expressed in a producer cell.

[0069] In one embodiment, the prodrug and/or the activating polypeptide of the prodrug are expressed in a target cell.

Imaging Agents

[0070] The nanomembrane delivery complexes may also be labeled with one or more positive markers that can be used to monitor over time the number or concentration of nanomembrane delivery complexes in vivo. Suitable fluorescent compounds include those that are approved by the Food & Drug Administration for human use including but not limited to fluorescein, indocyanin green, and rhodamine B. For example, producer cells or nanomembrane delivery complexes may be non-specifically labeled with fluorescein isothiocyanate (FITC; Bratosin et al., Cytometry 46:351-356 (2001)). For example, a solution of FITC-labeled lectins in phosphate buffered saline (PBS) with 0.2 mM

phenylmethysulfonyl fluoride (PMSF) is added to an equal volume of producer cells or isolated nanomembrane delivery complexes in the same buffer. The cells or complexes are incubated with the FITC-labeled lectins for 1 h at 4° C. in the dark. The lectins bind to sialic acids and beta-galactosyl residues on the surface of the producer cells or nanomembrane delivery complexes.

[0071] Other dyes may be useful for tracking nanomembrane delivery complexes in vivo. A number of reagents may be used to non-specifically label a nanomembrane delivery complex. For example, producer cells or nanomembrane delivery complexes may be labeled with PKH26 Red (See, e.g., Bratosin, et al., (1997) Cytometry 30:269-274). Producer cells (e.g. l-3x l0⁷ cells) are suspended in 1 ml of diluent and rapidly added to 1 ml or 2 μΜ PKH26 dissolved in the same diluent. The mixture is mixed by gentle pipetting and incubated at 25° C. for 2-5 min with constant stirring. The labeling may be stopped by adding an equal volume of human serum or compatible protein solution (e.g., 1% bovine serum albumin). After an additional minute, an equal volume of cell culture medium is added and the cells are isolated by centrifugation at 2000 ^xg for 5 min. Cells or complexes are washed three times by repeated suspension in cell culture medium and centrifugation. PHK26-labeled

nanomembrane delivery complexes may be monitored with a maximum excitation wavelength of 551 nm and a maximum emission wavelength of 567 nm.

[0072] Nanomembrane delivery complexes may be tracked in vivo using VivoTag 680 (VT680; VisEn Medical, Woburn, Mass., USA), a near-infrared fluorochrome with a peak excitation wavelength of 670±5 nm and a peak emission wavelength of 688±5 nm. VT680 also contains an amine reactive NHS ester which enables it to cross-link with proteins and peptides. The surface of producer cells or of nanomembrane delivery complexes may be labeled with VT680 (See, e.g., Swirski, et al, (2007) PloS ONE 10:el075). For example, 4x l0⁶ cells/ml are incubated with VT680 diluted in complete culture medium at a final concentration of 0.3 to 300 μg/ml for 30 min at 37° C. The cells are washed twice with complete culture medium after labeling. Cells or complexes may be non-specifically labeled based on proteins expressed on the surface of the producer cell or the nanomembrane delivery complex. Alternatively, a specific surface polypeptide (e.g. a receiver polypeptide) may be labeled with VT680. In some embodiments, a protein or peptide may be directly labeled with VT680 ex vivo and subsequently either attached to the surface of the cell or incorporated into the interior of the cell or complex using methods described herein. In vivo monitoring may, for example, be performed using the dorsal skin fold. Laser scanning microscopy may be performed using, for example, an Olympus IV 100 in which VT680 is excited with a red laser diode of 637 nm and detected with a 660/LP filter. Alternatively, multiphoton microscopy may be performed using, for example, a BioRad Radiance 2100 MP centered around an Olympus BX51 equipped with a 20x/0.95 NA objective lens and a pulsed Ti:Sapphire laser tuned to 820 nm. The latter wavelength is chosen because VT680 has a peak in its two- photon cross-section at 820 nm.

[0073] Alternatively or in addition, a nanomembrane delivery complex may be labeled with other red and/or near-infrared dyes including, for example, cyanine dyes such as Cy5, Cy5.5, and Cy7 (Amersham Biosciences, Piscataway, N.J., USA) and/or a variety of Alexa Fluor dyes including Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700 and Alexa Fluor 750 (Molecular Probes-Invitrogen, Carlsbad, Calif, USA). Additional suitable fluorophores include IRD41 and IRD700 (LI- COR, Lincoln, Nebr., USA), NIR-1 and lC5-OSu (Dejindo, Kumamotot, Japan), LaJolla Blue (Diatron, Miami, Fla., USA), FAR-Blue, FAR-Green One, and FAR-Green Two (Innosense, Giacosa, Italy), ADS 790-NS and ADS 821-NS (American Dye Source,

Montreal, Calif). Quantum dots (Qdots) of various emission/excitation properties may also be used for labeling nanomembrane delivery complexes (See, e.g., Jaiswal et al, Nature Biotech. 21 :47-51 (2003)). Many of these fluorophores are available from commercial sources either attached to primary or secondary antibodies or as amine-reactive succinimidyl or monosuccinimidyl esters, for example, ready for conjugation to a protein or proteins either on the surface or inside the nanomembrane delivery complex.

[0074] Magnetic nanoparticles may be used to track nanomembrane delivery complexes in vivo using high resolution MRI (Montet-Abou et al, Molecular Imaging 4: 165-171 (2005)). Magnetic particles may be internalized by several mechanisms. Magnetic particles may be taken up by a producer cell or by a nanomembrane delivery complex through fluid- phase pinocytosis or phagocytosis. Alternatively, the magnetic particles may be modified to contain a surface agent such as, for example, a membrane translocating HIV TAT peptide which promotes internalization. In some instances, a magnetic nanoparticle such as, for example, Feridex IV®, an FDA approved magnetic resonance contrast reagent, may be internalized into a producer cell or nanomembrane delivery complex in conjunction with a transfection agent such as, for example, protamine sulfate (PRO), polylysine (PLL), and lipofectamine (LFA).

Surface Molecules or Markers

[0075] In some embodiments, the nanomembrane delivery complex comprises polypeptides on its surface selected from CD47, CD55, CD40, CD63, CD9, CD133 and CD59. In some embodiments, the nanomembrane delivery complex is modified to contain the one or more polypeptides. In some embodiments, the producer cell is modified to contain the one or more polypeptides. In some embodiments, the producer cell naturally contains the one or more polypeptides and nanomembrane delivery complexes derived therefrom also contain the polypeptides. The surface polypeptides may confer different functionalities to the nanomembrane delivery complex, e.g. specific targeting capabilities, delivery functions (e.g. fusion molecules), enzymatic functions, increased or decreased half-life in vivo, etc.

[0076] In some embodiments, the surface polypeptide may, e.g., stabilize the

nanomembrane delivery complex, target the nanomembrane delivery complex to particular cells and tissues, engage the reticulo-endothelial system, protect the nanomembrane delivery complex from macrophages and other phagocytic cells, and/or evade other components of the innate immune system. Suitable polypeptides include, e.g., complement regulatory polypeptides, inhibitors of cell-mediated degradation (e.g., CD47, CD55, CD40, CD63, CD9, CD 133 and CD59), and anti-inflammatory polypeptides. Alternatively or in addition, such polypeptides may shorten or control the half-life of the complex, including targeting to macrophages or other phagocytic cells. Suitable polypeptides may promote apoptosis or otherwise trigger opsonization.

[0077] For example, CD40 is a costimulatory protein found on antigen presenting cells and is required for their activation; CD63 is a cell surface glycoprotein that forms a complex with integrins; CD 133 is thought to act as an organizer of cell membrane topology; and CD9 is a member of the transmembrane 4 superfamily, also known as the tetraspanin family that mediates signal transduction events.

[0078] As many drugs are systemically delivered to the blood circulatory system, the answer to the problem of effective drug delivery often focuses on maintaining the drug in the blood for extended periods of time. Thus, the development of long-circulating (long half-life) therapeutics that remain biologically available in the blood for extended time periods is an unmet need. The nanomembrane delivery complexes described herein can be modified to increase or decrease their half-life in circulation. In some embodiments, the half-life of the payload in circulation may be modified by altering the half-life of the nanomembrane delivery complex. In some instances, the half-life is increased and the increase may be, for instance from about 1.5-fold to 20-fold for a therapeutic agent payload maintained in the nanomembrane delivery complex when compared to a therapeutic agent not contained in the nanomembrane delivery complex and the half-life being measured in a serum-containing solution.

[0079] Residency of the nanomembrane delivery complex and/or the payload in the circulatory system, in certain embodiments, is determined by the presence or absence of certain polypeptides on the nanomembrane delivery complex. For example, the

nanomembrane delivery complex may comprise a CD47, CD55, or CD59 polypeptide or a functional fragment thereof.

[0080] CD47 is a membrane protein that interacts with the myeloid inhibitory

immunoreceptor SIRPa (also termed CD172a or SHPS-1) that is present, e.g., on

macrophages. Engagement of SIRPa by CD47 provides a down-regulatory signal that inhibits host cell phagocytosis. For example, high levels of CD47 allow cancer cells to avoid phagocytosis despite the presence pro-phagocytic signals, such as high levels of calreticulin. CD47 also has further roles in cell adhesion, e.g., by acting as an adhesion receptor for THBS1 on producer cells and in the modulation of integrins. CD47 interaction with SIRPa further prevents maturation of immature dendritic cells, inhibits cytokine production by mature dendritic cells. CD47 interaction with SIRPy mediates cell-cell adhesion, enhances superantigen-dependent T-cell-mediated proliferation and co-stimulates T-cell activation.

[0081] CD47 is a 50 kDa membrane receptor that has extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. There are four alternatively spliced isoforms of CD47 that differ only in the length of their cytoplasmic tail. In some embodiments, the nanomembrane delivery complex may comprise a CD47 or a functional fragment thereof comprising one or more of: the extracellular N-terminal IgV domain, one, two, three, four, or five transmembrane domains, and/or the short C-terminal intracellular tail. The cytoplasmic tail can be found as four different splice isoforms ranging from 4 to 36 amino acids. The 16 amino acid form 2 is expressed in all cells of hematopoietic origin and in endothelial and epithelial cells. The 36 amino acid form 4 is expressed primarily in neurons, intestine, and testis. The 4 amino acid form 1 is found in epithelial and

endothelial cells. The expression pattern of the 23 amino acid form 3 resembles that of form 4. In some embodiments, the nanomembrane delivery complex comprises CD47 or a functional fragment thereof that is of one of form 1, from 2, form 3, or from 4. In some embodiments, the nanomembrane delivery complex does not comprise form 2. In some embodiments, the nanomembrane delivery complex comprises a modified CD47, such as a conformational change. For example, a conformational change in CD47 is introduced so that the modified CD47 is capable of interacting with TSP-1. In one embodiment, the modified CD47 comprising the conformational change creates a different binding site for SIRPa. In some embodiments, the nanomembrane delivery complex comprises a modified CD47 polypeptide or a functional polypeptide fragment thereof comprising a conformational change. In certain embodiments, the nanomembrane delivery complex comprises a fusion of a CD47 isoform to the extracellular domain of a native producer cell polypeptide. For example, the N- or C- terminus of a native polypeptide of a producer cell may be fused to the CD47 polypeptide or functional fragment thereof, which may lead to a reduction of the SIRPa-mediated signal to macrophages to phagocytose the nanomembrane delivery complex.

[0082] In some embodiments, the producer cells naturally express CD47. In some embodiments, the natural levels of CD47 are altered in the producer cell, e.g., by over- expression or inhibition of CD47 expression using any suitable method, such as the introduction of exogenous nucleic acids (e.g., expression vectors, CD47 mRNA, CD47 siRNA, and the like).

[0083] For example, nanomembrane delivery complexes that are administered to a subject may comprise elevated CD47 levels when compared to native levels of a suitable control. Elevated CD47 levels may be achieved, e.g., by exogenous expression by the producer cell line of CD47 from an exogenous nucleic acid, by loading of CD47 mRNA into the producer cell or directly into the nanomembrane delivery complex, or by conjugating CD47 polypeptide to the surface of the producer cell or directly to the surface of the nanomembrane delivery complex. Elevated CD47 levels are useful to increase the half-life of the population of nanomembrane delivery complexes in the circulatory system of the subject. The nanomembrane delivery complexes comprise a payload (such as a therapeutic agent) and optionally a receiver and increasing the half-life of the nanomembrane delivery complex may increase the half-life of the payload in circulation. This potentially increases the therapeutic window in which payload is active. In one instance, a population of 10¹¹ nanomembrane delivery complexes comprises an adenosine deaminase payload and an exogenous CD47 polypeptide on its surface. When administered to a subject with an enzyme deficiency, such as ADA-SCID, the half-life of the nanomembrane delivery complex is extended beyond that of a complex not comprising exogenous CD47 polypeptide and the subject requires less frequent dosing. Half-life extension is a particular advantage when compared to current enzyme therapies not involving nanomembrane delivery complexes.

[0084] In some embodiments, CD47 is altered by heparin and/or chondroitin sulfate glycosaminoglycan (GAG) chains. In some embodiments, the nanomembrane delivery complex comprises CD47 as a proteoglycan. In some embodiments, the nanomembrane delivery complex comprises a CD47 proteoglycan that is conjugated to the complex. In one embodiment, the CD47 proteoglycan comprises heparin and/or chondroitin sulfate glycosaminoglycan (GAG) chains. In one embodiment, that CD47 proteoglycan has a size of greater than 150 kDa, 200 kDa, or greater than 250 kDa. In one embodiment, CD47 comprises one or more GAG chains at Ser64.

[0085] In some embodiments, the residency of a nanomembrane delivery complex generated using producer cells can be further modulated by changing the amount or number of oxidized lipids on the membrane of the nanomembrane delivery complex. In one embodiment, the nanomembrane delivery complex comprises oxidized lipids in an amount effective to shorten its half-life. In some embodiments, the amount of oxidized lipids in the membrane are altered such that mobility of CD47 is increased or decreased, thereby aiding or hindering, respectively the ability of CD47 to cluster on the membrane. (See, Olsson, Department of Integrative Medical Biology, Section for Histology and Cell Biology, Umea University, Umea, Sweden, 2008).

[0086] CD55, also known as complement decay-accelerating factor or DAF, is a 70 kDa membrane protein. CD55 recognizes C4b and C3b fragments of the complement system that are created during C4 (classical complement pathway and lectin pathway) and C3 (alternate complement pathway) activation. It is thought that interaction of CD55with cell-associated C4b and C3b proteins interferes with their ability to catalyze the conversion of C2 and factor B to active C2a and Bb and thereby prevents the formation of C4b2a and C3bBb, the amplification convertases of the complement cascade. CD55 is thought to block the formation of membrane attack complexes. CD55 may prevent lysis by the complement cascade. In some embodiments, the nanomembrane delivery complex comprises CD55 polypeptide or a functional polypeptide fragment thereof. In some embodiments, the nanomembrane delivery complex comprises an exogenous CD55 polypeptide and an exogenous CD47 polypeptide or functional polypeptide fragments thereof.

[0087] CD59 glycoprotein also known as MAC-inhibitory protein (MAC-IP), membrane inhibitor of reactive lysis (MIRL), protectin, or HRF is a protein that attaches to host cells via a glycophosphatidylinositol (GPI) anchor. When complement activation leads to deposition of C5b678 on host cells, CD59 can prevent C9 from polymerizing and forming the complement membrane attack complex. CD59 may prevent lysis by the complement cascade. In some embodiments, the nanomembrane delivery complex comprises CD59 polypeptide or a functional polypeptide fragment thereof. In some embodiments, the nanomembrane delivery complex comprises an exogenous CD59 polypeptide and an exogenous CD47 polypeptide or functional polypeptide fragments thereof.

[0088] In some embodiments, the nanomembrane delivery complex comprises one or more of an exogenous CD55 polypeptide, an exogenous CD59 polypeptide and/or an exogenous CD47 polypeptide or functional polypeptide fragments thereof in a desired amount, copy number and/or ratio sufficient to regulate the residency of the nanomembrane delivery complex in circulation.

[0089] Effective amounts of CD47, CD55, and CD59 include 10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁹ polypeptides per nanomembrane delivery complex. Alternatively, an effective amount is the amount capable of extending the nanomembrane delivery complex's half-life by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 400%, 800%, 1,000%, or 10,000% relative to the half- life that the nanomembrane delivery complex would exhibit without the polypeptides.

Receivers

[0090] Optionally, the nanomembrane delivery complex comprises a receiver. A receiver polypeptide comprises or consists essentially of a polypeptide. In some embodiments, a receiver comprises or consists essentially of a carbohydrate, a nucleic acid, a lipid, a small molecule, or a combination thereof. The receiver is synthetic. For example, the receiver is an exogenous polypeptide or molecule, or expressed from an exogenous nucleic acid.

[0091] In some embodiments the receiver functions to "target", e.g., aggregate around, concentrate itself in, home to, undergo a transformation near, or otherwise engage a target molecule, cell or tissue of interest. In some embodiments, a receiver is capable of interacting with a target, e.g., to associate with, bind to, or fuse with a target, such as a target cell in sufficient proximity and for a sufficient duration for the nanomembrane delivery complex to bring about delivery of the payload to the target.

[0092] In other embodiments, the interaction of the receiver with a target comprises altering an activity of the target. In other embodiments, the interaction of the receiver with a target comprises altering the composition of the target. In other embodiments, the interaction of the complex with a target comprises reducing an activity of the target. In other

embodiments, the interaction of the complex with a target comprises inactivating the target.

[0093] In other embodiments, the interaction of the receiver with a target comprises altering the R A composition of the target. In other embodiments, the interaction of the complex with a target comprises inducing translation in the target of a payload RNA.

[0094] In some embodiments, receivers comprise polypeptides. Receiver polypeptides may range in size from 6 amino acids to 3000 amino acids and may exceed 6, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or may exceed 500 amino acids.

Receiver polypeptides may range in size from about 20 amino acids to about 500 amino acids, from about 30 amino acids to about 500 amino acids or from about 40 amino acids to about 500 amino acids.

[0095] In some embodiments, the receiver polypeptide comprises a chimeric or fusion protein which may comprise two or more distinct protein domains. These chimeric receivers are heterologous or exogenous in the sense that the various domains are derived from different sources, and as such, are not found together in nature and can be encoded e.g., by exogenous nucleic acids. Receiver polypeptides can be produced by a number of methods, many of which are well known in the art and also described herein. For example, receiver polypeptides can be obtained by extraction (e.g., from isolated cells), by expression of an exogenous nucleic acid encoding the receiver polypeptide, or by chemical synthesis. Receiver polypeptides can be produced by, for example, recombinant technology, and expression vectors encoding the polypeptide introduced into host cells (e.g., by transformation or transfection) for expression of the encoded receiver polypeptide.

[0096] There are a variety of conservative changes that can generally be made to an amino acid sequence without altering activity. These changes are termed conservative substitutions or mutations; that is, an amino acid belonging to a grouping of amino acids having a particular size, charge or other characteristic can be substituted for another amino acid. Substitutions for an amino acid sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, methionine, and tyrosine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations are not expected to substantially affect apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point. Conservative substitutions also include substituting optical isomers of the sequences for other optical isomers, specifically D amino acids for L amino acids for one or more residues of a sequence. Moreover, all of the amino acids in a sequence may undergo a D to L isomer substitution. Exemplary conservative substitutions include, but are not limited to, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free ~ OH is maintained; and Gin for Asn to maintain a free NH₂. Moreover, point mutations, deletions, and insertions of the polypeptide sequences or corresponding nucleic acid sequences may in some cases be made without a loss of function of the polypeptide or nucleic acid fragment. Substitutions may include, e.g., 1, 2, 3, or more residues. Any teaching of a specific amino acid sequence or an exogenous nucleic acid encoding the polypeptide or teaching of the name of the name thereof includes any conservative substitution point mutations, deletions, and insertions of those polypeptide sequences or corresponding nucleic acid sequences and any sequence depositied for the protein or gene in a database that can be made without a loss of function of the polypeptide or nucleic acid fragment.

[0097] Any of the methods described herein may be used to generate any of the polypeptides described herein (e.g. therapeutic polypeptides and surface or maker

polypeptides) and application of these methods is not restricted to receiver polypeptides.

[0098] In some embodiments, the receiver polypeptide is associated with the membrane of the nanomembrane delivery complex. In other embodiments, the receiver polypeptide is not associated with the membrane of the nanomembrane delivery complex.

[0099] In one embodiment, the receiver comprises a polypeptide that comprises an amino acid sequence derived from an antibody. The antibody receiver may be expressed as a full-length protein or a fragment thereof. In one embodiment, the receiver comprises an antibody amino acid sequence that is specific for a desired target. In some embodiments, the antibody is a scFv. In other embodiments, the antibody is a nanobody. [00100] In one embodiment, the receiver comprises a polypeptide that comprises an amino acid sequence derived from a scFv antibody. The scFv antibody receiver may be expressed as a full-length protein or a fragment thereof. The scFv antibody may be expressed as a fusion protein. Suitable scFv receiver polypeptides include, but are not limited to, those listed in Table 2.

[00101] The production of scFvs is known in the art. The scFv receiver may be made specific to any target molecule including, but not limited to, those in Table 1.

[00102] In certain embodiments, the receiver comprises a camelid-derived nanobody. Nanobodies are usually 12-15 kDa. They are considerably smaller than antibodies and scFv. Nanobodies may thus be easier to transfect, and the nanobody receiver will be more easily expressed, translated and or transported to the cell surface in a producer cell and ultimately the nanomembrane delivery complex derived therefrom. In certain embodiments, nanobody receivers are employed to minimize immunogenic effects caused by a specific receiver. Nanobodies because of their small size will offer reduced immunogenic potential. In certain embodiments, receiver nanobodies are employed because they have an increased ability to recognize hidden or uncommon epitopes compared to standard antibodies. For example, they can bind to small enzymatic cavities of a target and modulate the molecular behavior of the target.

[00103] In some embodiments, receivers comprise a protein-binding partner or a receptor on the surface of the nanomembrane delivery complex, which functions to target the nanomembrane delivery complex to a specific tissue space or to interact with a specific moiety on a target cell, either in vivo or in vitro. Suitable protein-binding partners include antibodies and functional fragments thereof, scaffold proteins, or peptides.

[00104] In some embodiments, the receiver is a molecule that promotes endocytosis in the target cell, e.g. by engaging receptors that stimulate receptor-mediated endocytosis. Suitable receivers for this purpose include, but are not limited to, transferrin, insulin, growth factors, epidermal growth factor, ligands for receptor tyrosine kinases, mannose, somatostatin, hormones, and ligands of scavenger receptors.

[00105] In some embodiments, the receiver can be a molecule that promotes

nanomembrane delivery complex fusion to the target cell, e.g. the target cell plasma membrane, the endosomal membrane, or the lysosomal membrane, thus transferring the payload to the cytoplasm of the target cell. In some embodiments, the receiver is a coat protein, e.g. clathrin, coat protein complex (COP)l, COP2; or a soluble N-ethylmaleimide- sensitive factor attachment protein receptor (SNARE), e.g. synaptobrevin, syntaxin, Tlglp, SNAP -25, Vam3p, Vam7p; or a membrane fusion protein, e.g. a bacterial membrane fusion protein, a dynamin, DynA of Bacillus subtilis, HlyD; or a cell-penetrating polypeptide, e.g. a microbial pore forming protein, a poly-arginine polypeptide, an anti-microbial peptide, a microbial exotoxin, or a microbial endotoxin.

[00106] In other embodiments, the receiver that promotes membrane fusion is an adhesion molecule (e.g., ICAM1), integrins (e.g., betal and beta2 integrins), tetraspanins (e.g.

transferrin), phosphatidylserine, or MFGE.

[00107] In some embodiments, the receiver mediates tissue targeting of the

nanomembrane delivery complex. In some embodiments, the receiver mediates

extravasation, intravasation, or tissue penetration of the nanomembrane delivery complex. In certain embodiments, the receiver that mediates tissue targeting is a small peptide. In other embodiments, the receiver mediates tissue or cell penetration of the nanomembrane delivery complex.

[00108] In some embodiments, the receiver is a targeting molecule. In certain

embodiments the targeting molecule may be an aptamer, an scFV, an antibody, a nanobody, a homing peptide, a folic acid, a cyclodextrin, a transferrin, a luteinizing hormone-releasing hormone, or a glycoprotein.

