METHODS OF MAKING TARGETED VESICLES, AND COMPOSITIONS MADE THEREBY

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of and priority to U.S. Provisional Application No. 62/824,901 filed March 27, 2019, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. 1R01CA204846-01A1 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted as a text file named

“UGA_2019_147_PCT_ST25.txt,” created on March 27, 2020, and having a size of 1,349 bytes is hereby incorporated by reference pursuant to 37 C.F.R § 1.52(e)(5).

FIELD OF THE INVENTION

This application is generally in the field of drug delivery, and more specifically methods of making and using lipid-based delivery vesicles for delivery of cargo including nucleic acids such as miRNA.

BACKGROUND OF THE INVENTION

Since their discovery in 1983 by two distinct research groups (Pan & Johnstone, Cell. 33(3):967-78 (1983), Harding & Heuser, J Cell Biol.

97(2):329-39 (1983)), exosomes have been well characterized in diverse areas of research. Several recent studies have outlined their significance as key regulators of cell-to-cell communication, biomarker vehicles in conditions such as Alzheimer’s disease and cancer (Iranifar et ak, J Cell Physiol. 234(3):2296-305 (2019) doi: 10.1002/jcp.27214, Mitchell et ak, Proc Natl Acad Sci U SA. 105(30):10513-8 (2008)), or as carriers for therapeutics (Di et ak, Artif Cells Nanomed Biotechnol. 46(Sup3): S564- S570 (2018) doi: 10.1073/pnas.0804549105). While exosomes require purification before being utilized as carriers, they can hold diverse cargo such as microRNA (miRNA), protein and mRNA, and widely access all cell types through circulation, including crossing the blood-brain barrier (Das et al., Mol Pharm. 16(1): 24-40 (2018) doi:

10.1021/acs.molpharmaceut.8b00901). Exosomes are increasingly being utilized as efficient drug carriers, for example paclitaxel loaded into exosomes showed increased accumulation in both drug-sensitive and - resistant lung cancer cells (Kim et al., Nanomedicine. 12(3):655-64 (2016) doi: 10.2217/nnm- 2016-0237), and exosomally delivered miR-9 in breast cancer fibroblasts and miR-122 served as a chemo-sensitizer in

hepatocellular carcinoma (Baroni et al., Cell Death Dis. 7(7):e2312 (2016) doi: 10.1038/cddis.2016.224 , Lou et al., J Hematol Oncol. 8:122 (2015) doi: 10.1186/sl3045-015-0220-7).

miRNAs, small 20-22 nucleotide RNAs, have also been at the forefront of therapeutics research, especially in cancer, due to their ability to effectively inhibit gene expression in cancer cells by binding mRNA and inhibiting protein translation (Bartel, Cell.116(2): 281-97 (2004) doi:

10.1016/S0092-8674(04)00045-5). For example, miR-26a-containing exosomes delivered to hepatocellular carcinoma cells were shown to significantly slow cell migration and proliferation due to increased miRNA levels (Liang et al., Int J Nanomedicine. 13:585-99 (2018) doi:

10.2147/IJN.S154458). miRNAs have also been considered as key candidates for circulating biomarkers indicative of disease state and, in some cases, tumor staging and progression (Lu et al., Nature 435(7043):834-8 (2005) doi: 10.1038/nature03702, Calin & Croce, Nat Rev Cancer 6(11):857- 66 (2006) doi:10.1038/nrcl997 ). For example, a recent study revealed the significance of miR-489-3p in ovarian cancer development and its ability to target and repress MEK1, a prominent oncogene relevant in several malignancies (Kuppa et al., Cancer Lett. 432:84-92 (2018) doi:

10.1016/j.canlet.2018.05.037). miR-34 has also been well classified as a tumor suppressor in several cancers in the past, and reached phase 1 clinical trials in 2017 (Rupaimoole & Slack, Nat Rev Drug Discov. 16(3):203-22 (2017) doi: 10.1038/nrd.2016.246, Agostini & Knight, Oncotarget. 5(4):872- 81 (2014) DOI: 10.18632/oncotarget.l825). Miraversin, which is an anti- sense miRNA inhibitor that targets overexpression of miR-122, reached phase 2 clinical trials for Hepatitis C virus infections (van der Ree et al., Aliment Pharmacol Ther. 43(1): 102- 13 (2016) doi: 10.1111/apt.l3432).

Methods for engineering the surfaces of extracellular vesicles are discussed in Antes, et al., Journal of Nanobiotechnology, 16:61 (2018), https://doi.org/10.1186/sl2951-018-0388-4. However, there remains a need for improved lipid-based delivery vesicles and methods for making them, particularly for use in the delivery of nucleic acid cargo such as miRNAs.

For example, a significant barrier for implementing exosomes as a therapeutic vehicle of microRNAs in the clinic is their propensity to cause off-target effects due to their versatile target range and the difficulties of bioengineering them without disrupting them, which ultimately affects their endocytotic efficiency.

Thus, it is an object of the invention to provide methods of making versatile and customizable lipid-based delivery vehicles that are effective at specifically targeting cells and delivering cargo, including nucleic acids, with improved efficiency, and the lipid vehicles formed therefrom.

SUMMARY OF THE INVENTION

Functionalized lipid vesicles having a lipid membrane and a functional element, and methods of making them are provided. The methods of making the vesicles typically include mixing lipid vesicles formed of one or more lipids with one or more lipid conjugates and dialyzing the mixture for an effective amount (i.e., sufficient amount) of time and under conditions suitable for the lipid conjugate to insert into the membrane of lipid vesicles and form functionalized lipid vesicles that include the lipid conjugate as a functional element. The lipid conjugates typically include a lipid component conjugated or otherwise linked to a functional element.

For example, a method of making functionalized lipid vesicles can include mixing, in the presence of detergent, lipid vesicles including one or more lipids with one or more types of lipid conjugates and dialyzing the mixture for an effective amount (i.e., sufficient amount) of time for the lipid conjugate(s) to insert into the lipid vesicles and form functionalized lipid vesicles. Typically the dialysis removes the remaining detergent.

Some of the methods further include preparing the lipid conjugate. The methods can include one or more, preferably all, of the following steps: (i) mixing or otherwise suspending the lipid component, or a precursor thereof, in a solution including a concentration of detergent near the critical micelle concentration to form a suspension,

(ii) dialyzing the suspension to remove excess detergent, and encourage formation of stable micelles in the suspension,

(iii) adding, mixing, or otherwise contacting the suspension with the functional element under conditions suitable for the functional element to conjugate, or otherwise link, to the lipid component to form the lipid conjugate. Step (ii) can be before or after step (iii). In some embodiments, a method of making a lipid conjugate proceeds in the order of step (i), (ii), and (iii).

The first dialysis may remove excess detergent, but leave an effective amount suitable for stabilizing the hydrophobic regions of the lipid component, or precursor thereof, in a semi-aqueous solution. The detergent, which can be selected by the practitioner, should be one suitable for doing so. An exemplary detergent is n - do dec y 1 - b - D - m a 1 to s i de (DDM), optionally at a concentration of about 0.1%.

When the lipid conjugate and vehicle are mixed to form

functionalized lipid vehicles, dialysis of the mixture (which can also be a second dialysis following the first dialysis during preparation of the lipid conjugant) removes the remaining detergent, and facilitates formation of functionalized lipid vesicle product.

The initial lipid vesicle can be naturally occurring, for example, isolated or otherwise collected from cultured or uncultured tissue, cells, or fluid. In some embodiments, the fluid is one derived from or conditioned by cultured cells, or is blood, plasma, lymph liquid, malignant pleural effusion, amniotic liquid, breast milk, semen, saliva or urine. In some embodiments, the cells are peripheral blood mononuclear cells. The lipid vesicles can be, for example, apoptotic bodies and/or blebs (AB), microvesicles (MV), exosomes, or tunneling nanotubes (TNT).

In some embodiments, the lipid vesicles are synthetic. Synthetic vesicles include, for example, niosomes and liposomes.

In some embodiments, the lipid vesicles are exosomes or exosome mimics, preferably between about 30-150 nm. The lipid conjugate typically includes a functional element conjugated to or otherwise linked, directly or indirectly, to a lipid (also referred to as the lipid component of the conjugate). The functional element can be a small molecule, protein or polypeptide, carbohydrate, nucleic acid or a combination thereof. In preferred embodiments, at least one of the functional elements is a targeting moiety that increases attachment, binding, or association of the functionalized lipid vesicle to a target cell(s), tissues(s), and/or microenvironment(s) relative to the lipid vesicle. Additionally or alternatively the targeting moiety can increase attachment, binding, or association of the functionalized lipid vesicle to a target cell(s), tissues(s), and/or microenvironment(s) relative non-targeted cell(s), tissue(s), and/or microenvironment(s).

In some embodiments, the targeting moiety targets cancer cells. In more specific embodiments, the targeting moiety targets CD44 or

CD29/Integrin beta-1.

In some embodiments, the targeting moiety targets hepatocytes. In more specific embodiments, the targeting moiety targets asialoglycoprotein receptor 1/HL-l (ASGR1).

In some embodiments, the functional element is an antibody. Thus, in some embodiments, the targeting moiety is an antibody that binds to CD44 or CD29/Integrin beta-1 or I-CAM. In other embodiments, the targeting moiety is antibody that binds to asialoglycoprotein receptor 1/HL-l (e.g., Anti-ASGRl antibody).

In other embodiments, the functional element is a detectable label such as a fluorophore, radiolabel, magnetic label, or a contrast agent.

The methods of making the functionalized lipid vesicles can include loading the lipid vesicles or functionalized lipid vesicles with an active agent. The loading of the vesicles typically includes mixing vesicles and active agent alone or in combination with incubation, freeze-thaw cycling, sonication, extrusion, chemical transfection, electroporation, or a combination thereof.

The active agent can be, for example, a therapeutic, nutritional, diagnostic, prophylactic compound, or a combination thereof. The active agent can also include or be a protein, peptide, carbohydrate, polysaccharide, nucleic acid molecule, and/or organic small molecule.

In some embodiments, the active agent is one or more nucleic acid molecules selected from antisense, siRNA, miRNA, anti-miRNA, primary transcript miRNA (pri-miRNA), aptamers, ribozymes, external guide sequences for ribonuclease P, triplex forming agents, and CRIPSR/Cas component(s), or a polynucleotide encoding any of the foregoing. The miRNA can be a pri-miRNA, precursor miRNA (pre-miRNA), mature miRNA, miRNA mimic, or a fragment or variant thereof that retains the biological activity of the miRNA. In some embodiments, the nucleic acid such as miRNA, targets an oncogene.

In a particular embodiment, the active agent is a nucleic acid such as an miRNA loaded by a method that includes electroporation.

Functionalized lipid vesicles made according to the disclosed methods, and pharmaceutical compositions formed therefrom, are also provided, as are methods of using the functionalized lipid vesicles and pharmaceutical compositions. For example, the functionalized lipid vesicles can be used to deliver active agent(s) to cells in vitro and in vivo. In preferred in vivo methods, the active agent-loaded functionalized lipid vesicles are administered to a subject in need thereof in an effective amount to treat a disease or disorder. Diseases and disorders include, but are not limited to, cancer, infectious diseases, autoimmune diseases, genetic diseases, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a flow diagram illustrating an exemplary method of isolating naturally-occurring exosomes. First, peripheral blood mononuclear cells (PBMCs) are obtained from a human blood donor and cultured (e.g., for 24-48 hours) to allow exosomes to accumulate in the culture medium.

Exosomes are then isolated and purified using, e.g., an isolation kit such as the Exiqon Exosome Isolation Kit. Figure IB is flow diagram illustrating an exemplary method of making functionalized exosomes. For example, an antibody can be covalently linked through amine functional groups to a fatty acid optionally with a polyethylene glycol (PEG) tail, e.g., a Polyethylene glycol 2000 (PEG(2000)). This conjugate is inserted into exosomal membrane by removing hydrophobicity- stabilizing detergent through dialysis and forms antibody-labeled exosomes (“Abi-exosomes”). Figure 1C is an illustration of a model of exosome delivery of cargo to cells. Following loading (e.g., electroporation) of cargo (e.g., miRNA (R)) into bioengineered exosome (e.g., Abi-exosomes), a targeting moiety (e.g., antibodies) on the exosomes binds to the targets, which are on or near the cell surface. This is followed by exosome internalization. Once internalized, the exosomes are degraded and the cargo is released from the particle. In the illustration, the cargo is miRNA that upon release finds and inhibits its target mRNA.

Figures 2A-2B are line graphs showing that dynamic light scattering confirms a particle size increase indicative of Ab-exosome formation.

Dynamic light scattering provides a means to measure particle size in solution and deduce whether Ab-exosomes were successfully formed.

Figure 2A illustrates that attachment of the antibody-label to exosomes causes a size peak shift to the right, indicating a larger particle size compared to exosomes without the antibody-label, confirming the attachment. Figure 2B provides additional details, comparing the average particle sizes of exosomes (-D-), Abi-exosomes (exosomes labeled with antibodies) (-□-), a mixture of 100 mM l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol)-2000] (DSPE-PEG 2000) (fatty acid with DDM and 100 pM (NBD-DSPE) solubilized in 0.1% w/v of the detergent N- dodecyl- -D-maltoside (DDM) at the critical micelle concentration (CMC) (Fatty Acids, -o-), and 0.1% w/v DDM (-¨-). The Malvern Zetasizer Nano ZS DLS instrument (Malvern Pananalytical Ltd., United Kingdom) with a 105.251-QS ultra-micro fluorescence quartz cuvette (Hellma USA,

Plainview, NY) was used to perform the DLS experiments to determine sizes and distributions of the particles. The DLS chromatograms were analyzed with Zetasizer 7.03 software using standard refractive indices for a protein in aqueous solution.

Figure 3A is a bar graph showing the effect of electroporation on miRNA levels in exosomes as well as delivery into cells compared to standard protocol of transfection, or incubation of miRNA with exosomes without electroporation. Electroporation of miRNA into exosomes can result in 700,000-fold higher miRNA levels in the exosomes compared to purified exosomes. This in turn results in higher miRNA uptake efficiency into cells as well compared to transfection (miRNA alone) (*p<0.05) or miRNA incubated with exosomes without electroporation (**p<0.01). Figure 3B is a bar graph comparing miRNA transfection (miRNA alone) to miR

electroporated into either purified exosomes or Abi-exosomes. Figure 3C is a flow diagram illustrating two different strategies for loading (e.g., electroporation) exosomes with cargo (e.g., miRNA): before (“Strategy #1”) and after (“Strategy #2”) functionalization. Figure 3D is a bar graph showing the effect of electroporation of miRNA into completed Abi- exosomes (Strategy #2) compared to miRNA electroporation into naked exosomes before particle construction (Strategy #1).

Figure 4A is an image of an electrophoretic gel showing the detection of CD44 and CD29 on the surface of MD A- MB -231, and other cell types. Figure 4B is a bar graph comparing to the use of antibodies autotaxin, CD44 and CD29 in Abi-exosomes construction.

Figure 5 is a bar graph showing miR-21-5p targets and inhibits PTEN expression in MDA-MB-231 cells. miR-21-5p has long been characterized as having the ability to target the prominent oncogene PTEN, which is responsible for controlling cell growth and survival, among other roles.

Figure 6 is a bar graph showing the efficiency of mmu-miR-298-5p incorporation into Hep-G2 cells determined from qRT-PCR analysis. The columns from left to right: Hep-G2 cells only (Cells Only); Hep-G2 cells and DharmaFECT (DharmaFECT only); Hep-G2 cells, 3.8 pg mmu-miR-298-5p, and DharmaFECT (miR-298); Abi-exosomes with the anti-ASGR-1 antibodies and miR-298-5p (Abi-exosomes+miR-298); exosomes with mmu- miR-298-5p (Exosomes+miR-298). The column values represent an average, and the error bars represent the standard deviation from quadruplicates. Statistics: ** p-value<0.01; * p-value<0.05

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

“Active agent” as used herein refers to a physiologically or pharmacologically active substance that acts locally and/or systemically in the body. An active agent is a substance that is administered to a patient for the treatment (e.g., therapeutic agent), prevention ( e. g. , prophylactic agent), or diagnosis (e.g., diagnostic agent) of a disease or disorder.

“Hydrophobic” as used herein refers to a non-polar molecule or part of a molecule that cannot form energetically favorable interactions with water molecules and therefore does not dissolve in water.

“Hydrophilic” as used herein describes a polar molecule or part of a molecule that forms enough energetically favorable interactions with water molecules to dissolve readily in water.

“Amphiphilic” as used herein describes a molecule having both hydrophobic and hydrophilic regions, such as in a phospholipid or a detergent molecule.

“Effective amount” and“suitable amount” as used herein with respect to a therapeutic agent is at least the minimum concentration required to effect a measurable improvement or prevention of any symptom or a particular condition or disorder, to effect a measurable enhancement of life expectancy, or to generally improve patient quality of life. The effective amount may vary depending on such factors as the disease or condition being treated, the active agent(s) (e.g., particular targeted constructs, etc.) being administered, the size of the subject, or the severity of the disease or condition. With regard to cancer, an effective amount can refer to an amount of the active agent that reduces or inhibits tumor growth or tumor burden.

The effective amount can be in the context of the delivery systems disclosed herein. For example, in some embodiments,“therapeutically effective amount” refers to an amount of the therapeutic agent that, when incorporated into and/or onto particles described herein, produces some desired effect at a reasonable benefit/risk ratio applicable to any medical treatment. One of ordinary skill in the art may empirically determine the effective amount of a particular compound without necessitating undue experimentation.

“Pharmaceutically acceptable” as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. “Antibody” as used herein refers to natural or synthetic antibodies that bind a target antigen. The term includes polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term“antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that bind the target antigen.

“Single chain Fv” and“scFv” as used herein means a single chain variable fragment that includes a light chain variable region (VL) and a heavy chain variable region (VH) in a single polypeptide chain joined by a linker which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and Vi.of the single polypeptide chain to associate with one another to form a Fv). The VL and VH regions may be derived from the parent antibody or may be chemically or recombinantly synthesized.

“Individual,”“host,”“subject,” and“patient” as used herein are used interchangeably to refer to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. The subject can be a human or veterinary patient.

“Treatment” as used herein refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

II. Lipid-Based Delivery Vehicles

To make the targeting of exosomes and other lipid vesicles more specific, customizable functionalized lipid-based vesicles including, but not limited to, antibody-labeled exosomes, that can be used as vehicles to deliver cargo such as nucleic acids to cells are provided. Exosome vesicles functionalized with an antibody (i.e., antibody-labeled exosomes) can also be referred to as“Abi-exosomes.”

Preferred methods of making the functionalized vesicles are also provided.

The experiments below illustrate an exemplary method where the vesicles (e.g., exosomes) are bioengineered as a tool for delivery of cargo such as miRNA using a targetable molecular cell surface marker to increase cargo uptake. The exosomes can be purified from a variety of cell types including, but not limited to, mesenchymal stem cells (MSCs), or peripheral blood mononuclear cells (PBMCs) from a group of blood donors, or made synthetically. For example, in the experiments below, exosomes were functionalized without disrupting their structural integrity or interfering with their endocytic machinery. A functional element, exemplified with an antibody, was attached to a long polyethylene glycol (PEG) chain and a fatty acid (FA) and the antibody-PEG-FA complex was passively inserted into exosomes by dialysis.

Upon removal of the stabilizing n - do dec y 1 - b - D - m a 1 to s i de (DDM) detergent, the tail of the pegylated fatty acid conjugate formed a covalent linkage to the antibody, which was inserted into the exosomal membrane to maintain its hydrophobicity. The addition of an antibody attachment drove the functionalized exosomes, initially purified from peripheral blood mononuclear cells obtained from a human blood donor and loaded with miRNA cargo (e.g., MiR-21-5p, which inhibits expression of the phosphatase and tensin homolog (PTEN) gene and has been implicated in many cancers), to target specific cell surface proteins corresponding to the antibody, thereby increasing exosomal uptake.

To monitor the insertion process, a fluorescently-labeled FA can be added with the antibody-PEG-FA complex, which quenches upon insertion into the exosome.

Through electroporation, the miR-21-5p cargo was internalized into the Abi-exosome without disrupting its structure. Subsequently, cells treated with Abi-exosomes containing miR-21-5p and antibodies targeting triple- negative breast cancer (TNBC) cells showed 700,000-fold higher specificity for TNBC cells than unlabeled exosomes with miR-21-5p or transfected with miR-21-5p alone. Cellular internalization of the miR-21-5p leads to significant reduction of PTEN expression, thus demonstrating feasibility of genetic modulation by exosome delivered miR.

Functionalized vesicles can be customized with a wide range of functional elements and cargo to target specific cells and induce specific biological, chemical, physiological, pharmacological, etc., results. As discussed in more detail below, functional elements, lipids, lipid vehicles, cargos, detergents, dialysis membranes and other features can be combined, exchanged, or substituted, to generate customized functional vesicles for use in a variety of applications, including the treatment of diseases such as cancer.

A. Lipid-based Vesicles

1. Vesicle Structure

The disclosed lipid-based vesicles (also referred to herein as vehicles) are modified to include a targeting moiety, typically conjugated to a lipid that inserts into, or otherwise forms part of, the lipid-based vesicle. Such targeting conjugates can be added to the vesicle during synthesis of the vesicle using, for example synthetic techniques that are known in the art. However, preferably, the conjugates are added to the vesicle after initial vesicle synthesis. Thus, lipid vesicle prior to the addition of the targeting conjugate can be referred to as initial lipid vesicle, starting lipid vesicle materials, or in any other way suitable to distinguish the lipid vesicle before and after the insertion or other addition of the targeting conjugate. For example, as discussed in more detail below, a particularly preferred technique includes dialysis of a mixture including initial lipid vesicles and lipid conjugates to form functionalized lipid vesicles that include the targeting conjugate.

