Mitochondrial optogenetics-based gene therapies and their methods of use

Abstract

The present disclosure is directed to compositions comprising mitochondrial optogenetics-based gene therapies and their methods of use. In some embodiments, a composition described herein comprises an expression vector comprising a first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein. In some cases, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence. In some instances, the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS). In some implementations, when the expression vector is expressed, the luciferase protein is localized to the cytosol. In some cases, the IMM-MLS comprises a leading sequence from a mitochondrial inner membrane protein selected from ABCB10, ABCB140, Cytochrome C, and renal outer medullary potassium channel (ROMK).

Description

T2025-099 (069596.00097)MITOCHONDRIAL OPTOGENETICS-BASED GENE THERAPIES AND THEIR METHODS OF USECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 723,791, filed November 22, 2024, which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under R01 CA262028 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING OR TABLE

[0003] The material in the accompanying sequence listing is hereby incorporated by reference in its entirety into this application. The accompanying file, named 069596-00097, was created on November 20, 2025 and is 99 kB.FIELD

[0004] The present invention relates to mitochondrial optogenetics-based gene therapies.BACKGROUND

[0005] Mitochondria have emerged as a promising target for cancer treatment, given their role in cell signaling, cell growth, and cell function. Numerous mitochondrial-targeted anticancer therapies, such as mitocans, mitochondriotropics, and mitochondriotoxics, have been developed. However, these therapies typically target specific signaling pathways or proteins, such as hexokinase, Bcl-2 family proteins, thiol redox, and VDAC / ANT. These pathways and proteins may undergo unpredicted mutations during long-term treatment, leading to drug resistance and reduced efficacy.

[0006] A profound and sustained dissipation of the electrical potential gradient across the inner mitochondrial membrane may be a trigger for cell death. Chemical uncouplers and permeability transition pore activators have also been used to depolarize the membrane potentialT2025-099 (069596.00097) of the inner mitochondrial membrane. However, the lack of specificity for cancer cells limits their in vivo utility, as mitochondrial activity is important for all cells. Genetic methods can modulate mitochondrial function in specific tissues but do not directly target membrane and often cause irreversible side effects.

[0007] Thus, there is a need for cancer treatment approaches that can directly trigger cell death of cancer cell via their mitochondria, such as disrupting the cancer cell membrane potential.SUMMARY

[0008] Compositions comprising mitochondrial optogenetics-based gene therapies and their methods of use are described herein.

[0009] In one aspect, a composition described herein comprises an expression vector or expression plasmid comprising a first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein. In some instances, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence. In some implementations, the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS). It is to be understood that in some cases, the channelrhodopsin protein comprises at least two domains, a channelrhodopsin ion channel domain that can change the mitochondrial membrane potential (Am) when light and / or bioluminescence is present and an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS) that can effectively target the channelrhodopsin fusion protein to an inner mitochondria membrane. It is to be understood that in some embodiments, when the expression vector is expressed, the luciferase protein is localized to the cytosol. An optogenetic tool referred herein as mitochondrial luminoptogenetics (“mLumiOpto” or “cmLumiOpto”) is described herein. In one aspect, the mLumiOpto genes disclosed herein comprise an expression vector comprising: a first nucleic acid sequence encoding a channelrhodopsin fusion protein; and a second nucleic acid sequence encoding a luciferase protein, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence; wherein the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-T2025-099 (069596.00097) mitochondrial localization signal (IMM-MLS); and when the expression vector is expressed, the luciferase protein is localized to the cytosol.

[0010] In some cases, the luciferase protein comprises hRluc (a humanized version of Renilla reniformis luciferase), hGluc (humanized version of Gaiissia princeps luciferase), NLuc (also, NanoLuc® which is an engineered luciferase derived from Oplophorus gracilirostris), M23hGluc (a mutant version of hGluc with five amino acid changes compared to wild-type), or sbGluc (a slow bum variant of Gaussict luciferase with glow-type kinetics). In some cases, the luciferase protein comprises a variant of hRluc, hGluc, Nluc, M23hGluc, or sbGluc. In some cases, the luciferase protein comprises NLuc or a variant thereof. In some embodiments, the IMM-MLS comprises a leading sequence from a mitochondrial inner membrane protein selected from ABCB10, ABCB 140, Cytochrome C, and renal outer medullary potassium channel (ROMK). In some embodiments, the nucleic acid sequence encoding a channelrhodopsin fusion protein and the nucleic acid sequence encoding a luciferase protein are separated by a self- cleavable linker. In some embodiments, the self-cleavable linker is 2A.

[0011] In some instances, the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are operably linked to an expression control sequence. In some embodiments, the expression control sequence is the same expression control sequence. Moreover, in some implementations, the expression control sequence comprises a cancer-specific promoter. In some cases, the cancer specific promoter is cfos.

[0012] It is to be understood that any expression vector or expression plasmid not inconsistent with the technical objectives of the present disclosure may be used in a composition disclosed herein. In some cases, the expression vector or expression plasmid may comprise a viral vector. Any viral vector not inconsistent with the technical objectives of the present disclosure may be used. For example, in some cases, the viral vector may be an adenoviral vector. In some implementations, the viral vector may be an adeno-associated virus (AAV) vector.

[0013] In another aspect, any composition described herein comprises a nanoparticle. In some embodiments, a nanoparticle as described herein comprises and / or encapsulates any expression vector or expression plasmid described herein. In some cases, a nanoparticle described herein further comprises an antibody disposed on its surface. In some instances, theT2025-099 (069596.00097) antibody is specific for a tumor antigen. In some implementations, the tumor antigen comprises CD276, EGFR, or a combination thereof. Additionally, in some embodiments, the antibody is attached to the surface of the nanoparticle via or through a linker. In some further embodiments, the linker comprises DMPE-PEG or DSPE-PEG.

[0014] Any nanoparticle not inconsistent with the technical objectives of the current disclosure may be used. In some instances, a nanoparticle comprises an adenovirus. In some implementations, a nanoparticle comprises a liposome. In some embodiments, a nanoparticle comprises an extracellular vesicle; in some cases, the extracellular vesicle comprises an exosome.

[0015] In yet another aspect, methods of treating a disease in a human patient or subject in need thereof are described. In some embodiments, a method comprises administering to a patient a therapeutically effective amount of a composition described herein. The composition may comprise any expression vector or expression plasmid disclosed herein. The composition may further comprise any nanoparticle described herein. In some embodiments, the disease is cancer. In some embodiments, the cancer comprises a heterogeneous cancer and / or a metastatic cancer. In some embodiments, the cancer comprises non-small cell lung cancer, glioblastoma multiforme (GBM) or triple-negative breast cancer (TNBC).

[0016] In yet another aspect, methods of upregulating tumoral immunity in a human patient in need thereof are described. In some embodiments, a method comprises administering a therapeutically effective amount of a composition described herein. The composition may comprise any expression vector or expression plasmid disclosed herein. The composition may further comprise any nanoparticle described herein.

[0017] In yet another aspect, methods of reducing the mass of cancer stem cells in a human patient in need thereof are described. In yet another aspect, methods of inducing differentiation of cancer stem cells in a human patient in need thereof are described. In some embodiments, the method comprises administering a therapeutically effective amount of a composition described herein. The composition may comprise any expression vector or expression plasmid disclosed herein. The composition may further comprise any nanoparticle described herein. In some embodiments, the cancer stem cells are non-small cell lung cancer stem cells, glioblastoma multiforme (GBM) stem cells, or a triple-negative breast cancer (TNBC) stem cells.T2025-099 (069596.00097)

[0018] Moreover, it is to be understood that the method of administration of any composition disclosed herein is not limited. For example, in some cases, administration of the composition may be done intravascularly or intravenously. In some embodiments, administration may be done intracerebroventricularly.

[0019] In a variation of any aspect or embodiment, a method disclosed herein further comprises administering a luciferase substrate. In some cases, the luciferase substrate is administered simultaneously with or sequentially to a composition disclosed herein. In some cases, the luciferase substrate is administered after a composition disclosed herein. In another variation of any aspect or embodiment, a method disclosed herein further comprises administering a chemotherapeutic. In some variations, the chemotherapeutic is administered simultaneously with or sequentially to a composition disclosed herein.

[0020] In yet another aspect, disclosed herein is a kit comprising (a) instructions for treating a cancer, (b) a luciferase substrate and (c) a composition disclosed herein. The composition may comprise any expression vector or expression plasmid disclosed herein. The composition may further comprise any nanoparticle described herein. In some embodiments the composition disclosed herein is co-packaged with the luciferase substrate. In some embodiments, the composition disclosed herein is co-formulated with the luciferase substrate. In one embodiment, the kit further comprises at least one chemotherapeutic agent. In some embodiments, the kit comprises a plurality of dosage forms, the plurality of dosage forms comprising one or more doses. In one embodiment, each dose comprises a therapeutically effective amount of the composition disclosed herein and / or the luciferase substrate. In one embodiment, the dosage form of the composition is formulated for intravenous administration and the dosage form of the luciferase substrate is formulated for parenteral administration. In one embodiment, the kit further comprises at least one chemotherapeutic.

[0021] The foregoing embodiments and other embodiments are further described in the detailed description which follows.BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1A. is a cartoon drawing of one embodiment of an mLumiOpto expression vector. In this variation, luciferase (e.g., NLuc) and mitochondrial rhodopsin (e.g., ABCB- CoChR, where CoChR is a blue light-gated channelrhodopsin from Chloromonas oogama) genesT2025-099 (069596.00097) are linked via a cleavable 2A linker. FIG. IB is a graphical representation of photostimulation with LED light (0.5 mW / mm2, 24 hours) induced mitochondrial depolarization (measured by Mito View dye) in ABCB-ChR2- and ABCB-CoChR-expressing cells, with a more significant effect in ABCB-CoChR cells. FIG. 1C is a graphical representation of NLuc and Rlu radiance for varying concentrations over time, wherein NLuc emitted much stronger luminescence than RLuc when coupled with ViviRen™. FIG. ID is a diagrammatic mechanism of cmLumiOpto gene therapy. FIG. IE shows confocal images showing high-level mitochondrial CoChR expression in HeLa and MDA-MB-231 cells, indicated by strong overlap of eYFP (fused to CoChR) and MitoTracker dye. FIG. IF shows confocal images showing NLuc-GFP and CoChR-mCherry coexpression in mLumiOpto-transfected MDA-MB-231 cells. FIG. 1G shows In Vivo Imaging System (IVIS)images showing ViviRen™-induced dose-dependent luminescent responses in mLumiOpto transfected cancer cells and luminescence at different concentrations of ViviRen™. FIG. 1H shows confocal images showing ViviRen™-induced dose-dependent mitochondrial depolarization in mLumiOpto-transfected MDA-MB-231 cells and a graph of normalized mitochondrial membrane potential (A m) at different concentrations of ViviRen. FIG. II illustrates a plot of ViviRen™-indUCed dose-dependent cell death in mLumiOpto-transfected MDA-MB-231 cells, with no cytotoxic effect in mock-transfected cells. FIG.1 J illustrates a plot of ViviRen™ cytotoxicity in mLumiOpto-transfected cancer cell lines, including GBM cells (U251 and U87) and TNBC cells (BT-20 and MDA-MB-468). *: P<0.05 vs. control. # P<0.05 vs. ChR2. n=4-6 / group.

[0023] FIG. 2A illustrates a plot of cell viability (%) for Z-VADFMK (pan-caspase inhibitor), Z-DEVD-FMK (caspase 3 inhibitor) and Z-LEHD-FMK (caspase 9 inhibitor) significantly alleviated mLumiOpto-mediated cytotoxicity in MDA-MB-231 cells, whereas Nec- 1 (necrosis inhibitor) and Z-IETD-FMK (caspase 8 inhibitor) had no effect. FIG. 2B shows expression of apoptotic markers cleaved caspase 3 (Casp-3) and PARP significantly increased in mLumiOpto-treated cells compared to controls. FIG. 2C shows Caspase 3 activity significantly increased in mLumiOpto-treated cells compared to controls. FIG. 2D shows the number of TUNEL-positive cells was significantly higher in mLumiOpto-treated cancer cells compared to controls. FIG. 2E shows immunofluorescence imaging showed cytochrome C release in mLumiOpto-treated cancer cells but not in controls. FIG. 2F shows Syto24 staining revealed mLumiOpto-induced substantial DNA damage. FIG. 2G shows Western blotting confirmed DNAT2025-099 (069596.00097) damage by increased Y-H2AX expression in mLumiOpto-treated cells. FIG. 2H shows cyclosporin A (CsA) and MitoQ had no significant effect on mLumiOpto-mediated cancer cell death. *: P<0.05 vs. mLumiOpto (a) or control (else). n=4 / group.

[0024] FIGS. 3 A-H shows characterization of an AAV-mediated mLumiOpto gene delivery in a GBM xenograft mouse model. FIG. 3A shows TEM imaging showed AAV DJ / 8 particles with correct morphology and size (~20 nm). FIG. 3B shows Western blotting confirming AAV viral capsid proteins VP1, VP2, and VP3. FIG. 3C shows ViviRen™ (30 pM) induced strong NLuc luminescence in mLumiOpto AAV-transduced GBM U87cells. FIG. 3D shows mLumiOpto (AAV+ViviRen™) induced dramatic mitochondrial depolarization in U87 cells, as measured by MitoView. FIG. 3E shows mLumiOpto killed 90-99% of GBM U251, U251-TMZ, LN229, and GL261 cells within 72 hours, while AAV or ViviRen™ alone had no significant cytotoxic effect. FIG. 3F shows AAV administered through i.c.v. injection led to remarkably higher mLumiOpto gene (NLuc) expression in GBM tumors compared to those treated through i.v. injection. FIG. 3G shows ViviRen™ elicited strong luminescence in mLumiOpto AAV- transduced GBM xenografts. FIG. 3H shows ex vivo IVIS imaging confirming mLumiOpto expression in GBM xenografts but not in normal organs. n=4-6 / group.

[0025] FIGS. 4A-G represent evaluations of anti-cancer efficacy of mLumiOpto in GBM U87 xenograft mouse model. FIG.4A is a graphical representation of percent survival, showing mLumiOpto treatment significantly extended the survival of GBM xenograft mice compared to AAV and saline. FIG. 4B is a graphical representation of body weight trends among the control and treatment groups. FIG. 4C shows H&E staining of the paraffin section slides of GBM xenograft demonstrating tumor burden reduction by mLumiOpto. FIG. 4D shows IHC staining of tumor slides with antibodies of cleaved caspase 3 and Ki67 indicating apoptosis-induced cell death and inhibition of proliferation, respectively, post-treatment. FIG. 4E shows H&E staining did not detect obvious injury or toxicity in normal organs of the treatment group. FIG. 4F shows IVIS imaging revealed > 10-fold reduction in GBM tumor size in mLumiOpto-treated groups compared to controls (saline and AAV only). Vertical dividing lines indicate mice from different source images. FIG. 4G shows MRI images taken in the late stage of the survival study (i.e., 23 days after the last ViviRen™ injection) confirmed a significant reduction of GBM tumor burden in mLumiOpto-treated groups. *: P<0.0001 vs. Saline. n=8-10 / group.T2025-099 (069596.00097)

[0026] FIGS. 5A-F represent evaluation of anti -cancer efficacy of mLumiOpto in GBM PDX xenograft mouse model. FIG. 5A shows MRI at the endpoint (14 weeks post PDX xenograft) showing reduced tumor burden in mLumiOpto-treated mice compared to controls. FIG. 5B shows H&E staining confirmed reduced tumor cells in GBM PDX xenografts. FIG. 5C shows mLumiOpto treatment significantly extended survival of GBM PDX xenograft mice compared to saline. FIG. 5D is a graphical representation showing body weight profiles were comparable between groups. FIG. 5E shows IHC staining with cleaved caspase 3 and Ki67 antibodies indicated apoptosis activation and proliferation inhibition. FIG. 5F shows H&E staining showed no injury or toxicity in normal organs of the treated group as compared to control group. *: P<0.0001 vs. Saline. n=10-15 / group.

[0027] FIGS 6A-H represent construction and in vitro characterization of one mAb-Exo- AAV FIG. 6A shows a schematic description of mAb-Exo-AAV, in which an anti-EGFR mAb is tagged to the surface of Exo- AAV via linking with a DMPE-PEG-NHS moiety. FIG. 6B shows mAb-Exo-AAV size distribution determined by NanoSight. FIG. 6C shows TEM imaging showing expected mAb-Exo-AAV morphology and size. FIG. 6D shows representation of flow cytometry showing that mAb-Exo-AAV bound strongly to TNBC cells (MDA-MB-468 and MDA-MB-231). FIG. 6E shows confocal imaging confirmed that mAb-Exo-AAV-Cy5.5 is bound to the surface and internalized by MDA-MB-468 cells. FIG. 6F shows confocal imaging showed that AAV-Cy3 transduced over 95% of TNBC cells and started accumulating around the nucleus 30 minutes after incubation. FIG. 6G shows IVIS imaging confirmed ViviRen™-induced bright NLuc luminescence in mAb-Exo-AAV-transduced MDA-MB-231 cells. FIG. 6H shows complete blood cell count demonstrated low peripheral toxicity of mAb-Exo-AAV compared to saline and AAV in BALB / cJ mice. *: p<0.05 vs. Saline. n=5 / group.

[0028] FIGS. 7A-G represent evaluation of anti-cancer efficacy of mAb-Exo-AAV-delivered mLumiOpto in human TNBC MDA-MB-231 xenograft mouse model. FIG. 7A shows live animal IVIS imaging revealed NLuc luminescence overlay with TNBC xenograft. FIG. 7B shows ex vivo IVIS imaging showed mAb-Exo-AAV accumulation in TNBC xenografts, not in normal organs. FIG. 7C shows CoChR gene expression was detected in tumor of mLumiOpto mAb-Exo-AAV mice but not in saline controls. FIG. 7D shows high CoChR expression in TNBC tumors and undetectable expression in normal tissues of mAb-Exo-AAV mice. FIG. 7E shows tumor volume reduced in mLumiOpto-treated TNBC xenograft mice compared to control groupsT2025-099 (069596.00097)(saline, AAV and ViviRen™ alone). FIG. 7F shows H&E staining revealed significant cell death in tumors of mLumiOpto-treated mice. FIG. 7G shows H&E staining showed no injury in normal organs of mLumiOpto-treated mice. *:P<0.05 vs. controls. n=6 / group.

[0029] FIG. 8A-G represent evaluation of anti-cancer efficacy of mAb-Exo-AAV-delivered mLumiOpto in mouse 4T1 immunocompetent xenograft mouse model. FIG. 8 A shows ViviRen™ triggered strong NLuc bioluminescence in mAb-Exo-AAV mice. FIG. 8B shows prolonged ViviRen™ administration effectively inhibited tumor growth in mAb-Exo-AAV mice compared to controls (saline). FIG. 8C shows terminal tumor wet weight in mAb-Exo-AAV mice was significantly lower than controls. FIG. 8D shows flow cytometry showed enriched CD11C+DC (left) and CD8+T (right) cells in the TME of mLumiOpto-treated mice compared with controls (saline). FIGS. 8E-8G show Luminex assays of tumor tissues showing significant upregulation of cytokines TFN-y, IL-ip, IL-2, IL-4, IL-13 and IL-12p70 and minimal changes in immune suppressors IL- 17A and IL-23. *P<0.05 vs. Saline. n=4-6 / group.

[0030] FIG. 9 shows imaging showing neither mLumiOpto plasmid transfection nor ViviRen™ induction alone significantly affected cancer cell ATm.

[0031] FIG. 10 is a graphical representation of normalized ATm.

[0032] FIG. 11 A shows increased cytochrome C release in mLumiOpto-treated cells compared to controls by elevated cytosolic cytochrome C expression. FIG. 11B shows the expression of the autophagy marker LC3B also increased in treated TNBC cells. FIG. 11 C shows the necrosis marker HMGB remained unchanged. FIG. 1 ID shows in GBM U251 cells the apoptosis inhibitor attenuated mLumiOpto-induced cytotoxicity, but necrosis inhibition was trivial. FIG. HE shows intracellular flow cytometry showing the expression of cleaved caspase- 9, cleaved caspase-3 and LC3B, but not the necrosis marker HMGB1, significantly increased following mLumiOpto treatment.

[0033] FIG. 12A shows the cfos promoter mediated higher GFP expression in U87 and MDA-MB-231 compared to non-cancerous normal human astrocytes (NHA) and mammary epithelial cells 184B5, confirming its high cancer selectivity. FIGS. 12B-C shows ViviRen™ induction did not significantly affect the viability of mLumiOpto AAV co-cultured NHA and 184B5 cells.T2025-099 (069596.00097)

[0034] FIG. 13 is a graphical representation of CoCHR relative levels showing that icv (intracerebroventricular) injection achieved better GBM tumor-specific mLumiOpto gene delivery compared to iv (intravenous) injection with higher levels of CoChR expression.

[0035] FIGS. 14A-B shows flow cytometry revealed 80-90% of NLuc+ GBM and TNBC cells in GBM U251 xenograft and TNBC MDA-MB-231 xenograft respectively indicating high in vivo infection efficiency.

[0036] FIGS. 15A-B shows immunofluorescence assay images revealing evident cytochrome C release in the treated group.

[0037] FIG. 16 is a graphical representation of GBM tumor flux (a.u.) showing a reduction in GBM tumor volume in mLumiOpto treated groups compared to saline and AAV only.

[0038] FIG. 17A-B show immunofluorescence staining that detected cytochrome C release from mitochondria to the cytoplasm following mLumiOpto treatment.

[0039] FIG. 18A-G represent evidence mAb-Exo-AAV is a safe vehicle for delivering mLumiOpto genes and mLumiOpto technology has minimal toxicity in healthy animals. FIG. 18A shows mice maintained normal body weight, indicating no major toxicity at the tested dosages. FIGS. 18B-D show whole blood analysis comprising normal counts of erythrocytes (FIG. 18B), leukocytes (FIG. 18C) and thrombocytes (FIG. 18D). FIG. 18E shows H&E staining of major organs revealed no apparent inflammation, apoptosis or necrosis. FIG. 18F shows histology and fractional shortening showing normal cardiac function. FIG. 18G is a graphical representation of serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine between saline and mAb-Exo-AAV-treated BALB / cJ mice.

[0040] FIG. 19 shows NLuc expression in tumor tissue of mAb-Exo-AAV treated MDA- MB-231 xenograft mice.

[0041] FIGS. 20A-B show analysis of immunosuppressive markers CD33 (FIG. 20A) and CD39 (FIG. 20B) revealing slight downregulation of Tregs (14.2-16.9% in saline group vs. 7.9- 15.1% in mAb-Exo-AAV group) and minimal change in myeloid-derived suppressor cells (MDSCs) (7.3-11.6% in saline group vs. 8.39-11.3% in mAb-Exo-AAV treatment group).

[0042] FIGS. 21A-B represent a mechanism study of anti-GBM by cmLumiOpto. FIG. 21A shows scRNA-seq analysis showing reduction of classical (>90%), oligodendrocyte, astrocyte, and radial glial cells (50-90%) and slight increase of fibroblast and pericyte. FIG. 21B is a flowT2025-099 (069596.00097) cytometry graph representing that cmLumiOpto induced reduction of sternness markers in GBM cells.

[0043] FIGS. 22A-B show analysis of GSC treated by cmLumiOpto. FIG. 22A shows flow cytometry showed reduction of sternness markers (Nanog, Nestin, OCT4 & SA100A) and induced GSC cell death in vitro. FIG. 22B shows MRI images on Day 10 showing reduced mass in vivo.

[0044] FIGS. 23A-D represent reduction of TNBC metastasis. FIG. 23 A shows IVIS showing CD276 mAb-Exo-AAV reduced or cleared metastasis in distal metastasis models. FIG. 23B shows IHC staining of CD276+ TNBC xenograft and lung metastasis. Scale bar: 20 pm. FIG. 23C shows images showing lung metastasis reduction. FIG. 23D shows cmLumiOpto destroyed the tumor microenvironment.

[0045] FIGS. 24A-C represent a mechanism study of TNBC treatment with cmLumiOpto. FIG. 24A shows RNA-seq demonstrated that cmLumiOpto downregulated multiple metastasis signaling pathways such as Wnt, Hippo, TGF-0, IL-6 / JAK. / STAT3, IGF-IGFR, and FGF-FGFR. FIG. 24B shows downregulation of DNA damage response, methylation, kinase and GTPase. FIG. 24C shows upregulation of tumor immunity (T) and IL 12 post treatment.

[0046] FIGS. 25A-B illustrate one synthesis of liposomes and exosomes. FIG. 25 A is an illustrative representation of the synthesis of liposomes. The mixture of cholesterol, dioleoylphosphatidylcholine (DOPC), DSPE-PEG-NHS and DSPE-mPEG was evaporated at 60°C and 465 mbar for 2 hours, followed by hydration to form multilamellar particles with diameter of >1 pm and sonication to generate homogeneous liposomes. FIG. 25B is an illustrative representation of biomanufacturing of exosomes in a 2 L stirred tank fed-batch operated bioreactor. The exosomes were harvested at cell viability <40%, filtered via depth filtration and purified using a 300 kDa SEC column. Further buffer exchange was performed to replace elution buffer, and the exosomes were concentrated and stored at -80°C.

[0047] FIGS. 26A-I represent construction of mAb-LNPs, evaluation of their anticancer efficacy, and targeting specificity in vitro and in vivo. FIG. 26A shows TNBC-targeting mAb was tagged to the surface of liposomes via the integrated DSPE-PEG-NHS linker and combined chemotherapies (i.e., gemcitabine (GC) and mertansine (DM1),) were packed into the mAb- LNPs. FIG. 26B shows NanoSight showed homogenous distribution of liposomes with diameter of 103±23 nM. FIG. 26C shows transmission electron microscopy (TEM) image indicated theT2025-099 (069596.00097) high purity and morphology of synthesized liposomes. FIG. 26D shows flow cytometry analysis showed that EGFR mAb has high surface binding rate to TNBC cells (MDA-MB-231 and MDA- MB-468) while low binding to normal breast cells (184B5). FIG. 26E shows live-cell microscopy imaging showed surface binding and internalization of EGFR mAb-LNPs-Cy5 into MDA-MB-468 cells. FIG. 26F shows live-animal IVIS imaging showed in vivo TNBC targeting by EGFR mAb-LNPs-Cy7 in MDA-MB-231-FLuc xenograft in NSG mice. FIG. 26G shows ex vivo images of tumor and organs, including brain, heart, lung, spleen and liver, also confirmed tumor targeting of mAb. FIG. 26H shows dosage effect of mAb-LNPs-drugs on body weight of mice. FIG. 261 shows tumor volume changes post treatment with PBS, mAb-Lipo, and mAb- Lipo-GC / DMl (data represent mean SEM, n = 5).