[00109] In some embodiments, the receiver mediates nanomembrane delivery complex chemotaxis. In this aspect, the nanomembrane delivery complex is able to migrate to target tissue in response to cytokine or chemokine gradients.

[00110] In some embodiments, the receiver mediates angiogenesis. In some

embodiments, angiogenesis mediated by the receiver enables improved tissue distribution or PK of the nanomembrane delivery complex.

Targets

[00111] A suitable receiver may be chosen to interact with a specific target. Suitable targets include entities that are associated with a specific disease, disorder, or condition. However, targets may also be chosen independent of a specific disease, disorder, or condition. [00112] In some embodiments, the nanomembrane delivery complex does not comprise a receiver and the nanomembrane delivery complex is capable of interacting with a target in the absence of a receiver.

[00113] In some embodiments, the target is a bacterium, for example Enterococcus, Streptococcus, or Mycobacteria, Rickettsia, Mycoplasma, Neisseria meningitides, Neisseria gonorrheoeae, Legionella, Vibrio cholerae, Streptococci, Staphylococcus aureus,

Staphylococcus epidermidis, Pseudomonas aeruginosa, Corynobacteria diphtheriae,

Clostridium spp., enterotoxigenic Eschericia coli, and Bacillus anthracis. Other pathogens for which bacteremia has been reported at some level include the following: Rickettsia,

Bartonella henselae, Bartonella quintana, Coxiella burnetii, chlamydia, Mycobacterium leprae, Salmonella; shigella; Yersinia enterocolitica; Yersinia pseudotuberculosis; Legionella pneumophila; Mycobacterium tuberculosis; Listeria monocytogenes; Mycoplasma spp.; Pseudomonas fluorescens; Vibrio cholerae; Haemophilus influenzae; Bacillus anthracis; Treponema pallidum; Leptospira; Borrelia; Corynebacterium diphtheriae; Francisella;

Brucella melitensis; Campylobacter jejuni; Enterobacter; Proteus mirabilis; Proteus; and Klebsiella pneumoniae.

[00114] In some embodiments, the target is a virus, including but limited to, those whose infection involves injection of genetic materials into host cells upon binding to cell surface receptors, viruses whose infection is mediated by cell surface receptors. Non-limiting examples of these viruses can be selected from Paramyxoviridae (e.g., pneumovirus, morbillivirus, metapneumovirus, respirovirus or rubulavirus), Adenoviridae (e.g.,

adenovirus), Arenaviridae (e.g., arenavirus such as lymphocytic choriomeningitis virus), Arteriviridae (e.g., porcine respiratory and reproductive syndrome virus or equine arteritis virus), Bunyaviridae (e.g., phlebovirus or hantavirus), Caliciviridae (e.g., Norwalk virus), Coronaviridae (e.g., coronavirus or torovirus), Filoviridae (e.g., Ebola-like viruses),

Flaviviridae (e.g., hepacivirus or flavivirus), Herpesviridae (e.g., simplexvirus, varicellovirus, cytomegalovirus, roseolovirus, or lymphocryptovirus), Orthomyxoviridae (e.g., influenza virus or thogotovirus), Parvoviridae (e.g., parvovirus), Picomaviridae (e.g., enterovirus or hepatovirus), Poxviridae (e.g., orthopoxvirus, avipoxvirus, or leporipoxvirus), Retroviridae (e.g., lentivirus or spumavirus), Reoviridae (e.g., rotavirus), Rhabdoviridae (e.g., lyssavirus, novirhabdovirus, or vesiculovirus), and Togaviridae (e.g., alphavirus or rubivirus). Specific examples of these viruses include human respiratory coronavirus, influenza viruses A-C, hepatitis viruses A to G, and herpes simplex viruses 1-9. [00115] In some embodiments, the target is a parasite, including but not limited to, for example, intestinal or blood-borne parasites, protozoa, trypanosomes; haemoprotozoa and parasites capable of causing malaria; enteric and systemic cestodes including taeniid cestodes; enteric coccidians; enteric flagellate protozoa; filarial nematodes; gastrointestinal and systemic nematodes and hookworms.

[00116] In some embodiments, the target is a fungus, including but not limited to, for example, Candida albicans, Candida glabrata, Aspergillus, T. glabrata, Candida tropicalis, C. krusei, and C. parapsilosis.

[00117] In some embodiments, the target is a lipid, lipid complex, or proteolipid complex.

[00118] In some embodiments, the target is a LFA (e.g., lymphocyte function-associated antigen 1), intercellular adhesion molecules (e.g., ICAMl), extracellular matrix proteins (e.g., fibronectin), phosphatidylserine receptors (e.g., T cell immunoglobulin domain, mucin domain proteins, TIM1/TIM4), lactaherin, or integrins (e.g., avb3 or avb5).

[00119] In some embodiments, the target is an inflammatory molecule, a cytokine or a chemokine.

[00120] In some embodiments, the target is a carbohydrate, polysaccharide, or amino acid.

[00121] In some embodiments, the target is a virus, a viral antigen, an envelope antigen or a capsid antigen.

[00122] In some embodiments, the target is a bacterium, a bacterial antigen, a bacterial surface antigen, a secreted bacterial toxin, or a secreted bacterial antigen.

[00123] In some embodiments, the target is a fungus, a fungal antigen, a fungal cell surface antigen, a secreted fungal toxin, or a secreted fungal antigen.

[00124] In some embodiments, the target is DNA or R A.

[00125] In some embodiments, the target is a circulating cell, an inflammatory cell, a tumor cell, or a metastatic cancer cell.

[00126] In some embodiments, the target is a mammalian cell, including but not limited to, for example, a human cell, a circulating cell, an immune cell, a neutrophil, an eosinophil, a basophil, a lymphocyte, a monocyte, a B cell, a T cell, a CD4+ T cell, a CD8+ T cell, a gamma-delta T cell, a regulatory T cell, a natural killer cell, a natural killer T cell, a macrophage, a Kupffer cell, a dendritic cell, a cancer cell, a cancer stem cell, a circulating tumor cell, a cancer cell from one of the following cancers including, but not limited to, ACUTE lymphoblastic leukaemia (ALL), ACUTE myeloid leukaemia (AML), anal cancer, bile duct cancer, bladder cancer, bone cancer, bowel cancer, brain tumours, breast cancer, cancer of unknown primary, cancer spread to bone, cancer spread to brain, cancer spread to liver, cancer spread to lung, carcinoid, cervical cancer, choriocarcinoma, chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML), colon cancer, colorectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational trophoblastic tumours (GTT), hairy cell leukaemia, head and neck cancer, hodgkin lymphoma, kidney cancer, laryngeal cancer, leukaemia, liver cancer, lung cancer, lymphoma, melanoma skin cancer, mesothelioma, men's cancer, molar pregnancy, mouth and oropharyngeal cancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer, non Hodgkin lymphoma (NHL), oesophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, rare cancers, rectal cancer, salivary gland cancer, secondary cancers, skin cancer (non melanoma), soft tissue sarcoma, stomach cancer, testicular cancer, thyroid cancer, unknown primary cancer, uterine cancer, vaginal cancer, and vulval cancer.

[00127] In some embodiments, the target is a non-circulating cell or tissue. In some embodiments, the target is a specific tissue including, but not limited to, endothelial tissues, connective tissues, muscle tissue, nervous tissue, and epithelial tissue. In some embodiments, the target is a specific organ systems based on an affinity for ligands associated with the tissues therein, including, but not limited to, the brain, liver, kidneys, gastrointestinal system, pancreas, spleen, and lungs.

Pharmaceutical Compositions and Dosage Forms

[00128] Provided herein are pharmaceutical compositions comprising nanomembrane delivery complexes that are suitable for administration to a subject. The pharmaceutical compositions generally comprise a plurality of nanomembrane delivery complexes and a pharmaceutically-acceptable excipient or carrier in a form suitable for administration to a subject. Pharmaceutically-acceptable excipients or carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions comprising a plurality of nanomembrane delivery complexes. (See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18th ed. (1990)). The pharmaceutical compositions are generally formulated sterile and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[00129] In some embodiments, the pharmaceutical composition comprises one or more therapeutic agents and the nanomembrane delivery complex described herein. In some embodiments, the nanomembrane delivery complexes are co-administered with of one or more separate therapeutic agents, wherein co-administration includes administration of the separate therapeutic agent before, after or concurrent with administration of the

nanomembrane delivery complex.

[00130] Pharmaceutically-acceptable excipients include excipients that are generally safe (GRAS), non-toxic, and desirable, including excipients that are acceptable for veterinary use as well as for human pharmaceutical use.

[00131] Examples of carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or compound is incompatible with the nanomembrane delivery complexes described herein, use thereof in the compositions is contemplated.

Supplementary therapeutic agents may also be incorporated into the compositions. Typically, a pharmaceutical composition is formulated to be compatible with its intended route of administration. The nanomembrane delivery complexes can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intradermal, transdermal, rectal, intracranial, intraperitoneal, intranasal; intramuscular route or as inhalants. In one

embodiment, the pharmaceutical composition comprising nanomembrane delivery complexes is administered intravenously, e.g. by injection. The nanomembrane delivery complexes can optionally be administered in combination with other therapeutic agents that are at least partly effective in treating the disease, disorder or condition for which the nanomembrane delivery complexes are intended.

[00132] Solutions or suspensions can include the following components: a sterile diluent such as water, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediammetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and compounds for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00133] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders. For intravenous

administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The composition is generally sterile and fluid to the extent that easy syringeability exists. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal compounds, e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. If desired, isotonic compounds, e.g., sugars, polyalcohols such as manitol, sorbitol, sodium chloride can be added to the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition a compound which delays absorption, e.g., aluminum monostearate and gelatin.

[00134] Sterile injectable solutions can be prepared by incorporating the nanomembrane delivery complexes in an effective amount and in an appropriate solvent with one or a combination of ingredients enumerated herein, as desired. Generally, dispersions are prepared by incorporating the nanomembrane delivery complexes into a sterile vehicle that contains a basic dispersion medium and any desired other ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The nanomembrane delivery complexes can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner to permit a sustained or pulsatile release of the nanomembrane delivery complexes.

[00135] Systemic administration of compositions comprising nanomembrane delivery complexes can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, e.g., for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the modified nanomembrane delivery complexes are formulated into ointments, salves, gels, or creams as generally known in the art.

[00136] The nanomembrane delivery complexes can also be prepared as pharmaceutical compositions in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[00137] In one embodiment the pharmaceutical composition comprising nanomembrane delivery complexes is administered intravenously into a subject that would benefit from the pharmaceutical composition. In other embodiments, the composition is administered to the lymphatic system, e.g., by intralymphatic injection or by intranodal injection (see e.g., Senti et al, 2008 PNAS 105(46): 17908), or by intramuscular injection, by subcutaneous administration, by direct injection into the thymus, or into the liver.

[00138] In one embodiment, the pharmaceutical composition comprising nanomembrane delivery complexes is administered as a liquid suspension. In one embodiment the

pharmaceutical composition is administered as a formulation that is capable of forming a depot following administration, and in a preferred embodiment slowly release nanomembrane delivery complexes into circulation, or remain in depot form.

[00139] Typically, pharmaceutically acceptable compositions are highly purified to be free of contaminants, are biocompatible and not toxic, and are suited to administration to a subject. If water is a constituent of the carrier, the water is highly purified and processed to be free of contaminants, e.g., endotoxins.

[00140] The pharmaceutically acceptable carrier may be lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginates, gelatin, calcium silicate, micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, and/or mineral oil, but is not limited thereto. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavor enhancer, an emulsifying agent, a suspension agent, and/or a preservative.

[00141] The pharmaceutical compositions described herein comprise a nanomembrane delivery complex and optionally a pharmaceutically active or therapeutic agent. The therapeutic agent can be a biological agent, a small molecule agent, or a nucleic acid agent. [00142] Dosage forms are provided that comprise a pharmaceutical composition comprising a nanomembrane delivery complex described herein. In some embodiments, the dosage form is formulated as a liquid suspension for intravenous injection.

[00143] Medical devices are provided that comprise a container holding a pharmaceutical composition comprising a nanomembrane delivery complex described herein and an applicator for intravenous injection of the pharmaceutical composition to a subject.

[00144] Medical kits are provided that comprise a pharmaceutical composition

comprising a nanomembrane delivery complex described herein and a medical device for intravenous injection of the pharmaceutical composition to a subject.

[00145] A pharmaceutically acceptable suspension of nanomembrane delivery complexes can be packaged in a volume of approximately 1 ml to approximately 500 ml. The packaging can be a syringe or an IV bag suitable for transfusions. Administration of the suspension is carried out, e.g., by intravenous or intra-arterial injection, optionally using a drip from an IV bag or the like. The administration is typically carried out intravenously in the arm or via a central catheter. For administrations exceeding 50 ml use of a drip is preferred.

[00146] In one embodiment, the preparation of nanomembrane delivery complexes is subjected to radiation, e.g., X rays, gamma rays, beta particles, alpha particles, neutrons, protons, elemental nuclei, UV rays in order to damage residual replication-competent nucleic acids.

[00147] In one embodiment, the preparation of nanomembrane delivery complexes is subjected to gamma irradiation using an irradiation dose of more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, or more than 100 kGy.

[00148] In one embodiment, the preparation of nanomembrane delivery complexes is subjected to X-ray irradiation using an irradiation dose of more than 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, or greater than 10000 mSv.

Methods of Making Nanomembrane Delivery Complexes

[00149] Provided herein are method for producing isolated nanomembrane delivery complexes and pharmaceutical preparations thereof.

[00150] In some embodiments, the methods comprise: a) providing a producer cell capable of generating a nanomembrane delivery complex, b) obtaining from the producer cell the nanomembrane delivery complex, c) modifying the nanomembrane delivery complex with a payload, and d) isolating the modified nanomembrane delivery complex.

[00151] In some embodiments, the methods comprise: a) providing a producer cell capable of generating a nanomembrane delivery complex, b) modifying the producer cell with a payload, c) obtaining from the producer cell the nanomembrane delivery complex, and d) isolating the modified nanomembrane delivery complex.

[00152] Optionally, the isolated nanomembrane delivery complexes may be formulated into a pharmaceutical composition described herein. If desired, the activity of the

pharmaceutical composition is tested or analyzed. Testing can include one or more of: i) analyzing the presence or activity of the payload, ii) analyzing the potency of the

nanomembrane delivery complex in a phenotypic or functional assay iii) detecting the presence or absence of one or more biomarkers from the producer cell, iv) analyzing the size distribution of the nanomembrane delivery complexes, and/or v) analyzing the membrane composition of the nanomembrane delivery complexes.

[00153] If desired, the producer cell can be modified to comprise a receiver. Alternatively or in addition, the nanomembrane delivery complex is modified to comprise a receiver.

[00154] In some embodiments, the nanomembrane delivery complexes are released by the producer cells into a culture medium. The nanomembrane delivery complexes may be generated through a variety of cellular mechanisms including the endosomal sorting complexes required for transport I and II (ESCRT I and II), alternate endosome production pathways derived thereof, or mechanistic perturbation or disruption of the producer cell membrane, such as microfluidic compression or lysis, exposure to chemical stresses such as pH, or apoptosis.

[00155] In some embodiments, the producer cell is a mammalian cell that is isolated or derived from a mammalian cell line. The nanomembrane delivery complexes may be derived from various cell lines, including eukaryotes, prokaryotes, archae, fungi and protists.

[00156] In some embodiments, generating a nanomembrane delivery complex comprises using isolated optionally cultured producer cells that are autologous and/or allogeneic to the subject in which the nanomembrane delivery complex is administered.

[00157] The producer cell may be cultured. Cultured producer cells can be scaled up from bench-top scale to bioreactor scale. For example, the producer cells are cultured until they reach saturation density, e.g., lxlO⁵, lxlO⁶, lxlO⁷, or greater than lxlO⁷ per ml. Optionally, upon reaching saturation density, the producer cells can be transferred to a larger volume of fresh medium. The producer cells may be cultured in a bioreactor, such as, e.g., a Wave-type bioreactor, a stirred-tank bioreactor. Various configurations of bioreactors are known in the art and a suitable configuration may be chosen as desired. Configurations suitable for culturing and/or expanding populations of producer cells can easily be determined by one of skill in the art without undue experimentation. The bioreactor can be oxygenated. The bioreactor may optionally contain one or more impellers, a recycle stream, a media inlet stream, and control components to regulate the influx of media and nutrients or to regulate the outflux of media, nutrients, and waste products.

[00158] In some embodiments, the bioreactor is a Wave bioreactor or a impeller-driven agitator. The bioreactor may be aerated by means of a sparger. In one embodiment, the bioreactor is disposable. In one embodiment, the bioreactor is CIP (cleaned in place). The final number of producer cells that may be obtained in a bioreactor setting as described herein can be greater than 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³ or greater than 10¹³ cells. The density of producer cells may be monitored during culture by measuring cell density by hemacytometer counting or by optical density reading at 600 nm. Optionally, the culture process is monitored for pH levels, oxygenation, agitation rate, and/or recycle rate.

[00159] In some embodiments, the producer cells may be treated with chemicals, hormones, metabolites, nucleic acids, proteins, enzymes, lipids, nutrients, micronutrients, or any other molecule to affect the cell's phenotype or profile of nanomembrane delivery complexes.

[00160] In some embodiments, the producer cells may be treated with a molecule, e.g., a DNA molecule, an RNA molecule, a mRNA, an siRNA, a microRNA, a IncRNA, a shRNA, a hormone, or a small molecule, that activates or knocks down one or more genes.

[00161] Producer cell inputs, including but not limited to, nutrients, micronutrients, metabolites, amino acids, sugars, and fatty acids may be increased or decreased. For example, producer cells may be grown in hypoxic conditions prior to and during isolation of nanomembrane delivery complexes.

[00162] The producer cells may be treated with physical stimuli, including but not limited to, irradiation, pressure, shear stress, mixing, turbulence, and shaking.

[00163] In one embodiment, the producer cell is differentiated from a starter or precursor cell. In this embodiment, the differentiation state of the producer cell is assessed by an in vitro assay. Suitable in vitro assays include measuring the number of cells, protein content or expression level, e.g., of a biomarker (e.g. differentiation marker), mR A content or expression level, e.g., of a biomarker (e.g. a differentiation marker), lipid content, partition of a substrate, catalytic activity, or metabolic activity.

[00164] In some embodiments, the produser cells are cultured and the differentiation state of the cells and/or resulting nanomembrane delivery complexes is assessed at multiple time points over the course of the culture process.

[00165] In certain embodiments, a producer cell expresses (naturally or upon

modification) a polypeptide (e.g. a receiver polypeptide, a therapeutic polypeptide and/or a surface marker polypeptide). In some embodiments, nanomembrane delivery complexes derived from the producer cells comprise the polypeptide that is expressed by the producer cell. The polypeptide may be exhibited on the surface of the nanomembrane delivery complex or may reside within the interior space of the nanomembrane delivery complex.

[00166] In certain embodiments, the polypeptide (e.g. a receiver polypeptide, a therapeutic polypeptide and/or a surface marker polypeptide) is conjugated to the producer cell or the nanomembrane delivery complex. The polypeptide usually is conjugated to the surface of the producer cell or nanomembrane delivery complex. Conjugation may be achieved chemically or enzymatically, by methods known in the art.

[00167] In certain embodiments, the polypeptide (e.g. a receiver polypeptide, a therapeutic polypeptide and/or a surface marker polypeptide) is loaded into the producer cell or nanomembrane delivery complex. In some embodiments, the polypeptide is not loaded into or onto the producer cell or nanomembrane delivery complex.

[00168] In some embodiments, the nanomembrane delivery complex comprises a polypeptide (e.g. a receiver polypeptide, a therapeutic polypeptide and/or a surface marker polypeptide) that is optionally i) expressed in the producer cell from an exogenous nucleic acid, ii) conjugated to the producer cell or the nanomembrane delivery complex, iii) loaded into or onto the producer cell or the nanomembrane delivery complex, and any combination of i), ii) and iii).

[00169] A non-polypeptide payload (e.g. a nucleic acid, such as an R A, e.g. siR A, miR A, shRNA, etc., a therapeutic small molecule or a toxin) may be i) expressed in the producer cell from an exogenous nucleic acid, ii) conjugated to the producer cell or the nanomembrane delivery complex, iii) loaded into or onto the producer cell or the nanomembrane delivery complex, and any combination of i), ii) and iii), as applicable for the respective payload.

[00170] In some embodiments, the nanomembrane delivery complex is generated by contacting a suitable producer cell with an exogenous nucleic acid encoding the payload, receiver of surface marker. In some embodiments, the nucleic acid is a DNA. In other embodiments, the nucleic acid is a RNA.

[00171] A payload, receiver or surface marker may be expressed by a producer cell from a transgene or mRNA introduced into a producer cell by electroporation, chemical or polymeric transfection, viral transduction, mechanical membrane disruption, or other method. The producer cells may be modified, e.g., by transfection of single or multiple copies of genes, transduction with a virus, or electroporation in the presence of DNA or RNA. In some embodiments, the nanomembrane delivery complex derived from the producer cell comprises the payload, receiver or surface marker that is expressed by the producer cell.

[00172] A payload may be expressed by a target cell from a transgene or mRNA introduced into a nanomembrane delivery complex by electroporation, chemical or polymeric transfection, viral transduction, mechanical membrane disruption, or other method when the target cell is contacted with the nanomembrane delivery complex.

[00173] In some instances, the exogenous nucleic acid is an RNA molecule, or a DNA molecule that encodes for an RNA molecule, that silences or represses the expression of a target gene. For example, the molecule can be a small interfering RNA (siRNA), an antisense RNA molecule, or a short hairpin RNA (shRNA) molecule.

[00174] Messenger RNA may be derived from in vitro transcription of a cDNA plasmid construct containing the coding sequence corresponding to the payload, surface marker or receiver polypeptide. For example, the cDNA sequence corresponding to the polypeptide (e.g. a receiver polypeptide, a therapeutic polypeptide and/or a surface marker polypeptide) may be inserted into a cloning vector containing a promoter sequence compatible with specific RNA polymerases. For example, the cloning vector ZAP Express® pBK-CMV (Stratagene, La Jolla, Calif, USA) contains T3 and T7 promoter sequence compatible with T3 and T7 RNA polymerase, respectively. For in vitro transcription of sense mRNA, the plasmid is linearized at a restriction site downstream of the stop codon(s) corresponding to the end of the coding sequence of the receiver polypeptide. The mRNA is transcribed from the linear DNA template using a commercially available kit such as, for example, the R AMaxx® High Yield Transcription Kit (from Stratagene, La Jolla, Calif, USA). In some instances, it may be desirable to generate 5'-m7GpppG-capped mRNA. As such, transcription of a linearized cDNA template may be carried out using, for example, the mMESSAGE mMACHINE High Yield Capped RNA Transcription Kit from Ambion (Austin, Tex., USA). Transcription may be carried out in a reaction volume of 20-100 μΐ at 37° C. for 30 min to 4 h. The transcribed mRNA is purified from the reaction mix by a brief treatment with DNase I to eliminate the linearized DNA template followed by precipitation in 70% ethanol in the presence of lithium chloride, sodium acetate or ammonium acetate. The integrity of the transcribed mRNA may be assessed using electrophoresis with an agarose-formaldehyde gel or commercially available Novex pre-cast TBE gels (e.g., Novex, Invitrogen, Carlsbad, Calif, USA).

[00175] Methods for transferring expression vectors into producer cells that are suitable to produce the nanomembrane delivery complexes described herein include, but are not limited to, viral mediated gene transfer, liposome mediated transfer, transformation, gene guns, transfection and transduction, e.g., viral mediated gene transfer such as the use of vectors based on DNA viruses such as adenovirus, adenoassociated virus and herpes virus, as well as retroviral based vectors. Examples of modes of gene transfer include e.g., naked DNA, CaP0₄ precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, and cell microinjection.

[00176] Viral gene transfer may be used to trans feet the producer cells with DNA encoding a payload (e.g. polypeptide or RNA), surface marker polypeptide or receiver polypeptide. A number of viruses may be used as gene transfer vehicles including Moloney murine leukemia virus (MMLV), adenovirus, adeno-associated virus (AAV), herpes simplex virus (HSV), lentiviruses such as human immunodeficiency virus 1 (HIV 1), and

spumaviruses such as foamy viruses, for example (See, e.g., Osten et al, HEP 178: 177-202 (2007)). Retroviruses, for example, efficiently transduce mammalian cells including human cells and integrate into chromosomes, conferring stable gene transfer.