The lipid vesicles can be any form of naturally-occurring or artificial or synthetic lipid-based vesicles. Such vesicles include, but are not limited to, apoptotic bodies and/or blebs (AB), microvesicles (MV), exosomes, tunneling nanotubes (TNT), niosomes, and liposomes. In preferred embodiments, the vesicles are exosomes. Exosomes are small cell-derived vesicles that serve as conveyors of cellular information and have caused considerable excitement for their potential to deliver therapeutics. As a drug delivery vehicle, they are advantageous because they possess the surface proteins that promote endocytosis and they have the potential to deliver macromolecules. Also, if the exosomes are obtained from the same individual as they are delivered to, the exosomes will be immunotolerant.

Exosomes are vesicles with the size of 30-150 nm, often 40-100 nm, and are observed in most cell types. Exosomes are often similar to MVs with an important difference: instead of originating directly from the plasma membrane, they are generated by inward budding into multivesicular bodies (MVBs). The formation of exosomes includes three different stages: (1) the formation of endocytic vesicles from plasma membrane, (2) the inward budding of the endosomal vesicle membrane resulting in MVBs that consist of intraluminal vesicles (ILVs), and (3) the fusion of these MVBs with the plasma membrane, which releases the vesicular contents, known as exosomes.

Exosomes have a lipid bilayer with an average thickness of ~5 nm (see e.g., Li, Theranostics, 7(3):789-804 (2017) doi: 10.7150/thno.l8133). The lipid components of exosomes include ceramide (sometimes used to differentiate exosomes from lysosomes), cholesterol, sphingolipids, and phosphoglycerides with long and saturated fatty-acyl chains. The outer surface of exosomes is typically rich in saccharide chains, such as mannose, polylactosamine, alpha-2,6 sialic acid, and N-linked glycans.

Many exosomes contain proteins such as platelet derived growth factor receptor, lactadherin, transmembrane proteins and lysosome associated membrane protein-2B, membrane transport and fusion proteins like annexins, flotillins, GTPases, heat shock proteins, tetraspanins, proteins involved in multivesicular body biogenesis, as well as lipid-related proteins and phospholipases. These characteristic proteins therefore serve as good biomarkers for the isolation and quantification of exosomes. Another key cargo that exosomes carry is nucleic acids including deoxynucleic acids (DNA), coding and non-coding ribonucleic acid (RNA) like messenger RNA (mRNA) and microRNA (miRNA).

Although exosomes are preferred, other extracellular vesicles can also be used.

ABs are heterogenous in size and originate from the plasma membrane. They can be released from all cell types and are about 1-5 pm in size.

MVs with the size of 20 nnr ¹ pm are formed due to blebbing with incorporation of cytosolic proteins. In contrast to ABs, the shape of MVs is homogenous. They originate from the plasma membrane and are observed in most cell types.

TNT are thin (e.g., 50-700 nm) and up to 100 pm long actin containing tubes formed from the plasma membrane.

As used herein, the terms AB, MV, exosomes, and TNT refer to naturally occurring lipid vesicles. They can be isolated from tissue, cells, and fluid directly from a subject, including cultured and uncultured tissue, cells, or fluids, and fluid derived or conditioned by cultured cells (e.g., conditioned media). For example, exosomes are present in physiological fluids such as plasma, lymph liquid, malignant pleural effusion, amniotic liquid, breast milk, semen, saliva and urine, and are secreted into the media of cultured cells.

As used herein, liposomes and niosomes refer to synthetic lipid vesicles.

Liposomes are a spherical vesicle composed of at least one bilayer of amphipathic molecules which forms a membrane separating an intravesicular medium from an external medium. The intravesicular medium constitutes the internal aqueous core of the liposome. Hydrophilic molecules or components, can be encapsulated inside the internal aqueous core of the liposome via active methods of encapsulation known in the art and described below. Hydrophobic molecules or components can be entrapped inside the membrane. The liposomes can be, for example, multilamellar vesicles (MLV), small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), or cochleate vesicles. In some embodiments, the delivery composition is a micelle, or another lipid-based delivery vehicle. See, for example, Torchilin, et ak, Advanced Drug Delivery Reviews, 58(14): 1532-55 (2006) doi: 10.1016/j.addr.2012.09.031, which is specifically incorporated by reference herein in its entirety.

Niosomes are non-ionic surfactant-based vesicles. Niosomes are formed most typically by non-ionic surfactant and cholesterol incorporation as an excipient, however, other excipients can also be used. Niosomes have more penetrating capability than the previous preparations of emulsions.

They are structurally similar to liposomes in having a bilayer, but the fatty acids within the noisome have a single hydrophobic tail rather than two.

Synthetic vesicles can be designed to have some or all characteristics (e.g., size, shape, lipid content, etc.) that are similar or the same as naturally occurring counterparts including AB, MV, exosomes, and TNT. Thus, in some embodiments, liposomes that are more specifically designed to mimic a naturally occurring counterpart and can then be referred to as synesthetic or artificial exosomes, AB, MV, or TNT, etc.

2. Lipids

The disclosed lipid-based vesicles and the lipid conjugates disclosed herein typically include one or a combination of two or more lipids that can be neutral, anionic, or cationic at physiologic pH. The vesicles include, or otherwise can be formed from, any suitable lipid or combination of lipids. Likewise, the conjugates can include or otherwise be formed of any suitable lipid. In some embodiments, a combination of two, three, four, five, or more different lipid conjugates (e.g., different lipids and the same target moiety, different lipids and different targeting moieties, or the same lipid and different targeting moiety) can be inserted or otherwise added to the same lipid vesicle.

Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids,

lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyl-glycero-3- phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2-dioleylphosphoethanolamine (DOPE), 1 ,2- dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2-dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1 ,2-diacyl-.s77-glycero-3-phosphocholines, 1 -acyl-2-acyl-.v/7-glycero-3- phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. The compositions can be generated from a single type of lipid, or a combination of two or more lipids.

The vesicles and conjugates may include a sphingomyelin metabolite. Sphingomyelin metabolites include, without limitation, ceramide, sphingosine, or sphingosine 1-phosphate (SIP). The concentration of the sphingomyelin metabolites included in the lipids of the vesicles can range from, for example, about 0.1 mol % to about 10 mol %, or from about 2.0 mol % to about 5.0 mol %, or can be in a concentration of about 1.0 mol %.

Suitable cationic lipids include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Other cationic lipids also include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[l-(2,3-dioloyloxy)propyl]- N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dialkyloxy-3-dimethylammonium propanes,

dioctadecylamidoglycylspermine (DOGS), 3 -[N-(N',N'-dimethylamino- ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl- 1 -propanaminium trifluoro- acetate (DOSPA), b-alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diCi4-amidine, N-ferf-butyl-N'-tetradecyl-3-tetradecylamino- propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio- acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)- propylamide (DOSPER), and N , N , N' , N'-tetramethyl- , N'-bis(2- hydroxylethyl)-2,3-dioleoyloxy-l ,4-butanediammonium iodide. In some embodiments, the cationic lipids can be l-[2-(acyloxy)ethyl]2- alkyl(alkenyl)-3-(2-hydroxyethyl)-imidazolinium chloride derivatives, for example, l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2- (hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imida zolinium chloride (DPTIM). In some embodiments, the cationic lipids can be 2,3- dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3- dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl- 3 -dimethyl-hydroxy ethyl ammonium bromide (DORIE), 1 ,2- dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide (DORIE- HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide (DORIE-Hpe), 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DSRIE).

The compositions can be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE.

In some embodiments, the vesicles and/or conjugates include or are formed of one or more of 1, 2-distearoyl-.s77-glycero-3-phosphatidylcholine (DSPC), 1, 2-distearoyl-.s77-glycero-3-phosphatidylethanolamine (DSPE), and 1, 2-distearoyl-.s77-glycero-3-phosphoethanolamine-A ^, -| poly

(ethyleneglycol) 2000 (DSPE-PEG) and can include a sterol.

The vesicles and conjugates can include a sterol component. For example, a sterol component may be included to confer the vesicle suitable physicochemical and biological behavior. Such a sterol component may be selected from cholesterol or its derivative e.g., ergosterol or cholesterolhemisuccinate, but it is preferably cholesterol. Cholesterol is often used in lipidic formulation of liposomes because it is generally recognized that the presence of cholesterol decreases their permeability and protects them from the destabilizing effect of plasma or serum proteins.

In some embodiments, particularly where the lipid is used in synthetic liposomes or niosomes, or as the lipid domain for the disclosed conjugates, the lipid can include a synthetic polymer poly-(ethylene glycol) (PEG) in liposome composition (see, e.g., Paphajopoulos, et al., PNAS, 88(24): 11460-11464 (1991) doi: 10.1073/pnas.88.24.11460). The presence of PEG on the surface of the liposomal carrier has been shown to extend blood-circulation time while reducing mononuclear phagocyte system uptake. Further, by synthetic modification of the terminal PEG molecule, stealth liposomes can be actively targeted with monoclonal antibodies or ligands. Liposomes, including long circulating liposomes and stealth liposomes are reviewed in Immordino, et al, Int J Nanomedicine, 1(3):297- 315 (2006) doi: 10.2217/17435889.1.3.297), which is specifically incorporated by reference herein in its entirety.

In preferred embodiments, the lipid includes a phospholipid- PEG conjugate. In liposomes composed of phospholipids and cholesterol, the ability of PEG to increase the circulation lifetime of the vesicles has been found to depend on both the amount of grafted PEG and the length or molecular weight of the polymer (Allen, et al., Biochim Biophys Acta., 1066(1):29— 36 (1991) doi: 10.1016/0005-2736(91)90246-5). In most cases, the longer-chain PEGs have produced the greatest improvements in blood residence time. For example, Allen et al reported that blood levels were higher for SM/PC/CHOL/DSPE-PEG liposomes with longer molecular weight PEG (i.e., PEG 1900 and PEG 5000) than for liposomes containing PEG-lipid with a shorter chain PEG (i.e., PEG 750 and PEG 120). The presence of PEG 2000 doubled the amount of lipid remaining in the plasma compared to formulations containing PEG 350 to 750. In some

embodiments, the PEG is about PGE 350 to about PEG 5000, or between about PEG 750 and about PEG 5000, or between about PEG 1000 and PEG 3000. In a particular embodiment, the PEG is PEG 2000. In addition to modulating the lipid composition, size, and charge of the vesicle to increase in vivo circulation, liposomal surfaces can be modified, for example, with glycolipids or sialic acid or PEG. In some embodiments, the lipid vehicle can be a“long circulating” or“sterically stabilized” or“stealth” lipid vehicle.

Long-circulating sterically-stabilized liposomes (SSL) have the ability to stably encapsulate drugs and facilitate dmg delivery (Muggia, et al., Current Oncol. 2001; 3(2): 156-62 doi: 10.1007/sl 1912-001-0016-5; Zhu, et al., J Pharm Sci. 2011; 100(8): 3146-59 doi: 10.1002/jps.22580; Marra, et al., Biotechnology advances. 2012; 30(1): 302-9. doi:

10.1016/j.biotechadv.2011.06.018). They can alter the pharmacokinetics of the drug, especially compared to free dmg and sometimes enhance their pharmacological activity (Muggia, et al., Current Oncol. 2001; 3(2): 156-62. doi:10.1007/sll912-001-0016-5). Tumor specific drug delivery using lipid- based nanoparticulate dmg carriers, such as SSL, have been used to encapsulate and release drugs, often with higher efficiency compared to free dmg (Gabizon, et al., Horiz Biochem Biophys. 1989; 9: 185-211 PMID: 2656476). Differences in the half-life and/or tissue and tumor distribution are believed to be primary drivers for these actions. Additionally, SSL are also believed to decrease off-targeted toxicity (Lasic, et al., Biochimica et biophy ica acta. 1991; 1070(1): 187-92 doi: 10.1016/0005-2736(91)90162- 2, Sharma, et al., Pharm Res. 1997; 14(8): 992-8;

doi: 10.1023/A: 1012136925030). DOXIL® is an example of a clinically approved nanoparticle-encapsulating the anti-cancer dmg doxombicin. In addition to their ability to stabilize drugs and enhance their bio-distribution, SSL accumulate passively in solid tumors due to the enhanced permeability and retention effect mediated by defects in the vasculature and lack of functional lymphatics (Maeda, et al., J Control Release. 2000; 65: 271-84 doi: 10.1016/S0168-3659(99)00248-5 , Yuan, et al., Cancer research. 1994; 54: 3352-6 PMID: 8012948).

B. Conjugates

The disclosed vesicles include a functional element conjugated or otherwise linked to a lipid (also referred to herein as lipid component). Suitable lipids include, but are not limited to, those discussed above with respect to the lipid vesicles. In some embodiments, the lipid component of the conjugate is different from the other lipid(s) that form the lipid vesicle. In particular embodiments, the lipid component of the conjugate is a fatty acid or a pegylated fatty acid. As discussed above, the lipid can include a PEG molecule. In some embodiments the lipid is the same as at least one of the lipids that forms the lipid vesicle. In particular embodiments the lipid is l,2-disteroyl-sn-glycero-3-phosphoethanolamine (DSPE) with or without a PEG molecule. The lipid component can also be formed from a precursor that includes a chemical moiety that facilitates conjugation, attachment, or another suitable linkage with a functional element.

The functional element can be, for example, a small molecule, protein or polypeptide, carbohydrate, nucleic acid or a combination thereof. The functional moieties can serve a variety of different functions; such as enhancing targeting of the vehicle, inducing intracellular uptake of the target cell, endosome disruption in the target cell, tracking or otherwise monitoring or identifying the vehicle, or a combination thereof. The lipid vesicles can include a combination of two or more of the same or different types of moieties.

1. Targeting Moieties

In particularly preferred embodiments, the conjugate is a targeting moiety. The targeting moiety typically increases attachment, binding, or association of the lipid vesicle to a target cell(s), tissues(s), and/or microenvironment(s) relative other (e.g., non-targeted) cell(s), tissue(s), and/or microenvironment(s). Additionally, or alternatively, the conjugate can enhance cell penetration.

Typically, targeting moieties include a targeting domain and a lipid. Targeting moieties can also include additional domains. For example, the targeting moiety can include one or more linker domains. The targeting domain is conjugated or otherwise linked directly or indirectly to the lipid domain. In some embodiments the targeting domain is conjugated or linked to the lipid domain through a linker.

A targeting domain typically includes or consists of one or more targeting molecules. Exemplary target molecules can include proteins, peptides, nucleic acids, saccharides, or polysaccharides that bind to one or more targets associated with an organ, tissue, cell, extracellular matrix, etc.

In some embodiments, the targeting molecule may preferentially bind to a specific type of tumor or infected cell.

The degree of specificity with which the disclosed lipid vesicles are targeted can be modulated through the selection of a targeting molecule with the appropriate affinity and specificity. For example, a targeting molecule can be a polypeptide, such as an antibody that specifically recognizes a tumor marker that is present exclusively or in higher amounts on a malignant cell (e.g. , a tumor antigen). Suitable targeting molecules that can be used to direct lipid vesicles to cells and tissues of interest, for example cancerous tissue, that are known in the art.

For example, antibodies are very specific. These can be polyclonal, monoclonal, fragments, recombinant, or single chain, many of which are commercially available or readily obtained using standard techniques.

The antibodies can be fragment of a full-length antibody, or a fusion protein formed for segments of the antibody. Exemplary antibody fragments and fusions include, but are not limited to, single chain antibodies, single chain variable fragments (scFv), di-scFv, tri-scFv, diabody, triabody, teratbody, disulfide-linked Fvs (sdFv), Fab', F(ab')2, Fv, and single domain antibody fragments (sdAb).

Examples of moieties include, for example, targeting moieties which provide for the delivery of molecules to specific cells, e.g., antibodies to hematopoietic stem cells, CD34 ⁺ cells, T cells or any other preferred cell type, as well as receptors and ligands expressed on the preferred cell type. Preferably, the moieties target hematopoeitic stem cells.

Targeting molecules can also include neuropilins and endothelial targeting molecules, integrins, selectins, and adhesion molecules.

Examples of molecules targeting extracellular matrix (“ECM”) include glycosaminoglycan (“GAG”) and collagen.

Other useful ligands attached to lipids include pathogen-associated molecular patterns (PAMPs). PAMPs target Toll-like Receptors (TLRs) on the surface of the cells or tissue, or signal the cells or tissue internally, thereby potentially increasing uptake. PAMPs conjugated to the particle surface or co-encapsulated may include: unmethylated CpG DNA (bacterial), double- stranded RNA (viral), lipopolysacharride (bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial), zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial), flagellin (bacterial) poly(inosinic- cy tidy lie) acid (bacterial), lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).

An immunoglobulin molecule containing an Fc portion (targeting Fc receptor), heat shock protein moiety (HSP receptor), phosphatidylserine (scavenger receptors), and lipopolysaccharide (LPS) are additional receptor targets on cells or tissue.

Lectins can render lipid vesicles target- specific to the mucin and mucosal cell layer include lectins isolated from Abrus precatroius, Agaricus bisporus, Anguilla, Arachis hypogaea, Pandeiraea simplicifolia, Bauhinia purpurea, Caragan arobrescens, Cicer arietinum, Codium fragile, Datura stramonium, Dolichos biflorus, Erythrina corallodendron, Erythrina cristagalli, Euonymus europaeus, Glycine max, Elelix aspersa, Helix pomatia, Lathyrus odoratus, Lens culinaris, Limulus polyphemus,

Lysopersicon esculentum, Maclura pomifera, Momordica charantia, Mycoplasma gallisepticum, Naja mocambique, as well as the lectins

Concanavalin A, Succinyl-Concanavalin A, Triticum vulgaris, Ulex europaeus I, II and III, Sambucus nigra, Maackia amurensis, Limax fluvus, Homarus americanus, Cancer antennarius, and Lotus tetragonolobus.

The choice of targeting molecule will depend on the cells or tissues to be targeted. The targeting molecule may generally increase the binding affinity of the vesicles for cell or tissues or may target the vesicles to a particular cell type in a tissue.

In some embodiments, the targeting domain includes or is a positively charged molecule such as avidin, polyethyleneimine or polylysine, that increases the binding of the vesicles to a negatively charged surface or substrate such as extracellular matrix or mucus layers.

Epithelial cell targeting molecules include monoclonal or polyclonal antibodies or bioactive fragments thereof that recognize and bind to epitopes displayed on the surface of epithelial cells. Epithelial cell targeting molecules also include ligands that bind to a cell surface receptor on epithelial cells.

A variety of receptors on epithelial cells may be targeted by epithelial cell targeting molecules. Examples of suitable receptors to be targeted include, but are not limited to, IgE Fc receptors, EpCAM, selected carbohydrate specificites, dipeptidyl peptidase, and E-cadherin.

Additional strategies and exemplary targets for targeting domains are provide below and are particularly useful for the treatment of cancer.

a. Molecular classes of targeting domains i. Ligands and receptors

In one embodiment, tumor or tumor-associated neovasculature targeting domains are ligands that bind to cell surface antigens or receptors that are specifically expressed on tumor cells or tumor-associated neovasculature or are overexpressed on tumor cells or tumor-associated neovasculature as compared to normal tissue. Tumors also secrete a large number of ligands into the tumor microenvironment that affect tumor growth and development. Receptors that bind to ligands secreted by tumors, including, but not limited to, growth factors, cytokines and chemokines, including the chemokines discussed above, are suitable as targeting domains for the vesicles disclosed herein. Ligands secreted by tumors can be targeted using soluble fragments of receptors that bind to the secreted ligands.

Soluble receptor fragments are fragments of polypeptides that may be shed, secreted or otherwise extracted from the producing cells and include the entire extracellular domain, or fragments thereof.

ii. Antibodies

In another embodiment, tumor or tumor-associated neovasculature targeting domains are antibodies, for example, single polypeptide antibodies that bind to cell surface antigens or receptors that are specifically expressed on tumor cells or tumor-associated neovasculature or are overexpressed on tumor cells or tumor-associated neovasculature as compared to normal tissue.

iii. Fc domains

In another embodiment, tumor or tumor-associated neovasculature targeting domains are Fc domains of immunoglobulin heavy chains that bind to Fc receptors expressed on tumor cells or on tumor-associated

neovasculature. As defined herein, the Fc region includes polypeptides containing the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM. In a preferred embodiment, the Fc domain is derived from a human or murine

immunoglobulin. In a more preferred embodiment, the Fc domain is derived from human IgGl or murine IgG2a including the C _H 2 and C _H 3 regions.

b. Antigens, ligands, and receptors to target i. Tumor-specific and tumor-associated antigens

In some embodiments, the targeting domain specifically binds to an antigen that is expressed by tumor cells. The antigen expressed by the tumor may be specific to the tumor, or may be expressed at a higher level within the tumor cells than non- tumor cells. Antigenic markers such as serologically defined markers known as tumor associated antigens, which are either uniquely expressed by cancer cells or are present at markedly higher levels (e.g., elevated in a statistically significant manner) in subjects having a malignant condition relative to appropriate controls, are known.

Tumor-associated antigens may include, for example, cellular oncogene-encoded products or aberrantly expressed proto-oncogene-encoded products (e.g., products encoded by the neu, ras, trk, and kit genes), or mutated forms of growth factor receptor or receptor-like cell surface molecules (e.g., surface receptor encoded by the c-erbB gene). Other tumor- associated antigens include molecules that may be directly involved in transformation events, or molecules that may not be directly involved in oncogenic transformation events but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melanoma associated antigens, etc.)

