Methods and compositions for two-stage microbubble delivery of active agents - Patents.com

Abstract

In various aspects, the present disclosure provides methods, compositions, and kits for treating target tissue with one or more active agents. In some embodiments, the delivery of active agents to target tissue comprises administration of microbubbles and ultrasound.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was written to a file named 053151-516001WO_ST25.txt, created on January 9, 2022, and is 18,298 bytes in size. [Background technology]

[0002] background Cancer is the number one cause of death and is responsible for increasing medical costs. Traditionally, cancer has been treated using chemotherapy, radiotherapy, and surgical methods. However, the plasticity and heterogeneity of tumor cells continue to be a challenge to the effective treatment of many cancers. In addition, traditional treatments may have shortcomings, such as insufficient specificity, unacceptable toxicity, and too low efficacy. Challenges may also arise when trying to direct treatment to certain tissues, especially those that are more difficult to target, such as the brain. Moreover, such challenges are not limited to the treatment of cancer, and inefficient targeting to the tissue of interest may limit the effectiveness of therapeutic agents and / or increase side effects, or limit diagnostic methods using imaging agents. Summary of the Invention

[0003] Quick Overview In view of the above, there is a need for improved targeting of active agents (including therapeutic and imaging agents, for "theranostic" applications in which both therapeutic and imaging agents are delivered simultaneously), as well as improved targeting in cancer treatment. The present disclosure provides methods and compositions that address this need, and also provides additional benefits.

[0004] In some aspects, the present disclosure provides a method for administering an active agent to a target tissue. In some embodiments, the active agent is a therapeutic agent or an imaging agent. In some embodiments, the method includes: (a) administering a first microbubble composition to a subject, the first microbubble composition containing a first microbubble and not containing an active agent; (b) administering a first ultrasound wave to the target tissue, destroying the first microbubbles; (c) administering a second microbubble composition to the subject after administering the first ultrasound wave, the second microbubble composition containing a second microbubble complexed with an active agent; and (d) administering a second ultrasound wave to the target tissue, destroying the second microbubbles and releasing the active agent to the target tissue.

[0005] In some embodiments, the method described herein is called focused ultrasound (FUS) dual microbubble (FUS-DMB) delivery. In some embodiments, FUS-DMB is applied to treat brain cancer, such as those originating in the brain (e.g., glioblastoma multiforme or GBM), and metastatic tumors in the brain whose primary tumor site is outside the brain. In some embodiments, FUS-DMB involves first using focused ultrasound in the target tissue to temporarily open the blood-brain barrier (BBB) ​​using microbubbles (MBs) that lack the active agent to be delivered (e.g., empty MBs), and then applying ultrasound-targeted microbubble-destruction (UTMD) technology, in which an active agent (e.g., adenovirus or therapeutic protein) is complexed with the microbubbles, which are administered systemically (or directly), and released in the target tissue (e.g., brain or pancreas) by FUS. In some embodiments, benefits include treatment of primary GBM, recurrent GBM, or secondary brain cancer (due to metastasis from other sites in the body) without the need for surgery. In some embodiments, the direct addition of an active agent (e.g., a virus) complexed with MB to a tumor undergoing debulking surgery and application of FUS is used to enhance therapeutic activity.

[0006] In some aspects, the present disclosure provides kits for use in treating a target tissue with an active agent. In some embodiments, the kit includes first and second microbubble compositions, where (i) the active agent is a therapeutic agent or an imaging agent, (ii) the first microbubble composition contains a first microbubble and no active agent, and (iii) the second microbubble composition contains a second microbubble complexed with an active agent.

[0007] In some embodiments, use of the kit includes (a) administration of a first microbubble composition, (b) administration of a first ultrasound wave directed to the target tissue to destroy the first microbubbles, (c) administration of a second microbubble composition following administration of the first ultrasound wave, and (d) administration of a second ultrasound wave directed to the target tissue to destroy the second microbubbles. [Brief description of the drawings]

[0008] [Figure 1]1A-1C show a focused ultrasound (FUS) dual microbubble (FUS-DMB) delivery strategy for delivering adenovirus to the brain, according to one embodiment. Figure 1A provides a schematic of the FUS-DMB delivery protocol. Briefly, 100 μl of diluted microbubbles (MB)+Ad.5 / 3-CMV-Luc was injected via the tail vein and allowed to circulate for 15 seconds. After 15 seconds, the mouse brains were sonicated for 1 minute using focused ultrasound (FUS) (group 1; left panel of FIG. 1B and first bar of FIG. 1C). The second group (group 2; right panel of FIG. 1B and second bar of FIG. 1C) was injected with diluted microbubbles (empty) and sonicated (FUS) in the brain region for 1 minute, then IV injected with 100 μl of MB containing Ad.5 / 3-CMV-Luc, and MB was allowed to circulate for 15 seconds before sonicating (FUS) in the brain region for 1 minute (FUS-DMB delivery approach). The next day, the animals were imaged using an IVIS imager. FIG. 1B shows a representative photograph. FIG. 1C shows the relative luciferase intensity measured in individual animals at a single time point, with the average value from three mice plotted. *Statistically significant. [Figure 2A]2A and 2B show focused ultrasound (FUS) dual MB (FUS-DMB) delivery and efficient delivery of therapeutic virus to the brain according to an embodiment. FIG. 2A shows a schematic diagram of an example of an experimental protocol. Mice were anesthetized via intraperitoneal (IP) administration of ketamine (40 mg / kg) and xylazine (3 mg / kg) and immobilized in a stereotaxic frame. Intracerebral injection of 5 μl of DMEM medium containing 10,000 glioma cells (GBM-6-Luc) was performed over 10 minutes using a Hamilton syringe. The skull opening was closed using sterile bone wax and the skin incision was closed using sterile surgical staples or surgical glue. After one week, the mice were imaged and randomized into three groups based on tumor growth (IVIS Image). These tumors were treated with intravenous (IV) injection of microbubbles (MB) containing either Ad.5 / 3-null or Ad.5 / 3-CTV with or without BBB opening (using FUS). Figure 2B shows representative photographs (upper panel) and relative luciferase intensity measurements (lower panel) in individual animals at a single time point, with the average values ​​from three mice plotted. *Statistically significant. [Figure 2B] See legend to Figure 2A. [Diagram 3]Figure 3A shows an image of a preparation of His-MDA-7 protein complexed with microbubbles (MBs) (MDA-7-MB). To confirm the association of His-MDA-7 with MBs, His-MDA-7 labeled with Alexa Fluor 488 was mixed and incubated with MB lyophilisates overnight. Unincorporated labeled His-MDA-7 was removed by centrifugation, and the MBs (white layer) were mixed with 1 ml of PBS and observed under a fluorescent microscope. Scale; 1 μm. The labeled His-MDA-7 (identified by green fluorescence) was associated with the lipid shell of MBs. Figure 3B shows tumor-specific delivery of Alexa Fluor-His-MDA-7-encapsulated MBs combined with ultrasound-targeted MB destruction (UTMD). DU-145 human prostate cancer cells were established as xenografts in the left flank of nude mice. Alexa Fluor-labeled His-MDA-7 / MB complexes were injected through the tail vein of nude mice and sonoporated for 10 min in xenografted (DU-145) tumors implanted in the left flank by handheld ultrasound (SonoSite Micro-Maxx US platform) with a L25 linear array transducer set at mechanical index 0.7 and MPa 1.8 (UTMD approach). Fluorescent images were acquired using a Xenogen IVIS Spectrum. Release and tumor-specific delivery of labeled His-MDA-7 were mainly localized to the left flank where ultrasound was applied. Brighter colors indicate higher fluorescence intensity. [Figure 4] Figure 4 shows that after clearance of circulating blood bubbles, MBs decorated with covalently attached cVEGF adhere to mouse MC38 tumor vasculature (left panel). Sequoia Cadence CPS imaging mode. MBs decorated with anti-VCAM-1 antibody (nanobody) fragments (right panel, upper frame) or control antibody fragments (lower frame). Comparison with control shows accumulation of targeted MBs in MC38 mouse tumor vasculature. [Figure 5A]Figures 5A-5D show site-specific delivery of Ad.5 / 3-CMV-luc using targeted or decorated microbubbles (D-MB) in immunocompetent prostate cancer Hi-myc mice and breast cancer MMTV-PyMT mice. Biotinylated anti-V-CAM-1 (B-VCAM-1) (100 μg) was incubated with streptavidin microbubbles (MB-SA) (approximately 109 MB particles) to form biotin-anti-V-CAM-1-streptavidin-MB (MB-SA-B-anti-VCAM-1; D-MB) complexes. To validate the preparation of D-MB, both D-MB and simple MB-SA were mixed with avidin-FITC and flow cytometry was performed to confirm the formation of D-MB (Figure 5A). D-MB and simple MB complexed with Ad.5 / 3-CMV-luc were injected systemically via the tail vein, and sonoporation was performed as indicated by the dashed circle in Hi-myc (Figure 5B) and MMTV-PyMT (Figure 5C) using FUS 6 min after injection of MB / Ad.5 / 3-CMV-luc. Bioluminescence imaging (BLI) was performed 72 h after injection of D-MB / Ad.5 / 3-CMV-luc using IVIS Spectrum. BLI images of dissected tumors and organs of MMTV-PyMT mice injected with D-MB / ad.luc and then subjected to ultrasound targeted MB destruction (UTMD) at the site of the tumor (Figure 5D). [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 6A]Figures 6A and 6B show specific delivery of adenovirus (Ad) by targeted MB (anti-PSMA-MB) according to an embodiment. Figure 6A shows that tumor xenografts developed after subcutaneous (sc) injection of PC-3 in the left flank and PC-3-PIP (PC-3 overexpressing PSMA) in the right flank. After tumor formation, Ad.5 / 3-CMV-luc alone or Ad.5 / 3-CMV-luc complexed with plain (undecorated) MB or targeted (decorated) MB (anti-PSMA-MB) was injected via tail vein injection, and 10 minutes after injection, sonoporation was performed for 10 minutes in the right flank of the mice by using an ultrasound transducer. Figure 6B shows image acquisition 72 hours after sonoporation using an IVIS system, and the images were analyzed by Living Image 4.3.1. It was clearly shown that targeted (decorated) MBs provide more specific delivery of Ad without nonspecific delivery to adjacent organs, whereas intact (undecorated) MBs deliver Ad to adjacent tissues or organs (liver, spleen) in addition to the tumor site where ultrasound was applied. [Figure 6B] See legend to Figure 6A. [Figure 7A]Figures 7A-7D show that focused ultrasound (FUS) dual MB (FUS-DMB) delivery of Ad.5 / 3-CTV significantly extends survival of mice bearing human GBM tumors. Figure 7A; Schematic showing FUS-DMB-Ad.5 / 3-CTV administration. Figure 7B; Primary human GBM6 tumors expressing luciferase were established in nude mice. Mice were treated by intravenous injection of either Ad.5 / 3-null with FUS-DMB (left panel), Ad.5 / 3-CTV with DMB (no FUS / middle panel), or Ad.5 / 3-CTV with FUS-DMB (right panel). Representative BLI images are shown. Figure 7C; Kaplan-Meier analysis showing percent survival of GBM6-implanted mice treated as in B. *p<0.001 compared to control / DMB-CTV (no FUS). (Figure 7D) Mice were euthanized upon reaching the IACUC endpoint and brains were collected and fixed in formalin. FFPE tissue sections were stained for MDA-7 / IL-24 (transgene expression), Ki-67 (proliferation marker), CD-31 (angiogenesis marker), and GRP-78 (established downstream target of MDA-7 / IL-24). As expected, only FUS-DMB CTV treatment enhanced MDA-7 and GRP-78 expression and decreased Ki-67 and CD31 expression. [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 8]Figure 8A and Figure 8B show that FUS-DMB or direct intracranial delivery of Ad.5 / 3-CTV significantly and comparably prolongs the survival of mice bearing glioma stem cell (GSC) tumors. Luciferase-expressing GSC-8-11 tumors were established in nude mice. Figure 8A, mice were treated with either direct intracranial injection of Ad.5 / 3-CTV or intravenous injection of Ad.5 / 3-CTV with FUS-DMB, and mice were observed using IVIS imaging. Representative images of BLI are shown. Figure 8B, Kaplan-Meier analysis showing survival analysis of mice implanted with GSC-8-11 treated with FUS-DMB-Ad.5 / 3-CTV or IC-Ad.5 / 3-CTV. In both treatment protocols, significant prolongation of survival was observed. These observations are intriguing and highlight the power of the FUS-DMB approach as a novel treatment option for treating GBM patients non-surgically and non-invasively. * p<0.001 compared to control IC; @ p<0.001 compared to control. [Figure 9] Figures 9A and 9B show that multiple injections of Ad.5 / 3-CTV further prolong the survival of mice bearing tumors driven by GSCs. Figure 9A, mice were treated with a single intravenous injection of Ad.5 / 3-CTV with FUS-DMB or multiple injections of Ad.5 / 3-CTV with FUS-DMB and observed using IVIS imaging. Representative images of BLI are shown. Figure 9B, Kaplan-Meier analysis showing survival analysis of mice transplanted with GSC-8-11 treated as in A. This study establishes that Ad.5 / 3-CTV with FUS-DMB can be administered multiple times to animals without toxic effects, and that multiple administrations of Ad.5 / 3-CTV with FUS-DMB further enhance the survival of mice bearing gliomas than a single administration. The FUS-DMB approach involves non-invasive intravenous administration of Ad.5 / 3-CTV without surgery. *p<0.001 compared to control. [Figure 10]FIG. 10 shows that the FUS-DMB approach can specifically target the "theranostic" TCTV virus to the brain and non-invasively image GBM in mouse brain. GBM6 cells were intracranially injected and treated with FUS-DMB containing Ad.5-TCTV. Mice were imaged 24 hours after treatment using an IVIS imager. Representative BLI images are shown. Left panel, mice were intravenously injected with DMB-Ad.5-TCTV but no FUS was applied. Right panel, mice were intravenously injected with DMB-Ad.5-TCTV and FUS was applied in the brain region (FUS-DMB approach). TCTV is a unique tripartite theranostic virus that utilizes three separate promoters to uniquely target viral replication, cytokine production, and imaging potential to cancer cells. The conditional replication of TCTV is controlled by a cancer-selective (truncated PEG-3) promoter, the therapeutic component MDA-7 / IL-24 is under a ubiquitous (CMV) promoter, and finally, the imaging potential is synchronized by another cancer-selective (truncated tCCN1) promoter. This study suggests that FUS-DMB-TCTV can non-invasively image and treat gliomas in mouse brains. [Figure 11] Figure 11 shows a schematic diagram illustrating the FUS-DMB approach in the pancreas. The FUS-DMB approach can be used to deliver viruses systemically to the pancreas using focused ultrasound, and theoretically, to other organ sites in the body. The FUS-DMB approach is more effective in delivering viruses than the use of a single FUS MB approach (UTMD - ultrasound targeted microbubble destruction approach). [Figure 12]Figure 12A and Figure 12B show systemic administration of Ad using FUS-DMB to target the pancreas. KPC (Pdx-1-Cre / K-rasLSL-G12D / p53fl / fl) homozygous mice were injected with Ad.5 / 3-Luc / MB complex via tail vein and MB was allowed to circulate for 15 seconds. FUS was applied to the pancreatic region for 1 minute by a water immersion transducer using an amplitude of 10 dB, a frequency of 1 MHz, and a power of 3.5 mV. 48 hours after delivery, mice were imaged using a Xenogen IVIS Spectral Imager. The indicated organs were collected (Li-liver, Lu-lung, P-pancreas, Sp-spleen) and ex vivo imaging was performed by BLI. Figure 12A, IV (intravenous) injection of Ad.5 / 3-Luc alone, without FUS, resulted in accumulation of luciferase signal mainly in the liver. FIG. 12B, IV (intravenous) injection of Ad.5 / 3-Luc with FUS-DMB in the pancreatic region results in accumulation of luciferase primarily in the pancreas. [Figure 13]Figure 13A and Figure 13B show that systemic administration of Ad expressing shRNA with FUS-DMB in pancreas specifically reduces target gene expression in pancreas. MB / Ad (Ad.shmda-9) was injected systemically (100 μl) into KPC mice via tail vein with FUS-DMB. Mice were maintained for 48 hours, euthanized, and organs (spleen, pancreas, lungs, and liver) were collected. These organs were lysed and RNA / proteins were isolated using standard protocols. Figure 13A, Equal amounts of RNA were used to synthesize cDNA and real-time PCR was performed to check MDA-9 / syntenin / SDCBP mRNA levels. Mouse GAPDH was used as a transcriptional control. *p<0.05 (compared to control). Figure 13B, Western blotting analysis of MDA-9 / syntenin / SDCBP. β-actin was used as a loading control. This study establishes that Ad.shMDA-9 with FUS-DMB can be administered intravenously and inhibit MDA-9 / syntenin / SDCBP levels specifically in the pancreas compared to other organs. It also shows that Ad.shMDA-9 with FUS-DMB can be used to target the pancreas, which is not evident in naive mice (not receiving FUS). [Figure 14]Figure 14A and Figure 14B show that the dual MB approach is more efficient than the single MB approach. MB / Ad (Ad.shmda-9) was injected systemically (100 μl) into KPC mice via the tail vein using either single MB or FUS-DMB. Mice were euthanized and organs (spleen, pancreas, lungs, and liver) were collected at the indicated time points. These organs were lysed and RNA / proteins were isolated using standard protocols. Figure 14A, Equal amounts of RNA were used to synthesize cDNA and real-time PCR was performed to check MDA-9 / syntenin / SDCBP mRNA levels. Mouse GAPDH was used as a transcriptional control. *p<0.05 compared to control. Figure 14B, Western blotting analysis of MDA-9 / syntenin / SDCBP. β-actin was used as a loading control. This study establishes that Ad.shMDA-9 with FUS-DMB is administrable and more efficient in inhibiting MDA-9 / syntenin / SDCBP levels in the pancreas compared to FUS-MB (single microbubble delivery). [Figure 15] Figure 15 shows that FUS-DMB delivery of Ad.5 / 3-shMDA-9 significantly extends the survival of mice bearing pancreatic tumors. First, empty microbubbles were injected, and FUS was applied for 1 min using the same parameters as described in Figures 12A and 12B. After 1 min, MB / Ad.shMDA-9 treated with complement was injected systemically (100 μl) into KPC homozygous mice via the tail vein and sonoporated with FUS. These mice then received intraperitoneal injections of gemcitabine (20 mg / Kg) 24 and 48 hours after Ad.shMDA-9 injection. The same process was repeated twice (total injections of AdhMDA-9 + FUS-DMB: 3 times; gemcitabine: 6 times). These mice were observed for survival and Kaplan-Meier analysis was performed. * p<0.01 (compared to control); ** p<0.001 (compared to control). This study establishes that Ad.shMDA-9 with FUS-DMB can be administered multiple times to mice without known toxicity and can be combined with chemotherapeutic agents for better therapeutic efficacy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] Detailed Description While various embodiments and aspects of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments and aspects are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized in the practice of the invention.

[0010] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including but not limited to patents, patent applications, articles, books, manuals, and treatises, are expressly incorporated herein by reference in their entirety in nature.