[00177] Optionally, a fluorescent tracking molecule such as, for example, green fluorescent protein (GFP) may be trans fected using a viral-based approach (Tao et al, Stem Cells 25:670-678 (2007)). Ecotopic retroviral vectors containing DNA encoding the enhanced green fluorescent protein (EGFP) or a red fluorescent protein (e.g., DsRed-Express) are packaged using a packaging cell such as, for example, the Phoenix-Eco cell line

(distributed by Orbigen, San Diego, Calif). Packaging cell lines stably express viral proteins needed for proper viral packaging including, for example, gag, pol, and env. Supernatants from the Phoenix-Eco cells into which viral particles have been shed are used to transduce producer cells. In this instance, the percentage of cells expressing EGFP or DsRed-Express may be assessed by FACS. Other reporter genes that may be used to assess transduction efficiency include, for example, beta-galactosidase, chloramphenicol acetyltransferase, and luciferase as well as low-affinity nerve growth factor receptor (LNGFR), and the human cell surface CD24 antigen (Bierhuizen et al., Leukemia 13:605-613 (1999)).

[00178] Nonviral vectors may be used to introduce genetic material into suitable producer cell to generate nanomembrane delivery complexes. Nonviral-mediated gene transfer differs from viral-mediated gene transfer in that the plasmid vectors contain no proteins, are less toxic and easier to scale up, and have no host cell preferences. A number of delivery methods may be used to transfer nonviral vectors into suitable producer cells including chemical and physical methods.

[00179] Nonviral vectors may be introduced into suitable producer cells using synthetic macromolecules such as cationic lipids and polymers (Papapetrou et al., Gene Therapy 12:S118-S130 (2005)). Cationic liposomes, for example form complexes with DNA through charge interactions. The positively charged DNA/lipid complexes bind to the negative cell surface and are taken up by the cell by endocytosis. For example, the plasmid DNA

(approximately 0.5 μg in 25-100 of a serum free medium, such as, for example, OptiMEM (Invitrogen, Carlsbad, Calif.)) is mixed with a cationic liposome (approximately 4 μg in 25 μΐ_^ of serum free medium) such as the commercially available transfection reagent

Lipofectamine™ (Invitrogen, Carlsbad, Calif.) and allowed to incubate for at least 20 min to form complexes. The DNA/liposome complex is added to suitable producer cell and allowed to incubate for 5-24 h, after which time transgene expression may be assayed. Alternatively, other commercially available liposome tranfection agents may be used (e.g., In vivo

GeneSHUTTLE™, Qbiogene, Carlsbad, Calif).

[00180] Alternatively or in addition, a cationic polymer such as, for example,

polyethylenimine (PEI) may be used to transfect producer cells. Plasmid DNA is incubated with branched or linear PEIs varying in size from 0.8 K to 750 K (Sigma Aldrich, Saint Louis, Mo., USA; Fermetas, Hanover, Md., USA). PEI is prepared as a stock solution at 4.2 mg/ml distilled water and slightly acidified to pH 5.0 using HCl. The DNA may be combined with the PEI for 30 min at room temperature at various nitrogen/phosphate ratios based on the calculation that 1 μg of DNA contains 3 nmol phosphate and 1 μΐ of PEI stock solution contains 10 nmol amine nitrogen. The producer cells are seeded with the DNA/cationic complex, centrifuged at 280^xg for 5 min and incubated in culture medium for 4 or more hours until transgene expression is assessed.

[00181] A plasmid vector may be introduced into a producer cell or a nanomembrane delivery complex using a physical method such as particle-mediated transfection, "gene gun", biolistics, or particle bombardment technology (Papapetrou, et al, (2005) Gene Therapy 12:S118-S130). In this instance, exogenous nucleic acid is absorbed onto gold particles and administered to cells or complexes by a particle gun. A reporter gene such as, for example, beta-galactosidase, chloramphenicol acetyltransferase, luciferase, or green fluorescent protein may be used to assess efficiency of transfection.

[00182] Optionally, electroporation methods may be used to introduce a plasmid vector into suitable producer cell or nanomembrane delivery complex. Electroporation creates transient pores in the cell membrane, allowing for the introduction of various molecules into the cells and complexes including, for example, DNA and RNA as well as polypeptides and non-polypeptide therapeutic agents (e.g. therapeutic small molecules). Electroporation may be done using, for example, an ECM 600 electroporator (Genetronics, San Diego, Calif, USA). A number of alternative electroporation instruments are commercially available and may be used for this purpose (e.g., Gene Pulser Xcell™, BioRad, Hercules, Calif.; Cellject Duo, Thermo Science, Milford, Mass.).

[00183] In some embodiments, an episomal vector which may persist in the host nucleus as autonomously replicating genetic units without integration into chromosomes (Papapetrou et al, Gene Therapy 12:S118-S130 (2005)) may be used to modify producer cells. These vectors exploit genetic elements derived from viruses that are normally extrachromosomally replicating in cells upon latent infection such as, for example, EBV, human polyomavirus BK, bovine papilloma virus- 1 (BPV-1), herpes simplex virus- 1 (HSV) and Simian virus 40 (SV40). Mammalian artificial chromosomes may also be used for nonviral gene transfer (Vanderbyl et al, Exp. Hematol. 33: 1470-1476 (2005)).

[00184] Exogenous nucleic acids encoding payloads, receiver or surface molecules may be assembled into expression vectors by standard molecular biology methods known in the art, e.g., restriction digestion, overlap-extension PCR, and Gibson assembly.

[00185] In some embodiments, the nanomembrane delivery complex comprises a payload, receiver or surface marker that is chemically conjugated. Chemical conjugation may be accomplished by covalent bonding of the payload, receiver or surface marker to another molecule, with or without use of a linker. The formation of such conjugates is within the skill of artisans and various techniques are known for accomplishing the conjugation, with the choice of the particular technique being guided by the materials to be conjugated. The addition of amino acids to the polypeptide (C- or N-terminal) which contain ionizable side chains, e.g., aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, or tyrosine, and are not contained in the active portion of the polypeptide sequence, serve in their

unprotonated state as a potent nucleophile to engage in various bioconjugation reactions with reactive groups attached to polymers, e.g., homo- or hetero-bi-functional PEG (e.g., Lutolf and Hubbell, Biomacromolecules 2003;4:713-22, Hermanson, Bioconjugate Techniques, London. Academic Press Ltd; 1996). Conjugation is not restricted to polypeptides but is applicable also for non-polypeptides, e.g., lipids, carbohydrates, nucleic acids, and small molecules.

[00186] In one embodiment, the payload, receiver or surface marker may be bound to the surface of a nanomembrane delivery complex through a biotin-streptavidin bridge. Any surface membrane proteins of a nanomembrane delivery complex may be biotinylated using an amine reactive biotinylation reagent such as, for example, EZ-Link Sulfo-NHS-SS-Biotin (sulfosuccinimidyl 2-(biotinamido)-ethyl-l,3-dithiopropionate; Pierce-Thermo Scientific, Rockford, III, USA; See, e.g., Jaiswal et al, Nature Biotech. 21 :47-51 (2003)). For example, nanomembrane delivery complexes may be incubated for 30 min at 4° C. in 1 mg/ml solution of sulfo-NHS-SS in phosphate-buffered saline. Excess biotin reagent is removed by washing the complexes with Tris-buffered saline. The biotinylated complexes are then reacted with the biotinylated payload, receiver or surface marker in the presence of streptavidin to form the conjugated nanomembrane delivery complex.

[00187] In some embodiments, the nanomembrane delivery complex comprises a payload, receiver or surface marker that is enzymatically conjugated, including using transpeptidases, sortases, and isopeptidases. These methods include enzymatic strategies such as, e.g., transpeptidase reaction mediated by a sortase enzyme to connect one polypeptide containing the acceptor sequence LPXTG or LPXTA with a polypeptide containing the N-terminal donor sequence GGG, see e.g., Swee et al, PNAS 2013. The methods also include combination methods, such as e.g., sortase-mediated conjugation of Click Chemistry handles (an azide and an alkyne), respectively, followed by a cyclo-addition reaction to chemically bond the antigen to the cell, see e.g., Neves et al, Bioconjugate Chemistry, 2013. [00188] In certain embodiments, the payload, receiver or surface marker is loaded into the producer cell or nanomembrane delivery complex. A number of methods may be used to load a payload, receiver or surface marker into a producer cell or nanomembrane delivery complex. Suitable methods include, for example, hypotonic lysis, hypotonic dialysis, osmosis, osmotic pulsing, osmotic shock, ionophoresis, electroporation, sonication, microinjection, calcium precipitation, membrane intercalation, lipid mediated transfection, detergent treatment, viral infection, diffusion, receptor mediated endocytosis, use of protein transduction domains, particle firing, membrane fusion, freeze-thawing, mechanical disruption, and filtration. Any one such method or a combination thereof may be used to load nanomembrane delivery complexes or producer cells.

[00189] Generally, any method that induces controlled injury may be used to load an agent, e.g. a payload, receiver or surface marker into or onto a producer cell or

nanomembrane delivery complex. The controlled injury of the membrane of the producer cell or nanomembrane delivery complex can be caused by, for example, pressure induced by mechanical strain or shear forces, subjecting the cell to deformation, constriction, rapid stretching, rapid compression, or pulse of high shear rate. The controlled injury leads to uptake of material, e.g., a payload, receiver or surface marker into the interior of the nanomembrane delivery complex or the cytoplasm of the producer cell from the surrounding cell medium. Any suitable controlled injury method may be used to generate the

nanomembrane delivery complexes described herein.

[00190] Controlled cell injury as used herein includes: i) virus-mediated transfection (e.g., Herpes simplex virus, Adeno virus, Adeno-associated virus, Vaccinia virus, or Sindbis virus), ii) chemically-mediated transfection, e.g., cationic polymer, calcium phosphate, cationic lipid, polymers, and nanoparticles, such as cyclodextrin, liposomes, cationic liposomes, DEAE-dextran, polyethyleneimine, dendrimer, polybrene, calcium phosphate, lipofectin, DOTAP, lipofectamine, CTAB/DOPE, DOTMA; and iii) physically-mediated transfection, including direct injection, biolistic particle delivery, electroporation, laser-irradiation, sonoporation, magnetic nanoparticles, and controlled deformation (e.g., cell squeezing), as exemplified by micro-needle, nano-needle, femtosyringe, atomic-force microscopy (AFM) tip, gene gun (e.g., gold nanoparticles), Amaxa Nucleo fector, phototransfection (multi-photon laser), impalefection, and magnetofection, and other suitable methods known in the art.

[00191] For hypotonic lysis producer cells or nanomembrane delivery complexes are exposed to low ionic strength buffer causing them to burst allowing loading of an agent, e.g. a payload, receiver or surface marker. Alternatively, controlled dialysis against a hypotonic solution to swell the cells or complexes and create pores in the cell or complex membrane is used. The cells or complexes are subsequently exposed to conditions that allow resealing of the membrane.

[00192] For electroporation, producer cells or nanomembrane delivery complexes are exposed to an electrical field which causes transient holes in the cell or complex membrane, allowing loading of an agent, e.g. a payload, receiver or surface marker.

[00193] For sonication, producer cells or nanomembrane delivery complexes are exposed to high intensity sound waves, causing transient disruption of the cell or complex membrane allowing loading of an agent, e.g. a payload, receiver or surface marker.

[00194] For detergent treatment, producer cells or nanomembrane delivery complexes are treated with a mild detergent which transiently compromises the cell or complex membrane by creating holes allowing loading of an agent, e.g. a payload, receiver or surface marker. After cells or complexes are loaded, the detergent is washed away thereby resealing the membrane.

[00195] For receptor mediated endocytosis, producer cells or nanomembrane delivery complexes that have a surface receptor which upon binding of the receiver or payload (e.g., therapeutic agent) induces internalization of the receptor and the associated receiver or payload.

[00196] For mechanical firing, producer cells or nanomembrane delivery complexes may be bombarded with a payload, receiver or surface marker attached to a heavy or charged particle such as, for example, gold microcarriers and are mechanically or electrically accelerated such that they traverse the cell membrane. Microparticle bombardment may be achieved using, for example, the Helios Gene Gun (from, e.g., Bio-Rad, Hercules, Calif, USA).

[00197] In some embodiments, producer cells or nanomembrane delivery complexes may be loaded with a payload, receiver or surface marker by fusion with a synthetic vesicle such as, for example, a liposome. In this instance, the vesicles themselves are loaded with the payload, receiver or surface marker using one or more of the methods described herein or known in the art. Alternatively, the payload, receiver or surface marker may be loaded into the vesicles during vesicle formation. The loaded vesicles are then fused with the producer cells or nanomembrane delivery complexes under conditions that enhance membrane fusion. Fusion of a liposome, for example, may be facilitated using various inducing agents such as, for example, proteins, peptides, polyethylene glycol (PEG), and viral envelope proteins or by changes in medium conditions such as pH.

[00198] For filtration, producer cells or nanomembrane delivery complexes and the payload, receiver or surface marker may be forced through a filter of pore size smaller than the cell or complex causing transient disruption of the cell membrane and allowing the payload, receiver or surface marker to enter the cell or complex.

[00199] For freeze thawing, producer cells or nanomembrane delivery complexes are subjected to several freeze thaw cycles, resulting in cell membrane disruption allowing loading of an agent, e.g. a payload, receiver or surface marker.

[00200] In certain embodiments, generating a nanomembrane delivery complex comprises the step of contacting an isolated membrane derived from a producer cell with a payload (e.g., a therapeutic agent). In some embodiments, generating a nanomembrane delivery complex comprises the step of contacting an isolated membrane derived from a producer cell with a receiver. In some embodiments, generating a nanomembrane delivery complex comprises the step of contacting an isolated membrane derived from a producer cell with a payload (e.g., a therapeutic agent) and a receiver

Methods of Isolating Nanomembrane Delivery Complexes

[00201] The nanomembrane delivery complexes may be isolated from the producer cells. It is contemplated that all known manners of isolation of nanomembrane delivery complexes are deemed suitable for use herein. For example, physical properties of nanomembrane delivery complexes may be employed to separate them from a medium or other source material, including separation on the basis of electrical charge (e.g., electrophoretic separation), size (e.g., filtration, molecular sieving, etc), density (e.g., regular or gradient centrifugation), Svedberg constant (e.g., sedimentation with or without external force, etc). Alternatively, or additionally, isolation may be based on one or more biological properties, and include methods that may employ surface markers (e.g., for precipitation, reversible binding to solid phase, FACS separation, specific ligand binding, non-specific ligand binding, etc.). In yet further contemplated methods, the nanomembrane delivery complexes may also be fused using chemical and/or physical methods, including PEG-induced fusion and/or ultrasonic fusion. [00202] Isolation (and enrichment) can be done in a general and non-selective manner (typically including serial centrifugation). Alternatively, isolation and enrichment can be done in a more specific and selective manner (e.g., using producer cell-specific surface markers). For example, specific surface markers may be used in immunoprecipitation, FACS sorting, bead-bound ligands for magnetic separation etc.

[00203] In some embodiments, size exclusion chromatography can be utilized to isolate the nanomembrane delivery complexes. Size exclusion chromatography techniques are known in the art. Exemplary, non-limiting techniques are provided herein. In some embodiments, a void volume fraction is isolated and comprises the nanomembrane delivery complexes of interest. Further, in some embodiments, the nanomembrane delivery complexes can be further isolated after chromatographic separation by centrifugation techniques (of one or more chromatography fractions), as is generally known in the art. In some embodiments, for example, density gradient centrifugation can be utilized to further isolate the nanomembrane delivery complexes. Still further, in some embodiments, it can be desirable to further separate the producer cell-derived nanomembrane delivery complexes from nanomembrane delivery complexes of other origin. For example, the producer cell- derived nanomembrane delivery complexes can be separated from non-producer cell-derived nanomembrane delivery complexes by immunosorbent capture using an antigen antibody specific for the producer cell

[00204] In some embodiments, the isolation of nanomembrane delivery complexes may involve combinations of methods that include, but are not limited to, differential

centrifugation, size-based membrane filtration, concentration and/or rate zonal centrifugation.

Methods of Characterizing Nanomembrane Delivery Complexes

[00205] In some embodiments, nanomembrane delivery complexes are isolated and characterized by metrics including, but not limited to, size, shape, morphology, or molecular compositions such as nucleic acids, proteins, metabolites, and lipids.

[00206] Nanomembrane delivery complexes may be assessed by methods known in the art including, but not limited to, transcriptomics, sequencing, proteomics, mass spectrometry, or HPLC. Nanomembrane delivery complexes may further be assessed by methods that include, but are not limited to, electron microscopy, flow cytometry and Western blotting.

[00207] The composition of nucleotides associated with an isolated nanomembrane delivery complex composition (including RNAs and DNAs) can be measured using a variety of techniques that are well known to those of skill in the art (e.g., quantitative or semiquantitative RT-PCR, Northern blot analysis, solution hybridization detection). In a particular embodiment, the level of at least one RNA is measured by reverse transcribing RNA from the nanomembrane delivery complex composition to provide a set of target

oligodeoxynucleotides, hybridizing the target oligodeoxynucleotides to one or more RNA- specific probe oligonucleotides (e.g., a microarray that comprises RNA-specific probe oligonucleotides) to provide a hybridization profile for the nanomembrane delivery complex composition, and comparing the nanomembrane delivery complex composition hybridization profile to a hybridization profile generated from a control sample. An alteration in the signal of at least one RNA in the test sample relative to the control sample is indicative of the RNA composition.

[00208] Also, a microarray can be prepared from gene-specific oligonucleotide probes generated from known RNA sequences. The array may contain two different oligonucleotide probes for each RNA, one containing the active, mature sequence and the other being specific for the precursor of the RNA (for example miRNA and pre-miRNAs). The array may also contain controls, such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs and other RNAs (e.g., rRNAs, mRNAs) from both species may also be printed on the microchip, providing an internal, relatively stable, positive control for specific hybridization. One or more appropriate controls for non-specific hybridization may also be included on the microchip. For this purpose, sequences are selected based upon the absence of any homology with any known RNAs.

[00209] The microarray may be fabricated using techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5'-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GeneMachine OmniGrid.TM. 100 Microarrayer and Amersham CodeLink.TM. activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g., 6x SSPE/30% formamide at 25°C. for 18 hours, followed by washing in 0.75x TNT at 37°C. for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary R As, in the nanomembrane delivery complex preparation. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Image intensities of each spot on the array are proportional to the abundance of the corresponding R A in the nanomembrane delivery complexes.

[00210] The identity of the producer cells or nanomembrane delivery complexes can be assessed by in vitro assays. For example, the identity of the producer cells or nanomembrane delivery complexes is assessed by counting the number of cells or complexes in a population, e.g., by microscopy, by flow cytometry, or by hemacytometry. Alternatively or in addition, the identity of the producer cells or nanomembrane delivery complexes is assessed by analysis of protein content of the cell or complex, e.g., by flow cytometry, Western blot, immunoprecipitation, fluorescence spectroscopy, chemiluminescence, mass spectrometry, or absorbance spectroscopy. In one embodiment, the protein content assayed is a surface protein, e.g., a differentiation marker, a receptor, a co-receptor, a transporter, a glycoprotein. In some embodiments, the identity of the producer cells or nanomembrane delivery complexes is assessed by analysis of the receiver content of the cell or complex, e.g., by flow cytometry, Western blot, immunoprecipitation, fluorescence spectroscopy,

chemiluminescence, mass spectrometry, or absorbance spectroscopy. For example, the identity of the producer cells or nanomembrane delivery complexes can be assessed by the mR A content of the cells or complexes, e.g., by RT-PCR, flow cytometry, or northern blot. The identity of the producer cells can be assessed by nuclear material content, e.g., by flow cytometry, microscopy, or southern blot, using, e.g., a nuclear stain or a nucleic acid probe. Alternatively or in addition, the identity of the producer cells or nanomembrane delivery complexes is assessed by lipid content of the cells or complexes, e.g by flow cytometry, liquid chromatography, or by mass spectrometry.

Methods of Using Nanomembrane Delivery Complexes

[00211] Provided are compositions, methods, kits, and reagents for treatment or prevention of diseases or conditions in humans and other mammals. In some embodiments, pharmaceutical compositions comprising nanomembrane delivery complexes may be used for therapeutic purposes, such as the treatment or prevention of disease, disorder or condition.

[00212] Provided herein are methods of targeting a cell or tissue to treat or prevent a disease, disorder or condition. The subject may suffer from the disease, disorder or condition or may be at risk of developing the disease, disorder or condition. The methods provided herein include the administration of a suitable nanomembrane delivery complex described herein in an amount effective to substantially deliver the payload to the target cell or tissue, thereby preventing or treating the disease, disorder or condition. In some embodiments, the nanomembrane delivery complex is formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated for intravenous injection to the subject. The compositions may be administered once to the subject. Alternatively, multiple administrations may be performed over a period of time. For example, two, three, four, five, or more administrations may be given to the subject. In some embodiments, administrations may be given as needed, e.g., for as long as symptoms associated with the disease, disorder or condition persist. In some embodiments, repeated administrations may be indicated for the remainder of the subject's life. Treatment periods may vary and could be, e.g., no longer than a year, six months, three months, two months, one month, two weeks, one week, three days, two days, or no longer than one day.

[00213] In some embodiments, the pharmaceutical composition is administered at a frequency sufficient to effectively increase the concentration of payload in the target cell or tissue above a level that is associated with a symptom of the disease, disorder or condition.

[00214] In some embodiments, the time interval between administrations within a treatment period is no longer than the period in which the number of nanomembrane delivery complexes in circulation is reduced to less than about 5%, 10%, 15%, 20%, 25%, 30%>, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the number of nanomembrane delivery complexes present in the administered pharmaceutical composition.

[00215] An effective amount of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nanomembrane delivery complex (e.g., size, and in some cases the extent of molecules to be delivered), and other determinants. In general, an effective amount of the composition provides efficient cellular response of the target cell. Increased efficiency may be

demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nanomembrane delivery complex constituents), increased cellular response, or reduced innate immune response of the host subject.

[00216] The dosing and frequency of the administration of the nanomembrane delivery complexes and pharmaceutical compositions thereof can be determined, e.g., by the attending physician based on various factors such as the severity of disease, the patient's age, sex and diet, the severity of any inflammation, time of administration, and other clinical factors. In one example, an intravenous administration is initiated at a dose which is minimally effective, and the dose is increased over a pre-selected time course until a positive effect is observed. Subsequently, incremental increases in dosage are made limiting to levels that produce a corresponding increase in effect while taking into account any adverse affects that may appear.

[00217] Each dose of nanomembrane delivery complexes can be administered at intervals such as once daily, once weekly, twice weekly, once monthly, or twice monthly.

[00218] In some embodiments, the compositions are administered at least twice over a treatment period such that the disease, disorder or condition is treated, or a symptom thereof is decreased. In some embodiments, the compositions are administered at least twice over a treatment period such that the disease, disorder or condition is treated, or a symptom thereof is prevented. In some embodiments, the pharmaceutical composition is administered a sufficient number of times over a treatment period such that a sufficient amount of payload is delivered to the target cell or tissue during the treatment period. In some embodiments, the pharmaceutical composition is administered a sufficient number of times over a treatment period such that a sufficient amount of payload is delivered to the target cell or tissue during the treatment period such that one or more symptoms of the disease, disorder or condition is prevented, decreased or delayed. In some embodiments, increasing the payload concentration in the target cell or tissue includes increasing the peak concentration, while in others it includes increasing the average concentration. In some embodiments, a substantial increase during the treatment period can be determined by comparing a pretreatment or post-treatment period in the human subject, or by comparing measurements made in a population undergoing treatment with a matched, untreated control population.

[00219] In some embodiments, the pharmaceutical composition is administered a sufficient number of times a treatment period such that the concentration of payload in the target cell or tissue is increased for at least about one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, or greater than six months. In some embodiments, the pharmaceutical composition is administered a sufficient number of times a treatment period such that the concentration of payload in the target cell or tissue is increased for a period of time at least as long as the treatment period.