(see, e.g., U.S. Patent No. 6,699,475; Jager, et ah, Int. J. Cancer, 106:817-20 (2003) doi: 10.1002/ijc.11292 ; Kennedy, et a , Int. Rev. Immunol., 22:141- 72 (2003) doi: 10.1080/08830180305222; Scanlan, et al. Cancer Immun., 4:1 (2004) PMID: 14738373). Genes that encode cellular tumor associated antigens include cellular oncogenes and proto-oncogenes that are aberrantly expressed. In general, cellular oncogenes encode products that are directly relevant to the transformation of the cell, so these antigens are particularly preferred targets for oncotherapy and immunotherapy. An example is the tumorigenic neu gene that encodes a cell surface molecule involved in oncogenic

transformation. Other examples include the ras, kit, and trk genes. The products of proto-oncogenes (the normal genes which are mutated to form oncogenes) may be aberrantly expressed (e.g., overexpressed), and this aberrant expression can be related to cellular transformation. Thus, the product encoded by proto-oncogenes can be targeted. Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules that are expressed on the tumor cell surface. An example is the cell surface receptor encoded by the c-erbB gene. Other tumor-associated antigens may or may not be directly involved in malignant transformation. These antigens, however, are expressed by certain tumor cells and may therefore provide effective targets. Some examples are carcinoembryonic antigen (CEA), CA 125 (associated with ovarian carcinoma), and melanoma specific antigens.

In ovarian and other carcinomas, for example, tumor associated antigens are detectable in samples of readily obtained biological fluids such as serum or mucosal secretions. One such marker is CA125, a carcinoma associated antigen that is also shed into the bloodstream, where it is detectable in serum (e.g., Bast, et ak, N. Eng. J. Med., 309:883 (1983) doi:

10.1056/NEJM198310133091503; Lloyd, et ak, Int. J. Cane., 71:842 (1997) doi: 10.1002/(SICI) 1097-0215(19970529)71 :5<842::AID-IJC24>3.0.CO;2- 8. CA125 levels in serum and other biological fluids have been measured along with levels of other markers, for example, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN mucin (STN), and placental alkaline phosphatase (PLAP), in efforts to provide diagnostic and/or prognostic profiles of ovarian and other carcinomas (e.g., Sarandakou, et ak, Acta Oncol., 36:755 (1997) doi: 10.3109/02841869709001350; Sarandakou, et ak, Eur. J. Gynaecol. Oncol., 19(l):73-77 (1998) PMID: 9476065; Meier, et ak, Anticancer Res. , 17(4B):2945 (1997) PMID: 9329571; Kudoh, et ak, Gynecol. Obstet. Invest., 47:52 (1999) doi: 10.1159/000010062). Elevated serum CA125 may also accompany neuroblastoma (e.g., Hirokawa, et al., Surg. Today, 28 (3):349- 354 (1998) doi: 10.1007/s005950050139), while elevated CEA and SCC, among others, may accompany colorectal cancer (Gebauer, et ah, Anticancer Res., 17(4B):2939-2942 (1997) PMID: 9329569).

The tumor associated antigen mesothelin, defined by reactivity with monoclonal antibody K-l, is present on a majority of squamous cell carcinomas including epithelial ovarian, cervical, and esophageal tumors, and on mesotheliomas (Chang, et ah, Cancer Res., 52(1): 181-186 (1992) PMID: 1727378; Chang, et ah, Int. J. Cancer, 50:373 (1992) doi:

10.1002/ijc.2910500308; Chang, et ah, Int. J. Cancer, 51:548 (1992) doi:

10.1002/ijc.2910510408; Chang, et ah, Proc. Natl. Acad. Sci. USA, 93:136 (1996) doi: 10.1073/pnas.93.1.136; Chowdhury, et ah, Proc. Natl. Acad. Sci. USA, 95:669 (1998) doi: 10.1073/pnas.95.2.669). Using MAb K-l, mesothelin is detectable only as a cell-associated tumor marker and has not been found in soluble form in serum from ovarian cancer patients, or in medium conditioned by OVCAR-3 cells (Chang, et ah, Int. J. Cancer,

50:373 (1992) doi: 10.1002/ijc.2910500308). Structurally related human mesothelin polypeptides, however, also include tumor-associated antigen polypeptides such as the distinct mesothelin related antigen (MRA) polypeptide, which is detectable as a naturally occurring soluble antigen in biological fluids from patients having malignancies (see WO 00/50900).

A tumor antigen may include a cell surface molecule. Tumor antigens of known structure and having a known or described function, include the following cell surface receptors: HER1 (GenBank Accession NO: U48722), HER2 (Yoshino, et ah, J. Immunol., 152 (5):2393-2400 (1994) PMID: 8133050; Disis, et ah, Cane. Res., 54 (1): 16-20 (1994) PMID: 7505195; GenBank Ace. Nos. X03363 and M17730), HER3 (GenBank Ace. Nos. U29339 and M34309), HER4 (Plowman, et ah, Nature, 366:473 (1993) doi: 10.1038/366473a0; GenBank Ace. Nos. L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank Ace. Nos. U48722, and K03193), vascular endothelial cell growth factor (GenBank NO: M32977), vascular endothelial cell growth factor receptor (GenBank Ace. Nos. AF022375, 1680143, U48801 and X62568), insulin-like growth factor-I (GenBank Acc. Nos. X00173, X56774, X56773, X06043, European Patent No. GB 2241703), insulin-like growth factor-II (GenBank Acc. Nos.

X03562, X00910, M17863 and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981) doi:

10.1073/pnas.78.5.3039; GenBank Acc. Nos. X01060 and Ml 1507), estrogen receptor (GenBank Acc. Nos. M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank Acc. Nos. X51730, X69068 and M15716), follicle stimulating hormone receptor (FSH-R) (GenBank Acc. Nos. Z34260 and M65085), retinoic acid receptor (GenBank Acc. Nos. L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes, et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989) doi:

10.1073/pnas.86.18.7159; GenBank Acc. Nos. M65132 and M64928) NY- ESO-1 (GenBank Acc. Nos. AJ003149 and U87459), NA 17-A (PCT Publication NO: WO 96/40039), Melan- A/MART- 1 (Kawakami, et al., Proc. Nat. Acad. Sci. USA, 91:3515 (1994) doi: 10.1073/pnas.91.9.3515; GenBank Acc. Nos. U06654 and U06452), tyrosinase (Topalian, et al., Proc. Nat.

Acad. Sci. USA, 91:9461 (1994) doi:10.1073/pnas.91.20.9461; GenBank Acc. NO: M26729; Weber, et al., J. Clin. Invest, 102:1258 (1998) doi:10.1172/JCI4004.), Gp-100 (Kawakami, et al., Proc. Nat. Acad. Sci.

USA, 91:3515 (1994) doi: 10.1073/pnas.91.9.3515; GenBank Acc. NO: S73003, Adema, et al., J. Biol. Chem., 269 (31):20126-20133 (1994) PMID: 7519602), MAGE (van den Bruggen, et al., Science, 254:1643 (1991) doi: 10.1126/science.1840703); GenBank Acc. Nos. U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339,

LI 8920, U03735 and M77481), BAGE (GenBank Acc. NO: U19180; U.S. Pat. Nos. 5,683,886 and 5,571,711), GAGE (GenBank Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143 and U19142), any of the CTA class of receptors including in particular HOM- MEL-40 antigen encoded by the SSX2 gene (GenBank Acc. Nos. X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA, Gold and Freedman, J. Exp. Med., 121:439 (1965) doi: 10.1084/jem.l21.3.439;

GenBank Acc. Nos. M59710, M59255 and M29540), and PyLT (GenBank Acc. Nos. J02289 and J02038); p97 (melanotransferrin) (Brown, et al., 7. Immunol. , 127 (2):539-546 (1981) PMID: 6166674; Rose et al., Proc. Natl. Acad. Sci. USA, 83:1261-61 (1986) doi: 10.1073/pnas.83.5.1261).

Additional tumor associated antigens include prostate surface antigen (PSA) (U.S. Pat. Nos. 6,677,157; 6,673,545); b-human chorionic gonadotropin b- HCG) (McManus, et al., Cancer Res., 36 (9 PT 2):3476-3481 (1976) PMID: 975106; Yoshimura, et al., Cancer, 73:2745-52 (1994) doi:/10.1002/1097- 0142( 19940601)73 : 11<2745 : : AID-CNCR2820731116>3.0.CO ;2- V;

Yamaguchi, et al., Br. J. Cancer, 60:382-84 (1989) doi:

10.1038/bjc.l989.289: Alfthan, et al., Cancer Res., 52:4628-33 (1992)

PMID: 1324787); glycosyltransferase b-1,4-N- acetylgalactosaminyltransferases (GalNAc) (Hoon, et al., Int. J. Cancer, 43:857-62 (1989) doi: 10.1002/ijc.2910430520; Ando, et al., Int. J. Cancer, 40:12-17 (1987) doi: 10.1002/ijc.2910400104; Tsuchida, et al., J. Natl. Cancer, 78:45-54 (1987) doi: 10.1002/1097-

0142(19890315)63:6<1166: :AID-CNCR2820630621>3.0.CO;2-5; Tsuchida, et al., J. Natl. Cancer, 78:55-60 (1987) doi: 10.1093/jnci/78.1.55); NUC18 (Lehmann, et al., Proc. Natl. Acad. Sci. USA, 86:9891-95 (1989) doi:

10.1073/pnas.86.24.9891; Lehmann, et al., Cancer Res., 47:841-45 (1987) PMID: 3542195); melanoma antigen gp75 (Vijayasardahi, et al., J. Exp.

Med. 171:1375-80 (1990) doi: 10.1084/jem.l71.4.1375; GenBank Accession NO: X51455); human cytokeratin 8; high molecular weight melanoma antigen (Natali, et al., Cancer, 59:55-63 (1987) doi: 10.1002/1097- 0142(19870101)59: 1<55::AID-CNCR2820590115>3.0.CO;2-R; keratin 19 (Datta, et al., J. Clin. Oncol. 12:475-82 (1994)

doi: 10.1200/JCO.1994.12.3.475).

Tumor antigens of interest include antigens regarded in the art as cancer/testis (CT) antigens that are immunogenic in subjects having a malignant condition (Scanlan, et al., Cancer Immun. , 4:1 (2004) PMID: 14738373). CT antigens include at least 19 different families of antigens that contain one or more members and that are capable of inducing an immune response, including, but not limited to, MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA 17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).

Additional tumor antigens that can be targeted, including a tumor- associated or tumor-specific antigen, include, but are not limited to, a- actinin-4, Bcr-Abl fusion protein, Casp-8, b-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR- fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml- RARoc fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, Mage- Al,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-l/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr vims antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP- 180, MAGE-4, MAGE-5, MAGE-6, pl85erbB2, pl80erbB-3, c-met, nm- 23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, b- Catenin, CDK4, Mum-1, pl6, TAGE, PSMA, PSCA, CT7, telomerase, 43- 9F, 5T4, 791Tgp72, a-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7- Ag, MOV18, NBY70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS. Other tumor-associated and tumor- specific antigens are known to those of skill in the art and are suitable for targeting by the disclosed fusion viruses.

ii. Antigens associated with tumor neovasculature

Cancer therapeutics can be more effective in treating tumors by targeting to blood vessels of the tumor. Tumor-associated neovasculature provides a readily accessible route through which viral therapeutics can access the tumor. In some embodiments the targeting domain specifically binds to an antigen that is expressed by neo vasculature associated with a tumor.

The antigen may be specific to tumor neovasculature or may be expressed at a higher level in tumor neovasculature when compared to normal vasculature. Exemplary antigens that are over-expressed by tumor- associated neovasculature as compared to normal vasculature include, but are not limited to, VEGF/KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin and <¾b3 integrin/vitronectin. Other antigens that are over expressed by tumor-associated neovasculature as compared to normal vasculature are known to those of skill in the art and are suitable for targeting by the disclosed vesicles.

iii. Chemokines/chemokine receptors

In another embodiment, the targeting domain is engineered to specifically bind to a chemokine or a chemokine receptor. Chemokines are soluble, small molecular weight (8-14 kDa) proteins that bind to their cognate G-protein coupled receptors (GPCRs) to elicit a cellular response, usually directional migration or chemotaxis. Tumor cells secrete and respond to chemokines, which facilitate growth that is achieved by increased endothelial cell recruitment and angiogenesis, subversion of immunological surveillance and maneuvering of the tumoral leukocyte profile to skew it such that the chemokine release enables the tumor growth and metastasis to distant sites. Thus, chemokines are vital for tumor progression.

Based on the positioning of the conserved two N-terminal cysteine residues of the chemokines, they are classified into four groups: CXC, CC, CX3C and C chemokines. The CXC chemokines can be further classified into ELR+ and ELR- chemokines based on the presence or absence of the motif‘glu-leu-arg (ELR motif)’ preceding the CXC sequence. The CXC chemokines bind to and activate their cognate chemokine receptors on neutrophils, lymphocytes, endothelial and epithelial cells. The CC chemokines act on several subsets of dendritic cells, lymphocytes, macrophages, eosinophils, natural killer cells but do not stimulate neutrophils as they lack CC chemokine receptors except murine neutrophils. There are approximately 50 chemokines and only 20 chemokine receptors, thus there is considerable redundancy in this system of ligand/receptor interaction.

Chemokines elaborated from the tumor and the stromal cells bind to the chemokine receptors present on the tumor and the stromal cells. The autocrine loop of the tumor cells and the paracrine stimulatory loop between the tumor and the stromal cells facilitate the progression of the tumor.

Notably, CXCR2, CXCR4, CCR2 and CCR7 play major roles in

tumorigenesis and metastasis. CXCR2 plays a vital role in angiogenesis and CCR2 plays a role in the recruitment of macrophages into the tumor microenvironment. CCR7 is involved in metastasis of the tumor cells into the sentinel lymph nodes as the lymph nodes have the ligand for CCR7, CCL21. CXCR4 is mainly involved in the metastatic spread of a wide variety of tumors.

iv. Targets from Experiments

In some embodiments, the targeting moiety specifically targets a target exemplified in the experiments below, including, for example, ENPP2/autotaxin, CD44, CD29/Integrin beta-1, 1-CAM, or

Asialoglycoprotein Receptor 1/HL-l antibody.

c. Exemplary Targeting Moieties

Suitable antibodies, fragments thereof, and other targeting moieties are known in the art and can be used in the disclosed compositions and methods. See, for example, Firer and Gellerman, J Hematol Oncol., 5: 70 (2012), doi: 10.1186/1756-8722-5-70, Lambert and Berkenblit, Annu Rev Med., 69:191-207 (2018), doi: 10.1146/annurev-med-061516-121357, Diamantis and Banerji, Br J Cancer, 114(4):362-7 (2016). doi:

10.1038/bjc.2015.435. Epub 2016 Jan 7, and Chiavenna, et ak, J Biomed Sci. 2017; 24: 15, Published online 2017 Feb 20. doi: 10.1186/sl2929-016-0311- y, each of which is specifically incorporated by reference herein in its entirety.

Specific, non-limiting examples, of antibodies and fragment and fusion proteins thereof that can be used in the disclosed compositions and methods for the targeting and treatment of cancer, include, but are not limited to, those utilized in the experiments provided herein including:

• Anti-ENPP2/autotaxin (Invitrogen Product No. PA5-12478

• Anti-CD44 (Cell Signaling Technologies Product No. #37259S) · Anti-CD29/Integrin beta-1 (Cell Signaling Technologies Product No.

#4706S)

• Anti-I-CAM (Cell Signaling Technologies Product #4915S)

• Anti- Asialoglycoprotein Receptor 1/HL-l (abCAM, Product

Number: ab49355).

Specific, non-limiting examples, of antibodies and fragment and fusion proteins thereof that can be used in the disclosed compositions and methods for the targeting and treatment of cancer, include, but are not limited to, those described in Tables 1-4.

Table 1: Target antigens in solid tumors (Antibody-Drug Conjugates (ADC)) (adapted from Diamantis and Banerji, Br J Cancer, 114(4):362-7 (2016))

Abbreviations: ADC=antibody-drug conjugate; ALL=acute lymphocytic leukemia; AML=acute myelogenous leukemia; CLL=chronic lymphocytic leukemia; DLBCL=diffuse large B-cell lymphoma;

HL=Hodgkin lymphoma; NHL=non-Hodgkin lymphoma; NSCLC=non- small cell lung cancer; SCLC=small cell lung cancer; TNBC=triple-negative breast cancer.

Table 2: Antibody-drug conjugates (ADCs) marketed, in clinical trials, or in phase II development for treating hematologic malignancies (adapted from Lambert and Berkenblit, Annu Rev Med. , 69: 191-207 (2018), doi: 10.1146/annurev-med-061516- 121357)

^a Antibody abbreviations: huIgG, humanized IgG; chlgG, chimeric IgG. ^b Although these antibodies were humanized, changes in naming methodology at International Nonproprietary Names resulted in the“ximab” suffix of chimeric antibodies. ^c Abbreviations: ALCL, anaplastic large cell lymphoma; AML, acute myeloid leukemia; B-ALL, B cell acute lymphoblastic leukemia; DLBCL, diffuse large cell lymphoma; FDA, United States Food and Drug Administration;

Table 3: Antibody-drug conjugates (ADCs) marketed, in pivotal clinical trials, or in phase II development for treating solid tumors

(adapted from Lambert and Berkenblit, Annu Rev Med. , 69: 191-207 (2018), doi: 10.1146/annurev-med-061516- 121357)

aAntibody abbreviations: hu!gG, humanized IgG; chlgG, chimeric IgG; tg mouse, transgenic mouse with human Ig repertoire. ^b Although these antibodies were humanized, changes in naming methodology at International Nonproprietary Names resulted in the“ximab” suffix of chimeric antibodies. Abbreviations: DLL3, delta- like protein 3; EGFR, epidermal growth factor receptor; ENPP3, ectonucleotide pyrophosphatase/phosphodiesterase 3; FOLR1 or FRa, folate receptor alpha; gpNMB, glycoprotein nonmetastatic B; FL, follicular lymphoma; mBC, metastatic breast cancer; NSCLC, non small cell lung cancer; TNBC, triple-negative breast cancer; PSMA, prostate-specific membrane antigen; MMAE/F, mono methyl auristatin E/mono methyl auristatin F; PBD, pyrrolobenzodiazepine; DM1, N2'- deacetyl-N2'-(3-mercapto- l-oxopropyl)-maytansine; DM4, N2'-deacetyl- N2'-(4-mercapto-4-methyl- l-oxopentyl)-maytansine; SMCC, succinimidyl- 4-(N-maleimidomethyl)cyclohexane-l-carboxylate; SPDB, N-succinimidyl- 4-(2-pyridyldithio)butyrate; sSPDB, N-succinimidyl 4-(2-pyridyldithio)-2- sulfobutanoate; vc, valine-citmlline; va, valine-alanine.

Table 4: Summary of FDA approved monoclonal antibodies for treatment of solid tumors (adapted from Chiavenna, et al., J Biomed Sci.

2017; 24: 15, Published online 2017 Feb 20. doi: 10.1186/sl2929-016-0311- y)

2. Tracking, Imaging, and Diagnostic Moieties

In some embodiments, the functional element include or is a tracking, imaging, or diagnostic moiety.

Exemplary agents include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides, x-ray imaging agents, and contrast agents.

For imaging, radioactive materials such as Technetium99 ( ^99m Tc) or magnetic materials such as Fe ₂ 0 ₃ could be used. Examples of other materials include gases or gas emitting compounds, which are radioopaque. The most common imaging agents for brain tumors include iron oxide and gadolinium. Diagnostic agents can be radioactive, magnetic, or x-ray or ultrasound-detectable. Other detectable labels include, for example, radioisotopes, fluorophores (e.g., fluorescein isothiocyanate (FITC), phycoerythrin), enzymes (e.g., alkaline phosphatase, horseradish

peroxidase), element particles (e.g., gold particles) or contrast agents.

For example, a fluorescent label can be chemically conjugated to a lipid to yield a fluorescently labeled lipid as exemplified below. In other embodiments the label is a contrast agent. A contrast agent refers to a substance that enhances the contrast of structures or fluids within the body in medical imaging. Contrast agents are known in the art and include, but are not limited to agents that work based on X-ray attenuation and magnetic resonance signal enhancement. Suitable contrast agents include iodine and barium.

C. Active Agents

Agents to be delivered include therapeutic, nutritional, diagnostic, and prophylactic compounds. Proteins, peptides, carbohydrates,

polysaccharides, nucleic acid molecules, and organic molecules, as well as diagnostic agents, can be delivered.

Exemplary materials to be incorporated are drugs and imaging agents. Therapeutic agents include antibiotics, antivirals, anti-parasites (helminths, protozoans), anti-cancer (referred to herein as

"chemotherapeutics", including cytotoxic drugs such as doxorubicin, cyclosporine, mitomycin C, cisplatin and carboplatin, BCNU, 5-FU, methotrexate, adriamycin, camptothecin, epothilones A-F, and taxol), antibodies and bioactive fragments thereof (including humanized, single chain, and chimeric antibodies), antigen and vaccine formulations, peptide drugs, anti-inflammatories, nutraceuticals such as vitamins, and nucleic acid drugs (including DNA, RNAs including mRNAs, antisense, siRNA, miRNA, anti-miRNA, piwi-interacting RNA (piRNA), aptamers, ribozymes, external guide sequences for ribonuclease P, and triplex forming agents such as“tail- clamp” peptide nucleic acids (tcPNAs)). In some embodiments, the active agent is a vector, plasmid, or other polynucleotide encoding a nucleic acid such as those discussed above.