[0048] FIGS. 27A-H represent construction of dual targeting mAb-Exo, evaluation of their anti cancer efficacy, and targeting specificity in vitro and in vivo. FIG. 27A is a scheme showing the structure of anti-EGFR / CD47 mAb-EV- Verrucarin-A. Purified exosomes were surface tagged with anti-EGFR and anti-CD47 mAb via DSPE-PEG-NHS. FIG. 27B shows size distribution analysis by NanoSight. FIG. 27C shows Western blotting of exosome biomarkers (CD63, HSP70, GAPDH). FIG. 27D shows live-cell confocal microscopy imaging of internalization of EGFR mAb-EV-Cy5 and CD47 mAb-EV-Cy5. Cytoplasm of MDA-MB-468 TNBC cells was labelled with GFP, and mAb-EV was labelled with fluorescent dye Cy5. Scale bar equals 10 pm. FIG. 27E shows live-animal and ex vivo IVIS imaging confirming TNBC targeting ofEGFR / CD47 mAb-EV-Cy7. FIG. 27F shows ICso curves ofVer-A in TNBC cells with GC as control. FIG. 27G is a graphical representation of tumor volume change in 4T1 xenograft models treated with targeting delivered Ver-A compared to GC as control (n = 5-6). FIG. 27H is a graphical representation of body weight change of mice in groups of PBS, Exo, 0.5 mg / kg of EGFR mAb-EV- Ver-A, 0.5 mg / kg of CD47 mAb-EV- Ver-A, 0.5, 1.5, 2.0 and 2.5 mg / kg of EGFR / CD47 mAb-EV- Ver-A.

[0049] FIGS. 28A-D represent CD276 receptor expression in TNBCs. FIG. 28A shows TCGA dataset analysis of CD276 transcript in ER+ / PR+, HER2+ and ER- / PR- / HER2- breast cancers as compared to normal breast tissue. FIG. 28B shows Western blotting analyses of surface CD276 expression in human TNBC cell lines and normal breast epithelial cells. FIG. 28C shows IHC staining of TMA of TNBC patients (n=110). FIG. 28D shows representative IHC images of breast tissues with minimal (normal), low, medium and high CD276 expressions.T2025-099 (069596.00097)

[0050] FIGS. 29A-C represent TNBC-targeting of CD276 mAb. FIG. 29A shows flow cytometry analysis of CD276 surface binding to MDA-MB-231, MDA-MB-468 and 4T1 cells. FIG. 29B shows representative live-animal and ex vivo IVIS images demonstrating CD276 mAb- Cy5.5 accumulation in human TNBC xenografts 24 hours post tail vein injection. FIG. 29C shows representative IVIS imaging showing CD276 mAb-Cy5.5 accumulation in mouse TNBC xenografts.

[0051] FIGS. 30A-F show construction and characterizations of a CD276 mAb-Exo-AAV. FIG. 30A represents the structure of mAb-Exo-AAV. FIG. 30B represents production of Exo- AAV from Viral Production Cells (VPC) in 2-L stirred-tank bioreactor at Temp 37°C, pH 7.0, Agt 210 rpm, and DO 40%. FIG. 30C shows NanoSight Pro assay for mAb-Exo-AAV size distribution and titration. FIG. 30D shows TEM images of mAb-Exo-AAV and free AAV. FIG. 30E shows Western blotting analysis confirming exosome biomarkers of CD9, CD63 and HSP70 and negative marker of Calnexin. FIG. 30F shows an example of a schematic process flow diagram of large-scale Exo- AAV biomanufacturing.

[0052] FIGS. 31 A-F represent in vitro evaluations of CD276 mAb-Exo-AAV. FIG. 31 A shows flow cytometry analysis of TNBC surface binding of Cy7 labelled mAb-Exo-AAV in MDA-MB-231, MDA-MB-468 and 4T1 cells. FIG. 3 IB shows representative confocal images showing internalization of mAb-Exo-AAV-Cy7 in MDA-MB-468 cells. FIG. 31C is a graphical representation of effect of harvest viability on Exo-AAV production yield. FIG. 3 ID is a graphical representation of an anti-cancer mechanism study using MDA-MB-231 cells treated with saline (control), cmLumiOpto, and cmLumiOpto plus inhibitors of Z-VAD-FMK (pancaspase inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), necrostatin (necrosis inhibitor) or Z-IETD-FMK (caspase-8 inhibitor). FIG. 3 IE is a graphical representation of assessing the synergism of cmLumiOpto (0-1,000,000 multiplicity of infection (MOI)) and poly (ADP-ribose) polymerase inhibitor (PARPi) (0-20 pM) using MDA-MB-231 cells. FIG. 3 IF is a graphical representation of the cytotoxic effects of cmLumiOpto, PARPi and cmLumiOpto / PARPi in MDA-MB-231 and MDA-MB-468 cells, n = 3 / group. *P<0.005.

[0053] FIGS. 32A-E represent evaluation of anti-TNBC efficacy in MDA-MB-231 xenografted NSG mouse models. FIG. 32Ais a graphical representation of tumor volume profiles treated with i.v. administration of cmLumiOpto at doses of 2xlO10ptc / kg (low), 10xl010ptc / kg (medium) or 30xl010ptc / kg (high) following Q7Dx3 as indicated by arrow; olaparib (aT2025-099 (069596.00097)PARPi) at dose of 50 mg / kg; cmLumiOpto (medium dose, l Oxl O10ptc / kg-BW) in combination with Olaparib (50 mg / kg); and saline (control). Data were presented as mean ± SEM, n = 6. *P<0.05 vs. saline using ANOVA followed by Dunnett’s t-test. FIG. 32B shows H&E staining of tumor tissues harvested on Day 19. Scale bar equals 20 pm. FIG. 32C shows IHC stained tumor tissues with Ki67 (proliferation marker) antibody and CCasp3 antibody (apoptosis marker). Scale bar equals 20 pm. FIG. 32D shows flow cytometry analysis of dissociated MDA-MB-231 xenograft, n = 4. IxlO6cells were co-stained with 1 pg of AF488 labelled Ki67 antibody and 1 pg of AF647 labelled NLuc antibody. FIG. 32E is a graphical representation of body weight profiles of the mice.

[0054] FIGS. 33A-C represent an assessment of anti-TNBC efficacy in 4T1 metastatic models. Mice were treated with cmLumiOpto (medium dose lOxlO10ptc / kg, i.v. injection on Days 0 and 7) / 01aparib (50 mg / kg, oral administration) and saline (control), n = 5. FIG. 33A shows IVIS imaging of BALB / cJ mice carrying metastatic 4Tl-FLuc. FIG. 33B shows H&E staining of lung tissues with TNBC metastasis. Scale bar equals 20 pm. FIG. 33C shows IHC staining of tumors spot treatment using markers of cell proliferation (Ki67), apoptosis (CCasp3), activated T / NK (CD45), and macrophage cells (F4 / 80). Scale bar equals 20 pm.

[0055] FIGS. 34A-F represent an investigation of anti-cancer mechanism of TNBC treatment with cmLumiOpto. FIG. 34A shows an immunofluorescence assay of cytochrome C release in 4T1 xenografts. FIG. 34B is a graphical representation of a seahorse assay to analyze the mitochondrial function in MDA-MB-231 post treatment, n = 4. FIG. 34C is a graphical representation of Luminex assay analysis of tumor tissues with 4T1 metastasis. FIG. 34D shows RNA-seq demonstrating cmLumiOpto downregulated metastasis signaling of Wnt, TGF- , IL-6 and FGFR. FIG. 34E shows upregulation of tumor immunity (T cells) and IL12. FIG. 34F shows downregulation of DNA damage response and histone lysine methylation.

[0056] FIGS. 35A-D represent an assessment of anti-TNBC efficacy in PDX models. Mice were treated with cmLumiOpto (medium dose) / PARPi (50 mg / kg) and saline (control), n = 4-5. FIG. 35Ais a graphical representation of tumor volume profiles following treatment as indicated with arrow. FIG. 35B is a graphical representation of body weight profiles of treated mice. FIG. 35C shows H&E staining of tumor tissue post treatment to confirm cell death. FIG. 35D shows IHC stained tumor tissues with Ki67 (proliferation marker) antibody and CCasp3 antibody (apoptosis marker).T2025-099 (069596.00097)

[0057] FIG. 36A shows representative THC images of human normal organs stained with the humanized CD276 mAh. Scale bar equals 70 gm. Evaluation of tumor-selectivity of CD276 mAh in mouse. FIG. 36B shows representative IHC images of mouse normal organs stained with a humanized anti-human / mouse CD276 mAb. n=3. Mouse malignancy was used as control. FIG. 36C is a graphical representation of CD276 mAb produced in a stirred-tank bioreactor with volumetric titer of -80-120 mg / L. FIG. 36D is a graphical representation of CD276 mAb purified using a protein A column.

[0058] FIG. 37 shows H&E staining of major organs (brain, heart, lungs, liver, spleen, kidneys) with no signs of inflammation, apoptosis, or necrosis.

[0059] FIGS. 38A-C are representative toxicity assessment in 4T1 metastasis models. FIG. 38A shows IHC staining of lung tissues carrying TNBC metastasis. FIG. 38B is a graphical representation of mice treated with saline, cmLumiOpto and cmLumiOpto / Olaparib. FIG. 38C shows H&E staining of major organs (brain, heart, liver, spleen, kidneys) detected no toxicity or tissue damage in treatment groups.

[0060] FIGS. 39A-D represent the efficacy of cmLumiOpto / PARPi in suppressing TNBC metastasis across distinct preclinical models. FIG. 39A shows IVIS imaging revealed that cmLumiOpto / PARPi treatment significantly reduced TNBC metastases. FIG. 39B is a graphical representation showing therapeutic administration did not impact mouse body weight. FIG. 39C shows H&E staining of lung tissues demonstrated lower metastatic burden three weeks posttreatment.

[0061] FIG. 40 shows H&E staining of major organs (brain, heart, lungs, liver, spleen, kidneys) revealed no signs of inflammation or necrosis, confirming the safety of the therapy described in FIG. 35.

[0062] FIGS. 41A-B are graphical representations of complete blood cell count demonstrating low peripheral toxicity of cmLumiOpto / PARPi in BALB / cJ mice. n=2.

[0063] FIGS. 42A-E represent an anti-CD276 mAb production and evaluation. FIG. 42A is a graphical representation of viable cell density (VCD) and viability of the anti-CD276 mAb after production in a shaker flask at 37°C and 130 rpm. FIG. 42B shows flow cytometric analysis of TNBC cells (MDA-MB-231, MDA-MB-468, and 4T1) surface binding by CD276 mAb. FIG. 42C shows TNBC targeting and internalization of CD276 mAb labelled with AF647 at 2 or 24 hrs. FIGS. 42D-E show live animal and ex vivo imaging confirming the in vivo TNBC-specificT2025-099 (069596.00097) targeting of CD276 mAb-Cy5.5 in 4Tl-FLuc xenografted BALB / cJ models (FIG. 42D) or in MDA-MB-468-FLuc xenografted NSG models (FIG. 42E). Images were captured at 24 hrs. post tail vein injection.

[0064] FIG. 43 A shows an antibody conjugated to a chemotherapeutic (DM1) via a linker (Sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate). FIGS. 43B-G represent the characterization and evaluation of the CD276 antibody drug conjugate (ADC) and CD276 mAb-Exo-AAV. FIG. 43B shows SDS-PAGE of CD276 mAb and mAb-DMl ADC. M: marker, 1 : mAb, 2: ADC. FIG. 43 C shows ADC conjugation confirmation in HPLC equipped with a MAbPac™ HIC-Butyl column. FIG. 43D shows cytotoxicity analysis of DM1 in TNBC MDA-MB-231 cells (■), MDA-MB-468 cells (•) and 4T1 cells (A). FIG. 43E shows cytotoxicity analysis of CD276 ADC in TNBC MDA-MB-231 cells (■), MDA-MB-468 cells (•) and 4T1 cells (A). FIG. 43F shows confocal images showing the internalization of Exo- AAV into MDA-MB-468 cells 24 hrs after incubation. FIG. 43G shows Nanosight Pro analysis of the concentration and particle size distribution of Exo- AAV.

[0065] FIGS. 44A-F represent in vivo anti-TNBC efficacy of CD276 ADC in immunocompetent models and TNBC PDX models. FIG. 44Ais a graphical representation of tumor volume post treatment. FIG. 44B is a graphical representation of changes in mouse body weight (BW) after treatment. FIG. 44C shows HE staining of tumor tissues indicating low tumor intensity and cell death. Scale bar equals to 50 pm. FIG. 44D shows IHC staining of tumor section demonstrating upregulation of tumor immunity, apoptosis, and inhibition of proliferation. Scale bar equals to 50 pm. FIG. 44E is a graphical representation of PDX tumor volume profile. TNBC PDX xenografted in female NSG mice treated with CD276 mAb-DMl and saline following schedule of Q4Dx4 as indicated by the black arrow, n = 7. FIG. 44F is a graphical representation of normalized mouse body weight.

[0066] FIGS. 45A-D represent the synergism of combined cmLumiOpto and CD276 ADC. FIG. 45Ais a graphical representation of the cytotoxic effects of saline, cmLumiOpto, ADC and cmLumiOpto+ADC in MDA-MB-231 and MDA-MB-468 cells, n = 3. FIG. 45B is a graphical representation of the seahorse assay indicating oxygen consumption rate of TNBC MDA-MB- 231 cells post treatment, n = 3. FIGS. 45C-D are graphical representation of multi-plex Luminex assay results identifying several enhanced cytokines in tumor microenvironment by synergism of cmLumiOpto and ADC. n = 4.T2025-099 (069596.00097)

[0067] FIGS. 46A-C represent in vivo anti-TNBC synergy of cmLumiOpto and CD276 ADC in immunocompetent metastatic models. Mice were treated with saline (control), CD276 ADC (low dose of 8 mg / kg-BW), cmLumiOpto (low dose of lOxlO10ptc / kg-BW), and combination of cmLumiOpto with CD276 ADC (same dose as monotherapy), n = 5. FIG. 46A shows IVIS imaging of BALB / cJ mice carrying metastatic 4Tl-FLuc. FIG. 46C shows H&E staining of harvested lung tissue from the mouse with strongest bioluminescent signal. Scale bar equals to 2 mm. FIG. 46C shows H&E staining of lung tissue with TNBC tumor. Scale bar equals to 70 pm.

[0068] FIGS. 47A-C represent anti-cancer mechanisms of combined cmLumiOpto and CD276 ADC using syngeneic models of 4T1 xenografted BALB / cJ mice. FIG. 47A shows RNA- seq demonstrating cmLumiOpto downregulated metastasis signaling of Wnt, TGF-P, IL-6 and FGFR. FIG. 47B shows upregulation of tumor immunity (T cells) and IL12. FIG. 47C shows downregulation of DNA damage response and histone lysine methylation, n = 4 / group.

[0069] FIGS. 48A-D represent in vivo anti-TNBC synergy of CD276 ADC and cmLumiOpto in PDX model. Mice were treated with low dose of combined cmLumiOpto (lOxlO10ptc / kg) and CD276 ADC (8 mg / kg-BW) and saline (control), n = 5. FIG. 48Ais a graphical representation of tumor volume profiles. FIG. 48B is a graphical representation of body weight. FIG. 48C shows H&E staining of tumor. Scale bar equals 70 pm. FIG. 48D shows H&E staining of major organs harvested including brain, heart, lungs, liver, spleen and kidneys. Scale bar equals 70 pm.

[0070] FIGS. 49A-B represent evaluation of toxicity of anti-CD276 mAb and ADC in BALB / cJ mice without TNBC tumor xenograft. FIG. 49A shows H&E staining of major organs (brain, heart, liver, kidney, lung, and spleen) at 28 days post i.v. injection of 20 mg / kg ADC via tail vein. Scale bar equals to 100 pm. n = 7. FIG. 49B is a graphical representation of whole blood analysis showing minimal effect of anti-CD276 mAb on blood cell count and peripheral immunity, n = 7. WBC: white blood cell; NE: neutrophils; LY: lymphocyte; RBC: red blood cell; Hb: hemoglobin; PTL: platelet.

[0071] FIGS. 50A-F represent in vivo anti-TNBC efficacy of CD276 ADC in immunocompromised models. FIG. 50A is a graphical representation of tumor volume post treatment which was started on Day 4 indicated by the black arrow. Data were presented as mean ± SEM. FIG. 50B is a graphical representation of body weight (BW) after treatment. FIG. 50C is a graphical representation of tumor weight on the endpoint. FIG. 50D shows HE staining ofT2025-099 (069596.00097) tumor tissues indicated low tumor intensity and cell death. Scale bar equals to 70 pm. FIG. 50E shows IHC staining of tumor section demonstrated improvement of tumor immunity, apoptosis, and inhibition of proliferation. Scale bar equals to 70 pm. FIG. 50F shows HE staining of major organs.

[0072] FIGS. 51 A-B represent a toxicity evaluation of combined cmLumiOpto and CD276 ADC in immunocompetent metastasis models. FIG. 51A is a graphical representation of mouse body weight profdes during treatment. FIG. 5 IB shows H&E staining of major organs harvested including brain, heart, liver, spleen and kidneys. Scale bar equals 70 pm.DETAILED DESCRIPTION

[0073] Embodiments described herein can be understood more readily by reference to the following detailed description. Elements, apparatus, and methods described herein, however, are not limited to the specific embodiments presented in the detailed description. It should be recognized that these embodiments are merely illustrative of the principles of the present disclosure. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the disclosure.

[0074] In addition, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1.0 to 10.0” should be considered to include any and all subranges beginning with a minimum value of 1.0 or more and ending with a maximum value of 10.0 or less, e.g., 1.0 to 5.3, 1 to 4, 3 to 7, 4.7 to 10.0, 3.6 to 7.9, or 5 to 8.

[0075] All ranges disclosed herein are also to be considered to include the end points of the range, unless expressly stated otherwise. For example, a range of “between 5 and 10,” “from 5 to 10,” or “5-10” should generally be considered to include the end points 5 and 10.

[0076] Further, when the phrase “up to” is used in connection with an amount or quantity, it is to be understood that the amount is at least a detectable amount or quantity (that is, the amount is a non-zero amount). For example, a material present in an amount “up to” a specified amount can be present from a detectable (or non-zero) amount and up to and including the specified amount.T2025-099 (069596.00097)

[0077] Additionally, in any disclosed embodiment, the terms “substantially,” “approximately,” and “about” may be substituted with “within [a percentage] of’ what is specified, where the percentage includes 0.1, 1, 5, and 10 percent.

[0078] It is also to be understood that the article “a” or “an” refers to “at least one,” unless the context of a particular use requires otherwise.

[0079] Compositions and methods of preparation and uses thereof are disclosed. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. The terminology used in the description of the subject matter herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the subject matter.

[0080] The term “effective amount,” as used herein, refers broadly to that amount of a recited composition effective to treat, prevent, or reduce the severity or progression of a disorder in a subject, such as a human subject. This includes improving the subject’s condition (e.g., in one or more symptoms), delaying or reducing the progression of the disease and / or disorder, preventing or delaying the onset of the disorder, and / or changing clinical parameters, disease or illness, etc., as would be well known in the art.

[0081] For example, an effective amount can refer to the amount of a composition that improves a condition in a subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.

[0082] As used herein, the terms “treating,” “treatment,” and the like are used to mean obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disorder or sign or symptom thereof, and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder, or the relief or elimination of a symptom thereof. Thus, treatment includes preventing or protecting against the disease, that is, causing the clinical symptoms not to develop; and / or inhibiting the disease or disorder, that is, arresting or suppressing the development of clinical symptoms; and / or relieving the disease or disorder that is, causing the regression of clinical symptoms; and / or reducing the metastasis of the primary tumor or cancer.T2025-099 (069596.00097)

[0083] Terms such as “administering” or “administration” include acts such as prescribing, dispensing, giving, or taking a substance such that what is prescribed, dispensed, given, or taken is actually contacts the patient’s body externally or internally (or both). Administering when used in reference to a cell includes exposing the cell to a therapy or agent and / or incorporating the therapy or agent into the cell.

[0084] The term “therapeutic dosage” refers to a commonly used dose in clinical practice for the treatment of a disease or condition.

[0085] The “patient” or “subject” treated as disclosed herein is, in some embodiments, a human patient, although it is to be understood that the principles of the presently disclosed subject matter indicate that the presently disclosed subject matter is effective with respect to all vertebrate species, including mammals, which are intended to be included in the terms “subject” and “patient.” Suitable subjects are generally mammalian subjects. The subject matter described herein finds use in research as well as veterinary and medical applications. The term “mammal” as used herein includes, but is not limited to, humans, non-human primates, cattle, sheep, goats, pigs, horses, cats, dog, rabbits, rodents (e.g., rats or mice), monkeys, etc. Human subjects include neonates, infants, juveniles, adults and geriatric subjects. The subject “in need of’ the methods disclosed herein can be a subject that is experiencing a disease state and / or is anticipated to experience a disease state, and the methods and compositions of the invention are used for therapeutic and / or prophylactic treatment.

[0086] Described herein are compositions comprising mitochondrial optogenetics-based gene therapies and their methods of use in the treatment of disease.I. General

[0087] Mitochondria, the cell’s powerhouses, are signaling organelles that regulate cellular processes important for cell growth and function. Mitochondrial genetics and biochemical metabolisms are implicated in various aspects of the cancer cell metastatic cascade, including motility, invasion, microenvironment modulation, plasticity, and colonization. Given their role, mitochondria have emerged as a promising target for cancer treatment. Numerous mitochondrial- targeted anti -cancer therapies, such as mitocans, mitochondriotropics, and mitochondriotoxics, have been developed. However, these therapies typically target specific signaling pathways or proteins, such as hexokinase, Bcl-2 family proteins, thiol redox, and VDAC / ANT, which mayT2025-099 (069596.00097) undergo unpredicted mutations during long-term treatment, leading to drug resistance and reduced efficacy. Consequently, the clinical translation of mitochondrial-targeted therapies has not yet succeeded.

[0088] The mitochondrion consists of two membranes, a relatively permeable outer membrane and a highly folded and impermeable inner membrane (IMM). Proper mitochondrial function relies on maintaining the electrical potential gradient across the IMM, known as ATm. A profound and sustained dissipation of A m is a trigger for cell death. Chemical uncouplers (e g., FCCP and CCCP) or permeability transition pore (mPTP) activators (e.g., Atr and ployP) have been used to depolarize ATm. However, lack of cancer specificity limits their in vivo utility, as mitochondrial activity is important for all cells. Genetic methods can modulate mitochondrial function in specific tissues but do not directly target A m and often cause irreversible side effects. Thus, there is currently a dearth of approaches that can directly and specifically disrupt cancer cell A'Pm.

[0089] Triple-negative breast cancers (TNBCs; HER2-, ER-, PR-) are highly aggressive, metastatic, and heterogeneous, accounting for 15-20% of all breast cancer cases. Despite advances in oncology, TNBCs remain among the most challenging malignancies to treat, with recurrence rates exceeding 50% and poor survival outcomes following primary therapy. Metastasis constitutes over 90% death of cancer patients. Standard chemotherapeutic regimens, such as anthracycline-taxane-based treatments, remain the cornerstone of TNBC management1'*-4’1-4However, these therapies frequently fail to achieve long-term disease control, leading to tumor relapses and therapeutic resistance. Single-agent therapies, such as chemotherapy and monoclonal antibodies (mAbs), have shown limited efficacy in recurrent and metastatic TNBCEx'4’5-8. in contrast, combinatorial strategies have shown greater promise. Notably, the integration of immunotherapy with chemotherapy, exemplified by the combination of Atezolizumab (a PD-L1 immune checkpoint inhibitor) and Abraxane (nab-paclitaxel), has introduced a novel therapeutic avenue for PD-L1+ TNBCEx-4-9'1highlighting the potential of immune-based approaches. In addition, the FDA-approved antibody-drug conjugate sacituzumab govitecan, an anti-Trop-2 monoclonal antibody linked to the topoisomerase I inhibitor SN-38, has emerged as an effective option for patients with refractory TNBCsEx 4’12-14. Despite these advances, current conventional and targeted therapies remain insufficient for highly aggressive TNBCs. The primary challenges include early metastatic spreadEx 4’1?, tumor heterogeneityEx 4’16-T2025-099 (069596.00097)18, low response rates, and the emergence of drug resistance over prolonged treatment durationsEx 4 19-20. Given these obstacles, there is a critical need for innovative therapeutic strategies that can improve patient outcomes.

[0090] Mitochondrial genetics and biochemical metabolism changes are associated with cancer cell metastatic cascade and multiple hallmarks such as motility and invasion, microenvironment modulation, plasticity, and colonization. Not intending to be bound by theory, it is believed that targeting mitochondria could effectively manage TNBC metastasis progression.