[00220] In some embodiments, the nanomembrane delivery complexes are administered, e.g. intravenously to the circulatory system or a tissue of a mammalian subject, such as a human. In some embodiments, the nanomembrane delivery complexes provide a natural barrier between a payload (e.g., therapeutic agent) and the immune system. In some embodiments, the nanomembrane delivery complexes are capable of residing in the circulatory system or tissue of a subject for an extended period of time allowing delivery of a more efficient therapeutic effect than what can be achieved by delivery through other methods currently used.

[00221] Nanomembrane delivery complexes may interact with a target cell in a tissue or circulatory system of the subject. In some embodiments, the composition or phenotype of the target cell is modified subsequent to its interaction with the complex. In some embodiments, the modification of the target cell leads to a reduction in disease burden, may alleviate a symptom of the disease or has some other treatment effect.

[00222] In some embodiments, nanomembrane delivery complexes interact with a target cell and increase the concentration of a therapeutic agent in the target cell. In some embodiments, a therapeutic agent is delivered to the cytoplasm of the target cell. In some embodiments, the therapeutic agent is a functional mRNA which may be translated in the cytoplasm of the target cell. A resulting polypeptide may be functional and modulate signaling or regulatory behavior, morphology, or growth of the target cell.

[00223] Provided are methods of treating a disease, disorder or condition comprising administering to a subject in need thereof a pharmaceutical composition comprising the nanomembrane delivery complexes described herein, optionally in form of the dosage form described herein, in an amount effective to treat the disease, disorder or condition.

[00224] In some embodiments, the preparations comprise nanomembrane delivery complexes comprising a payload that is capable, upon contact, of killing or restoring the functionality of an infected, impaired or dysregulated cell or tissue that is associated with the disease, disorder or condition. In some embodiments, the nanomembrane delivery complex facilitates the contacting of the infected, impaired or dysregulated cell or tissue with the payload in sufficient proximity and for a sufficient duration to bring about the killing or restoring the functionality of the infected, impaired or dysregulated cell or tissue. In some embodiments, an infected or dysregulated cell or tissue is killed thereby treating the disease, disorder or condition. In other embodiments an impaired or dysregulated cell or tissue is restored thereby treating the disease, disorder or condition. For example, an impaired enzyme function may be restored or a dysregulated enzyme function regulated.

[00225] In some embodiments, pharmaceutical compositions comprising nanomembrane delivery complexes may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis);

inflammatory disorders (e.g. arthritis, pelvic inflammatory disease); infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis);

neurological disorders (e.g. Alzheimer's disease, Huntington's disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g. atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration);

proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g. fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g. depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia).

[00226] In one embodiment, the nanomembrane delivery complex is administered to a subject in need thereof to treat cancers. Such cancers include, but are not limited to, pancreatic cancers, biliary tract cancer, liver cancer, breast cancer, glioma, lung cancer, leukemias, gastrointestinal cancers, neuroendocrine tumors, throat cancers, melanoma, colon cancer, prostate cancer, ovarian cancer, testicular cancer, ocular cancer, and kidney cancer.

[00227] In one embodiment, the nanomembrane delivery complex is administered to a subject in need thereof treat autoimmune disease. Such autoimmune diseases include, but are not limited to, multiple sclerosis, peripheral neuritis, Sjogren's syndrome, rheumatoid arthritis, graft versus host disease, alopecia, Autoimmune pancreatitis, Behcet's disease, Bullous pemphigoid, Celiac disease, Devic's disease (neuromyelitis optica),

Glomerulonephritis, IgA nephropathy, assorted vasculitides, scleroderma, diabetes, arteritis, vitiligo, ulcerative colitis, irritable bowel syndrome, psoriasis, uveitis, and systemic lupus erythematosus.

[00228] In one embodiment, the nanomembrane delivery complex is administered to a subject in need thereof to treat neurodegenerative diseases and brain-related conditions. Such indications include, but are not limited to, Parkinson's disease, Alzheimer's, stroke, aneurysms, neuroencephalitis, and ALS.

[00229] In one embodiment, the nanomembrane delivery complex is administered to a subject in need thereof to treat a disease, disorder or condition selected from Table 3 and Table 4.

[00230] Diseases, disorders and conditions associated with target cells or tissues that may be treated or prevented by administering nanomembrane delivery complexes include, but are not limited to: diseases associated with infectious agents or pathogens (e.g., bacterial, fungal, viral, parasitic infections), disease associated with toxic proteins, diseases associated with the accumulation of lipids, diseases associated with apoptotic, necrotic, aberrant or oncogenic mammalian cells, and metabolic diseases.

[00231] In some embodiments, provided are methods of treating diseases, including, but not limited to, metabolic diseases, cancers, clotting and anti-clotting diseases. The methods include administering to a subject in need thereof a pharmaceutical composition of nanomembrane delivery complexes in an amount sufficient to treat the metabolic disease, the cancer, the clotting disease or anti-clotting disease of the subject.

[00232] Diseases, disorders and conditions associated with targets in the circulatory system that may be treated or prevented by administering nanomembrane delivery complexes are described herein.

[00233] Diseases, disorders and conditions associated with targets in the circulatory system that may be treated or prevented by administering nanomembrane delivery complexes include, but are not limited to:, diseases associated with infectious agents or pathogens (e.g., bacterial, fungal, viral, parasitic infections), , diseases associated with apoptotic, necrotic, aberrant or oncogenic mammalian cells, and metabolic diseases.

[00234] Provided herein, in some embodiments, are methods for the treatment or prevention of diseases or conditions that are associated with molecules or entities that reside, at least in part, in specific target cells or tissues. The methods comprise, in certain

embodiments, administering to a subject in need thereof nanomembrane delivery complexes in an amount effective to treat or prevent the disease or condition that is associated with molecules or entities that reside, in specific target cells or tissues.

[00235] Provided herein are methods of inducing in vivo delivery of nanomembrane delivery complexes in a mammalian subject in need thereof. Therein, an effective amount of a composition containing a nanomembrane delivery complex is administered to the subject using the delivery methods described herein. The nanomembrane delivery complex is provided in an amount such that the nanomembrane delivery complex is localized into a cell of the subject. The cell in which the nanomembrane delivery complex is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nanomembrane delivery complex administration.

[00236] Provided herein are methods of transplanting cells containing or producing nanomembrane delivery complexes to a mammalian subject. Administration of cells to mammalian subjects is known to those of ordinary skill in the art, such as local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), as is the formulation of cells in pharmaceutically acceptable carrier.

[00237] Provided are methods of inducing a cellular response using the nanomembrane delivery complexes described herein. Such response can be in vivo, ex vivo, in culture, or in vitro. For example, a target cell population is contacted with an effective amount of a composition containing a nanomembrane delivery complex. The population is contacted under conditions such that the nanomembrane delivery complex is localized into one or more cells of the cell population.

[00238] In one embodiment, the nanomembrane delivery complex is administered as part of a treatment regimen that further includes administration of a second, standard-of-care therapy.

[00239] In certain embodiments, the administered nanomembrane delivery complex directs up-regulation of (via expression in the cell, delivery in the cell, or induction within the cell) of one or more polypeptides that provide a functional activity which is substantially absent in the target cell to which the polypeptide is delivered. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature. In related embodiments, the administered nanomembrane delivery complex directs up-regulation of one or more polypeptides that increases (e.g., synergistically) a functional activity which is present but substantially deficient in the target cell in which the polypeptide is up-regulated.

[00240] In other embodiments, the administered nanomembrane delivery complex directs up-regulation of (via expression in the cell, delivery in the cell, or induction within the cell) of one or more polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the target cell in which the polypeptide is up-regulated. Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof. In some embodiments, the polypeptide increases the level of an endogenous protein in the cell to a desirable level; such an increase may bring the level of the endogenous protein from a subnormal level to a normal level, or from a normal level to a super-normal level.

[00241] Alternatively, the polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Usually, the activity of the endogenous protein is deleterious to the subject, for example, due to mutation of the endogenous protein resulting in altered activity or localization. Additionally, the polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell. Examples of antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins. Additionally, the antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.

Modulation of Cell Fate

[00242] Provided are methods of inducing an alteration in cell fate in a target mammalian cell. The target mammalian cell may be a precursor cell and the alteration may involve driving differentiation into a lineage, or blocking such differentiation. Alternatively, the target mammalian cell may be a differentiated cell, and the cell fate alteration includes driving de-differentiation into a pluripotent precursor cell, or blocking such de- differentiation, such as the dedifferentiation of cancer cells into cancer stem cells. In situations where a change in cell fate is desired, effective amounts of nanomembrane delivery complexes encoding a cell fate inductive molecule or signal as a payload is introduced into a target cell under conditions such that an alteration in cell fate is induced. In some

embodiments, the nanomembrane delivery complexes are useful to reprogram a subpopulation of cells from a first phenotype to a second phenotype. Such a reprogramming may be temporary or permanent. Optionally, the reprogramming induces a target cell to adopt an intermediate phenotype.

[00243] Additionally, the methods can be used to generate induced pluripotent stem cells (iPS cells). The use of iPS cells generated using the methods described herein is expected to have a reduced incidence of teratoma formation.

[00244] Also provided are methods of reducing cellular differentiation in a target cell population. For example, a target cell population containing one or more precursor cell types is contacted with a composition having an effective amount of a nanomembrane delivery complex composition, under conditions such that the nanomembrane delivery complex reduces the differentiation of the precursor cell. In non-limiting embodiments, the target cell population contains injured tissue in a mammalian subject or tissue affected by a surgical procedure. The precursor cell is, e.g., a stromal precursor cell, a neural precursor cell, or a mesenchymal precursor cell.

Targeting diseased cells

[00245] Provided herein are methods for targeting pathogenic or diseased cells or tissues, including cancer cells, using nanomembrane delivery complexes that deliver cytotoxic or cytostatic molecules. The molecule may be delivered into the target pathogenic cell exclusively or preferentially to reduce off-target effects of the therapeutic. Receivers described herein may be used that are capable of targeting the nanomembrane delivery complexes preferentially to the target pathogenic cell.

Methods of gene silencing

[00246] The nanomembrane delivery complex compositions described herein are useful to silence (e.g., prevent or substantially reduce) expression of one or more target genes in a target cell population. A nanomembrane delivery complex containing or encoding a polypeptide capable of directing sequence-specific histone H3 methylation is introduced into the target cells under conditions such that the polypeptide is translated and reduces gene transcription of a target gene via histone H3 methylation and subsequent heterochromatin formation. In some embodiments, the silencing mechanism is performed on a cell population present in a mammalian subject. By way of non- limiting example, a useful target gene is a mutated Janus Kinase-2 family member, wherein the mammalian subject expresses the mutant target gene suffers from a myeloproliferative disease resulting from aberrant kinase activity.

[00247] Administration of nanomembrane delivery complexes carrying siRNAs are also provided herein. As has been previously demonstrated in yeast, sequence-specific trans silencing is an effective mechanism for altering cell function. While this mechanism functions in cis- with centromeric regions of DNA, sequence-specific trans silencing is possible through co-transfection with double-stranded siRNAs for specific regions of DNA and concomitant RNAi-directed silencing of the siRNA ribonuclease Eril (Buhler et al. Cell 2006, 125, 873-886).

Modulation of biological pathways

[00248] The efficient delivery of molecules (payloads) via nanomembrane delivery complexes into cells provides a desirable mechanism of modulating target biological pathways. Such modulation includes antagonism or agonism of a given pathway. In one embodiment, a method is provided for antagonizing a biological pathway in a target cell by contacting the cell with an effective amount of a nanomembrane delivery complex composition comprising a polypeptide or comprising a functional nucleic acid (e.g., mRNA) which encodes a polypeptide, under conditions such that the peptide is localized into the target cell or the polypeptide is capable of being translated in the cell from the nucleic acid, wherein the polypeptide inhibits the activity of another polypeptide functional in the biological pathway. Exemplary biological pathways are those defective in an autoimmune or inflammatory disorder such as multiple sclerosis, rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis colitis, or Crohn's disease; in particular, antagonism of the IL-12 and IL-23 signaling pathways are of particular utility. (See Kikly K, Liu L, Na S, Sedgwick J D (2006) Curr. Opin. Immunol. 18 (6): 670-5). Further, provided are modified nucleic acids encoding an antagonist for chemokine receptors; chemokine receptors CXCR-4 and CCR-5 are required for, e.g., HIV entry into host cells (Arenzana-Seisdedos F et al. (1996) Nature 383:400).

[00249] Alternatively, provided are methods of agonizing a biological pathway in a target cell. Exemplary agonized biological pathways include pathways that modulate cell fate determination. Such agonization is reversible or, alternatively, irreversible. [00250] In some embodiments, contacting a target cell with a nanomembrane delivery complex modulates a biological pathway that causes a cytotoxic cellular response. In some embodiments, the polypeptide is a protein cytotoxic to the target cell.

[00251] In other embodiments, nanomembrane delivery complexes may similarly carry metabolites, lipids, or small molecules that modulate the activity of a biological pathway. Such molecules may be recombinant, synthesized or natively isolated.

Methods of nucleic acid delivery

[00252] Methods are provided to enhance nucleic acid delivery from nanomembrane delivery complexes into a cell population, in vivo, ex vivo, or in culture. For example, a cell culture containing a plurality of target cells (e.g., eukaryotic cells such as yeast or

mammalian cells) is contacted with a composition comprising a nanomembrane delivery complex having at least one nucleic acid, which optionally has a translatable region. The nucleic acid within the nanomembrane delivery complex composition exhibits enhanced retention in the target cell relative to a corresponding free nucleic acid. The retention of the nucleic acid within the nanomembrane delivery complex composition is greater than the retention of the free nucleic acid. In some embodiments, it is at least about 50%, 75%, 90%>, 95%, 100%, 150%, 200% or more than 200% greater than the retention of the free nucleic acid. Such retention advantage may be achieved by one round of transfection with the nucleic acid within a nanomembrane delivery complex composition, or may be obtained following repeated rounds of transfection.

[00253] In some embodiments, the nucleic acid within the nanomembrane delivery complex composition is delivered to a target cell population with one or more additional nucleic acids. Such delivery may be at the same time, or the nucleic acid within the nanomembrane delivery complex composition is delivered prior to delivery of the one or more additional nucleic acids. The additional one or more nucleic acids may be within the same or within a separate nanomembrane delivery complex composition or free nucleic acids. It is understood that the initial presence of the nucleic acid within a nanomembrane delivery complex composition does not substantially induce an innate immune response of the target cell population and, moreover, that the innate immune response will not be activated by the later presence of the additional nucleic acids. In this regard, the nucleic acid within the nanomembrane delivery complex composition may not itself contain a translatable region, if the protein desired to be present in the target cell population is translated from the additional nucleic acid.

[00254] The nucleic acid within the nanomembrane delivery complex composition may have at least one nucleoside modification or may be unmodified.

Definitions

[00255] "Administration," "administering" and variants thereof means introducing a composition, such as a nanomembrane delivery complex, or agent into a subject and includes concurrent and sequential introduction of a composition or agent. The introduction of a composition or agent into a subject is by any suitable route, including orally, pulmonarily, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or

subcutaneously), rectally, intralymphatically, or topically. Administration includes self- administration and the administration by another. A suitable route of administration allows the composition or the agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject.

[00256] As used herein, the term "antibody" encompasses an immunoglobulin whether natural or partly or wholly synthetically produced, and fragments thereof. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partly or wholly synthetically produced. "Antibody" further includes a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. Use of the term antibody is meant to include whole antibodies, polyclonal, monoclonal and recombinant antibodies, fragments thereof, and further includes single-chain antibodies, humanized antibodies; murine antibodies; chimeric, mouse-human, mouse-primate, primate-human monoclonal antibodies, anti-idiotype antibodies, antibody fragments, such as, e.g., scFv, (scFv)2, Fab, Fab', and F(ab')2, F(abl)2, Fv, dAb, and Fd fragments, diabodies, and antibody-related polypeptides. Antibody includes bispecific antibodies and multispecific antibodies so long as they exhibit the desired biological activity or function.

[00257] The term "antigen binding fragment" used herein refers to fragments of an intact immunoglobulin, and any part of a polypeptide including antigen binding regions having the ability to specifically bind to the antigen. For example, the antigen binding fragment may be a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, or a scFv fragment, but is not limited thereto. A Fab fragment has one antigen binding site and contains the variable regions of a light chain and a heavy chain, the constant region of the light chain, and the first constant region CHI of the heavy chain. A Fab' fragment differs from a Fab fragment in that the Fab' fragment additionally includes the hinge region of the heavy chain, including at least one cysteine residue at the C-terminal of the heavy chain CHI region. The F(ab')2 fragment is produced whereby cysteine residues of the Fab' fragment are joined by a disulfide bond at the hinge region. A Fv fragment is the minimal antibody fragment having only heavy chain variable regions and light chain variable regions, and a recombinant technique for producing the Fv fragment is well known in the art. Two-chain Fv fragments may have a structure in which heavy chain variable regions are linked to light chain variable regions by a non- covalent bond. Single-chain Fv (scFv) fragments generally may have a dimer structure as in the two-chain Fv fragments in which heavy chain variable regions are covalently bound to light chain variable regions via a peptide linker or heavy and light chain variable regions are directly linked to each other at the C-terminal thereof. The antigen binding fragment may be obtained using a protease (for example, a whole antibody is digested with papain to obtain Fab fragments, and is digested with pepsin to obtain F(ab')2 fragments),and may be prepared by a genetic recombinant technique. A dAb fragment consists of a VH domain. Single-chain antibody molecules may comprise a polymer with a number of individual molecules, for example, dimmer, trimer or other polymers.

[00258] "Applicator" refers to any device used to connect to a subject. This includes, e.g., needles, cannulae, catheters, and tubing, as well as containers attached thereto.

[00259] "Associated with" when used to describe the relationships among multiple compounds or molecules encompasses such as, e.g., any interaction between a receiver and a target or between a nanomembrane delivery complex and a target. This includes enzymatic interaction, ionic binding, covalent binding, non-covalent binding, hydrogen bonding, London forces, van der Waals forces, hydrophobic interaction, lipophilic interactions, magnetic interactions, electrostatic interactions, and the like.

[00260] "Associated with" when used to describe the relationships among a target, entity, compound, agent, or molecule and a disease, disorder, condition, symptom or phenotype is any link that may reasonably be made between them, including a causal link, or a statistical significant link, an empirically established link, a suggested link, whether or not causative of the disease, disorder, condition, symptom or phenotype. [00261] "Binding" describes an interaction among compounds or molecules, e.g., between a receiver and a target or between a nanomembrane delivery complex and a target, that comes about by covalent binding or non-covalent binding, including ionic binding, electrostatic interactions, hydrogen bonding, London forces, van der Waals forces, hydrophobic interaction, lipophilic interactions, and similar.

[00262] As used herein, the term "biological sample" refers to any type of material of biological origin isolated from a subject, including, for example, DNA, R A, lipids, carbohydrates, and protein. The term "biological sample" includes tissues, cells and biological fluids isolated from a subject. Biological samples include, e.g., but are not limited to, whole blood, plasma, serum, semen, saliva, tears, urine, fecal material, sweat, buccal, skin, cerebrospinal fluid, bone marrow, bile, hair, muscle biopsy, organ tissue or other material of biological origin known by those of ordinary skill in the art. Biological samples can be obtained from, e.g., biopsies of internal organs or from cancers. Biological samples can be obtained from subjects for diagnosis or research or can be obtained from healthy subjects, as controls or for basic research.

[00263] The "circulatory system of a subject," as used herein, encompasses the space occupied by whole blood and optionally the lymphatic system in a human, inclusive of plasma and all circulating cells and molecules, and distributed throughout arteries, veins, capillaries, and lymphatic vessels of all tissues.

[00264] A "complex" as used herein comprises an association of two or more entities. A complex may comprise one or more polypeptides, nucleic acid, lipids, carbohydrates, inorganic compounds, organic compounds, and the like. A complex can be functional (multiunit polypeptides) or non-functional (e.g., aggregates or precipitates) and may have beneficial or detrimental properties (e.g., immune complexes). Complexes may be naturally occurring or may be man-made or synthetic. Synthetic complexes include higher order entities, e.g., subcellular structures and cells if they comprise a synthetic compound or molecule. The nanomembrane delivery complex is a complex as defined herein.

[00265] The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase

"consisting of excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.

[00266] "Decrease," in the context of a symptom of a treated disease, disorder or condition, refers to a reduction in measurable or conveyable parameters associated with the disease or condition that manifest as symptoms. Examples of measurable parameters are a reduction in the subject's body temperature, a reduction in the concentration of targets in a sample taken from the subject, reduction in the intensity of inflammation or size of an inflamed area, reduction in the number of infiltrating cells, reduction in the number of episodes associated with the disease, disorder or condition, increase/decrease in organ size, weight gain/loss, etc. Examples of conveyable parameters are, e.g., the subject's own assessment of well being and quality of life.

[00267] "Different polypeptide origin" refers to the organism or species from which a genetic sequence encoding the polypeptide, the polypeptide, or portion thereof, is sourced.

[00268] A "domain" is a part of a polypeptide, such as a receiver polypeptide that is generally having a 3 -dimensional structure and may exhibit a distinct activity, function, such as, e.g., a catalytic, an enzymatic, a structural role, or a binding function.

[00269] An "epitope" includes any segment on an antigen to which an antibody or other ligand or binding molecule binds. An epitope may consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. In some embodiments, receivers comprise specific epitopes. In some embodiments, targets comprise specific epitopes.

[00270] As used herein, the term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" encompasses any of the agents approved by a regulatory agency of the US Federal government or listed in the US Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogate the biological activity and properties of the administered compound. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe, non-toxic, and desirable. [00271] The term "exogenous" when used in the context of nucleic acid includes a transgene and recombinant nucleic acids.

[00272] As used herein, the term "expression" refers to the process to produce a polypeptide including transcription and translation. Expression may be, e.g., increased by a number of approaches, including: increasing the number of genes encoding the polypeptide, increasing the transcription of the gene (such as by placing the gene under the control of a constitutive promoter), increasing the translation of the gene, knocking out of a competitive gene, or a combination of these and/or other approaches.

[00273] "Fusion or chimera" is defined as a polypeptide sequence, or corresponding encoding nucleotide sequence, that is derived from the combination of two or more sequences that are not found together in nature. This may be a combination of separate sequences derived from separate genes within the same genome, or from heterologous genes derived from distinctly different species' genomes.

[00274] As used herein, the term "increase," "enhance," "stimulate," and/or "induce" (and like terms) generally refers to the act of improving or increasing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[00275] As used herein, the term "inhibit," "suppress," "decrease," "interfere," and/or "reduce" (and like terms) generally refers to the act of reducing, either directly or indirectly, a concentration, level, function, activity, or behavior relative to the natural, expected, or average, or relative to a control condition.

[00276] As used herein, the terms "isolate", "isolated," and "isolating" or "purify", "purified" and "purifying" as well as "extracted" or 'extracting" are used interchangeably and refer to the state of a preparation (e.g., a plurality of known or unknown amount and/or concentration) of desired nanomembrane delivery complexes, that have undergone one or more processes of purification, e.g., a selection or an enrichment of the desired

nanomembrane delivery complex preparation. In some embodiments, isolating or purifying as used herein is the process of removing, partially removing (e.g. a fraction) of the

nanomembrane delivery complexes from a sample containing producer cells, or substantially removing the nanomembrane delivery complexes from a sample (e.g. substantially in its entirety). In some embodiments, an isolated nanomembrane delivery complex composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In other embodiments, an isolated nanomembrane delivery complex composition has an amount and/or concentration of desired nanomembrane delivery complexes at or above an acceptable amount and/or concentration. In other embodiments, the isolated nanomembrane delivery complex composition is enriched as compared to the starting material (e.g., biological material collected from tissue, bodily fluid, or producer cell preparations) from which the composition is obtained. This enrichment may be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as compared to the starting material. Isolated nanomembrane delivery complex preparations are substantially free of residual biological products. In some embodiments, the isolated nanomembrane delivery complex preparations are 100% free, 99% free, 98%> free, 97% free, 96%> free, or 95% free of any contaminating biological matter. Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites. Substantially free of residual biological products may also mean that the nanomembrane delivery complex composition contains no detectable producer cells and that only

nanomembrane delivery complexes are detectable.

[00277] As used herein, "a mammalian subject" includes all mammals, including without limitation, humans, domestic animals (e.g., dogs, cats and the like), farm animals (e.g., cows, sheep, pigs, horses and the like) and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea pigs and the like). The terms "individual," "subject," "host," and "patient," are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments the subject is a human.