Exemplary drugs to be delivered include anti- angiogenic agents, antiproliferative and chemotherapeutic agents such as rampamycin.

Representative classes of diagnostic materials include paramagnetic molecules, fluorescent compounds, magnetic molecules, and radionuclides. Exemplary materials include, but are not limited to, metal oxides, such as iron oxide, metallic particles, such as gold particles, etc. Biomarkers can also be conjugated to the surface for diagnostic applications.

One or more active agents may be formulated alone or with excipients or encapsulated on, in or incorporated into the nanocarriers.

Active agents include therapeutic, prophylactic, neutraceutical and diagnostic agents. Any suitable agent may be used. These include organic compounds, inorganic compounds, proteins, polysaccharides, nucleic acids or other materials that can be incorporated using standard techniques.

Alternatively, vesicles may encapsulate cellular materials, such as for example, cellular materials to be delivered to antigen presenting cells as described below to induce immunological responses.

Prophylactics can include compounds alleviating swelling, reducing radiation damage, and anti-inflammatories·

Exemplary agents for imaging including radioactive materials, fluorescent label, and contrast agents are discussed above.

Active agents can be selected based on the type of treatment being employed. Exemplary active agents for treating cancer, ischemia, and injury.

Active agents include synthetic and natural proteins (including enzymes, peptide-hormones, receptors, growth factors, antibodies, signaling molecules), and synthetic and natural nucleic acids (including RNA, DNA, anti-sense RNA, triplex DNA, inhibitory RNA (RNAi), and nucleic acids), and biologically active portions thereof. Suitable active agents have a size greater than about 1,000 Da for small peptides and polypeptides, more typically at least about 5,000 Da and often 10,000 Da or more for proteins. Nucleic acids are more typically listed in terms of base pairs or bases (collectively "bp"). Nucleic acids with lengths above about 10 bp are typically used in the present method. More typically, useful lengths of nucleic acids for probing or therapeutic use will be in the range from about 20 bp (probes; inhibitory RNAs, etc.) to tens of thousands of bp for genes and vectors. The active agents may also be hydrophilic molecules, and optionally have a low molecular weight.

Thus, in some embodiments, the active agent can be a functional nucleic acid. Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction. As discussed in more detail below, functional nucleic acid molecules can be divided into the following non- limiting categories:

antisense molecules, siRNA, miRNA, aptamers, ribozymes, triplex forming molecules, RNAi, and external guide sequences. The functional nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

Functional nucleic acid molecules can interact with any

macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functional nucleic acids can interact with the mRNA or the genomic DNA of a target polypeptide or they can interact with the polypeptide itself. Often functional nucleic acids are designed to interact with other nucleic acids based on sequence homology between the target molecule and the functional nucleic acid molecule. In other situations, the specific recognition between the functional nucleic acid molecule and the target molecule is not based on sequence homology between the functional nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place. a. Functional Nucleic Acids

i. miRNA

A particularly interesting therapeutic macromolecule to deliver with the disclosed vesicles are microRNAs. They represent a class of non-coding RNAs that can modulate cellular activities on a genetic level. Thus, the functional nucleic acid can be a microRNA (miRNA or miRs) molecule. miRNAs represent a class of small, 18- to 28-nucleotide-long, noncoding RNA molecules (Tanase, et al., Molecular Pathology of Pituitary Adenomas, Chapter 8, MicroRNAs, pg. 91-96 (2012)). More than 900 members of the family have been identified in humans. Their major role is in the

posttranscriptional regulation of protein expression, and their involvement has been confirmed in normal and in pathological cellular processes including, but not limited to, cell differentiation, cell cycle progression, and apoptosis. miRNAs are“multivalent,” with one miRNA able to target multiple genes, thus regulating the expression of several proteins.

miRNAs are generated from large RNA precursors (termed pri- miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs, which fold into imperfect stem-loop structures (Lee, Y., et al., Nature (2003) 425(6956):415-9) doi: 10.1038/nature01957. The pre- miRNAs undergo an additional processing step within the cytoplasm where mature miRNAs of 18-25 nucleotides in length are excised from one side of the pre-miRNA hairpin by an RNase III enzyme, Dicer (Hutvagner, G., et al., Science (2001) 12:12 doi: 10.1126/science.l062961 and Grishok, A., et al., Cell (2001) 106(l):23-34 doi: 10.1016/S0092-8674(01)00431-7). miRNAs have been shown to regulate gene expression in two ways. First, miRNAs that bind to protein-coding mRNA sequences that are exactly complementary to the miRNA induce the RNA-mediated interference (RNAi) pathway. Messenger RNA targets are cleaved by ribonucleases in the

ribonucleoprotein complex known as the RNA-induced silencing complex (RISC) complex. This mechanism of miRNA-mediated gene silencing has been observed mainly in plants (Hamilton, A.J. and D.C. Baulcombe,

Science (1999) 286(5441):950-2 doi: 10.1126/science.286.5441.950 and Reinhart, B.J., et al., miRNAs in plants. Genes and Dev. (2002) 16:1616- 1626 doi: 10.1101/gad.l004402), but an example is known from animals (Yekta, S., I.H. Shih, and D.P. Bartel, Science (2004) 304(5670):594-6 doi: 10.1126/science.1097434). In the second mechanism, miRNAs that bind to imperfect complementary sites on messenger RNA transcripts direct gene regulation at the posttranscriptional level but do not cleave their mRNA targets. MiRNAs identified in both plants and animals use this mechanism to exert translational control of their gene targets (Bartel, D.P., Cell (2004) 116(2):281-97 doi: 10.1016/S0092-8674(04)00045-5).

Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumor suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. The two approaches can be referred to as miRNA replacement or restoration therapy and miRNA reduction or inhibition therapy. See, e.g., Chakraborty, et ak, Oncotarget, 9:10164-10174 (2018),

doi.org/10.18632/oncotarget.24309, which, along with references cited therein, are specifically incorporated by reference herein their entireties. miRNA replacement or restoration therapy employs the reuse of miRNAs that are deleted or downregulated in cancers. For example, miR-15a and miR-16-1 have been shown induce apoptosis, block proliferation and control prostate cancer by targeting numerous oncogenic activities. Systemic delivery of tumor suppressor miRNAs using miR-34a and let-7 decreased the tumor load in a KRAS (K-ras; a proto-oncogene)-activated non-small cell lung cancer mouse model. Systemic delivery of tumor suppressor miR-34a and miR-16 repressed the development of prostate and colon cancer and let- 7a in a KRAS mutant mouse model controlled the growth of lung cancer in a xenograft mouse model or murine lung tumor model. MRX34 is a liposome- based miR-34 mimic that can be intravenously injected, and is under investigation for treatment of advanced hepatocellular carcinoma patients. miRNA reduction or inhibition therapy can inactivate those miRNAs that are overexpressed or upregulated in cancers, especially in tumors.

Several miRNA inhibitory agents have been studied over time. Some are locked nucleic acid or LNA, antisense anti-miR oligonucleotides, small molecule inhibitors of miRNAs, and miRNA sponges. Locked nucleic acids (LNA)-anti-miR intravenous injections were shown to block miR-122 and miR92a-3p. LNA inhibition of miR92a-3p encouraged apoptosis and stoped cell propagation in human acute leukemia. Commercial LNA-anti-miR-122 is being tested in clinical trials to manage hepatitis C virus (HCV). Several small molecules have also been reported to inhibit miRNAs including miR-21, which over expressed in different types of human cancers and HeLa cells. miRNA sponges antagonize miRNA, which has RNA transcripts with multiple tandem repeats. It has been noted that sponge RNAs enclose binding sites opposite to a miRNA. A long non coding RNA (IncRNA), IncRNA H19, was also shown to act as miRNA sponges in colorectal cancer Naro, et ak, Bioorg Med Chem Lett., 25:4793- 96 (2015), doi.org/10.1016/j.bmcl.2015.07.016 PMID:26220158, and a miRNA sponge was shown to hinder miR-9 in extremely malignant cells. This sponge is used in the pulmonary micro-metastasis in murine models, which slows metastasis development. miR-9 intensity is linked to MYCN amplification, metastatic status and tumor ranking (Ma, et ak, Nat Cell Biol., 12:247-56 (2010)).

Other targets include miR-205, miR-129, and miR-145.

Combination therapy with miRNA along with a chemotherapeutic agent for the treatment of cancer have also been reported:

miR-205 + gemcitabine used to treat the pancreatic cancer. It inhibited tumor growth in gemcitabine resistant pancreatic cancer cells (MIA PaCa-2(R) and CAPAN-l(R) cells).

miR-34a + paclitaxel used to treat cancers where miR-34a was integrated jointly with paclitaxel into solid lipid nanoparticles (miSLNs- 34a/PTX).

miR-34a + doxorubicin inhibited prostate cancer metastasis and progenitor cells. It hindered prostate cancer metastasis through repressing CD44.

miR-129 + fluorouracil (5-FU) was used to treat colorectal tumor mouse model.

miR-145 + fluorouracil (5-FU) was used to treat both breast cancer cells as well as the breast cancer mouse model.

miR-34a + Docetaxel was used to treat metastatic breast cancer. See, also e.g., Rupaimoole and Slack, Nature Reviews Drug

Discovery, 16:203-222 (2017) doi: 10.1038/nrd.2016.246., Zeng, et al., Methods of Enzymology, 392:371-380 (2005) doi: 10.1016/S0076- 6879(04)92022-8, and Chakraborty, et ah, Molecular Therapy: Nucleic Acids, 8:132-143 (2017) doi: 10.1016/j.omtn.2017.06.005, each of which is specifically incorporated by reference herein in its entirety.

Suitable miRNA molecules and other cargo for miRNA regulation for use in the compositions and methods described herein include, but are not limited to, pri-miRNA, pre-miRNA, mature miRNA, miRNA mimics, or fragments or variants thereof that retain the biological activity of the miRNA and DNA encoding a pri-miRNA, pre-miRNA, mature miRNA, miRNA mimic or fragment or variant thereof, or DNA encoding regulatory elements of the miRNA, miRNA sponges, long non-coding RNAs, and small molecules, alone or in combination with other therapeutic agents such as chemotherapeutic drugs.

In some embodiments the nucleic acid encoding the miRNA molecule is on a vector. These vectors include a sequence encoding a mature microRNA and in vivo expression elements. In a preferred embodiment, these vectors include a sequence encoding a pre-miRNA and in vivo expression elements such that the pre-miRNA is expressed and processed in vivo into a mature miRNA. In another embodiment, these vectors include a sequence encoding the pri-miRNA gene and in vivo expression elements. In this embodiment, the primary transcript is first processed to produce the stem- loop precursor miRNA molecule. The stem- loop precursor is then processed to produce the mature microRNA. Vectors include, but are not limited to, plasmids, cosmids, phagemids, viruses, other vesicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the nucleic acid sequences for producing the microRNA, and free nucleic acid fragments which can be attached to these nucleic acid sequences.

miRNA sequences are known in the art available at miRBase, some accession numbers for which are provided herein and expressly incorporated by reference in their entireties. Exemplary miRNA sequences that can be used as cargo, or to create miRNA expression constructs, include those used in the experiments below and others including, but not limited to:

• miR-489-3p: GUGACAUCACAUAUACGGCAGC (SEQ ID NO: 1).

• miR-21-5p: UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO:2)

• miR-298-5p: GGCAGAGGAGGGCUGUUCUUCCC (SEQ ID

NOG)

• miR-298: AGCAGAAGCAGGGAGGUUCUCCCA (SEQ ID NO:4)

• miR-101: CAGUUAUCACAGUGCUGAUGCU (SEQ ID NOG).

ii. Antisense

The functional nucleic acids can be antisense molecules. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAse H mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule. Exemplary methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. It is preferred that antisense molecules bind the target molecule with a dissociation constant (K _d ) less than or equal to 10 ⁶ , 10 ⁸ , 10 ¹⁰ , or 10 ¹² .

iii. Aptamers

The functional nucleic acids can be aptamers. Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem- loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with K _d s from the target molecule of less than 10 ¹² M. It is preferred that the aptamers bind the target molecule with a Kd less thanlO ⁶ M, 10 ⁸ , 10 ¹⁰ , or 10 ¹² M. Aptamers can bind the target molecule with a very high degree of specificity and affinity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower (or higher affinity) than the K _d with a background binding molecule. It is preferred when doing the comparison for a molecule such as a polypeptide, that the background molecule be a different polypeptide.

iv. Ribozymes

The functional nucleic acids can be ribozymes. Ribozymes are functional nucleic acid molecules that are capable of catalyzing a chemical reaction, either intramolecularly or intermolecularly. There are a number of different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions which are based on ribozymes found in natural systems, such as hammerhead ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Preferred ribozymes cleave RNA or DNA substrates, and more preferably cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates through recognition and binding of the target substrate with subsequent cleavage. This recognition is often based mostly on canonical or non-canonical base pair interactions.

This property makes ribozymes particularly good candidates for targeting specific sites on nucleic acids for cleavage because recognition of the target substrate is based on the target substrates sequence.

v. Triplex Forming Oligonucleotides

The functional nucleic acids can be triplex forming molecules.

Triplex forming functional nucleic acid molecules are molecules that can interact with either double- stranded or single- stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a K _d less than 10 ⁶ , 10 ^s , 10 ¹⁰ , or 10 ¹² M.

vi. External Guide Sequences

The functional nucleic acids can be external guide sequences.

External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, which is recognized by RNase P, which then cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukarotic cells. Representative examples of how to make and use EGS molecules to facilitate cleavage of a variety of different target molecules are known in the art.

vii. RNA Interference

In some embodiments, the functional nucleic acids induce gene silencing through RNA interference. Gene expression can also be effectively silenced in a highly specific manner through RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11 doi: 10.1038/35888; Napoli, et al. (1990) Plant Cell 2:279-89 doi: 10.1105/tpc.2.4.279; Hannon, (2002) Nature, 418:244-51 doi:10.1038/418244a). Once dsRNA enters a cell, it is cleaved by an RNase III -like enzyme, Dicer, into double stranded short interfering RNAs (siRNA) 21-23 nucleotides in length that contains 2 nucleotide overhangs on the 3’ ends (Elbashir, et al. (2001) Genes Dev., 15:188-200 doi: 10.1101/gad.862301; Bernstein, et al. (2001) Nature, 409:363-6 doi: 10.1038/35053110; Hammond, et al. (2000) Nature, 404:293- 6 doi: 10.1038/35005107). In an ATP dependent step, the siRNAs become integrated into a multi- subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21 doi:

10.1016/S0092-8674(01)00547-5). At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases (Martinez, et al. (2002) Cell, 110:563-74 doi: 10.1016/S0092-8674(02)00908-X). However, the effect of miRNA or siRNA or their use is not limited to any type of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, a siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02/44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, herein incorporated by reference for the method of making these siRNAs.

Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double- stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, et al. (2001) Nature, 411:494 498 doi: 10.1016/S0248-4900(03)00079-0) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82 doi: 10.1016/S0014-5793(00)01883-4). siRNA can be chemically or in vz ^' /ro-synthesized or can be the result of short double- stranded hairpin like RNAs (shRNAs) that are processed into siRNAs inside the cell.

Synthetic siRNAs are generally designed using algorithms and a

conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The

Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion’ s SILENCER® siRNA Construction Kit.

The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAse (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex’s GENESUPPRESSOR™ Construction Kits and Invitrogen’s BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors.

In some embodiment, the functional nucleic acid is siRNA, shRNA, miRNA. In some embodiments, the composition includes a vector expressing the functional nucleic acid. Methods of making and using vectors for in vivo expression of functional nucleic acids such as antisense oligonucleotides, siRNA, shRNA, miRNA, EGSs, ribozymes, and aptamers are known in the art.

viii. Other Gene Editing Compositions

In some embodiments the functional nucleic acids are gene editing compositions. Gene editing compositions can include nucleic acids that encode an element or elements that induce a single or a double strand break in the target cell’s genome, and optionally a polynucleotide.

1. Strand Break Inducing

Elements

CRISPR/Cas

In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a CRISPR/Cas system. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15 :339(6121):819— 823 (2013) doi: 10.1126/science.l231143. and Jinek, et ak, Science, 337(6096):816-21 (2012)) doi: 10.1126/science.1225829. By transfecting a cell with the required elements including a Cas gene and specifically designed CRISPRs, the organism's genome can be cut and modified at any desired location. Methods of preparing compositions for use in genome editing using the CRISPR/Cas systems are described in detail in WO 2013/176772 and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.

In general,“CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer- direct repeat) can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.

In some embodiments, a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural

crRNAdracrRNA duplex as described in Cong, Science, 15:339(6121):819- 823 (2013) doi: 10.1126/science.1231143. and Jinek, et ak, Science, 337(6096):816-21 (2012)) doi: 10.1126/science.1225829. A single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)). Within an sgRNA, the crRNA portion can be identified as the‘target sequence’ and the tracrRNA is often referred to as the‘scaffold’.

There are many resources available for helping practitioners determine suitable target sites once a desired DNA target sequence is identified. For example, numerous public resources, including a bioinformatically generated list of about 190,000 potential sgRNAs, targeting more than 40% of human exons, are available to aid practitioners in selecting target sites and designing the associate sgRNA to affect a nick or double strand break at the site. See also, CRISPRs web server

(https://crispr.i2bc.paris-saclay.fr/), a tool designed to help scientists find CRISPR targeting sites in a wide range of species and generate the appropriate crRNA sequences.

In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a target cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. While the specifics can be varied in different engineered CRISPR systems, the overall methodology is similar. A practitioner interested in using CRISPR technology to target a DNA sequence can insert a short DNA fragment containing the target sequence into a guide RNA expression plasmid. The sgRNA expression plasmid contains the target sequence (about 20 nucleotides), a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter and necessary elements for proper processing in eukaryotic cells. Such vectors are commercially available (see, for example, Addgene). Many of the systems rely on custom, complementary oligos that are annealed to form a double stranded DNA and then cloned into the sgRNA expression plasmid. Co-expression of the sgRNA and the appropriate CRISPR-associated (Cas) enzyme from the same or separate plasmids in transfected cells results in a single or double strand break (depending of the activity of the Cas enzyme) at the desired target site.

Zinc Finger Nucleases

In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a zinc finger nucleases (ZFNs). ZFNs are typically fusion proteins that include a DNA-binding domain derived from a zinc- finger protein linked to a cleavage domain.

The most common cleavage domain is the Type IIS enzyme Fokl. Fokl catalyzes double-stranded cleavage of DNA, at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other. See, for example, U.S. Pat. Nos. 5,356,802; 5,436, 150 and 5,487,994; as well as Li et al. Proc., Natl. Acad. Sci. USA 89 (1992):4275- 4279 doi: 10.1073/pnas.89.13.5847; Li et al. Proc. Natl. Acad. Sci. USA, 90:2764-2768 (1993) doi: 10.1073/pnas.90.7.2764; Kim et al. Proc. Natl. Acad. Sci. USA. 91:883-887 (1994) doi: 10.1073/pnas.91.3.883; Kim et al. J. Biol. Chem. 269 (50): 31978-31982 (1994) PMID: 7989374. One or more of these enzymes (or enzymatically functional fragments thereof) can be used as a source of cleavage domains.

The DNA-binding domain, which can, in principle, be designed to target any genomic location of interest, can be a tandem array of Cys2His2 zinc fingers, each of which generally recognizes three to four nucleotides in the target DNA sequence. The Cys2His2 domain has a general structure: Phe (sometimes Tyr)-Cys-(2 to 4 amino acids)-Cys-(3 amino acids)- Phe(sometimes Tyr)-(5 amino acids)-Leu-(2 amino acids)-His-(3 amino acids)-His. By linking together multiple fingers (the number varies: three to six fingers have been used per monomer in published studies), ZFN pairs can be designed to bind to genomic sequences 18-36 nucleotides long.

Engineering methods include, but are not limited to, rational design and various types of empirical selection methods. Rational design includes, for example, using databases including triplet (or quadruplet) nucleotide sequences and individual zinc finger amino acid sequences, in which each triplet or quadruplet nucleotide sequence is associated with one or more amino acid sequences of zinc fingers which bind the particular triplet or quadruplet sequence. See, for example, U.S. Pat. Nos. 6, 140,081; 6,453,242; 6,534,261; 6,610,512; 6,746,838; 6,866,997; 7,067,617; U.S. Published Application Nos. 2002/0165356; 2004/0197892; 2007/0154989;

2007/0213269; and International Patent Application Publication Nos. WO 98/53059 and WO 2003/016496.

Transcription Activator-Like Effector Nucleases

In some embodiments, the element that induces a single or a double strand break in the target cell’s genome is a nucleic acid construct or constructs encoding a transcription activator- like effector nuclease

(TALEN). TALENs have an overall architecture similar to that of ZFNs, with the main difference that the DNA-binding domain comes from TAL effector proteins, transcription factors from plant pathogenic bacteria. The DNA-binding domain of a TALEN is a tandem array of amino acid repeats, each about 34 residues long. The repeats are very similar to each other; typically they differ principally at two positions (amino acids 12 and 13, called the repeat variable diresidue, or RVD). Each RVD specifies preferential binding to one of the four possible nucleotides, meaning that each TALEN repeat binds to a single base pair, though the NN RVD is known to bind adenines in addition to guanine. TAL effector DNA binding is mechanistically less well understood than that of zinc-finger proteins, but their seemingly simpler code could prove very beneficial for engineered- nuclease design. TALENs also cleave as dimers, have relatively long target sequences (the shortest reported so far binds 13 nucleotides per monomer) and appear to have less stringent requirements than ZFNs for the length of the spacer between binding sites. Monomeric and dimeric TALENs can include more than 10, more than 14, more than 20, or more than 24 repeats. Methods of engineering transcription activator-like (TAL) to bind to specific nucleic acids are described in Cermak, et al, Nucl. Acids Res. 1-11 (2011) doi: 10.1093/nar/gkr218. US Published Application No.