[0091] To achieve in vivo targeting of mitochondria, an optogenetic tool referred herein as mitochondrial luminoptogenetics (“mLumiOpto” or “cmLumiOpto”) is described. In one aspect, the mLumiOpto genes disclosed herein comprise an expression vector comprising: a first nucleic acid sequence encoding a channelrhodopsin fusion protein; and a second nucleic acid sequence encoding a luciferase protein, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence; and wherein the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS). In one embodiment, the expressed channelrhodopsin protein is in the IMM of a cell and the expressed emission spectrum-matched luciferase is in the cytosol of the cell. Introduction of a luciferase substrate to the cell triggers luciferase bioluminescence, which in turn activates the channelrhodopsin, leading to mitochondrial depolarization and cancer cell death. In one embodiment, CoChR, a blue light-gated channelrhodopsin from Chloromonas oogama is expressed in the IMM and nanoluciferase (NLuc), an emission spectrum-matched luciferase from the deep-sea shrimp Oplophorus graciHrostris. is expressed in the cytosol of the same cells. In one embodiment, a cancer-enhanced promoter (cfos) was used to selectively express mLumiOpto genes in tumor cells. In another embodiment, a monoclonal antibody-tagged exosome-associated adeno- associated virus (mAb-Exo-AAV) vehicle was used to deliver mLumiOpto genes to cancer cells, such as tumor cells in vivo. Not intending to be bound by theory, it is believed that mLumiOpto effectively induces cancer mitochondrial depolarization and cytotoxicity with the synthesized endogenous bioluminescence. Furthermore, not intending to be bound by theory, it is believed that the mAb-Exo-AAV can facilitate cancer-specific gene delivery and functional expression of mLumiOpto, allowing targeted elimination of cancer cells with minimal side effects viaT2025-099 (069596.00097) synergizing mitochondrial depolarization-mediated cell death and mAb or AAV-mediated in situ immunity within the tumor microenvironment (TME).

[0092] In one aspect, a method is disclosed to selectively deliver an expression vector disclosed herein to cancer cells in vivo by an adeno-associated virus. In another aspect, a method is disclosed to selectively deliver an expression vector disclosed herein to cancer cells in vivo using a cancer-targeted monoclonal-antibody-tagged exosome-associated adeno-associated virus carrying the expression vector.

[0093] Moreover, herein, the ability of mLumiOpto to induce mitochondrial depolarization and cytotoxicity across different cancer cell types, including glioblastoma (GBM) and triplenegative breast cancer (TNBC), was examined. Additionally, the cancer-specific surface binding, internalization, transduction efficiency, biodistribution, and tumor-specific expression of mLumiOpto was assessed. The therapeutic efficacy of mLumiOpto, delivered via AAV or mAb- Exo-AAV, was evaluated in preclinical mouse models with GBM or TNBC xenograft. Herein, the results demonstrated that mLumiOpto effectively induces cancer cell death and significantly reduces tumor burden without impairing normal organs or tissues.

[0094] The sustained and irreversible collapse of the inner mitochondrial membrane (IMM) potential ((A'Pm) drives cells toward apoptosisEx 4,22. Disclosed herein is an advanced cancer mitochondrial-targeted luminoptogenetics system, cmLumiOpto, utilizing endogenous Nanoluciferase (NLuc) bioluminescence to activate light-gated cationic rhodopsin channels (CoChR) in the IMM.Ex 4’23In one embodiment, the system enables controlled, dose-dependent mitochondrial depolarization via administration of a luciferase substrate, e.g. ViviRen™ luciferin, with prolonged Amcollapse leading to persistent DNA damage and apoptotic cell death. As disclosed herein, this approach demonstrated remarkable efficacy in reducing tumor burden and inducing tumor cell death in glioblastoma and TNBC xenograft mouse models.

[0095] To enhance tumor specificity for the methods of treatment disclosed herein, in one embodiment, a tumor antigen-targeted monoclonal antibody -tagged exosome-associated adeno- associated virus (mAb-Exo-AAV) carrying an expression vector is disclosed herein, optionally with a tumor-selective cfos promoter. In some instances, the tumor antigen comprises NK-1R, MET, HER2, Trop-2, Nectin-4, SSTR2, CD19, CD20, CD22, CD33, CD47, CD276 or EGFR or a combination thereof. CD276 (also known as B7-H3, Uniprot: Q5ZPR3)Ex 4,24an immune checkpoint molecule that suppresses natural killer (NK) and T cells responsesEx 4’25'27, isT2025-099 (069596.00097) overexpressed in >80% of breast cancersEx 4’28‘30, making it an attractive target for TNBC- specific therapy. An anti-CD276 mAb, e.g. such as disclosed in WO 2025 / 019340, is capable of binding the extracellular domain of transmembrane CD276, enabling precise TNBC targeting. This mAb exhibits affinity, high cross-species reactivity, cancer specificity, and plasma stability, making it a highly effective therapeutic vector.Ex 431To enhance tumor specificity for the methods of treatment disclosed herein, in one embodiment, a CD276-targeted monoclonal antibody -tagged exosome-associated adeno-associated virus (mAb-Exo-AAV) carrying an expression vector is disclosed herein, optionally with a tumor-selective cfos promoter. In another embodiment, a EGFR-targeted monoclonal antibody-tagged exosome-associated adeno- associated virus (mAb-Exo-AAV) carries an expression vector as disclosed herein, optionally comprising a tumor-selective cfos promoter. Without being bound by theory, such a system not only ensures cancer specific targeting, e.g. TNBC-specific targeting, but also enhances tumoral immunity, creating a dual-action therapeutic strategy providing an improved gene delivery vehicle, particularly with regard to protein- mAb-Exo-AAV, where the protein, such as EGFR, is highly expressed in normal human tissues, such as esophagus, placenta, pancreas, kidney, liver, intestine, and reproductive organs complicating off-target risks and raising safety concerns.

[0096] Mitochondrial depolarization in cancer cells and other cell types induces DNA damage, consistent with blocking DNA damage repair, and can completely eliminate cells treated according to the methods disclosed herein. Given the high prevalence of BRCA1 / 2 inactivation in TNBCs, the FDA has approved poly (ADP-ribose) polymerase inhibitors (PARPi) such as olaparib, which disrupts DNA repair mechanisms in tumor cellsEx 433'35, for TNBC treatment.Ex'4’36’37Despite promising clinical trial results in BRCA1 / 2 wild-type TNBC (NCT02158507)EX 4,34, olaparib alone or in combination with EGFR inhibitor achieves only a moderate 24% response rate. As disclosed herein in one embodiment, cmLumiOpto delivered with CD276 mAb-Exo-AAV in addition to PARPi, such as for example olaparib, leads to a synergistic effect, amplifying DNA damage accumulation and apoptotic cell death through the combined action of cmLumiOpto and PARPi, while activating tumoral immunity via CD276 mAb. Together, these mechanisms enable a multi-pronged attack to eliminate the tumor and ultimately improve treatment outcomes for a cancer, such as for example TNBC.

[0097] In some embodiments, the cancer comprises non-small cell lung cancer, glioblastoma multiforme (GBM), or breast cancer, including triple-negative breast cancer (TNBC). In someT2025-099 (069596.00097) embodiments, the cancer comprises melanoma, leukemia, astrocytoma, lymphoma, or Hodgkin's lymphoma. For each exemplary cancer, the monoclonal antibody-tagged exosome-associated adeno-associated virus (mAb-Exo-AAV) carrying an expression vector as disclosed herein, optionally comprising a tumor-selective cfos promoter, can be prepared with one or more mAb targeting the cells of the cancer of interest.II. Expression Vectors

[0098] In some embodiments, an expression vector or expression plasmid comprising a first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein is disclosed. In some cases, the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane- mitochondrial localization signal (IMM-MLS). In some implementations, the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence.

[0099] It is to be understood that in some cases, when the expression vector is expressed, the channelrhodopsin protein in the mitochondrial membrane of a cell is light-sensitive. In some cases, the luciferase protein is localized in the cytosol of the cell. In some instances, light and / or bioluminescence is produced by the luciferase protein in the presence of a luciferase substrate, such as for example, after administration of a luciferase substrate to the cell. It is also to be understood that in some implementations, light and / or bioluminescence induces channelrhodopsin protein-mediated depolarization (A m) in the mitochondria. Moreover, in some implementations, this depolarization is independent of endogenous proteins (e.g., mitochondrial permeability transition pores). In some cases, this depolarization produces disruption in the inner mitochondrial membrane. In some embodiments, this disruption may produce cell death. As shown herein, the light produced by luciferase, such as for example NLuc, more strongly triggers channelrhodopsin protein-mediated depolarization when the luciferase is in the cytosol, as compared to when the luciferase is localized to the mitochondrial membrane, such as the outer mitochondrial membrane.

[0100] Turning to more detail about the expression vector or expression plasmid, it is to be understood that any expression vector or expression plasmid not inconsistent with the technical objectives of the present disclosure may be used. In some cases, the expression vector orT2025-099 (069596.00097) expression plasmid may comprise a viral vector. Any viral vector not inconsistent with the technical objectives of the present disclosure, including for example, delivering and expressing a channelrhodopsin fusion protein and luciferase protein in a cancer cell as disclosed herein, may be used. For example, in some cases, the viral vector may be an adenoviral vector. In some implementations, the viral vector may be an adeno-associated virus (AAV) vector. In some instances, the viral vector may be a modified AAV vector. For example, in some embodiments, the modified AAV vector may have enhanced tissue tropism. In some cases, the modified AAV vector may be modified to be capable of delivering and expressing the disclosed channelrhodopsin fusion protein and the luciferase protein in the cell of a mammal. In some instances, the modified AAV vector may be based on one or more of several capsid types, including AAV 1, AAV2, AAV5, AAV6, AAV8, AAV9, and AAV11. Moreover, in some embodiments, the capsid proteins of the viral vector may be altered and / or engineered.

[0101] Turning to other viral vectors, in some embodiments, the viral vector may be a retroviral vector or a lentiviral vector. In some embodiments, the lentiviral vector may be based on Human Immunodeficiency Viruses (e.g., HIV-1 and HIV-2) or the Simian Immunodeficiency Virus (SIV). Other viral vectors may be contemplated. For example, vectors derived from viruses such as vaccinia virus, Epstein-Barr virus, sindbis virus, cytomegalovirus, and herpes simplex virus may be employed.

[0102] In some cases, the expression vector or expression plasmid may comprise a nucleotide sequence encoding a first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein. In some embodiments, an expression vector described herein may comprise other genes, as described below. It is to be understood that in some instances, the nucleotide sequence encoding a channelrhodopsin fusion protein, the nucleotide sequence encoding the luciferase protein, and other genes on the expression vector or expression plasmid described herein (i.e., an expression control sequence) may be located on a single expression vector or expression plasmid. However, in some cases, genes described herein may be placed on a plurality of expression vectors or expression plasmids.

[0103] Turning to particular components of expression vectors or expression plasmids described herein, in some embodiments, expression vectors described herein comprise a first nucleic acid sequence encoding a channelrhodopsin fusion protein. In some cases, theT2025-099 (069596.00097) channelrhodopsin fusion protein has at least two domains, an inner mitochondrial membrane- mitochondrial localization signal (IMM-MLS) that can effectively target the fusion protein to an inner mitochondria membrane and a channelrhodopsin ion channel domain that can change the mitochondrial membrane potential (Am) when light is present. For reference purposes herein, it is to be understood that an inner-mitochondrial membrane-mitochondrial localization signal is a leading sequence from a mitochondrial inner membrane protein. The inner mitochondrial membrane-mitochondrial localization signal may be any inner mitochondrial membrane- mitochondrial localization signal not inconsistent with the technical objectives of this disclosure. In some implementations, the IMM-MLS comprises ABCB10. In some embodiments, ABCB10 has the nucleic acid sequence of SEQ. ID No. 1. In some cases, the IMM-MLS comprises ABCB140. In some implementations, ABCB140 has the nucleic acid sequence of SEQ. ID No. 2. In some embodiments, the IMM-MLS comprises the Cytochrome C MLS. In some instances, Cytochrome C MLS has the nucleic acid sequence of SEQ. ID No. 3. In some cases, the IMM- MLS comprises the human renal outer medullary potassium channel (ROMK) MLS. In some embodiments, ROMK MLS has the nucleic acid sequence of SEQ. ID No. 4.

[0104] Turning to the channelrhodopsin ion channel domain, any channelrhodopsin ion channel not inconsistent with the technical objectives of this disclosure may be used. In some embodiments, the channelrhodopsin ion channel domain comprises Chloromonas oogama channelrhodopsin (CoChR). In some cases, Chloromonas oogama channelrhodopsin (CoChR) has an amino acid sequence of SEQ. ID No. 5. In some embodiments, Chloromonas oogama channelrhodopsin (CoChR) has a nucleic acid sequence of SEQ. ID No. 6. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 5 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70- 98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75- 90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85- 99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95- 98%, 98-100%, 98-99%, or 99-100%.

[0105] In some cases, the channelrhodopsin ion channel domain comprises ChR2(hl34R). In some embodiments, ChR2(hl34R) has an amino acid sequence of SEQ. ID No. 7. In some implementations, ChR2(hl34R) has a nucleic acid sequence of SEQ. ID No. 8. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 7T2025-099 (069596.00097) of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75- 95%, 75-90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85- 100%, 85-99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95- 99%, 95-98%, 98-100%, 98-99%, or 99-100%.

[0106] In some cases, the channelrhodopsin ion channel domain comprises CHIEF. In some embodiments, CHIEF has an amino acid sequence of SEQ. ID No. 9. In some implementations, CHIEF has a nucleic acid sequence of SEQ. ID No. 10. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 9 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70- 90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75-90%, 75-85%, 75- 80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85-99%, 85-98%, 85- 95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95-98%, 98-100%, 98- 99%, or 99-100%.

[0107] In some cases, the channelrhodopsin ion channel domain comprises ChrimsonR. In some embodiments, ChrimsonR has an amino acid sequence of SEQ. ID No. 11. In some implementations, ChrimsonR has a nucleic acid sequence of SEQ. ID No. 12. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 11 of 65- 100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70- 99%, 70-98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75- 95%, 75-90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85- 100%, 85-99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95- 99%, 95-98%, 98-100%, 98-99%, or 99-100%.

[0108] In some cases, the channelrhodopsin ion channel domain comprises Chronos. In some embodiments, Chronos has an amino acid sequence of SEQ. ID No. 13. In some implementations, Chronos has a nucleic acid sequence of SEQ. ID No. 14. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 13 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70- 98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75- 90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85-T2025-099 (069596.00097)99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95- 98%, 98-100%, 98-99%, or 99-100%.

[0109] In some cases, the channelrhodopsin ion channel domain comprises CsChR. In some embodiments, CsChR has an amino acid sequence of SEQ. ID No. 15. In some implementations, CsChR has a nucleic acid sequence of SEQ. ID No. 16. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 15 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70- 90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75-90%, 75-85%, 75- 80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85-99%, 85-98%, 85- 95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95-98%, 98-100%, 98- 99%, or 99-100%.

[0110] In some cases, the channelrhodopsin ion channel domain comprises hChR2(C128A). In some embodiments, hChR2(C128A) has an amino acid sequence of SEQ. ID No. 17. In some implementations, hChR2(C128A) has a nucleic acid sequence of SEQ. ID No. 18. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 17 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70- 100%, 70-99%, 70-98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75- 98%, 75-95%, 75-90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80- 85%, 85-100%, 85-99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95- 100%, 95-99%, 95-98%, 98-100%, 98-99%, or 99-100%.

[0111] In some cases, the channelrhodopsin ion channel domain comprises hChR2(C128S). In some embodiments, hChR2(C128S) has an amino acid sequence of SEQ. ID No. 19. In some implementations, hChR2(C128S) has a nucleic acid sequence of SEQ. ID No. 20. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 19 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70- 100%, 70-99%, 70-98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75- 98%, 75-95%, 75-90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80- 85%, 85-100%, 85-99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95- 100%, 95-99%, 95-98%, 98-100%, 98-99%, or 99-100%.

[0112] In some cases, the channelrhodopsin ion channel domain comprises VChRl. In some embodiments, VChRl has an amino acid sequence of SEQ. ID No. 21. In some implementations,T2025-099 (069596.00097)VChRlhas a nucleic acid sequence of SEQ. ID No. 22. Tn some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 21 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70- 90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75-90%, 75-85%, 75- 80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85-99%, 85-98%, 85- 95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95-98%, 98-100%, 98- 99%, or 99-100%.

[0113] In some cases, the channelrhodopsin ion channel domain comprises Cl VI. In some embodiments, C1V1 has an amino acid sequence of SEQ. ID No. 23. In some implementations, C1V1 has a nucleic acid sequence of SEQ. ID No. 24. In some instances, the channelrhodopsin ion channel domain has a sequence identity with SEQ. ID No. 23 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70- 90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75-98%, 75-95%, 75-90%, 75-85%, 75- 80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80-85%, 85-100%, 85-99%, 85-98%, 85- 95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95-100%, 95-99%, 95-98%, 98-100%, 98- 99%, or 99-100%.

[0114] In some embodiments described herein, expression vectors or expression plasmids described herein comprise a second nucleic acid sequence encoding a luciferase protein. In some implementations, when the expression vector is expressed, the luciferase protein is preferably localized to the cytosol. Stated differently, in some implementations described herein, the luciferase protein does not comprise and / or lacks a targeting peptide and / or targeting sequence. Thus, as understood by one of ordinary skill in the art, in some embodiments, a luciferase protein is not a luciferase fusion protein. Moreover, in some cases, a luciferase protein comprises only a luciferase domain. In some cases, the luciferase protein is not localized to nor target the outer mitochondrial membrane.

[0115] As understood by one of ordinary skill in the art, in some cases, a luciferase protein produces a bioluminescence in the presence of a luciferase substrate. Any luciferase substrate not inconsistent with the technical objectives of the current disclosure may be used to produce a bioluminescence from or by a luciferase protein described herein. Non-limiting examples of a luciferase substrate include coelenterazine (CTZ) and CTZ analogues, including but not limited to ViviRen™, EnduRen™, furimazine, fluorofurimazine,and diphenylterazine (DTZ, dehydro-T2025-099 (069596.00097) coelenterazine). The structures of coelenterazine, EnduRen™ and ViviRen™ (by Promega Corporation (Madison, WI)) are:ViviRen™

[0116] In some embodiments, the luciferase substrate is coelenterazine. In some preferred embodiments, the luciferase substrate is ViviRen™. As disclosed herein, there are advantages to choosing a luciferase substrate that efficiently and effectively binds to the chosen luciferase in the cytosol, the luciferase itself is chosen to efficiently and effectively produce light / bioluminescence of the correct wavelength and intensity to trigger the associated lightdependent channelrhodopsin in the mitochondrial membrane, thereby leading to depolarization and cell death.

[0117] Many luciferase proteins and / or luciferase domains may be contemplated by one skilled in the art. In some embodiments, a luciferase protein comprises hRluc. In some instances, hRluc has the amino acid sequence of SEQ. ID No. 25. In some implementations, hRluc has the nucleic acid sequence of SEQ. ID No. 26. In some embodiments, a luciferase protein comprises hGluc. In some instances, hGluc has the amino acid sequence of SEQ. ID No. 27. In some implementations, hGluc has the nucleic acid sequence of SEQ. ID No. 28. In some implementations, a luciferase protein comprises NLuc. In some instances, NLuc has the amino acid sequence of SEQ. ID No. 29. In some implementations, NLuc has the nucleic acid sequenceT2025-099 (069596.00097) of SEQ. ID No. 30. In some cases, a luciferase protein comprises M23hGluc. In some instances, M23hGluc has the amino acid sequence of SEQ. ID No. 31. In some implementations, M23hGluc has the nucleic acid sequence of SEQ. ID No. 32. In some instances, a luciferase protein comprises sbGluc. In some instances, sbGluc has the amino acid sequence of SEQ. ID No. 33. In some implementations, sbGluc has the nucleic acid sequence of SEQ. ID No. 34.

[0118] In some instances, the luciferase protein and / or luciferase domain has a sequence identity with any of SEQ. ID No. 25, SEQ. ID No. 27, SEQ. ID No. 29, SEQ. ID No. 31, or SEQ. ID No. 33 of 65-100%, 65-99%, 65-98%, 65-95%, 65-90%, 65-85%, 65-80%, 65-75%, 65-70%, 70-100%, 70-99%, 70-98%, 70-95%, 70-90%, 70-85%, 70-80%, 70-75%, 75-100%, 75-99%, 75- 98%, 75-95%, 75-90%, 75-85%, 75-80%, 80-100%, 80-99%, 80-98%, 80-95%, 80-90%, 80- 85%, 85-100%, 85-99%, 85-98%, 85-95%, 85-90%, 90-100%, 90-99%, 90-98%, 90-95%, 95- 100%, 95-99%, 95-98%, 98-100%, 98-99%, or 99-100%.

[0119] In some implementations, the luciferase enzyme is smaller than 25, 24, 23, 22, 21, 20, 19, 18 or 17 kDa. In some implementations, the luciferase enzyme has an amino acid sequence that is no more than 250, 225, 200, or 175 amino acids. In some implementations, the luciferase enzyme has a nucleic acid sequence that is no more than 550, 540, 530, 520, 510, 500, 490 or 480 base pairs. In some implementations, the luciferase enzyme’s emission spectrum is matched to or overlaps effectively with the selected channelrhodopsin protein.

[0120] Moreover, in some embodiments, the first nucleic acid sequence encoding a channelrhodopsin fusion protein and a second nucleic acid sequence encoding a luciferase protein are operably linked to one or more expression control sequences. For reference purpose herein, it is to be understood that in some cases, “operably linked” indicates that the expression of a nucleic acid sequence is under the control of an expression control sequence and / or promoter that the sequence is spatially connected to. For example, in some embodiments, the expression control sequence may be positioned at the 5' end of the sequence or 3' end of a sequence under its control. In some cases, the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are operably linked to one expression control sequence and / or the same expression control sequence. However, in some embodiments, the nucleic acid sequence encoding a channelrhodopsin fusion protein and the nucleic acid sequence encoding a luciferase protein are operably linked to different expression control sequences.T2025-099 (069596.00097)

[0121] Turning to the identity of the expression control sequence, in some embodiments, the expression control sequence comprises a promoter. It is to be understood for reference purposes herein, that in some cases, a “promoter” comprises a synthetic or naturally-derived sequence which is capable of conferring, activating or enhancing expression of a polynucleotide in a cell. In some cases, a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of same. A promoter may also comprise distal enhancer or repressor elements, which may be located as much as several thousand base pairs from the start site of transcription. Moreover, in some instances, a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals. In some embodiments, a promoter may regulate the expression of a gene component constitutively or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents. Non-limiting examples of constitutive promoters include but are not limited to the cytomegalovirus (CMV) early enhancer / promoter, the hybrid CMV enhancer / chicken P-actin (CBA) promoter, and the “CAG promoter,” a synthetic promoter constructed from the CMV early enhancer element, the promoter, the first exon and the first intron of chicken P-actin gene, and the splice acceptor of the rabbit P-globin gene. In some embodiments, the CMV promoter has the nucleic acid sequence of SEQ. ID NO. 37. In some instances, the CAG promoter has the nucleic acid sequence of SEQ. ID NO. 38.

[0122] In some cases, the promoter comprises a tissue-specific promoter. Any tissue-specific promoter not inconsistent with the technical objectives of this disclosure may be used. Nonlimiting examples of tissue-specific promoters include but are not limited to the human synapsin I (Synl) gene promoter, the hNSE promoter, the Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, and the chromogranin A (CgA) promoter. In some instances, the Synl promoter has the nucleic acid sequence of SEQ. ID NO. 39. In some implementations, the hNSE promoter has the nucleic acid sequence of SEQ. ID NO. 40. In some embodiments, the CaMKII promoter has the nucleic acid sequence of SEQ. ID NO. 41. In some cases, the CgA promoter has the nucleic acid sequence of SEQ. ID NO. 42.

[0123] In some implementations, the promoter comprises a cancer-specific promoter. Any cancer-specific promoter not inconsistent with the technical objectives of this disclosure may beT2025-099 (069596.00097) used. For example, in some embodiments, a cancer-specific promoter comprises the cfos promoter. In some instances, the cfos promoter has the nucleic acid sequence of SEQ. ID NO.43. In some instances, a cancer-specific promoter comprises the promoter of the insulinoma- associated 1 (INSM1) gene. In some cases, the INSMI promoter has the nucleic acid sequence of SEQ. ID NO. 44

[0124] In some cases, the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are separated by a self-cleavable linker. It is to be understood that for reference purposes herein, in some implementations, a “linker,” such as used to link nucleic acids, is a peptide and / or protein sequence added between proteins to add physical space between proteins to increase the likelihood that the proteins fold independently and / or properly (i.e., not misfolded). In some cases, a linker described herein may comprise a cleavage site. In some cases, this cleavage site may be for proteases or chemical agents. In some implementations, the linker is a self-cleaving peptide. In some instances, the self-cleaving peptide comprises the 2A self-cleaving peptide (2A). It is to be understood that in some embodiments, the 2A self-cleaving peptide is an oligopeptide (usually 19-22 amino acids) located between two proteins in some members of the picornavirus family. Additional non-limiting examples of 2A self-cleaving peptides include are not limited to the porcine teschovirus- 1 2A (P2A), Thoseci asignci virus 2A (T2A), equine rhinitis A virus 2A (E2A), Bombyx mori cytoplasmic polyhedrosis virus (BmCPV 2A), and Bombyx mori infectious flacherie virus (BmIFV 2A). In some embodiments, the 2A self-cleaving peptide has the nucleic acid sequence of SEQ. ID NO. 35 (P2A). In some instances, the 2A self-cleaving peptide has the nucleic acid sequence of SEQ. ID NO. 36 (T2A).

[0125] Moreover, in some cases, a nucleic acid linker may comprise an internal ribosome entry site (IRES) element. It is to be understood that in some cases, an IRES element can be used to create multigene, or polycistronic, messages. In some embodiments, IRES elements are able to bypass the ribosome scanning model of 5' methylated cap dependent translation and begin translation at internal sites. Non-limiting examples of IRES elements include but are not limited to those of the picornavirus family (polio and encephalomyocarditis) and the IRES elements from mammals.

[0126] In some embodiments, the nucleic acid sequences of the components disclosed herein are selected such that the resulting expression vector does not exceed the packing capacity of theT2025-099 (069596.00097) selected AAV. For example, in some embodiments a combination of the following: the nucleic acid sequence encoding a channelrhodopsin fusion protein, comprising one or two domains, an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS) and a channelrhodopsin ion channel domain; the nucleic acid sequence encoding a luciferase protein; one or more expression control sequences, including for example, a cancer-specific promoter, such as for example cfos; a self-cleaveable linker, such as for example 2A, and one or more optional sequences disclosed herein, cumulatively does not exceed the packing capacity of the selected AAV. In some embodiments, the expression vector does not exceed 5.3, 5.2, 5.1 or 5.0 kb base pairs. In some cases, the expression vector does not exceed 4.9 kb base pairs. In some cases, the expression vector does not exceed 4.8, 4.7, 4.6, or 4.5 base pairs.III. Nanoparticles

[0127] In one aspect, compositions comprising nanoparticles are described herein. In some embodiments, nanoparticles described herein comprise, encapsulate, or contain any expression vector or expression plasmid disclosed herein, such as in Section II.