[00278] "Medical device" refers to any device, apparatus or machine used to deliver a dose of a nanomembrane delivery complex and/or a therapeutic agent. This includes containers, bottles, vials, syringes, bags, cartridges, cassettes, magazines, cylinders, or canisters.

[00279] "Medical kit" refers to a packaged unit that includes a medical device, applicator, appropriate dosage of nanomembrane delivery complex optionally including a therapeutic agent, and relevant labeling and instructions. [00280] As used herein, the term "modulate," "modulating", "modify," and/or

"modulator" generally refers to the ability to alter, by increase or decrease, e.g., directly or indirectly promoting/stimulating/upregulating or interfering with/inhibiting/downregulating a specific concentration, level, expression, function or behavior, such as, e.g., to act as an antagonist or agonist. In some instances a modulator may increase and/or decrease a certain concentration, level, activity or function relative to a control, or relative to the average level of activity that would generally be expected or relative to a control level of activity.

[00281] "Membrane" as used herein is a boundary layer that separates an interior space from an exterior space comprising one or more biological compounds, typically lipids, and optionally polypeptides. Membranes can be lipid bilayers. Included in the definition of membrane are cell membranes comprising, e.g., a phospholipid bilayer and cell membrane associated polypeptides. The nanomembrane delivery complex comprises a membrane as defined herein.

[00282] "Nanomembrane delivery complex" as used herein is a small (between 10-1000 nm in diameter) particle comprising a membrane that encloses an internal space. The complex comprises at least one lipid or fatty acid and polypeptide and optionally comprises a payload (e.g. a therapeutic agent or imaging agent), a receiver (e.g. a targeting moiety), a polynucleotide (e.g. a nucleic acid, RNA or DNA), a sugar (e.g. a simple sugar,

polysaccharide or glycan) or other molecules. The nanomembrane delivery complex can be naturally occurring or synthetic. The nanomembrane delivery complex may be derived from a producer cell.

[00283] The phrase "nucleic acid molecule" refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. It includes chromosomal DNA and self- replicating plasmids, vectors, m NA, tRNA, siRNA, etc. which may be recombinant and from which exogenous polypeptides may be expressed when the nucleic acid is introduced into a cell.

[00284] A "payload" as used herein is a therapeutic agent that acts on a target (e.g. a target cell) that is contacted with the nanomembrane delivery complex. Payloads that may be introduced into a nanomembrane delivery complex and/or a producer cell include therapeutic agents such as, nucleotides (e.g. nucleotides comprising a detectable moiety or a toxin or that disrupt transcription), nucleic acids (e.g. DNA or mRNA molecules that encode a

polypepetide such as an enzyme, or RNA molecules that have regulatory function such as miR A, dsDNA, IncRNA, siRNA), amino acids (e.g. amino acids comprising a detectable moiety or a toxin or that disrupt translation), polypeptides (e.g. enzymes), lipids,

carbohydrates, and small molecules (e.g. small molecule drugs and toxins). The payload may comprise nucleotides, e.g. nucleotides that are labeled with a detectable or cytotoxic moiety (e.g. a radiolabel).

[00285] The term "pharmaceutically-acceptable" and grammatical variations thereof, refers to compositions, carriers, diluents and reagents capable of administration to or upon a subject without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[00286] As used herein, the term "pharmaceutical composition" refers to one or more of the compounds described herein, such as, e.g., a nanomembrane delivery complex mixed or intermingled with, or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients. One purpose of a pharmaceutical composition is to facilitate administration of preparations of nanomembrane delivery complexes to a subject.

[00287] A "producer cell" (or "parent cell") is any cell from which a nanomembrane delivery complex can be isolated. A producer cell is a cell which serves as a source for the nanomembrane delivery complex membrane. A producer cell may share a protein, lipid, sugar, or nucleic acid component with the nanomembrane delivery complex. In some embodiments the producer cell is a modified or synthetic cell. In some embodiments, the producer cell is a cultured or isolated cell.

[00288] A "receiver," as used herein, is an entity (or targeting moiety) capable of interacting with a target, e.g., to associate with or bind to a target. A receiver can comprise or can consist essentially of a polypeptide. In some embodiments, the receiver comprises a polypeptide, a carbohydrate, a nucleic acid, a lipid, a polysaccharide (e.g., a glycan moiety), a small molecule, or a combination thereof. A receiver is a synthetic molecule. In some embodiments, a receiver polypeptide is encoded by an exogenous nucleic acid (e.g. DNA or mRNA). In some embodiments, producer cells may comprise a receiver. In some

embodiments, the nanomembrane delivery complexes may comprise a receiver.

[00289] As used herein, the term "substantially" or "substantial" refers, e.g., to the presence, level, or concentration of an entity in a particular space, the effect of one entity on another entity, or the effect of a treatment. For example, an activity, level or concentration of an entity is substantially increased if the increase is 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50- fold, 100-fold, or 1000-fold relative to a baseline. An activity, level or concentration of an entity is also substantially increased if the increase is 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, or 500% relative to a baseline.

[00290] "Synthetic" refers to a compound or molecule that is either man-made and non- naturally occurring, or if it is naturally occurring is placed in a context or location that it would not naturally exist, or if it naturally exists in the context or location is in a state of purity, or is present in an amount, concentration or number that it would not naturally be present in the context or location. Synthetic entities can be isolated or purified compounds that are optionally chemically or enzymatically modified from their natural state, exogenous nucleic acids, exogenous (heterologous) receivers, payloads (therapeutic agents) and the like. The presence of a synthetic compound or molecule, as defined herein, in any entity renders the entire entity "synthetic". For example, a producer cell or nanomembrane delivery complex comprising a payload and/or a receiver is synthetic.

[00291] A "target cell" (or "recipient cell") refers to a cell that interacts with, binds or associates with, receives a payload or uptakes a nanomembrane delivery complex. This may occur in vitro or in vivo, e.g. in a subject. More generally, a "target," as used herein, is an entity capable of interacting with a receiver and/or a nanomembrane delivery complex without a receiver. A target includes, but is not limited to a polypeptide (e.g., an antibody or antibody-related polypeptide, a complement constituent, an amyloid protein, a pathogen, a toxin, a prion), a molecule (e.g., a metabolite, a steroid, a hormone, a carbohydrate; an oligosaccharide; a chemical; a polysaccharide, a DNA; an R A; a lipid, an amino acid, an element, a toxin or pathogen), a complex (e.g., an immune complex), or a cell (e.g., a cancer cell, a macrophage, a bacterium, a fungus, a virus, or a parasite). A target is intended to be detected, diagnosed, impaired, destroyed or altered (e.g., functionally complemented) by the methods provided herein.

[00292] A "therapeutic agent" or "therapeutic molecule" includes a compound or molecule that, when present in an effective amount, produces a desired therapeutic effect, pharmacologic and/or physiologic effect on a subject in need thereof. It includes any compound, e.g., a small molecule drug, or a biologic (e.g., a polypeptide drug or a nucleic acid drug) that when administered to a subject has a measurable or conveyable effect on the subject, e.g., it alleviates or decreases a symptom of a disease, disorder or condition. [00293] "Transgene" or "exogenous nucleic acid" refers to a foreign or native nucleotide sequence that is introduced into a nanomembrane delivery complex. Transgene and exogenous nucleic acid are used interchangeably herein and encompass recombinant nucleic acids.

[00294] As used herein, "treat," "treating," and/or "treatment" are an approach for obtaining beneficial or desired clinical results, pharmacologic and/or physiologic effect, e.g., alleviation of the symptoms, preventing or eliminating said symptoms, and refer to both therapeutic treatment and prophylactic or preventative treatment of the specific disease, disorder or condition. Beneficial or desired clinical results, pharmacologic and/or physiologic effect include, but are not limited to, preventing the disease, disorder or condition from occurring in a subject that may be predisposed to the disease, disorder or condition but does not yet experience or exhibit symptoms of the disease (prophylactic treatment), alleviation of symptoms of the disease, disorder or condition, diminishment of extent of the disease, disorder or condition, stabilization (i.e., not worsening) of the disease, disorder or condition, preventing spread of the disease, disorder or condition, delaying or slowing of the disease, disorder or condition progression, amelioration or palliation of the disease, disorder or condition, and combinations thereof, as well as prolonging survival as compared to expected survival if not receiving treatment.

[00295] The term "therapeutically effective amount" or "effective amount" is an amount of an agent being administered to a subject sufficient to effect beneficial or desired clinical results, pharmacologic and/or physiologic effects. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. The effective amount thus refers to a quantity of an agent or frequency of administration of a specific quantity of an agent sufficient to reasonably achieve a desired therapeutic and/or prophylactic effect. For example, it may include an amount that results in the prevention of, treatment of, or a decrease in, the symptoms associated with a disease or condition that is being treated, e.g., the diseases or medical conditions associated with a target. The amount of a therapeutic composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, pathologic conditions, diets, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. Further, the effective amount will depend on the methods of formulation and administration used, e.g., administration time, administration route, excretion speed, and reaction sensitivity. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

[00296] A "variant" is a polypeptide which differs from the original protein by one or more amino acid substitutions, deletions, insertions, or other modifications. These modifications do not significantly change the biological activity of the original protein. In many cases, a variant retains at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the biological activity of original protein. The biological activity of a variant can also be higher than that of the original protein. A variant can be naturally- occurring, such as by allelic variation or polymorphism, or be deliberately engineered. The amino acid sequence of a variant is substantially identical to that of the original protein. In many embodiments, a variant shares at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99%, or more global sequence identity or similarity with the original protein. Sequence identity or similarity can be determined using various methods known in the art, such as Basic Local Alignment Tool (BLAST), dot matrix analysis, or the dynamic programming method. In one example, the sequence identity or similarity is determined by using the Genetics Computer Group (GCG) programs GAP (Needleman-Wunsch algorithm). The amino acid sequences of a variant and the original protein can be substantially identical in one or more regions, but divergent in other regions.

[00297] As used herein, the term "vector" is a nucleic acid molecule, preferably self- replicating, which transfers and/or replicates an inserted nucleic acid molecule, such as a transgene or exogenous nucleic acid into and/or between host cells. It includes a plasmid or viral chromosome into whose genome a fragment of recombinant DNA is inserted and used to introduce recombinant DNA, or a transgene, into a nanomembrane delivery complex.

EXAMPLES

[00298] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the invention in any way. The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); Green & Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition (Cold Spring Harbor Laboratory Press, 2012); Colowick & Kaplan, Methods In Enzymology (Academic Press); Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, 2012); Sundberg & Carey, Advanced Organic Chemistry: Parts A and B, 5th Edition (Springer, 2007).

Example 1 : Contacting of Producer Cells or Nanomembrane Delivery Complexes

Nucleic Acid - Transfection

[00299] The nucleic acid of interest is scaled up to provide approximately 5 ug nucleic acid per 10^A5 producer cells or complexes to be loaded. The nucleic acid is diluted in Opti- MEM Medium (Life Technologies) at a ratio of 1 ug to 50 uL medium. The diluted nucleic is then combined with a transfection reagent (Trans-IT for DNA, Trans-IT mRNA for mRNA, Trans-IT siRNA for siRNA, Minis Bio) at a 1 : 1 volume ratio and allowed to form complexes for 5 minutes at room temperature. The nucleic acid complex is added to cells for 12-24 hours. Optionally, after this period of time, the media can be exchanged with fresh media such that the transfection reagents are no longer present.

Nucleic Acid - Viral Transduction

[00300] The gene of interest is cloned into the multiple cloning site of lentivirus vector pCDH with the MSCV promoter sequence from System Biosciences. Lentivirus is produced in 293T cells by transfecting the cells with lipofectamine. 5χ10^Λ6 293T cells (Lenti-X 293T Cell Line, Clontech catalog #632180) are plated in a P10 petri dish the day before

transfection. Cell confluency should be around 70%. One plate is transfected per construct. 20 μΐ (10 μg) pPACKHl (System Biosciences) plasmid mix + 2 μg lenti construct + 20 μΐ Plus reagent (LifeTechnologies, Catalog # 11514-015) are combined in 400 μΐ Optimem and incubated 15 min at RT. 30 μΐ of LF2000 (LifeTechnologies, Catalog # 11668-019) is diluted into 400 μΐ Optimem, added dropwise to DNA mix, and incubated for 15 min RT. DNA mix is added to cells (cells are in 9 ml of Optimem). Cells are incubated for 6 hours and then the medium is changed to DMEM/10% FBS. The virus supernatant is collected 48 hours post- transfection by centrifugation at 1,500 rpm for 5 minutes. The supernatant is collected and frozen in 1 ml aliquots at -80°C. Producer cells are transduced at day 3-7 of the culture process described herein. 5xlO^A5 cultured cells are plated in 500 μΐ, of medium containing 20 μg/mL polybrene in a 24-well plate. For each virus, cells are transduced in triplicate wells. Virus supernatant is added in another 500 μΐ_^ of medium and the sample is mixed by pipetting. Infection is achieved by spinoculation, spinning the plate at 2000 rpm for 90 minutes at room temperature. After spinoculation, the cells are incubated at 37C overnight, and the next day 1 mL of fresh IMDM medium with appropriate cytokines is added.

Nucleic Acid - Cationic Polymer

[00301] An mRNA ecoding the transgene of interest and including an upstream promoter sequence and a downstream poly A tail can be purchased from multiple commercial vendors (e.g., IDT-DNA, Coralville IA). RNA transfections are carried out using RNAIMax

(Invitrogen, Carlsbad, Calif.) or TRANSIT-mRNA (Mims Bio, Madison, Wis.) cationic lipid delivery vehicles. RNA and reagent are first diluted in Opti-MEM basal media (Invitrogen, Carlsbad, Calif). 100 ng/uL RNA is diluted 5 x and 5 μΐ,, of RNAIMax per μg of RNA is diluted 10^χ. The diluted components are pooled and incubated 15 minutes at room temperature before they are dispensed to culture media. For TRANSIT-mRNA transfections, 100 ng/uL RNA is diluted 10* in Opti-MEM and BOOST reagent is added (at a

concentration of 2 μΕ, per μg of RNA), TRANSIT-mRNA is added (at a concentration of 2 μί, per μg of RNA), and then the RNA-lipid complexes are delivered to the culture media after a 2-minute incubation at room temperature. RNA transfections are performed in Nutristem xenofree hES media (STEMGENT®, Cambridge, Mass.) or Opti-MEM plus 2% FBS. Successful introduction of the mRNA transcript into producer cells can be monitored using various known methods, such as a fiuorescent label or reporter protein, such as Green Fluorescent Protein (GFP). Successful transfection of a modified mRNA can also be determined by measuring the protein expression level of the target polypeptide by e.g., Western Blotting or immunocytochemistry. Similar methods may be followed for large volume scale-up to multi-liter (5-10,000 L) culture format following similar RNA-lipid complex ratios.

Nucleic Acid - Electroporation

[00302] mRNA ecoding the transgene of interest and including an upstream promoter sequence and a downstream poly A tail can be purchased from multiple commercial vendors (e.g., IDT-DNA, Coralville IA). Electroporation parameters are optimized by transfecting producer cells with mRNA transcripts and measuring transfection efficiency by quantitative RT-PCR with primers designed to specifically detect the exogenous transcripts. For certain cells preparations, discharging a 150 uF capacitor into 2.5>< 10^A6 cells suspended in 50 μΐ of Opti-MEM (Invitrogen, Carlsbad, Calif.) in a standard electroporation cuvette with a 2 mm gap is sufficient for repeated delivery in excess of 10,000 copies of modified mRNA transcripts per cell, as determined using the standard curve method, while maintaining high viability (>70%). Cell density may vary from 1 x 10^Λ6 cell/50 μΐ to a density of 2.5 x 10^Λ6 cells/500 and require from 110V to 145 V to trans feet cells with similar efficiencies measured in transcript copies per cell. Large multi-liter (5-10,000 L) electroporation may be performed similar to large volume flow electroporation strategies similar to methods described with the above described constraints (Li et al, 2002; Geng et al, 2010).

Polypeptide - Liposome

[00303] Producer cells and nanomembrane delivery complexes can be loaded with exogenous protein on their surface and in their interior space. The loading of proteins can be performed using liposomes. Lipids (Pro-Ject reagent, Pierce) in organic solvent are dried under nitrogen into a thin film in glass scintillation vial. Approximately 2 uL lipids are used per 10^A5 cells. Polyclonal mouse IgG (Abeam) is labeled with Dylight-650 (Pierce) per manufacturer's instructions. Protein solution at 0.1 mg/mL in PBS is added to the dried lipid mixture. The solution is pipetted several times, incubated for 5 minutes at room temperature, then vortexed vigorously to generate encapsulating liposomes. Serum-free medium is added to bring the total volume to 500 uL per 10^A5 cells. The liposomal mixture is then incubated with the cells for 3-4 hours at 37C.

Polypeptide - Surface conjugation

[00304] The producer cell or nanomembrane delivery complex surface is treated with Traut's reagent (2-iminothiolane HC1, Pierce) to thiolate primary amines. Traut's reagent is dissolved in Tris buffer pH 8 with EDTA to prevent oxidation of sulfhydryls. Approximately 1 pmol Traut's reagent is used to treat 10^A6 cells or complexes. Incubate Traut's reagent with cells or complexes for 1 hour at room temperature. Remove excess or unreacted reagent by centrifugation and washing the cells. The number of available sulfhydryl groups can be measured using Ellman's Reagent. Polypeptides suitable for conjugation are treated with amine-to -sulfhydryl crosslinker, such as SMCC (Pierce) according to manufacturer's instructions. Excess crosslinking reagent is removed by desalting. The maleimide- functionalized protein is then incubated with the thiolated cells for several hours. Unreacted protein is separated from the conjugated cells by centrifugation and washing.

Polypeptide - Lipid insertion into membrane

[00305] Traut's reagent (Thermo Fisher) is used to generate sulfhydryl groups on an amine-containing suitable polypeptides following manufacturer's protocol. The reaction mixture is incubated for 1 h at room temperature (RT) on a shaker and washed through a spin desalting column (Zeba, MWCO 7K, Thermo Scientific) following the manufacturer's instructions to remove the unreacted Traut's reagent. The generation of sulfhydryl groups on the modified polypeptide is quantified using Ellman's Reagent (Pierce) based on the manufacturer's protocol. DSPE-PEG3₄oo-mal (1 x 10^A- 3 M in PBS, 4 μ L, molar ratio lipid:Polypeptide = 1 : 1) (all lipids purchased from Avanti Polar Lipids and stored as chloroform solution under argon at -20 C) are added to the desalted polypeptide solution and incubated at RT on a shaker. After 1 h, the sample solution is filtered using a centrifugal filter device (Microcon, Millipore Co.) at 14 000 g for 15 min at 4 ° C to remove the small molecules and suspended in 600 μ L PBS (1 mg/rnL polypeptide). 200 μ L of producer cells or nanomembrane delivery complexes are suspended in 1000 μ L PBS and spun at 1500 g for 30 s, repeated four times and suspended in 800 μ L PBS. The conjugation of producer cells or nanomembrane delivery complexes with DSPE-PEG-polypeptide is prepared by mixing the suspensions and various amounts of DSPE-PEG-Polypeptide solution (1 mg per mL) followed by incubation for 15-30 min at 37 ° C. The mixture is kept for 5 min at room temperature, then washed three times in PBS and resuspended to a final cell or complex concentration of 5 x 10^A8 per mL. An automated cell counter (Countess, Invitrogen) is used to measure the cell or complex concentration.

Polypeptide - Cell-penetrating peptide

[00306] The manufacture of protamine-conjugated polypeptide is known in the art, see e.g., Kwon et al. 2009 J Contr Rel 139(3): 182. 5 mg/ml of Low Molecular Weight Protamine (LMWP) in 50 mM HEPES buffer (pH 8) is mixed with the heterobifunctional cross-linker 3- (2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP, Sigma-Aldrich) at a 1 : 10 molar ratio in DMSO and shaken for 1 h at room temperature. The reaction mixture is then treated with 50 mM dithiothreitol (DTT, Sigma-Aldrich) and the thiolated LMWP is purified by HPLC on a heparin affinity column. The product is collected by ultrafiltration, lyophilized, and stored at -20°C until further use. For conjugation, 5 mg/ml suitable polypeptide is mixed with SPDP (40 μΐ of 0.1 M SPDP in ethanol to 1 ml protein solution) in phosphate buffer, and stirred at room temperature for 1 h. Unreacted SPDP is removed by rapid desalting and buffer exchange by FPLC with 0.1 M phosphate buffer (pH 7.4).

Activated polypeptide is then conjugated with a 10-fold molar excess of the above -prepared LMWP-SH for 24 h at 4°C. The LMWP -polypeptide conjugates are isolated by ion-exchange chromatography using a heparin affinity column followed by five rounds of centrifugal filtration (molecular weight cut-off: 5,000 Da). Pooled LMWP -polypeptide conjugates are concentrated, and the degree of conjugation determined by MALDI-TOF mass spectroscopy. For uptake experiments, producer cells or nanomembrane delivery complexes are incubated with a 0.5 mg/ml solution of the LMWP -polypeptide conjugates for 30 min at room temperature under gentle shaking, the washed and stored at 2-8 C.

Polypeptide - Enzymatic conjugation

[00307] Cell surface enzymatic conjugations with sortase are known in the art, see e.g., Shi et al PNAS 2014 111(28): 10131. To label the N-terminus of a surface polypeptide of the producer cell or nanomembrane delivery complex with polypeptide, 30 uL of 500 uM S aureus sortase and 1 mM polypeptide with LPETGG at the C terminus is preincubated in 50 mM Tris pH 7.5, 150 mM NaCl, on ice for 15 minutes and added to 5xlO^A7 producer cells or nanomembrane delivery complexes in a suitable buffer. The sortase and cell or complex mixture is incubated on ice for 30 min with occasional gentle mixing, then spun at 500 xg for 2 min at 4C to remove buffer, then washed three times with 1 mL of ice-cold PBS.

Small Molecule (producer cell cytoplasm or nanomembrane delivery complex interior)

[00308] Liposomal Project reagent (Pierce) is dried under nitrogen into a thin film in glass scintillation vials. Approximately 2 uL reagent is needed per 10^A5 producer cells or nanomembrane delivery complexes. Solution of small molecule of interest in PBS is added to the dried liposome reagent. The solution is pipetted several times, incubated for 5 minutes at room temperature, then vortexed vigorously to generate encapsulating liposomes. Serum-free medium is added to bring the total volume to 500 uL per 10^A5 cells or complexes. The liposomal mixture is incubated with the cells for 3-4 hours at 37°C.

Small Molecule (producer cell or nanomembrane delivery complex surface)

[00309] The conjugation of small molecules to the surface of producer cells or

nanomembrane delivery complexes using chemical functionalities is well known in the art, see e.g., Hermanson GT, Bioconjugation Techniques 2^nd Ed, ISBN 978-0123705013. Briefly, the small molecule of interest is provided with an amine-reactive functional group, such as NHS ester, for example NHS ester biotin (Pierce). The small molecule of interest is stored in organic solvent to prevent hydrolysis of the NHS ester functional group. The small molecule of interest is incubated with cells or complexes in aqueous medium in large molar excess (at least 10 pmol for 10^A6 cells) to drive conjugation to primary amines on the cell surface. After 1 hr incubation, the excess unreacted molecule is removed by centrifugation and washing of the cells or complexes.

Example 2: Assessment of Polypeptide Presence Cell surface proteins

[00310] For cell surface proteins, the level of protein expression can be detected as early as 2 days after transfection by flow cytometry with antibodies specific for the protein or for a co-expressed epitope tag, such as HA-tag. Producer cells are modified with a transgene that is introduced by lentiviral transduction. Two days after transduction, cells are collected, washed in PBS buffer, and stained with 1 :50 dilution of mouse anti-HA antibody (Abeam) for 1 hr. Cells are washed and then stained with a 1 : 100 dilution of alexa 488-labeled goat anti-mouse secondary antibody (Life Technologies) for 30 minutes on ice. Cells are washed and analyzed on a flow cytometer (Attune, Life Technologies). Transduction efficiency is assessed as the percentage of alexa 488-positive cells in the population.