2011/0145940, which discloses TAL effectors and methods of using them to modify DNA. Miller et al. Nature Biotechnol 29: 143 (2011) doi:

10.1038/nbt.l755 reported making TALENs for site-specific nuclease architecture by linking TAL truncation variants to the catalytic domain of Fokl nuclease. The resulting TALENs were shown to induce gene modification in immortalized human cells. General design principles for TALEN binding domains can be found in the patent titled,“Tal Effector- mediated DNA Modification (WO 2011/072246)” for example.

2. Gene Altering Polynucleotides

The nuclease activity of the genome editing systems described herein cleave target DNA to produce single or double strand breaks in the target DNA. Double strand breaks can be repaired by the cell in one of two ways: non-homologous end joining, and homology- directed repair. In non- homologous end joining (NHEJ), the double-strand breaks are repaired by direct ligation of the break ends to one another. As such, no new nucleic acid material is inserted into the site, although some nucleic acid material may be lost, resulting in a deletion. In homology-directed repair, a donor polynucleotide with homology to the cleaved target DNA sequence is used as a template for repair of the cleaved target DNA sequence, resulting in the transfer of genetic information from a donor polynucleotide to the target DNA. As such, new nucleic acid material can be inserted/copied into the site.

Therefore, in some embodiments, the genome editing composition optionally includes a donor polynucleotide. The modifications of the target DNA due to NHEJ and/or homology-directed repair (HDR) can be used to induce gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc.

Accordingly, cleavage of DNA by the genome editing composition can be used to delete nucleic acid material from a target DNA sequence by cleaving the target DNA sequence and allowing the cell to repair the sequence in the absence of an exogenously provided donor polynucleotide. Alternatively, if the genome editing composition includes a donor polynucleotide sequence that includes at least a segment with homology to the target DNA sequence, the methods can be used to add, i.e., insert or replace, nucleic acid material to a target DNA sequence (e.g., to“knock in” a nucleic acid that encodes for a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6xHis, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g., promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation), and the like. As such, the

compositions can be used to modify DNA in a site- specific, i.e.,“targeted”, way, for example gene knock-out, gene knock-in, gene editing, gene tagging, etc. as used in, for example, gene therapy.

In applications in which it is desirable to insert a polynucleotide sequence into a target DNA sequence, a polynucleotide including a donor sequence to be inserted is also provided to the cell. By a“donor sequence” or“donor polynucleotide” or“donor oligonucleotide” it is meant a nucleic acid sequence to be inserted at the cleavage site. The donor polynucleotide typically contains sufficient homology to a genomic sequence at the cleavage site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the cleavage site, e.g., within about 50 bases or less of the cleavage site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the cleavage site, to support homology-directed repair between it and the genomic sequence to which it bears homology. The donor sequence is typically not identical to the genomic sequence that it replaces. Rather, the donor sequence may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the genomic sequence, so long as sufficient homology is present to support homology-directed repair. In some embodiments, the donor sequence includes a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region.

b. Nucleic Acid Composition

The functional nucleic acids can be DNA or RNA nucleotides which typically include a heterocyclic base (nucleic acid base), a sugar moiety attached to the heterocyclic base, and a phosphate moiety which esterifies a hydroxyl function of the sugar moiety. The principal naturally-occurring nucleotides comprise uracil, thymine, cytosine, adenine and guanine as the heterocyclic bases, and ribose or deoxyribose sugar linked by phosphodiester bonds.

In some embodiments, the nucleic acids are composed of nucleotide analogs that have been chemically modified to improve stability, half-life, or specificity or affinity for a target receptor, relative to a DNA or RNA counterpart. The chemical modifications include chemical modification of nucleobases, sugar moieties, nucleotide linkages, or combinations thereof.

As used herein‘modified nucleotide” or“chemically modified nucleotide” defines a nucleotide that has a chemical modification of one or more of the heterocyclic base, sugar moiety or phosphate moiety constituents. In some embodiments, the charge of the modified nucleotide is reduced compared to DNA or RNA nucleic acids of the same nucleobase sequence. For example, the nucleic acid can have low negative charge, no charge, or positive charge.

Typically, nucleoside analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequence-specific fashion between the nucleic acid analog molecule and bases in a standard polynucleotide (e.g., single- stranded RNA or single-stranded DNA). In some embodiments, the analogs have a substantially uncharged, phosphorus containing backbone.

i. Heterocyclic Bases

The principal naturally-occurring nucleotides include uracil (U), thymine (T), cytosine (C), adenine (A) and guanine (G) as the heterocyclic bases. The nucleic acids can include chemical modifications to their nucleobase constituents. Chemical modifications of heterocyclic bases or heterocyclic base analogs may be effective to increase the binding affinity or stability in binding a target sequence. Chemically-modified heterocyclic bases include, but are not limited to, inosine, 5-(l-propynyl) uracil (pU), 5- (1-propynyl) cytosine (pC), 5-methylcytosine, 8-oxo-adenine,

pseudocytosine, pseudoisocytosine, 5 and 2-ami no-5-(2’-deoxy-P-D- ribofuranosyl)pyridine (2-aminopyridine), and various pyrrolo- and pyrazolopyrimidine derivatives.

ii. Sugar Modifications

Nucleic acids can also contain nucleotides with modified sugar moieties or sugar moiety analogs. Sugar moiety modifications include, but are not limited to, 2’-0-aminoetoxy, 2’-0-amonioethyl (2’-OAE), 2’-0- methoxy, 2’-0-methyl, 2-guanidoethyl (2’-OGE), 2'-0,4’-C-methylene, 2'-0- (methoxyethyl) (2'-OME) and 2'-0-(N-(methyl)acetamido) (2'-OMA). 2'-0- aminoethyl sugar moiety substitutions are especially preferred because they are protonated at neutral pH and thus suppress the charge repulsion between the triplex forming oligonucleotide (TFO) and the target duplex. This modification stabilizes the C3'-endo conformation of the ribose or dexyribose and also forms a bridge with the i- 1 phosphate in the purine strand of the duplex.

In some embodiments, the functional nucleic acid is a morpholino nucleic acid. Morpholino nucleic acids are typically composed of two more morpholino monomers containing purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, which are linked together by phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one monomer to the 5' exocyclic carbon of an adjacent monomer. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.

Important properties of the morpholino-based subunits typically include: the ability to be linked in a oligomeric form by stable, uncharged backbone linkages; the ability to support a nucleotide base (e.g. adenine, cytosine, guanine, thymidine, uracil or inosine) such that the polymer formed can hybridize with a complementary-base target nucleic acid, including target RNA, with high T _m , even with oligomers as short as 10-14 bases; the ability of the oligomer to be actively transported into mammalian cells; and the ability of an oligomerRNA heteroduplex to resist RNAse degradation.

In some embodiments, nucleic acids employ morpholino-based subunits bearing base-pairing moieties, joined by uncharged linkages, as described above.

iii. Internucleotide Linkages

Nucleic acids connected by an internucleotide bond that refers to a chemical linkage between two nucleoside moieties. Modifications to the phosphate backbone of DNA or RNA nucleic acids may increase the binding affinity or stability nucleic acids, or reduce the susceptibility of nucleic acids nuclease digestion. Cationic modifications, including, but not limited to, diethyl-ethylenediamide (DEED) or dimethyl- aminopropylamine (DMAP) may be especially useful due to decrease electrostatic repulsion between the nucleic acid and a target. Modifications of the phosphate backbone may also include the substitution of a sulfur atom for one of the non-bridging oxygens in the phosphodiester linkage. This substitution creates a phosphorothioate internucleoside linkage in place of the phosphodiester linkage. Nucleic acids containing phosphorothioate intemucleoside linkages have been shown to be more stable in vivo.

Examples of modified nucleotides with reduced charge include modified intemucleotide linkages such as phosphate analogs having achiral and uncharged intersubunit linkages (e.g. Verma et a , Annual Review of Biochemistry, 67(1): 99-134 doi: 10.1146/annurev.biochem.67.1.99), and uncharged morpholino-based polymers having achiral intersubunit linkages (see, e.g., U.S. Pat. No. 5,034,506), as discussed above. Some

internucleotide linkage analogs include morpholidate, acetal, and polyamide- linked heterocycles.

In another embodiment, the nucleic acids are composed of locked nucleic acids. Locked nucleic acids (LNA) are modified RNA nucleotides (see, for example, Braasch, et ak, Chem. Biol. , 8(1): 1-7 (2001) doi:

10.1016/S1074-5521(00)00058-2). LNAs form hybrids with DNA which are more stable than DNA/DNA hybrids, a property similar to that of peptide nucleic acid (PNA)/DNA hybrids. Therefore, LNA can be used just as PNA molecules would be. LNA binding efficiency can be increased in some embodiments by adding positive charges to it. Commercial nucleic acid synthesizers and standard phosphoramidite chemistry are used to make LNAs.

In some embodiments, the nucleic acids are composed of peptide nucleic acids. Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the phosphate backbone of the nucleic acid is replaced in its entirety by repeating N-(2-aminoethyl)-glycine units and phosphodiester bonds are typically replaced by peptide bonds. The various heterocyclic bases are linked to the backbone by methylene carbonyl bonds. PNAs maintain spacing of heterocyclic bases that is similar to conventional DNA nucleic acids, but are achiral and neutrally charged molecules. Peptide nucleic acids are comprised of peptide nucleic acid monomers.

Other backbone modifications include peptide and amino acid variations and modifications. Thus, the backbone constituents of nucleic acids such as PNA may be peptide linkages, or alternatively, they may be non-peptide peptide linkages. Examples include acetyl caps, amino spacers such as 8-amino-3,6-dioxaoctanoic acid (referred to herein as O-linkers), amino acids such as lysine are particularly useful if positive charges are desired in the PNA, and the like. Methods for the chemical assembly of PNAs are well known. See, for example, U.S. Patent Nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,736,336, 5,773,571 and 5,786,571.

Nucleic acids optionally include one or more terminal residues or modifications at either or both termini to increase stability, and/or affinity of the nucleic acid for its target. Commonly used positively charged moieties include the amino acids lysine and arginine, although other positively charged moieties may also be useful. Nucleic acids may be end capped to prevent degradation using a propylamine group. Procedures for 3' or 5' capping nucleic acids are well known in the art.

In some embodiments, the functional nucleic acid can be single stranded or double stranded. III. Methods of Making Functionalized Vesicles

A. Sources of Vesicles

1. Methods of Isolating Naturally-Occurring Vesicles

Methods of isolating extracellular vesicles from tissue, cells, and fluid directly from a subject, including cultured and uncultured tissue, cells, or fluids, and fluid derived or conditioned by cultured cells (e.g., conditioned media) are known in the art.

See, for example, Li, Thernaostics, 7(3):789-804 (2017) doi:

10.7150/thno.l8133, Ha, et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016/j.apsb.2016.02.001, Skotland, et al., Progress in Lipid Research, 66:30-41 (2017) doi: 10.1016/j.plipres.2017.03.001, Phinney and Pittenger, Stem Cells, 35:851-858 (2017) doi: 10.1002/stem.2575, each of which is specifically incorporated by reference, and describes of isolating extracellular vesicles, particularly exosomes.

For example, extracellular vesicles, particularly exosomes, can be isolated using differential centrifugation, flotation density gradient centrifugation, filtration, high performance liquid chromatography, and immunoaffinity-capture.

One of the most common isolation technique for isolating exosomes from cell culture is differential centrifugation, whereby large particles and cell debris in the culture medium are separated using centrifugal force between 200-100, OOOxg and the exosomes are separated from supernatant by the sedimenting exosomes at 100, OOOxg. Purity can be improved, however, by centrifuging the samples using flotation density gradient centrifugation with sucrose or Optiprep. Tangential flow filtration combined with deuterium/sucrose-based density gradient ultracentrifugation was employed to isolate therapeutic exosomes for clinical trials.

Ultrafiltration and high performance liquid chromatography (HPLC) are additional methods of isolating exosomes based on their size differences. Exosomes prepared by HPLC are highly purified.

Hydrostatic filtration dialysis has been used for isolating extracellular vesicles from urine.

Another common technique for exosome isolation is a monoclonal antibody-based method. Antibodies against exosome-associated antigens— such as cluster of differentiation (CD) molecules CD63, CD81, CD82, CD9, epithelial cell adhesion molecule (EpCAM), and Ras-related protein (Rab5)— are used for affinity-based separation. The antibodies can be immobilized in different media conditions and combined with magnetic beads, chromatographic matrix, plates, and microfluidic devices for separation. Non-exosomes vesicles that carry the antigens also bind to the antibody, and may also be isolated in this way.

Microfluidics-based devices have also been used to rapidly and efficiently isolate exosomes, tapping on both the physical and biochemical properties of exosomes at microscales. In addition to size, density, and immunoaffinity, sorting mechanisms such as acoustic, electrophoretic and electromagnetic manipulations can be implemented.

Methods of characterizing exosomes are also known in the art.

Exosomes can be characterized based on their size, protein content, and lipid content. Exosomes are sphere-shaped structures with sizes between 40-100 nm and are much smaller compared to other systems, such as a microvesicle, which has a size range from 100-500 nm. Several methods can be used to characterize exosomes, including flow cytometry, nanoparticle tracking analysis, dynamic light scattering, western blot, mass spectrometry, and microscopy techniques. Exosomes can also be characterized and marked based on their protein compositions, with integrins and tetraspanins being the two most abundant proteins found in exosomes. Other protein markers include TSG101, ALG-2 interacting protein X (ALIX), flotillin 1, and cell adhesion molecules. Similar to proteins, lipids are major components of exosomes and can be utilized to characterize them.

An advantage of utilizing vesicles such as exosome that are isolated from natural sources includes avoidance of immunogenicity that can be associated with artificially produced lipid vesicles.

In some embodiments, the vesicles are isolated from cells, tissue, or fluid of the subject to be treated. Vesicles obtained in this manner, for example from human blood, and can thus be used for personalized medicine with a patient’ s own lipid-based vesicles after they are functionalized. Other promising sources include, for example, immune cells and stromal cells. 2. Methods of Making Synthetic and Artificial

Vesicles

Methods of making liposomes and niosomes are also known in the art. Liposomes typically have an aqueous core. The aqueous core can contain water or a mixture of water and alcohol. Suitable alcohols include, but are not limited to, methanol, ethanol, propanol, (such as isopropanol), butanol (such as n-butanol, isobutene, seobutanol, iari-butanol, pentane (such as amyl alcohol, isobutyl carbinol), hexanol (such as 1-hexanol, 2- hexanol, 3-hexanol), heptanol (such as 1-heptanol, 2-heptanol, 3-heptanol and 4-heptanol) or octanol (such as 1-octanol) or a combination thereof.

The liposomes have either one or several aqueous compartments delineated by either one (unilamellar) or several (multilamellar) phospholipid bilayers (Sapra, et al., Curr. Drug Deliv., 2, 369-81 (2005) doi:

10.2174/156720105774370159). Multilamellar liposomes have more lipid bilayers for hydrophobic therapeutic agents to associate with. Thus, potentially greater amounts of therapeutic agent are available within the liposome to reach the target cell.

Depending on the mode of preparation, the size and the degree of lamellarity of the vesicles can be tuned. Several methods for preparing unilamellar lipidic vesicles have been described in the literature: reverse phase evaporation (Szoka et ak, PNAS, 1978; 75(9):4191-4198 doi:

0.1073/pnas.75.9.4194), ethanol injection (Pons et al. International Journal of Pharmaceutics, 1993; 95(1 -3):51-56 doi: 10.1016/0378-5173(93)90389- W), heating method (Mozafari et ak, Journal of Biotechnology, 2007;

129:604-613 doi: 10.1016/j.jbiotec.2007.02.005), but the most simple is the lipid film hydration method (Bangham et ak, J. Mol. Bio., 1965; 13:238-252 doi: 10.1016/S0022-2836(65)80093-6).

Briefly, in the lipid film hydration method, lipids are solubilized in an organic solvent such as chloroform. After homogenization of the solution, the organic solvent is evaporated under a nitrogen stream. The as-obtained dried lipid film is then hydrated by an aqueous medium at a temperature above the main phase transition temperature T _m , leading to the formation of multilamellar vesicles with sizes ranging from 100 to 800 nm (Mills J. K. et al. Methods in Enzymology 2004; 387:82-113 doi: 10.1016/S0076- 6879(04)87006-X). Cycles of dehydration and rehydration, by respectively freezing (in liquid nitrogen) and thawing the solution (at a temperature above T _m ), allow increasing the aqueous internal volume by forming unilamellar vesicles. A process allowing vesicles size calibration can be applied to obtain a homogeneous size distribution. Sonication produces Small

Unilamellar Vesicles (SUV) with size ranging from 20 to 50 nm, whereas extrusion process through a filter membrane produces LUVs with size ranging from 50 to 500 nm depending on the size of the filter pores. Both processes, sonication and extrusion, are performed at a temperature above Tm-

Niosomes can be prepared by various methods, many of which are similar to preparing their liposomal counterparts, including, but not limited to ether injection method (EIM), hand shaking method (HSM), reverse phase evaporation method (REV), trans membrane pH gradient, the“Bubble” method, microfluidization method, formation of niosomes from

proniosomes, thin-film hydration method (TFH), heating method (HM), freeze and thaw method (FAT), and dehydration rehydration method (DRM) (Moghassemi and Hadjizadeh, J. Contr. Release, 185:22-36 (2014)).

B. Methods of Making Lipid Conjugates

The disclosed conjugates can be made using any suitable means, including through cross-linking reactions. Conjugation of proteins, peptides, nucleic acids, and small molecules are known in the art (see, e.g., “Conjugation of Proteins, Peptides, & Drugs to Liposomes,” by Anvanti Polar Lipids, Inc.).

Common examples include, but are not limited to, amide conjugation, disulfide/theioether conjugation, and coupling systems such as

biotin/streptavidin binding.

Amide Conjugation: phospholipids with either amine or carboxyl functional groups, for example, carboxyacyl derivatives of

phosphatidylethanolamine (PE) can be used for conjugation with

proteins/peptides, or drugs containing amine, carboxyl, or hydroxy groups.

Various acyl chain lengths, including, but not limited to those ranging from 4 to 22 carbons, and can be utilized. Disulfide/Thioether Conjugation : Lipids including, but not limited to, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2- pyridyldithio)propionate] (PDP-PE) and l,2-dioleoyl-sn-glycero-3- phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide] (MPB-PE) or

1.2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p- maleimidomethyl)cyclohexane-carboxamide] (MCC-PE) can be used for disulfide or thioether based conjugation of thio-containing proteins or peptides.

Maleimide-containing lipids, including, but not limited to, MPB-PE, can also be used. For example, the experiments below utilized a lipid including a maleimide. Maleimide is a reactive group that irreversibly reacts with sulfhydryl groups on proteins linking whatever the maleimide group is attached. This is a well-known crosslinking reaction. Lipids having a terminal maleimide are commercially available and can be used with any polypeptide having at least one sulfhydryl (e.g., a cysteine), such as an antibody, to form a lipid-polypeptide conjugated through thioether bond.

1.2-disteroyl-sn-glycero-3-phosphoethanolamine-N- [maleimide(poly ethylene glycol)-2000], DSPE-2000 was used as a substrate for antibody conjugation.

A comparison of pyridyldithiopropionate (PDP) and

maleimidophenylbutyramide (MPB) conjugates showed the MPB formed more stable complexes that survive in serum longer. The

maleimidomethylcyclohexane-carboxamide (MCC) contains a more stable maleimide function group toward hydrolysis in aqueous reaction

environments, due to the proximity of an aliphatic cyclohexane ring rather than the aromatic phenyl group of MPB.

Any peptide or protein initially lacking the necessary amino acid to facilitate conjugations (e.g., lysine, cysteine, etc.) can be recombinantly modified to add one or more additional amino acids, for example to the N- or C-terminus.

Coupling Systems : The lipid domain and targeting domain can also be linked through a coupling systems. For example, biotinylated lipids can be used and linked to the targeting domain via, for example, streptavidin or neutravidin. The lipids can be linked directly to the biotin, or have a spacer, e.g., a 6-carbon spacer between the biotin and the lipid. Biotin PE has been used for bilayer stabilization, temperature/pH sensitive liposomal drug delivery, tumor imaging, two-dimensional crystallization on lipid bilayers, immobilization of liposomes on gel beads for chromatographic analysis of drug-membrane partitioning, and vivo targeting applications. Other coupling systems include, glutathione-S-transferase/glutathione, maltose binding protein/amylase and maltose binding protein/maltose.

In some embodiments, the lipid conjugate is formed without a coupling system, using, for example, one of the methods described above.

In particular embodiments, synthesis is performed where fatty acids are either conjugated to the N-terminus, or to the side-chain of a lysine or a cysteine. Commonly used fatty acids are: Caprylic acid (C8), Capric acid (CIO), Laurie acid (Cl 2), Myristic acid (Cl 4), Palmitic acid (Cl 6) or Stearic acid (Cl 8).

In some embodiments, cholesterol is conjugated to a peptide via an N- or C-terminal inserted cysteine. For this, a cholesterol derivative that has been modified with a cysteine-reactive 2-bromoacetyl moiety can be used. See, for example, Pepscan.