[0011] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J.Wiley & Sons (New York, NY 1994); and Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012).Similar or equivalent methods, devices and materials to those described herein can be used in the practice of the present invention.The following definitions are provided to facilitate understanding of some terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0012] As used herein, the term "about" refers to a range of values, including the specified value, that would be considered by those skilled in the art to be reasonably similar to the specified value. In some embodiments, the term "about" refers to within a range of standard deviation using measurements generally accepted in the art. In some embodiments, about refers to a range that covers + / - 10% of the specified value. In some embodiments, about refers to the specified value.

[0013] It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude optional elements. As such, this statement is intended to support the use of exclusive terminology in the claims, such as "solely," "only," and the like, with respect to the recitation of claim elements, or "negative" limitations, such as "absent of [a particular feature or element]," or "excluding [a particular feature or element]," or "not present (e.g., not including) [a particular feature or element]."

[0014] The terms "nucleic acid," "nucleic acid molecule," "nucleic acid oligomer," "oligonucleotide," "nucleic acid sequence," "nucleic acid fragment," and "polynucleotide" are used interchangeably and include, but are not limited to, polymeric forms of nucleotides, either deoxyribonucleotides or ribonucleotides, covalently linked together, which may have various lengths, or analogs, derivatives, or modifications thereof. Different polynucleotides may have different three-dimensional structures and may perform various functions, known or unknown. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (e.g., but not limited to, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of a sequence, isolated RNA of a sequence, nucleic acid probes, and primers. Polynucleotides useful in the methods of the present disclosure may include naturally occurring nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of such sequences. Polynucleotides may include one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

[0015] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid, but function in a manner similar to naturally occurring amino acids. The terms "non-naturally occurring amino acids" and "unnatural amino acids" refer to amino acid analogs, synthetic amino acids, and amino acid mimetics that are not found in nature.

[0016] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted one-letter codes.

[0017] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include amino acid polymers that have been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. A "fusion protein" refers to a chimeric protein comprising two or more separate protein sequences that are recombinantly expressed as a single moiety.

[0018] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity in a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity, when compared and aligned for maximum correspondence in a comparison window or specified region) as measured using a sequence comparison algorithm (optionally with default parameters) or by manual alignment and visual inspection. In some embodiments, sequences that are "substantially identical" are at least 80%, 90%, 95%, 99% or more identical. In the case of nucleic acids, percent identity can also refer to or be applied to the complement of a test sequence. As described below, preferred algorithms can take into account gaps, etc. In certain embodiments, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0019] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, where the portion of the comparison window of the polynucleotide or polypeptide sequence may include additions or deletions compared to the reference sequence (not including the additions or deletions) due to the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (e.g., with respect to the reference sequence), and multiplying the result by 100 to obtain the percentage of sequence identity. Programs for determining sequence identity are known to those skilled in the art, and include, but are not limited to, BLAST (see, for example, the NCBI website www.ncbi.nlm.nih.gov / BLAST, etc., optionally with default parameters), Needleman-Wunsch algorithm (see, for example, the EMBOSS Needle aligner available at www.ebi.ac.uk / Tools / psa / emboss_needle / , optionally with default settings).

[0020] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Because of deletions, insertions, truncations, fusions, etc. that must be considered when determining optimal alignment, in general, the amino acid residue number of a test sequence, determined by simple counting from the N-terminus, is not necessarily the same as the number of the corresponding position in the reference sequence. For example, if a variant has a deletion relative to the aligned reference sequence, the amino acid corresponding to the position of the reference sequence at the site of the deletion is not present in the variant. If there is an insertion into the aligned reference sequence, the insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence. An amino acid mutation may be identified by a notation that identifies the original amino acid (e.g., as in the wild-type sequence or the reference sequence), the position of the mutation, and the amino acid that is changed from the original amino acid. For example, "K122R relative to SEQ ID NO:2" indicates a mutation of lysine at position 122 of SEQ ID NO:2 to arginine. Nucleotide variations can also use a similar notation scheme.

[0021] The term "numbered with respect to" or "corresponding to," when used in reference to the numbering of a given amino acid or polynucleotide sequence, refers to the numbering of residues in a specified reference sequence when comparing the given amino acid or polynucleotide sequence to a reference sequence. Whether an amino acid corresponds to a particular position in a reference sequence, optionally at a different position (e.g., the K122R mutation relative to SEQ ID NO:2), can be determined by sequence alignment. Generally, an alignment showing the identity of one or more amino acids adjacent to a indicated position in a reference sequence allows the corresponding position of the query sequence to be locally located with respect to the reference sequence to confirm that the corresponding amino acid mutation is present, optionally at the shifted number position in the query sequence. In some embodiments, a region containing at least 3-15 amino acids that includes the mutation position is locally aligned with the corresponding reference sequence with a relatively high percent identity (e.g., at least about 90%, 95%, or 100% identity), excluding the position of the mutation amino acid on the query sequence. In certain embodiments, an amino acid of a query MDA-7 / IL-24 protein sequence corresponds to a particular position in a reference sequence if the polypeptide of the query sequence is aligned to that particular position in the reference sequence when the two sequences are optimally aligned using the BLASTP alignment algorithm with default parameters.

[0022] The term "MDA-7", "IL-24", or "MDA-7 / IL-24" refers to a protein having MDA-7 activity, including homologs, isoforms, and functional fragments thereof. The term includes recombinant or naturally occurring forms of MDA-7, or variants, homologs, or isoforms thereof that maintain MDA-7 activity (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wild-type MDA-7). In some embodiments, the variant, homolog, or isoform has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of a contiguous 50, 100, 150, or 200 amino acids) compared to a naturally occurring MDA-7 protein. In some embodiments, the MDA-7 protein is substantially identical to the protein identified by accession number NP_006841, or a variant or homologue with substantial identity thereto. In some embodiments, the MDA-7 protein is substantially identical to the protein identified by UniProt Q13007, or a variant or homologue with substantial identity thereto. In some embodiments, the IL-24 gene is substantially identical to the nucleic acid sequence listed in RefSeq(mRNA)NM_006850, or a variant or homologue with substantial identity thereto. In some embodiments, the IL-24 gene is substantially identical to the nucleic acid sequence listed in Ensembl reference number ENSG00000162892, or a variant or homologue with substantial identity thereto. In some embodiments, the amino acid sequence or nucleic acid sequence is a sequence known at the time of filing of this application. In some embodiments, the protein is a precursor form that includes a signal sequence. In some embodiments, the signal sequence is not the native MDA-7 signal sequence, but rather is, for example, a modified native signal sequence, an unmodified signal sequence from another gene (e.g., the insulin gene), or a modified signal sequence from another gene.In some embodiments, the protein is a mature form of MDA-7 that lacks a signal sequence at the N-terminus of the precursor form of the protein. The mature form may be produced post-translationally from a precursor form that contains a signal sequence, or may be directly translated from a polynucleotide that encodes the mature form that does not contain a signal sequence N-terminal to the sequence of mature MDA-7. In some embodiments, the MDA-7 / IL-24 protein comprises SEQ ID NO:4, or a variant, homolog, or isoform thereof that maintains or enhances MDA-7 activity. In some embodiments, the MDA-7 / IL-24 protein comprises SEQ ID NO:3, or a variant, homolog, or isoform thereof that maintains or enhances MDA-7 activity. In some embodiments, the MDA-7 / IL-24 protein does not comprise the first 49 amino acids of SEQ ID NO:2. In some embodiments, the MDA-7 / IL-24 protein comprises SEQ ID NO:18, or a variant, homolog, or isoform thereof that maintains or enhances MDA-7 activity. Further non-limiting examples of MDA-7 polynucleotide and polypeptide sequences are described in US20200354745A1, which is incorporated herein by reference.

[0023] The term "MDA-9", "syntenin", "syndesin-binding protein", "SDCBP", or "MDA-9 / syntenin" refers to a protein having MDA-9 activity, including homologs, isoforms, and functional fragments thereof. The term includes recombinant or naturally occurring forms of MDA-9, or variants, homologs, or isoforms thereof that maintain MDA-9 activity (e.g., within at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity compared to wild-type MDA-9). In some embodiments, the variant, homolog, or isoform has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a portion of 50, 100, 150, or 200 consecutive amino acids) compared to a naturally occurring MDA-9 protein. In some embodiments, the MDA-9 protein is substantially identical to the protein identified by accession number NP_005616, or a variant or homologue with substantial identity thereto. In some embodiments, the MDA-9 protein is substantially identical to the protein identified by UniProt O00560, or a variant or homologue with substantial identity thereto. In some embodiments, the MDA-9 gene is substantially identical to the nucleic acid sequence listed in RefSeq(mRNA)NM_005625, or a variant or homologue with substantial identity thereto. In some embodiments, the SDCBP gene is substantially identical to the nucleic acid sequence listed in Ensembl reference number ENSG00000137575, or a variant or homologue with substantial identity thereto. In some embodiments, the amino acid sequence or nucleic acid sequence is a sequence known at the time of filing of this application. In some embodiments, the protein is a precursor form that includes a signal sequence. Further non-limiting examples of MDA-9 polynucleotide and polypeptide sequences are described in WO2017120439, which is incorporated herein by reference.

[0024] The terms "signal sequence" and "signal peptide" refer to a polypeptide sequence capable of directing the secretion of a protein that includes the signal peptide. Typically, a signal peptide is at or near the N-terminus of a protein. A signal peptide may be directly adjacent to the protein to be secreted or may be joined by a linker of one or more amino acids. In eukaryotes, secretion typically involves directing the protein to the endoplasmic reticulum and may involve cleavage to remove part or all of the signal peptide before being secreted outside the cell. In bacteria, the protein may be secreted into the periplasm or medium. A signal peptide can direct the secretion of a protein that includes the signal peptide if, when attached to a protein of interest (e.g., MDA-7 / IL-24 protein), more of the protein of interest is secreted from the cell than in the absence of the signal peptide. In some embodiments, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the protein of interest is secreted. In some embodiments, at least 50% of the protein of interest is secreted. Secretion can be measured in any suitable system, such as cultured cells as described herein. In some embodiments, a signal sequence is joined to the protein of interest such that the entire signal sequence is removed by cleavage during the secretion process.

[0025] Those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences that modify, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" if the modification results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present disclosure. The following eight groups each contain amino acids that are conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins (1984)).

[0026] Any number of amino acids in a protein structure can be replaced by other amino acids without any appreciable loss of tumoricidal effect. Because it is the ability and nature of protein interaction that defines the biological functional activity of a protein, any number of amino acid substitutions can be made in the protein sequence, and of course in its DNA coding sequence, to nevertheless obtain a protein with similar properties. Thus, it is contemplated that various changes can be made in the polypeptide sequence of the present disclosure, or the corresponding DNA sequence encoding said polypeptide, while retaining at least a portion of the biological activity. Such biological activity can be assessed by various techniques, such as the assays described in the examples herein.

[0027] The term "purified" when applied to a nucleic acid or protein means that the nucleic acid or protein is essentially free of one or more other cellular components with which it is associated in its native state or in a whole cell lysate. It may, for example, be in a homogenous state or mixed with one or more other compounds, and may be in either a dry or aqueous solution. For example, the MDA-7 / IL-24 protein (or a polynucleotide or vector encoding it) may be purified from a cell lysate and then combined with one or more other agents (e.g., microbubbles and, optionally, an anti-cancer agent). Thus, a composition comprising a purified MDA-7 / IL-24 protein (or a polynucleotide or vector encoding it) may contain additional compounds, but generally is free of or has reduced levels of one or more impurities present in the lysate or medium from which the MDA-7 / IL-24 protein (or a polynucleotide or vector encoding it) was originally isolated. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A molecule that is the predominant species present in a preparation is substantially purified.

[0028] As used herein, the term "cancer" refers to all kinds of cancers, neoplasms, or malignant tumors found in mammals (e.g., humans), such as leukemia, lymphoma, carcinoma, and sarcoma.Exemplary cancers that can be treated by the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease, and non-Hodgkin's lymphoma.Exemplary cancers that can be treated by the compounds or methods provided herein include thyroid, endocrine system, brain, breast, cervix, colon, head and neck, liver, kidney, lung, ovary, pancreas, rectum, stomach, and uterus cancer. Further examples include thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma (PDAC), cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, squamous cell carcinoma of the head and neck, invasive breast cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, neoplasms of the endocrine or exocrine pancreas, medullary thyroid cancer, In some embodiments, the cancer is a cancer that has metastasized to bone. In some embodiments, the cancer is a prostate cancer, e.g., a bone metastasis from a prostate cancer.

[0029] As used herein, the terms "metastasis," "metastatic," and "metastatic cancer" may be used interchangeably and refer to the spread of a proliferative disease or disorder, e.g., cancer, from one organ or other non-adjacent organ or body part. "Metastatic cancer" is also referred to as "Stage IV cancer." Cancer begins in an origin site, e.g., the prostate, which is called the primary tumor, e.g., primary prostate cancer. Some of the cancer cells of the primary tumor or origin site acquire the ability to penetrate and invade the surrounding normal tissue in a localized area, and / or penetrate the walls of the lymphatic or vascular system and circulate through the system to other sites and tissues in the body. A second clinically detectable tumor formed from the cancer cells of the primary tumor is called a metastatic tumor or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if prostate cancer metastasizes to bone, the secondary tumor at that site of bone will consist of abnormal prostate cells, not abnormal bone cells. Secondary tumors of bone are called metastatic bone cancer.Therefore, the phrase metastatic cancer refers to disease in which a subject has or has had a primary tumor and has one or more secondary tumors.The phrase subject with non-metastatic cancer or non-metastatic cancer refers to disease in which a subject has a primary tumor but does not have one or more secondary tumors.For example, metastatic lung cancer refers to disease in a subject who has or has had a history of a primary lung tumor and has one or more secondary tumors in a second location or multiple locations, such as bone.

[0030] As used herein, a "subject" can be a mammal, e.g., a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In some embodiments, the subject is a human. In some embodiments, the subject is a mammal (e.g., a human) that has or may have a cancer described herein, e.g., a metastatic cancer. In some embodiments, the subject is a mammal (e.g., a human) that is at risk of developing a cancer described herein, e.g., a metastatic cancer.

[0031] "Treating" or "treatment", as used herein, broadly includes any approach to obtain a beneficial or desired outcome, e.g., clinical outcome, for a condition of a subject. Beneficial or desired clinical outcome includes, but is not limited to, alleviation or amelioration of one or more symptoms or conditions, whether partial or complete, detectable or undetectable, reduction in the extent of disease, stabilization of the disease state (i.e., no progression), delay or slowing of disease progression, improvement or palliation of the disease state, reduction in recurrence of disease, and remission. In some aspects, the subject has previously been treated for the disease. In other words, "treatment", as used herein, includes any cure or amelioration of the disease. Treatment can completely or partially alleviate symptoms of the disease, remove the underlying cause of the disease, shorten the duration of the disease, or a combination thereof. In the case of cancer, treatment can include slowing, stopping, or reversing cancer cell proliferation (e.g., in tumor growth measured by tumor size or rate of change).

[0032] "Preventing," as used herein, refers to a reduction in the occurrence or incidence of one or more disease symptoms in a patient. Prevention may be complete (no detectable symptoms) or may be partial, with fewer symptoms observed than would likely occur in the absence of treatment. Prevention includes prophylactic treatment.

[0033] The length of treatment period depends on various factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used in treatment, or a combination thereof.It is also understood that the effective dosage of the agent used for treatment or prevention may increase or decrease during the course of a particular treatment or prevention regimen.Changes in dosage may occur and be evident by standard diagnostic assays known in the art.In some cases, chronic administration may be required.For example, the composition is administered to the subject in an amount and for a period sufficient to treat the patient.In some embodiments, administration of the composition of the present disclosure treats the subject's cancer (e.g., metastatic bone cancer) and prevents further disease systems (e.g., metastasis, e.g., bone metastasis).

[0034] The compositions described herein may be used in combination with each other or with other active agents known to be useful in the treatment of cancer, such as anti-cancer agents. "Anti-cancer agent" is used according to its plain and ordinary meaning and refers to a composition (e.g., a compound, drug, antagonist, inhibitor, modulator) that has anti-tumor properties or the ability to inhibit the growth or proliferation of cancer cells. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an agent identified herein that has utility in the method of treating cancer. In some embodiments, the anti-cancer agent is an agent approved by the FDA or similar regulatory agency of a country other than the United States to treat cancer.

[0035] As used herein, the term "administering" includes oral administration, administration as a suppository, topical contact, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intrathecal administration, intranasal administration, or subcutaneous administration to a subject, or implantation of a sustained release device, such as a mini-osmotic pump. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarteriolar, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. "Co-administering" means that the compositions described herein are administered simultaneously with, immediately prior to, or immediately after the administration of one or more additional therapies, such as cancer therapies, such as chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. The compounds of the present invention may be administered to a patient alone or simultaneously. Simultaneous administration is intended to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). Thus, the preparation may be combined with other active agents, if desired (e.g., to reduce metabolic degradation). In some embodiments, "administering" a protein or a composition comprising a protein refers to administering the protein itself (e.g., MDA-7 / IL-24 protein), rather than the polynucleotide encoding the protein.

[0036] An "effective amount" is an amount of a compound sufficient to achieve a stated purpose (e.g., achieve the effect of its administration, treat a disease, reduce enzyme activity, increase enzyme activity, reduce a signal transduction pathway, or reduce one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom of a disease, which may also be referred to as a "therapeutically effective amount". A "reduction" of a symptom (and grammatical equivalents of this phrase) means a reduction in the severity or frequency of the symptom, or the elimination of the symptom. A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, has an intended prophylactic effect, for example, prevents or delays the onset (or recurrence) of an injury, disease, pathology, or condition, or reduces the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition, or a symptom thereof. A complete prophylactic effect does not necessarily occur by a single administration, but may occur only after a series of several administrations. Thus, a prophylactically effective amount can be administered by one or more administrations. "Activity-reducing amount" as used herein refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of antagonist. "Function-destroying amount" as used herein refers to the amount of antagonist required to destroy the function of an enzyme or protein compared to the absence of antagonist. The exact amount depends on the purpose of treatment and can be ascertained by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0037] For any compound described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration is the concentration of active compound that can achieve the methods described herein, as measured using methods described herein or known in the art.

[0038] The therapeutically effective amount for use in humans can also be determined from animal models.For example, the dose for humans can be formulated to achieve the concentration found to be effective in animals.The dosage in humans can be adjusted by monitoring the effectiveness of the compound and adjusting the dosage upwards or downwards as described above.The adjustment of dosage to achieve maximum effectiveness in humans based on the above and other methods is well within the capabilities of those skilled in the art.

[0039] The term "therapeutically effective amount" as used herein refers to an amount of a therapeutic composition sufficient to ameliorate a disorder as described above. For example, for a given parameter, a therapeutically effective amount shows an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect compared to a control.

[0040] Dosage may vary depending on the patient's needs and the compound utilized. In the context of the present disclosure, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose is also determined by the existence, nature, and extent of adverse side effects. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages that are less than the optimal dose of the compound. Thereafter, the dosage is increased by small increments until the optimal effect is reached under the circumstances. Dosage and dosage intervals can be individually adjusted to provide a level of the compound administered that is effective for the particular clinical indication being treated. This provides a treatment regime commensurate with the severity of the individual's condition.