Intracellular proteins

[00311] For intracellular proteins, the level of protein expression can be detected as early as 8-12 hours after transfection by Western Blot. Producer cells are modified with a transgene comprising an HA-tag that is introduced by lentiviral transduction.Two days after

transduction, cells are collected, washed in PBS buffer, and lysed in RIPA cell lysis buffer (Pierce). Cell lysate is denatured by boiling in 100 mM DTT, then loaded onto a NuPage SDS-PAGE pre-cast gel. After electrophoresis and transfer to nitrocellulose membrane, protein bands are developed by staining with 1 :5000 dilution of mouse anti-HA antibody (Abeam) followed by 1 :5000 dilution of goat anti-mouse HRP (Pierce), and subsequent treatment with HRP substrate (SuperSignal, Pierce). Images are captured using an Amersham imager (GE healthcare).

Example 3 : Western blot and separation of proteins from nanomembrane delivery complexes

[00312] For western blot analysis, cells or nanomembrane delivery complex preparations are lysed in lysis buffer [20 mM Tris-HCl (pH 7.4); 140 mM NaCl; 2 mM EDTA; 50 mM NaF; 1% Nonidet P-40; 0.5% Na deoxycholate; 100 μΜ Na3V04; 2 μg ml-lantipain, pepstatin and leupeptin; 1% aprotinin and 1 mM phenylmethylsulfonylfluoride] for 20 min at 4°C. Nuclei and cell debris are removed by centrifugation. Nanomembrane delivery complexes solubilized in lysis buffer or post-nuclear lysates are quantified by Bradford assay, solubilized in Laemmli loading buffer and analyzed under reducing or non-reducing (for tetraspanin) conditions by SDS-PAGE followed by electrob lotting on Immobilon P membrane (Millipore). The same amount of proteins, as measured by Bradford assay, from control cells and pellets of the successive centrifugations are separated on 12% SDSP, transferred to Immobilon P membrane (Millipore) and incubated with specific antibodies followed by HRP-conjugated secondary antibodies and detected using an enhanced chemiluminescence kit (Roche Diagnostics, Meylan, France).

Example 4: Transduction of a Producer Cell with and HA-tagged Tansgene and Isolation of a Nanomembrane delivery complex.

[00313] The experiment was designed to show the ability of a modified, mammalian producer cell line to produce nanomembrane delivery complexes that exhibit an exogenous molecular components of the producer cell. A K562 erythroleukemic cell line was transgenically modified using a lentiviral vector containing an HA transgene that resulted in intracellular expression. Nanomembrane delivery complexes from the modified producer cell line were separated and enriched using standard ultracentrifugation techniques. The isolated nanomembrane delivery complexes were lysed and protein extracts prepared. An ELISA was conducted to detect HA antigen in the nanomembrane delivery complexes derived from the modified producer cell line. The ELISA data suggest that the nanomembrane delivery complexes comprised the HA antigen expressed in the producer cells. Further, it appeared that the HA content was enriched in the nanomembrane delivery complexes relative to the producer cell.

Example 5 : RNA Quantification

[00314] Total RNA is extracted from nanomembrane delivery complexes using TRIzol reagent (Invitrogen), and from the supernatant fraction using mirVana PARIS kit (Ambion, Austin, TX). Reverse transcription reactions are performed with 1 mg total RNA using HiFlex miSCRIPT RTII kit (Qiagen, Hilden, Germany) after DNase I treatment (Invitrogen). Mature miR-223 and 2 selected mRNAs are detected by quantitative PCR (qPCR) using miScript Primer Assay kit and SYBR Green (Qiagen). Small nuclear RNA U6 (RNU6) (for miR-223) and glyceraldehyde-3 -phosphate dehydrogenase (for mRNAs) are used as reference genes for relative quantitation using the 2^A-DDCt method known in the art.

Example 6: RNAseq for miRNAs

[00315] Nanomembrane delivery complex RNA samples isolated from producer cell preparations are prepared using the Small RNA Sample Preparation Kit (Illumina, Inc, San Diego, CA). Sequencing is performed on the Illumina GAIIx with a read length of 36 nt. R A sequences are identified using miRanalyzerl6 based on the bowtie architecture. The number of unique reads are counted, normalized to transcript size, and expressed relative to the total number of reads.

Example 7: RNAseq for RNAs

[00316] PAXgene, cell-free and nanomembrane delivery complex RNA is converted into cDNA libraries using the Ion Total RNA-Seq Kit V2 (Life Technologies, Australia) and prepared for deep sequencing as described previously (Cheng L, Sun X, Scicluna BJ, Coleman BM, Hill AF. Characterization and deep sequencing analysis of nanomembrane delivery complex and non-nanomembrane delivery complex miRNA in human urine. Kidney Int. 2013). Pooled libraries with unique barcodes are loaded on 318 sequencing chips and run on the Ion Torrent Personal Genome Machine (Life Technologies, Australia). The Torrent Suite 3.4.1 is used to manage the Ion Torrent PGM to process raw signals and perform base calling. The sequences are then assessed for quality and primer-adapter sequences are trimmed by the Torrent Suite software, followed by alignment to the human reference genome (HG19). The trimmed and aligned data is transferred to Partek Genomics Suite and mapped to known RNA. The number of reads for each RNA is normalized to reads per million (RPM) across all samples. Samples containing less than 5 RPM are removed. Partek Genomics suite and statistical package are used to perform statistical analysis, hierarchical clustering and to identify unique RNA in each sample type.

Example 8: qRT-PCR miRNA

[00317] miRNA levels of nanomembrane delivery complexes are assessed by quantitative reverse-transcription (qRT)-polymerase chain reaction (PCR) with the use of microRNA LNA Primer sets (Exiqon) specific for hsa-miR-16, -22, -126, -185, -320b, 423-5p, U6 small nuclear RNA, and cel-miR-39-5p. mRNA levels for intercellular adhesion molecule (ICAM- 1) and cyclophilin are analyzed with TaqManAssays (Life Technologies).

Example 9: Proteomic analysis of nanomembrane delivery complexes

LC-MS/MS

[00318] Proteins from nanomembrane delivery complex compositions are prepared from lysates and separated on 4-12% SDS-PAGE gels, fixed and stained with Coomassie blue (Bio-Rad, Hercules, CA). Each gel lane is trimmed in pieces and each was subjected to trypsin digestion. The recovered peptides are subsequently vacuum-dried. Nanoflow LC- MS/MS analysis is performed using a nanoflow UPLC system coupled to a QTOF Premier mass spectrometer (Waters, Manchester, UK). Samples are loaded in 1% aqueous formic acid (FA) and analyzed by reverse phase LC-MS/MS in a UPLC reverse phase chromatography system (Waters). Tryptic peptides are desalted on a Symmetry CI 8 trapping cartridge (Waters) and further separated on an analytical column (Atlantis CI 8, 75 μιη id x 3 μιη, Waters) with an integrated electrospray ionization emitter tip (SilicaTips for Micromass ZSpray NanoFlow, 10 μιη diameter, New Objective). Peptides are eluted at a flow rate of 250 nL/min from the analytical column directly to electrospray ionization emitter tip by using a 30 min gradient from 0 to 30% solvent B (solvent A: 1% aqueous FA and solvent B: 100% acetonitrile, 1%FA). Data is acquired in the data dependent acquisition mode (DDA), in which a full scan mass spectrum (m/z: 300-1500) was followed by MS/MS (m/z: 50-1995) in the three most abundant multi-charged ions (+2 and +3) every 4 s. Argon is used as the collision gas. Collision energies are interpolated linearly as a function of a charge state and m/z of each peptide. Dynamic exclusion is incorporated for 30 seconds. A scan of the reference compound (Glufibrinopeptide B) is acquired every ten scans of the analyte through the entire run. Raw data is processed using ProteinLynx Global Server v2.2.5 (Waters). The resulting pkl file is searched against v52 of SwissProt sequence database (Rattus: 5830 sequences) with rat as taxonomy using an in-house Mascot server (Version 2.2.03, Matrix Sciences, London, UK). One miss cleavage is allowed; carbamidomethyl is chosen as fixed modification and oxidation as variable modification. A peptide mass tolerance of 20 ppm and 0.1 Da of fragment mass tolerance are allowed. Only proteins with at least one specific peptide (with a Mascot colour code of red and bold) are included.

GO slim categorization

[00319] Gene ontology (GO) annotations for proteins in UniProt knowledgebase

(http://www.ebi.ac.uk/GOA/index.html) are used for categorization of the proteins identified by mass spectrometry. In order to calculate the statistical significance of enrichment or depletion of specific GO categories all human proteins from the UniProt knowledgebase are extracted from the same database used for the mass spectrometry data search. The probabilities are calculated using a binomial distribution. Example 10: Co-culture with breast cancer lines to detect nanomembrane delivery complex uptake

[00320] Nanomembrane delivery complexes are stained with green PKH67 fluorescent dye (Sigma- Aldrich). After staining, nanomembrane delivery complexes are washed with phosphate-buffered saline and centrifuged at 120,000g for 70 minutes. One microgram of PKH67-labeled nanomembrane delivery complexes are incubated with 1 x 105 breast cancer cells at 37 °C or 4 °C for 4 hours. The uptake of PKH67-labeled nanomembrane delivery complexes is analyzed using flow cytometry and confocal fluorescence microscopy.

Example 11 : Transfer of modified nanomembrane delivery complexes comprising GFP to ES cells

[00321] Immediately after passaging, 200 ml of ESCs (~10⁶ cells/mL) are transferred to a culture tube in serum- free medium with 1 ml of Vybrant DiD (Molecular Probes). The cells are incubated with nanomembrane delivery complexes for 20 minutes at 37°C, and then washed 2X with pre-warmed DMEM. After the last wash, the cells are resuspended in 200 ml serum- free medium and separated into two tubes. An additional 100 ml of serum- free medium is added to one tube serving as control and 100 ml of nanomembrane delivery complexes containing GFP (collected from 3-4 T175 flasks) are added to the second tube. Both tubes are incubated at 37°C for 3 hrs prior to imaging the cells on a Leica TCS-SP inverted confocal microscope. Images are taken with a 100X, 1.4 NA plan pochromatic objective. Excitation is achieved with the 488nm laser line for GFP and the 633nm laser line for DiD. The spectral detector is set to 498-53 lnm for GFP and 650-714nm for DiD detection. All images are taken with the pinhole set to 1 airy unit. The DiD image is overlayed on the GFP image after digitization using the Leica confocal software or Adobe Photoshop CS2.

Example 12: Transfer of nanomembrane delivery complex miRNAs to embryonic fibroblasts

[00322] Approximately 1.6xl0⁵ cells/well of gamma-irradiated MEFs are pre-plated on 24 well plates at least 2 days prior to initiation of the experiment. Isolated nanomembrane delivery complexes are divided evenly between all samples and co-incubated with the pre- plated MEFs. Growth-arrested MEFs are used to maintain a constant ratio of nanomembrane delivery complexes to MEFs between each experiment. Each experimental series includes a negative control (MEF only) and 4 experimental time points (MEF+nanomembrane delivery complexes). MEFs are collected after 1, 12, 36, and 54 hours. To collect the cells while removing free-floating nanomembrane delivery complexes, MEFs are washed with PBS twice prior to dissociation. Cells are then dissociated from the plates and washed two more times with PBS. After each wash, cells are spun down at 3,500xg for 5 minutes so that any residual nanomembrane delivery complexes would float in the supernatant and be discarded. MEF total R A is isolated and real time quantitative RT-PCR is performed with a subset of miRNAs. As an indirect measure of miRNA transfer, the difference in Ct values is determined between the negative control and each experimental time point; a positive value indicates transfer.

Example 13: Exogenous miRNA Delivery via Nanomembrane Delivery Complexes

[00323] miRNA is loaded into nanomembrane delivery complexes via secretion from the producer cell line. miR-150 contained in the nanomembrane delivery complexes is shown to be delivered into HMEC endothelial cells and functionally represses endogenous protein expression. To demonstrate the direct repression of c-Myb by nanomembrane delivery complexes through elements in the 3 ' UTR, the entire c-Myb 3 ' UTR is cloned into a luciferase reporter plasmid, and the resulting plasmid is transfected into HMEC-1 cells combined with or without treatment of nanomembrane delivery complexes. Producer cells are labeled with Dil-C16 (red) and then cultured in RPMI 1640 medium supplemented with 10% FBS. After 4 hr, the supernatants are collected and centrifuged to isolate nanomembrane delivery complexes. The nanomembrane delivery complexes are resuspended in MCDB-131 medium and incubated with HMEC-1 cells at 4°C or 37°C, respectively. After incubation for 2 hr, HMEC-1 cells are washed, fixed, and observed under confocal microscopy.

Endothelial Cell Migration Assay

[00324] The migration ability of HMEC-1 is tested in a Transwell Boyden Chamber (6.5 mm, Costar). The polycarbonate membranes (8 mm pore size) on the bottom of the upper compartment of the Transwells are coated with 0.1% gelatin matrix. Cells are suspended in serum- free MCDB-131 culture medium at a concentration of 4 X 10⁵ cells/ml, treated with or without nanomembrane delivery complexes for 2 hr and then added to the upper chamber (4X10⁴ cells/well). 0.5 ml of MCDB-131 with 10% FBS is added to the lower compartment, and the Transwell-containing plates are incubated for 4 hr in a 5% C0₂ atmosphere saturated with H₂0. At the end of the incubation, cells that enter the lower surface of the filter membrane are fixed with 90% ethanol for 15 min at room temperature, washed three times with distilled water, and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (non-migrant) are scraped off gently with a cotton swab. Images of migrant cells are captured by a photomicroscope (BX51, Olympus). Cell migration is quantified by blind counting of the migrated cells on the lower surface of the membrane, with five fields per chamber.

Example 14: Administration of nanomembrane delivery complexes to mice to determine localization to endothelium and delivery of RNA

[00325] At 8 weeks of age, mice received tail-vein injections of saline or normal nanomembrane delivery complexes or DiI-C16-labeled nanomembrane delivery complexes. After 6 hr, murine thoracic aorta endothelium is isolated, washed with PBS five times to remove contaminated nanomembrane delivery complexes, and then viewed under fluorescence microscopy. For measurement of RNA levels, total RNA is extracted from thoracic aorta by using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions.

Example 15: Administration of let-7a-containing nanomembrane delivery complexes in a human tumor xenograft model

[00326] Luciferase-expressing HCC70 cells (2x 10⁶) are injected subcutaneously into the mammary fat pads of 5-week-old RAG2-/- mice. Four weeks after transplantation, tumors are sized using an IVIS (Xenogen, Hopkinton, MA). Producer cells are transfected with synthetic let-7a. Let-7a-containing nanomembrane delivery complexes are purified from culture supernatants of the producer cells and intravenously injected (1 ug of purified nanomembrane delivery complexes, once per week for 4 weeks) into mice with transplanted luciferase-expressing HCC70 cells. Let-7a levels in the nanomembrane delivery complex samples are evaluated using TaqMan miRNA assays and real-time PCRs.

In vivo imaging offluorescentlv labeled exosomes

[00327] A stock solution of the lipophilic near-infrared dye XenoLight DiR (Caliper Life Sciences, Hopkinton, MA) is prepared in ethanol. A 300-umol/l working solution is prepared in diluent-C solution (Sigma-Aldrich). Nanomembrane delivery complexes isolated from culture supernatant-derived producer cells are incubated with 2 μιηοΐ/ΐ DiR for 30 minutes. The nanomembrane delivery complexes are then washed with 10 ml of phosphate-buffered saline, subjected to ultracentrifugation, and injected intravenously into RAG2-/- mice (4 μg of exosomes/mouse). Migration of fluorescently labeled nanomembrane delivery complexes in murine organs is detected using an IVIS 24 hours post injection.

In vivo imaging of xenograft tumors

[00328] Mice are anesthetized via isoflurane inhalation, and intraperitoneally injected with 100 ul of 7.5 mg/ml luciferin solution (Promega). Bioluminescence imaging is initiated with an IVIS (Xenogen) 10 minutes post injection. The region of interest is defined manually, and bioluminescence data are expressed as photon flux values (photons/s/cm²/steradian). Background photon flux is defined using an area of the tumor that does not receive an intraperitoneal injection of luciferin. All bioluminescence data is collected and analyzed using an IVIS.

Example 16: Gene expression analysis on target cell line

[00329] Total RNA is isolated from target cell line cultured in presence or absence of producer cell-derived nanomembrane delivery complexes using the miRNeasy Mini kit (QIAGEN) according to the manufacturer's directions.

Microarray

[00330] Microarray gene expression profile is determined using a Human Genome U133 2.0 Plus chip, human transcriptome complete (Affymetrix).

Real-time RT-PCR

[00331] RNA is converted to cDNA using the High-Capacity cDNA Reverse

Transcription kit (Invitrogen). Gene expression of human GAPDH and HBG1/HBG2, mouse GAPDH and TLR2 (primers and probes from Invitrogen), using the TaqMan Gene

Expression Master Mix (Invitrogen), is assessed with real-time PCR (Applied Biosystems 7900 HT Fast Real-Time PCR System with SDS Version 2.2.2 software; Invitrogen).

Example 17: Targeting RNA to brain in vivo

Nanomembrane delivery complexes are generated from a producer cell comprising a receiver that targets the complex to a specific tissue for payload delivery. In this example, the producer cell is transfected with a receiver comprising a membrane protein linked to a neuron-specific peptide, which targets isolated nanomembrane delivery complexes to the brain tissue. Recipient neurons specifically receive a functional RNA payload. Example 18: Producer cell isolation, transfection and nanomembrane delivery complex purification

[00332] Fetal calf serum (FCS) used for nanomembrane delivery complex production is spun at 25,000xg for 90 min before preparation of medium. Primary producer cells are harvested from murine bone marrow and cultured in DMEM Glutamax (Gibco-BRL), 10% FCS and antibiotics, supplemented with 10 ng/ml murine GM-CSF (Sigma-Aldrich).

Producer cells are transfected 4 d after harvesting with 5 μg of pLamp2b derivative plasmids and 5 μΐ of TransIT LTl transfection reagent (Mirus Bio) as per manufacturer's instructions in six-well plates with 3 x 10⁶ cells per well. Cell culture medium is changed on day 7 and cell culture supernatant harvested 24 h thereafter, spun at 12,000xg for 30 min to remove cell debris, then spun again at 120,000xg for lh to pellet nanomembrane delivery complexes. The isolated nanomembrane delivery complexes are then resuspended in 0.1 M ammonium acetate with a 27G syringe. The yield of nanomembrane delivery complexes is about 20-30 μg (based on Bradford measurement) per well.

Other Embodiments

[00333] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[00334] Many modifications and other embodiments of the inventions set forth herein will easily come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[00335] All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. APPENDIX: TABLES

Table 1. Targets

General Classes of Targ ets

Amino

Microbes Polypeptides DNA Acids

Fungi Toxins RNA Prions

Bacteria Lipids Parasites Cytokines

Virus Cells Cellular Debris

Infectious Disease-Related Targets

Cell invasion Secreted effector

Lipopolysaccharides protein Intermedilysin protein sptP

Cholera Invasion protein

Zona occludens toxin enterotoxin sipA Seeligeriolysin

Actin polymerization Iota toxin

protein RickA Cysteine protease component la Serine protease

Actin polymerization Cytolethal

protein RickA distending toxin Ivanolysin Shiga toxin

Adenosine

monophosphate -protein

transferase vopS Cytolysin LepB Sphingomyelinase

Cytotoxic

adenylate cyclase necrotizing factor Lethal factor Staphylokinase

Adenylate cyclase ExoY Cytotoxin Leukotoxin Streptokinase

ADP-ribosyltransferase Dermonecrotic

enzymatic component toxin Listeriolysin Streptolysin

Microbial

Aerolysin Deubiquitinase collagenase Streptopain

Outer membrane

protein IcsA

Alpha-toxin Diphtheria toxin autotransporter Suilysin

Panton-Valentine

Alveolysin Enterohemolysin Leucocidin F Superantigen

T3SS secreted

Alveolysin Enterotoxin Perfringolysin effector EspF

Epidermal cell

differentiation

Anthrolysin 0 inhibitor Pertussis toxin Tetanus toxin

Arp2/3 complex-activating

protein rickA Exoenzyme Phospholipase Tir

Binary ADP- ribosyltransferase CDT Plasminogen

toxin Exotoxin activator TolC

G-nucleotide Toxic shock

Botulinum neurotoxin exchange factor Pneumolysin syndrome toxin

Guanine

nucleotide

exchange factor Zink-

C2 toxin, component II sopE Protective antigen carboxypeptidase Heat stable Zink-

CagA enterotoxin Protein kinase carboxypeptidase

IgA-specific

serine

Calmodulin-sensitive endopeptidase Zn-dependent adenylate cyclase autotransporter Pyolysin peptidase

Inositol

phosphate

Cell cycle inhibiting factor phosphatase sopB RTX toxin

Lipid & Cell Targets

very low density

Circulating tumor cells lipid (VLDL) triglycerides Fatty acids

high density

Metastases lipoprotein chylomicrons Cholesterol low density

Eukaryotic cells lipoprotein apolipoproteins

Table 2. Polypeptide Payloads and Receivers

General Classes

[phosphorylase] Carboxylic-Ester

phosphatase Hydrolases fumarylacetoacetase phosphodiesterase I carboxymethylenebut fusarinine-C phosphoglycerate

1 ,4-lactonase enolidase ornithinesterase phosphatase

11-cis-retinyl- cellulose- phosphoglycolate palmitate hydrolase polysulfatase galactolipase phosphatase l-alkyl-2- acetylglycerophosph cephalosporin-C phosphoinositide ocholine esterase deacetylase gluconolactonase phospholipase C

2'-hydroxybiphenyl-

2-sulfinate glucose- 1- desulfinase cerebroside-sulfatase phosphatase phospho lipase Al

2-pyrone-4,6- dicarboxylate cetraxate glucose-6- lactonase benzylesterase phosphatase phospholipase A2

3', 5'-bisphosphate chlorogenate glutathione

nucleotidase hydrolase thiolesterase phospholipase C

3- hydroxyisobutyryl- glycerol- 1- CoA hydrolase chlorophyllase phosphatase phospholipase D

glycerols- phosphonoacetaldehyd

3 '-nucleotidase cholinesterase phosphatase e hydrolase

glycerophosphochol

3-oxoadipate enol- ine phosphonoacetate lactonase choline-sulfatase phosphodiesterase hydrolase

Glycosidases, i.e.

enzymes that

hydrolyse 0- and S- choloyl-CoA glycosyl phosphonopyruvate

3-phytase hydrolase compounds hydrolase

4-hydroxybenzoyl- phosphoprotein CoA thioesterase chondro-4-sulfatase glycosulfatase phosphatase

4- methyloxaloacetate Phosphoric-diester esterase chondro-6-sulfatase Glycosylases hydrolases

citrate-lyase histidinol- Phosphoric-monoester

4-phytase deacetylase phosphatase hydrolases

hormone-sensitive Phosphoric-triester

4-pyridoxolactonase cocaine esterase lipase hydrolases

Hydrolysing N- glycosyl phosphoserine

5 '-nucleotidase cutinase compounds phosphatase

Hydrolysing S- poly(3-

6-acetylglucose cyclamate glycosyl hydroxybutyrate) deacetylase sulfohydrolase compounds depolymerase

6- poly(3- phosphogluconolact Cysteine hydroxyacylglutathi hydroxyoctanoate) onase endopeptidases one hydrolase depolymerase a-amino-acid Cysteine-type hydroxybutyrate- polyneuridine- esterase carboxypeptidases dimer hydrolase aldehyde esterase a-Amino-acyl- hydroxymethylgluta protein-glutamate peptide hydrolases D-arabinonolactonase ryl-CoA hydrolase methylesterase

quorum-quenching N- acetoacetyl-CoA deoxylimonate A- iduronate-2- acyl-homoserine hydrolase ring-lactonase sulfatase lactonase

acetoxybutynylbithi inositol-phosphate retinyl-palmitate ophene deacetylase dGTPase phosphatase esterase

Serine dehyrdatase or acetylajmaline dihydrocoumarin juvenile-hormone serine hydroxymethyl esterase hydrolase esterase transferase

acetylalkylglycerol

acetylhydrolase Dipeptidases kynureninase Serine endopeptidases serine-

L- ethanolaminephosphat acetylcholinesterase Dipeptide hydrolases arabinonolactonase e phosphodiesterase

Dipeptidyl-peptidases

acetyl-CoA and tripeptidyl- limonin-D-ring- Serine -type hydrolase peptidases lactonase carboxypeptidases

Diphosphoric- S -formy lglutathione acetylesterase monoester hydrolases lipoprotein lipase hydrolase

acetylpyruvate disulfoglucosamine- L-rhamnono- 1 ,4- sialate 0- hydrolase 6-sulfatase lactonase acetylesterase

dodecanoyl-[acyl- acetylsalicylate carrier-protein]

deacetylase hydrolase lysophospholipase sinapine esterase

Site specific

Endodeoxyribonuclea endodeoxyribonucleas ses producing 3'- mannitol-1- es: cleavage is not acetylxylan esterase phosphomonoesters phosphatase sequence specific

Site-specific

Endodeoxyribonuclea endodeoxyribonucleas ses producing 5'- Metallocarboxypept es that are specific for acid phosphatase phosphomonoesters idases altered bases .