In some embodiments, the lipid is conjugated to a fluorophore. For example, a range of fluorescent and biotinylated analogs of the five naturally occurring lipid classes: phospholipids, sphingolipids (including ceramides), fatty acids, triglycerides and steroids are commercially available. See, e.g., Molecular Probes™ Handbook, A Guide to Fluorescent Probes and Labeling Technologies, 11 ^th Edition (2010), Chapter 13, Probes for Lipids and Membranes,

C. Methods of Introducing Lipid Conjugates into Delivery Vesicles

The function element can be introduced into a lipid delivery vehicle that includes a lipid amenable to its conjugation. In other words, a vesicle such as a liposome includes a lipid having an element (such as those described above), available for a conjugation reaction with a functional element after formation of the vesicle. Alternatively, the functional element can be reacted with the lipid and later used alone or in combination with non- functionalized lipid to form the vesicle. However, these methods have drawbacks and limitations.

First, intermicellar crosslinking can occur. Furthermore, both of these methods present difficulty for vesicles such as exosomes that are naturally-occurring and thus pre-formed in the absence of conjugate.

Lipid conjugates can be introduced into synthetic liposomes and niosomes at the time these vesicles are formed.

Performing crosslinking and introduction of the lipid conjugate in a separate step provides advantages. In this process, the lipid vesicle are made artificially using standard procedures (i.e., liposomes) or purified from natural sources (i.e., exosomes). To functionalize the lipid vesicle, a functionalized lipid (e.g., fatty acid- antibody) is passively inserted into a lipid vesicle through dialysis. This passive insertion approach of labeling the lipid vesicle avoids disrupting the membrane of the lipid vesicle unlike other methods for integrating antibodies such as sonication.

In an exemplary method, lipid that will form the foundation of the lipid-conjugate (e.g., the lipid component of the lipid-conjugate, or a precursor thereof) is suspended in a concentration of detergent near the critical micelle concentration (0.1% DDM), and then dialyzed to remove excess detergent, and encourage formation of stable micelles in solution. The detergent should be one that is suitable for stabilizing the hydrophobic regions of the lipid in a semi-aqueous solution. It will be removed upon formation of the final product.

Next, the lipid that forms the foundation of the lipid-conjugate (e.g., the lipid component, or a precursor thereof) is mixed with a functional element, for example a targeting moiety such as an antibody, under concentrations and conditions suitable for conjugation (e.g., covalent attachment) of the functional element to the lipid component, to form a lipid- conjugate. When the functional element is an antibody, the lipid-conjugate can also be referred to as an antibody-label.

This lipid-conjugate is mixed with pre-formed lipid vesicle, for example exosomes such as those obtained from cultured human peripheral blood mononuclear cells and dialyzed using a suitable dialysis system. The dialyzed product, containing the functionalized lipid-conjugate inserted into the lipid vesicle membrane, is now a functionalized lipid vesicle. In a more specific embodiment, pegylated lipid (which are commercially available) is resuspended in 0.1% DDM, which is close to the critical micelle concentration, and then dialyzed to remove excess DDM, and encourage formation of stable micelles in solution. The DDM detergent is responsible for stabilizing the hydrophobic regions of the pegylated fatty acid in a semi-aqueous solution, and will be removed upon formation of the final product. Then, the pegylated fatty acid is mixed with antibody at a 2:1 fatty acid: antibody concentration ratio and incubated for 1 h at room temperature, resulting in covalent attachment of antibody to lipid, named the antibody-label or lipid conjugate. This lipid conjugate is mixed with 100-150 uL of pre-formed lipid vesicle obtained from cultured human peripheral blood mononuclear cells and dialyzed using a suitable dialysis system.

For example, in the experiments below, lipid conjugate solution was dialyzed using a Slide- A-Lyzer MINI Dialysis units with a 10,000 Da molecular weight cut-off (Thermo Fisher) against 2L of lx PBS for 2 h in a 4°C cold room to integrate the lipid conjugate into the exosomal membrane and remove any detergent remaining from lipid conjugate preparation from solution. This molecular weight cut-off ensures that the components required to build these functionalized lipid vesicles remain in solution, but excess detergent (e.g., DDM) and buffer can be removed by dialysis to form a functionalized lipid vesicle product.

The described methods can be scaled-up by using a larger dialysis membrane that holds more volume, and using larger quantities of each component to produce large amounts of the final functionalized lipid vesicles. Functional elements, lipids, lipid vehicles, detergents, dialysis membranes and other features can be mixed or substituted, for example, as described herein.

The functionalized lipid vesicles can be precipitated using any suitable means. For example, in the experiments below, the antibody-labeled exosomes (referred to as Abi-exosomes) were incubated overnight with Exosome Precipitation Buffer from the Exosome Isolation Kit per manufacturer instructions (Exiqon). The solution was then centrifuged at 50,000 rpm (>250,000 g’s) for 1 h at 20°C to pellet the Abi-exosomes, the supernatant was removed and the final product was resuspended in lx PBS before the introduction of cargo (e.g., electroporated with miRNA).

D. Loading with Active Agent

Active agents can be loaded into the lipid vesicles using any suitable means.

The disclosed lipid vesicles most typically have an aqueous solution core surrounded by a hydrophobic membrane, often in the form of a lipid bilayer. Hydrophilic solutes dissolved in the core cannot readily pass through the bilayer. Hydrophobic chemicals associate with the bilayer. The vesicles can thus be loaded with hydrophobic and/or hydrophilic molecules. To deliver the molecules to a site of action, the lipid bilayer can fuse with other bilayers such as the cell membrane or be internalized by endocytosis.

Synthetic vesicles can be loaded with active agent by preparing (i.e., forming) the vesicles in a solution containing the active agent.

Naturally occurring and other pre-formed vesicles can also be loaded with drug. For example, methods of loading drug into pre-formed vesicles including exosomes are known in the art and reviewed in Ha, et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi:

10.1016/j.apsb.2016.02.001, and discussed in Yang, et al., J Control Release, 243:160-171 (2016). doi: 10.1016/j.jconrel.2016.10.008. Briefly, small molecules have been loaded by mixing and incubation and through complexation with, for example, surface elements. Proteins and peptides have been loaded by incubation, with or without a permeabilizer such as saponin, through freeze-thaw cycling, sonication, and extrusion procedures. Nucleic acids have been load by chemical transfection and electroporation. See also Table 2 of Ha, et al., Acta Pharmaceutica Sinica B, 6(4):287-296 (2016) doi: 10.1016/j.apsb.2016.02.001, and the references cited therein.

In a particular preferred embodiment, nucleic acid active agents, such as as miRNA, are loaded by electroporation. In electroporation experiments, Bio-Rad Gene Pulser X-Cell CE was used to electroporate miRNA in an exponential decay model into the exosomes at 150 Volts, 125 pF of capacitance for 10-15 microseconds in a 4mm cuvette, which were subsequently incubated at room temperature for 30 min prior to treating cells. With these parameters, there is a 700,000-fold higher internalization of the macromolecule microRNA (miRNA) into exosomes (Fig. 3A, last column).

IV. Pharmaceutical Compositions

Pharmaceutical compositions including for lipid based vesicles are also provided. Pharmaceutical compositions can be for administration by parenteral (intramuscular (IM), intraperitoneal (IP), intravenous (IV) or subcutaneous injection (SubQ)), transdermal (either passively or using iontophoresis or electroporation), or transmucosal (nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible inserts and can be formulated in dosage forms appropriate for each route of administration·

In some embodiments, the compositions are administered

systemically, for example, by intravenous or intraperitoneal administration, in an amount effective for delivery of the compositions to targeted cells. Other possible routes include trans-dermal and oral.

In certain embodiments, the compositions are administered locally, for example, by injection directly into a site to be heated. For example, in some embodiments such as for the treatment of cancer, the compositions are injected or otherwise administered directly to one or more tumors.

Typically, local injection causes an increased localized concentration of the compositions which is greater than that which can be achieved by systemic administration. In some embodiments, the compositions are delivered locally to the appropriate cells by using a catheter or syringe. Other means of delivering such compositions locally to cells include using infusion pumps (for example, from Alza Corporation, Palo Alto, Calif.) or incorporating the compositions into polymeric implants (see, for example, P. Johnson and J. G. Lloyd-Jones, eds., Drug Delivery Systems: Fundamentals and Techniques (Chichester, England: Ellis Horwood Ltd., 1988 ISBN-10: 0895735806), which can effect a sustained release of the drug to the immediate area of the implant.

The liposome compositions can be provided to the cell either directly, such as by contacting it with the cell, or indirectly, such as through the action of any biological process. For example, the vesicles can be formulated in a physiologically acceptable carrier, and injected into a tissue or fluid surrounding the cell. As further studies are conducted, information will emerge regarding appropriate dosage levels for treatment of various conditions in various patients, and the ordinary skilled worker, considering the therapeutic context, age, and general health of the recipient, will be able to ascertain proper dosing. The selected dosage depends upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment desired. Generally, dosage levels of 0.001 to 10 mg/kg of body weight daily are administered to mammals. Generally, for intravenous injection or infusion, dosage may be lower. Generally, the total amount of the active agent administered to an individual using the disclosed vesicles can be less than the amount of unassociated active agent that must be administered for the same desired or intended effect and/or may exhibit reduced toxicity.

A. Formulations for Parenteral Administration

In a preferred embodiment the compositions are administered in an aqueous solution, by parenteral injection such as IM, IP, IV or SubQ.

The formulation can be in the form of a suspension or emulsion. In general, pharmaceutical compositions are provided including effective amounts of one or more active agents optionally include pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions can include diluents, sterile water, buffered saline of various buffer content (e.g., Tris-HCl, acetate, phosphate) at various pHs and ionic strengths; and optionally, additives such as detergents and solubilizing agents (e.g., TWEEN® 20, TWEEN® 80 also referred to as polysorbate 20 or 80), anti-oxidants (e.g., ascorbic acid, sodium

metabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate. The formulations may be lyophilized and

redissolved/resuspended immediately before use. The formulation may be sterilized by, for example, filtration through a bacterium retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. B. Other Formulations

The compositions can be applied topically. Topical administration can include application to the lungs, nasal, oral (sublingual, buccal), vaginal, or rectal mucosa. These methods of administration can be made effective by formulating the vesicle with transdermal or mucosal transport elements. For transdermal delivery, such elements may include chemical enhancers or physical enhancers such as electroporation or microneedle delivery. For mucosal delivery PEGylation of the vesicle or addition of chitosan or other mucosal permeants or pH protective elements for oral delivery is preferred.

Vesicles such as liposomes can be delivered to the lungs (Taylor and Newton, Thorax. 1992 Apr; 47(4): 257-259 PMID: 1585287). A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent® nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acom® II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin® metered dose inhaler (Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler® powder inhaler (Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all have inhalable insulin powder preparations approved or in clinical trials where the technology could be applied to the formulations described herein.

Formulations for administration to the mucosa will typically be spray dried drug particles, which may be incorporated into a tablet, gel, capsule, suspension or emulsion. Standard pharmaceutical excipients are available from any formulator.

Lipid vesicles such as liposomes have also been formulated for oral delivery (Woodley, Crit Rev Ther Drug Carrier Syst. 1985;2(1): 1-18 PMID: 3913528; Hua, Front Pharmacol. 2014; 5: 138 doi:

10.3389/fphar.2014.00138, etc.). Oral formulations may be in the form of chewing gum, gel strips, tablets, capsules, or lozenges. Oral formulations may include excipients or other modifications to the particle which can confer enteric protection or enhanced delivery through the GI tract, including the intestinal epithelia and mucosa (see Samstein, et al., Biomaterials, 29(6):703-8 (2008) doi: 10.1016/j.biomaterials.2007.10.026.

Transdermal formulations may also be prepared. These will typically be ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations can include penetration enhancers. Chemical enhancers and physical methods including

electroporation and microneedles can work in conjunction with this method. V. Methods of Use

The lipid vesicles can be used to deliver active agents in vitro and in vivo. The methods of treatment typically include using the disclosed vesicles loaded with one or more active agents, to deliver the one or more active agents into cells, or to a cell’s microenvironment. The methods typically include contacting the active agent-loaded vesicle with one more cells. The contacting can occur in vivo or in vitro.

In some embodiments, the vesicles include two or more active agents. For example, using two or more different miRNAs may result in additive target inhibition and subsequent cytotoxic effects of the miRNA in different cell types.

In some embodiments, the vesicles include multiple antibody attachments on vesicles. Having multiple antibodies may assist the functionalized vesicles in navigating the tumor microenvironment to specifically target a cell type overexpressing multiple surface proteins.

A. In vivo Methods

A typical in vivo method of treatment includes administering to a subject in need thereof an effective amount of an active agent-loaded lipid vesicle composition to reduce one or more symptoms of a disease or disorder.

1. Drug delivery

The vesicles can be used to deliver an effective amount of one or more therapeutic, diagnostic, and/or prophylactic agents to an individual in need of such treatment. The amount of agent to be administered can be readily determined by the prescribing physician and is dependent on the age and weight of the patient and the disease or disorder to be treated. The vesicles are useful in drug delivery (as used herein“drug” includes therapeutic, nutritional, diagnostic and prophylactic agents), whether injected intravenously, subcutaneously, or intramuscularly, administered to the nasal or pulmonary system, injected into a tumor milieu, administered to a mucosal surface (vaginal, rectal, buccal, sublingual), or encapsulated for oral delivery. The vesicles may be administered as a dry powder, as an aqueous suspension (in water, saline, buffered saline, etc.), in a hydrogel, organogel, in capsules, tablets, troches, or other standard pharmaceutical excipient

As discussed herein, compositions can be used as delivery vehicles for a number of active agents including small molecules, nucleic acids, proteins, and other bioactive agents. The active agent or agents can be encapsulated within, dispersed within, and/or associated with the surface of the vehicles. In some embodiments, the vehicles packages two, three, four, or more different active agents for simultaneous delivery to a cell.

2. Transfection

The disclosed compositions can be for cell transfection of polynucleotides. As discussed in more detail below, the transfection can occur in vitro or in vivo, and can be applied in applications including gene therapy and disease treatment. The compositions can be more efficient, less toxic, or a combination thereof when compared to a control.

The particular polynucleotide delivered by the vesicles can be selected by one of skill in the art depending on the condition or disease to be treated. The polynucleotide can be, for example, a gene or cDNA of interest, a functional nucleic acid such as an inhibitory RNA, a tRNA, an rRNA, or an expression vector encoding a gene or cDNA of interest, a functional nucleic acid a tRNA, or an rRNA. In some embodiments two or more

polynucleotides are administered in combination.

In some embodiments, the polynucleotide is not integrated into the host cell’s genome (i.e., remains extrachromosomal). Such embodiments can be useful for transient or regulated expression of the polynucleotide, and reduce the risk of insertional mutagenesis. Therefore, in some embodiments, the vesicles are used to deliver mRNA, siRNA, miRNA, etc., or non integrating expression vectors that are expressed transiently in the host cell. In some embodiments, the polynucleotide is integrated into the host cell’s genome. For example, gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes: (a) a normal gene can be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common; (b) an abnormal gene can be swapped for a normal gene through homologous recombination; (c) an abnormal gene can be repaired through selective reverse mutation, which returns the gene to its normal function; (d) the regulation (the degree to which a gene is turned on or off) of a particular gene can be altered.

Gene therapy can include the use of viral vectors, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus, AIDS vims, neuronal trophic vims, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Gene targeting via target recombination, such as homologous recombination (HR), is another strategy for gene correction.

In some embodiments, the polynucleotide encodes a protein.

B. Diseases to be Treated

The disclosed compositions can be used to treat a broad range of diseases and disorders including, but not limited to, cancer, infections, genetic disorders, etc. The targeting domain and active agent can be selected based on the disease or disorder to be treated.

For example, in some embodiments, the disclosed compositions and methods of treatment thereof are used for treating cancer, including tumor therapy. The methods typically include administering a subject in need there of an effective amount to the composition to reduce one or more symptoms of cancer. For example, therapeutically effective amounts of the disclosed compositions used in the treatment of cancer will generally kill tumor cells or inhibit proliferation or metastasis of the tumor cells or a combination thereof. Symptoms of cancer may be physical, such as tumor burden, or biological such as apoptosis of cancer cells. For example, the composition can be administered in an amount effective to kill cancer cells, improve survival of a subject with cancer, or a combination thereof. The actual effective amounts of composition can vary according to factors including the specific, the particular composition formulated, the mode of administration, and the age, weight, condition of the subject being treated, as well as the route of administration and the disease or disorder.

An effective amount of the composition can be compared to a control. Suitable controls are known in the art. A typical control is a comparison of a condition or symptom of a subject prior to and after administration of the composition. The condition or symptom can be a biochemical, molecular, physiological, or pathological readout. In another embodiment, the control is a matched subject that is administered a different therapeutic agent.

Accordingly, the compositions disclosed here can be compared to other art recognized treatments for the disease or condition to be treated. In a preferred embodiment, the results achieved with a composition including a delivery vehicle and drug is compared to the results achieved by free drug (e.g., drug without delivery vehicle), or with delivery vehicles having or not having a targeting moiety.

In a mature animal, a balance usually is maintained between cell renewal and cell death in most organs and tissues. The various types of mature cells in the body have a given life span; as these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is so regulated that the numbers of any particular type of cell remain constant. Occasionally, though, cells arise that are no longer responsive to normal growth-control mechanisms. These cells give rise to clones of cells that can expand to a considerable size, producing a tumor or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant. The term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis. In this process, small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate.

In this way, a primary tumor at one site can give rise to a secondary tumor at another site. The compositions and methods described herein are useful for treating subjects having benign or malignant tumors by delaying or inhibiting the growth of a tumor in a subject, reducing the growth or size of the tumor, inhibiting or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.

Tumors, for example malignant tumors, which may be treated are classified herein according to the embryonic origin of the tissue from which the tumor is derived. Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. The disclosed compositions are particularly effective in treating carcinomas. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage. Leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated with the provided

compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colo-rectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterus.

In the most preferred embodiments, the cancer is prostate cancer or breast cancer.

In some embodiments, the disclosed compositions are used to treat multiple cancer types concurrently. The compositions can also be used to treat metastases or tumors at multiple locations. For example, breast cancer metastasizes to the brain, bone, liver and lungs, where it would be beneficial to utilize this method.

Exemplary targeting moieties and active agents that can be used in the treatment of cancer are discussed above, and exemplified below.

C. Dosage Regimens

The frequency of administration of a method of treatment can be, for example, one, two, three, four or more times daily, weekly, every two weeks, or monthly. In some embodiments, the composition is administered to a subject once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In some embodiments, the frequency of administration is once, twice or three times weekly, or is once, twice or three times every two weeks, or is once, twice or three times every four weeks. In some embodiments, the composition is administered to a subject 1-3 times, preferably 2 times, a week.

D. Combination Therapies

Combination therapies are also disclosed. The disclosed

compositions can include, or can be administered to a subject in need thereof alone or in combination with one or more additional therapeutic agents. The additional therapeutic agents are selected based on the condition, disorder or disease to be treated. For example, the lipid vesicles composition can be co administered with one or more additional agents that treat cancer. In a preferred embodiment the additional therapeutic agent targets a different pathway so that the combined effect of the therapies is greater than each alone.

The term“combination” or“combined” is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents. Therefore, the combinations can be administered either

concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). The additional therapeutic agents can be administered locally or systemically to the subject, or coated or incorporated onto, or into a device or graft. The additional agent(s) can be part of the same vesicle, added to different lipid vesicles or other delivery vehicles such as polymeric nanoparticles, or administered as free-drug.

Additional therapeutic agents include conventional cancer therapeutics such as chemotherapeutic agents, cytokines, chemokines, and radiation therapy. The majority of chemotherapeutic drugs can be divided into: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. All of these drugs affect cell division or DNA synthesis and function in some way. Additional therapeutics include monoclonal antibodies and the new tyrosine kinase inhibitors, e.g., imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors).

Representative chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,

cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epipodophyllo toxins, epirubicin, etoposide, etoposide phosphate, fludarabine, fluorouracil, gemcitabine,

hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin , lomustine, mechlorethamine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, teniposide, tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, taxol and derivatives thereof, trastuzumab (HERCEPTIN®), cetuximab, and rituximab (RITUXAN® or

MABTHERA®), bevacizumab (AVASTIN®), and combinations thereof. Representative pro-apoptotic agents include, but are not limited to, fludarabinetaurosporine, cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2), and combinations thereof.

VI. Kits

Dosage units including the disclosed composition, for example, in a pharmaceutically acceptable carrier for shipping and storage and/or administration are also disclosed. Components of the kit may be packaged individually and can be sterile. In some embodiments, a pharmaceutically acceptable carrier containing an effective amount of the composition is shipped and stored in a sterile vial. The sterile vial may contain enough composition for one or more doses. The composition may be shipped and stored in a volume suitable for administration, or may be provided in a concentration that is diluted prior to administration. In another embodiment, a pharmaceutically acceptable carrier containing drug can be shipped and stored in a syringe. Kits containing syringes of various capacities or vessels with deformable sides (e.g., plastic vessels or plastic-sided vessels) that can be squeezed to force a liquid composition out of an orifice are provided. The size and design of the syringe will depend on the route of administration·

Any of the kits can include instructions for use.

The disclosed compositions and methods can be further understood through the following numbered paragraphs.

1. A method of making functionalized lipid vesicles comprising a lipid membrane, the method comprising mixing in the presence of detergent lipid vesicles comprising one or more lipids with one or more types of lipid conjugates, each type of lipid conjugate comprising a lipid component and a functional element

and dialyzing the mixture for an effective amount of time for the lipid conjugate(s) to insert into the lipid vesicles and form functionalized lipid vesicles.