[0128] In some embodiments, a nanoparticle described herein comprises, consists of, consists essentially of, or is a virus and / or a viral particle. In some instances, a virus and / or a viral particle comprises a lentivirus. Any lentivirus not inconsistent with the technical objectives of the current disclosure may be used. Moreover, in some cases, a virus and / or viral particle comprises an adeno-associated virus and / or adenovirus. Any adeno-associated virus (AAV) and / or adenovirus not inconsistent with the technical objectives of the current disclosure may be used.

[0129] In some embodiments, a nanoparticle described herein comprises, consists of, consists essentially of, or is an extracellular vesicle (EV). It is to be understood that in some cases, an extracellular vesicle comprises a closed or bound double-layered membrane lipid vesicle formed from lysosomes and secreted by cells. It is also to be understood that in some implementations, an extracellular vesicle is able to transport and / or carry a payload, such as a molecule or a plurality of molecules on their surface and / or within their lumen. In some implementations, an EV comprises an apoptotic body or cancer-related oncosome. In some instances, apoptotic bodies or cancer related onocosomes have an average size of 1-10 pm. In some embodiments, an EV comprises a microvesicle. In some cases, a microvesicle has anT2025-099 (069596.00097) average size of 100-1000 nm. Tn some instances, an extracellular vesicle comprises, consists of, consists essentially of, or is an exosome. In some implementations, the population of exosomes has an average size of 30-150 nm. In some implementations, the exosomes have an average size of 100-300 nm, 100-200 nm, or 125-190 nm; in some cases the average size of exosome, such as mAh -Exo- AAV is 130±75 nm or 165±25 nm. In some implementations, exosomes have an average size of 150-250 nm, 160-240 nm, 170-230 nm, 180-220 nm, or 190-210 nm. In some embodiments, the exosomes have an average size of 200 nm. In some cases, the exosome contains an adeno-associated virus and / or adenovirus disclosed herein. In some embodiments, the population of extracellular vesicles, such as exosomes, containing one or more AAV carrying the expression vectors as disclosed herein, have an average particle size of 150-250 nm, 160-240 nm, 170-230 nm, 180-220 nm, or 190-210 nm. In some embodiments, the exosomes containing AAV - expression vectors as disclosed herein, have an average size of 200 nm. In some embodiments disclosed herein, the loading of an exosome with AAV-encapsulated expression vectors disclosed in Section I is maximized. In some embodiments, the loading of the extracellular vesicle, such as an exosome, is limited by the upper size limit of the exosome. In some cases, the extracellular vesicle, such as an exosome, has an AAV packing rate of at least 10, 20, 30, 40, 50, 60, 70 or 80 vg / ptc. In some cases, the extracellular vesicle, such as an exosome, has an AAV packing rate of 10-60, 20-50, or 25-35 vg / ptc. In some cases, the AAV packing rate is 30 vg / ptc.

[0130] Extracellular vesicles disclosed herein may be produced from any cell line and / or cell type not inconsistent with the technical objectives of the current disclosure. For example, in some cases, extracellular vesicles described herein may be produced by immune cells (e.g., B lymphocytes, T lymphocytes, or dendritic cells). In some embodiments, extracellular vesicles described herein may be produced by glioma cells, platelets, reticulocytes, neurons, intestinal epithelial cells and tumor cells. Moreover, extracellular vesicles described herein, in some cases, may be produced by dendritic cells, HEK293 cells, HEK293T cells, CHO cells, or human ESC- derived mesenchymal stem cells. Additionally, extracellular vesicles described herein, in some embodiments, may be produced by any autologous patient-derived, heterologous haplotype- matched or heterologous stem cells.

[0131] Extracellular vesicles produced by cells may be isolated from culture media by any method not inconsistent with the technical objectives of the current disclosure. In some cases,T2025-099 (069596.00097) culture media may be centrifuged, fdtered, or some combination thereof to obtain the extracellular vesicles described herein. Any synthetic technique not inconsistent with the technical objectives of the present disclosure may be used to form an extracellular vesicle, including an exosome. In some embodiments, extracellular vesicles comprising the disclosed channelrhodopsin fusion protein and the disclosed luciferase protein are produced by culturing cells expressing the proteins and subsequently isolating the extracellular vesicles from the culture media.

[0132] In some embodiments, a nanoparticle described herein comprises, consists of, consists essentially of, or is a liposome. Any liposome not inconsistent with the technical objectives of the present disclosure may be used. In some embodiments, a liposome comprises cholesterol, l,2-dioleoyl-sn-glycero-3 -phosphocholine, or a combination thereof. In some cases, a liposome described herein may have an average size of 75-125 nm, 75-100 nm, or 100-125 nm.

[0133] In some cases, a nanoparticle described herein further comprises an antibody disposed on its surface. Any antibody not inconsistent with the technical objectives of the current disclosure may be used. In some embodiments, up to 25, 50, 75, 100, 125, 150, 175, 200, 225, or 250 copies of each mAb are on each nanoparticle, such as for example, each Exo-AAV particle. In some embodiments, 10-200 or 20-100 copies of each mAb are on each nanoparticle, such as for example, each Exo-AAV particle. In some embodiments, the antibody is specific for a tumor antigen. Any tumor antigen not inconsistent with the technical objectives of the current disclosure may be used. In some instances, the tumor antigen comprises NK-1R, MET, HER2, Trop-2, Nectin-4, SSTR2, CD 19, CD20, CD22, CD33, CD47, CD276 or EGFR or a combination thereof. In some instances, the tumor antigen comprises NK-1R, MET, HER2, Trop-2, Nectin-4, SSTR2, CD 19, CD20, CD22, CD33, CD47, or combinations thereof. In some instances, the tumor antigen comprises NK-1R, MET, HER2, Trop-2, Nectin-4, CD19, CD20, CD22, CD33, CD47, or combinations thereof. In some instances, the tumor antigen comprises MET, Trop-2, Nectin-4, or combinations thereof. In some instances, the tumor antigen comprises CD276, EGFR, or a combination thereof.

[0134] In some implementations, the antibody is attached and / or immobilized to the surface of the nanoparticle through a linker. It is to be understood that for reference purposes herein, in some implementations, a “linker,” such as used to link an antibody and a nanoparticle, comprises a chemical moiety with a one or more termini configured to bind to an antibody and one or moreT2025-099 (069596.00097) termini configured to bind to a nanoparticle. Any linker not inconsistent with the technical objectives of the current disclosure may be used. In some embodiments, the linker comprises DSPE ( l ,2-distearoyl-w-glycero-3- )hosphoethanolainine), DMPE (1,2-dimyristoyl-sn-glycero- 3 -phosphoethanolamine), PEG (polyethylene glycol), mPEG (methoxy -polyethylene glycol) or combinations thereof In some embodiments, the linker comprises mPEG-DSPE, mPEG-DMPE, PEG-DMPE, PEG-DSPE or combinations thereof. It is to be understood that in some cases, a PEG-DSPE linker comprises a linear PEG phospholipid with a saturated C18 stearoyl fatty acid (octadecanoic acid), which comprises hydrophilic and hydrophobic phospholipid PEG conjugates. It is to be understood that in some cases, a PEG-DMPE linker comprises a linear PEG phospholipid with saturated C14 myristoyl fatty acid (tetradecanoic acid). In some cases, a PEG terminus comprises an activated carboxylic acid N-hydroxylsuccinimide (NHS) ester, which reacts with a primary amine, such as in an antibody or protein. In some embodiments, the nanoparticle surface comprises mAb-linker, wherein the linker comprises DMPE-PEG.

[0135] In some embodiments, the surface of the nanoparticle further comprises a stabilizing moiety (stabilizer). Any stabilizer not inconsistent with the technical objectives of the current disclosure may be bound to the surface of the nanoparticle, such as for example a mAb-Exo- AAV nanoparticle. In particular, a stabilizer bound to the surface of the nanoparticle should not elicit a significant immunogenicity response in the cell and the size of the stabilizer should not interfere with effective penetration of the nanoparticle into a disease cell, particularly a cancer cell. In some embodiments, a group such as DMPE-mPEG, DSPE-mPEG, or a combination thereof, is bound to the surface of the nanoparticle and is not bound to a mAb. In such cases, and without being bound by theory, DMPE-mPEG and / or DSPE-mPEG, are believed to stabilize the mAb-Exo-AAV particle. In some variations, one or more DSPE-mPEG are bound to the surface of the mAb-Exo-AAV.

[0136] In some variations, disclosed herein is an exosome comprising an expression vector comprising: a first nucleic acid sequence encoding a channelrhodopsin fusion protein; and a second nucleic acid sequence encoding a luciferase protein, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence; and wherein the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM- MLS). In some cases, the expression vector is bound to an AAV. In some cases, the surface of theT2025-099 (069596.00097) nanoparticle (Exo-AAV) comprises an mAb bound via a linker, such as for example, a linker comprising DMPE-PEG. In such cases, the product can be designated ‘mAb -Exo- AAV’ In some cases, the mAb targets a cancer surface receptor, such as for example CD276. In some cases, the surface of the exosome is further bound to a stabilizer, such as DSPE-mPEG. In some embodiments, the exosome further comprises a luciferase substrate disclosed herein, including a coelenterazine analogue, such as for example, ViviRen™.

[0137] In some cases, the disclosed nanoparticles may further comprise an agent, such as a therapeutic agent. In some embodiments, the nanoparticles deliver the agent to a target cell. Agents described herein may include but are not limited to therapeutic drugs (e.g., small molecule drugs), therapeutic proteins, and therapeutic nucleic acids (e.g., therapeutic RNA).IV. Methods of Use and Methods of Treating a Disease

[0138] In one aspect, a method of treating a disease in a human patient or subject in need thereof is described herein. In some embodiments, the method comprises administering to a patient a therapeutically effective amount of a composition is described herein. The composition may comprise any expression vector or expression plasmid disclosed herein, including in Section II. The composition may comprise any nanoparticle described herein, including in Section III. Nanoparticles disclosed herein, such as for example exosomes, may comprise any expression vector or expression plasmid described herein in Section II. In some embodiments, the disease is cancer. The type of cancer is not particularly limited. In some embodiments, the cancer comprises a heterogeneous cancer. In some embodiments, the cancer comprises a metastatic cancer. In some embodiments the cancer is both heterogenous and metastatic. In some embodiments, the cancer is a recurrent cancer. In some embodiments, the cancer comprises lung cancer, such as for example, non-small cell lung cancer, glioma, such as for example, glioblastoma multiforme (GBM), or breast cancer, including triple-negative breast cancer (TNBC). In some embodiments, the cancer comprises melanoma, leukemia, astrocytoma, lymphoma, or Hodgkin's lymphoma.

[0139] In yet another aspect, a method of upregulating tumoral immunity in a human patient in need thereof is described herein. The type of tumor cancer is not particularly limited. In some embodiments, the tumor cancer comprises glioma, including GBM, lung cancer, including non- small cell lung cancer, or breast cancer, including TNBC. In some embodiments, the cancerT2025-099 (069596.00097) comprises melanoma, leukemia, astrocytoma, lymphoma, or Hodgkin's lymphoma. In some embodiments, the method comprises administering a therapeutically effective amount of a composition described herein. The composition may comprise any expression vector or expression plasmid disclosed herein, including in Section II. The composition may comprise any nanoparticle described herein, including in Section III. Nanoparticles disclosed herein, such as for example exosomes, may comprise any expression vector or expression plasmid described herein in Section II. Upregulation of tumoral immunity can be evaluated by a variety of methods, including but not limited to, single-cell RNA sequencing, flow cytometry, immunohistochemistry, multiplex Luminex or combinations thereof. In some embodiments, the methods disclosed herein improve tumor immunity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the methods disclosed herein improve tumor immunity by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the methods disclosed herein improve tumor immunity in a range, 5%-50%, 10%-45%, 15%- 40%, 20%-35% or 25%-30%.

[0140] In yet another aspect, methods of reducing the mass of cancer stem cells, methods of inducing differentiation of cancer stem cells or combinations thereof in a human patient in need thereof are described. In some embodiments, the methods comprise administering a therapeutically effective amount of a composition described herein. The composition may comprise any expression vector or expression plasmid disclosed herein, including in Section II. The composition may comprise any nanoparticle described herein, including in Section III. Nanoparticles disclosed herein may comprise any expression vector or expression plasmid described herein in Section II. In some embodiments the cancer stem cells are heterogenous and / or metastatic cancer cells. In some embodiments, the cancer stem cells are recurrent cancer stem cells. In some embodiments, the cancer stem cells are lung cancer stem cells, such as for example, non-small cell lung cancer cells, glioma stem cells, such as GBM stem cells, or breast cancer stem cells, such as TNBC stem cells. In some embodiments, the stem cells comprise melanoma stem cells, leukemia stem cells, astrocytoma stem cells, lymphoma stem cells, or Hodgkin's lymphoma stem cells.

[0141] Moreover, it is to be understood that in some cases, a “therapeutically effect amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms. The specific therapeutically effective dose level for any particularT2025-099 (069596.00097) patient will depend upon a variety of factors including the disorder being treated and the severity of the disease; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition employed and like factors well known in the medical arts.

[0142] Compositions disclosed herein may be administered to a patient in any route not inconsistent with the objectives of the current disclosure. In some cases, compositions described herein will be administered by any one of the following routes: intranasal, parenteral (e.g., intravascular, intramuscular, intravenous, intratumoral, intracerebroventricular, intrathecal or subcutaneous) administration. In some cases, administration may be done intravascularly or intravenously. In some embodiments, administration may be done intracerebroventricularly. Other non-limiting examples of routes of administration include but are not limited to intracerebral administration, subcutaneous administration, or transdermal administration.

[0143] In a variation of any aspect or embodiment disclosed herein, a method further comprises administering a luciferase substrate. Any luciferase substrate not inconsistent with the technical objectives of the current disclosure may be used. For example, non-limiting examples of a luciferase substrate include coelenterazine (CTZ) and CTZ analogues, including but not limited to ViviRen™, EnduRen™, furimazine, fluorofurimazine, and diphenylterazine (DTZ, dehydro-coelenterazine), as well as their variants.

[0144] Analogues of coelenterazine have lower autoluminescence compared to the parent compound and have a higher inherent light output in the presence of luciferase, as well as longer kinetics than coelenterazine. Without being bound by theory, the addition of small blocking groups protect the reaction sites in coelenterazine, reducing the rate of degradation and the spontaneous emission of light that can lead to high background luminescence. The blocking groups can be cleaved by esterases and lipases within cells, thereby enabling binding to and light emission from luciferase. The type of coelenterazine analogue, particularly the identity of possible blocking groups is not particularly limited. In some implementations, methods described herein comprise NLuc as the luciferase and ViviRen™ as the substrate; the pair-generated bioluminescence activates the mitochondrial CoChR channel, leading to A m depolarization without external light.T2025-099 (069596.00097)

[0145] Administration of the expression vector in the nanoparticles of the present application, including mAb-Exo-AAV, can enable expression of the encoded proteins in the targeted cancer cells for some length of time. In some cases, expression of the proteins in the targeted cancer cells is robust after 1, 2, 3, 4, 5, 6, or 7 days, or after 2, 4, 6, or 8 weeks, or after 3, 4, 5, 6, 7, 8, 9 or 10 months.

[0146] In some cases, the luciferase substrate is administered simultaneously or sequentially to a composition disclosed herein. In some cases, the luciferase substrate is administered at the same time as a composition disclosed herein.

[0147] In some cases, a composition disclosed herein is administered to a patient and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days later, the patient is administered both a luciferase substrate and a composition disclosed herein. In some cases, the luciferase substrate is administered with a composition disclosed herein. In some cases, the luciferase substrate is formulated within a nanoparticle disclosed herein, such as a mAb-Exo-AAV.

[0148] In some cases, the luciferase substrate is administered after a composition comprising an expression vector or nanoparticle comprising such an expression vector, as disclosed herein. In some cases, the luciferase substrate is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the composition. In some cases, the luciferase substrate is administered to a patient once after 2, 4, 6, or 8 weeks, or after 3, 4, 5, 6, 7, 8, 9 or 10 months after a composition disclosed herein is administered to a patient; in some cases, the luciferase substrate is administered 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times in such a window of time. In some embodiments, the luciferase substrate is formulated for intranasal, transdermal or parenteral (e.g., intravascular, intramuscular, intravenous, intratumoral, intracerebroventricular, intrathecal or subcutaneous) administration. In some cases, the luciferase substrate is administered intramuscularly, subcutaneously, or transdermally. In some cases, the luciferase substrate is selfadministered by the patient.

[0149] Based on the responsiveness of the disease, particularly cancer, to the administration of a composition disclosed herein and the luciferase substrate, variations in the treatment regimen are possible.

[0150] It is to be understood that in some cases, the methods described herein may further comprise administering one or more chemotherapeutics. Any chemotherapeutic agent not inconsistent with the technical objectives of the current disclosure may be used. Non-limitingT2025-099 (069596.00097) examples of chemotherapeutics include but are not limited to paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, decarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, aspargainase erwinia chyrsanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylatem, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfdzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octretide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, BCG, temsirolimus, bendamustine hydrochloride, triptorelin, aresnic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid. In some implementations, methods of treating a disease in a patient described herein may further comprise administering a poly (ADP-ribose) polymerase inhibitor (PARPi). In some variations, the PARPi is selected from the group consisting of pamiparib (BGB-290), veliparib (ABT-888), olaparib (Lynparza®), rucaparib (Rubraca®), CEP 9722, niraparib (Zejula®), rucaparib, talazoparib (Talzenna®, BMN-673), 4-amino-l,8-naphthalimide, and E7016. In some variations, the PARPi is olaparib. In some variations, the chemotherapeutic is administered simultaneously or sequentially to a composition disclosed herein.T2025-099 (069596.00097)

[0151] In some embodiments, the chemotherapeutic is an antibody drug conjugate (ADC). Any ADC not inconsistent with the technical objectives of the current disclosure may be used. In some cases, the ADC comprises an antibody that binds to or targets a protein or receptor on the surface of cancer cells. In some cases, the antibody targets tumor cells. In some cases, the antibody targets EGFR, NK-1R, MET, HER2, Trop-2, Nectin-4, SSTR2, CD 19, CD20, CD22, CD33, CD47, or CD276.

[0152] In some embodiments, a method disclosed herein comprises administering an FDA- approved chemotherapeutic, wherein the chemotherapeutic monotherapy has a response rate of less than 25%, less than 20%, or less than 15%. As shown herein, administration of a composition disclosed herein with a luciferase substrate in combination with an FDA approved chemotherapeutic having a low response rate yields synergistic therapeutic effect.IV. Kits

[0153] In one aspect, disclosed herein is a kit comprising (a) instructions for treating a cancer, (b) a luciferase substrate and (c) a composition disclosed herein. The composition may comprise any expression vector or expression plasmid disclosed herein, including in Section II. The composition may further comprise any nanoparticle described herein, including in Section III. Nanoparticles disclosed herein, such as exosomes, may comprise any expression vector or expression plasmid described herein in Section II. In one embodiment, the kit further comprises at least one chemotherapeutic disclosed herein.

[0154] In one embodiment, the kit comprises co-packaged composition and luciferase substrate. In another embodiment, the kit comprises a plurality of dosage forms, the plurality of dosage forms comprising one or more doses; wherein each dose comprises a therapeutically effective amount of the luciferase substrate or any composition disclosed herein. In yet another embodiment, the kit comprises co-formulated composition and luciferase substrate. In yet another embodiment, the kit comprises a plurality of dosage forms, the plurality of dosage forms comprising one or more doses; wherein each dose comprises a therapeutically effective amount of the luciferase substrate and any composition disclosed herein. In some cases, any composition disclosed herein is formulated for parenteral (e.g., intravascular, intramuscular, intravenous, intratumoral, intracerebroventricular, intrathecal or subcutaneous) administration; in some cases, a composition is formulated for intravenous, intravascular, or intracerebroventricularT2025-099 (069596.00097) administration. Tn some cases, the luciferase substrate is formulated for parenteral (e.g., intravascular, intramuscular, intravenous, intratumoral, intracerebroventricular, intrathecal or subcutaneous) administration; in some cases, luciferase substrate is formulated for subcutaneous or intramuscular, or for transdermal administration.

[0155] Some embodiments are further illustrated in the following non-limiting Examples.EXAMPLESExample 1Materials and Methods

[0156] The animal studies in this non-limiting Example were conducted according to the Institutional Animal Care and Use Committee (IACUC) Protocols IACUC-2022A00000035 and IACUC-2022A00000029, which were approved by the Institutional Biosafety Committee at Ohio State University.Cell lines and culture media

[0157] Viral Production Cells 2.0 (VPC) (Gibco, Cat# A49784, RRID: RRID:CVCL_0045, Grand Island, NY) were maintained in a chemically defined viral production medium supplemented with 4 mM GlutaMAX in shaker flasks on an orbital shaker at 135 rpm. The human cervical cancer cell line HeLa (CLS, Cat# 300194 / p772_HeLa, RRID:CVCL_0030) were cultured in DMEM supplemented with 10% (v / v) Fetal Bovine Serum (FBS) and 2 mM L- glutamine. The TNBC cell lines MDA-MB-231 (ATCC, Cat# HTB-26, RRID:CVCL_0062, Manassas, VA), MDA-MB-231 -FLuc (GenTarget, Cat# SC059-Puro, RRID:CVCL_YZ80, San Diego, CA), MDA-MB-468 (ATCC, Cat# HTB-132, RRID:CVCL_0419) and BT-20 (ATCC, Cat# HTB-19. RRID :CVCL 0178), human GBM LN-229 cells (ATCC, Cat# CRL-2611, RRID:CVCL_0393), and mouse GMB GL261 cells (Creative Bioarray, Cat# NCL-2108P28, RRID:CVCL_Y003, Shirley, NY) were cultured in DMEM / F12 medium supplemented with 10% FBS, 4 g / L glucose, 4 mM L-glutamine, 100 U / mL Penicillin and 100 pg / mL Streptomycin. The mouse TNBC cell line 4Tl-FLuc (ATCC, Cat# CRL- 2539-LUC2, RRID:CVCL_5I85) was cultivated in RPMI-1640 medium supplemented with 10% FBS and 1% P / S. The human GBM cell lines U87 (ATCC, Cat# HTB-14, RRID: CVCL 0022) and U87-FLuc (ATCC, Cat# HTB- 14-LUC2, RRID:CVCL_UR33) were maintained in EMEM with 10% FBS and 8 pg / mL Blasticidin. Human GBM U251 (MilliporeSigma, Cat# 09063001, RRID:CVCL_0021, Burlington, MA) and drug-resistant U251-TMZ (in-house developed) cells were maintained inT2025-099 (069596.00097)EMEM with immune suppression markers IL-1 P, IL-17A and IL-23, 2 mM L-glutamine, 1% non-essential amino acids (NEAA), 1 mM sodium pyruvate, and 10% FBS. All cell lines were incubated at 37°C and 5% or 8% CO2 in a humidified incubator (Eppendorf, Enfield, CT). All media, supplements, and bioreagents used in this study were purchased from Fisher Scientific (Waltham, MA) unless otherwise specified. All cell lines or PDX lines, except those specified, were commercially purchased, authenticated via polymorphic short tandem repeat analysis at University Genomics Core, and confirmed mycoplasma-free with in-house PCR targeting 16S rRNA genes. The time between cell thaw and use in the experiments was 2-3 weeks.Plasmid Construction

[0158] CMV-ABCB-CoChR-eYFP. The ABCB, CoChR and eYFP gene fragments were amplified from CAG-ABCB-ChR2-eYFP, AAV-Syn-CoChR-GFP (RRID:Addgene_59070), and pcDNA3.1 -PsChR2-eYFP (RRID:Addgene_69057), respectively. The PCR primers are ABCB- forward, ABCB-reverse, CoChR-forward, CoChR-reverse, eYFP l -forward, and eYFP l- reverse (Table 1). These gene fragments were cloned into pcDNA3.1-PsChR2- eYFP backbone vector using the HiFi Assembly Kit (New England Biolabs, Ipswich, MA).

[0159] CMV-ABCBAPoChR-mCheiTy. The ABCB-CoChR gene fragments were amplified from CMV-ABCB-CoChR-eYFP and cloned into pcDNA3.0-Magneto2.0-p2A-mCherry (RRID:Addgene_74308) backbone vector. The primers ABCB-CoChR l -forward and AB CBCoChR l -reverse are listed in Table 1.

[0160] CMV-NLuc-2A-ABCB-CoChR-mCherry. The NLuc, 2A, and ABCB-CoChR-mCherry gene fragments were PCR amplified from pNL-CMV-NLuc (Promega #N1091), pcDNA3.0- Magneto2.0-p2A-mCherry (RRID:Addgene 74308), and CMV-ABCB-CoChR-mCherry, respectively. The amplified genes were cloned into the CMV-ABCB-CoChR-mCherry vector. The PCR primers are NLuc_l -forward, NLuc_l -reverse, 2A-forward, 2A-reserve, ABCB- CoChR_2-forward, and ABCBCoChR_2-reserve (Table 1).