Acting on acid

anhydrides to

catalyse Site-specific transmembrane Endopeptidases of endodeoxyribonucleas movement of unknown catalytic Metalloendopeptida es: cleavage is substances mechanism ses . sequence specific

Acting on acid

anhydrides to

facilitate cellular Endoribonucleases methylphosphothio

and subcellular producing 3'- glycerate sphingomyelin movement phosphomonoesters phosphatase phosphodiesterase

Acting on GTP to

facilitate cellular Endoribonucleases

and subcellular producing 5'- methylumbelliferyl- S -succiny lglutathione movement phosphomonoesters acetate deacetylase hydrolase Endoribonucleases

that are active with

either ribo- or

Acting on deoxyribonucleic

phosphorus-nitrogen acids and produce 3'- monoterpene e- bonds phosphomonoesters lactone hydrolase steroid-lactonase

Endoribonucleases

that are active with

either ribo- or

deoxyribonucleic N-

Acting on sulfur- acids and produce 5'- acetylgalactosamine

nitrogen bonds phosphomonoesters -4-sulfatase sterol esterase

N- actinomycin Enzymes acting on acetylgalactosamine

lactonase acid anhydrides -6-sulfatase steryl-sulfatase

N- acylcarnitine Enzymes Acting on acetylgalactosamino succinyl-CoA hydrolase carbon-carbon bonds glycan deacetylase hydrolase

Enzymes acting on

carbon-nitrogen N- bonds, other than acetylglucosamine- sucrose-phosphate acyl-CoA hydrolase peptide bonds 6-sulfatase phosphatase

Enzymes acting on

carbon-phosphorus N-sulfoglucosamine

acylglycerol lipase bonds sulfohydrolase sugar-phosphatase acyloxyacyl Enzymes acting on oleoyl-[acyl-carrier- Sulfuric-ester hydrolase carbon-sulfur bonds protein] hydrolase hydrolases acylpyruvate Enzymes Acting on

hydrolase ether bonds Omega peptidases tannase

Enzymes acting on orsellinate-depside

ADAMTS13 halide bonds hydrolase Thioester hydrolases

Enzymes acting on Thioether and

Adenosine peptide bonds trialkylsulfonium deaminase (peptidases) oxaloacetase hydrolases adenylyl- [glutamate— Enzymes acting on

ammonia ligase] phosphorus-nitrogen palmitoyl[protein] Threonine hydrolase bonds hydrolase endopeptidases

ADP-dependent

medium-chain-acyl- Enzymes acting on palmitoyl-CoA thymidine

CoA hydrolase sulfur-nitrogen bonds hydrolase phosphorylase

ADP-dependent

short-chain-acyl- Enzymes acting on

CoA hydrolase sulfur-sulfur bonds pectinesterase trehalose-phosphatase

ADP- phosphoglycerate Peptidyl peptide

phosphatase Ether hydrolases . hydrolases triacetate-lactonase

Exodeoxyribonucleas

es producing 5'- Peptidyl-amino-acid Triphosphoric- alkaline phosphatase phosphomonoesters hydrolases monoester hydrolases Exonucleases that are

active with either

ribo- or Peptidylamino-acid

deoxyribonucleic hydrolases or

all-trans-retinyl- acids and produce 3'- acylamino-acid

palmitate hydrolase phosphomonoesters hydrolases trithionate hydrolase

Exonucleases that are

active with either

ribo- or

deoxyribonucleic

aminoacyl-tRNA acids and produce 5'- Peptidyl- hydrolase phosphomonoesters dipeptidases tropinesterase

Exoribonucleases

producing 3'- phenylacetyl-CoA

Aminopeptidases phosphomonoesters hydrolase ubiquitin thiolesterase

Exoribonucleases

producing 5'- Phenylalanine UDP-sulfoquinovose arylesterase phosphomonoesters . ammonia lyase synthase

Phenylalanine

arylsulfatase Factor IX hydroxylase uricase

Asparaginase Factor VIII pheophorbidase uronolactonase

Aspartic fatty-acyl-ethyl-ester

endopeptidases synthase phloretin hydrolase wax-ester hydrolase phorbol-diester xylono-1,4- b-diketone hydrolase hydrolase lactonase

Childhood Carcinoma Ureter,

Involving NUT Gene Transitional Cell

Cancer

Atypical

Teratoid/Rhabd

oid Tumor,

Childhood Esophageal Cancer Molar pregnancy Retinoblastoma

Basal Cell Esthesioneuroblastoma, Mouth and Rhabdomyosarco

Carcinoma Childhood oropharyngeal cancer ma

Multiple Endocrine

Neoplasia Syndromes, Salivary Gland

Bile duct cancer Ewing sarcoma Childhood Cancer

Multiple

Extragonadal Germ Cell Myeloma/Plasma Cell

Bladder cancer Tumor Neoplasm Sarcoma

Extrahepatic Bile Duct

Bone cancer Cancer Mycosis Fungoides Secondary cancers

Myelodysplasia

Bowel cancer Eye Cancer Syndromes Sezary Syndrome

Brain Stem Myelodysplastic/Myel

Glioma, oproliferative

Childhood Gallbladder Cancer Neoplasms Skin Cancer

Myeloproliferative Skin cancer (non

Brain tumours Gastric cancer Disorders, Chronic melanoma)

Nasal Cavity and

Gastrointestinal Carcinoid Paranasal Sinus Small Cell Lung

Breast cancer Tumor Cancer Cancer

Bronchial

Tumors, Nasopharyngeal Small Intestine

Childhood Germ Cell Tumor cancer Cancer

Burkitt Gestational trophoblastic Soft Tissue

Lymphoma tumours (GTT) Neuroblastoma Sarcoma

Cancer of

unknown Non-Hodgkin Squamous Cell primary Glioma Lymphoma Carcinoma

Squamous Neck

Cancer with

Cancer spread Non- Small Cell Lung Occult Primary, to bone Hairy cell leukaemia Cancer Metastatic

Cancer spread Stomach (Gastric) to brain Head and neck cancer Oesophageal cancer Cancer

Cancer spread

to liver Heart Cancer, Childhood Oral Cancer Stomach cancer

T-Cell

Lymphoma, Cutaneous - see

Mycosis

Cancer spread Hepatocellular (Liver) Fungoides and to lung Cancer Oral Cavity Cancer Sezary Syndrome Carcinoid Histiocytosis, Langerhans

Tumor Cell Oropharyngeal Cancer Testicular cancer

Carcinoma of

Unknown Osteosarcoma (Bone

Primary Hodgkin Lymphoma Cancer) Throat Cancer

Cardiac (Heart) Osteosarcoma and Thymoma and

Tumors, Malignant Fibrous Thymic

Childhood Hypopharyngeal Cancer Histiocytoma Carcinoma

Central Nervous

System Atypical

Teratoid/Rhabd

oid Tumor,

Childhood Intraocular Melanoma Ovarian Cancer Thyroid Cancer

Central Nervous

System Transitional Cell

Embryonal Islet Cell Tumors, Cancer of the

Tumors, Pancreatic Neuroendocrine Renal Pelvis and

Childhood Tumors Pancreatic Cancer Ureter

Pancreatic

Central Nervous Neuroendocrine

System, Tumors (Islet Cell Unknown primary Childhood Kidney cancer Tumors) cancer

Ureter and Renal

Pelvis,

Langerhans Cell Papillomatosis, Transitional Cell

Cervical cancer Histiocytosis Childhood Cancer

Chordoma,

Childhood Laryngeal Cancer Paraganglioma Urethral Cancer

Choriocarcinom Uterine Cancer, a Leukemia Parathyroid Cancer Endometrial

Chronic

Lymphocytic

Leukemia

(CLL) Lip and Oral Cavity Cancer Penile Cancer Uterine Sarcoma

Chronic

myeloid

leukaemia

(CML) Liver cancer Pharyngeal Cancer Vaginal cancer

Chronic

Myeloproliferati Lobular Carcinoma In Situ

ve Disorders (LCIS) Pheochromocytoma Vulvar Cancer

Waldenstrom

Low Malignant Potential Macroglobulinemi

Colon cancer Tumor Pituitary Tumor a

Plasma Cell

Neoplasm/Multiple

Lymphoma Lung Cancer Myeloma Wilms Tumor

Complement and Immune Complex-Related Diseases

Age-related ANCA-associated vasculitis Glomerulonephritis - MYH9-related macular (Includes Pauci-immune) sparse hair - disease degeneration telangiectasis

Atypical

hemolytic Anti-glomerular basement

uremic membrane disease Goodpasture's Nail-patella syndrome (Goodpasture's) sndrome syndrome

Autoimmune Granulomatosis with

hemolytic polyangiitis (ANCA Nail-patella-like anemia Arthus Reaction and Wegeners) renal disease

CI inhibitor Guillain-Barre

deficiency Asthma syndrome Nephritis

Non-amyloid monoclonal

Atypical hemolytic uremic Hemolytic immunoglobulin

Clq deficiency syndrome angioedema (HAE) deposition disease

Autoimmune inner ear

disease (AIED) Henoch-Schonlein Pauci-immune

Clr deficiency Sensorineural hearing loss purpura glomerulonephritis

Pediatric systemic lupus

Cls deficiency Autoimmune uveitis HIVICK erythematosus

Autosomal dominant

intermediate Charcot- Hypersensitivty

C2 deficiency Marie-Tooth disease type E vasculitis Pierson syndrome

Hypocomplementemic

C3 deficiency Behcet disease urticarial vasculitis Polyarteritis

Idiopathic

membranous polyarteritis

C4 deficiency Berger (IgA) Nephropathy glomerulonephritis nodosa

Idiopathic nephrotic Polymyalgia

C5 deficiency Buergers disease syndrome rheumatica

Central nervous system IgA nephropathy

C6 deficiency vasculitis (Berger's disease) Polymyositis

IgA

nephropathy/vasculitis

(Henoch- S chonlein Polymyositis/ derm

C7 deficiency Choroiditis purpura) atomyositis

Chronic demyelinating Immune Poststaphilococcal

C8 deficiency polyneuropathy (CIDP) thrombocytopenia glomerulonephritis

Immunobullous Poststeptococcal

C9 deficiency Churg-strauss syndrome diseases glomerulonephritis

Primary

Immunotactoid or membranoprolifer

CD55 fibrillary ative

deficiency Cogan's syndrome glomerulopathy glomerulonephritis

Rapidly progressive

CD59 Collagen type III Infection-related glomerulonephritis deficiency glomerulopathy glomerulonephritis (Crescentic) Rapidly

Complement progressive Factor I Congenital and infantile Inflammatory glomerulonephritis deficiency nephrotic syndrome myopathies (RPGN)

Congenital membranous

Complement nephropathy due to

factor-H related maternal anti-neutral

l(CFHRl) endopeptidase Juvenile Rasmussen deficiency alloimmunization dermatomyositis syndrome

Complement

factor-H related

3(CFHR3) Cryoglobulinaemia/Cold

deficiency agglutinin diease Juvenile polymyositis Reactive arthritis

CR3/CR4

defieciency

(leukocyte

adhesion Relapsing deficiency 1) Cryoglobulinemic vasculitis Kawasaki disease polychondritis

Factor B Lipoprotein

deficiency Cutaneous vasculitis glomerulopathy Renal amyloidosis

Factor D Demyelinating myopathies Reynolds deficiency (paraprotein associated) Lupus nephritis syndrome

Factor H Rheumatoid deficiency Denys-Drash syndrome Lupus nephropathy arthritis

Sarcoidosis (Nesnier Boeck

Factor I Schuamann deficiency Dermatomyositis May Hegglin anomaly Disease)

Ficolin 3 Membranoglomerular Schimke immuno- deficiency Dermatomyositis nephritis osseous dysplasia

MASP2 Membranoproliferativ

deficiency Diabetic nephropathy e glomerulonephritis Scleroderma

Membranoproliferativ

e glomerulonephritis

Drug-induced immune Type I (MPGN Type Sebastian

MBL deficiency complex vasculitis I) syndrome

Membranoproliferativ

Eosinophilic e glomerulonephritis

granulomatosis with Type II (Dense

Non-alcoholic polyangiitis (Churgg- Deposit Disease, Secondary steatohepatitis Strauss) MPGN Type II) amyloidosis

Membranoproliferativ

Paroxysmal e glomerulonephritis Severe or nocturnal Type III (MPGN Type recurring C diff hemoglobinuria Epstein Syndrome HI) colitis

Properdin Essential mixed Membranouse Sjogren's deficency cryoglobulinemia glomerulonephritis syndrome

Action Staphylococcal or myoclonus - Familial Mediterranean streptococcal renal failure fever Menieres disease sepsis syndrome

Acute

respiratory

disease

syndrome

(ARDS)/Severe

acute

respiratory

syndrome Microscopic Stiff person

(SARS) Familial renal amyloidosis polyangiitis syndrome

Familial steroid-resistant

Acute serum nephrotic syndrome with Minimal change Systemic lupus sickness sensorineural deafness disease erythematosus

Adult-onset Still Mixed connective

disease Farmer's lung tissue disease Systemic sclerosis

Age-related

macular Mostly large vessel

degeneration Fechtner Syndrome vasculitis Takayasu arteritis

Toxic epidermal necrolysis mostly mediu m (Stevens Johnson

AL amyloidosis Fibronectin glomerulopathy vessel vasculitis syndrome)

Transplantation/re

Alport's Mostly small vessel perfusion (solid syndrome Fibrosing alveolitis vsculitis organ)

Alzheimer's Muckle-Wells

disease Focal segmental glomerular syndrome Vasculitis

Amyloidosis

(AL, AA, Focal segmental Wegener's

MIDD, Other) glomerulosclerosis Myasthenia gravis granulomatosis

Giant cell Galloway-Mowat

arteritis Frasier syndrome syndrome

Type 1 diabetes Myasthenia gravis Graves' disease Pernicious anemia thrombocytopenic Primary biliary

Crohn's disease alopecia areata purpura cirrhosis

Ulcerative Guillain-Barre

colitis autoimmune hepatitis syndrome Psoriasis

Inflammatory autoimmune Autoimmune Rheumatoid bowel syndrome deramtomyositis myocarditis arthritis

Multiple Autoimmune

sclerosis Juvenile idiopathic arthritis pemphigus Vitiligo

Enzyme Deficiencies & Vascular Diseases

2,4-dienoyl- CoA reductase Fabry disease (1 :80,000 to Isobutyryl-CoA Peripheral deficiency 1 : 117,000) dehydrogenase neuropathy

2-Methyl-3- Familial Peroxisomal hydroxy butyric hypercholesterolemia disorders aciduria (1 :500) Isovaleric acidemia (1 :50,000; e.g., Zellweger

syndrome, neonatal adrenoleukodystro phy, Refsum's disease)

2- methylbutyryl- CoA Familial myocardial Lactase deficiency

dehydrogenase infarct/stroke (common) Phenylketonuria

3-hydroxy-3- methylglutaryl Fatty acid oxidation Lesch-Nyhan Primary

(HMG) aciduria disorders (1 : 10,000) syndrome hyperoxaluria

3- methylglutaconi Lipoprotein lipase Propionic c aciduria Galactokinase deficiency deficiency (rare) acidemia

3-oxothiolase long-chain 1-3- deficiency hdroxyacyl-CoA

(1 : 100,000) Galactose epimerase dehydrogenase Recurrent emesis

4- Short-chain acyl- hydroxybutyric Lysinuric protein CoA

aciduria Galactosemia intolerance (rare) dehydrogenase

5,10- methylenetetrah

ydrofolate

reductase

deficiency Lysinuric protein Sucrase-isomaltase

(common) Galactosemia (1 :40,000) intolerance (rare) deficiency (rare)

5-Oxoprolinuria

(pyroglutamic Symptoms of aciduria) Gaucher's disease Malonic acidemia pancreatitis

Transferase deficient galactosemia

Abetalipoprotei Maple syrup urine (Galactosemia nemia (rare) Glutaric acidemia type I disease type 1)

Acute

Intermittent Medium chain acyl- Trifunctional

Porphyria Glutaric acidemia Type II CoA dehydrogenase protein deficiency

Glutathione Synthetase Medium/short chain

Deficiency w/ 5- L-3 -hydroxy acyl- Tyrosinemia type

Alkaptonuria oxoprolinuria CoA dehydrogenase 1

Glutathione Synthetase

Deficiency w/o 5- Medum-chain Tyrosinemia type

Argininemia oxoprolinuria ketoacyl-coA thiolase 2

Metachromatic

argininosuccinat Glycogenolysis disorders leukodystrophy Tyrosinemia type e aciduria (1 :20,000) (1 : 100,000) 3

Benign Glycogenosis, type I Metachromatic Upward gaze hyperphenylalan (1 :70,000) leukodystrophy paralysis inemia (1 : 100,000)

beta Very long chain ketothiolase Hemolytic anemia due to Methylmalonic acyl-CoA deficiency adenylate kinase deficiency acidemia (Cbl C) dehydrogenase

Biopterin

cofactor Hemolytic anemia due to

biosynthesis deficiency in Glucose 6 Methylmalonic

defects phosphate dehydrogenase acidemia (Cbl D) Wilson Disease

Biopterin Aicardi-Goutieres cofactor Hemolytic anemia due to Methylmalonic Syndrome (may be regeneration diphosphoglycerate mutase acidemia (vitamin bl2 an allelic form of defects deficiency non-responsive) CLE)

biotin- unresponsive 3- methylcrotonyl- CoA Hemolytic anemia due to Methylmalonic

carboxylase erythrocyte adenosine acidemia w/0 Cutaneous lupus deficiency deaminase overproduction homocystinuria erythematosus

Carbamoyl Hemolytic anemia due to Methylmalonic

phosphate glucophosphate isomerase aciduria and Dermatitis synthetase deficiency homocystinuria herpetiformis

Carnitine Hemolytic anemia due to

acylcarnitine glutathione reductase Mitochondrial

translocase deficiency disorders (1 :30,000) hemophilia A

Mitochondrial

disorders (1 :30,000;

e.g., cytochrome-c

oxidase deficiency;

Carnitine Hemolytic anemia due to MELAS syndrome;

palmitoyltransfe glyceraldehyde-3 -phosphate Pearson's syndrome

rase I dehydrogenase deficiency [all rare]) hemophilia B

Idiopathic steroid

Mitochondrial sensitive nephrotic disorders (1 :30,000; syndrome (same as

Carnitine Hemolytic anemia due to e.g., Leigh disease, focal segmental palmitoyltransfe pyrimidine 5' nucleotidase Kearns-Sayre glomerulaosclerosi rase II deficiency syndrome [rare]) s)

Mitochondrial

disorders (1 :30,000;

Hemolytic anemia due to e.g., lipoamide Immune

Carnitine uptake red cell pyruvate kinase dehydrogenase thrombocytopenic defect deficiency deficiency [rare]) purpura

Mitochondrial

disorders (1 :30,000;

citrullinemia e.g., Pearson's

type I HHH syndrome (rare) syndrome [rare]) Myasthenia gravis

Multiple carboxylase

Citrullinemia (holocarboxylase Oligoarticular type II homocysteinuria synthetase) juvenile arthritis Multiple carboxylase

deficiency (e.g.,

holocarboxylase

Congenital synthetase [rare]) and

disorders of biotinidase

glycosylation deficiencies

(rare) Homocystinuria (1 :200,000) (1 :60,000) Scleroderma

Solar urticaria

D-2- hyperammonemia/ornithine (maybe hydroxyglutaric mia/citrullinemia (ornithine Muscle protophyria aciduria transporter defect) cramps/ spasticity erythema)

D-2- Myoadenylate Thrombotic hydroxyglutaric Hyperlipoproteinemia, deaminase deficiency thrombocytopenic aciduria (rare) types I and IV (rare) (1 : 100,000) purpura

Hypermethioninemia due to Tubulointerstitial

Enteropeptidase glycine N-methyltransferase Niemann-Pick nephritis with deficiency (rare) deficiency disease, type C (rare) Uveitis/ ATIN

Hypermethioninemia

Ethylmalonic encephalopathy due to Nonketotic Von willebrand encephalopathy adenosine kinase deficiency hyperglycinemia disease

Hyperprolinemia

Infectious Diseases & Infectious Agents

Infection-induced

immune complex

Acinetobacter Dengue haemorrhagic fever vasculitis Sepsis

Arcobacter

butzleri Disseminated infection with

infection - blood mycobacterium avium

infection complex - blood infection Klebsiella Serratia

Arcobacter

cryaerophilus

infection - blood Leprosy/Hansen's Staphylococcus infection E. coli disease Aureus

Arcobacter Stenotrophomonas infection - blood maltophilia - blood infection Enterobacter Malaria infection

Streptococcal Group A invasive disease - blood

Bacteremia Enterococcus Meningococcus infection

Methicillin Resistant

Bacterial Staphylococcus Streptococcus endocarditis Glanders - blood infection Aureus pneumoniae

Campylobacter

fetus infection - Streptococcus blood infection Gonorrhea Pseudomonas pyogenes

Campylobacter

jejuni infection - Rhodococcus equi - blood infection Hepatitis blood infection Trypanosomiasis Human Immunodeficiency

Candida Virus Salmonella Yellow fever

Coagulase-negative Staphylococcus

Ab hemolytic anemia B, and/or glycophorin C, Rh antibody against antigen glycophorins and/or

Rh antigen

Complemen Age-related macular a suitable complement

t degeneration regulatory protein active complement complement factor H, or a

Complemen Atypical hemolytic suitable complement

t uremic syndrome regulatory protein active complement

Complemen Autoimmune a suitable complement

t hemolytic anemia regulatory molecule active complement

Complement factor I, a

Complemen Complement Factor I suitable complement

t deficiency regulatory protein active complement

Complemen Non-alcoholic a suitable complement

t steatohepatitis regulatory molecule active complement

Complemen Paroxysmal nocturnal a suitable complement

t hemoglobinuria regulatory protein active complement

3- hydroxyvalerylcarniti ne, 3- methylcrotonylglycin e (3-MCG) and 3-

3 -methylcrotonyl-Co A 3 -methylcrotonyl-Co A hydroxyisovaleric

Enzyme carboxylase deficiency carboxylase acid (3-HIVA)

Acute Intermittent

Enzyme Porphyria Porphobilinogen deaminase Porphobilinogen

Acute lymphoblastic

Enzyme leukemia Asparaginase Asparagine

Acute lymphocytic

leukemia, acute

Enzyme myeloid leukemia Asparaginase Asparagine

Acute myeloblastic

Enzyme leukemia Asparaginase Asparagine

Adenine

phosphoribosyltransfer adenine Insoluble purine 2,8-

Enzyme ase deficiency phosphoribosyltransferase dihydroxyadenine

Adenosine deaminase

Enzyme deficiency Adenosine deaminase Adenosine

Enzyme Afibrinogenemia FI enzyme replacement

Alcohol

Enzyme Alcohol poisoning dehydrogenase/oxidase Ethanol

Enzyme Alexander's disease FVII enzyme replacement

Enzyme Alkaptonuria homogentisate oxidase homogentisate

Enzyme Argininemia Ammonia monooxygenase ammonia

argininosuccinate

Enzyme aciduria Ammonia monooxygenase ammonia

Enzyme citrullinemia type I Ammonia monooxygenase ammonia

Enzyme Citrullinemia type II Ammonia monooxygenase ammonia

Enzyme Complete LCAT Lecithin-cholesterol Cholesterol deficiency, Fish-eye acyltransferase (LCAT)

disease,

atherosclerosis,

hypercholesterolemia

Thiosulfate-cyanide

Enzyme Cyanide poisoning sulfurtransferase Cyanide

Enzyme Diabetes Hexokinase, glucokinase Glucose

Enzyme Factor II Deficiency FII enzyme replacement

Familial

Enzyme hyperarginemia Arginase Arginine

Fibrin Stabilizing

Enzyme factor Def. FXIII enzyme replacement

3 -hydroxy glutaric

Glutaric acidemia type and glutaric acid

Enzyme I lysine oxidase (C5-DC), lysine

Enzyme Gout Uricase Uric Acid

Uric acid (Urate

Enzyme Gout - hyperuricemia Uricase crystals)

Enzyme Hageman Def. FXII enzyme replacement

Hemolytic anemia due

to pyrimidine 5'

nucleotidase

Enzyme deficiency pyrimidine 5' nucleotidase pyrimidines

Thrombin (factor II

Enzyme Hemophilia A Factor VIII a) or Factor X

Factor XIa or Factor

Enzyme Hemophilia B Factor IX X

Enzyme Hemophilia C FXI enzyme replacement

Hepatocellular

Enzyme carcinoma, melanoma Arginine deiminase Arginine

Enzyme Homocystinuria Cystathionine B synthase homocysteine

hyperammonemia/orni

thinemia/ citrullinemia

(ornithine transporter

Enzyme defect) Ammonia monooxygenase Ammonia

Leucine metabolizing

Enzyme Isovaleric acidemia enzyme leucine

d-aminolevulinate

Enzyme Lead poisoning dehydrogenase lead

Lesch-Nyhan

Enzyme syndrome Uricase Uric acid

Maple syrup urine Leucine metabolizing

Enzyme disease enzyme Leucine

Methylmalonic

acidemia (vitamin b 12

Enzyme non-responsive) methylmalonyl-CoA mutase methylmalonate

Mitochondrial

neurogastrointestinal

Enzyme encephalomyopathy thymidine phosphorylase thymidine Mitochondrial

neurogastrointestinal

encephalomyopathy

Enzyme (MNGIE) Thymidine phosphorylase Thymidine

Enzyme Owren's disease FV enzyme replacement

Serine dehyrdatase or serine

Enzyme p53-null solid tumor hydroxymethyl transferase serine

Pancreatic

Enzyme adenocarcinoma Asparaginase asparagine

Phenylalanine hydroxylase,

phenylalanine ammonia

Enzyme Phenylketonuria lyase Phenylalanine

Enzyme Primary hyperoxaluria Oxalate oxidase Oxalate

Propionate conversion

Enzyme Propionic acidemia enzyme? Proprionyl coA

Purine nucleoside

phosphorylase Purine nucleoside

Enzyme deficiency phosphorylase Inosine, dGTP

Enzyme Stuart-Power Def. FX enzyme replacement

Thrombotic ultra-large von

Thrombocytopenic willebrand factor

Enzyme Purpura ADAMTS13 (ULVWF)

Transferase deficient

galactosemia Galactose- 1-

Enzyme (Galactosemia type 1) galactose dehydrogenase phosphate

Enzyme Tyrosinemia type 1 tyrosine phenol-lyase tyrosine

von Willebrand

Enzyme disease vWF enzyme replacement

IC

clearance IgA Nephropathy Complement receptor 1 Immune complexes

IC

clearance Lupus nephritis Complement receptor 1 immune complex

IC Systemic lupus

clearance erythematosus Complement receptor 1 immune complex

Anthrax (B. anthracis) an an antibody-like binder to

Infectious infection B. anthracis surface protein B. anthracis

an an antibody-like binder to

Infectious C. botulinum infection C. botulinum surface protein C. botulinum

an antibody- like binder to C.