2. The method of paragraph 1, wherein the lipid conjugate is formed by one or more of the steps of

(i) mixing or otherwise suspending the lipid component, or a precursor thereof, in a solution comprising a concentration of detergent near the critical micelle concentration to form a suspension,

(ii) dialyzing the suspension to remove excess detergent, and encourage formation of stable micelles in the suspension,

(iii) adding, mixing, or otherwise contacting the suspension with the functional element, under conditions suitable for the functional element to conjugate, or otherwise link, to the lipid component to form the lipid conjugate.

3. The method of paragraph 2, wherein the detergent is of a type and amount suitable for stabilizing the hydrophobic regions of the lipid component, or precursor thereof, in a semi-aqueous solution.

4. The method of paragraphs 2 or 3, wherein dialysis of the mixture removes the detergent.

5. The method of any one of paragraphs 1-4, wherein the detergent is n -dodec y 1 - b- D - m a 1 to s i de (DDM), optionally at a concentration of about 0.1%. 6. The method of any one of paragraphs 1-5, wherein the lipid vesicle is naturally occurring.

7. The method of any one of paragraphs 1-6, wherein the lipid vesicles are isolated from cultured or uncultured tissue, cells, or fluid.

8. The method of paragraph 7, wherein the fluid derived from, or conditioned by, cultured cells.

9. The method of paragraph 7, wherein the fluid is blood, plasma, lymph liquid, malignant pleural effusion, amniotic liquid, breast milk, semen, saliva or urine.

10. The method of any one of paragraphs 1-9, wherein the lipid vesicles are apoptotic bodies and/or blebs (AB), microvesicles (MV), exosomes, or tunnelling nanotubes (TNT).

11. The method of any one of paragraphs 1-5 wherein the lipid vesicles are synthetic.

12. The method of paragraph 11, wherein the synthetic lipid vesicles are niosomes or liposomes.

13. The method of any one of paragraphs 1-12, wherein the lipid vesicles are between about 30-150 nm.

14. The method of any one of paragraphs 1-13, wherein the functional element is a small molecule, protein or polypeptide, carbohydrate, nucleic acid or a combination thereof.

15. The method of paragraphs 14, wherein the functional element is a targeting moiety that increases attachment, binding, or association of the functionalized lipid vesicle to a target cell(s), tissues(s), and/or

microenvironment(s) relative to the lipid vesicle.

16. The method of any one of paragraphs 1-15, wherein the functional element is a targeting moiety that increases attachment, binding, or association of the functionalized lipid vesicle to a target cell(s), tissues(s), and/or microenvironment(s) relative non-targeted cell(s), tissue(s), and/or microenvironment(s).

17. The method of paragraphs 15 or 16, wherein the targeting moiety targets cancer cells.

18. The method of any one of paragraphs 15-17, wherein the targeting moiety targets CD44 or CD29/Integrin beta-1. 19. The method of any one of paragraphs 15-18, wherein the functional element is an antibody.

20. The method of any one of paragraphs 1-19 wherein the functional element comprise or is a detectable label.

21. The method of paragraph 20, wherein the detectable label is a fluorophore, radiolabel, magnetic label, or a contrast agent.

22. The method of any one of paragraphs 1-21 further comprising loading the lipid vesicles or functionalized lipid vesicles with an active agent.

23. The method of paragraph 22, wherein the active agent is selected from therapeutic, nutritional, diagnostic, prophylactic compounds, and combinations thereof.

24. The method of paragraphs 22 or 23, wherein the active agent is a protein, peptide, carbohydrate, polysaccharide, nucleic acid molecule, and or organic small molecule.

25. The method of paragraph 24, wherein the nucleic acid molecule is selected from antisense, siRNA, miRNA, anti-miRNA, piRNA, aptamers, ribozymes, external guide sequences for ribonuclease P, triplex forming agents, and CRIPSR/Cas component(s), or a polynucleotide encoding any of the foregoing.

26. The method of paragraph 25, wherein the nucleic acid molecule is an miRNA, anti-miRNA, or a polynucleotide encoding the foregoing.

27. The method of paragraph 26, wherein the miRNA is a pri- miRNA, pre-miRNA, mature miRNA, miRNA mimics, or fragments or variants thereof that retains the biological activity of the miRNA.

28. The method of paragraph 27, wherein the miRNA targets an oncogene.

29. The method of any one of paragraphs 22-28, wherein the loading of the lipid vesicles comprises mixing vesicles and active agent alone or in combination with one or more of incubation, freeze-thaw cycling, sonication, extrusion, chemical transfection, and electroporation.

30. The method of paragraph 29 wherein the active agent is an miRNA and the loading of the active agent comprises electroporation. 31. A functionalized lipid vesicle formed according to the method of any one of paragraphs 1-30.

32. A pharmaceutical composition comprising the functionalized lipid vesicles of paragraph 31.

33. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the functionalized lipid of paragraph 31 or the pharmaceutical composition of paragraph 32.

34. The method of paragraph 33, wherein the subject has cancer and the active agent treats the cancer.

35. A method of making functionalized lipid vesicles comprising a lipid membrane, the method comprising mixing in the presence of detergent, lipid vesicles comprising one or more lipids with one or more types of lipid conjugates, each type of lipid conjugate comprising a lipid component and a functional element,

and dialyzing the mixture for an effective amount of time for the lipid conjugate(s) to insert into the lipid vesicles and form functionalized lipid vesicles,

wherein the lipid vesicles are naturally occurring exosomes isolated from cultured or uncultured tissue, cells, or fluid.

36. The method of paragraph 35, wherein the functional element is an antibody.

37. The method of paragraph 36, wherein the antibody specifically targets a cancer antigen.

38. The method of paragraph 37, further comprising loading the lipid vesicles or functionalized lipid vesicles with an active agent.

39. The method of paragraph 38, wherein the active agent is a tumor suppressor miRNA or mimic thereof.

40. A functionalized exosome vesicle formed according to the method of paragraph 39.

41. A method of treating a subject in need thereof comprising administering to the subject an effective amount of the functionalized lipid of paragraph 40. 42. The method of paragraph 41, wherein the subject has cancer, the functional element targets cells of the cancer, and the active agent treats the cancer. Examples

Example 1: Construction and confirmation of antibody-labeled exosomes

Materials and Methods

Harvesting of human peripheral blood mononuclear cells (PBMCs)

Human donors were enrolled for blood collection in compliance with the guidelines of the World Medical Association’ s Declaration of Helsinki and the Human Research Protection Program and Institutional Review Board guidelines for human subject research at University of Georgia. Enrolled healthy volunteers signed the consent forms following appropriate introduction about the study. The human blood protocol (University of Georgia protocol no. 2012-10769) and the consent form were reviewed and approved by the Institutional Review Board of the University of Georgia. PBMCs were isolated from heparinized blood samples using the Histopaque 1077 Reagent (Sigma Aldrich, St. Louis, MO, USA) as per manufacturer instructions and as previously described (Sil et al., Inflamm Res. 66(3):227- 37 (2017) doi: 10.1007/s00011-016-1008-0). PBMCs were resuspended in RPMI without glutamine and phenol red (Coming, Corning, NY, USA) supplemented with 1M HEPES buffer solution (Sigma Aldrich). Cells were counted and plated at a density of 1 million cells per 25 cm ² in a 75 cm ² flask in RPMI (Coming, Coming, NY, USA) supplemented with 10% fetal bovine semm (FBS) (Atlanta Biologicals, Flowery Branch, GA, USA) and penicillin/streptomycin (Thermo Fisher, Waltham, MA, USA) in a humidified atmosphere of 5% C02 at 37°C for 24-48 h.

Preparation of fatty acids, antibody and exosomes

Two fatty acids, namely l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], or DSPE- PEG(2000) maleimide, and fluorescent l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(7-nitro-2-l,3-benzoxadiazol-4-yl), or NBD-DSPE (Avanti Polar Lipids, Alabaster, AL, USA), were obtained and dissolved in DMSO to yield a lmg/mL solution of fatty acids. DDM detergent (Sigma Aldrich) was resuspended in lx PBS to yield a 100 mg/mL solution.

Subsequently a 0.1% w/v solution of DDM was also prepared using lx PBS. DSPE-PEG 2000 Maleimide and fluorescent NBD-DSPE fatty acids were dissolved in 1 mL of 0.1% DDM at a concentration of IOOmM each. This solution was used to verify appropriate excitation and emission of the fluorescent NBD-DSPE using the SpectraMax M2 Plate Reader (Molecular Devices, Sunnyvale, CA, USA), with excitation at 445-460 nm with an expected emission at 540 nm (data not shown). This 1:1 mixture of the two fatty acids was used for Abi-exosomes construction.

PBMCs were plated in RPMI (Corning) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin/streptomycin (Thermo Fisher) as described above. Exosomes were isolated from PBMCs using the Exiqon Exosome Isolation Kit (Exiqon, Woburn, MA, USA) and stored at -80°C if required prior to use.

Invitrogen ENPP2/autotaxin antibody (Product No. PA5- 12478) (Thermo Fisher) as well as Cell Signaling Technologies CD44 (Product No. #37259S) and CD29/Integrin beta-1 (Product No. #4706S) antibodies were purchased and stored at -20°C prior to use (Cell Signaling Technologies, Danvers, MA, USA).

Construction of fatty acid-antibody anchored exosomes

The fatty acid solution containing 1 : 1 DSPE-PEG(2000)

Maleimide:NBD-DSPE in 0.1% DDM was dialyzed using Slide- A-Lyzer MINI Dialysis units with a 10,000- molecular weight cut-off (Thermo Fisher) against 2L of lx PBS for 2 h in a 4°C cold room. Following dialysis, presence of the fatty acid was deduced by exciting a 1:10 dilution of the sample between 445-460 nm and confirming an emission at 540 nm using SpectraMax M2 Plate Reader. The remaining sample was combined with antibody at a 2: 1 ratio to generate the antibody-label and the sample was centrifuged and incubated at room temperature for 1 h prior to addition of 100pL of purified exosomes from PBMCs. This solution was mixed by pipetting, briefly centrifuged, and incubated at room temperature for an additional 1 h. Then, the sample once again underwent dialysis using the 10,000 Dalton molecular weight cut-off dialysis units against 2L of lx PBS for 2 h in a 4°C cold room to integrate the fatty acid- attached antibody into the exosomal membrane and remove all DDM from solution.

The dialyzed product, containing the antibody-label integrated into the exosomal membrane, creating Abi-exosomes, was incubated overnight with Exosome Precipitation Buffer from the Exosome Isolation Kit per manufacturer instructions (Exiqon) to precipitate the Abi-exosomes. The solution was then centrifuged at 50,000 rpm for 1 h at 20°C to pellet the Abi- exosomes, the supernatant was removed and the final product was resuspended in lx PBS and electroporated with miRNA before addition to cells. Electroporation was performed using the BioRad Gene Pulser X-Cell CE was used to electroporate miRNA into the Abi-exosomes using an exponential decay exposure at 150 Volts and 125 pF of capacitance for 10- 15 microseconds in a 4mm cuvette.

Dynamic Light Scattering

The DLS technique is a powerful tool for estimating the sizes and distributions of particles (typically in submicron range) in a solution using their light scattering properties (Beme and Pecora, Dynamic Light

Scattering: With Applications to Chemistry, Biology, and Physics,

Unabridged edition, Dover Publications, Mineola, N.Y, 2000, Schmitz, Introduction to Dynamic Light Scattering by Macromolecules, Academic Press, Boston, 1990.). To determine whether antibodies were successfully anchored into the exosomal membrane to form Abi-exosomes, dynamic light scattering was used to determine particle size in solution compared to each individual component required for the final product. Undiluted antibody, purified exosomes and fatty acid at or below critical micelle concentration (CMC, H ₂ 0=0.0087% DDM) were prepared for this method. It was initially believed there could be an increase in particle size in the 10-20 nm range due to the small size of the fatty acid and -10-15 nm size of antibody attached to the exosome. Since the refractive index (RI) of water (RI: 1.337; viscosity: 0.887 cP; temperature: 25°C) differs from that of lipid vesicles (RI: 1.447; absorption: 0.001), the presence of the particle in solution was detectable (Matsuzaki et al. Biochim Biophys Acta., 1461(l):219-26 (2000)). The Zetasizer Nano ZS was used to perform dynamic light scattering and configured with a customized protocol to determine size of exosomes and Abi-exosomes (Malvern Panalytical, Malvern, United Kingdom). The Zetasizer software, provided by Malvern Panalytical, was used to obtain and analyze data.

Statistics

One-way analysis of variance (ANOVA) test was used to determine statistical significance between groups comparing miRNA uptake into cells. A confidence interval of 95% with all p-values less than 0.05 was considered significant (*). Student’s T-Test was also used to compare two groups to one another, also with a 95% confidence interval. Microsoft Excel and GraphPad Prism 7 were utilized for numerical data analysis and statistical significance.

Results

In order to bioengineer customizable Abi-exosomes to target a specific protein on cell surfaces, a fatty acid, autotaxin antibody, and purified exosomes from human peripheral blood mononuclear cells were utilized. PBMCs were cultured and exosomes therein extracted after obtained blood from a human donor (Fig. 1A). Then, the antibody and fatty acid were incubated together in the presence of 0.1 % DDM detergent to result in a covalent bond attaching the fatty acid to the antibody tail. Subsequently, the antibody labeled with fatty acid, now considered and referred to as the antibody-label, was incubated with exosomes and underwent dialysis to remove the DDM detergent stabilizing the hydrophobic fatty acid tail. This resulted in insertion of the fatty acid into the exosomal membrane to maintain its hydrophobicity, forming antibody-labeled exosomes (also referred to herein as Abi-exosomes). (Fig. IB). miRNA (R) was introduced into the Abi-exosomes by electroporation (Fig. 1C).

To confirm insertion of the antibody into the exosome, the size increase was monitored by the dynamic light scattering technique. (Stetefeld et al., Biophys Rev. 8(4):409-27 (2016) doi: 10.1007/sl2551-016-0218-6). During the process of engineering an Abi-exosome, insertion of the antibody with pegylated fatty acid into the exosome will significantly increase the size of the particle. The polyethylene glycol-2000 (PEG(2000)) and the antibody will contribute to the change in particle size. From previous dynamic light scattering experiments, PEG(2000) was found to have a hydrodynamic radius and diameter of 1.6 and 3.2 nm, respectively (Ling et al., Nanoscale Research Letters 8(1):538 doi: 10.1186/1556-276X-8-538). A generic IgG antibody was found to have an average hydrodynamic diameter of 11.3 nm by dynamic light scattering analysis (Nobbman et al., Biotechnology and Genetic Engineering Reviews 24(1): 117-128 (2007) doi:

10.1080/02648725.2007.10648095). The PEG(2000) and the IgG antibody have a combined hydrodynamic diameter of 14.5 nm. Therefore, the Abi- exosome diameter could be as much as 29 nm larger than an unlabeled exosome.

Results indicated that this was indeed the case; the Abi-exosomes had a particle size approximately 12 nm larger in size on average compared to purified exosomes without the antibody-label attachment, and was therefore deemed to be successfully constructed, possibly with multiple antibody attachments per exosome (Figs. 2A-2B). Autotaxin antibody was consistent with the 10-15 nm size expected, however, fatty acid micelles were unable to be broken up and generated large, inaccurate particle size measurements.

A first dynamic light scattering chromatogram in Figure 2A shows the size of the exosome before and after insertion of the antibody with pegylated fatty acid. Upon insertion of the antibody with pegylated fatty acid, the exosome diameter increases from 40 nm to 54 nm (corresponding to a rightward peak shift comparing exosomes alone and Abi-exosomes).

A second dynamic light scattering chromatogram in Figure 2B shows the particle size distribution DLS chromatograms determined from experiments in nm. Each of the samples showed a distinct average size and distributions. The fatty acids (i.e. a mixture of 100 mM DSPE-PEG 2000 and 100 pM NBD-DSPE) solubilized in 0.1% w/v DDM had a size distribution of 6.3 ± 2.0 nm. The size distribution of exosomes was considerably higher with a diameter of 39.8 ± 5.0 nm. This is in the typical size distribution range observed for exosomes (Doyle, et al., 8(7), 727 (2019)

doi.org/10.3390/cells8070727; Lim, et al., J. Nanobiotechnology. 17(1) (2019) doi.org/10.1186/sl2951-018-0433-3). The average diameter of Abi- exosomes was about 10 nm higher at 50.1 ± 8.0 nm. The size increase is consistent with antibody labelling within the Abi-exosomes (Reth, Nat. Immunol. 14 (2013) 765-767 doi.org/10.1038/ni.2621). In contrast, DDM detergent micelles were 5 -fold larger at the critical micelle concentration (CMC) with a size distribution of 251 ± 164 nm.

The experiments illustrated in Figures 2A and 2B both utilized ENPP2/autotaxin antibody.

Example 2: Cellular delivery of miRNA with Abi-exosomes

Materials and Methods

Electroporation of exosomes with miRNA

The miRNA mimic used is an miR-489-3p miRNA mimic having the sequence GUGACAUCACAUAUACGGCAGC (SEQ ID NO: l).

Exosomes were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher) as per manufacturer’s protocol. Under a sterile hood, 1 pg each of exosomes and miRNA mimic obtained from Thermo Fisher were combined in 400 pL of serum- free DMEM medium in a Gene Pulser Cuvette (Bio-Rad, Hercules, CA, USA). The Bio-Rad Gene Pulser X-Cell CE was used to electroporate miRNA into the Abi-exosomes using an exponential decay exposure at 150 Volts and 125 pF of capacitance for 10-15 microseconds in a 4mm cuvette. The electroporated product was subsequently incubated at room temperature for 30 min prior to treating cells.

Treatment of cell lines

MDA-MB-231 cells were grown and maintained in DMEM

(Coming) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals), penicillin/streptomycin (Thermo Fisher) and incubated in a humidified atmosphere of 5% C02 at 37°C. Cell lines were plated at 200,000 cells per well in a 6- well plate and incubated overnight. Media was refreshed the following day with 2.7 mL of fresh 10% DMEM or RPMI (complete medium) and treated with 300 uL of Abi-exosomes electroporated with miRNA. The plate was incubated for 48 or 72 h before protein or intracellular RNA and extracellular exosomal RNA were extracted.

Intracellular and exosomal RNA extraction and Quantitative real-time PCR

TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate intracellular RNA as per the manufacturer’s protocol. Exosomal RNA extraction was performed using the Exosome Isolation Kit followed by the miRCURY RNA Isolation Kit - Cell & Plant (Exiqon). All RNA was stored at -80°C.

Complementary DNA (cDNA) was prepared from intracellular and exosomal RNA using the Taqman microRNA Reverse Transcription Kit (Thermo Fisher). cDNA was ran in a 384-well format in a quantitative real time PCR (qRT-PCR) assay using Taqman Universal PCR MasterMix (Thermo Fisher) and the ABI 7900HT machine (Applied Biosystems, Foster City, CA, USA) as per manufacturer protocols. Microsoft Excel and GraphPad Prism 7 were used for data analysis and statistics.

Results

Experiments were designed to assess whether miRNA delivery into cells could be increased by encapsulating the miRNA into purified PBMC- derived exosomes compared to standard miRNA transfection. For this, 1 pg of exosomes were electroporated with 1 pg of miR-489-3p miRNA mimic, and added to cells for 72 h to allow for exosomal uptake. Then, RNA was extracted from cells and exosomes in cultured media, and miRNA levels were assessed by qPCR.

Exosomes alone or those incubated with miRNA but without electroporation had significantly lower miRNA levels in cells. In fact, electroporated exosomes contained up to 700,000-fold higher levels of miRNA compared to purified exosomes, and were successfully taken up by cells to result in higher miRNA concentrations compared to the transfection (*p<0.05) (Fig. 3A). Transfection is the standard method of miRNA delivery into cells involving encapsulating miRNA in a transfection reagent such as Dharmafect to ensure stable delivery into cells.

As attachment of an antibody-label to the exterior of the exosomal membrane may present some structural hindrances for exosomal uptake into cells, miRNA uptake with and without the antibody-label were measured. Strikingly, attachment of an antibody to exosomes increases miRNA uptake efficiency into the cells compared to both unlabeled exosomes and miRNA transfection. More specifically, Abi-exosome mediated miRNA delivery into cells is much more efficient than miRNA electroporation and delivery using plain exosomes (***p<0.001) without the antibody-label (Fig. 3B). Next, miRNA electroporation efficiency and subsequent stability in the Abi-exosomes were investigated to ensure successful uptake and delivery into cells. Electroporation of miRNA could occur either before (“Strategy #1”) or after (“Strategy #2”) construction of Abi-exosomes (Fig. 3C). Using qRT-PCR measuring miRNA expression, it was discovered that miRNA electroporation after Abi-exosome construction resulted in higher miRNA levels in the exosomes and subsequently, higher cellular concentrations. Comparatively, miRNA electroporation into naked exosomes before Abi- exosome construction resulted in much lower miRNA uptake (Fig. 3D). This result indicates that miRNA uptake efficiency into cells is increased when miRNA is electroporated into fully contrasted Abi-exosomes (***p<0.001). This shows that the Abi-exosome particle is able to withstand miRNA electroporation. All miRNA expression was normalized to U6 endogenous control expression.