[0161] CMV-NLuc-GFP-2A-ABCB-CoChR-mCherry. The NLuc, GFP, and 2A-ABCB- CoChRmCherry gene fragments were PCR amplified from pNL-CMV-NLuc, CMV-myc-mito- GFP (RRID:Addgene_71542), and CMV-NLuc-2A-ABCB-CoChR-mCherry, respectively. The amplified genes were cloned into CMV-NLuc-2A-ABCB-CoChR-mCherry vector. The primers used are NLuc_2-forward, NLuc_2-reserve, GFP-forward, GFP-reserve, 2A-ABCB- CoChRforward, and 2A-ABCB-CoChR-reserve (Table 1).T2025-099 (069596.00097)

[0162] AAV-DJ / 8-cfos-NJ.uc-2A-ABCB-CoChR. The NLuc-2A-ABCB-CoChR gene fragment was PCR amplified from CMV-NLuc-2A-ABCB-CoChR-mCherry and cloned into the pAAV-DJ / 8 expression vector (Cell Biolabs, San Diego, CA) following the manufacture’s instruction. The primers are NLuc-CoChR_2-forward and NLuc-CoChR_2-reverse (Table 1).Table 1. Primer Sequences.T2025-099 (069596.00097)mAb-Exo-AAV construction and titration

[0163] mA b-Exo-AAV construction. The biosimilar of Cetuximab, anti-epidermal growth factor receptor (EGFR) mAb (Bio X Cell, Lebanon, NH), was tagged to the surface of Exo- AAV via a mPEG-DSPE linker to generate the TNBC -targeting mAb-Exo-AAV. Following the procedure developed in a previous study (22,23), Exo-AAV was labeled with fluorescent dye Cy5.5 PE (only for IVIS imaging) and modified with mPEG-DSPE at a molar ratio of 1 : 10,000:6,000,000 (Exo- A AV: Cy 5.5 mPEG-DSPE). The Exo-AAV-PEG-Cy5.5 was then conjugated with anti-EGFR mAb via a DSPE-PEG-NHS linker with a molar ratio of 1 :2,680: 13,000.T2025-099 (069596.00097)

[0164] Titration and characterizations. The purified AAV was digested with DNAse! to extract ssDNA and titrated using RT-PCR with primers: SEQ. ID No. 71 (forward: 5'- ATTGTCCTGAGCGGTGAAA-3'), SEQ. ID No. 72 (reverse: 5'- CACAGGGTACACCACCTTAAA-3'). The size distribution, morphology, biomarkers and purity of mAb -Exo- AAV were characterized using NanoSight (Salisbury, UK), transmission electron microscopy (TEM), and Western blotting. The AAV packed in each exosome was titrated using RT-PCR with the same primers, and the Exo- AAV was titrated using NanoSight to calculate the AAV packing rate in the Exo (i.e., AAV copy per exosome particle). The surface binging of mAb-Exo-AAV to TNBC was determined using flow cytometry, as previously described (24).Confocal imaging

[0165] Mitochondrial AT / w measurement. Cells were stained with mitochondrial membrane potential fluorescent dye TMRM (100 nM) or Mitoview 633 (25 nM), as previously described (25). The fluorescence of TMRM and Mitoview 633 was imaged with a 543 nm and 635 nm laser, respectively, using an Olympus FV1000 confocal microscope (Olympus, Center Valley, PA). Images were analyzed offline using Imaged software (RRID:SCR_003070, National Institutes of Health, Bethesda, MD).

[0166] Colocalization analysis. Cells cultivated on a 15-mm glass-bottom dish were transfected with CMV-ABCB-CoChR-eYFP plasmid. Forty-eight hours after transfection, cells were loaded with MitoTracker Deep Red (250 nM) for 30 minutes. The CoChR-YFP and MitoTracker were simultaneously imaged with confocal microscopy. For NLuc and CoChR coexpression analysis, cells were transfected with CMV-NLuc-GFP-2A-ABCB-CoChR-mCherry plasmid. Forty-eight hours later, the expression of NLuc-GFP and CoChR-mCherry was simultaneously imaged with confocal microscopy. Images were processed offline using the Imaged software for colocalization analysis.

[0167] mAb-Exo-AAV and AAV transduction analysis. mAb-Exo-AAV and AAV carrying mLumiOpto genes were labelled with sulfo-Cyanine5.5 and sulfo-Cyanine3 (red) from Lumiprobe (Hunt Valley, MD), respectively. TNBC MDA-MB-468 cells were infected with BacMam GFP Transduction Control (green, Fisher) for 24 hours. Then, mAb-Exo-AAV-Cy5.5 was incubated with TNBC MDA-MB-468 cells at 37°C for 2 hours. The fluorescence of GFP and Cy5.5 was imaged with a 510 nm and 694 nm laser, respectively, to monitor theT2025-099 (069596.00097) internalization of mAb-Exo-AAV The AAV-Cy3 was incubated with MDA-MB-468 cells that were stained with DAPI for 20 minutes. The fluorescence of DAPI and Cy5.5 was imaged with a 461 nm and 694 nm laser, respectively.

[0168] Immunofluorescence. Cells cultivated on glass coverslips or tissue sections were fixed in 4% formaldehyde in PBS and treated with PBS containing 10% goat serum and 0.3% Triton X-100 to block nonspecific staining. Samples were then incubated overnight at 4°C with anti- TOMM20 (1 :200 dilution) (Abeam Cat# ab205486, RRID:AB_2943509) and anti-cytochrome C (1:200 dilution) (Cell Signaling Technology Cat#11940, RRID:AB_2637071) primary antibodies. Thereafter, samples were washed with 0.1% BSA in PBS, blocked again with blocking buffer (30 minutes at room temperature), and incubated with 1 :200 diluted secondary antibodies labeled with AF488 and AF647 in 1% BSA, 1% goat serum, and 0.3% Triton X-100 in PBS. Images were acquired using confocal microscopy.In vitro cytotoxicity assay

[0169] Cells were seeded onto 96-well plates at a density of 5xl04cells / mL and transfected with mLumiOpto plasmid (DNA:cells=1.2 pg: lxl06cells). Forty-eight hours later, ViviRen™ (0-60 pm), an engineered luciferin purchased from Promega (P1232), was added to the culture. Mock transfected cells subjected to the same treatment were used as control. Two days later, cell viability was measured using MTT assay (ThermoFisher, Waltham, MA) following the manufacturer’s instruction.

[0170] To delineate the mechanistic pathway underlying mLumiOpto-mediated cytotoxicity, cells were treated with mLumiOpto for 48 hours with or without the presence of a pan-caspase inhibitor Z-VAD-FMK (20 pM), a necroptosis inhibitor 7-Cl-O-Nec-l (100 pM), a caspase-9 specific inhibitor Z-DEVD-FMK (100 pM), a caspase-3 specific inhibitor Z-LEHD-FMK (20 pM), a caspase-8 specific inhibitor (Z-IETD-FMK, 20 pM), an mPTP opening inhibitor cyclosporin A (CsA, 10 pM), or a mitochondrial-specific antioxidant MitoQ (300 nM). Cell viability was measured using a TC20 automated cell counter (BioRad, Hercules, CA) or a MTT assay.Western blotting

[0171] Cell lysate or AAV samples were subjected to SDS-PAGE using NuPAGE 4-12% BisTris gels. After electrophoresis, the proteins were electro-transferred to a PVDF membrane (ThermoFisher) and subjected to immunoblot assay by primary antibodies followed byT2025-099 (069596.00097) secondary antibodies. Antibodies of cleaved caspase 3 (Cell Signaling Technology, Cat#9661 , RRID:AB_2341188), cleaved PARP (Cell Signaling Technology Cat# 9148,RRID:AB 10827981), LC3B (Cell Signaling Technology Cat# 8899, RRID:AB 2797680), cytochrome C (Cell Signaling Technology Cat# 11940, RRID:AB_2637071), and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025) were purchased from Cell Signaling Technology. TOMM20 (Abeam Cat#ab205486, RRID:AB_2943509) was purchased from Abeam. VP1, VP2 and VP3 (ARP American Research Products Cat# 03-61057, RRID:AB_1540382) were obtained from ARP American Research Products (Waltham, MA). Quantification analysis of blots was performed with the Imaged software. Targeted bands were normalized to GAPDH.Xenograft mouse models

[0172] Human GBM cell line xenograft model. Six-week-old nude (J:NU HOM Homozygous for Foxnl<nu>) mice (RRID:IMSR _JAX:007850), with an equal number of males and females, were stereotactically injected with human GBM cells, as previously described (22). Briefly, 0.5xl06U87 cells (ATCC, Cat# HTB-14, RRID: CVCL_0022), a widely used human GMB cell line (26,27), were suspended in 3-pL growth medium and implanted into the frontal region of the cerebral cortex at 0.4 pL / min using a Stoelting Just for Mouse Stereotaxic Instrument (Stoelting, Wood Dale, IL). The burr hole in the skull was closed with sterile bone wax, and 5 mg / kg carprofen was administrated immediately before surgery and every 12-24 hours for 48 hours post-surgery. The intracranially xenografted mice were monitored daily for 7 days and used for in vivo studies.

[0173] Human GBM PDX xenograft models. GBM PDX line was maintained at low passages(2-4) in NSG (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ) mice (RRID:IMSR_JAX:005557) as previously described (28). To establish the PDX model, 0.5xl06PDX cells were intracranially implanted into nude mice following the same process as U87 implantation. When the PDX tumor reached ~1 mm3, detected with MRI in -week 6, the xenografted mice were treated with saline (control) or mLumiOpto. The GBM PDX tumor growth was monitored using MRI until week 14 or tumor volume reached >3.5 mm3.

[0174] Human TNBC xenograft immunocompromised model. Five million MDA-MB-231- FLuc or MDA-MB-231 cells were orthotopically injected into the mammary fat pad of 6-week- old NSG (RRID:IMSR_JAX:005557) female mice. When tumor volume reached -75-100 mm3,T2025-099 (069596.00097) mice were intravenously (i.v.) administered anti-EGFR mAb-Exo-AAV (2x1013ptc / kg-BW), free AAV (60xl013vg / kg-BW), ViviRen™ (2 mg / kg-BW), or saline in 50 pL via tail vein injection (n=6 / group).Four days later, mice in the mAb-Exo-AAV group received daily ViviRen™ injections (2 mg / kg-BW) for three consecutive days. Tumor volumes were measured using an external caliper every two days. At the end of the treatment, tumor tissues and major organs were harvested for paraffin sectioning, H&E staining, and biochemical analysis.

[0175] Mouse TNBC xenograft immunocompetent model. Three million mouse 4T1-Fluc cells (ATCC, Cat# CRL-2539-LUC2, RRID:CVCL_5I85) were subcutaneously injected into the mammary fat pad of 6-week-old BALB / cJ female mice (RRID:IMSR_JAX:000651). When the tumor volume reached -75-100 mm3, mice were randomly divided into two groups (n=6 / group) and received saline or anti-EGFR mAb-Exo-AAV (2xl013ptc / kg-BW) on Day 0 (i.v. injection), followed by ViviRen™ administration (2 mg / kg-BW) via tail vein injection on Days 4-6. Tumor volumes were measured every two days using an external caliper, and the wet weight of the terminal tumors was recorded at the end of the study. Tumor tissues were used for flow cytometry analysis to detect immune cells infiltration in the TME.Bioluminescence imaging

[0176] In vitro imaging. Cells were seeded in clear-bottom black well-plates and transfected with RLuc or NLuc plasmid. After 48 hours, the medium was replaced with colorless medium containing ViviRen™ (0-30 pM). Bioluminescence was detected at 0, 2, 4, 6 and 20 hours using IVIS Lumina Series III (PerkinElmer, Waltham, MA) at 470 nm.

[0177] In vivo imaging. TNBC tumor-bearing mice received a single dose of mLumiOpto mAb- Exo-AAV (IxlO13ptc / kg-BW, i.v. injection) and ViviRen™ (2 mg / kg-BW, i.v. injection). Twenty-four hours later, mice were imaged with IVIS to measure NLuc luminescence. To evaluate tumor-specific targeting and biodistribution, TNBC MDA-MB-231-FLuc xenograft mice were injected with mAb-Exo-AAV (IxlO13ptc / kg-BW, i.v. injection). Mice were imaged after 24 hours with an exposure time of 10 seconds. Tumor-specific targeting was determined by: 1) analyzing the overlay of the NLuc luminescence and tumor; 2) ex vivo IVIS imaging of the harvested tumor and major organs; and 3) analyzing the transcripts of tumor and major organs. Magnetic Resonance Imaging (MRI)

[0178] MRI was performed using the BioSpect 94 / 30USR system (Bruker BioSpin, Billerica, MA) at the Ohio State University Small Animal Imaging Core Facility. T2-weighted scans wereT2025-099 (069596.00097) acquired with the following parameters: TR / TE: 2500 / 33 (ms), FA: 180 (degree), NEX: 2, FOV: 20 mm*15.313, matrix: 256*196, slice thickness: 1mm, slice distance: 1 mm and slices: 18.Mice were anesthetized, and a 0.2 mmole / kg Gadolinium-based contrast agent was administrated intraperitoneally before imaging. The mice were then secured on an animal bed and placed in the MRI scanner. A rectal thermometer was used to measure body temperature, and respiration and heart rate were monitored using the Small Animal Monitoring System (Small Animals Instruments, Stony Brook, NY) during the imaging session.Histological analysis

[0179] Hematoxylin and eosin (H&E) staining. The tissues were dehydrated in ethanol, cleared in xylene, embedded in paraffin, sectioned at 5 pm, and mounted on frosted microscope slides. The paraffin sectioned slides were dewaxed with xylene and gradient hydrated with 100%-50% ethanol and deionized water. The hydrated slides were stained with hematoxylin, rinsed with deionized water, dipped in 1% HC1 in 70% ethanol, immersed in 1% NFUOH for blue color development overnight, and stained with eosin for 30 seconds. The stained slides were dehydrated in 95% and 100% ethanol and cleared in xylene.

[0180] TUNEL assay. Apoptotic cells were assessed using the DeadEnd Fluorometric TUNEL System (Promega, Madison, WI) according to the manufacturer’s instruction. Briefly, the slides were mounted with Permount mounting medium, with the nuclei counterstained with DAPI. The slides were then dried and imaged with a fluorescent microscope. Data were analyzed with the ImageJ software.RNA isolation and transcript expression analysis

[0181] Total RNA was extracted and purified from frozen tissues using the RNeasy Mini Kit (Qiagen, Germantown, MD). The cDNA was synthesized from 500 ng of RNA using the QuantiTect Reverse Transcription Kit (Qiagen). Quantitative real-time PCR (RT-PCR) was performed using Select Master Mix (ThermoFisher) in a BioRad IQ5 detection system (Bio-Rad, Portland, ME). The transcript level of the CoChR and NLuc genes were normalized to the average levels of Gapdh and Rpl32. PCR primers are listed in Table 2, with specificity confirmed by 1% agarose gel electrophoresis and melt curves. Fold differences in mRNA expression were calculated using 2A Cl.T2025-099 (069596.00097)Table 2. PCR Primers.Flow cytometry analysis[00182J Analysis of AAV or mAb-Exo-AAV in vivo infection. AAV (3.3xl012vg / kg-BW) was intracerebroventricularly (i.c.v.) injected to ~2mm GBM U251 xenografted nude mice (n=4). EGFR mAb-Exo-AAV (2x1013 ptc / kg-BW) was i.v. injected to 100mm3 TNBC MDA-MB-231 xenografted NSG mice (n=4). Seven days post-infection, tumor samples were harvested and dissociated with a Tissue Dissociation Kit (lOlBio, Sunnyvale, CA). The IxlO6dissociated tumor cells were stained with 1 pg of anti-Ki-67 antibody labelled with AF488 (Cell Signaling Technology Cat# 9129, RRID:AB_2687446) and 1 pg of NLuc antibody (Promega Cat# N7000, RRID:AB_3095534) labelled with AF647 (Fisher) at 37°C for 30 minutes. The average fluorescence intensity was determined using a FACSCalibur Flow Cytometer (Becton-Dickinson, Franklin Lakes, NJ), and data were analyzed with FlowJo 7.6.1 software (RRID:SCR_008520, TreeStar, Ashland, OR).

[0183] Tumoral immunity analysis. Freshly isolated tumor tissues were dissociated with a tissue dissociation kit following the manufacturer’s instructions. The dissociated tumor cells were stained with an AF488 anti-CD8 antibody (BioLegend Cat# 100723, RRID:AB_389304) and an APC anti-CDl 1c antibody (BioLegend Cat# 100723, RRID:AB_389304) and analyzed by flow cytometry to assess infiltrated immune cells.Luminex assay

[0184] Chemokines and cytokines within the TME were measured using a Luminex -based multiplexing assay kit (Luminex Corporate, Austin, TX). A pre-configured 26-plex chemocytokine assay kit (EPX070-20835-901) was purchased from ThermoFisher. All assay reagents were prepared following manufacturer’s instructions. The Luminex assay wasT2025-099 (069596.00097) performed in 96-well plates provided with the kit, and the raw data of mean fluorescence intensity (MFI) were read using the Luminex MAGPIX with XPONENT software. Whole blood analysis

[0185] The blood samples were drawn from the heart for blood cell count using HemaVet 950FS (Drew Scientific, Miami Lakes, FL). The erythrocytes (red blood cells and hemoglobin) and leukocytes (lymphocytes, monocytes, white blood cells, and neutrophils) were titrated to analyze the general peripheral immune response.Statistical analysis

[0186] The experimental data are presented as mean ± standard error of the mean (SEM). Statistical comparisons among groups were performed using the two-way ANOVA Tukey’s multiple comparisons test or one-way ANOVA Holm- Sidak’s multiple comparisons test. P<0.05 was considered statistically significant. The distribution of data was tested using the Shapiro- Wilk normality test.Results mLumiOpto development

[0187] An expression vector to co-express light-gated rhodopsin in the IMM and an emission spectrum-matched luciferase in the cytoplasm of cancer cells was constructed. Specifically, the mLumiOpto (NLuc-2A-ABCB10-CoChR) was synthesized by cloning CoChR (peak L = 470 nm) and NLuc (peak Xem = 460 nm), which were fused through a cleavable 2A linker, into a pcDNA3.0 expression vector (Fig. 1 A). CoChR was used over the more commonly used ChR2 due to its higher (~ 10-fold) photocurrent and greater efficiency in inducing mOpto-mediated ATm depolarization (Fig. IB). NLuc was selected for its much brighter bioluminescence compared to other discovered blue light-emitting luciferases such as Renilla luciferase (RLuc) (29) when paired with ViviRen™, an engineered luciferin (Fig. 1C). The ABCB10 MLS was fused to the N-terminus of CoChR; without being bound by theory, this helps ensure mitochondrial expression. As represented in Fig. ID, and without being bound by theory, it is believed that the cmLumiOpto gene vector leads to the expression of the COChR protein with cancer-specific promoter (cfos) in the inner mitochondrial membrane (IMM) of cancer mitochondria. In one embodiment, the addition of ViviRen™ in cell cytoplasm causes NLuc’s bright endogenous luminescent signal, which activates the COChR protein channels and causes the protein channels to open. Depolarization of IMM potential of mitochondria causesT2025-099 (069596.00097) mitochondrial death and leads to cell death. ABCB10 MLS led to high-levels of CoChR and mitochondrial-specific CoChR expression across various tumor cell lines, including HeLa and TNBC MDA-MB-231 (Fig. IE), as indicated by the strong overlap between eYFP (fused with CoChR) and MitoTracker (a mitochondrial indicator). Confocal microscopy confirmed the coexpression of NLuc (fused with eGFP) and CoChR (fused with mCherry) in mLumiOpto- transfected MDA-MB-231 cells (Fig. IF). mLumiOpto induces cancer cell mitochondrial depolarization and cytotoxicity

[0188] To validate the functionality and efficiency of mLumiOpto in mediating cancer cell mitochondrial depolarization, MDA-MB-231 cells were transfected with the NLuc-2A- ABCBlO-CoChR plasmid and treated with varying doses (0-100 pM) of ViviRen™. ViviRen™ elicited intracellular NLuc luminescence (Fig. 1G) and ATm depolarization (Fig. 1H) in a dosedependent manner, confirming the functional expression of NLuc and CoChR proteins and the capability of mLumiOpto to depolarize mitochondria. Prolonged exposure to ViviRen™ (48 hours) caused a dose-dependent reduction in cell viability in NLuc-2A-ABCB10-CoChR- transfected MDA-MB-231 cells (Fig. II). This mLumiOpto-mediated cytotoxicity was also observed in multiple human GBM (U251 and U87) and TNBC (BT-20 and MDA-MB-468) cell lines, exhibiting substantial cell death with ViviRen™ induction (Fig. 1 J). Neither mLumiOpto plasmid transfection nor ViviRen™ induction alone significantly affected cancer cell ATm (Figs. 9 -10) and viability (Fig. ILU).

[0189] The mechanisms underlying mLumiOpto-mediated cancer cell death were investigated. The effect of apoptosis inhibitor Z-VAD-FMK and necroptosis inhibitor ecrostatin- 1 (Nec-1) on the viability of mLumiOpto-expressing MDA-MB-231 cells treated with ViviRen™ was examined. Z-VAD- FMK significantly reduced cell death, while Nec-1 had no noticeable effect (Fig. 2A), suggesting activation of caspase-dependent apoptosis. To determine whether the apoptotic pathway was intrinsic or extrinsic, cells were co-treated with ViviRen™ and caspasespecific inhibitors. Both caspase-9 inhibitor Z-DEVD-FMK and caspase-3 inhibitor Z-LEHD- FMK, but not caspase-8 inhibitor Z-IETD-FMK, effectively attenuated mLumiOpto-induced cytotoxicity (Fig. 2A), indicating activation of the intrinsic apoptotic pathway. Apoptosis activation was confirmed by increased expression of apoptosis markers cleaved caspase-3 and PARP (Fig. 2B), caspase-3 activity (Fig. 2C), and TUNEL-positive cells (Fig. 2D) in mLumiOpto-treated cells compared to controls (i.e., mock-transfected). Increased cytochrome CT2025-099 (069596.00097) release in mLumiOpto-treated cells was also observed compared to controls, revealed by punctuated cytochrome C staining disparate from TOMM20 (Fig. 2E) and confirmed by elevated cytosolic cytochrome C expression (Fig. 11 A). The expression of the autophagy marker LC3B also increased in treated TNBC cells (Fig. 11B), while the necrosis marker HMGB remained unchanged (Fig. 11C). Similar results were observed in GBM U251 cells, where the apoptosis inhibitor attenuated mLumOpto-induced cytotoxicity, but necrosis inhibition was trivial (Fig. 11D). Consistently, the expression of cleaved caspase-9, cleaved caspase-3 and LC3B, but not the necrosis marker HMGB1, significantly increased following mLumiOpto treatment, revealed by intracellular flow cytometry (Fig. 1 IE).

[0190] Further investigations revealed nuclear condensation and fragmentation in treated TNBC cells, as observed through Syto24 staining (Fig. 2F). Western blot analysis confirmed DNA damage in mLumiOpto-treated cells, indicated by significantly increased y-H2AX expression (Fig. 2G). The effects of the mPTP inhibitor Cyclosporin A (CsA) and mitochondrial specific antioxidant MitoQ on mLumiOpto-mediated cytotoxicity were also examined, and it was found that neither significantly influenced cell death (Fig. 2H). Not intending to be bound by theory, it is believed that these data indicate that mLumiOpto induces cancer cell cytotoxicity primarily through mitochondrial-mediated intrinsic apoptosis and DNA damage, independent of canonical mPTP opening and excessive oxidative stress.A A V construction and characterization for in vivo gene delivery

[0191] To deliver synthesized mLumOpto genes to cancer cells in vivo, an AAV expression vector was constructed using the commercial hybrid serotype AAV-DJ / 8 with a heparin-binding domain mutation, which shows high infection efficiency in vivo. Additionally, the cfos promoter was utilized to enhance cancer-selective gene expression. AAV was produced in a stirred-tank bioreactor and purified using ion-exchange liquid chromatography as previously described (32). The size (~20 nm) and morphology of purified AAV DJ / 8 were verified using TEM (Fig. 3A). Western blotting confirmed the expression of AAV capsid proteins VP1, VP2, and VP3 (Fig. 3B). The cfos promoter mediated remarkably higher GFP expression in U87 and MDA-MB-231 compared to non-cancerous normal human astrocytes (NHA) and mammary epithelial cells 184B5 (Fig. 12A), confirming its high cancer selectivity. ViviRen™ triggered robust luminescence (Fig. 3C) and substantial mitochondrial depolarization (Fig. 3D) in AAV- transduced U87 cells, demonstrating functional expression of mLumiOpto proteins. Along withT2025-099 (069596.00097) observed mitochondrial collapse, ViviRen™ induction caused dramatic cell death in various AAV-transduced GMB cell lines, including the drug-resistant U251-TMZ cells compared to the controls (saline, AAV only, and ViviRen™ only) (Fig. 3E). ViviRen™ induction did not significantly affect the viability of mLumiOpto AAV co-cultured NHA (Fig. 12B) and 184B5 cells (Fig. 12C).

[0192] To assess the in vivo gene delivery efficiency of AAV, i.v. and i.c.v. injections were compared in the GBM model. qRT-PCR analysis revealed that i.c.v. injection achieved GBM tumor-specific mLumiOpto gene delivery, with remarkably higher levels of NLuc (Fig. 3F) and CoChR (Fig. 13) expression than i.v. injection. Live-animal IVIS imaging (Fig. 3G) and ex vivo imaging of the isolated organs (Fig. 3H) confirmed functional mLumiOpto expression in AAV- transduced (via i.c.v. injection) GBM xenografts but not the normal organs. Flow cytometry revealed 80-90% of NLuc+GBM and TNBC cells in the xenografts (Fig. 14A-B), indicating high in vivo infection efficiency. Consequently, direct intracranial administration was used for subsequent anti-GBM efficacy studies.A A V-delivered mLumiOpto for GBM treatment

[0193] To evaluate mLumiOpto’s anti -tumor efficacy, U87 xenografted mice were randomly divided into four groups (n=8-10 / group) and received i.c.v. injection of saline (control), AAV only (3.3xl012vg / kg-BW), mLumiOpto 1 (AAV dose: 1.6xl012vg / kg-BW) and mLumiOpto 2 (AAV dose: 3.3xl012vg / kg-BW), respectively. Mice in the mLumiOpto groups received ViviRen™ (2 mg / kg-BW) through tail vein injection daily for 3 consecutive days following AAV administration. Survival of mice in mLumiOpto groups was significantly prolonged compared to controls (Fig. 4A), with similar body weight profiles across all groups (Fig. 4B). H&E staining showed a dramatic reduction in tumor burden with mLumiOpto treatment (Fig. 4C). IHC staining revealed increased cleaved caspase-3 and Ki67 expression in mLumiOpto tumors (Fig. 4D), indicating apoptosis induction and proliferation inhibition. Immunofluorescence assay revealed evident cytochrome C release in the treated group, implying mitochondrial depolarization and injury (Fig. 15A-B). No damage to normal organs (brain, heart, lung, liver, spleen, and kidney) was detected (Fig. 4E) and no behavioral changes were observed in the treated mice. IVIS imaging on Day 28 (i.e., 10 days after the last ViviRen™ administration) revealed a dramatic reduction in GBM tumor volume in mLumiOpto groups (Fig. 4F and Fig.T2025-099 (069596.00097)16). Endpoint MRI on Day 44 confirmed reduced tumor burden in both mLumiOpto groups (Fig. 4G).