[0041] "Pharmaceutically acceptable excipient" and "pharmaceutically acceptable carrier" refer to a substance that aids in the administration and absorption of an active agent to a subject and can be included in the composition of the present disclosure without causing significant adverse toxicological effects to the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohol, oils, gelatin, carbohydrates such as lactose, amylose, or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents, etc. Such preparations can be sterilized and, if desired, mixed with auxiliary agents that do not adversely react with the compounds of the present disclosure, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts that affect osmotic pressure, buffers, coloring agents, and / or aromatic substances. Those of skill in the art will recognize that other pharmaceutical excipients are useful in the present disclosure.

[0042] Pharmaceutical preparations are optionally in unit dosage form.In such form, the preparation is divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation, the package containing individual amounts of the preparation, for example, tablet, capsule, and powder in vial or ampoule.The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or any of these in the appropriate number packaged form.The unit dosage form can also be a frozen dispersion.

[0043] As used herein, the term "microbubble" generally refers to a spherical arrangement of lipids that creates an outer shell and an inner cavity. The lipid layer can be modified to stably bind molecules, for example, by incorporating an active agent as part of the outer shell during the formation of the microbubble, or by complexing the active agent with the shell (e.g., via non-covalent interactions) after the formation of the microbubble. In some embodiments, the use of microbubbles as vectors for the delivery of active agents utilizes the destruction of drug-loaded microbubbles by a focused ultrasound beam as the microvessel passes through the target area, which results in localized delivery upon destruction of the microbubble shell while sparing non-target areas (see, for example, US Patent Publication No. 2013204166). Ultrasound / microbubble targeted delivery (UMTD) has been used to deliver genes to cells in vitro and, more recently, in experimental animal models to treat diabetes and cardiovascular disease in vivo (Chen et al. (2007) Gene Ther. 14:1102-1110; Fujii et al. (2009) J. Am. Coll. Cardiol. Cardiovasc. Imaging 2:869-879). In some embodiments, the microbubbles are gene or molecular therapy vectors. The use of microbubbles as gene vectors has advantages over viral systems. In UMTD, intravenously injected microbubbles are destroyed as they pass through the microcirculation of the target site where the ultrasound beam is directed, and selective payload delivery can be functionally achieved without the need for invasive approaches such as direct intratumoral injection. In some embodiments, the lipid microbubbles we used for UMTD are administered repeatedly. In some embodiments, because the microbubbles are ultrasound contrast agents, one can also simultaneously image the passage of the microbubbles into the target tissue (e.g., tumor), thereby allowing more precise real-time guidance of active agent delivery. Any of a variety of techniques can be used in forming the microbubbles from a variety of suitable materials.For example, commercially available compositions useful for forming microbubbles include, but are not limited to, SONAZOID, OPTISON, SONOVUE, MICROMARKER, POLYSON, and other such ultrasound imaging agents. The microbubbles may include, but are not limited to, dipalmitoyl and distearoyl phosphatidic acids (DPPA, DSPA), dipalmitoyl and distearoyl phosphatidylserines (DPPS, DSPS), phosphatidylglycerols such as dipalmitoyl and distearoyl phosphatidylglycerol (DPPG, DSPG), 1,2-bis(10,12-tricosadinyl-sn-glycero-3-phosphokobune, La-phosphatidylcholine, PE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, PE-PEG2000-biotin, and combinations of one or more of these. The microbubbles may be formed from, but are not limited to, lipids. Non-lipids (e.g., proteins, and / or one or more active agents) may be included to form part of the outer shell of the microbubbles as a complex with one or more lipids of the shell. The lumen may be occupied by a suitable gas, e.g., air or perfluorobutane. Further non-limiting examples of gases are described in US20160243234A1, which is incorporated herein by reference. Generally, microbubbles have a mean or median diameter of about 0.1 microns to about 100 microns. Further non-limiting examples of compositions for the formation of microbubbles are described in US20160108429A1 and WO2020118271A1, which are incorporated herein by reference.

[0044] As used herein, the term "target tissue" refers to a collection of related or similar cells. Non-limiting examples of target tissue include connective tissue, muscle tissue, nerve tissue, or epithelial tissue. Target tissue may include epithelial tissue forming the surface of the skin, the respiratory tract, soft tissue, the reproductive system, and the lining of the digestive tract; fibrous connective tissue, skeletal connective tissue, humoral connective tissue, vasculature, bone, ligaments, tendons, blood, blood vessels, fat, areola, skeletal muscle, smooth muscle, cardiac muscle, nerve tissue of the brain, nerve tissue of the brain, nerve tissue of the spinal cord, nerve tissue of the cranial nerves, nerve tissue of the spinal nerves. Target tissue may be healthy or diseased (e.g., cancerous). Target tissue may be derived from a living organism or may be grown in vitro. Target tissue may be transplanted.

[0045] As used herein, the term "theranostic" refers to a combination of the terms therapeutic and diagnostic. A non-limiting example of a theranostic composition is one that can be used to both image and treat a target tumor in a subject.

[0046] As used herein, the term "active agent" refers to a compound that is a therapeutic agent, an imaging agent, or a theranostic agent.

[0047] As used herein, the term "active agent encapsulated in the microbubbles" refers to a compound that is a therapeutic agent, an imaging agent, or an agent that is both a therapeutic agent and an imaging agent, and that is encapsulated in the microbubbles.

[0048] As used herein, the term "active agent expelled from the microbubbles" refers to a compound that is expelled from the microbubbles and that is a therapeutic agent, an imaging agent, or an agent that is both a therapeutic agent and an imaging agent.

[0049] As used herein, "therapy involving microbubbles" refers to a therapy that includes treatment with one or more active agents and that also includes microbubbles.

[0050] As used herein, "microbubble-free therapy" refers to a therapy that includes treatment with one or more active agents and does not include microbubbles.

[0051] "Therapeutic agent" as used herein refers to an agent (e.g., a compound or composition described herein) that has an intended preventive effect, such as preventing or delaying the onset (or recurrence) of injury, disease, pathology, or condition, or reducing the likelihood of onset (or recurrence) of injury, disease, pathology, or condition, or symptoms thereof, or an intended therapeutic effect, such as treating or improving the objective or subjective parameters of injury, disease, pathology, or condition, or symptoms thereof, such as reducing symptoms; remission; shrinking, or improving the tolerability of injury, pathology, or condition by the patient; slowing down the rate of degeneration or decline; reducing the debilitating end point of degeneration; or improving the physical or mental well-being of the patient, when administered to a subject. In some embodiments, the therapeutic agent is an anti-cancer agent. In some embodiments, the therapeutic agent is a nucleic acid, a protein, or a vector (e.g., a plasmid or a virus).

[0052] ;Pentrozole;Perflubron;Perfosfamide;Perillyl alcohol;Phenazinomycin;Phenyl acetate;Phosphatase inhibitors;Picibanil;Pilocarpine hydrochloride;Pirarubicin;Piritrexim;Placetin A;Placetin B;Plasminogen activator inhibitors;Platinum complexes;Platinum compounds;Platinum-triamine complexes;Porfimer sodium;Porfiromycin;Prednisone;Propyl bis-acridone bis-acridone;prostaglandin J2;proteasome inhibitors;protein A-based immunomodulators;protein kinase C inhibitors;protein kinase C inhibitors, microalgae;protein tyrosine phosphatase inhibitors;purine nucleoside phosphorylase inhibitors;purpurins;pyrazoloacridines;pyridoxylated hemoglobin polyoxyethylene conjugates;raf antagonists;raltitrexed;ramosetron;ras farnesyl protein transferase inhibitors;ras inhibitors;ras-GAP inhibitors;demethylated retelliptine demethylated);rhenium Re186 etidronate;rhizoxin;ribozyme;RII retinamide;logretimide;rohitukine;romurtide;roquinimex;rubiginone B1;ruboxyl;safingol;saintopin;SarCNU;sarcophytol A;sargramostim;Sdi 1 mimetic;semustine;senescence derived inhibitor 1;sense oligonucleotide;signal transduction inhibitor;signal transduction modulator;single-chain antigen binding protein;sizofuran;sobuzoxane;sodium borocaptate;sodium phenylacetate;sorberol;somatomedin binding protein;sonermin;sparfosic acid;spicamycin D;spiromustine;splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium sodium);tegafur;tellurapyrylium;telomerase inhibitors;temoporfin;temozolomide;teniposide;tetrachlorodecaoxide;tetrazomine;thaliblastine;thiocoraline;thrombopoietin;thrombopoietin mimetics;thymalfasin;thymopoietin receptor agonists;thymotrinan;thyroid-stimulating hormone;tin ethyl etiopurpurin;tirapazamine;titanocene dichloride bichloride;topsentin;toremifene;totipotent stem cell factor;translation inhibitors;tretinoin;triacetyluridine;triciribine;trimetrexate;triptorelin;tropisetron;turosteride;tyrosine kinase inhibitors;tyrophostin system;UBC inhibitors;ubenimex;urogenital sinus-derived growth inhibitory factor;urokinase receptor antagonists;vapreotide;variolin B;vector system, red blood cell gene therapy;velaresol;veramine;verdins;verteporfin;vinorelbine;vinxaltine;vitaxin;vorozole;zanoterone;zeniplatin;zilascorb;zinostatin stimalamer, adriamycin, dactinomycin, bleomycin, vinblastine, cisplatin, acivicin;aclarubicin;acodazole hydrochloride;acronine;adozelesin;aldesleukin;altretamine;ambomycin;ametantrone acetate;aminoglutethimide;amsacrine;anastrozole;anthramycin;asparaginase;asperlin;azacitidine;azetepa;azotomycin;batimastat;benzodepa;bicalutamide;bisantrene hydrochloride;bisnafide dimesylate dimesylate;bizelesin;bleomycin sulfate;brequinar sodium;bropirimine;busulfan;cactinomycin;calusterone;caracemide;carbetimer;carboplatin;carmustine;carbicin hydrochloride;carzelesin;cedefingol;chlorambucil;cirolemycin;cladribine;crisnatol mesylate mesylate);cyclophosphamide;cytarabine;dacarbazine;daunorubicin hydrochloride;decitabine;dexormaplatin;dezaguanine;dezaguanine mesylate;diaziquone;doxorubicin;doxorubicin hydrochloride;droloxifene;droloxifene citrate;dromostanolone propionate;duazomycin;edatrexate;eflornithine hydrochloride;elsamitrucin;enloplatin;enpromate;epipropidine;epirubicin hydrochloride;erbulozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil;Fluorocitabine;Fosquidone;Fostriecin sodium sodium); gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; iimofosine; interleukin I1 (including recombinant interleukin II, or rlL.sub.2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-1a; interferon gamma-1b; iproplatin; irinotecan hydrochloride; lanreotide hydrochloride; letrozole; leuprolide hydrochloride; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; meno Galil;Mercaptopurine;Methotrexate;Methotrexate sodium;Metoprine;Meturedepa;Mitindomide;Mitocalcin;Mitochromin;Mitogillin;Mitomalcin;Mitomycin;Mitosper;Mitotane;Mitoxantrone hydrochloride;Mycophenolic acid;Nocodazoie;Nogalamycin;Ormaplatin;Oxisuran;Pegaspargase;Periomycin;Pentamustine;Peplomycin sulfate;Perfosfamide;Pipobroman;Piposulfan;Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol hydrochloride; Semustine; Simtrazene; S Parfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Talisomycin;Tecogalan sodium;Tegafur;Teloxantrone hydrochloride;Temoporfin;Teniposide;Te Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Toremifene citrate; Trestorone acetate; Triciribine phosphate; Trimetrexate; Trimetrexate glucuronate; Triptorelin; Tubrozole hydrochloride; Uracil mustard; Uredepa; Vapreotide ( vapreotide;verteporfin;vinblastine sulfate;vincristine sulfate;vindesine;vindesine sulfate;vinepidine sulfate;vinglycinate sulfate;vinleurosine sulfate;vinorelbine tartrate;vinrosidine sulfate;vinzolidine sulfate;vorozole;zeniplatin;zinostatin;Zorubicin hydrochloride, agents that arrest cells in the G2-M phase and / or modulate the formation or stability of microtubules (e.g., Taxol™ (i.e., paclitaxel), Taxotere™, compounds containing a taxane skeleton, Erbulozole (i.e., R-55104), Dolastatin 10 (i.e., DLS-10 and NSC-376128), Mibobulin isethionate (i.e., CI-980), Vincristine, NSC-639829, Discodermolide (i.e., NVP-XX-A-296), ABT-751 (Abbott, i.e., E-7010), Altorhyrtins (e.g., A altrilutin A and altrilutin C), spongistatins (e.g., spongistatin 1, spongistatin 2, spongistatin 3, spongistatin 4, spongistatin 5, spongistatin 6, spongistatin 7, spongistatin 8, and spongistatin 9), cemadotin hydrochloride (i.e., LU-103793 and NSC-D-669356), epothilones (e.g., epothilone A, epothilone B, epothilone C (i.e., desoxyepothilone A or dEpoA), epothilone D (i.e., KOS-862, dEpoB, and desoxyepothilone B), epothilone E, epothilone F, epothilone B N-oxide, epothilone A N-oxide, 16-aza-epothilone B, 21-aminoepothilone B (i.e., BMS-310705), 21-hydroxyepothilone D (i.e., desoxyepothilone F and dEpoF), 26-fluoroepothilone, auristatin PE (i.e., NSC-654663); Sobridotin (i.e., TZT-1027), LS-4559-P (Pharmacia, i.e., LS-4577), LS-4578 (Pharmacia, i.e., LS-477-P), LS-4477 (Pharmacia), LS-4559 (Pharmacia), RPR-112378 (Aventis), vincristine sulfate, DZ-3358 (Daiichi), FR-182877 (Fujisawa, i.e., WS-9885B), GS-164 (Takeda), GS-198 (Takeda), KAR-2 (Hungarian Academy of SAH-49960 (Lilly / Novartis), SDZ-268970 (Lilly / Novartis), AM-97 (Armad / Kyowa Hakko), AM-132 (Armad), AM-138 (Armad / Kyowa Hakko), IDN-5005 (Indena), Cryptophycin 52 (i.e., LY-355703), AC-7739 (Ajinomoto, i.e., AVE-8063A and CS-39.HCl), AC-7700 (Ajinomoto, i.e., AVE-8062, AVE-8062A, CS-39-L-Ser.HCl, and RPR-258062A), Vitilevuamide, Tubulysin A, Canadensol, Centaureidin (i.e., NSC-106969), T-138067 (Tularik, i.e., T-67, TL-138067, and TI-138067), COBRA-1 (Parker Hughes), H10 (Kansas State University), H16 (Kansas State University), Oncocidin A1 (i.e., BTO-956 and DIME), DDE-313 (Parker Hughes Institute), Fijianolide B, Laulimalide, SPA-2 (Parker Hughes Institute),Institute), SPA-1 (Parker Hughes Institute, i.e., SPIKET-P), 3-IAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-569), Narcosine (also known as NSC-5366), Nascapine, D-24851 (Asta Medica), A-105972 (Abbott), Hemiasterin, 3-BAABU (Cytoskeleton / Mt. Sinai School of Medicine, i.e., MF-191), TMPN (Arizona State University), Vanadocene acetylacetonate, T-138026 (Tularik), Monsatrol, Inanocin ) (i.e., NSC-698666), 3-IAABE (Cytoskeleton / Mt. Sinai School of Medicine), A-204197 (Abbott), T-607 (Tuiarik, i.e., T-900607), RPR-115781 (Aventis), eleutherobin series (e.g., desmethyleleutherobin, desaetyleleutherobin, isoeleutherobin A, and Z-eleutherobin), Caribaeoside, Caribaeolin, Halichondrin B, D-64131 (Asta Medica), D-68144 (Asta Medica), Diazonamide A, A-293620 (Abbott), NPI-2350 (Nereus), Taccalonolide A, TUB-245 (Aventis), A-259754 (Abbott), Diozostatin, (-)-Phenylahistin (i.e., NSCL-96F037), D-68838 (Asta Medica), D-68836 (AstaMedica), Myoseverin B, D-43411 (Zentaris, i.e., D-81862), A-289099 (Abbott), A-318315 (Abbott), HTI-286 (i.e., SPA-110, trifluoroacetate) (Wyeth), D-82317 (Zentaris), D-82318 (Zentaris), SC-12983 (NCI), Resverastatin sodium phosphate, BPR-OY-007 (National Health Research (Institutes, and SSR-250411 (Sanofi)), steroids (e.g., dexamethasone), finasteride, aromatase inhibitors, gonadotropin-releasing hormone agonists (GnRH), e.g., goserelin or leuprolide, corticosteroids (e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate, megestrol acetate, medroxyprogesterone acetate), estrogens (e.g., diethlystilbestrol, ethinyl estradiol), antiestrogens (e.g., cefotaxime ... tamoxifen), androgens (e.g., testosterone propionate, fluoxymesterone), antiandrogens (e.g., flutamide), immunostimulants (e.g., bacillus Calmette-Guerin (BCG), levamisole, interleukin 2, alpha-interferon, etc.), monoclonal antibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, and anti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33 monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonal antibody-Pseudomonas exotoxin conjugate, etc.), radioimmunotherapy (e.g., 111 In, 90 Y, or 131I, etc.), triptolide, homoharringtonine, dactinomycin, doxorubicin, epirubicin, topotecan, itraconazole, vindesine, cerivastatin, vincristine, deoxyadenosine, sertraline, pitavastatin, irinotecan, clofazimine, 5-nonyloxytryptamine, vemurafenib, dabrafenib, erlotinib, gefitinib, EGFR inhibitors, epidermal growth factor receptor (EGFR) targeted treatments or therapeutic agents (e.g., gefitinib (Iressa™), erlotinib (Tarceva™), cetuximab (Erbitux™), lapatinib (Tykerb™), panitumumab (Vec tibix™), vandetanib (Caprelsa™), afatinib / BIBW2992, CI-1033 / canertinib, neratinib / HKI-272, CP-724714, TAK-285, AST-1306, ARRY334543, ARRY-380, AG-1478, dacomitinib / PF299804, OSI-420 / desmetinib These include, but are not limited to, erlotinib, AZD8931, AEE788, pelitinib / EKB-569, CUDC-101, WZ8040, WZ4002, WZ3146, AG-490, XL647, PD153035, BMS-599626), sorafenib, imatinib, sunitinib, dasatinib, and the like.

[0053] An "imaging agent" is a compound that allows for the detection, imaging, and / or monitoring of the presence and / or progression of a condition, pathological disorder, and / or disease. Imaging agents include compounds that are detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Exemplary imaging agents include, but are not limited to, 32The radiolabels include P radionuclides, positron-emitting isotopes, fluorescent dyes, fluorophores, antibodies, bioluminescent molecules, chemiluminescent molecules, photoactive molecules, metals, electron-dense reagents, enzymes (e.g., those used in ELISA), magnetic contrast agents, quantum dots, nanoparticles (e.g., gold nanoparticles), biotin, digoxigenin, haptens, and proteins or other entities that can be made detectable, for example, by incorporating a radiolabel into an antibody or peptide specifically reactive with a target peptide. Any method known in the art for conjugating an antibody to a label can be utilized. Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, Alexa fluor®, Cy3, Cy5, BODIPY, and cyanine dyes. Exemplary radionuclides include fluorine-18, gallium-68, and copper-64. Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese. In some embodiments, the imaging moiety is a bioluminescent molecule.