Infectious C. difficile infection difficile surface protein C. difficile

an antibody-like binder to

Infectious Candida infection Candida surface protein Candida

an antibody-like binder to

Infectious E. coli infection E.coli surface protein E. coli

an antibody-like binder to

Infectious Ebola infection Ebola surface protein Ebola

Hepatitis B (HBV) an antibody-like binder to

Infectious infection HBV surface protein HBV

Infectious Hepatitis C (HCV) an antibody-like binder to HCV infection HCV surface protein

Human an antibody-like binder to

immunodeficiency HIV envelope proteins or

Infectious virus (HIV) infection CD4 or CCR5 or HIV

M. tuberculosis an antibody-like binder to M.

Infectious infection tuberculosis surface protein M. tuberculosis

Malaria (P. an antibody-like binder to P.

Infectious falciparum) infection falciparum surface protein P. falciparum

Hepatic lipase Lipoprotein, deficiency, intermediate density

Lipid hypercholesterolemia Hepatic lipase (LIPC) (IDL)

Hyperalphalipoprotein Cholesteryl ester transfer Lipoprotein, high

Lipid emia 1 protein(CETP) density (HDL)

an antibody-like binder to

low-density lipoprotein

Lipid hypercholesterolemia (LDL), LDL receptor LDL

an antibody-like binder to

high-density lipoprotein

Lipid hypercholesterolemia (HDL) or HDL receptor HDL

chilomicrons and lipoprotein lipase very low density

Lipid deficiency lipoprotein lipase lipoproteins (VLDL)

Lipoprotein lipase

deficiency, disorders

of lipoprotein Lipoprotein, very

Lipid metabolism lipoprotein lipase (LPL) low density (VLDL)

Lysosomal Aspartylglucosaminuri

storage a (208400) N-Aspartylglucosaminidase glycoproteins

Cerebrotendinous

xanthomatosis

Lysosomal (cholestanol lipidosis; lipids, cholesterol, storage 213700) Sterol 27-hydroxylase and bile acid

Ceroid lipofuscinosis

Lysosomal Adult form (CLN4, Palmitoyl-protein

storage Kufs^* disease; 204300) thioesterase-1 lipopigments

Ceroid lipofuscinosis

Infantile form (CLN1,

Lysosomal Santa vuori-Haltia Palmitoyl-protein

storage disease; 256730) thioesterase-1 lipopigments

Ceroid lipofuscinosis

Juvenile form (CLN3,

Batten disease, Vogt-

Lysosomal Spielmeyer disease; Lysosomal transmembrane

storage 204200) CLN3 protein lipopigments

Ceroid lipofuscinosis

Late infantile form

(CLN2, Jansky-

Lysosomal Bielschowsky disease; Lysosomal pepstatin- storage 204500) insensitive peptidase lipopigments Ceroid lipofuscinosis

Progressive epilepsy

Lysosomal with intellectual Transmembrane CLN8

storage disability (600143) protein lipopigments

Ceroid lipofuscinosis

Lysosomal Variant late infantile Transmembrane CLN6

storage form (CLN6; 601780) protein lipopigments

Ceroid lipofuscinosis

Variant late infantile

Lysosomal form, Finnish type Lysosomal transmembrane

storage (CLN5; 256731) CLN5 protein lipopigments

Cholesteryl ester

Lysosomal storage disease

storage (CESD) lisosomal acid lipase lipids and cholesterol

Congenital disorders

of N-glycosylation

CDG la (solely

neurologic and

neurologic-

Lysosomal multivisceral forms; N-glycosylated storage 212065) Phosphomannomutase-2 protein

Congenital disorders

Lysosomal of N-glycosylation Mannose (Man) phosphate N-glycosylated storage CDG lb (602579) (P) isomerase protein

Congenital disorders Dolicho-P-

Lysosomal of N-glycosylation Glc:Man9GlcNAc2-PP- N-glycosylated storage CDG Ic (603147) dolichol glucosyltransferase protein

Congenital disorders Dolicho-P-

Lysosomal of N-glycosylation Man:Man5GlcNAc2-PP- N-glycosylated storage CDG Id (601110) dolichol mannosyltransferase protein

Congenital disorders

Lysosomal of N-glycosylation Dolichol-P-mannose N-glycosylated storage CDG Ie (608799) synthase protein

Congenital disorders

Lysosomal of N-glycosylation Protein involved in mannose- N-glycosylated storage CDG If (609180) P-dolichol utilization protein

Congenital disorders Dolichyl-P-mannose:Man-7-

Lysosomal of N-glycosylation GlcNAc-2-PP-dolichyl-a-6- N-glycosylated storage CDG Ig (607143) mannosyltransferase protein

Dolichy 1-P-glucose : Glc- 1 -

Congenital disorders Man-9-GlcNAc-2-PP-

Lysosomal of N-glycosylation dolichyl-a-3- N-glycosylated storage CDG Ih (608104) glucosyltransferase protein

Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG Ii (607906) a-1 ,3-Mannosyltransferase protein

Congenital disorders Mannosyl-a-1,6-

Lysosomal of N-glycosylation glycoprotein-β- 1 ,2-N- N-glycosylated storage CDG Ila (212066) acetylglucosminyltransferase protein Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG lib (606056) Glucosidase I protein

Congenital disorders

of N-glycosylation

CDG lie (Rambam-

Lysosomal Hasharon syndrome; N-glycosylated storage 266265 GDP-fucose transporter- 1 protein

Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG lid (607091) β- 1 ,4-Galactosyltransferase protein

Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG He (608779) Oligomeric Golgi complex-7 protein

Congenital disorders

Lysosomal of N-glycosylation UDP-GlcNAc:dolichyl-P N-glycosylated storage CDG Ij (608093) NAcGlc phosphotransferase protein

Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG Ik (608540) β- 1 ,4-Mannosyltransferase protein

Congenital disorders

Lysosomal of N-glycosylation N-glycosylated storage CDG 11 (608776) a- 1 ,2-Mannosyltransferase protein

Congenital disorders

of N-glycosylation,

Lysosomal type I (pre-Golgi N-glycosylated storage glycosylation defects) a- 1 ,2-Mannosyltransferase protein

Lysosomal Cystinosin (lysosomal

storage Cystinosis cystine transporter) Cysteine

Lysosomal Fabry's disease Trihexosylceramide a- globotriaosylceramid storage (301500) galactosidase e

Farber's disease

Lysosomal (lipogranulomatosis;

storage 228000) Ceramidase lipids

Lysosomal fucose and complex storage Fucosidosis (230000) a-L-Fucosidase sugars

Galactosialidosis

(Goldberg's syndrome,

combined

neuraminidase and β-

Lysosomal galactosidase Protective protein/cathepsin

storage deficiency; 256540) A (PPCA) lysosomal content

Lysosomal Glucosylceramide β- storage Gaucher's disease glucosidase sphingolipids

Glutamyl ribose-5-

Lysosomal phosphate storage ADP-ribose protein glutamyl ribose 5- storage disease (305920) hydrolase phosphate

Lysosomal Glycogen storage

storage disease type 2 alpha glucosidase glycogen (Pompe's disease)

Lysosomal GM1 gangliosidosis, acidic lipid material, storage generalized Ganglioside β-galactosidase gangliosides

GM2 activator protein

deficiency (Tay-Sachs

Lysosomal disease AB variant,

storage GM2A; 272750) GM2 activator protein gangliosides

Lysosomal

storage GM2 gangliosidosis Ganglioside β-galactosidase gangliosides

Infantile sialic acid

Lysosomal storage disorder Na phosphate cotransporter,

storage (269920) sialin sialic acid

Lysosomal Krabbe's disease Galactosylceramide β- storage (245200) galactosidase sphingolipids

cholesteryl

Lysosomal Lysosomal acid lipase esters and triglycerid storage deficiency (278000) Lysosomal acid lipase es

Metachromatic

Lysosomal leukodystrophy

storage (250100) Arylsulfatase A sulfatides

Mucolipidosis ML II N-Acetylglucosaminyl- 1 -

Lysosomal (I-cell disease; phosphotransfeerase catalytic N-linked

storage 252500) subunit glycoproteins

Mucolipidosis ML III

Lysosomal (pseudo-Hurler's N-acetylglucosaminyl- 1 - N-linked

storage polydystrophy) phosphotransfeerase glycoproteins

Mucolipidosis ML III

(pseudo-Hurler's

Lysosomal polydystrophy) Type N-linked

storage III-A (252600) Catalytic subunit glycoproteins

Mucolipidosis ML III

(pseudo-Hurler's

Lysosomal polydystrophy) Type N-linked

storage III-C (252605) Substrate -recognition subunit glycoproteins

Mucopolysaccharidosi

s MPS I H/S (Hurler-

Lysosomal Scheie syndrome;

storage 607015) α-1-Iduronidase glycosaminoglycans

Mucopolysaccharidosi

Lysosomal s MPS I-H (Hurler's

storage syndrome; 607014) α-1-Iduronidase glycosaminoglycans

Mucopolysaccharidosi

Lysosomal s MPS II (Hunter's

storage syndrome; 309900) Iduronate sulfate sulfatase glycosaminoglycans

Mucopolysaccharidosi

s MPS III (Sanfilippo's

Lysosomal syndrome) Type III-A Heparan-S-sulfate

storage (252900) sulfamidase glycosaminoglycans

Lysosomal Mucopolysaccharidosi N-acetyl-D-glucosaminidase glycosaminoglycans

Lysosomal Sandhoff s disease;

storage 268800 β-Hexosaminidase B gangliosides

Saposin B deficiency

Lysosomal (sulfatide activator

storage deficiency) Saposin B sphingolipids

Saposin C deficiency

Lysosomal (Gaucher's activator

storage deficiency) Saposin C sphingolipids

Schindler's disease

Lysosomal Type I (infantile severe

storage form; 609241) N-Acetyl-galactosaminidase glycoproteins

Schindler's disease

Type II (Kanzaki

Lysosomal disease, adult-onset

storage form; 609242) N-Acetyl-galactosaminidase glycoproteins

Schindler's disease

Lysosomal Type III (intermediate

storage form; 609241) N-Acetyl-galactosaminidase glycoproteins

Lysosomal mucopolysaccharides storage Sialidosis (256550) Neuraminidase 1 (sialidase) and mucolipids

Lysosomal Sialuria Finnish type Na phosphate cotransporter,

storage (Salla disease; 604369) sialin sialic acid

UDP-N-acetylglucosamine- 2-epimerase/N-

Lysosomal Sialuria French type acetylmannosamine kinase,

storage (269921) sialin sialic acid

Lysosomal Sphingolipidosis Type

storage I (230500) Ganglioside β-galactosidase sphingolipids

Sphingolipidosis Type

Lysosomal II (juvenile type;

storage 230600) Ganglioside β-galactosidase sphingolipids

Sphingolipidosis Type

Lysosomal III (adult type;

storage 230650) Ganglioside β-galactosidase sphingolipids

Lysosomal Tay-Sachs disease;

storage 272800 β-Hexosaminidase A gangliosides

Lysosomal Winchester syndrome

storage (277950) Metalloproteinase-2 mucopolysaccharides

Lysosomal

storage Wolman's disease lysosomal acid lipase lipids and cholesterol a-Mannosidosis

Lysosomal (248500), type I carbohydrates and storage (severe) or II (mild) a-D-Mannosidase glycoproteins

Lysosomal β-Mannosidosis carbohydrates and storage (248510) β-D-Mannosidase glycoproteins

Toxic alpha hemolysin an antibody-like binder to

Molecule poisoning alpha hemolysin alpha hemolysin

Toxic an antibody-like binder to

Molecule antrax toxin poisoning anthrax toxin anthrax toxin Toxic bacterial toxin-induced an antibody-like binder to

Molecule shock bacterial toxin bacterial toxin

Toxic botulinum toxin an antibody-like binder to

Molecule poisoning botulinum toxin botulinum toxin

Toxic Hemochromatosis

Molecule (iron poisoning) iron chelator molecular iron

Toxic

Molecule Methanol poisoning Methanol dehdrogenase Methanol

Toxic

Molecule Nerve gas poisoning Butyryl cholinesterase Sarin

Toxic Prion disease caused an antibody-like binder to

Molecule by PRP prion protein PRP Prion protein PRP

Toxic Prion disease caused an antibody-like binder to

Molecule by PRPc prion protein PRPc Prion protein PRPc

Toxic Prion disease caused an antibody-like binder to

Molecule by PRPsc prion protein PRPsc Prion protein PRPsc

Toxic Prion disease cuased an antibody-like binder to

Molecule by PRPres prion protein PRPres Prion protein PRPres an antibody-like binder to

cytokines or Duffy antigen

Toxic Sepsis or cytokine receptor of chemokines

Molecule storm (DARC) cytokines

Toxic spider venom an antibody-like binder to

Molecule poisoning spider venom spider venom

Toxic

Molecule Wilson disease copper chelator molecular copper

miR-148b miR-452 miR-22* miR-491-5p miR-149 miR-453 miR-342-3p miR-382 miR-150 miR-454-3p miR-128 miR-583 miR-151 miR-454-5p miR-342-5p miR-874 miR-152 miR-483 miR-362-3p miR-516b miR-15b miR-484 miR-886-3p miR-518f miR-16 miR-485-5p miR-361-5p miR-622 miR-17-3p miR-486 miR-30a miR-K12-8 miR-17-5p miR-487b miR-223 miR-513a-3p miR-181a miR-494 miR-331-3p miR-UL36 miR-181b miR-500 miR-564 miR-141 * miR-181c miR-502 miR-425 miR-492 miR-181d miR-505 miR-502-3p miR-129-5p miR-182 miR-512-3p miR-590-5p miR-30c-2* miR-183 miR-513 miR-330-3p miR-486-5p miR-185 miR-517c miR-378 miR-631 miR-186 miR-519b miR-139-3p miR-184 miR-187 miR-521 miR-28-3p miR-145 miR-188 miR-522 miR-32 miR-628-5p miR-18a miR-526a miR-301a miR-BHRFl-1 miR-18b miR-527 miR-542-3p miR-518d-3p miR-190 miR-532 miR-34b* let-7d* miR-191 miR-550 miR-17 miR-93* miR-192 miR-557 miR-532-3p miR-548d-5p miR-193a miR-565 miR-140-3p miR-548c-5p miR-193b miR-571 miR-28-5p miR-770-5p miR-194 miR-574 miR-30e* miR-744* miR-196b miR-578 miR-532-5p miR-449a miR-197 miR-582 miR-146b-5p miR-548a-5p miR-198 miR-584 miR-503 miR-148a* miR-19a miR-585 miR-339-3p miR-624* miR-19b miR-590 miR-338-3p miR-219-5p miR-200a miR-593 miR-33a miR-16-2* miR-200b miR-594 miR-374b miR-29c* miR-200c miR-595 miR-30e miR-550* miR-202 miR-603 miR-362-5p miR-15b* miR-203 miR-608 miR-140-5p miR-15a* miR-205 miR-612 miR-151-3p miR-106a* miR-206 miR-625 miR-454 miR-196a miR-20a miR-628 miR-29c miR-138-2* miR-20b miR-629 miR-15a miR-33b miR-21 miR-634 miR-142-5p miR-301b miR-210 miR-637 miR-374a miR-7-1 * miR-214 miR-638 miR-193a-3p miR-30d* miR-22 miR-642 miR-151-5p miR-574-3p miR-220 miR-645 miR-744 miR-181a* miR-221 miR-647 miR-BART 19-3p miR-19b-l * miR-222 miR-649 miR-378* miR-20a* miR-224 miR-652 miR-340 miR-9* miR-23a miR-660 miR-21 * miR-7 miR-23b miR-663 miR-17* miR-431 * miR-24 miR-671 miR-142-3p miR-BART 12 miR-25 miR-765 miR-193a-5p miR-153 miR-26a miR-766 miR-936 miR-658 miR-26b miR-768-3p miR-193b* miR-122 miR-27a miR-768-5p miR-451 miR-939 miR-27b miR-769-3p miR-921 miR-181c* miR-28 miR-769-5p miR-Hl miR-885-5p miR-296 miR-801 miR-510 miR-BART 11 -5p miR-29a miR-9 miR-483-5p miR-BART 19-5p miR-29b miR-92 miR-150* miR-BHRFl-2*

What is claimed is:

Claims

1. An isolated nanomembrane delivery complex comprising a payload and a surface molecule selected from the group consisting of CD47, CD55, CD40, CD63, CD9, CD133 and CD59.

2. The isolated nanomembrane delivery complex of claim 1, wherein the payload is a therapeutic polypeptide, polynucleotide, polysaccharide, lipid, small molecule or toxin.

3. The isolated nanomembrane delivery complex of claim 1, wherein the payload is a polynucleotide selected from the group consisting of: mRNA, miRNA, shRNA, dsDNA, IncRNA, and siRNA.

4. The isolated nanomembrane delivery complex of claim 3, wherein the mRNA encodes a therapeutic polypeptide.

5. The isolated nanomembrane delivery complex of claim 4, wherein the therapeutic polypeptide has enzymatic activity.

6. The isolated nanomembrane delivery complex of claim 1, wherein the nanomembrane delivery complex has a diameter of 20 to 150 nanometers.

7. The isolated nanomembrane delivery complex of claim 1 further comprising an imaging agent.

8. The isolated nanomembrane delivery complex of claim 7, wherein the imaging agent is selected from the group consisting of a radionuclide, an isotope, a fluorescent/fluorochrome molecule, a dye, and a chemiluminescent molecule.

9. The isolated nanomembrane delivery complex of claim 1 further comprising a receiver polypeptide.

10. The isolated nanomembrane delivery complex of claim 9, wherein the receiver is capable of directing the nanomembrane delivery complex to a target.

11. A pharmaceutical composition comprising the nanomembrane delivery complex of any one of claims 1-10 and a pharmaceutically acceptable excipient or carrier.

12. The pharmaceutical composition of claim 11 further comprising a second therapeutic agent.

Ill

13. A dosage form comprising the pharmaceutical composition of claim 11 or 12 formulated as a sterile solution for intravenous injection.

14. A method of treating a disease, disorder or condition selected from Table 3 and Table 4, the method comprising administering to a subject in need thereof the pharmaceutical composition of claim 8 or 9, optionally in form of the dosage form of claim 10, in an amount effective to treat the disease, disorder or condition.

15. The method of claim 14, wherein the nanomembrane delivery complex comprises a payload and delivers the payload to a target cell or tissue that is associated with the disease, disorder or condition in an amount effective to treat the disease, disorder or condition.

16. The method of claim 15, wherein the target cell is, or the target tissue comprises an infected, impaired or dysregulated cell.

17. The method of claim 16, wherein the nanomembrane delivery complex facilitates the contacting of the infected, impaired or dysregulated cell with the payload in sufficient proximity and for a sufficient duration to bring about a substantial killing of the infected, impaired or dysregulated cell, thereby treating the disease, disorder or condition.

18. The method of claim 16, wherein the nanomembrane delivery complex facilitates the contacting of the infected, impaired or dysregulated cell with the payload in sufficient proximity and for a sufficient duration to bring about a substantial restoration of the functionality of the infected, impaired or dysregulated cell, optionally wherein an impaired enzyme function is restored or a dysregulated enzyme function regulated, thereby treating the disease, disorder or condition.

19. The method of any one of claims 14-18 further comprising administering a second, standard-of-care therapy.

20. A method for producing the isolated nanomembrane delivery complex of any one of claims 1-10, the method comprising: a) providing a producer cell capable of generating a nanomembrane delivery complex, b) obtaining from the producer cell the nanomembrane delivery complex,

c) modifying the isolated nanomembrane delivery complex with a payload,

d) isolating the modified nanomembrane delivery complex, and

e) optionally formulating the isolated nanomembrane delivery complex into a

pharmaceutical composition, wherein the producer cell and/or the isolated nanomembrane delivery complex either a) naturally comprise or b) are modified to comprise a surface molecule selected from the group consisting of CD47, CD55, CD40, CD63, CD9, CD133 and CD59.

21. The method of claim 20, wherein the nanomembrane delivery complexes are released by the producer cells into a culture medium.

22. The method of claim 20, wherein modifying the nanomembrane delivery complex is carried out by membrane perturbation.

23. The method of claim 20, wherein the producer cell is a mammalian cell that is isolated or derived from a cell line.

24. The method of claim 20 further comprising testing the activity of the pharmaceutical composition, wherein testing comprises one or more of: i) analyzing the presence or activity of the payload, ii) analyzing the potency of the nanomembrane delivery complex in a phenotypic or functional assay iii) detecting the presence or absence of one or more biomarkers from the producer cell, iv) analyzing the size distribution of the nanomembrane delivery complexes, and/or v) analyzing the membrane composition of the nanomembrane delivery complexes.

25. The nanomembrane delivery complex of any one of the preceding claims for use as a medicament.

26. The nanomembrane delivery complex of any one of the preceding claims for use in the treatment of any one of the diseases listed in Table 3 and Table 4.

27. A composition comprising any embodiment described in the specification.

28. A method comprising any embodiment described in the specification.