[0194] The mLumiOpto’s anti-cancer efficacy using a GBM PDX xenograft mouse model was also further investigated. Mice received i.c.v. AAV injections (3.3xl012vg / kg-BW) in weeks 6, 8, and 10, followed by ViviRen™ induction (2 mg / kg-BW). MRI at the endpoint showed remarkable tumor burden reduction in the mLumiOpto-treated group compared to the saline group (Fig. 5A). H&E staining confirmed reduced tumor cell density (Fig. 5B). mLumiOpto significantly extended survival compared to controls (Fig. 5C), without affecting mouse body weights (Fig. 5D). IHC staining with cleaved caspase-3 and Ki67 antibodies indicated mLumiOpto-induced apoptosis and proliferation inhibition (Fig. 5E). Similar to U87 xenograft models, immunofluorescence staining detected obvious cytochrome C release from mitochondria to the cytoplasm, suggesting mitochondrial depolarization following mLumiOpto treatment (Fig.17). H&E staining revealed no injury in major organs including the brain, heart, lung, liver, spleen, and kidney (Fig. 5F). Not intending to be bound by theory, it is believed that these evaluations demonstrate AAV-delivered mLumiOpto may be used to manage GBM through depolarizing mitochondria.Construction and characterization of mAb-Exo-AAV for targeting mLumiOpto delivery in vivo

[0195] To achieve highly efficient and targeted delivery of mLumiOpto genes to cancer cells in vivo, a mAb-Exo-AAV delivery vehicle was developed. A high-quality and high yielding Exo- AAV was produced using Viral Production Cells 2.0 in a 2-L stirred-tank bioreactor. Then, the purified Exo- AAV was surface tagged with an EGFR mAb, cetuximab, via mPEG-DSPE linker, creating TNBC -targeting mAb-Exo-AAV (Fig. 6A). NanoSight analysis showed a mAb-Exo- AAV size distribution of 133.4±74 nm (Fig. 6B), and TEM confirmed the morphology and particle size (Fig. 6C). The packed AAV measurements exhibited high AAV packing rates of 20- 50 vg / ptc of exosomes, with a mean of ~30 vg / ptc. Alexa Fluor 488 dye was used to label mAb and detect the tagging ratio, which revealed 20-100 copies of mAb on each Exo- AAV particle. Flow cytometry analysis showed strong surface binding of anti-EGFR mAb-Exo-AAV to EGFR+TNBC MDA-MB-231 (>60%) and MDA-MB-468 (>95%) cells (Fig. 6D). Confocal microscopy confirmed that Cy5.5-labeled mAb-Exo-AAV bound to the surface of MDA-MB-468 cells within 20 minutes of incubation (Fig. 6E) and internalized within 2 hours. The accumulation of Cy5.5- labeled AAV in >95% of cells 30 minutes post-transduction (Fig. 6F) was also observed,T2025-099 (069596.00097) indicating high transduction efficiency. ViviRen™ triggered strong NLuc bioluminescence in mAb-Exo-AAV-transduced MDA-MB-231 cells, demonstrating functional NLuc expression (Fig. 6G).

[0196] To evaluate the potential immune response of mAb-Exo-AAV in the TME and its effect on general immunity, mAb-Exo-AAV, free AAV, and saline (control) were administered to healthy BALB / cJ mice via tail vein injection. Two weeks later, whole blood samples were collected and analyzed. mAb-Exo-AAV had no significant effect on blood cell counts except for lymphocytes, which were approximately 30% higher than control (Fig. 6H) but within the normal range (0.9-9.3 K / pL). In contrast, free AAV significantly increased white blood cells and neutrophils while reducing monocytes (Fig. 6H). Not intending to be bound by theory, it is believed that these results suggest that mAb-Exo-AAV causes less peripheral immunity compared to free AAV. mAb-Exo-AAV mLumiOpto inhibits tumor growth in preclinical TNBC models

[0197] A comprehensive evaluation of mAb-Exo-AAV-delivered mLumiOpto was conducted for tumor treatment, including dosage tolerance, tumor targeting, biodistribution, and anti-tumor efficacy. To investigate the tolerated dosage and potential toxicity, various doses of mAb-Exo- AAV were i.v. injected into C57BL / 6J mice, followed by administration of ViviRen™ three days later to induce intracellular bioluminescence. The mice maintained normal body weight, indicating no major toxicity at the tested dosages (Fig. 18A). Whole blood analysis reported normal counts of erythrocytes, leukocytes and thrombocytes (Figs. 18B-D). Furthermore, H&E staining of major organs revealed no apparent inflammation, apoptosis or necrosis (Fig. 18E). Consistent with histology, echocardiograph showed normal cardiac function (Fig. 18F). No liver and kidney damage were observed in m Ab -Exo- A AV-treated BALB / cJ mice, indicated by similar serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine between the two groups (Fig. 18G). These findings suggest that mAb-Exo-AAV is a safe vehicle for delivering mLumiOpto genes and mLumiOpto technology has minimal toxicity in healthy animals.

[0198] To assess tumor-specific targeting and biodistribution, TNBC MDA-MB-231 xenograft NSG female mice were i.v. administered anti-EGFR mAb-Exo-AAV. Live-animal IVIS imaging demonstrated strong NLuc luminescence in the tumors (Fig. 7A), indicating that mAb- Exo-AAV specifically targeted tumors. Consistent with in vivo imaging, ex vivo imaging detectedT2025-099 (069596.00097) bright Cy7 fluorescence in tumors, not in normal organs (Fig. 7B). Further biodistribution analysis in MDA-MB-231 xenograft mice revealed significant CoChR (Fig. 7C) and NLuc (Fig. 19) expression in tumor tissue of mAb-Exo-AAV mice but not in that of controls. CoChR expression in normal organs (e.g., heart, brain, lung, spleen, kidney, intestine, pancreas, stomach, colon, and liver) of mAb-Exo-AAV mice was undetectable (Fig. 7D). Collectively, these data demonstrated that anti-EGFR mAb-Exo-AAV specifically targets tumors and delivers mLumiOpto genes to EGFR+TNBC in vivo.

[0199] To assess anti-cancer efficacy, anti-EGFR mAb-Exo-AAV was i.v. injected into MDA-MB-231 xenografts when tumor volume reached 25-50 mm3. Tumor-bearing mice given saline, free AAV, or ViviRen™ only served as controls. Remarkably, TNBC tumor stopped growing shortly after ViviRen™ induction, with tumor volume eventually shrinking in the treatment (i.e., mAb-Exo-AAV+ViviRen™) group. In contrast, tumors grew rapidly in all control groups (Fig. 7E). H&E staining of paraffin-sectioned TNBC tumors, harvested 4-5 days after the last ViviRen™ induction, revealed severe cell death and reduced tumor cell density in mLumiOpto-treated mice compared to controls (Fig. 7F). no apparent damage or injury was observed in normal organs of mLumiOpto-treated mice (Fig. 7G). Together, these results indicate that mAb-Exo-AAV-delivered mLumiOpto has high anti-tumor efficacy and minimal off-target toxicity. The anti-cancer effectiveness of mLumiOpto was further assessed in immunocompetent mouse models. EGFR+mouse TNBC 4T1 xenografted BALB / cJ female mice were administered either saline or anti-EGFR mAb-Exo-AAV on Day 0, followed by daily ViviRen™ injections on Days 4-6. IVIS imaging after the first ViviRen™ induction showed robust luminescent response in 4T1 xenografts (Fig. 8 A), confirming tumor-specific and functional NLuc expression. Similar to the MDA-MB-231 xenografts, 4T1 tumor growth was significantly inhibited in mAb-Exo- AAV mice compared to controls (Fig. 8B). Notably, the terminal tumor wet weight in mAb-Exo- AAV-treated mice was only 6-8% of that in the controls (Fig. 8C). Intriguingly, significant infiltration of CDllc+ dendritic cells (DC) and CD8+ T cells in the TME of mLumiOpto-treated mice compared to the controls was observed (Fig. 8D), suggesting enhanced tumoral immunity. To investigate the immune regulation effect of mAb-Exo-AAV carrying mLumiOpto, a Luminex multiplex assay was performed to measure the levels of chemokines and cytokines in the TME. There was significant upregulation of TFN-y, IL-2, IL-4, IL-13 and IL-12p70, while modulation of IL-10, IL-17A and IL-23 was minimal (Fig. 8E). Additional analysis of immunosuppressiveT2025-099 (069596.00097) markers CD33 and CD39 revealed slight downregulation of Tregs (14.2-16.9% in saline group vs. 7.9-15.1% in mAb-Exo-AAV group) and minimal change in myeloid-derived suppressor cells (MDSCs) (7.3-11.6% in saline group vs. 8.39-11.3% in mAb-Exo-AAV treatment group) (Fig. 20A-B).Discussion

[0200] Mitochondria have been considered as a promising therapeutic target for cancer treatment, but translating this concept into clinical practice has proven challenging. Although certain drugs targeting mitochondria have demonstrated promise in preclinical studies, their effectiveness in clinical trials has been limited due to the low efficacy, drug resistance and side effect from lack of specificity. As shown herein, mLumiOpto, a therapeutic strategy that can specifically and directly destroys cancer mitochondria to induce cancer cell death, is described. In some variations, either AAV or mAb-Exo-AAV platforms may be used to deliver mLumiOpto genes to cancer cells in vivo, which may maximize potency and minimize off-target effects. Preclinical mouse xenograft models demonstrate that AAV (i.c.v. administration) and mAb-Exo- AAV (i.v. administration)-delivered mLumiOpto effectively kill cancer cells and inhibit tumor growth without causing noticeable side effects.

[0201] As disclosed herein, mitochondrial -targeted luminoptogenetics, enables dynamic mitochondrial manipulation both in vitro and in vivo. In one variation, this approach utilizes endogenous bioluminescence from the NLuc-ViviRen™ pair. Luciferases emit light at specific wavelengths when paired with substrates like luciferin. For instance, RLuc emits blue light (kpeak -470 nm) with coelenterazine. NLuc is used herein for its dynamic and efficient mitochondrial control in vivo due to its monomeric structure, stability, high and sustained luminescence at low substrate doses, and non-toxicity to cells. NLuc emits bright, sustained bioluminescence at low ViviRen™ concentrations, and it is small (encoded by a 513-bp gene), ATP-independent, and uniformly distributed intracellularly (19). In vitro studies described herein show that NLuc- ViviRen™ pair-generated bioluminescence effectively activates the mitochondrial CoChR channel, leading to ViviRen™ dose-dependent A m depolarization without external light. Not intending to be bound by theory, it is believed that by harnessing endogenous bioluminescence, mLumiOpto overcomes the challenges of delivering external light to deep tissues and minimizes potential side effects on surrounding healthy tissues. This ability to manipulate mitochondrial function in freely moving animals renders mLumiOpto a tool for in vivo investigations.T2025-099 (069596.00097)

[0202] The capability of mLumiOpto in inducing cancer cell death was validated using in vitro cell lines and in vivo tumor xenograft mouse models. It was found that mLumiOpto induced cancer cell death in a ViviRen™ dose-dependent manner and was cytotoxic to all treated cancer cell types. Animal studies further demonstrated mLumiOpto’s ability to eliminate tumor cells in vivo, effectively inhibiting tumor growth in various xenograft mouse models, including multiple TNBC subtypes and intracranial GBM. This is notable given the lack of effective treatment options for those aggressive and recurrent cancers. Neither ViviRen™ nor mLumiOpto expression alone exhibited any deleterious effects on cancer cell mitochondria and viability. These findings highlight mLumiOpto as a robust and versatile approach for targeted cancer cell death in both preclinical and translational settings. Its ability to effectively combat tumor growth across diverse cancer types positions mLumiOpto as a therapeutic strategy for various malignancies.

[0203] Moreover, these studies provide valuable insights into the mechanisms of mLumiOpto-mediated cancer cell death. It was found that this cell death primarily occurs through the intrinsic apoptotic pathway, accompanied by DNA damage and autophagy, and is independent of mtROS and mPTP opening. These findings highlight mLumiOpto’s ability to eliminate cancer cells without relying on cancer-type-associated proteins or signaling pathways. It is worth noting that the activation of mitophagy in mLumiOpto-treated cancer cells was also detected. Not intending to be bound by theory, while mitophagy does not induce apoptotic cell death through cytochrome C release as a default pathway, under conditions of extreme stress, where mitochondrial damage is too extensive, it is believed that mitophagy can lead to cell death, either through the release of cytochrome C and subsequent apoptosis or through autophagydependent mechanisms.

[0204] A major challenge in current cancer treatment is the development of drug resistance, which compromises the efficacy of conventional therapies. Proposed mechanisms include cancer heterogeneity, the TME, cancer stem cells, inhibition of cell death pathways, and mutations in therapeutic targets (36, 37). mLumiOpto employs heterologous CoChR genes that do not target or rely on cancer-associated proteins or pathways, such as EGFR, VEGF, ALK, PI3K / AKT / mTOR or p53, which are often impaired during long-term treatment. Consequently, not intending to be bound by theory, it believed that mLumiOpto-based approaches may be lessT2025-099 (069596.00097) prone to developing drug resistance and more efficient in eliminating heterogeneous cancer cells compared with conventional therapies.

[0205] AAV vectors have gained considerable attention as a promising approach for delivering therapeutic genes. The FDA has approved AAV-delivered Luxtuma and Zolgensma for treating rare inherited blindness and spinal muscular atrophy, respectively, and numerous clinical studies are exploring AAV-based gene therapy for various diseases (39-41). However, free AAV administered through i.v. injection faces challenges in cancer treatment, including lack of specific targeting, relatively low infection efficiency, and pre-existing AAV neutralizing antibodies in a significant portion of the patients (42-45).

[0206] Exosomes, extracellular nanovesicles secreted by cells, are a vehicle for delivering therapeutic genes, including AAV vectors, due to their low antigenicity and toxicity. Compared to free AAV, Exo-AAV protects genetic material from degradation and increases circulation stability. Additionally, mAbs can bind to specific surface receptors on cancer cells, directing exosomes carrying therapeutic genes to target cells. This combination (mAb-Exo-AAV) allows for targeted gene delivery, enhancing the efficiency and specificity of mLumiOpto therapy. Herein, an mAb-Exo-AAV platform is established by surface tagging an anti-EGFR mAb (targeting 52-89% of TNBC) to Exo-AAV using a DMPE-PEG-NHS linker. Not intending to be bound by theory, it is believed that the integration of mPEG-DSPE into the exosome membrane improves circulation stability. Animal results demonstrated that mAb-Exo-AAV effectively targets tumors, achieving high-level and functional mLumiOpto expression specifically in tumor tissues with undetectable non-specific distribution in normal organs. Moreover, mAb-Exo-AAV allows for facile surface conjugation of various mAbs, including dual mAbs, to Exo-AAV, expanding the applicability of this approach to a broader range of patients and various cancer types or subtypes.

[0207] Herein, the highly efficient and cancer-specific gene delivery capability of anti-EGFR mAb-Exo-AAV is demonstrated, but also its ability to induce an anti-tumor immune response is shown in immunocompetent TNBC xenograft mouse models. Not intending to be bound by theory, it is believed that the mechanisms underlying this enhanced tumoral immunity are multifaceted. Herein, mAb-Exo-AAV, with a high mAb surface tagging rate, facilitates immune responses within the TME. Second, the immune response triggered by AAV capsid immunity can reactivate memory CD8+ T lymphocytes through histocompatibility (MHC) class I presentation,T2025-099 (069596.00097) leading to tumor destruction. Not intending to be bound by theory, it is believed that after specifically targeting tumor cells, mAb-Exo-AAV is internalized to release AAV intracellularly, and the AAV enters the nucleus while its capsid undergoes proteasomal degradation, enhancing MHC surface expression, CD8+T cell activation, and adaptive immunity within the TME. Third, the flow cytometry analysis described herein revealed an increase in DCs in freshly harvested tumor tissues, aligning with the “cross-priming mechanism.” Apoptotic cancer cells release tumor antigens into the TME, which are subsequently captured by antigen-presenting DC cells, facilitating CD8+T cell activation. Not intending to be bound by theory, it is believed these activated CD8+T cells selectively target cancer cells, creating a more favorable TME for immune cell infiltration and further boosting tumoral immunity.

[0208] In summary, this non-limiting Example introduces mLumiOpto, an innovative luminoptogenetic approach targeting cancer mitochondria to trigger cytotoxicity. mAb- Exo- AAV-delivered mLumiOpto is a strategy for inducing targeted cancer cell death and activating immune response activation in the TME. This novel therapeutic strategy addresses major cancer treatment challenges, including reduced drug resistance and enhanced efficacy. Moreover, mLumiOpto technology may be used to treat other challenging cancers, such as recurrent GBM and non-small cell lung cancers, by substituting the cancer-targeting mAb on the surface of Exo- AAV Finally, mitochondria play an essential role in turn ori genesis, metastasis, and sternness, making mLumiOpto a powerful tool in mechanistic studies due to its ability to dynamically modulate mitochondria.EXAMPLE 2

[0209] Herein, cancer mitochondria-targeted luminoptogenetics or cmLumiOpto, which is capable of directly depolarizing cancer cell ATm, reducing GBM heterogeneity and sternness, and inhibiting cancer metastasis, is described. Specifically, cmLumiOpto synthesizes and coexpresses a blue light-gated cationic channelrhodopsin from Chloromonas oogcima (CoChR, XPeak=470 nm) in IMM and an engineered deep-sea shrimp Nanoluciferase (NLuc) generating bright, sustained, endogenous, blue (LpCak=460 nm) bioluminescence in cytosol. Incorporating a tumor-specific promoter (cfos) and mitochondrial leading sequence (ABCB), cmLumiOpto genes (cfos-NLuc-2A-ABCB-CoChR) showed remarkable cancer infection rate in vivo, mitochondrial targeting, and functional gene expression. Induction with engineered NLuc luciferin (ViviRen™)T2025-099 (069596.00097) elicited robust endogenous bioluminescence, which activates mitochondrial CoChR, triggering permeability disruption of cancer cell IMM, mitochondrial damage, proliferation inhibition, and cell death in a dose-dependent manner.

[0210] As described herein, cmLumiOpto provides a gene therapy with several advantages to manage GBM sternness and control cancer metastasis in vivo. First, compared with traditional gene therapy, such as p53 miRNA, chemotherapy and biotherapy, cmLumiOpto directly depolarizes IMM potential to trigger tumor or cancer (such as TNBC) cell death through introducing heterologous genes. Second, without relying on any cell type or subtype-specific signature or signaling transduction, which is frequently impaired during cancer treatment, cmLumiOpto can be less prone to or bypass drug resistance development. Third, the use of recombinant AAV-DJ / 8 as gene delivery vehicle the chosen administration route, coupled with a tumor-specific promoter (cfos) and a mitochondrial leading sequence (ABCB), ensures high specificity for GBM mitochondria. In addition to cfos and ABCB, the application of cancer- targeted mAb-Exo-AAV as gene delivery vehicle enables cmLumiOpto to target metastatic cancer mitochondria with high specificity. Finally, since mitochondria play a role in modulating tumorigenicity, sternness, cancer metastasis, and resistance to radio and chemotherapy, not intending to be bound by theory, it is believed targeting mitochondria with cmLumiOpto overcomes these major challenges in GBM treatment and cancer metastasis clearance.

[0211] Not intending to be bound by theory, it is believed cmLumiOpto is helpful for the investigation of the mechanisms of cancer metastasis and sternness. First, the capability of cmLumiOpto to directly, dynamically, and partially or fully collapse mitochondria (depending on dose) enables the investigation of the mechanisms of cancer metastasis. For instance, cmLumiOpto may reveal the multifaceted cell death mechanisms that are caused by mitochondrial depolarization and understand how mitochondrial collapse or dysfunction downregulates the metastasis hallmarks of motility and vascular invasion, microenvironment modulation, and colonization and their correlated signaling. Second, cmLumiOpto may be employed to explore the multiple synergetic cell death mechanisms triggered by mitochondrial depolarization, the impact of mitochondrial function on GSC status and sternness (proliferation, self-renewal, differentiation), and changes of cell type distribution in the TME to address GBM heterogeneity. Third, the correlation between mitochondrial function and the core metabolism and signaling pathways in metastasis and / or drug resistance may be investigated. Fourth,T2025-099 (069596.00097) integrating cmLumiOpto, scRNA-seq and metabolic analysis may delineate the roles of cellular physiological processes, such as cell cycle and proliferation, ROS generation, apoptosis signaling, DNA damage and repair, necrosis factors and autophagy. The molecular, genetic and metabolic features in metastasis development and GSC inhibition may be delineated. cmLumiOpto in GBM treatmentHeterogeneity and sternness reduction.

[0212] scRNA-seq analysis demonstrated tumor heterogeneity regulation by cmLumiOpto, with decreases in classical glioblastoma cells (>90%), oligodendrocyte, astrocyte and radial glial cells (50-90%) (FIG. 21 A). Flow cytometry results revealed that cmLumiOpto induced reduction of sternness markers in GBM cells (FIG. 2 IB).

[0213] Flow cytometry data showed significant cell death and reduced levels of sternness markers (Nanog, Nestin, OCT4 and S100A4) in GSCs after cmLumiOpto treatment (FIG. 22A). MRI imaging showed that cmLumiOpto also inhibited the growth rate of GSCs by 60-90% within 10 days (FIG. 22B). These results collectively indicate that cmLumiOpto is effective to treat GBMs by reducing heterogeneity and sternness.Evaluation of cmLumiOpto in TNBC treatment cmLumiOpto reduced TNBC metastasis.

[0214] About IxlO64Tl-FLuc cells were injected into 6-week-old BALB / cJ female mice via the tail vein. Cancer metastasis was monitored with IVIS imaging twice a week. Upon metastasis detection, mice received a weekly i.v. injection of 5xl012ptc / kg (medium level of dose) of CD276 mAb-Exo-AAV for 3 wks, followed by administration of 1.85 mg / kg ViviRen™. IVIS revealed complete clearance of TNBC metastases in 7 out of 10 mice and ~ 90% reduction of metastases in 3 out of 10 mice compared to the saline control group within two weeks following delivery of the targeted cmLumiOpto (FIG. 23 A). The TNBC surface markers (HER27ER7PR’ / EGFR+ / CD27 6+) and proliferation markers such as Ki-67 and MCM2 were confirmed with IHC staining of the TNBC colonized in lung tissue (FIG. 23B). The major metastatic sites in this distant metastasis model are lung and liver, as observed in both live-animal IVIS (FIG. 23A) and the harvested organs (FIG. 23C). Further H&E staining of the lung tissues confirmed the metastatic TNBC tumor microenvironment was damaged by CD276 mAb-Exo-AAV carrying cmLumiOpto. Bone metastasis could be achieved with intracardiac injection.T2025-099 (069596.00097)Correlations of cancer metastasis and dysfunctional mitochondria.

[0215] The annotation of cell type (ACT), differential gene expression (DGE), gene set enrichment analysis (GSEA), GO pathway enrichment (GOPE), and functional analysis (FA) were performed to analyze the TNBC metastasis signaling pathways (FIGS. 24A-C).Specifically, the signaling pathways involved in cancer metastasis, including Wnt-P-catenin, TGF-P, PI3K / AKT / mTOR, RAF-MEK-ERK, SATA-3, IL-6 / JAK / STAT3, IGF-IGFR, JNK, OPG / RANK / RANKL, FGF-FGFR, Notch, JAK-STAT, and CaN / NFATCl, were analyzed.Conclusion

[0216] Herein, it is described that cmLumiOpto may be used to treat GBM by reducing heterogeneity and inhibiting / clearing GSCs and reducing cancer metastasis, as shown in TNBC as a model cancer. Not intending to be bound by theory, it is believed cmLumiOpto can provide an effective strategy to treat aggressive, heterogeneous, metastatic tumor and cancers. Moreover, not intending to be bound by theory, it is believed treatment with cmLumiOpto may improve the quality of life and survival rate of cancer patients.EXAMPLE 3

[0217] This study compared multiple such nanoparticle-based delivery vehicles. Cationic lipid nanoparticles (LNPs) and exosome nanoparticles (EV) were synthesized and characterized to verify their structure, size and morphology. Their cancer specificity was tested by surface tagging monoclonal antibody (mAb) developed to target the surface receptor overexpressed TNBCs. Then, the transfection / transduction efficiency of these nanoparticles was evaluated using multiple TNBC cell lines. Finally, the in vivo cancer-targeting and drug delivery were evaluated and compared.Materials and Methods

[0218] Construction ofLNPs - OPC (di oleoylphosphatidylcholine) and cholesterol were mixed with chemotherapy drugs gemcitabine (GC) and mertansine (DM1), and DSPE-mPEG (for stabilization of NPs) (FIG. 25A). DSPE-PEG-NHS linker was used to attach mAb to LNPs (FIGS. 26A-I).

[0219] Construction of Exosomes- HEK293 cells were cultured in a 2 L stirred tank-fed batch bioreactor at pH 7 with DO 50% and agitation at 70 rpm (FIG. 25B). Harvested and purified exosomes were incubated with chemo drugs for 24 hours at room temperature.T2025-099 (069596.00097)

[0220] TNBC targeting mAh and conjugation - EGFR / CD47 mAb were surface tagged via a DSPE-PEG-NHS linker (FIG. 27A-B). Flow cytometry and live-cell confocal imaging were employed to test anti-TNBC efficacy and targeting specificity (FIG. 27C-E).

[0221] In vitro characterization - MTT proliferation assays were utilized to verify anticancer cytotoxicity (FIG. 27F).

[0222] TNBC xenografts and in vivo targeting efficacy - MDA-MB-231-FLuc cells or 4T1- Fluc cells were subcutaneously xenografted in NSG or BALB / cJ female mice (FIGS. 27E-27G, FIGS. 28A-F).