Example 3: Investigation of antibody-label

Materials and Methods

Cell lines, Protein Extraction and SDS-PAGE gel electrophoresis

OVCAR-3, MeWo and BT-474 cells were grown in RPMI medium (Coming) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals) and penicillin/streptomycin (Thermo Fisher) and incubated in a humidified atmosphere of 5% C02 at 37°C. Cells were washed with lx PBS twice and centrifuged at 4000 rpm for 5 min to collect cell pellet before addition of RIPA Lysis and Extraction Buffer containing

Protease/Phosphatase Inhibitor Cocktail (lOOx) (Thermo Fisher). They were shaken on ice with occasional vortexing for 30 min, sonicated and centrifuged at 13,000 rpm for 10 min to collect the protein supernatant. Protein was stored at -80°C before quantification using the Pierce BCA Protein Assay Kit (Thermo Fisher) as per manufacturer’s protocol.

Samples were boiled with 6x Laemelli sample buffer at 95°C for 5-10 minutes prior to loading on an SDS-PAGE gel. The BioRad SDS-PAGE System and protocol were used to probe for CD44, CD29, and GAPDH antibodies at 1:1000 dilution overnight and HRP-conjugated anti-rabbit secondary antibodies at 1:10000 dilutions (BioRad; Cell Signaling

Technologies). The Flourchem Imager System (Protein Simple, San Jose,

CA, USA) was used to detect bands on the PVDF membranes and band quantification was performed using the ImageJ Software (National Institutes of Health, Rockville, MD, USA).

Antibodies were from Cell Signaling Technologies®: CD44 (Product No. 37259S); CD29/Integrin beta-1 (Product No. #4706S), GAPDH (Product No. #5174); I-CAM (Product #4915S).

The miRNA cargo was an miR-21-5p having the sequence

UAGCUU AUC AG ACU G AU GUU G A (SEQ ID NO:2) (miRBase Accession Number: MI0000077).

Results

Previous studies have shown that certain proteins, such as CD44, and CD29/Integrin s-1 are overexpressed on the surface of MDA-MB-231 cells (Lobba et al., Cytometry A. 81(12): 1084-91 (2012) doi:

10.1002/cyto.a.22220, Cahall et al., Breast Cancer (Auckl). 9(Suppl 1): 1-11 (2015) doi: 10.4137/BCBCR.S25461). Targeting these proteins might allow for more proximity of Abi-exosomes and higher uptake efficiency into cells. The expression of these markers on TNBC MDA-MB-231 cells, as well as ER+/PR+/HER2+ (triple positive) BT474, MeWo (melanoma) and OVCAR- 3 (high grade serous ovarian carcinoma) cell lines was measured (Fig. 4A). Results confirmed the overexpression of both CD44 and CD29 in MDA-MB- 231 cells compared to BT474 (triple-positive breast cancer) and MeWo (melanoma) cells. ICAM-1 expression was tested, but not confirmed, possibly due to low expression in all four cell lines. OVCAR-3 also had higher CD44 and CD29 expression, possibly related to the gynecological origin of this tumor type as well.

When CD44 (Cell Signaling Technologies® Product No. 37259S) and CD29/Integrin beta-1 (Cell Signaling Technologies® Product No.

#4706S) antibodies were used for Abi-exosome construction, miRNA expression after treatment in MDA-MB-231 cells showed that CD44- labeled-exosomes had the highest miR-21-5p miRNA delivery efficiency into cells (Fig. 4B). CD44-labeled Abi-exosomes are more effective at miRNA delivery than autotaxin-labeled Abi-exosomes (*p<0.05) and CD29- labeled Abi-exosomes. MiRNA concentration was normalized to U6 endogenous control expression in cells and exosomes.

CD29-labeled-exosomes achieved less efficient miRNA delivery, whereas autotaxin-labeled-exosomes were comparable to the standard miRNA transfection efficiency. This may be due to autotaxin not being on the cell surface like CD44, but simply proximal to the surface. Therefore, targeting CD44 results is more closeness of the Abi-exosomes to the cell surface, increasing their probability of being internalized.

ICAM-l-labeled-exosomes were also tested and comparable to the standard miRNA transfection efficiency, however, as noted above, ICAM- 1 expression was not confirm.

This data shows that while Abi-exosomes targeting an extracellular enzyme close to the cell surface can be slightly more efficient than miR transfection, directly targeting a marker on the cell surface can result in significantly higher miR uptake into cells (*p<0.05). Less efficient miR delivery (*p<0.05) by CD29-labelled exosomes was possibly due to inefficient targeting by the antibody to this surface marker.

Example 4: Functionality of miRNA delivery into TNBC cells

Materials and Methods

Generally as described above.

The antibody against CD44 was obtained from Cell Signaling Technologies®: Product No. 37259S.

The miRNA cargo was an miR-21-5p having the sequence

UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO:2) (miRBase Accession Number: MI0000077).

Results

To assess functionality of miRNA delivery into cells using Abi- exosomes, the expression of PTEN in MDA-MB-231 cells treated with miR- 21-5p containing anti-CD44 antibody-labeled Abi-exosomes was measured. PTEN has been previously reported in various studies to be a target of miR- 21-5p in MDA-MB-231 cells and is also implicated in other cancers such as gastric or colorectal cancer (Dai et a , Oncol Lett. 14(6):6929-36 (2017) doi: 10.3892Zol.2017.7007, Wu et ak, Cell Physiol Biochem. 43(3):945- 58 (2018) doi: 10.1159/000481648, Wang et al„ DNA Cell Biol. 37(l):38-45 (2018) doi: 10.1089/dna.2017.3922).

For this experiment, miR-21-5p was electroporated into Abi- exosomes (targeting CD44) and treated cells for 48 hours and then measured the ability of the miRNA to successfully inhibit its intracellular target.

Results show that Abi-exosomes increased miR-21-5p levels in the cells (**p<0.01) and were also able to inhibit expression of PTEN significantly in the cell (*p=0.0269). miRNA and PTEN expression were normalized to U6 and 18S endogenous control, respectively.

Fig. 1C illustrates a model of a possible mechanism of delivery of exosomes into cell. Cells treated with the Abi-exosomes are endocytosed and subsequently degraded to release miRNA. Using an antibody for Abi- exosome construction with a protein target on the cell surface is believed to increase proximity of the particles to the cells and further increase cellular uptake efficiency and miRNA levels in the cells.

The experiments above illustrate the development of customizable antibody-labeled exosomes (Abi-exosomes) that can be used as a vehicle to deliver miRNA cargo to cells utilizing exosomes derived from peripheral blood mononuclear cells. MiRNA was electroporated into the particle and incubated with cells to characterize uptake of the Abi-exosomes and miRNA release into the cells (Fig. 1C). Dynamic light scattering, a method often employed to determine particle sizes in solution (Stetefeld et ak, Biophys Rev. 8(4):409-27 (2016) doi: 10.1007/sl2551-016-0218-6), confirmed successful formation of Abi-exosomes by observing an increase in particle size with the addition of the antibody-label to exosomes (Figs. 2A-2B). Further, experiments comparing efficiency of Abi-exosomes to the standard of miRNA delivery, transfection, and unlabeled exosomes showed that antibody-label attachment results in more efficient uptake and miRNA delivery into cells (Fig. 3A-3B). Additionally, electroporation of miRNA after Abi-exosomes construction (Fig. 3C, Strategy #2) was found to be more efficient for miRNA delivery into cells (Fig. 3D).

When the Abi-exosomes were bioengineered with antibodies targeting markers overexpressed on the MDA-MB-231 cell surface such as CD44 and CD29, CD44-labeled-exosomes were found to be most efficient at uptake and miRNA delivery into cells (Fig. 4B). CD44 is highly

overexpressed in various cancer cells and named a prominent regulator of metastasis, especially in breast cancer cells (Senbanjo & Chellaiah, Front Cell Dev Biol. 5:18 (2017) doi: 10.3389/fcell.2017.00018). Utilizing this target for cargo delivery with CD44-labeled exosomes allowed the hijacking of a survival tactic for cancer cells to potentially target cancer cells especially with lethal miRNA cargo in the bioengineered exosomes.

Compared to autotaxin, which brings the Abi-exosomes close to the cell surface, targeting CD44 brings the Abi-exosomes onto the cell surface to target CD44, increasing the likelihood of their uptake. Functionality of this method was confirmed by treated MDA-MB-231 cells with miR-21-5p, which lead to inhibition of PTEN consistent with previous studies involving the same cell line (Dai et a , Oncol Lett. 14(6):6929-36 (2017) doi:

10.3892/ol.2017.7007, Fragni et ak, Naunyn Schmiedebergs Arch

Pharmacol. 389(5):529-38 (2016) doi: 10.1007/s00210-016-1224-8).

Long-term storage of the completed Abi-exosome at -80°C can reduce the ability of the constructed particle to deliver miRNA into cells. It is possible that the Abi-exosomes themselves are stable when stored at low temperatures, but that the miRNA is unable to be electroporated in or is subsequently exported out of the particle. In this case, electroporating higher concentrations of the miRNA into the Abi-exosome before treatment to cells may increase uptake efficiency. If the Abi-exosome itself is unstable upon storage at - 80°C, storage at -20°C or 4°C may also be considered.

While the literature outlines increased CD44 expression in MDA- MB-231 cells, CD44 expression is also relatively high in healthy peripheral blood mononuclear cells (Cahall et ak, Breast Cancer (Auckl). 9(Suppl 1):1- 11 (2015)). Since the exosomes used in the disclosed experiments are derived from peripheral blood mononuclear cells, it is possible that they also contain higher expression of CD44, but due to their small size and low concentration, CD44 expression could not be assessed. High CD44 expression on exosomal surfaces could result in the CD44- label targeting the surface of exosomes themselves instead of successfully integrating into the exosomal membrane to form Abi-exosomes. However, dynamic light scattering confirmed that the antibody label is in fact inserted into the exosome, and not targeting a protein on the surface due to the size distribution of Abi-exosomes compared to exosomes alone. In other words, if the antibody-label was not inserted into the exosome, the particle size increase would be up to two times higher based on the component sizes.

Example 5: Abi-exosomes that Target Hepatocytes

Materials and Methods

Anti-ASGRl antibody

Anti- Asialoglycoprotein Receptor 1/HL-l antibody (at>49355) from abCAM, Product Number: at>49355.

miR-298-5p cargo having a sequence:

GGCAGAGGAGGGCUGUUCUUCCC (SEQ ID NOG) (miRBase

Accession No. MI0000398)

The Abi-exosomes were bioengineered from exosomes, as described in the Examples above. The antibody used with the Abi-exosomes was the Anti-ASGRl antibody, which specifically targets a receptor found on the surface of liver cells. Ten pi (3.8 pg) of mouse miR-298-5p were electroporated into the 3.8 pg Abi-exosomes or 3.8 pg exosomes using the Bio-Rad (Hercules, CA) at 150 v, 125 pF capacitance. Afterward, the Abi- exosomes exosomes were suspended in 400 pi serum-free Eagle's Minimum Essential Medium (EMEM) medium.

A six-well plate was plated to a concentration of 200,000 Hep-G2 cells/well. These Hep-G2 cells were serum-starved for 24 hours after they were adherent to the plate to ensure that all the cells were in the G1 -arrest phase of the cell cycle. After 24 hours, the Hep-G2 cells were untreated, treated with the transfection reagent DharmaFECT (Thermo Fisher

Scientific, Waltham, MA), treated with DharmaFECT and mouse miR-298- 5p, treated with Abi-exosomes containing mmu-miR-298-5p, or treated with exosomes containing mouse miR-298-5p. The RNA was extracted 72 hours after the treatment using TRIzol Reagent (Invitrogen, Carlsbad, CA).

Complementary DNA (cDNA) was prepared from intracellular RNA using the Taqman microRNA Reverse Transcription Kit for mmu-miR-298-5p (Thermo Fisher Scientific, Waltham, MA). The cDNA was then run in a 384-well format in a qRT-PCR assay using Taqman Universal PCR MasterMix (Thermo Fisher Scientific, Waltham, MA) and the ABI 7900HT machine (Applied Biosystems, Foster City, CA,). The qRT-PCR

fluorescence results were analyzed using GraphPad Prism 7 and normalized against background fluorescence using control U6 small nuclear RNA (snRNA) and untreated cells.

Results

Abi-exosomes were bioengineered with the anti-asialoglycoprotein receptor 1/HL-l (Anti-ASGRl) antibody to target hepatocytes. The efficiency of these Abi-exosomes to deliver microRNA (miRNA) to hepatocytes was determined with the mouse 5’ miRNA called mmu-miR-298-5p, which will suppress mouse P-glycoprotein (Pgp) expression (Xie, et al., Front.

Neurosci. 12 (2018), doi.org/10.3389/fnins.2018.00602.

Haenisch, et al., Br. J. Clin. Pharmacol, 77: 587-596 (2014)

doi.org/10.ll ll/bcp.12251, Bao, et al., Targeted gene therapy of ovarian cancer using an ovarian-specific promoter, Gynecol. Oncol. 84: 228-234 (2002). doi.org/10.1006/gyno.2001.6490), in the immortalized human liver carcinoma cells called Hep-G2 cells (Donato, et al., Methods Mol. Biol. Clifton NJ. 1250: 77-93 (2015). doi.org/10.1007/978-l-4939-2074-7_5). Using mouse mmu-miR-298-5p to determine the efficiency of miRNA transfer to human hepatocytes is advantageous because Hep-G2 cells do not express mouse Pgp and are therefore insensitive to miR-298-5p. These experiments also demonstrate the potential for in vivo targeting of Pgp in the mouse liver.

Figure 6 shows the relative ratio determined from quantitative real time PCR (qRT-PCR) analysis of mouse mmu-miR-298-5p compared to the control Hep-G2 cells. The far two left columns are the negative controls. In the far left column, the Hep-G2 cells were untreated and demonstrate an absence of mouse mmu-miR-298-5p. The column to the right of this column shows mmu-miR-298-5p produced from qRT-PCR analysis in Hep-G2 cells with DharmaFECT, which is a transfection reagent. Again, qRT-PCR analysis of Hep-G2 cells treated only with DharmaFECT did not detect the nucleic acid of interest, which demonstrates they lack mmu-miR-298-5p. In the next column, adding rat mmu-miR-298-5p and DharmaFECT to Hep-G2 cells produced a relative quantitative amplification of the nucleic acid of 254.4 ± 32.1, indicating significant incorporation of 3.8 pg mouse mmu- miR-298-5p into Hep-G2 cells. The next column shows the results of incorporating mmu-miR-298-5p into Hep-G2 cells, facilitated by the hepatocyte targeting of Abi-exosomes. The relative quantification was about 8-fold greater than samples utilizing DharmaFECT or 2001.2 ± 134.8, showing that Abi-exosomes can more efficiently incorporate 3.8 pg of mmu- miR-298-5p. In contrast, the relative ratio of mmu-miR-298-5p reflecting miR incorporation into exosomes not containing anti-ASGRl (hepatocyte- targeting) antibodies is shown on the far right. The relative ratio normalized to control was 1266.8 ± 40.1, indicating that the exosomes were only half as efficient as Abi-exosomes at incorporating 3.8 pg of mmu-miR-298-5p.

Abbreviations used herein, particularly in the Examples

b-HCG: b-human chorionic gonadotropin

2;-OMA: 2'-0-(N-(methyl)acetamido)

2'-OAE: 2'-0-amonioethyl

2’-OGE: 2-guanidoethyl (2’-OGE),

2’-OME: 2'-0-(methoxy ethyl)

^99m Tc: Technetium99

A: adenine

AB : apoptotic bodies and/or blebs

Abi-exosome: antibody-labeled exosome

ALIX: ALG-2 interacting protein X

ANOVA: analysis of variance

antimiR: molecules targeting miRNA

ATX: autotaxin

Bp: base pairs

C: cytosine

Cas: CRISPR-associated

CD: cluster of differentiation

cDNA: complementary DNA

CEA: carcinoembryonic antigen CMC: critical micelle concentration

CRISPR: clustered regularly interspaced short palindromic repeats crRNA: pre-CRISPR RNA

CT: cancer/testes

CTAB : b-alanyl cholesterol, cetyl trimethyl ammonium bromide

DC-Chol: 3 -[N-(N',N'-dimethylamino-ethane)carbamoyl]cholesterol

DDM: n -dodec y 1 - b - D- m a 1 tos i de

DEED: diethyl-ethylenediamide

DHPE: 1 ,2-dihexadecylphosphoethanolamine

DLS: dynamic light scattering

DMAP: dimethyl- aminopropylamine

DMPC: 1 ,2-dimyristoylphosphatidylcholine

DMRIE: 1 ,2-dimyristyloxypropyl-3-dimethyl-hydroxylethyl ammonium bromide

DMTAP: dimyristoyl- N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salt

DNA: deoxynucleic acid

DOGS : dioctadecylamidoglycylspermine

DOPE: 1 ,2-dioleylphosphoethanolamine

DORI: 1 ,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide DORIE-HB: 1 ,2-dioleyl-oxy-propyl-3-dimethyl-hydroxybutyl ammonium bromide

DORIE-HP: 1 ,2-dioleyloxypropyl-3-dimetyl-hydroxypropyl ammonium bromide

DORIE-Hpe: 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammonium bromide

DORIE: 1 ,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

DOSPA: 2,3-dioleoyloxy-N-(2-(sperminecarboxamido)-ethyl)-N,N- dimethyl- l-propanaminium trilluoro-acetate

DOSPER: ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide

DOTAP: dioleoyl- N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium DOTIM: l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- hydroxyethyl)imidazolinium chloride

DOTMA : N- [ 1 - (2 , 3 -dioleyloxy)propyl] -N, N, N- trimethylammonium chloride DPPC: 1,2-dipalmitoyl phosphatidylcholine

DPRIE: 1 ,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

DPTAP: dipalmitoyl- N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salt

DPTIM: l-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2- hydroxyethyl)imidazolinium chloride

DRM: dehydration rehydration method

DSPC: 1 ,2-distearoylphosphatidylcholine

DSPE-PEG(2000) Maleimide: l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]

DSPE: l,2-disteroyl-sn-glycero-3-phosphoethanolamine

DSRIE: 1 ,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

dsRNA: double- stranded RNA

DSTAP: distearoyl- N-[l-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salt dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2- diacyloxy-3-trimethylammonium propanes, N-[l-(2,3-dioloyloxy)propyl]-

DODAP: N,N-dimethyl amine

ECM: extracellular matrix

EGFR: epidermal growth factor receptor

EGS: external guide sequence

EIM: ether injection method

ELR:“glu-leu-arg” motif

EpCAM: epithelial cell adhesion molecule

ESG: external guide sequences

FAT : freeze and thaw method

FBS: fetal bovine serum

FITC: fluorescein isothiocyanate

FSH-R: follicle stimulating hormone receptor

G: guanine GAG: glycosaminoglycan

GalNAc: glycosyltransferase -l,4-N-acetylgalactosaminyltransferases

GPCRs: G-protein coupled receptors

HA: hemagglutinin

HDR: homology-directed repair

HM: heating method

HPLC: high performance liquid chromatography

HSM: hand shaking method

IM: intramuscular

IP: intraperitoneal

IRES: internal ribosome entry sequence

IV: intravenous

K _d : dissociation constant

LNA: locked nucleic acids

LPS: lipopoly saccharide

LU V : large unilamellar vesicles

MALP-2: mycoplasmal lipoproteins

MCC : maleimidomethylcyclohexane-carboxamide

MCC-PE : 1 , 2-dioleoyl- sn-glycero- 3 -phosphoethanolamine-N - [4- (p- maleimidomethyl)cyclohexane-carboxamide]

miRNA: microRNA

MLV : multilamellar vescicles

MPB: maleimidophenylbutyramide

MPB-PE: l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p- maleimidophenyl)butyramide]

MRA: mesothelin related antigen

mRNA: messenger RNA

MV: microvesicles

NBD-DSPE: l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-(7-nitro- 2-l,3-benzoxadiazol-4-yl)

NHEJ: nonhomologous end joining

oncomiR: oncogenic miRNA

PAMPs: pathogen-associated molecular patterns PBMCs: peripheral blood mononuclear cells

PBS: phosphate buffered saline

pC: 5-(l-propynyl) cytosine

PC: phosphatidylcholine

PDGF: platelet derived growth factor receptor

PDP: pyridyldithiopropionate

PDP-PE: l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2- pyridyldithio)propionate]

PE: phosphatidylethanolamine

PEG: poly- (ethylene glycol)

PG: phosphatidylglycerol

PI: phosphatidylinositol

piRNA: piwi-interacting RNA

PNA: peptide nucleic acid

Pre-miRNA: precursor miRNA

Pri-miRNA: primary miRNA

PS: phosphatidylserine

PSA: prostate surface antigen

PTEN: phosphatase and tensin homolog

pU: 5-(l-propynyl) uracil

Rab5 : Ras-related protein

REV : reverse phase evaporation method

RI: refractive index

RISC: RNA-induced silencing complex

RNA: ribonucleic acid

RNAi: RNA interference

RVD: repeat variable diresidue

SIP: sphingosine- 1 -phosphate

scFv: single chain variable fragments

sdAb: single antibody antibody fragments

sdFv: disulfide-linked fragments

sgRNA: single-guide RNA

shRNA: short double- stranded hairpin-like RNAs

siRNA: short interfering RNA SSL: sterically-stabilized liposomes

SubQ: subcutaneous

SUV : small unilamellar vescicles

T: thymine

TAL: transcription activator-like

TALEN: transcription activator-like effector nuclease

tcPNA:“tail-clamp” peptide nucleic acids

TFH: thin-film hydration method

TFO: triplex forming oligonucleotide

TLRs: toll-like receptors

TMAG: diCi4-amidine, N-ferf-butyl-N'-tetradecyl-3-tetradecylamino- propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D-glutamate chloride

TNT: tunneling nanotubes

tracrRNA: trans-activating crRNA

tRNA: transfer RNA

U: uracil

ZFNs: zinc finger nucleases

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.