[0054] As used herein, the term "targeting moiety" and its equivalents refer to a molecule that recognizes and binds to a desired molecule or structure on the surface of a cell or tissue, thus directing the complex with which it is associated to accumulate at the target site preferentially over non-target sites. Non-limiting examples of targeting moieties include antibodies, antibody fragments, binding proteins, binding peptides, receptors, and ligands for receptors. A specific binding partner of a targeting moiety means that the targeting moiety binds to a target molecule or structure with higher specificity (e.g., at least 2-fold, 5-fold, 10-fold, 100-fold, or more specificity) than it binds to a molecule or structure other than the target. In some embodiments, the targeting moiety is a member of a known binding pair (e.g., antibody / antigen, ligand / receptor, and lectin / carbohydrate). In some embodiments, a complex containing a targeting moiety accumulates in a target tissue distal to the administration site to a greater extent than a comparable complex lacking the targeting moiety.

[0055] The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragment thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively.

[0056] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 amino acids or more that is primarily responsible for antigen recognition. The "variable heavy chain", "V H The terms "variable light chain," "VH," or "VH" refer to the variable region of an immunoglobulin heavy chain, including Fv, scFv, dsFv, or Fab; L The term "VL" or "VL" refers to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv or Fab.

[0057] Examples of functional fragments of antibodies include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (light chain variable region), VH (heavy chain variable region), Fab, F(ab)2', and any combination thereof, or any other functional portion of an immunoglobulin peptide capable of binding to a target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th ed. 2001). As will be appreciated by those of skill in the art, various antibody fragments can be obtained by a variety of methods, such as digestion of complete antibodies with enzymes, e.g., pepsin; or de novo synthesis. Antibody fragments are often synthesized de novo, either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodology (e.g., single chain Fv), or those identified using phage display libraries (see, e.g., McCafferty et al., J. Immunol. 1999, 143:131-132). al., (1990) Nature 348:552). The term "antibody" also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies. Bivalent and bispecific molecules are described, for example, in Kostelny et al. (1992) J. Immunol. 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579, Hollinger et al. (1993), PNAS. USA 90:6444, Gruber et al. (1994) J Immunol. 152:5368, Zhu et al. (1997) Protein Sci. 6:781, Hu et al. (1996) Cancer Res. 56:3055, Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995) Protein Eng. 8:301.

[0058] A "chimeric antibody" is an antibody molecule in which (a) the antigen binding site (variable region) has been modified, substituted or exchanged so that the constant region or a portion thereof is linked to a constant region of a different or modified class, effector function, and / or species, or to an entirely different molecule that confers new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or a portion thereof has been modified, substituted or exchanged with a variable region having a different or modified antigen specificity. Preferred antibodies of the invention and for use according to the invention include humanized and / or chimeric monoclonal antibodies.

[0059] The term "virus" refers to submicroscopic infectious agents that replicate only in host cells. The genetic material of a virus is either DNA or RNA.

[0060] The term "adenovirus" or "Ad" refers to viruses of the Adenoviridae family. Adenoviruses are non-enveloped, icosahedral, medium-sized (90-100 nm in diameter), double-stranded DNA viruses found in a wide range of vertebrate hosts.

[0061] The term "viral tropism" refers to the ability of a virus to infect a particular cell type and ultimately result in a successful infection.

[0062] The term "tropism-modified AdV" refers to adenoviruses that have been genetically modified to have a different tropism from their native tropism. For example, the tropism of HAd5 is primarily mediated by the interaction of the fiber / knob with the major adenovirus receptor, the coxsackie-adenovirus receptor (CAR). Many different approaches have been used in the past few years to bypass the dependency on CAR for adenovirus entry and replication. For example, several groups have genetically modified the knob domain of the fiber in an attempt to change the target of Ad vectors. Genetic modifications include virions containing chimeric fiber proteins composed of the tail and shaft domains of the adenovirus type 5 (Ad5) fiber and the knob domain of Ad3, or the exchange of the fiber for another serotype, such as Ad11 and Ad35. Because Ad3 viruses can bind desmogleins and CD46, these viruses with Ad.5 / 3 chimeric structures can bind CAR, desmogleins, and CD46, increasing their ability to infect cells with reduced levels of either of these receptors. Different approaches include the incorporation of a COOH-terminal polylysine sequence or an integrin-binding RGD motif at the COOH-terminus of the Ad5 fiber. Examples of tropism-modified adenoviruses include, but are not limited to, Ad.5 / 3-CTV, Ad5 / 3-C-RGD, Ad5 / 3-HI-RGD, Ad5 / 3-E2F-d24, and Ad5.RGD.pK7.

[0063] The term "CTV" or "cancer terminator virus" refers to a virus of the Adenoviridae family modified by modern microbial engineering techniques. Non-limiting examples of cancer-terminating viruses include theranostic tripartite CTVs (TCTVs) that selectively express three genes from three different promoters (e.g., Ad.5-TCTV, Ad.5 / 3-TCTV), as described in Bhoopathi, P., et al. (2021) Cancers, 13(4), 85, which is incorporated herein by reference, and tropism-modified cancer terminator viruses (e.g., Ad.5 / 3-CTV; Ad.5 / 3-CTV-M7), as described in WO2014093270A1, which is incorporated herein by reference.

[0064] Compositions and Kits In some aspects, the present disclosure provides compositions and kits for use in treating a target tissue with an active agent. In some embodiments, the kit includes a first and a second microbubble composition and an active agent. In some embodiments, the first microbubble composition does not include an active agent. In some embodiments, the second microbubble composition includes an active agent. In some embodiments, the active agent is a therapeutic agent. In some embodiments, the active agent is an imaging agent.

[0065] In some embodiments, the first and second microbubble compositions are formulated for intravenous administration. In some embodiments, the first composition is formulated for intravenous administration. In some embodiments, the second composition is formulated for intravenous administration.

[0066] In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 50 microns, about 1 micron to about 25 microns, about 1 micron to about 10 microns, or about 1 micron to about 5 microns. In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns. In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 2.5 microns to about 4 microns.

[0067] In some embodiments, the first microbubbles have a mean or median diameter of about 1 micron. In some embodiments, the first microbubbles have a mean or median diameter of about 2 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 3 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 4 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 5 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 1 micron to about 2 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 1 micron to about 3 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 1 micron to about 4 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 2 microns to about 3 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 2 microns to about 4 microns. In some embodiments, the first microbubbles have a mean or median diameter of about 3 microns to about 4 microns, hi some embodiments, the first microbubbles have a mean or median diameter of about 3 microns to about 5 microns.

[0068] In some embodiments, the second microbubbles have a mean or median diameter of about 1 micron. In some embodiments, the second microbubbles have a mean or median diameter of about 2 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 3 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 4 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 5 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 1 micron to about 2 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 1 micron to about 3 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 1 micron to about 4 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 2 microns to about 3 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 2 microns to about 4 microns. In some embodiments, the second microbubbles have a mean or median diameter of about 3 microns to about 4 microns, hi some embodiments, the second microbubbles have a mean or median diameter of about 3 microns to about 5 microns.

[0069] In some embodiments, the first and / or second microbubble comprises a targeting moiety. In some embodiments, the first microbubble contains a targeting moiety. In some embodiments, the second microbubble contains a targeting moiety. In some embodiments, the first and second microbubbles both contain targeting moieties, which may be the same or different. In some embodiments, the first and second microbubbles contain targeting moieties that bind to the same target. In some embodiments, the targeting moiety specifically binds to a particular protein, a particular cell, or a particular tissue.

[0070] In some embodiments, the microbubbles comprise a targeting moiety. In some embodiments, non-limiting examples of targeting moieties include antibodies, antibody fragments, binding proteins, binding protein fragments, receptors, receptor fragments, receptor ligands, peptides, polypeptides, polynucleic acids, polysaccharides, lipids, polymers, tumor associated antigens, tissue specific antigens, vascular associated antigens, or any combination of these molecules. In some embodiments, the targeting moiety is an antibody. In some embodiments, the targeting moiety is an antibody fragment. In some embodiments, the targeting moiety is a binding protein. In some embodiments, the targeting moiety is a binding protein fragment. In some embodiments, the targeting moiety is a receptor. In some embodiments, the targeting moiety is a receptor fragment. In some embodiments, the targeting moiety is a receptor ligand. In some embodiments, the targeting moiety is a peptide. In some embodiments, the targeting moiety is a polypeptide. In some embodiments, the targeting moiety is a polynucleic acid. In some embodiments, the targeting moiety is a polysaccharide. In some embodiments, the targeting moiety is a lipid. In some embodiments, the targeting moiety is a polymer. In some embodiments, the targeting moiety is a tumor associated antigen. In some embodiments, the targeting moiety is a tissue-specific antigen. In some embodiments, the targeting moiety is a vascular-associated antigen. In some embodiments, the targeting moiety is any combination of molecules described herein.

[0071] In some embodiments, the tumor associated antigen is selected from HER2, CEA, PSA, MUC1, PSMA, CA19-9, EpCAM, GPC3, mesothelin (MSLN), or EGFR. In some embodiments, the tumor associated antigen is HER2. In some embodiments, the tumor associated antigen is CEA. In some embodiments, the tumor associated antigen is PSA. In some embodiments, the tumor associated antigen is MUC1. In some embodiments, the tumor associated antigen is PSMA. In some embodiments, the tumor associated antigen is CA19-9. In some embodiments, the tumor associated antigen is EpCAM. In some embodiments, the tumor associated antigen is GPC3. In some embodiments, the tumor associated antigen is mesothelin (MSLN). In some embodiments, the tumor associated antigen is EGFR.

[0072] In some embodiments, the tissue specific antigen is selected from glycoprotein 2, cadherin-9, GFAP, nestin, Tuj-1, thymocyte antigen 1 (Thy1) / CD90, desmin, Cx43. In some embodiments, the tissue specific antigen is glycoprotein 2. In some embodiments, the tissue specific antigen is cadherin-9. In some embodiments, the tissue specific antigen is GFAP. In some embodiments, the tissue specific antigen is nestin. In some embodiments, the tissue specific antigen is Tuj-1. In some embodiments, the tissue specific antigen is thymocyte antigen 1 (Thy1) / CD90. In some embodiments, the tissue specific antigen is desmin. In some embodiments, the tissue specific antigen is Cx43.

[0073] In some embodiments, the targeting moiety comprises a VEGF polypeptide or its single chain variant that binds to VEGF receptor.In some embodiments, the targeting moiety is a VEGF polypeptide.In some embodiments, the targeting moiety is a single chain variant of VEGF.Non-limiting examples of VEGF polypeptides and their single chain variants that are useful as targeting moieties are provided in US20080312410A1, which is incorporated herein by reference.

[0074] In certain embodiments, the targeting moiety comprises a VCAM1 antibody or an epitope-binding fragment thereof. In some embodiments, the targeting moiety is a VCAM1 antibody. In some embodiments, the targeting moiety is a VCAM1 epitope-binding fragment of an antibody.

[0075] In some embodiments, the targeting moiety comprises a PSMA antibody or an epitope-binding fragment thereof. In some embodiments, the targeting moiety is a PSMA antibody. In some embodiments, the targeting moiety is a PSMA epitope-binding fragment of an antibody.

[0076] In some embodiments, the active agent is an anti-cancer agent. Several suitable anti-cancer agents are available, non-limiting examples of which are provided herein. In some embodiments, the active agent includes a vector, such as a plasmid or a virus. Non-limiting examples of viruses are adenovirus, cancer terminator virus (CTV), lentivirus, retrovirus, herpes virus, vaccinia virus, genetically modified HIV, or vesicular stomatitis virus. In some embodiments, the virus is a cancer terminator virus (CTV). In some embodiments, the virus is a lentivirus. In some embodiments, the virus is a retrovirus. In some embodiments, the virus is a herpes virus. In some embodiments, the virus is a vaccinia virus. In some embodiments, the virus is a genetically modified human immunodeficiency virus (HIV). In some embodiments, the virus is a vesicular stomatitis virus. In some embodiments, the virus is an adenovirus. In some embodiments, the replication of the virus is under the control of a cancer-selective promoter.

[0077] In some embodiments, the active agent is a theranostic virus. In some embodiments, the theranostic virus is an adenovirus. In some embodiments, the adenovirus is genetically modified. In some embodiments, the adenovirus is a tropism-modified virus. In some embodiments, the adenovirus comprises a tissue-selective promoter. In some embodiments, the adenovirus comprises a cancer-selective promoter. In some embodiments, the adenovirus comprises a tissue-selective terminator. In some embodiments, the adenovirus comprises a cancer-selective terminator. In some embodiments, the adenovirus comprises multiple cancer-selective promoters. In some embodiments, the adenovirus comprises multiple tissue-selective promoters. In some embodiments, the adenovirus comprises multiple cancer-selective terminators. In some embodiments, the adenovirus comprises multiple tissue-selective terminators. In some embodiments, the adenovirus is a tropism-modified cancer terminator virus.

[0078] In some embodiments, the active agent includes a protein or a nucleic acid. In some embodiments, the active agent is a protein. In some embodiments, the active agent is a nucleic acid. In some embodiments, the active agent is a small hairpin RNA (shRNA). In some embodiments, the active agent is a small interfering RNA (siRNA). In some embodiments, the active agent is an antisense RNA. In some embodiments, the active agent is a lncRNA. In some embodiments, the active agent is DNA. In some embodiments, the DNA encodes the shRNA or antisense RNA.

[0079] In some embodiments, the active agent includes an MDA-9 / syntenin inhibitor. The MDA-9 / syntenin inhibitor can inhibit MDA-9 / syntenin activity at the polypeptide or polynucleotide level. In some embodiments, the active agent includes a nucleic acid sequence that encodes a portion of the MDA-9 / syntenin DNA sequence. In some embodiments, the polynucleotide encodes a small hairpin RNA (shRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a small interfering RNA (siRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a microRNA (miRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a messenger RNA (mRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, or an antisense RNA that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a small hairpin RNA (shRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a small interfering RNA (siRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a microRNA (miRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes an antisense RNA that is complementary to a portion of the MDA-9 / syntenin sequence. MDA-9 / syntenin inhibitor compositions and methods are disclosed in International Patent Publications WO 2017 / 120439 and WO 2021 / 127305, which are incorporated herein by reference.

[0080] In certain embodiments, the active agent comprises an MDA-9 / syntenin polynucleotide sequence corresponding to SEQ ID NO:19 and / or SEQ ID NO:20. In some embodiments, the active agent comprises the nucleic acid sequence SEQ ID NO:19. In some embodiments, the active agent comprises the nucleic acid sequence SEQ ID NO:20.

[0081] In some embodiments, the active agent is a virus that includes a polynucleotide that encodes a portion of the MDA-9 / syntenin nucleic acid sequence. In some embodiments, the polynucleotide encodes a small hairpin RNA (shRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a small interfering RNA (siRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a microRNA (miRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, a messenger RNA (mRNA) that is complementary to a portion of the MDA-9 / syntenin sequence, or an antisense RNA that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a small hairpin RNA (shRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a small interfering RNA (siRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In some embodiments, the polynucleotide encodes a microRNA (miRNA) that is complementary to a portion of the MDA-9 / syntenin sequence. In certain embodiments, the polynucleotide encodes an antisense RNA complementary to a portion of the MDA-9 / syntenin sequence.

[0082] In certain embodiments, the active agent includes a virus containing an MDA-9 / syntenin polynucleotide sequence corresponding to SEQ ID NO:19 and / or SEQ ID NO:20. In some embodiments, the active agent includes the nucleic acid sequence SEQ ID NO:19. In some embodiments, the active agent includes the nucleic acid sequence SEQ ID NO:20.

[0083] In certain embodiments, the active agent includes a polynucleotide encoding an active RNA or protein, such as an MDA-7 / IL-24 protein. In some embodiments, the active agent includes a polynucleotide encoding an MDA-7 / IL-24 fusion protein. In some embodiments, the active agent includes a polynucleotide encoding an MDA- / IL-24 sequence variant.

[0084] In certain embodiments, the active agent is a virus containing a polynucleotide encoding the MDA-7 / IL-24 protein. In some embodiments, the active agent is an adenovirus containing a polynucleotide encoding the MDA-7 / IL-24 protein. In some embodiments, the active agent is a virus containing a polynucleotide encoding the MDA-7 / IL-24 fusion protein. In some embodiments, the active agent is an adenovirus containing a polynucleotide encoding the MDA-7 / IL-24 fusion protein. In some embodiments, the active agent is a virus containing a polynucleotide encoding a MDA-7 / IL-24 sequence variant. In some embodiments, the active agent is an adenovirus containing a polynucleotide encoding a MDA-7 / IL-24 sequence variant.

[0085] In some embodiments, the active agent is an MDA-7 / IL-24 protein. In some embodiments, the MDA-7 / IL-24 protein is a fusion protein. In some embodiments, the MDA-7 / IL-24 protein is a sequence variant. In some embodiments, the MDA-7 / IL-24 protein includes an insulin signal peptide.

[0086] In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence of SEQ ID NO:4 or a variant thereof. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:4. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 90% identical to SEQ ID NO:4. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 95% identical to SEQ ID NO:4.

[0087] In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80% or at least about 80% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 85% or at least about 85% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 90% or at least about 90% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 95% or at least about 95% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 96% or at least about 96% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 97% or at least about 97% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 98% or at least about 98% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 99% or at least about 99% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 100% identical to SEQ ID NO:4. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, over 25, 50, 75, or 100 consecutive amino acids of SEQ ID NO:4.

[0088] In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence of SEQ ID NO:18 or a variant thereof. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:18. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 90% identical to SEQ ID NO:18. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 95% identical to SEQ ID NO:18.

[0089] In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80% or at least about 80% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 85% or at least about 85% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 90% or at least about 90% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 95% or at least about 95% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 96% or at least about 96% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 97% or at least about 97% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 98% or at least about 98% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 99% or at least about 99% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 100% identical to SEQ ID NO:18. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, over 25, 50, 75, or 100 consecutive amino acids of SEQ ID NO:18.

[0090] In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence of SEQ ID NO:3 or a variant thereof. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:3. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 90% identical to SEQ ID NO:3. In some embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about or at least about 95% identical to SEQ ID NO:3.

[0091] In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80% or at least about 80% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 85% or at least about 85% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 90% or at least about 90% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 95% or at least about 95% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 96% or at least about 96% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 97% or at least about 97% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 98% or at least about 98% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 99% or at least about 99% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 100% identical to SEQ ID NO:3. In certain embodiments, the MDA-7 / IL-24 protein comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, over 50, 75, 100, or 150 consecutive amino acids of SEQ ID NO:3.

[0092] In certain embodiments, the MDA-7 / IL-24 protein includes a lysine to arginine mutation corresponding to a K122R change relative to SEQ ID NO:2, a K73R change relative to SEQ ID NO:3, or a K19R change relative to SEQ ID NO:4. SEQ ID NO:18 is an example of an amino acid sequence having a K122R mutation relative to SEQ ID NO:2. However, because SEQ ID NO:18 represents a shorter sequence than SEQ ID NO:2, the position of the mutation relative to SEQ ID NO:18 is amino acid 19. Nevertheless, optimal alignment between the two sequences shows that SEQ ID NO:18 aligns with a portion of SEQ ID NO:2 that is 100% identical except for position 19 of SEQ ID NO:18, which corresponds to position 122 of SEQ ID NO:2. Additionally, SEQ ID NO:18 represents the result of a K19R mutation relative to SEQ ID NO:4, where the two sequences are completely identical except for the position of the mutation.