[0223] Cationic lipid nanoparticles (LNPs) and exosome nanoparticles (EV) were synthesized and characterized to verify their structure, size, and morphology. Their cancer specificity was tested by surface tagging with monoclonal antibodies (mAb) that were used to target the surface receptor overexpressed TNBCs. Then, the transfection / transduction efficiency of these nanoparticles was evaluated using multiple TNBC cell lines. Finally, the in vivo cancertargeting and drug delivery methods were evaluated and compared. Taken together, this study provided a comparison of nanoparticles for cancer targeting and therapy.

[0224] Without being bound by theory, the combination of chemotherapies with different anti-cancer mechanisms (gemcitabine and mertansine in this study) has great potential to treat the highly aggressive TNBC.

[0225] The anti-cancer efficacy of mAb-LNPs and mAb-EV nanoparticles can be tested against the heterogenous and aggressive nature of TNBCs using in vivo patient derived xenograft (PDX) models.EXAMPLE 4

[0226] This study aimed to develop and evaluate a combinatorial therapeutic strategy for effectively targeting aggressive TNBCs. The specificity, gene packaging efficiency, and cytotoxic potential of CD276 mAb and mAb-Exo-AAV carrying cmLumiOpto were systematically characterized in vitro. To assess therapeutic efficacy, four TNBC mouse models were established to investigate the impact of cmLumiOpto / PARPi on tumor burden reduction and metastasis inhibition. Additionally, mechanistic insights were explored using Seahorse metabolic analysis, multiplex Luminex assays, RNA-Seq, and other analytical approaches.Without being bound by theory, the findings provide compelling evidence supporting the clinicalT2025-099 (069596.00097) translation of this targeted therapy and underscore its potential to improve treatment outcomes in patients with highly aggressive TNBC.Materials and MethodsCell lines and culture media

[0227] Viral Production Cell 2.0 (VPC) (Gibco, Cat# A49784, RRID: RRID:CVCL_0045, Grand Island, NY) was cultivated using viral production medium (VPM) supplemented with 4 mM GlutaMAX in shaker flask suspension culture at an agitation speed of 130 rpm. The human TNBC cell lines MDA-MB-231 (ATCC, Cat# HTB-26, RRID:CVCL_0062, Manassas, VA, USA), MDA-MB-468 (ATCC, Cat# HTB-132, RRID:CVCL_0419), and MDA-MB-231 -FLuc (GenTarget, Cat# SC059-Puro, RRID:CVCL_YZ80, San Diego, CA, USA) were maintained in DMEM with 10% fetal bovine serum (FBS, v / v) and 1% Pen / Strep in T-flasks. The mouse TNBC 4T1 (ATCC, Cat# CRL-2539, RRID:CVCL_0125) and 4Tl-FLuc (ATCC, Cat# CRL- 2539-LUC2, RRID:CVCL_5I85) were cultivated in RPMI-1640 with 10% FBS and 1% P / S. The seed culture for CD276 mAb production was kept in SFM medium with 4 mM L-glutamine and 6 g / L glucose31. All cell cultures were maintained at 37°C and 5% or 8% CO2 in a humidified incubator (Eppendorf, Enfield, CT, USA). All culture media and supplements were purchased from Fisher Scientific (Waltham, MA, USA) or Gibco, unless otherwise specified. The NSG (NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ) and BALB / cJ mice were purchased from Jackson Laboratory, Bar Harbor, ME). TNBC PDX lines were harvested from donor mice (Jackson Lab, Cat# J000103917, Bar Harbor, ME, USA) or the recipient mice carrying the passaged PDX, freshly frozen and stored in a liquid nitrogen. All commercial lines were authenticated via genetics profiling using polymorphic short tandem repeat analysis and tested in house for mycoplasma contamination using PCR amplification of 16S rRNA gene sequences. mA b-Exo-AA V production

[0228] Firstly, Exo- AAV was produced in a 2-L stirred-tank bioreactor (Distek, North Brunswick, NJ, USA) using Viral Production Medium (VPM) supplemented with 6 g / L glucose and 4 mM GlutaMAX at 37°C, pH 7.0, agitation 210 rpm, and DO 40% following previously established protocol s23,38'40. VPC cells (viable cell density of 3xl06cells / mL, viability of >95%) were co-transfected with three plasmids23, AAV-D S-cfos-NLuc^A-ABCB-CoChR, AAV-DJ / 8 Rep-Cap, and AAV-D / J8 Helper, at a 1 :1 :3 with a DNA-to-cell ratio of 0.5 pg per 106cells. Transfection was mediated using a viral -plex buffer (10% v / v), AAV-MAX transfection reagentT2025-099 (069596.00097)(0.6%), booster (0.3%), and enhancer (1%) (Gibco). Exo-AAV was harvested from the spent medium when culture viability declined to 60-80%, centrifuged at 3,000 x g for 20 min at 4°C, and clarified using a dual-layer regenerated cellulose depth filter (PDK5: 1.5-20 pm, PDE2: 0.2- 3.5 pm) (Cytiva, Marlborough, MA, USA). Exo-AAV was purified using liquid chromatography equipped with a 5-mL Cytiva Hiscreen Capto Core 400 column, followed with ultrafiltration using MilliporeSigma Amicon 100 kDa MWCO regenerated cellulose filters, as previously described23'41, 42. Secondly, CD276 mAb was produced in SFM medium using a 2-L stirred-tank bioreactor at 37°C, Agt 140 rpm, DO 40%, and pH 7.0. The CD276 mAb was purified using liquid chromatography (Bio-Rad, Hercules, CA, USA) with a Bio-Scale Mini UNOsphere SUPrA affinity column. The separated mAb was eluted using a two-phase buffer system, with Phase A (0.02 M Na3PO4, 0.02 M Na3CeH5O7, pH 7.5) and Phase B (0.1 M NaCl, 0.02 M NasCeHsO?, pH 3.0)31’43'49. Finally, Exo-AAV was modified using mPEG-DSPE and conjugated with CD276 mAb via a DSPE-PEG-NHS linker at a molar ratio of 1 :2, 680: 13,000 (Exo- AAVmAbdinker). The resulting mAb-Exo-AAV was purified and concentrated using Amicon 100 or 10 kDa MWCO regenerated cellulose filters and stored at -80°C in 125-mM trehalose formulation buffer.Immunohistochemistry (IHC) staining

[0229] A TNBC patient tissue microarray (TMA) (Cat# BRI 102, US Biomax, Derwood, MD, USA) was stained with an anti-CD276 antibody (Abeam, Rabbit monoclonal, Cat# ab226256, RRID:AB_3069232, 1 / 500 dilution) following a standard IHC protocol31. The stained TMA slide was scanned with Lionheart FX automated microscope (BioTek, Winooski, VT, USA), and images were processed offline with Image J. CD276 expression in each patient tissue sample was calculated using the formula: redintensity / bluemtensity of TNBC core / redintensity / blueintensity of positive core - 1) xlOO. Receptor expression levels were categorized as follows: high (>0.5, indicating >50% higher expression than the positive control), medium (-0.3- 0.5), and low or no (<-0.3, indicating 30% lower than positive control).Nanoparticle tracking analysis

[0230] mAb-Exo-AAV samples were buffer exchanged and diluted using PBS with dilution factors of 1 : 100. The particles were titrated and analyzed using NanoSight Pro (Malvern Panalytical, Malvern, UK). Samples were injected into microfluidics device at a pump perfusion rate of 3 pl . / min and imaged with parameters setup of 60 sec per capture, 5 captures per sample,T2025-099 (069596.00097)6 camera, detection threshold 5, and Temp 25°C. Each sample was titrated three times to analyze the size distribution and particle concentration.Transmission electron microscopy (TEM)

[0231] TEM imaging was performed to assess the morphology and size of mAb-Exo-AAV nanoparticle and free AAV following a published procedure with modifications23,41’50’51. Briefly, purified mAb-Exo-AAV samples were diluted at factors of 1 : 10, 1 : 100, and 1 : 1,000 in PBS buffer, and 10 pL of each sample was deposited onto carbon-coated Formvar grids. Prior to sample loading, grids were glow-discharged for 1 min using a K100X Glow Discharger (Electronic Microscope Sciences, Hatfield, PA, USA). Samples were then negatively stained with 2% uranyl acetate solution for 1 min, followed with two PBS washes and air drying. TEM images were acquired using a Tecnai T12 transmission electron microscope equipped with a CCD camera (Field Electron and Ion Company, Hillsboro, OR, USA) and processed with DigitalMicrograph software (Gatan, Pleasanton, CA, USA). qRT-PCR titration

[0232] The Exo- AAV samples were digested with DNase I to extract the packed ssDNA carrying cmLumiOpto gene. RT-PCR analysis was performed to titrate AAV, i.e. NLuc genome copy, using the following primers: SEQ. ID No. 81 (5'-ATTGTCCTGAGCGGTGAAA-3' (forward)) and SEQ. ID No. 81 (5'-CACAGGGTACACCACCTTAAA-3' (reverse)). The AAV packing rate in exosome was calculated using genome copy of NLuc gene per nanoparticle of Exo- AAV.Western blotting

[0233] As detailed in previous publications23,32, the lysates of TNBC cells or Exo- AAV samples were loaded to NuPAGE 4-12% gradient Bis-Tris gel for SDS-PAGE electrophoresis (Fisher). Proteins separated on gel were transferred to a methanol activated PVDF membrane with Bio-Rad power supply (Bio-Rad) and blocked using TBS buffer containing 0.1% Tween-20 and 5% fat-free milk. The primary antibodies of CD276 (Cat# ab 134161, Abeam), y-H2AX (Cat# ab2893, Abeam), cleaved PARP (Cat# 9148, Cell Signaling Technology, Danvers, MA, USA), cleaved caspase 3 (Cat# 9661, Cell Signaling), LC3B (Cat# 8899, Cell Signaling), [3-actin (Cat# sc-47778, Santa Cruz, CA, USA), and exosome panel of CD9, CD63, HSP70 and calnexin (Cat# ab275018, Abeam) with dilution factor of 1: 1,000 or 1:2,000 were applied. The horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) and HRPT2025-099 (069596.00097) substrate were used to detect the interested protein bands with Odyssey Fc imaging system (LL COR Biosciences, NE, USA).Flow cytometry

[0234] To assess the surface binding rate to TNBC cells, CD276 mAb and mAb-Exo-AAV were labeled with fluorescent dyes: Alexa Fluor™ 647 (Life Technologies, part of Fisher) and Sulfo-Cyanine 5.5 (Lumiprobe Life Science Solutions, Hunt Valley, MD, USA), respectively. About IxlO6TNBC cells (MDA-MB-231, MDA-MB-468, 4T1) were incubated with 1 pg of CD276 mAb-AF647 or 10xl06of mAb-Exo-AAV-Cy5.5 particles at room temperature for 30 mins. Surface binding was analyzed using a BD LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA), with a gating strategy set to exclude >0.5% fluorescent populations of unstained cells. Data were processed and analyzed using FlowJo V5.0 software to determine the surface binding rate.Live-cell confocal imaging

[0235] TNBC MDA-MB-468 cells expressing GFP were seeded at a density of IxlO5cells / mL in a 15-mm glass-bottom dish (Cellvis, Mountain View, CA, USA) and cultured for 6 hrs. About 10xl05particle / milliliter (ptc / mL) ofCD276 mAb-Exo-AAV, labelled with Cy5.5 fluorescent dye, was added to transfect the TNBC cells, followed by incubation at 37°C and 5% CO2 for 24 hrs. Live-cell images were acquired using a Nikon A1R-HD25 confocal microscope (Nikon, Melville, NY, USA) with 640 nm and 488 nm lasers for Cy5.5 and GFP fluorescence, respectively. Confocal images were analyzed with ImageJ to assess the internalization of mAb- Exo-AAV. To evaluate mitochondrial depolarization, cells treated with cmLumiOpto were stained with Mito View 63353(25 nmol / L) for 15 minutes and imaged at 635 nm using a Stellaris 5 Confocal microscope (Leica Camera, Teaneck, NJ).Seahorse assay

[0236] MDA-MB-231 cells treated with mAb-Exo-AAV at multiplicity of infection (MOI) of 100,000 were seeded in a 96- well microplate at a density of 2,000 cells per well. Upon reaching 90% confluence, the following treatments were applied: saline or ViviRen™ / PARPi.Mitochondrial activity in TNBC cells was assessed using the Seahorse XF Cell Mito Stress Test Kit and quantitated using a Seahorse XF Analyzer (Agilent, Santa Clara, CA, USA) following the manufacturer’s instructions.T2025-099 (069596.00097)Luminex assay

[0237] Chemokines and cytokines in the tumor microenvironment (TME) post treatment were titrated using a Procine Multiplex Luminex assay (Luminex Corporate, Austin, TX, USA). The pre-configured and customized 13-plex assay kit was purchased from R&D Systems (Minneapolis, MN, USA). Tumor tissues (w 4) were dissociated to extract the secreted chemocytokines to perform the assay following manufacturer’s procedure. Fluorescence intensity (MFI) was detected and quantitated using the Luminex MAGPIX (Luminex Corporate) and the raw data were analyzed using XPONENT software.In vitro anti-TNBC cytotoxicity assay

[0238] Approximately IxlO5TNBC (MDA-MB-468 and MDA-MB-231) cells were seeded in 200 pL of medium in 96-well plates. The cultures were treated with saline, CD276 mAb-Exo- AAV carrying cmLumiOpto (MOI: 100,000) and ViviRen™ (30 pM), PARPi (Olaparib, 20 pM), or cmLumiOpto / PARPi combination, and incubated at 37°C with 5% CO2 for three days. Cell growth and relative viability were assessed using the TACS MTT Cell Proliferation Assay54,5’. In vivo imaging system (IVIS)

[0239] Live-animal and ex vivo IVIS imaging was performed to evaluate TNBC targeting specificity of CD276-mAb. Briefly, when tumor volume reached 50-100 mm3, 50 pg of mAb labelled with Cy5.5 was intravenously (i.v.) injected through tail vein. After 24 hrs, luciferin was i.p. injected, and mice were imaged using IVIS Lumina Series III (PerkinElmer, Waltham, MA) with exposure time of 10 Secs. Both luminescence (FLuc) and fluorescence (Cy5.5) signals were captured. Additionally, major organs and tumors were harvested for ex vivo imaging to validate the biodistribution of CD276 mAb. Beyond distribution analysis, IVIS imaging was also used to monitor in vivo metastasis of TNBC cells expressing FLuc.TNBC cell line-derived xenograft model and in vivo treatment

[0240] A total of 5x l06TNBC MDA-MB-231 cells were injected into the mammary fat pad of 7-week-old NSG (NOD.Cg-Prkdc<scid> I12rg<tmlWjl> / SzJ) female mice. When tumor volume reached -75-100 mm3, the mice were randomized into six groups (n=6 / group). Group 1 received i.v. saline injection as a control. Groups 2-4 were i.v. administrated with CD276 mAb- Exo- AAV weekly at doses of 2xlOloptc / kg-BW (low), 10xl010ptc / kg-BW (medium) or 30xl010ptc / kg-BW (high), followed by i.v. injection of ViviRen™ (2 mg / kg-BW) daily for three consecutive days. Group 5 received oral administration of 50 mg / kg of Olaparib (PARPi) viaT2025-099 (069596.00097) water bottle feeding. Group 6 was treated with a combination of cmLumiOpto (low dose, 2x1010ptc / kg-BW) and Olaparib (50 mg / kg) following the same treatment regime as Groups 2-5.Tumor volumes were measured using a vernier caliper and mice body weight was monitored two or three times a week. Mice were sacrificed when tumor volume exceeded 1,000 mm3, body weight drop by >20%, or other early removal criteria, such as self-mutilation, inactivity, lethargy, poor response to stimuli, ataxia or ulcerative tumor, were met. In the end of animal study, tumor tissues and vital organs, including brain, heart, lungs, liver, spleen and kidneys, were harvested for H&E staining, IHC staining and biochemical analysis.Metastatic models and in vivo treatment

[0241] Approximately 2xl06mouse TNBC 4Tl-FLuc cells and human TNBC MDA-MB- 231-FLuc cells were i.v. injected through tail vein into 7-week-old BALB / cJ female mice and NSG female mice, respectively. In vivo metastasis progress was monitored by detecting FLuc signal using IVIS imaging. After metastasis was detected, mice (n=5 / group) were treated with saline, cmLumiOpto (1 Ox 1010ptc / kg-BW mAb-Exo-AAV and 2 mg / kg-BW ViviRen™), and cmLumiOpto (same dose) combined with PARPi (50 mg / kg-BW) following the treatment schedule outlined above. TNBC metastasis was assessed once a week using IVIS. Mice were sacrificed when body weight dropped by 20% or other early removal criteria were met. Major organs, including brain, heart, lungs, liver, spleen and kidneys, were harvested for H&E staining, IHC staining, mRNA sequencing, and other post treatment analyses.Patient derived xenograft (PDX) model and in vivo treatment

[0242] The CD276+TNBC PDX donor mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Once the volume of donor PDX reached 2,000-3,000 mm3, tumors were harvested, minced into Ixlxl mm3fragments, and implanted into the mammary fat pad of NSG mice using a 16G needle syringe (or snap frozen and stored in liquid nitrogen). Once the xenografted PDX reached 70-100 mm3, mice were divided into two groups (n=4-5) and treated with either saline or cmLumiOpto / PARPi (10xl010ptc / kg mAb-Exo-AAV, 2 mg / kg ViviRen™, 50 mg / kg PARPi). Tumor size and mouse body weight were monitored twice a week until tumor volume exceeded 1,000 mm3in the control group. Tumor tissue and major organs were then harvested for further post treatment analysis.T2025-099 (069596.00097)Bulk RNA sequencing

[0243] The lung tissues with TNBC metastasis were harvested, dissociated and lysed to extract total mRNA using RNeasy Fibrous Tissue Mini Kit (Qiagen, Germantown, MD, USA). The cDNA library construction and bulk RNA sequencing using Illumina HiSeq™ X Ten platform were carried out at Novogene America (Sacramento, CA, USA). Sequencing reads were mapped to the mouse reference genome (GRCm38) using Hierarchical Indexing for Spliced Alignment of Transcripts version 2 (HISAT2). Differentially expressed genes (DEGs) between treatment and saline groups were identified using edgeR (version 4.2.1) in R. The P-values were adjusted using Benjamini-Hochberg method to control the false discovery rate (FDR), with FDR < 0.05 set as the threshold for DEG selection. Gene Ontology (GO) enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA) method implemented in the clusterProfiler R package. The GO terms with a corrected P-value < 0.05 were considered significantly enriched.Paraffin section and hematoxylin and eosin (H&E) staining

[0244] The harvested tumor tissues and organs were dehydrated in 70% ethanol, cleaned with xylene and coated in paraffin. The paraffin embedded tissue blocks were sectioned at 5 pm thickness using a Leica microtome (Leica Biosystems, Deer Park, IL, USA). The sectioned slides were de-paraffinized with xylene, hydrated with 100-70% ethanol, washed with ddH?O, and stained with H&E as previously described23,31’40’51,52.Immun fluorescent staining

[0245] TNBC tumor tissue slides were incubated with rabbit anti-TOMM20 polyclonal antibody conjugated to AF488 (Abeam, Cat# ab205486) and anti-cytochrome C antibody- conjugated to AF647 (Biolegend, Cat# 612310) at a dilution of 1 :200. The IHC-stained slides were imaged using a Nikon A1R-HD25 confocal microscope (Nikon).Statistical analysis

[0246] The experimental data were presented as mean ± standard error of the mean (SEM) in this study. Statistical analysis and comparison were performed using two-tailed t test and oneway ANOVA followed by post-hoc (Dunnett’s) analysis with GraphPad Prism. / NO.05 was considered statistically significant for all tests.T2025-099 (069596.00097)ResultsCD276 overexpression in TNBCs

[0247] The Cancer Genome Atlas (TCGA) dataset analysis revealed that CD276 mRNA levels are significantly higher in TNBC (and other breast cancer) tissues compared to normal breast tissue (Fig. 28A). Western blot analysis confirmed high CD276 expression in TNBC lines MDA-MB-231 and MDA-MB-468, which represent mesenchymal stem-like (MSL), basal-like 2 (BL2), and luminal androgen receptor (LAR) subtypes. In contrast, CD276 expression was minimal in the normal breast epithelial cell line 184B5 (Fig. 28B). IHC staining of TNBC patient TMA (n=110) demonstrated that 23% of cases (25 / 110) exhibited high CD276 expression, 44% (47 / 110) had moderate expression, and 33% (35 / 110) showed minimal or no expression (Fig. 28C). Representative IHC images of normal breast tissue and TNBC cores with varying CD276 expressions levels are shown in Fig. 28D. Collectively and without being bound by theory, these findings highlight CD276 as a promising target for gene delivery in TNBC.CD276 mAb exhibiting high TNBC specificity

[0248] To assure the safety to deliver cmLumiOpto gene therapy via targeting CD276 receptor, the potential off-target in normal human organs was analyzed. The IHC staining of normal human tissue using the anti-human / mouse CD276 mAb did not detect significant binding in brain, heart, liver, spleen, lung, kidney, breast and pancreas (Fig. 36A). Similar IHC staining using mouse normal tissues (liver, kidney, lung, skeletal muscle, brain, heart, stomach, spleen, malignant mouse adrenal gland as positive control) did not detect obvious off-target of the CD276 mAb (Fig. 36B).

[0249] The CD276 mAb was produced in a stirred-tank bioreactor with volumetric titer of -80-120 mg / L from batch bioreactor (Fig. 36C) and subsequently purified using a protein A column (Fig. 36D). The TNBC targeting capability of CD276 mAb was evaluated and confirmed via flow cytometry analysis using human MDA-MB-231 and MDA-MB-468 cell lines and mouse 4T1 cell line (Fig. 29A). The surface binding rates were 98.5, 100 and 49.6% in these three lines, respectively, indicating cross-species reactivity of the mAb. Furthermore, the in vivo TNBC targeting ability of CD276 mAb was assessed using NSG mice xenografted with human MDA-MB-231-FLuc (Fig. 29B) and BALB / cI mice implanted with mouse 4Tl-FLuc tumors (Fig. 29C). Live-animal IVIS imaging conducted at 24 hrs post tail vein injection of 50 pg mAb showed strong overlap of TNBC tumors (FLuc) with CD276 mAb (Cy5.5). Ex vivo IVIST2025-099 (069596.00097) imaging of tumor and major organs, including the heart, liver, spleen, lungs, kidneys and brain, further confirmed the specific targeting of CD276 mAb to TNBC, with no detectable off- targeting accumulation in normal organs. Taken together and without being bound by theory, both in vitro and in vivo evaluations demonstrated the potential of the CD276 mAb as an effective TNBC-targeting agent for gene therapy delivery.Construction and characterizations of CD276 mAb-Exo-AAV

[0250] To achieve TNBC targeting delivery, the cmLumiOpto genes were packed in AAV, harvested and purified Exo-AAV secreted by VPC, and conjugated the CD276 mAb to the surface of exosomes using a DMPE-PEG-NHS linker23,51(Fig. 30A). To enhance circulation stability and reduce renal clearance, the mAb-Exo-AAV was further pegylated with mPEG- DSPE. As illustrated in Fig. 30B, high-yield production of Exo-AAV was achieved in 2-L stirred- tank bioreactor, yielding 9-10 xlO9particles / mL following an optimized protocol23,51. NanoSight Pro analysis revealed a size distribution of 100-300 nm, with an average diameter of 164±25 nm (Fig. 30C). TEM images confirmed the morphology of both Exo-AAV and free AAV particles (Fig. 30D). Western blot analysis detected the presence of key exosome markers CD9, CD63 and HSP70, while the absence of calnexin marker confirmed the high purity of Exo-AAV without endoplasmic reticulum contamination (Fig. 30E).

[0251] The production procedure of Exo-AAV was successfully scaled up from 30-mL to 300-mL culture in shaker flask and 2-L culture in bioreactor (Fig. 30F). Key factors influencing Exo-AAV yield and AAV quality included VPC transfection viability, nutrient supplementation, agitation rate, and harvest viability. The purification protocol was scaled up from a 5-mL to a 20- mL size-exclusion chromatography column, followed by ultrafiltration using a 100-kDa regenerated cellulose membrane, which further improved purity while maintaining a 90-95% recovery rate.In vitro evaluations of cmLumiOpto delivered with CD276 mAb-Exo-AAV

[0252] Flow cytometry analysis revealed high surface binding of CD276 mAb-Exo-AAV labelled with Cy7 in MDA-MB-231 (99.6%), MDA-MB-468 (99.5%) and 4T1 (97.1%) cells (Fig. 31 A), confirming strong TNBC-targeting capability. The TNBC transfection and internalization of CD276 mAb-Exo-AAV-Cy7 was validated using confocal microscope in GFP- expressing MDA-MB-468 cells, where the 84% overlay of GFP in cytoplasm and Cy7 signals in the internalized mAb-Exo-AAV indicated high transduction efficiency of the cmLumiOpto geneT2025-099 (069596.00097)(Fig. 3 IB). Functional expression of cmLumiOpto and the surface-bound mAb on Exo-AAV had been confirmed in previous study23. While AAV packaging efficiency in Exo-AAV harvested at 40% VPC viability was slightly higher than at 80% viability (18.32 vs. 15.80 gc-AAV / ptc-Exo- AAV, Fig. 31C), the production was improved by collecting Exo-AAV at 60-80% viability to balance yield and quality. Mechanism analyses further revealed that the apoptosis inhibitors Z- VAD-FMK (pan-caspase), Z-LEHD-FMK (caspase-9) and Z-IETD-FMK (caspase-8) reduced cell death, whereas the necrosis inhibitor necrostatin had no obvious effect (Fig. 3 ID). Without being bound by theory, cmLumiOpto induces caspase-dependent apoptosis rather than necrosis. To comprehensively evaluate the synergism between cmLumiOpto (0-lxl06MOI) and PARPi (0-20 pM), a design of experiments (DoE) was conducted. Assessment of MDA-MB-231 cell viability after two days of treatment, analyzed using the Bliss independence model, demonstrated synergistic effects (enhanced cell killing) across most combinations, with the strongest synergy observed at cmLumiOpto concentrations >lxl05MOI and PARPi concentrations >10 pM (Fig.3 IE). The cytotoxic effect of cmLumiOpto delivered with mAb-Exo-AAV, Olaparib, and combination were evaluated in MDA-MB-231 and MDA-MB-468. Cell viability was significantly reduced to 25.03-28.22%, 35.86-51.17%, and 4.47-13.32%, respectively, following treatment (Fig. 3 IF). Notably, cmLumiOpto gene therapy demonstrated higher cytotoxicity than Olaparib alone, and their combination further enhanced therapeutic potency against TNBC cells (Fig. 3 IF). Without being bound by theory, CD276 mAb-Exo-AAV is a promising gene delivery system, with high TNBC selectivity, transduction efficiency, and synergistic therapeutic potential when combined with PARP inhibition.Evaluation of anti-TNBC efficacy in immunocompromised models

[0253] The anti-cancer efficacy of cmLumiOpto at varying doses (low: 2, medium: 10 and high: 30 xlOloptc / kg-BW), PARPi monotherapy, and a combination of low dose cmLumiOpto with PARPi was evaluated in MDA-MB-231 xenografted NSG mouse models (n=6 / group). Following the first injection, tumor volume in groups receiving cmLumiOpto alone or in combination with PARPi decreased from 107-145 mm3to 42-78 mm3within one week (Fig. 32A). Medium and high doses of cmLumiOpto induced further tumor shrinkage (20-50 mm3) after the second injection (week 2) and achieved complete tumor regression after the 3rdinjection (week 3), with no recurrence observed through weeks 4-6. In contrast, tumors progressed to 815 mm3in control group (saline) and 386 mm3in PARPi monotherapy group, respectively, by weekT2025-099 (069596.00097)3, necessitating early sacrifice due to ulceration (>2 mm) before reaching 1 ,000 mm3. It should be noted that low-dose cmLumiOpto alone or cmLumiOpto / PARPi combination experienced minor tumor size rebound after treatment cessation, with volumes reaching 15-31 mm3(week 4), 34-35 mm3(week 5), and 48-61 mm3(week 6).