[0093] In some embodiments, the insulin signal peptide is human insulin signal peptide. In some embodiments, the insulin signal peptide comprises the amino acid sequence of SEQ ID NO:5 or a variant thereof. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 90% or at least about 90% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 95% or at least about 95% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide has 1, 2, 3, 4, or 5 amino acid substitutions relative to SEQ ID NO:5. In some embodiments, the insulin signal peptide is joined to the MDA-7 / IL-24 protein by a linker of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the linker is about 1-10, 2-8, 3-7, or 4-6 amino acids in length. In some embodiments, the insulin signal peptide is at the N-terminus of the fusion protein. In some embodiments, the insulin signal peptide is within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of the N-terminus of the fusion protein.

[0094] In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 80% or at least about 80% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 85% or at least about 85% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 90% or at least about 90% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 95% or at least about 95% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 96% or at least about 96% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 97% or at least about 97% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 98% or at least about 98% identical to SEQ ID NO:5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 99% or at least about 99% identical to SEQ ID NO: 5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 100% identical to SEQ ID NO: 5. In some embodiments, the insulin signal peptide comprises an amino acid sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 5, 10, 15, 20, or 24 consecutive amino acids of SEQ ID NO: 5.

[0095] In some embodiments, the inclusion of an insulin signal peptide in a fusion protein functions to increase mRNA transcript levels, protein levels, mature protein levels, mature protein fraction, secretion, and / or anti-cancer activity of the MDA-7 / IL-24 protein. In some embodiments, the function of the signal peptide is measured relative to a protein consisting of the amino acid sequence of SEQ ID NO:2 (or a polynucleotide or vector encoding same). In some embodiments, the function of the signal peptide is measured relative to a corresponding MDA-7 / IL-24 protein lacking the insulin signal peptide (or a polynucleotide or vector encoding same). In some embodiments, the mRNA transcript level, protein level, mature protein level, mature protein fraction, secretion, and / or anti-cancer activity of MDA-7 / IL-24 protein is increased by about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, or more, or at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, or more. In some embodiments, the increase is about 5-200%, 10-150%, 20-100%, or 40-75%. In some embodiments, the increase is at least about 5%. The relative change caused by insulin signal peptide can be measured in any suitable system, for example, in cultured cells as described herein.

[0096] In some embodiments, the polynucleotide encoding the fusion protein comprises a sequence described herein. In some embodiments, the polynucleotide comprises the nucleotide sequence of any one of SEQ ID NOs:6, 10-12, or 17, or a variant thereof. In some embodiments, the polynucleotide comprises a nucleotide sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs:6, 10-12, or 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to any one of SEQ ID NOs:6, 10-12, or 17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to any one of SEQ ID NOs:6, 10-12, or 17. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about or at least about 80% identical (eg, 90%, 95%, or 100% identical) to SEQ ID NO:17.

[0097] In some embodiments, the polynucleotide encoding the fusion protein comprises a nucleotide sequence that is about 80% or at least about 80% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 85% or at least about 85% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 96% or at least about 96% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 97% or at least about 97% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 98% or at least about 98% identical to SEQ ID NO:6. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 99% or at least about 99% identical to SEQ ID NO:6. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 100% identical to SEQ ID NO: 6. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about, or at least about, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 50, 100, 150, 200, 250, 300, or more contiguous nucleotides of SEQ ID NO: 6.

[0098] In some embodiments, the polynucleotide encoding the fusion protein comprises a nucleotide sequence that is about 80% or at least about 80% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 85% or at least about 85% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 96% or at least about 96% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 97% or at least about 97% identical to SEQ ID NO:10. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 98% or at least about 98% identical to SEQ ID NO:10. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 99% or at least about 99% identical to SEQ ID NO: 10. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 100% identical to SEQ ID NO: 10. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 50, 100, 150, 200, 250, 300, or more contiguous nucleotides of SEQ ID NO: 10.

[0099] In some embodiments, the polynucleotide encoding the fusion protein comprises a nucleotide sequence that is about 80% or at least about 80% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 85% or at least about 85% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 96% or at least about 96% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 97% or at least about 97% identical to SEQ ID NO:11. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 98% or at least about 98% identical to SEQ ID NO:11. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 99% or at least about 99% identical to SEQ ID NO:11. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 100% identical to SEQ ID NO:11. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 50, 100, 150, 200, 250, 300, or more contiguous nucleotides of SEQ ID NO:11.

[0100] In some embodiments, the polynucleotide encoding the fusion protein comprises a nucleotide sequence that is about 80% or at least about 80% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 85% or at least about 85% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 96% or at least about 96% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 97% or at least about 97% identical to SEQ ID NO:12. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 98% or at least about 98% identical to SEQ ID NO:12. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 99% or at least about 99% identical to SEQ ID NO: 12. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 100% identical to SEQ ID NO: 12. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 50, 100, 150, 200, 250, 300, or more contiguous nucleotides of SEQ ID NO: 12.

[0101] In some embodiments, the polynucleotide encoding the fusion protein comprises a nucleotide sequence that is about 80% or at least about 80% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 85% or at least about 85% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 90% or at least about 90% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 95% or at least about 95% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 96% or at least about 96% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 97% or at least about 97% identical to SEQ ID NO:17. In some embodiments, the polynucleotide comprises a nucleotide sequence that is about 98% or at least about 98% identical to SEQ ID NO:17. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 99% or at least about 99% identical to SEQ ID NO: 17. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 100% identical to SEQ ID NO: 17. In certain embodiments, the polynucleotide comprises a nucleotide sequence that is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical, or at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical over 50, 100, 150, 200, 250, 300, or more contiguous nucleotides of SEQ ID NO: 17.

[0102] In some embodiments, the MDA-7 / IL-24 protein retains biological activity. As a cytokine and a member of the IL-10 cytokine gene family, MDA-7 / IL-24 transmits negative signals through a receptor dimer consisting of R1-type and R2-type receptors (IL-20R1 and IL-20R2; IL-22R1 and IL-20R2; or a unique receptor pair, IL-20R1 and IL-22R1) to activate downstream signaling events. Assays for measuring such activity are available (see, e.g., WO2018089995A1). In some embodiments, the MDA-7 / IL-24 protein is a variant, homolog, or isoform that retains at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, or more of the biological activity of the MDA-7 / IL-24 protein of SEQ ID NO:2 or SEQ ID NO:3. In some embodiments, the MDA-7 / IL-24 protein retains at least 80% of the biological activity of the MDA-7 / IL-24 protein of SEQ ID NO: 3. In some embodiments, the MDA-7 / IL-24 protein retains at least 90% of the biological activity of the MDA-7 / IL-24 protein of SEQ ID NO: 3. In some embodiments, the MDA-7 / IL-24 protein is capable of activating the IL-20 / IL-22 receptor complex in a cancer cell of a subject or a reference cell line (e.g., DU-145 cells).

[0103] In some embodiments, the native signal peptide of the MDA-7 / IL-24 protein is recombinantly replaced with an insulin signal peptide. In such cases, the polynucleotide does not encode the native signal peptide of the MDA-7 / IL-24 protein. In some embodiments, the polynucleotide does not encode amino acids 1-49 of SEQ ID NO:2. In some embodiments, the MDA-7 / IL-24 protein expressed from the polynucleotide, after intracellular processing for secretion, is a mature MDA-7 / IL-24 protein that lacks the insulin signal peptide that was originally translated with the MDA-7 / IL-24 protein. In some embodiments, the MDA-7 / IL-24 protein is a truncated version of the MDA-7 / IL-24 protein that retains biological activity. For example, the MDA-7 / IL-24 protein may lack the first 54 amino acids of SEQ ID NO:3.

[0104] In some aspects, the disclosure provides a vector comprising any of the polynucleotides described herein. In some embodiments, the vector is an expression vector, such that the inserted polynucleotide is operably linked to a control (e.g., transcription and / or translation control) sequence. In this context, the term "operably linked" means that the polynucleotide of interest is inserted into the vector, such that the control sequence in the vector, when expressed, for example, in a cell, performs its intended function of controlling the transcription and / or translation of the polynucleotide. The vector and expression control sequence are selected to be compatible with the intended host or target cell. Examples of control sequences include, but are not limited to, promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Non-limiting examples of control sequences for use in expressing proteins in mammalian cells include promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP) and polyoma); non-viral control sequences, such as the ubiquitin promoter or the β-globin promoter; and sequences derived from different sources, such as the SRα promoter system, which contains sequences derived from the SV40 early promoter and sequences derived from the human T-cell leukemia virus type 1 long terminal repeat.

[0105] In some embodiments, the vector is a plasmid vector. In some embodiments, the vector is a viral vector, such as an adenovirus vector (Ad), an adeno-associated viral vector (AAV), a lentivirus vector, a retrovirus vector, a herpes virus, a vaccinia virus, a genetically modified HIV, a vesicular stomatitis virus, or other suitable viral vector. In some embodiments, the virus is an adenovirus. A variety of suitable adenoviruses are available. Non-limiting examples of adenoviruses that can be used in expressing MDA-7 / IL-24 protein include those described in WO2018089995A1, WO2017062708A1, US20180243382A1, US20160008413A1, and Dash et al., Cancer Res 2014;74:563-74. In some embodiments, the virus (e.g., adenovirus) is a replication-deficient adenovirus, so that viral replication in target cells is reduced or eliminated compared to the corresponding wild-type virus.In some embodiments, viral replication is under the control of a cancer-specific promoter, so that viral replication is higher in cancer cells than in non-cancer cells.A non-limiting example of a cancer-specific promoter is the cancer-selective Progression Elevated Gene-3 (PEG-3) promoter.Adenovirus replication can be made cancer-specific, for example, by placing E1A and E1B genes under the control of the PEG-3 promoter.

[0106] In some aspects, the present disclosure provides a kit for use in the methods disclosed herein. In some embodiments, the use of the kit includes administering a first microbubble composition, administering a first ultrasound wave to a target tissue that destroys the first microbubbles, administering a second microbubble composition after administering the first ultrasound wave, and administering a second ultrasound wave to a target tissue that destroys the second microbubbles. In some embodiments, the use of the kit includes administering a first microbubble composition, and administering a first ultrasound wave to a target tissue that destroys the first microbubbles. In some embodiments, the use of the kit includes administering a second microbubble composition, and administering a second ultrasound wave to a target tissue that destroys the second microbubbles.

[0107] In certain embodiments, administration of the second microbubble composition is within about 60 minutes, within about 30 minutes, within about 10 minutes, or within about 5 minutes of administration of the first microbubble composition. In some embodiments, administration is within about 60 minutes. In some embodiments, administration is within about 30 minutes. In some embodiments, administration is within about 10 minutes. In some embodiments, administration is within about 5 minutes.

[0108] In some embodiments, the target tissue comprises a tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the tumor is located in the brain, breast, lung, skin, gastrointestinal system, bone, abdominal cavity, pancreas, head, neck, oral cavity, spinal cord, or intestine of the subject. In some embodiments, the tumor is located in the brain. In some embodiments, the tumor is located in the breast. In some embodiments, the tumor is located in the lung. In some embodiments, the tumor is located in the gastrointestinal system. In some embodiments, the tumor is located in the bone. In some embodiments, the tumor is located in the abdominal cavity. In some embodiments, the tumor is located in the pancreas. In some embodiments, the tumor is located in the intestine. In some embodiments, the tumor is located in the oral cavity. In some embodiments, the tumor is located in the spinal cord. In some embodiments, the tumor is located in the head. In some embodiments, the tumor is located in the neck. In some embodiments, the tumor is located in the skin or on the skin.

[0109] In some embodiments, the tumor includes glioblastoma, melanoma, breast cancer, bone cancer, pancreatic cancer, liver cancer, colon cancer, oral cancer, head and neck cancer, spinal cancer, neuroblastoma, kidney cancer, or lung cancer. In some embodiments, the tumor is a glioblastoma. In some embodiments, the tumor is a melanoma. In some embodiments, the tumor is a breast cancer. In some embodiments, the tumor is a bone cancer. In some embodiments, the tumor is a pancreatic cancer. In some embodiments, the tumor is a liver cancer. In some embodiments, the tumor is a colon cancer. In some embodiments, the tumor is an oral cancer. In some embodiments, the tumor is a head and neck cancer. In some embodiments, the tumor is a spinal cancer. In some embodiments, the tumor is a neuroblastoma. In some embodiments, the tumor is a kidney cancer. In some embodiments, the tumor is a lung cancer.

[0110] In some embodiments, the target tissue is located in the brain, pancreas, stomach, intestine, bone, skin, oral cavity, head, neck, spinal cord, lung, kidney, or liver of a subject. In some embodiments, the target tissue is located in the brain. In some embodiments, the target tissue is located in the pancreas. In some embodiments, the target tissue is located in the stomach. In some embodiments, the target tissue is located in the intestine. In some embodiments, the target tissue is located in the bone. In some embodiments, the target tissue is located in the skin. In some embodiments, the target tissue is located in the oral cavity. In some embodiments, the target tissue is located in the head. In some embodiments, the target tissue is located in the neck. In some embodiments, the target tissue is located in the spinal cord. In some embodiments, the target tissue is located in the lung. In some embodiments, the target tissue is located in the kidney. In some embodiments, the target tissue is located in the liver.

[0111] In some embodiments, the administration of the first microbubble composition is in an amount effective to increase delivery of the active agent to the target tissue.

[0112] In some embodiments, the administration of the first microbubble composition is in an amount effective to increase delivery of the active agent across the blood-brain barrier.

[0113] method In some aspects, the present disclosure provides a method for administering an active agent to a target tissue. In some embodiments, the method comprises administering one or more compositions described herein, such as one or more compositions of a kit described herein. In some embodiments, the active agent is a therapeutic agent or an imaging agent. In some embodiments, the method comprises administering a first microbubble composition to a subject, the first microbubble composition containing a first microbubble and not containing an active agent; administering a first ultrasound wave to the target tissue, which destroys the first microbubbles; administering a second microbubble composition to the subject after administering the first ultrasound wave, the second microbubble composition containing a second microbubble complexed with an active agent; and administering a second ultrasound wave to the target tissue, which destroys the second microbubbles and releases the active agent to the target tissue.

[0114] In certain embodiments, the method includes administering the second microbubble composition within about 60 minutes, within about 30 minutes, within about 10 minutes, and within about 5 minutes of administration of the first microbubble composition. In some embodiments, the second microbubble composition is administered within about 60 minutes of administration of the first microbubble composition. In some embodiments, the second microbubble composition is administered within about 30 minutes of administration of the first microbubble composition. In some embodiments, the second microbubble composition is administered within about 10 minutes of administration of the first microbubble composition. In some embodiments, the second microbubble composition is administered within about 5 minutes of administration of the first microbubble composition.

[0115] In certain embodiments, the method includes administering the first microbubble composition, the second microbubble composition, or both, by intravenous administration. In some embodiments, the administration of the first microbubble composition is by intravenous administration. In some embodiments, the administration of the second microbubble composition is by intravenous administration. In some embodiments, the administration of the first microbubble composition and the second microbubble composition are both by intravenous administration.

[0116] In some embodiments, the first and / or second microbubbles are microbubbles as described herein, e.g., with respect to the first and / or second microbubbles of the kits described herein. In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 50 microns, about 1 micron to about 25 microns, about 1 micron to about 10 microns, or about 1 micron to about 5 microns. In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns. In some embodiments, the first and / or second microbubbles have a mean or median diameter of about 2.5 microns to about 4 microns.

[0117] In some embodiments, the first and / or second microbubble comprises a targeting moiety. In some embodiments, the first microbubble contains a targeting moiety. In some embodiments, the second microbubble contains a targeting moiety. In some embodiments, the first and second microbubbles both contain targeting moieties, which may be the same or different. In some embodiments, the first and second microbubbles contain targeting moieties that bind to the same target. In some embodiments, the targeting moiety specifically binds to a particular protein, a particular cell, or a particular tissue.

[0118] In some embodiments, the targeting moiety comprises a VEGF polypeptide or its single chain variant that binds to VEGF receptor.In some embodiments, the targeting moiety is a VEGF polypeptide.In some embodiments, the targeting moiety is a single chain variant of VEGF.Non-limiting examples of VEGF polypeptides and their single chain variants that are useful as targeting moieties are provided in US20080312410A1, which is incorporated herein by reference.

[0119] In certain embodiments, the targeting moiety comprises a VCAM1 antibody or an epitope-binding fragment thereof. In some embodiments, the targeting moiety is a VCAM1 antibody. In some embodiments, the targeting moiety is a VCAM1 epitope-binding fragment of an antibody.

[0120] In some embodiments, the targeting moiety comprises a PSMA antibody or an epitope-binding fragment thereof. In some embodiments, the targeting moiety is a PSMA antibody. In some embodiments, the targeting moiety is a PSMA epitope-binding fragment of an antibody.

[0121] In some embodiments, the target tissue comprises a tumor. In some embodiments, the tumor is a metastatic tumor. In some embodiments, the tumor is located in the brain, breast, lung, skin, gastrointestinal system, bone, abdominal cavity, pancreas, head, neck, oral cavity, spinal cord, or intestine of the subject. In some embodiments, the tumor is located in the brain. In some embodiments, the tumor is located in the breast. In some embodiments, the tumor is located in the lung. In some embodiments, the tumor is located in the gastrointestinal system. In some embodiments, the tumor is located in the bone. In some embodiments, the tumor is located in the abdominal cavity. In some embodiments, the tumor is located in the pancreas. In some embodiments, the tumor is located in the intestine. In some embodiments, the tumor is located in the oral cavity. In some embodiments, the tumor is located in the spinal cord. In some embodiments, the tumor is located in the head. In some embodiments, the tumor is located in the neck. In some embodiments, the tumor is located in the skin or on the skin.

[0122] In some embodiments, the tumor comprises glioblastoma, melanoma, breast cancer, bone cancer, pancreatic cancer, liver cancer, colon cancer, oral cancer, head and neck cancer, spinal cancer, neuroblastoma, kidney cancer, or lung cancer. In some embodiments, the tumor is glioblastoma. In some embodiments, the tumor is melanoma. In some embodiments, the tumor is breast cancer. In some embodiments, the tumor is bone cancer. In some embodiments, the tumor is pancreatic cancer. In some embodiments, the tumor is liver cancer. In some embodiments, the tumor is colon cancer. In some embodiments, the tumor is oral cancer. In some embodiments, the tumor is head and neck cancer. In some embodiments, the tumor is spinal cancer. In some embodiments, the tumor is neuroblastoma. In some embodiments, the tumor is kidney cancer. In some embodiments, the tumor is lung cancer.

[0123] In some embodiments, the target tissue is located in the brain, pancreas, stomach, intestine, bone, skin, oral cavity, head, neck, spinal cord, lung, kidney, or liver of a subject. In some embodiments, the target tissue is located in the brain. In some embodiments, the target tissue is located in the pancreas. In some embodiments, the target tissue is located in the stomach. In some embodiments, the target tissue is located in the intestine. In some embodiments, the target tissue is located in the bone. In some embodiments, the target tissue is located in the skin. In some embodiments, the target tissue is located in the oral cavity. In some embodiments, the target tissue is located in the head. In some embodiments, the target tissue is located in the neck. In some embodiments, the target tissue is located in the spinal cord. In some embodiments, the target tissue is located in the lung. In some embodiments, the target tissue is located in the kidney. In some embodiments, the target tissue is located in the liver.

[0124] In some embodiments, the target tissue is in a subject. In some embodiments, the subject is being treated for cancer. In some embodiments, the subject previously had cancer. In some embodiments, the subject previously went into remission from cancer.

[0125] In some embodiments, the first microbubble composition is administered in an amount effective to increase delivery of the active agent to the target tissue, for example, after the first administration of the first microbubble composition and the first ultrasound in the target tissue. In some embodiments, the first microbubble composition is administered in an amount effective to increase delivery of the active agent through the blood-brain barrier, for example, after the first administration of the first microbubble composition and the first ultrasound in the target tissue. In some embodiments, the first microbubble composition is administered in an amount effective to increase delivery of the active agent to the pancreas, for example, after the first administration of the first microbubble composition and the first ultrasound in the target tissue. In some embodiments, the increase in delivery is compared to administration of the second microbubble composition and the second ultrasound in the absence of administration of the first microbubble composition and the first ultrasound. In some embodiments, administration of a first microbubble composition and administration of a first ultrasound increases delivery of an active agent delivered by administration of a second microbubble composition and a second ultrasound by about or more than about 25%, 50%, 100%, 200%, 300%, or more. In some embodiments, delivery is increased by about or more than about 50%. In some embodiments, delivery is increased by about or more than about 100%. In some embodiments, delivery is increased by about or more than about 200%.

[0126] In some embodiments, the active agent is an active agent described herein, for example, with respect to the kits described herein. In some embodiments, the active agent comprises a protein or a nucleic acid. In some embodiments, the active agent is a protein. In some embodiments, the active agent is a nucleic acid. In some embodiments, the active agent is a nucleic acid. In some embodiments, the active agent is a small hairpin RNA (shRNA). In some embodiments, the active agent is a small interfering RNA (siRNA). In some embodiments, the active agent is an antisense RNA. In some embodiments, the active agent is a DNA. In some embodiments, the DNA encodes an shRNA or an antisense RNA. In some embodiments, the active agent is an anti-cancer agent. In some embodiments, the active agent inhibits expression of MDA-9 / syntenin (MDA-9 / syntenin inhibitor). In some embodiments, the active agent is an MDA-7 / IL-24 protein (e.g., a fusion protein) or a variant thereof, a polynucleotide encoding same, or a vector comprising such a polynucleotide, non-limiting examples of which are described herein. In some embodiments, the active agent comprises a virus, e.g., an adenovirus. In some embodiments, viral replication is under the control of a cancer-specific promoter.

[0127] In some embodiments, administration of a composition comprising MDA-7 / IL-24 protein includes administration to a target tissue, e.g., a tumor, a site where a tumor has been surgically removed, and / or a bone of a subject. In some embodiments, administration to a target tissue includes injection into or near the target tissue, or local application to the target tissue. In some embodiments, the composition is delivered distal to the target tissue, but is formulated to transport the MDA-7 / IL-24 protein (or a polynucleotide or vector encoding the protein) to the target tissue. In some embodiments, a moiety that transports to a particular tissue, e.g., cancer tissue and / or bone tissue, is complexed with the MDA-7 / IL-24 protein (or a polynucleotide or vector encoding the protein). Complexation may be direct with the targeting moiety, e.g., by covalent or non-covalent interactions. Complexation may be indirect, such that the MDA-7 / IL-24 protein (or a polynucleotide or vector encoding the protein) and the targeting moiety are separated by one or more other molecules that join the two via covalent or non-covalent interactions. In general, a targeting moiety is a moiety that can bind or otherwise associate with a biological entity (e.g., a membrane component, a cell surface receptor, a cell-specific membrane antigen, etc.) with a higher affinity than one or more non-target biological entities (e.g., a cell surface component of one or more different tissues). A targeting moiety typically allows a cargo (e.g., a polynucleotide, a vector, or a protein) to localize to a specific targeting site to a higher degree than elsewhere in the subject's body, or to a higher degree than would be achieved in the absence of the targeting moiety. Non-limiting examples of targeting moieties include antibodies, antigen-binding antibody fragments, aptamers, peptides, hormones, growth factors, ligands (e.g., receptor ligands), small molecules, and the like. Illustrative examples of targeting moieties that transport to bone are described in US20120028350A1, US20160052968A1, US20040038946A1, and US20180208650A1.In some embodiments, the microbubbles are conjugated to a targeting moiety that transports the microbubbles to a particular tissue, for example, cancer tissue, cancer vasculature, or bone tissue.

[0128] In some embodiments, administration of a composition comprising an MDA-9 / syntenin inhibitor includes administration to a target tissue, e.g., a tumor, a site where a tumor has been surgically removed, and / or the pancreas of a subject. In some embodiments, administration to a target tissue includes injection into or near the target tissue, or local application to the target tissue. In some embodiments, the composition is delivered distal to the target tissue, but is formulated to transport the MDA-9 / syntenin inhibitor to the target tissue. In some embodiments, a moiety that transports to a particular tissue, e.g., cancer tissue and / or pancreatic tissue, is complexed with the MDA-9 / syntenin inhibitor. Complexation may be direct with the targeting moiety, by covalent or non-covalent interactions. Complexation may be indirect, such that the MDA-9 / syntenin inhibitor and the targeting moiety are separated by one or more other molecules that join the two together, by covalent or non-covalent interactions. In general, a targeting moiety is a moiety that can bind or otherwise associate with a biological entity (e.g., a membrane component, a cell surface receptor, a cell-specific membrane antigen, etc.) with a higher affinity than one or more non-target biological entities (e.g., a cell surface component of one or more different tissues). A targeting moiety typically allows a cargo (e.g., a polynucleotide, a vector, or a protein) to localize to a specific targeting site to a higher degree than elsewhere in the subject's body, or to a target site to a higher degree than would be achieved in the absence of the targeting moiety. Non-limiting examples of targeting moieties include antibodies, antigen-binding antibody fragments, aptamers, peptides, hormones, growth factors, ligands (e.g., receptor ligands), small molecules, and the like. In some embodiments, microbubbles are complexed with a targeting moiety that transports the microbubbles to a specific tissue, e.g., cancer tissue, cancer vasculature, or pancreatic tissue.

[0129] In some embodiments, the method of administering an active agent or imaging agent to a target tissue includes a third microbubble composition, hi some embodiments, the third microbubble composition is administered after the administration of the second ultrasound.

[0130] In some embodiments, the method of administering an active agent or imaging agent to a target tissue includes a fourth microbubble composition, hi some embodiments, the fourth microbubble composition is administered after the administration of a third ultrasound wave.

[0131] In some embodiments, the third microbubble composition comprises a second active agent. In some embodiments, the fourth microbubble composition comprises a third active agent. In some embodiments, the second active agent is the same as the first active agent. In some embodiments, the third active agent is the same as the first active agent. In some embodiments, the third active agent is the same as the second active agent.

[0132] In some embodiments, the third microbubble composition comprises a second imaging agent. In some embodiments, the fourth microbubble composition comprises a third imaging agent. In some embodiments, the second imaging agent is the same as the first imaging agent. In some embodiments, the third imaging agent is the same as the first imaging agent. In some embodiments, the third imaging agent is the same as the second imaging agent.

[0133] In some embodiments, the microbubble composition is complexed with multiple active agents. In some embodiments, the microbubble composition is complexed with multiple imaging agents. In some embodiments, the microbubble composition is complexed with two active agents. In some embodiments, the microbubble composition is complexed with two imaging agents. In some embodiments, the microbubble composition is complexed with an active agent and an imaging agent.

[0134] In one embodiment, there is a method of treating a subject in need thereof comprising administering an additional active agent or imaging agent that is not complexed to the microbubbles.

[0135] In some embodiments, there is a method of treating a subject in need thereof, comprising administering an additional anti-cancer agent that is not complexed with microbubbles. In some embodiments, the anti-cancer agent is selected from chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. In some embodiments, the anti-cancer agent is chemotherapy. In some embodiments, the anti-cancer agent is hormonal therapy. In some embodiments, the anti-cancer agent is radiation therapy. In some embodiments, the anti-cancer agent is immunotherapy.

[0136] In some embodiments, the anti-cancer agent is selected from, but is not limited to, an alkylating agent, an antimetabolite, a natural product, a chemotherapeutic agent, a hormone, a polypeptide, or a small molecule having utility in methods of treating cancer. In some embodiments, the anti-cancer agent is an alkylating agent. In some embodiments, the anti-cancer agent is an antimetabolite. In some embodiments, the anti-cancer agent is a natural product. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is a hormone. In some embodiments, the anti-cancer agent is a polypeptide. In some embodiments, the anti-cancer agent is a small molecule having utility in methods of treating cancer.

[0137] In some embodiments, the anti-cancer agent is gemcitabine. In some embodiments, the anti-cancer agent is temozolomide.

[0138] In some embodiments, the anticancer agent further comprises a pharma- ceutically acceptable excipient.

[0139] Experimental protocol Complexation of microbubbles with active / imaging agents: Microbubbles (MB) are reconstituted with a buffer solution (e.g., PBS) containing an active agent (AA). MB and active agent are incubated at a temperature (e.g., 4°C) for a period of time (e.g., 2 hours). After incubation, non-encapsulated active agent is inactivated and / or removed. MB / AA can be added to an appropriate buffer solution (e.g., PBS) for administration to a subject.

[0140] Generation of focused ultrasound (FUS) waves and treatment of mice: Focused ultrasound (FUS) utilizes the same concept of acoustic wave propagation as more widely known ultrasound diagnostic applications. FUS may utilize a concave transducer with a single geometric focus or use a phased array to electronically steer the ultrasound. The output of the FUS is delivered during sonication to induce mechanical effects, thermal effects, or both. A FUS transducer (e.g., 2.25 MHz, 0.50 inch element diameter, standard case style, straight UHF connector, purchased from Olympus America Inc.) is used to perform sonication immediately (e.g., 15 seconds) after bubble administration. The transducer is driven by a function generator (e.g., AGI-E4436B, Agilent Technologies, Palo Alto, CA, USA) through a power amplifier (e.g., E&I 3100LA, ENI Inc., Rochester, NY, USA). A cone filled with degassed distilled water is attached to the transducer system. FUS is applied to the subject (eg, 3.5 mV, 10 dB, 1 MHz) after the microbubbles are administered.

[0141] Intravenous FUS-DMB Targeted Study: Diluted microbubbles (without active agent or imaging agent) are injected into a subject (e.g., via the tail vein of a mouse) and allowed to circulate for a period of time (e.g., 15 seconds). After circulation, the subject is sonicated (FUS) for a period of time (e.g., 1 minute) in selected regions (e.g., brain, pancreas, liver, kidney). Optionally, a second microbubble aliquot (with active agent or imaging agent) is injected into the subject, the bubbles are allowed to circulate, and then sonicated for a period of time (e.g., 1 minute). Optionally, a third microbubble aliquot (with active agent or imaging agent) is injected into the subject, the bubbles are allowed to circulate, and then sonicated for a period of time (e.g., 1 minute). Optionally, a fourth microbubble aliquot (with active agent or imaging agent) is injected into the subject, the bubbles are allowed to circulate, and then sonicated for a period of time (e.g., 1 minute). Optionally, any number of microbubble aliquots (with active agent or imaging agent) can be injected into a subject using the protocols described herein (more than 4). Subjects may be imaged using an IVIS imager and followed for survival, toxicity, or efficacy analysis.

[0142] Intracranial FUS-DMB Targeted Study - Tumor Formation: The subject is anesthetized and immobilized in a stereotaxic frame. A needle attached to a syringe is inserted into the right basal ganglia, which has ample space for tumor cell accumulation. The entry point is in the skull near the bregma. Intracerebral injection of cancer cells (e.g., 30,000 glioma cells) can initiate tumor formation. The skull opening is sealed with sterile bone wax, and the skin incision is closed using sterile surgical staples.

[0143] In some aspects, the disclosure provides for the use of a composition or kit described herein in the manufacture of a medicament for the treatment of cancer in a subject in need thereof. In some embodiments, the composition comprises a polynucleotide, vector, cell, or composition described herein.

[0144] array SEQ ID NO:1 (nucleotide sequence encoding the MDA-7 / IL-24 protein) TIFF2024503090000001.tif77170

[0145] SEQ ID NO:2 (amino acid sequence of MDA-7 / IL-24 protein) TIFF2024503090000002.tif26170

[0146] SEQ ID NO:3 (amino acid sequence of MDA-7 / IL-24 protein) TIFF2024503090000003.tif19170

[0147] SEQ ID NO:4 (Amino acid sequence of MDA-7 / IL-24 protein) TIFF2024503090000004.tif11169

[0148] SEQ ID NO:5 (amino acid sequence of insulin signal peptide (sp)) TIFF2024503090000005.tif3128

[0149] SEQ ID NO:6 (encoding the insulin (sp)-MDA-7 sequence) TIFF2024503090000006.tif83166

[0150] SEQ ID NO:7 (encoding the Flt3(sp)-MDA-7 sequence) TIFF2024503090000007.tif84166

[0151] SEQ ID NO:8 (encoding the BM40(sp)-MDA-7 sequence) TIFF2024503090000008.tif76166

[0152] SEQ ID NO:9 (encoding the IL-2(sp)-MDA-7 sequence) TIFF2024503090000009.tif77166

[0153] SEQ ID NO:10 (encoding the MDA-7(K22R) sequence) TIFF2024503090000010.tif91165

[0154] SEQ ID NO:11 (encoding insulin (sp)-MDA-7-K122R) TIFF2024503090000011.tif84166

[0155] SEQ ID NO:12 (encoding insulin (sp)-M4) TIFF2024503090000012.tif62166

[0156] SEQ ID NO:13 (encoding IL2(sp)-M4) TIFF2024503090000013.tif47166

[0157] SEQ ID NO:14 (encoding Flt-3(sp)-M4) TIFF2024503090000014.tif55165

[0158] SEQ ID NO:15 (encoding MDA-7(sp)-M4) TIFF2024503090000015.tif69166

[0159] SEQ ID NO:16 (encoding Flt-3(sp)-M4(K122R) TIFF2024503090000016.tif55165

[0160] SEQ ID NO:17 (encoding insulin (sp)-M4(K122R)) TIFF2024503090000017.tif63166

[0161] SEQ ID NO:18 (Amino acid sequence of MDA-7 / IL-24 protein (K122R)) TIFF2024503090000018.tif11168

[0162] SEQ ID NO:19 (specific hairpin short interfering (siRNA) oligonucleotide used against Ad.shMDA-9), sense strand TIFF2024503090000019.tif18169

[0163] SEQ ID NO:20 (specific hairpin short interfering (siRNA) oligonucleotide used against Ad.shMDA-9), antisense strand TIFF2024503090000020.tif18170

[0164] P form 1. A method for administering an active agent to a target tissue, the active agent being a therapeutic agent or an imaging agent, the method comprising: (a) administering to a subject a first microbubble composition, the first microbubble composition comprising first microbubbles and no active agent; (b) administering a first ultrasound wave to the target tissue, the first ultrasound wave destroying the first microbubbles; (c) administering to the subject a second microbubble composition after administration of the first ultrasound, the second microbubble composition comprising the second microbubble composition complexed with an active agent; and (d) administering a second ultrasound wave to the target tissue, which destroys the second microbubbles and releases the active agent into the target tissue: A method comprising:

[0165] 2. The method of embodiment P1, wherein the second microbubble composition is administered within about 60 minutes, 30 minutes, 10 minutes, or 5 minutes of administration of the first microbubble composition.

[0166] 3. The method of embodiment P1 or P2, wherein the step of administering the first microbubble composition, the second microbubble composition, or both comprises intravenous administration.

[0167] 4. The method of any one of aspects P1-P3, wherein the first and / or second microbubbles have a mean or median diameter of from about 1 micron to about 5 microns, or from about 2.5 microns to about 4 microns.

[0168] 5. The method of any one of embodiments P1-P4, wherein the first and / or second microbubbles comprise a targeting moiety.

[0169] 6. The method of embodiment P5, wherein the targeting moiety comprises (a) a VEGF polypeptide or a single chain variant thereof, (b) a VCAM1 antibody or an epitope-binding fragment thereof, or (c) a PSMA antibody or an epitope-binding fragment thereof.

[0170] 7. The method of any one of aspects P1-P8, wherein the target tissue comprises a tumor.

[0171] 8. The method of embodiment P9, wherein the tumor is a metastatic tumor.

[0172] 9. The method of embodiment P9 or P10, wherein the tumor is located in the brain, breast, lung, gastrointestinal system, bone, abdominal cavity, pancreas, or intestine of the subject.

[0173] 10. The method of any one of aspects P9-P11, wherein the tumor comprises glioblastoma, melanoma, breast cancer, or lung cancer.

[0174] 11. The method of any one of aspects P1-P12, wherein the target tissue is located in the brain, pancreas, stomach, intestine, bone, or liver of the subject.

[0175] 12. The method of any one of aspects P1-P13, wherein the target tissue is in the brain of the subject.

[0176] 13. The method of any one of aspects P1-P14, wherein the first microbubble composition is administered in an amount effective to increase delivery of the active agent to the target tissue.

[0177] 14. The method of embodiment P16, wherein the first microbubble composition is administered in an amount effective to increase delivery of the active agent across the blood-brain barrier.

[0178] 15. The method of any one of aspects P1-P17, wherein the active agent comprises a protein or a nucleic acid, and optionally, the nucleic acid comprises shRNA, siRNA, RNA, or DNA.

[0179] 16. The method of any one of aspects P1-P17, wherein the active agent comprises an anti-cancer agent.

[0180] 17. The method of any one of aspects P1-P17, wherein the active agent comprises a virus.

[0181] 18. The method of embodiment P21, wherein the virus is an adenovirus.

[0182] 19. The method of embodiment P21 or P22, wherein replication of the virus is under the control of a cancer-specific promoter.

[0183] 20. The method of any one of aspects P21-P23, wherein the virus comprises a polynucleotide encoding an shRNA, siRNA, or antisense RNA.

[0184] 21. The method of any one of aspects P21 to P23, wherein the virus comprises a polynucleotide encoding the MDA-7 / IL-24 protein.

[0185] 22. The method of any one of aspects P1-P17, wherein the active agent comprises an MDA-7 / IL-24 protein, or a polynucleotide encoding an MDA-7 / IL-24 protein.

[0186] 23. The method of embodiment P25 or P26, wherein the MDA-7 / IL-24 protein is a fusion protein.

[0187] 24. Any one of the methods P25 to P27, wherein the MDA-7 / IL-24 protein comprises an insulin signal peptide.

[0188] 25. The method of any one of aspects P25 to P28, wherein the MDA-7 / IL-24 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or 4.

[0189] 26. The method of any one of aspects P25 to P29, wherein the MDA-7 / IL-24 protein comprises a mutation corresponding to (a) a K73R change relative to SEQ ID NO:3, or (b) a K19R change relative to SEQ ID NO:4.

[0190] 27. A kit for use in treating a target tissue with an active agent, the kit comprising first and second microbubble compositions; (i) the active agent is a therapeutic agent or an imaging agent; (ii) the first microbubble composition comprises first microbubbles and does not comprise an active agent; and (iii) the second microbubble composition comprises second microbubbles complexed with an active agent; kit.

[0191] 28. The kit of embodiment P58, wherein the first microbubble composition, the second microbubble composition, or both, are formulated for intravenous administration.

[0192] 29. The kit of embodiment P58 or P59, wherein the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns, or about 2.5 microns to about 4 microns.

[0193] 30. The kit of any one of embodiments P58-P60, wherein the first and / or second microbubbles comprise a targeting moiety.

[0194] 31. The kit of embodiment P61, wherein the targeting moiety comprises (a) a VEGF polypeptide or a single chain variant thereof, (b) a VCAM1 antibody or an epitope-binding fragment thereof, or (c) a PSMA antibody or an epitope-binding fragment thereof.

[0195] 32. The kit of any one of aspects P58-P63, wherein the active agent comprises a protein or a nucleic acid, and optionally, the nucleic acid comprises shRNA, siRNA, RNA, or DNA.

[0196] 33. The kit of any one of aspects P58-P63, wherein the active agent comprises an anti-cancer agent.

[0197] 34. The kit of any one of aspects P58-P63, wherein the active agent comprises a virus.

[0198] 35. The kit of embodiment P66, wherein the virus is an adenovirus.

[0199] 36. The kit of embodiment P66 or P67, wherein replication of the virus is under the control of a cancer-specific promoter.

[0200] 37. The kit of any one of aspects P66 to P68, wherein the virus comprises a polynucleotide encoding an shRNA, siRNA, or antisense RNA.

[0201] 38. The kit of any one of aspects P66 to P68, wherein the virus comprises a polynucleotide encoding the MDA-7 / IL-24 protein.

[0202] 39. The kit of any one of aspects P58 to P63, wherein the active agent comprises an MDA-7 / IL-24 protein, or a polynucleotide encoding an MDA-7 / IL-24 protein.

[0203] 40. The kit of embodiment P70 or P71, wherein the MDA-7 / IL-24 protein is a fusion protein.

[0204] 41. The kit of any one of aspects P70 to P72, wherein the MDA-7 / IL-24 protein comprises an insulin signal peptide.

[0205] 42. The kit of any one of aspects P70 to P73, wherein the MDA-7 / IL-24 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or 4.

[0206] 43. The kit of any one of aspects P70 to P74, wherein the MDA-7 / IL-24 protein comprises a mutation corresponding to (a) a K73R change relative to SEQ ID NO:3, or (b) a K19R change relative to SEQ ID NO:4.

[0207] N form 1. 1. A method of administering an active agent to a target tissue, the active agent being a therapeutic agent or an imaging agent, the method comprising: (a) administering to a subject a first microbubble composition, the first microbubble composition comprising first microbubbles and not comprising the active agent; (b) administering a first ultrasound wave to the target tissue, the first ultrasound wave destroying the first microbubbles; (c) administering to the subject a second microbubble composition after the administration of the first ultrasound, the second microbubble composition comprising second microbubbles complexed with the active agent; and (d) administering a second ultrasound wave toward the target tissue, which destroys second microbubbles and releases the active agent into the target tissue. A method comprising: 2. The method of embodiment N1, wherein the second microbubble composition is administered within about 60 minutes, 30 minutes, 10 minutes, or 5 minutes of administration of the first microbubble composition. 3. The method of embodiment N1 or N2, wherein the step of administering the first microbubble composition, the second microbubble composition, or both comprises intravenous administration. 4. The method of any one of embodiments N1-N3, wherein the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns, or about 2.5 microns to about 4 microns. 5. The method of any one of embodiments N1-N4, wherein the first and / or second microbubbles comprise a targeting moiety. 6. The method of embodiment N5, wherein the targeting moiety is a molecule selected from an antibody, an antibody fragment, a binding protein, a binding protein fragment, a receptor, a receptor fragment, a receptor ligand, a peptide, a polypeptide, a polynucleic acid, a polysaccharide, a lipid, a polymer, a tumor-associated antigen, a tissue-type-associated antigen, a vascular-associated antigen, or any combination of such molecules. 7. The method of embodiment N6, wherein said targeting moiety binds to a tissue-specific antigen or a tumor-associated antigen. 8. The method of embodiment N6, wherein said targeting moiety comprises (a) a VEGF polypeptide or a single chain variant thereof, (b) a VCAM1 antibody or an epitope-binding fragment thereof, or (c) a PSMA antibody or an epitope-binding fragment thereof. 9. The method of any one of embodiments N1-N8, wherein said target tissue comprises a tumor. 10. The method of embodiment N9, wherein said tumor is a metastatic tumor. 11. The method of embodiment N9 or N10, wherein said tumor is located in the brain, breast, lung, gastrointestinal system, bone, abdominal cavity, oral cavity, pancreas, intestine, skin, head, neck, spinal cord, or liver of said subject. 12. The method of any one of embodiments N9-N11, wherein said tumor comprises glioblastoma, melanoma, breast cancer, bone cancer, pancreatic cancer, liver cancer, colon cancer, oral cancer, head and neck cancer, spinal cancer, neuroblastoma, renal cancer, or lung cancer. 13. The method of any one of aspects N1-N12, wherein the target tissue is located within the brain, pancreas, stomach, intestine, bone, skin, oral cavity, head, neck, spinal cord, lung, kidney, or liver of the subject. 14. The method of embodiment N13, wherein said target tissue is in the brain of said subject. 15. The method of embodiment N13, wherein said target tissue is in the pancreas of said subject. 16. The method of any one of aspects N1-N15, wherein the first microbubble composition is administered in an amount effective to increase delivery of the active agent to the target tissue. 17. The method of embodiment N16, wherein the first microbubble composition is administered in an amount effective to increase delivery of said active agent across the blood-brain barrier. 18. The method of any one of aspects N1-N17, wherein said active agent comprises a protein or a nucleic acid, and optionally, the nucleic acid comprises an shRNA, siRNA, miRNA, lncRNA, mRNA, RNA, a vector, a plasmid, DNA, or any combination thereof. 19. The method of any one of embodiments N1-N17, wherein the active agent comprises an anti-cancer agent. 20. The method of embodiment N19, wherein said anticancer agent is selected from an alkylating agent, an antimetabolite, a natural product, a chemotherapeutic agent, a hormone, a polypeptide, or a small molecule having utility in a method of treating cancer. twenty one. The method of any one of embodiments N1-N17, wherein the active agent comprises a virus. twenty two. The method of embodiment N21, wherein said virus is selected from adenovirus, a tropism-modified adenovirus, a cancer terminator virus (CTV), a lentivirus, a retrovirus, a herpes virus, a vaccinia virus, a genetically modified HIV, a tripartite theranostic cancer terminator virus (TCTV), an avian associated virus (AAV), and / or a vesicular stomatitis virus. twenty three. The method of embodiment N21 or N22, wherein replication of said virus is under the control of a cancer-selective promoter. twenty four. The method of any one of aspects N21 to N23, wherein said virus comprises a polynucleotide encoding an shRNA, siRNA, miRNA, sense RNA, antisense RNA, or lncRNA. twenty five. The method of any one of aspects N21 to N23, wherein said virus comprises a polynucleotide encoding an MDA-7 / IL-24 protein. 26. The method of any one of embodiments N1-N17, wherein said active agent comprises MDA-7 / IL-24 protein, or a polynucleotide encoding MDA-7 / IL-24 protein. 27. The method of embodiment N25 or N26, wherein the MDA-7 / IL-24 protein is a fusion protein. 28. The method of any one of embodiments N25-N27, wherein the MDA-7 / IL-24 protein comprises an insulin signal peptide. 29. The method of any one of embodiments N25-N27, wherein the MDA-7 / IL-24 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or 4. 30. The method of any one of embodiments N25 to N28, wherein the MDA-7 / IL-24 protein comprises (a) a K122R change relative to SEQ ID NO:2, (b) a K73R change relative to SEQ ID NO:3, (c) a K19R change relative to SEQ ID NO:4, or (d) a mutation corresponding to SEQ ID NO:18. 31. The method of any one of embodiments N1-N17, wherein the active agent comprises an MDA-9 / syntenin inhibitor. 32. The method of any one of aspects N21 to N23, wherein said virus comprises a polynucleotide encoding an MDA-9 / syntenin inhibitor. 33. The method of embodiment N32, wherein said polynucleotide sequence comprises a sequence encoding a MDA-9 / syntenin siRNA, shRNA, miRNA, lncRNA, or antisense RNA sequence. 34. The method of embodiment N33, wherein said sequence comprises SEQ ID NO:20. 35. (e) administering to the subject a third microbubble composition after the administration of the second ultrasound, the third microbubble composition comprising second microbubbles complexed with a second active agent or imaging agent; and (f) administering a third ultrasound wave toward the target tissue, which destroys a third microbubble and releases the second active agent or imaging agent into the target tissue. The method of any one of aspects N1-N34, further comprising: 36. The method of embodiment N35, wherein the second active agent or imaging agent is the same as the first active agent or imaging agent. 37. (g) administering to the subject a fourth microbubble composition after the administration of the third ultrasound, the fourth microbubble composition comprising third microbubbles complexed with a third active agent or imaging agent; and (h) administering a fourth ultrasound wave toward the target tissue, which destroys a fourth microbubble and releases a third active or imaging agent into the target tissue. The method of any one of embodiments N35-N36, further comprising: 38. The method of embodiment N37, wherein the third active agent or imaging agent is the same as the first active agent or imaging agent. 39. The method of embodiment N37, wherein the third active agent or imaging agent is the same as the second active agent or imaging agent. 40. The method of embodiment N37, wherein the third active agent or imaging agent is the same as the first and second active agents or imaging agents. 41. The method of any one of embodiments N1-N40, wherein the microbubble composition comprises two or more active agents. 42. The method of any one of embodiments N1-N41, wherein said subject has or is at risk of having cancer. 43. The method of embodiment N42, wherein said cancer is a solid tumor cancer. 44. The cancer may be brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, Hodgkin's disease and non-Hodgkin's lymphoma, thyroid cancer, endocrine system cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, stomach cancer, uterine cancer, thyroid cancer, bile duct cancer, pancreatic adenocarcinoma, pancreatic ductal adenocarcinoma (PDAC), skin cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, The method of embodiment N43, wherein the cancer is invasive breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesion, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical carcinoma, endocrine or exocrine pancreatic neoplasms, medullary thyroid cancer, medullary thyroid carcinoma, melanoma, colorectal cancer, papillary thyroid carcinoma, hepatocellular carcinoma, or prostate cancer. 45. The method of embodiment N42, wherein said cancer is brain cancer. 46. The method of embodiment N42, wherein said cancer is pancreatic cancer. 47. The method of embodiment N42, wherein said cancer is a metastatic cancer. 48. The method of embodiment N42, wherein said subject has previously been treated for cancer. 49. The method of embodiment N42, wherein said subject was previously in remission. 50. The method of any one of embodiments N1-N49, wherein the imaging agent is selected from a radionuclide, a positron emitting isotope, a fluorophore, an antibody, a bioluminescent molecule, a chemiluminescent molecule, a photoactive molecule, a metal, an electron-dense reagent, an enzyme, a magnetic contrast agent, a quantum dot, a nanoparticle, biotin, digoxigenin, a hapten, or a protein or other entity capable of being made detectable. 51. A method of treating cancer in a subject in need thereof comprising administering the method of any one of embodiments N1-N50 and administering an anti-cancer therapy that does not include microbubbles. 52. The method of embodiment N51, wherein the anti-cancer treatment not comprising microbubbles is chemotherapy, hormonal therapy, radiation therapy, or immunotherapy. 53. The method of embodiment N51, wherein the microbubble-free anti-cancer treatment is a microbubble-free anti-cancer agent. 54. The method of embodiment N53, wherein the microbubble-free anti-cancer agent is selected from an alkylating agent, an antimetabolite, a natural product, a chemotherapeutic agent, a hormone, a polypeptide, or a small molecule having utility in a method of treating cancer. 55. The method of embodiment N54, wherein the microbubble-free anticancer drug is gemcitabine. 56. The method of embodiment N54, wherein the microbubble-free therapeutic agent is temozolomide. 57. The method of any one of embodiments N51-N56, wherein the microbubble-free active agent further comprises a pharma- ceutically acceptable excipient. 58. 1. A kit for use in treating a target tissue with an active agent, the kit comprising first and second microbubble compositions; (i) the active agent is a therapeutic agent or an imaging agent; (ii) the first microbubble composition comprises a first microbubble and does not comprise the active agent; and (iii) a second microbubble composition comprising second microbubbles complexed with the active agent; kit. 59. The kit of embodiment N58, wherein the first microbubble composition, the second microbubble composition, or both, are formulated for intravenous administration. 60. The kit of embodiment N58 or N59, wherein the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns, or about 2.5 microns to about 4 microns. 61. The kit of any one of embodiments N58-N60, wherein the first and / or second microbubbles comprise a targeting moiety. 62. The kit of embodiment N61, wherein the targeting moiety is a molecule selected from an antibody, an antibody fragment, a binding protein, a binding protein fragment, a receptor, a receptor fragment, a receptor ligand, a peptide, a polypeptide, a polynucleic acid, a polysaccharide, a lipid, a polymer, a tumor-associated antigen, a tissue-specific antigen, or a vascular-associated antigen, or any combination of such molecules. 63. The kit of embodiment N62, wherein the targeting moiety comprises (a) a VEGF polypeptide or a single chain variant thereof, (b) a VCAM1 antibody or an epitope-binding fragment thereof, or (c) a PSMA antibody or an epitope-binding fragment thereof. 64. The kit of any one of aspects N58 to N63, wherein the active agent comprises a protein or a nucleic acid, and optionally, the nucleic acid comprises an shRNA, siRNA, miRNA, mRNA, RNA, a vector, a plasmid, DNA, or any combination thereof. 65. The kit of any one of embodiments N58-N63, wherein the active agent comprises an anti-cancer agent. 66. The kit of any one of embodiments N58 to N63, wherein the active agent comprises a virus. 67. The kit of embodiment N66, wherein said virus is a tropism modified adenovirus. 68. The kit of embodiment N66 or N67, wherein replication of said virus is under the control of a cancer-selective promoter. 69. The kit of any one of aspects N66 to N68, wherein said virus comprises a polynucleotide encoding an shRNA, siRNA, or antisense RNA. 70. The kit of any one of aspects N66 to N68, wherein the virus comprises a polynucleotide encoding an MDA-7 / IL-24 protein. 71. The kit of any one of embodiments N58 to N63, wherein said active agent comprises an MDA-7 / IL-24 protein, or a polynucleotide encoding an MDA-7 / IL-24 protein. 72. The kit of embodiment N70 or N71, wherein the MDA-7 / IL-24 protein is a fusion protein. 73. The kit of any one of embodiments N70 to N72, wherein the MDA-7 / IL-24 protein comprises an insulin signal peptide. 74. The kit of any one of aspects N70 to N73, wherein the MDA-7 / IL-24 protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:3 or 4. 75. The kit of any one of embodiments N70 to N73, wherein the MDA-7 / IL-24 protein comprises (a) a K122R change relative to SEQ ID NO:2, (b) a K73R change relative to SEQ ID NO:3, (c) a K19R change relative to SEQ ID NO:4, or (d) a mutation corresponding to SEQ ID NO:18. 76. The kit of any one of embodiments N58 to N63, wherein the active agent comprises an MDA-9 / syntenin polynucleotide inhibitor. 77. The kit of any one of aspects N66 to N68, wherein the virus comprises a polynucleotide encoding an MDA-9 / syntenin inhibitor. 78. The kit of embodiment N76 or N77, wherein the MDA-9 / syntenin polynucleotide sequence comprises a sequence encoding a siRNA, shRNA, miRNA, lncRNA, or antisense RNA sequence. 79. The kit of embodiment N78, wherein said sequence comprises SEQ ID NO:20. 80. A kit for use of any one of aspects N58 to N79, comprising: (a) administering a first microbubble composition; (b) administering a first ultrasound wave to the target tissue, the first ultrasound wave destroying the first microbubbles; (c) administering a second microbubble composition following the administration of the first ultrasound wave; and (d) administering a second ultrasound wave to the target tissue, the second ultrasound wave destroying the second microbubbles. Including the kit. 81. The kit for use of embodiment N80, wherein administration of the second microbubble composition is within about 60 minutes, 30 minutes, 10 minutes, or 5 minutes of administration of the first microbubble composition. 82. The kit for use of embodiment N80 or N81, wherein said target tissue comprises a tumor. 83. The kit for use in embodiment N82, wherein said tumor is a metastatic tumor. 84. The kit for use of embodiment N82 or N83, wherein said tumor is located in the brain, breast, lung, gastrointestinal system, bone, abdominal cavity, pancreas, intestine, skin, head, neck, oral cavity, spinal cord, or liver of the subject. 85. The kit for use of embodiment N82 or N83, wherein said tumor comprises glioblastoma, melanoma, breast cancer, pancreatic cancer, liver cancer, prostate cancer, colon cancer, oral cancer, head and neck cancer, spinal cancer, neuroblastoma, renal cancer, or lung cancer. 86. The kit for use of embodiment N80, wherein said target tissue is located in the brain, pancreas, stomach, intestine, bone, skin, oral cavity, abdominal cavity, spinal cord, head, neck, kidney, or liver of a subject. 87. The kit for use of any one of embodiments N82-N86, wherein the target tissue is in the subject's brain. 88. The kit for use of any one of embodiments N82-N86, wherein the target tissue is in the pancreas of the subject. 89. The kit for use of any one of embodiments N80-N88, wherein administration of the first microbubble composition is in an amount effective to increase delivery of the active agent to the target tissue. 90. The kit for use of embodiment N89, wherein administration of the first microbubble composition is in an amount effective to increase delivery of the active agent across the blood-brain barrier. EXAMPLES

[0208] It is understood that the examples and embodiments described herein are for illustrative purposes only, and in light of this, various modifications or changes will be suggested to those skilled in the art that are within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0209] Example 1: Dual treatment with MB (FUS-DMB delivery) approach We used FUS together with naked MBs (microbubbles lacking active agents) to temporarily open the BBB to allow access of luciferase-expressing adenovirus (later delivered in MBs and released by FUS using UTMD). As shown in Figure 1A-1C, FUS (by UTMD) temporarily opens the BBB, allowing the luciferase-expressing adenovirus (Ad.CMV-Luc incorporated in MBs) to enter and then be released into the brain after a second round of FUS. This protocol did not cause any obvious toxic effects, as the mice survived without any notable symptoms for at least 3 weeks after the procedure.

[0210] Next, we tested the feasibility of targeted delivery of therapeutic adenovirus (Ad.5 / 3-CTV) to brain tumors using FUS and UTMD using a focused ultrasound (FUS) dual MB (FUS-DMB) delivery approach. GBM-6, a highly invasive primary human glioblastoma cell line that recapitulates human disease (highly invasive, with fingerlike projections of tumor cells in the brain) when injected intracranially into mouse brains, was used in this study. MB-encapsulated GBM-6-Luc (a luciferase-expressing GBM-6 clone) containing Ad.5 / 3-CTV was delivered systemically to brain tumor-bearing mice, and therapeutic response was monitored through IVIS. The experimental group that underwent FUS and UTMD with empty MBs prior to therapeutic delivery of MB-Ad.5 / 3-CTV with FUS and UTMD (FUS-DMB) delivery strategy) demonstrated robust tumor suppression, as indicated by reduced fluorescence and enhanced animal survival (Figures 2A and 2B).

[0211] These findings demonstrate that after opening the BBB by naked MB and FUS, it is possible to administer a therapeutic adenovirus (Ad.5 / 3-CTV) in a "stealth manner" into the circulation and deliver it to the target GBM tumor in the brain by MB+FUS. This strategy resulted in significant therapeutic activity in animals bearing GBM-6 tumors without the need for surgery. For patients with GBM, this strategy allows for systemic administration of MB containing a therapeutic virus and application of FUS in patients after opening the BBB by naked MB and FUS using the FUS-DMB delivery approach without the need for tumor debulking surgery (see, for example, Figure 2B). In principle, this approach can be used for primary GBM as well as recurrent GBM (which occurs in the majority of patients after primary tumor removal and chemotherapy and / or radiotherapy) without the need for surgery. This approach can also be applied to treat metastatic tumors (from other sites such as breast or melanoma) that are established in the brain.

[0212] In addition to systemic delivery of therapeutic viruses, we also tested the possibility of using the MB and UTMD approach to deliver therapeutic proteins in vivo. His-tagged MDA-7 / IL-24 (His-MDA-7) was purified by using Ni-NTA column chromatography. 1 mg / ml His-MDA-7 solution was mixed with Alexa Fluor 488 containing 0.1 M sodium bicarbonate, followed by centrifugation using a centricon (3 kD cutoff MW) to remove unincorporated dye. Confirmation of His-MDA-7 labeling (Alexa Fluor-His-MDA-7) was obtained spectrofluorimetrically based on emission spectrum changes. Alexa Fluor-His-MDA-7 was incubated with MBs overnight, and the localization of the labeled protein complex within the MBs was monitored by green fluorescence and found to be associated with the lipid shell of the MBs (Figure 3A). To test the in vivo delivery of labeled Alexa Fluor-His-MDA-7 complexed with MB using the UTMD approach, DU-145 tumor xenografts were established in the left flank of nude mice. Alexa Fluor-His-MDA-7 complexed with MB was administered into the tail vein of the mice. As shown in Figure 3B, UTMD Alexa Fluor-MDA-7 was primarily located within the tumors in the left flank of the mice. These studies confirm the utility of MB to deliver therapeutic proteins in a target-specific manner in vivo when administered intravenously and then subjected to ultrasound.

[0213] Example 2: Enhanced delivery of MB to tumors, metastases, and tumor vasculature A variety of molecular markers are overexpressed on the vascular endothelium of tumor vasculature. To facilitate targeting of MB, MB can be decorated with targeting moieties (also called targeting ligands), such as antibodies. Targeting moieties can be attached to the surface of MB via biotin-streptavidin spacers or via direct chemical coupling, preferably directional coupling via thiol-maleimide. In direct therapeutic relevance, the latter approach offers a successful strategy for clinical translation.

[0214] Successful MB targeting to VEGFR2 was achieved by attaching single-chain VEGF molecules to the microbubble surface. Approximately less than 100,000 targeting ligand molecules per bubble are sufficient to ensure successful targeting to tumor vasculature overexpressing VEGFR2 (Figure 4, left panel). Another target for MB targeting to tumor vasculature is VCAM-1. Anti-VCAM-1 antibody fragments were successfully placed on the MB surface and targeted imaging was achieved in an in vivo mouse tumor animal model (Figure 4, right panel).

[0215] To generate targeted MBs, we functionalized them with mouse anti-V-CAM-1 antibody using biotin-streptavidin conjugation chemistry, as confirmed by flow cytometry data (Figure 5A). The targeted or decorated MBs (D-MBs) were further validated for site-specific delivery in two transgenic animals, Hi-Myc (prostate cancer) (Figure 5B) and PyMT (breast cancer) (Figure 5C). Ad.luc complexed with MBs was administered through IV route, followed by sonoporation at the corresponding tumor site. 72 hours after delivery, BLI imaging was performed using IVIS spectrum (Figure 5B and Figure 5C). In both models, Ad.luc was shown to be delivered to the site of sonoporation. Trace BLI signal was obtained from the liver. Improved delivery may be achieved by using next-generation sonoporation devices that may provide more directed FUS. Besides the liver, little evidence of Ad.luc delivery was obtained in secondary tumors (non-sonoporated mammary tumors in the PyMT model, Figure 5D) or kidneys, demonstrating the utility of the targeted UTMD approach for systemic delivery of Ad and thus, in principle, providing an effective means of systemic delivery of therapeutic viruses with enhanced payload delivery.

[0216] This approach can be used to facilitate the delivery of therapeutic viruses, recombinant proteins, and chemotherapy not only to GBM or metastatic tumors in the brain (using the dual MB UTMD approach of the present invention), but also to primary tumors, metastases, and tumor vasculature at different anatomical sites in the body.

[0217] We generated targeted (decorated) MBs (d-MBs) that specifically bind prostate-specific membrane antigen (PSMA). The targeted (decorated) MBs, called PE-anti-PSMA-MBs, specifically bound to PC-3-PIP cells (PC-3 cells overexpressing PSMA) (Figures 6A and 6B), whereas the binding of non-targeted (decorated) MBs (PE-IgG-MBs) showed limited binding compared to the decorated MBs (data not shown). We next examined the efficacy of d-MB delivery in an in vivo nude mouse model containing PC-3 and PC-3-PIP tumor xenografts. To observe the site-specific delivery of genes or therapeutic Ad by MBs combined with UTMD technology, PC-3 and PC-3-PIP cells were injected sc into the left and right flanks of nude mice, respectively (Figures 6A and 6B). There was no difference in tumor growth in both flanks, with tumor sizes in both flanks reaching approximately 100 mm. 3After reaching the target area, MB was injected into the mice by tail vein injection. Ad.5 / 3-CMV-luc conjugated to MB (simple MB) and Ad.5 / 3-CMV-luc conjugated to anti-PSMA-MB (targeted MB) and free Ad.5 / 3-CMV-luc were injected, and sonoporation was performed in the right flank of the mice (tumor bearing PC-3-PIP) using UTMD approach, and the mice were imaged 72 hours after injection of Ad.5 / 3-CMV-luc delivery. It was found that both simple MB and targeted MB could deliver Ad to the target site of sonoporation. In the case of simple MB, the delivery of Ad was simply due to release from MB in the area where ultrasound was applied, and the signal intensity of BLI was low compared to targeted MB. Furthermore, the signal in the case of simple MB was in a wider area compared to the convergent release of Ad by targeted MB, indicating that the delivery of Ad by targeted MB was more specific. More specific delivery of Ad by targeted MB may be due to the active binding of targeted MB to the surface of tumor cells and the subsequent release of Ad by applying ultrasound at the target site. Thus, the use of targeted MB limited the non-specific delivery of Ad in the surrounding tumor area. Delivery effect may be further enhanced by the combination of targeted MB and dual MB approach as described herein.

[0218] Example 3: Focused ultrasound (FUS) dual MB (FUS-DMB) delivery of Ad.5 / 3-CTV significantly extends survival of mice bearing human GBM tumors Intracranial injection of GBM6 cells: Mice were anesthetized via ip administration of (ketamine, 40 mg / kg; xylazine, 3 mg / kg) and immobilized in a stereotaxic frame. A 24-gauge needle attached to a Hamilton syringe was inserted into the right basal ganglia to a depth of 3.5 mm and then withdrawn 0.5 mm to create space for tumor cell accumulation. The entry point of the skull was 2 mm lateral and 1 mm dorsal to the bregma. Intracerebral injection of 5 μl of DMEM medium containing 30,000 glioma cells (GBM6 / GBM6-Luc / GSC-8-11-Luc) was performed over 10 min. The skull opening was sealed with sterile bone wax and the skin incision was closed using sterile surgical staples.

[0219] Ad.5 / 3-CTV procedure: Ten days after tumor cell implantation, adenoviral vectors were administered via stereotactic injection into intracranial tumors using the same anesthesia technique and stereotactic frame coordinates as above. Viral vectors suspended in 2 μl of phosphate-buffered saline (PBS) were delivered by slow infusion over 6 minutes. These mice were then imaged weekly until the IACUC endpoint and used for survival analysis.

[0220] Microbubble-adenovirus complex: Perfluorocarbon MB, 1 x 10 of the indicated adenovirus 11 The MB / Ad were reconstituted in 1 ml of PBS containing viral particles and incubated for 2 h at 4°C. After incubation, non-encapsulated surface-associated Ad was inactivated by treatment with 20% FBS for 3 h at 4°C and washed twice to remove unbound adenovirus. Finally, MB / Ad were dissolved in 1 ml of PBS before treatment. Complement-treated MB / Ad were injected systemically via the tail vein and sonoporated (using focused ultrasound).

[0221] Generation of Focused Ultrasound (FUS) Waves and Treatment of Mice to Cross the Blood-Brain Barrier (BBB): Focused ultrasound (FUS) utilizes the same concept of acoustic wave propagation as the more widely known ultrasound diagnostic applications. However, instead of acquiring and displaying echoes generated at several tissue interfaces for imaging, FUS generally utilizes a concave transducer with a single geometric focus or using a phased array to electronically steer it, with the majority of its power delivered during sonication to induce mechanical effects, thermal effects, or both. A FUS transducer (2.25 MHz, 0.50 inch element diameter, standard case style, straight UHF connector, purchased from Olympus America Inc.) is used to perform sonication immediately (15 seconds) after bubble administration. The transducer is driven by a function generator (AGI-E4436B, Agilent Technologies, Palo Alto, CA, USA) through a power amplifier (E&I 3100LA, ENI Inc., Rochester, NY, USA). A cone filled with degassed distilled water is attached to the transducer system. Mice were injected with 100 μl of diluted microbubbles and immediately after IV, FUS was applied (3.5 mV, 10 dB, 1 MHz) and BBB opening was observed by Evans Blue experiments.

[0222] Example 4: Systemic Administration of Ad Using FUS-DMB FUS-DMB systemic delivery: 100 μl of diluted MB was injected via tail vein and allowed to circulate for 15 seconds. After 15 seconds, the mice were sonicated (ultrasonicated) for 1 minute as described herein. Next, 100 μl of microbubbles containing Ad were injected I / V into the mice, the bubbles were allowed to circulate for 15 seconds, and then sonicated for 1 minute. The animals were imaged using an IVIS imager and followed for survival analysis.

[0223] Example 5: Systemic administration of Ad using FUS-DMB to target the pancreas Pancreatic delivery of shMDA-9 in the KPC mouse model: The KPC mouse model of pancreatic ductal adenocarcinoma (PDAC) was first described in 2005 and incorporates conditional activation of endogenous mutant alleles of Kras and Trp53 genes by Cre-Lox technology. Specifically, an activating point mutation in Kras (G12D) and a dominant-negative mutation in Trp53 (R172H) are conditionally activated in the mouse pancreas by crossing LSL-KrasG12D / +;LSL-Trp53R172H / + mice with Pdx-1-Cre mice expressing Cre recombinase under the expression of the pancreas-specific Pdx-1 promoter. Cre-mediated recombination acts to excise the loxP-flanked stop codon (LSL), an event that occurs only in cells expressing Cre, thereby resulting in conditional expression of the mutant Kras and Trp53 genes specifically in the mouse pancreas.

[0224] Intravenous FUS-DMB: 100 μl of diluted MB was injected via tail vein and allowed to circulate for 15 seconds. After 15 seconds, the mice were sonicated (ultrasonicated) in the pancreatic region for 1 minute as described above. Next, 100 μl of microbubbles containing Ad(Luc / shMDA-9) were injected I / V into the mice, the bubbles were allowed to circulate for 15 seconds, and then sonicated for 1 minute. The animals were imaged using an IVIS imager and followed for survival analysis.

[0225] Western blot analysis: KPC homozygous mice were first injected with empty microbubbles, FUS was applied for 1 min using the parameters described above, and 1 min later, complement-treated MB / Ad was injected systemically via the tail vein (100 μl) and sonoporated with FUS. The mice were observed, imaged, or euthanized at the indicated time points and various organs (spleen, pancreas, lungs, and liver) were collected. The organs were lysed and proteins were isolated using standard protocols, and equal amounts of protein were separated using SDS-PAGE. Western blot analysis was performed using MDA-9 specific antibodies. β-actin was used as a loading control.

[0226] Real-time PCR analysis: In KPC homozygous mice, empty microbubbles were first injected, FUS was applied for 1 min using the parameters described above, and 1 min later, complement-treated MB / Ad was injected systemically via the tail vein (100 μl) and sonoporated using FUS. The mice were observed, imaged, or euthanized at the indicated time points and different organs (spleen, pancreas, lungs, and liver) were collected. The organs were lysed, RNA was isolated using standard protocols, and equal amounts of RNA were used to synthesize cDNA according to the manufacturer's protocol. Real-time PCR was performed to check MDA-9 mRNA levels. Mouse GAPDH was used as a transcriptional control.

[0227] statistical analysis All data represent the mean ± SD from three independent experiments. Statistical analysis was performed using either Student's t-test (Microsoft excel) or Pearson's correlation (GraphPad prism software). P < 0.05 was considered significant.

[0228] Survival analysis: Kaplan-Meier curves were generated using PRISM Graph PAD software and used to estimate survival functions. Gemcitabine was given at a dose of 20 mg / kg via intraperitoneal injection.

[0229] References TIFF2024503090000021.tif182170TIFF2024503090000022.tif55165

Claims

1. An active agent for use in treating or imaging a subject, the treating or imaging comprising administering the active agent to a target tissue, the active agent being a therapeutic or imaging agent, the treating or imaging comprising: (a) administering to the subject a first microbubble composition, the first microbubble composition comprising first microbubbles and not comprising the active agent; (b) administering to the subject a first ultrasound wave directed at the target tissue that destroys the first microbubbles; (c) administering to the subject a second microbubble composition after the administration of the first ultrasound, the second microbubble composition comprising second microbubbles complexed with the active agent; and (d) administering a second ultrasound wave to the subject, directed at the target tissue, that destroys second microbubbles and releases the active agent into the target tissue. The active agent comprises

2. 2. The method of claim 1, wherein the second microbubble composition is administered within about 60 minutes, 30 minutes, 10 minutes, or 5 minutes of administration of the first microbubble composition.

3. 3. The method of claim 1, wherein the step of administering the first microbubble composition, the second microbubble composition, or both, comprises intravenous administration.

4. 4. The method of claim 1, wherein the first and / or second microbubbles have a mean or median diameter of about 1 micron to about 5 microns, or about 2.5 microns to about 4 microns.

5. 5. The active agent for use according to any one of claims 1 to 4, wherein the first and / or second microbubble comprises a targeting moiety, the targeting moiety being a molecule selected from an antibody, an antibody fragment, a binding protein, a binding protein fragment, a receptor, a receptor fragment, a receptor ligand, a peptide, a polypeptide, a polynucleic acid, a polysaccharide, a lipid, a polymer, a tumor-associated antigen, a tissue-type-associated antigen, a vascular-associated antigen, or any combination thereof. (i) the targeting moiety binds to a tissue-specific antigen or a tumor-associated antigen; or (ii) the targeting moiety comprises (a) a VEGF polypeptide or a single chain variant thereof, (b) a VCAM1 antibody or an epitope-binding fragment thereof, or (c) a PSMA antibody or an epitope-binding fragment thereof; 6. An active agent for use according to claim 5.

7. The method according to any one of claims 1 to 6, wherein the target tissue comprises a tumor. (i) the tumor is a metastatic tumor; (ii) the tumor is located in the brain, breast, lung, gastrointestinal system, bone, abdominal cavity, oral cavity, pancreas, intestine, skin, head, neck, spinal cord, or liver of the subject; or (iii) the tumor comprises glioblastoma, melanoma, breast cancer, bone cancer, pancreatic cancer, liver cancer, colon cancer, oral cancer, head and neck cancer, spinal cancer, neuroblastoma, renal cancer, or lung cancer; 8. An active agent for use according to claim 7.

9. 9. The method of claim 1, wherein the target tissue is located in the brain, pancreas, stomach, intestine, bone, skin, mouth, head, neck, spinal cord, lung, kidney, or liver of the subject.

10. The active agent for use according to any one of claims 1 to 9, wherein the first microbubble composition is administered in an amount effective to increase delivery of the active agent to the target tissue.

11. 11. The active agent for use according to claim 10, wherein the first microbubble composition is administered in an amount effective to increase delivery of the active agent across the blood-brain barrier.

12. The active agent (i) a protein or a nucleic acid, or (ii) Anticancer drugs 12. An active agent for use according to any one of claims 1 to 11, comprising:

13. The active agent for use according to claim 12, wherein the active agent comprises a nucleic acid including shRNA, siRNA, miRNA, lncRNA, mRNA, RNA, vector, plasmid, DNA, or any combination thereof.

14. the active agent comprises a virus; (i) the virus is selected from adenovirus, tropism-modified adenovirus, cancer terminator virus (CTV), lentivirus, retrovirus, herpesvirus, vaccinia virus, genetically modified human immunodeficiency virus, tripartite theranostic CTV (TCTV), adeno-associated virus, and / or vesicular stomatitis virus; (ii) replication of the virus is under the control of a cancer-selective promoter; (iii) the virus comprises a polynucleotide encoding an shRNA, siRNA, miRNA, sense RNA, antisense RNA, or lncRNA; or (iv) the virus comprises a polynucleotide encoding an MDA-7 / IL-24 protein; An active agent for use according to any one of claims 1 to 13.

15. The method of any one of claims 1 to 14, wherein the active agent comprises an MDA-7 / IL-24 protein or a polynucleotide encoding an MDA-7 / IL-24 protein.

16. The active agent (i) an MDA-9 / syntenin inhibitor; or (ii) a virus comprising a polynucleotide encoding an MDA-9 / syntenin inhibitor; 16. The active agent for use according to any one of claims 1 to 15, comprising:

17. An active agent for use according to any one of claims 1 to 16, wherein the treatment is a method for treating cancer in a subject in need thereof, the treatment further comprising the step of administering an anti-cancer treatment that does not contain microbubbles.

18. A method of treating cancer in a subject in need thereof, wherein the treating or imaging comprises: (e) administering to the subject a third microbubble composition after the administration of the second ultrasound, the third microbubble composition comprising third microbubbles complexed with a second active agent or imaging agent; and (f) administering a third ultrasound wave to the subject, directed at the target tissue, to destroy the third microbubbles and release the second active agent or imaging agent into the target tissue.

18. The active agent for use according to any one of claims 1 to 17, further comprising:

19. 19. The active agent for use according to any one of claims 1 to 18, wherein the imaging agent is selected from radionucleotides, positron emitting isotopes, fluorophores, antibodies, bioluminescent molecules, chemiluminescent molecules, photoactive molecules, metals, electron-dense reagents, enzymes, magnetic contrast agents, quantum dots, nanoparticles, biotin, digoxigenin, and haptens.

20. 1. A kit for use in treating a target tissue with an active agent, the kit comprising first and second microbubble compositions; (i) the active agent is a therapeutic agent or an imaging agent; (ii) the first microbubble composition comprises a first microbubble and does not comprise the active agent; and (iii) a second microbubble composition comprises second microbubbles complexed with the active agent; kit.

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