[0254] To assess whether the observed tumor size increase in low dose cmLumiOpto and cmLumiOpto / PARPi groups resulted from recurrence, the harvested tumor tissues were evaluated with histological analysis. H&E staining revealed severe tumor cell death and reduced cancer cell density in the cmLumiOpto group, while the cmLumiOpto / PARPi group exhibited TME disruption characterized by the fluid-like tissue (Fig. 32B). Without being bound by theory, the slight increase of tumor volume between Days 22-41 was unlikely due to TNBC recurrence but may involve non-cellular factors. Potential contributions include inflammatory responses, necrotic core expansion, vascular remodeling, and stromal changes. IHC staining showed that the percentage of Ki67 (.positive cells (a proliferation marker) ranged from 12-58% in the saline and PARPi groups, compared to 2-6% in the cmLumiOpto and cmLumiOpto / PARPi groups.Similarly, cleaved caspase-3 -positive cells (an apoptosis marker) were observed at 3-8% in the saline and PARPi groups, versus 25-55% in the cmLumiOpto and cmLumiOpto / PARPi groups. These results confirmed significant apoptotic activity and proliferation inhibition in treatment groups (Fig. 32C). While PARPi monotherapy slowed tumor progression, H&E and IHC staining revealed no significant tumor cell death or growth inhibition (Figs. 32B-C). Without being bound by theory, combining gene therapy with chemotherapy shows synergistic anti-cancer effects.

[0255] To further evaluate in vivo infection efficiency, fresh tumor tissues were analyzed on Day 5 post-injection. Flow cytometry of dissociated tumor cells stained for NLuc and Ki67 revealed that -75.9% of TNBC cells were successfully infected with CD276 mAb-Exo-AAV and expressed functional cmLumiOpto gene. In some instances, multiple administrations and chemotherapy integration may be required to enhance therapeutic efficacy (Figs. 32A-B).

[0256] Importantly, no significant body weight changes were observed across treatment groups (Fig. 32E), indicating minimal systemic toxicity. Furthermore, H&E staining of major organs (brain, heart, lungs, liver, spleen, kidneys) showed no signs of inflammation, apoptosis, or necrosis (Fig. 37), confirming the safety profile of cmLumiOpto and cmLumiOpto / PARPi therapy at the tested doses.T2025-099 (069596.00097)Evaluation of anti-TNBC efficacy in distant metastatic models

[0257] Metastasis inhibition in immunocompetent models. Following the detection of TNBC (4Tl-FLuc) metastasis via IVIS imaging, immunocompetent mouse models (n=5 / group) were treated with saline, cmLumiOpto (10xl010ptc / kg-BW) and cmLumiOpto / PARPi (10xl010ptc / kg- BW, 50 mg / kg-BW) via i.v. injection or drinking water supplementation. PARPi monotherapy was not included due to its limited efficacy in primary tumor models (Fig. 32). By week 3, IVIS imaging revealed extensive cancer metastasis in the saline group, moderate metastasis reduction in the cmLumiOpto group (two mice were lost before final imaging due to an accident), and complete blockage or elimination of metastases in the combination treatment group (Fig. 33A). Lung tissues with extensive TNBC metastasis were collected for histological and mechanism of action (MOA) analyses. Whole-slide scanning revealed dense tumor colonies in the lung tissue of saline-treated mice (Fig. 33B), whereas treatment with cmLumiOpto or cmLumiOpto / PARPi led to tumor necrosis, despite the presence of metastatic lesions. Microscopic imaging further highlighted remarkable differences between groups, with widespread tumor cell death and necrosis in treated mice, while tumors in saline-treated controls remained intact and proliferative (Fig. 33B).

[0258] IHC staining of tumor tissues (Fig. 33C) demonstrated significant suppression of proliferation marker Ki67 and an increase in apoptosis marker cleaved caspase-3 in treatment groups. Specifically, the percentage of Ki67-positive cells were 85%, 45% and 15%, while those of CCasp3 -positive cells were <5%, 40% and 40% in the saline, cmLumiOpto, and cmLumiOpto / PARPi groups, respectively. These data confirmed the ability of the therapies to induce cancer cell death and inhibit metastatic proliferation. Additionally, tumor-infiltrating NK cells (CD45+) were enriched from <5% in the saline group to 35% and 75% in the cmLumiOpto and cmLumiOpto / PARPi treatment groups, respectively. Macrophage phagocytosis (F4 / 80+)- positive cells were also enhanced from <5% in the saline group to 45% and 55% in the cmLumiOpto and cmLumiOpto / PARPi treatment groups, respectively. Without being bound by theory, this histological analysis suggests an immune-mediated tumor clearance mechanism. Furthermore, IHC staining of lung tissues harboring metastatic TNBC was performed with antibodies against CD8 (T cells), NK1.1 (NK cells) and NKp46 (NK cells). The percentages of positive cells were as follows: CD8, 2% and 65%; NK1.1, 5% and 75%; and NKp46, 1% and 55% in the saline and cmLumiOpto / PARPi groups, respectively (Fig. 38A). Without being boundT2025-099 (069596.00097) by theory, it is believed that immune cell infiltration in tumor microenvironment by cmLumiOpto treatment. Additionally, without being bound by theory, it is believed that tumor immune regulation contributed to the therapeutic effects, which can be confirmed through mechanism-of-action (MOA) studies.

[0259] Importantly, no significant changes in body weight were observed across groups (Fig. 38 A), and H&E staining of major organs (brain, heart, liver, spleen, kidneys) detected no toxicity or tissue damage in treatment groups (Fig. 38B), consistent with the safety profile observed in primary TNBC models (Fig. 37).

[0260] Metastasis inhibition in immunocompromised models. To further validate the anti- metastatic efficacy of cmLumiOpto / PARPi, a similar study was conducted in a second mouse model using immunocompromised NSG female mice bearing metastatic human TNBC (MDA- MB-231-FLuc). IVIS imaging revealed that cmLumiOpto / PARPi treatment significantly reduced TNBC metastases (Fig. 39A). Consistent with the immunocompetent model, therapeutic administration did not impact mouse body weight (Fig. 39B), and H&E staining of lung tissues demonstrated lower metastatic burden three weeks post-treatment (Fig. 39C). Without being bound by theory, cmLumiOpto / PARPi is efficacious in suppressing TNBC metastasis across distinct preclinical models.Anti-cancer mechanisms

[0261] To elucidate the anti-cancer mechanism of action (MOA) of combined cmLumiOpto / PARPi, its impact on TNBC mitochondrial structure and function was investigated. Immunofluorescence staining of TNBC tumors with TOM20 (outer mitochondrial membrane marker) and cytochrome c antibodies revealed intact mitochondrial architecture in untreated controls, with co-localized TOM20 and cytochrome c (Fig. 34A, left). In contrast, treated samples exhibited >66% of cytochrome c release from the mitochondria, indicative of mitochondrial injury and collapse (Fig. 34A, right), consistent with previous findings on cmLumiOpto monotherapy31. Mitochondrial functional impairment was quantified using the Seahorse assay in MDA-MB-231 cells treated with cmLumiOpto / PARPi for 48 hours (70-80% viability; Fig. 34B). Oxygen consumption rate (OCR) analysis demonstrated significant reductions in basal respiration (49.4 to 15.2 pmol / min), maximal respiration (50.4 to 14.0 pmol / min), and ATP production (38.2 to 13.6 pmol / min). Without being bound by theory, severeT2025-099 (069596.00097) mitochondrial bioenergetic disruption lead to mitochondria dysfunction and irreversible cancer cell death, by the targeted delivered cmLumiOpto.

[0262] Beyond immunohistochemical analysis of immune function and immune cell infdtration (Fig. 34C), lung tissues harboring TNBC (4T1) metastases were harvested and dissociated. The extract was applied to perform a multiplex Luminex assay to quantify cytokines secretion within the TME (Fig. 7C). The cmLumiOpto / PARPi markedly upregulated several key cytokines, including IL-la (2.41-fold; 355 to 1,211 pg / mL) and CCL2 (2.24-fold; 927 to 3,008 pg / mL). Additionally, IFN-y (1.87-fold; 31 to 58 pg / mL) and IL-12 p70 (1.67-fold; 17 to 29 pg / mL) were elevated, while IL-6 (7.18-fold; 117 to 843 pg / mL) and IL-4 (10.47-fold; 70 to 739 pg / mL). Herein the secretion of anti-tumor cytokines IFN-y and IL-12 and the dual-role cytokines IL-la, IL-6 and IL-4 was significantly increased by the cmLumiOpto / PARPi combination. The higher level of pro-tumor CCL2 was also observed in treatment group. Without being bound by theory, these data, together with the detected immune cells activation and infiltration (Figs. 6C) in tumor microenvironment, suggested immunomodulatory response, which could benefit anti-tumor efficacy of gene therapy-chemotherapy.

[0263] Bulk RNA-Seq analysis of lung tissues provided further insights into TNBC metastasis regulation. Differential gene expression (DGE) analysis revealed suppression of multiple metastasis-associated pathways by cmLumiOpto / PARPi (Fig. 34D). Canonical and non- canonical Wnt signaling, key drivers of epithelial-mesenchymal transition, invasion, angiogenesis and colonization, were inhibited. Downregulation of fibroblast growth factor receptor (FGFR) signaling curtailed downstream RAS / MAPK and PI3K / AKT activation, while reduced transforming growth factor 0 (TGF-0) expression correlated with diminished metastatic potential. Without being bound by theory, the observation of metastasis signaling in two mouse models and the downregulation of these metastatic signaling pathways indicated that targeting cancer mitochondria can effectively inhibit, reduce or eliminate the metastasis of aggressive cancers. In addition, the Hippo signaling, a tumor suppressor, was upregulated by treatment. In the TME, immune activation was evident through upregulated immune response and enhanced 0 T-cell activity and IL- 12 production (Fig. 34E). These results were consistent with the observed immunity upregulation and immune cells infiltration in tumor microenvironment (Figs. 33 and 34). Without being bound by theory, the immune function caused by CD276 mAb-Exo- AAC offers additional therapeutic benefits in targeted cancer treatment. Finally, DNA damageT2025-099 (069596.00097) responses methylation, and helicase activity, known to restrict proliferation and metastasis, were significantly reduced (Fig. 34F). Cancer mitochondria depolarization and mitochondrial inner membrane potential collapse cause persistent DNA damage.23The FDA approved PARPi can disrupt the repair of DNA damage in breast cancers. The RNA-Seq data underscored the multifaceted anti-metastatic impact of the combination of cmLumiOpto / PARPi, underscoring the multifaceted anti-metastatic impact of the combination of cmLumiOpto / PARPi.Efficacy of cmLumiOpto / PARPi in PDX models

[0264] To evaluate the therapeutic efficacy of cmLumiOpto / PARPi in a clinically relevant setting, PDX xenografted mouse models were established to preserve the heterogeneity and TME of human TNBC. When tumors reached -100 mm3, mice received two administrations of cmLumiOpto / PARPi (10xl010ptc / kg-BW, 50 mg / kg-BW), as indicated by arrows (Fig. 35A). By Day 25, tumor volume in the treatment group was 50% lower than in the saline group (776 vs 1,551 mm3). Body weight profiles showed no significant difference between treatment and control groups (Fig. 35B), and H&E staining of major organs (brain, heart, lungs, liver, spleen, kidneys) revealed no signs of inflammation or necrosis, confirming the safety of the therapy (Fig. 40). In addition, five doses (2xlO10, 5xl010, 10xl010, 2OxlO10and 50xl010ptc / kg BW) of cmLumiOpto combined with 50 mg / kg of Olaparib (PARPi) were administered to BALB / cJ mice via tail vein (n=2), with saline serving as the control. Blood samples were collected two weeks after treatment for complete blood count analysis (Fig. 41A-B), which demonstrated minimal hematological toxicity. Taken together, the cancer mitochondrial-targeted cmLumiOpto in combination with PARPi does not cause toxicity at the tested doses. Histological analysis of tumor tissues demonstrated widespread cell death within the TME following cmLumiOpto / PARPi treatment, while tumors in the saline group remained intact (Fig. 35C). Furthermore, IHC staining revealed that the combined gene-chemotherapy induced significant apoptosis and proliferation inhibition (Fig. 35D), with CCasp3-positive cells at <5% and >50% and Ki67 positive cells at 70% and <5%, in saline control and cmLumiOpto / PARPi treatment group, respectively, underscoring its potential to effectively suppress heterogeneous TNBC tumors in PDX models.Discussion

[0265] Conventional chemotherapeutics for TNBC is hampered by low response rates and synthetic lethality, prompting the need for more effective strategies. The cancer mitochondria-T2025-099 (069596.00097) targeting luminoptogenetics system (cmLumiOpto) disclosed herein induces severe mitochondrial dysfunction, DNA damage and subsequent cell death. Leveraging prior development of CD276 mAb for drug delivery to TNBC cells31, mAb-Exo-AAV were engineered to construct to deliver cmLumiOpto gene specifically to TNBC cells in vivo. Without being bound by theory, this approach selectively targets TNBC cells, depolarizes cancer mitochondria, and triggers severe mitochondrial dysfunction, culminating in robust tumor cell death and collapse of the TME without detectable off-target toxicity. Hence cmLumiOpto was evaluated as a monotherapy and in combination with PARPi across four TNBC animal models, demonstrating elimination of primary tumors and significant suppression or complete inhibition of metastasis when combined with PARPi.

[0266] Without being bound by theory, the cmLumiOpto / PARPi combination represents a synergistic, translatable therapeutic paradigm with distinct advantages over traditional TNBC treatments such as chemotherapy1 2and gene therapy (e.g., p5356'57). First, cmLumiOpto harnesses sustained intracellular bioluminescence from Nanoluciferase to activate light-gated mitochondrial rhodopsin channels, directly collapsing the ATm and triggering DNA damage. Unlike subtype-specific therapies reliant on endogenous signaling, which are often disrupted in TNBC, cmLumiOpto directly targets mitochondrial function, reducing the likelihood of resistance in heterogeneous tumors. Second, PARPi exploits cmLumiOpto-induced DNA repair deficiencies, amplifying cancer cell lethality. Without being bound by theory, thisdual mechanism integrates direct cell death, apoptosis, and autophagy (via cmLumiOpto) with DNA damage and metabolic suppression (via mitochondrial depolarization and PARPi), effectively targeting metastatic TNBC cells. In immunocompetent models and without being bound by theory, this synergy eradicated tumors and activated tumoral immunity, partly through CD276 mAb-mediated neutralization of immunosuppressive signals25’27 58-59, enhancing T / NK cell infiltration.

[0267] Mitochondria are pivotal regulators of cancer metastasis, modulating ATP production, reactive oxygen species and signaling pathways critical for proliferation, genome stability, and immune evasion60’61. Alterations in mitochondrial genetics and metabolism underpin metastatic cascades62’64, while mitochondrial stress responses confer resistance to cytotoxic therapies65’69. Without being bound by theory, cmLumiOpto-induced mitochondrial collapse repressed metastasis-related signaling, including proliferation, vascular invasion, and TME modulation, asT2025-099 (069596.00097) confirmed by bulk RNA sequencing in distant metastatic mouse models, where lung metastasis was inhibited or eliminated; this underscores the potential of mitochondria- targeted therapies to manage metastatic TNBC.

[0268] AAV is a promising delivery vehicle of therapeutic genes70due to its advantages, including long-term transgene expression, high stability and ability to infect a broad range of cell types. Eight AAV-based therapies, such as Luxturna, Roctavian and Zolgensma, have been approved to date, and approximately 300 clinical trials for treating Alzheimer, Parkinson and other diseases are on-going71’74. Despite these achievements, several major clinical challenges limit their applications, including pre-existing immunity from AAV-neutralizing antibodies, immunogenicity, and high-dose-induced hepatotoxicity, neurotoxicity and cardiotoxicity73,76In addition to AAV, FDA also approved lentivirus vectors for ex vivo genetic modification of cells, such as Zynteglo, Skysona and Abecma, for treating lymphoma, myeloma and rare diseases. The lack of targeted delivery has limited the in vivo application especially for cancer treatment. Unlike AAV and lentivirus, the mAb-Exo-AAV disclosed herein provides an effective targeted delivery vehicle for therapeutic genes, enabling gene therapy to treat low-grade, advanced and metastatic cancers. The vehicle disclosed herein comprises a cancer-selective cfos promoter, TNBC-targeting anti-CD276 mAb and the packed AAV carrying the cmLumiOpto genes.Compared to free AAV, mAb-Exo-AAV enables targeted delivery, high circulation stability, repeated dosing, and escape from AAV neutralization.77’80CD276, overexpressed in 67% of patients across subtypes, is an ideal target for primary and metastatic TNBC. Without being bound by theory, CD276 mAb not only facilitates delivery but also restores effector immune functions in the TME by neutralizing inhibitory signaling31. Importantly, the CD276 mAb exhibits favorable drug delivery parameters and plasma stability. TME modulation is characterized by the increased T / NK cell infiltration and activation31. Without being bound by theory,, the mAb-Exo-AAV not only targets cancer but also enhances tumoral immunity; and furthermore, the Exo- AAV platform supports conjugation with additional mAbs (e.g., targeting EGFR81’85, Trop-212’14,86’87, NK-1R88or MET89’91), broadening its applicability to heterogeneous TNBCs and potentially other cancers, such as non-small cell lung cancer. The established biomanufacturing process for mAb-Exo-AAV, utilizing stirred-tank bioreactor and liquid chromatography, enables large-scale production.40, 51T2025-099 (069596.00097)

[0269] In summary, this study discloses a combinatorial gene-chemotherapy using mitochondria-targeted cmLumiOpto in concert with PARPi. The integration of direct mitochondrial disruption, enhanced DNA damage, and immune activation offers an approach for overcoming the challenges posed by heterogeneous and metastatic TNBC.EXAMPLE 5

[0270] Three gene delivery vehicles, cationic lipid nanoparticles (LNPs) using traditional DOTAP and DOPE, RALA peptide nanoparticles (PNPs), and exosome nanoparticles (Exo), were compared.

[0271] The three vehicles were synthesized and characterized with NanoSight, Zeta and TEM. The gene packing rate for each vehicle for mitochondria-targeting mLumiOpto genes was compared using RT-PCR. The cancer specificity was tested by surface tagging each vehicle with a monoclonal antibody that was developed to target the surface receptor overexpressed in triplenegative breast cancers (TNBCs). The transfection / transduction efficiency and functional gene expression were then evaluated using multiple TNBC cell lines. Finally, the in vivo cancer targeting and gene delivery were evaluated and compared.

[0272] Taken together, this study provided a full comparison of three nanoparticles for cancer targeting and gene therapy.EXAMPLE 6

[0273] Disclosed herein is a cancer-specific luminoptogenetics-based gene therapy that causes severe mitochondrial dysfunction, thereby causing cancer cell death. A gene delivery vehicle, anti-CD276 mAb-tagged exosome-associated adeno-associated virus (mAb-Exo-AAV), has also been developed to target and kill TNBC cells in vivo. Novel gene therapy was evaluated as monotherapy, in combination with a chemotherapy, poly (ADP -Ribose) polymerase inhibitor (PARPi), and in combination with an antibody drug conjugate (ADC), to treat TNBCs.

[0274] The anti-TNBC efficacy was assessed in multiple animal models, including primary xenograft, metastatic, and PDX mice models.

[0275] The results showed that the CD276 mAb-Exo-AAV specifically targeted TNBC cells in vivo, the delivered gene therapy effectively depolarized cancer mitochondria; and TNBC tumor burden was eliminated with severe cancer cell death and the collapse of the tumorT2025-099 (069596.00097) microenvironment. Metastasis was significantly reduced by this gene therapy in distant metastatic models. Long-term treatment using combined therapies showed that TNBC recurrence was blocked. Taken together, the innovative gene therapy and the disclosed delivery vehicle were developed successfully to treat TNBCs.EXAMPLE 7

[0276] Triple-negative breast cancers (TNBCs) are aggressive and heterogeneous in nature with a recurrence rate observed in over 50% of patients treated with a primary round of conventional chemotherapy. Previous studies showed that CD276 (also known as B7-H3), an immune checkpoint molecule that suppresses natural killer (NK) and T cells responses, is overexpressed in >80% of breast cancers. Mitochondria play a central role in cellular metabolism, proliferation, and apoptosis, making them a promising therapeutic target in cancer. Herein is a combined treatment strategy, i.e. antibody-drug conjugates (ADCs) and gene therapy, to effectively treat CD2761TNBC. First, an anti-CD276 mAb (IgG2b / kappa) that targets the extracellular domain of surface receptor was developed and used to construct antibody - mertansine (DM1) conjugate. Further flow cytometry analysis, confocal microscopy imaging, and IVIS imaging demonstrated the TNBC specificity of the CD276 mAb. The high anti-cancer cytotoxicity and efficacy of CD276 ADC were revealed in three cell lines (MDA-MB-468, MDA-MB-231, 4T1) in vitro and in two TNBC xenografted mouse models in vivo, respectively. Second, an advanced cancer mitochondrial-targeted luminoptogenetics technology (cmLumiOpto) was prepared and a gene delivery vehicle, CD276-targeted monoclonal antibody- tagged exosome-associated adeno-associated virus (mAb-Exo-AAV) constructed. The cmLumiOpto reduced TNBC tumor burden and inhibited metastasis effectively by depolarizing cancer mitochondria and causing severe cancer cell death. The therapies were evaluated as monotherapies and in combination to evaluate combined potency and synergy. Three models, including cell line-based xenograft models, metastasis model and patient-derived xenograft (PDX) models, were used for the in vivo evaluations. Combining CD276-targeted ADC and cmLumiOpto provides targeted therapy for TNBC patients.

[0277] Anti-CD276 mAb was prepared in a shaker flask at 37°C and 130 rpm (Fig. 42A). Flow cytometric analysis of TNBC cells (MDA-MB-231, MDA-MB-468, and 4T1) showed surface binding by CD276 mAb. (Fig. 42B) TNBC targeting and internalization of CD276 mAbT2025-099 (069596.00097) labelled with AF647 was seen at 2 or 24 hrs (Fig. 42C). Live animal and ex vivo imaging confirmed the in vivo TNBC-specific targeting of CD276 mAb-Cy5.5 in 4Tl-FLuc xenografted BALB / cJ models (Fig. 42D) or in MDA-MB-468-FLuc xenografted NSG models (Fig. 42E). Images were captured at 24 hrs post tail vein injection.

[0278] mAb-Exo-AAV was prepared by conjugating CD276 mAh with Exo-AAV via DMPE-PEG-NHS according to methods disclosed herein. (See FIG. 30A)

[0279] CD276-DM1 ADC was prepared by conjugating CD276 mAh conjugated to a microtubulin inhibitor mertansine via sulfo-SMCC linker at the lysine sites on the mAh (see for example, WO 2025 / 019334). The CD276-DM1 ADC (Fig. 43A) and CD276 mAb-Exo-AAV were characterized and evaluated in vitro. FIG. 43B shows SDS-PAGE of CD276 mAh and mAb-DMl ADC. M: marker, 1 : mAh, 2: ADC. FIG. 43C shows ADC conjugation confirmation in HPLC equipped with a MAbPac HIC-Butyl column. The cytotoxicity of DM1 (Fig. 43D) and CD276 ADC (Fig. 43E) were evaluated in TNBC MDA-MB-231 cells, MDA-MB-468 cells and 4T1 cells. FIG. 43F shows confocal images showing the internalization of Exo-AAV into MDA- MB-468 cells 24 hrs after incubation. FIG. 43G shows Nanosight Pro analysis of the concentration and particle size distribution of Exo-AAV.

[0280] FIGS. 44A-F represent in vivo anti-TNBC efficacy of CD276 ADC in immunocompetent models and TNBC PDX models. Fig. 44Ais a graphical representation of tumor volume post treatment. The mouse TNBC 4Tl-FLuc line xenografted female BALB / cJ mice treated with CD276 mAb-DMl, CD276 mAh and saline (controls) following schedule of Q4Dx4 as indicated by the black arrow, n = 7. Data were presented as mean ± SEM. *P<0.05 vs. saline using ANOVA followed by Dunnett’s t-test. FIG. 44B is a graphical representation of changes in mouse body weight (BW) after treatment. FIG. 44C shows HE staining of tumor tissues indicating low tumor intensity and cell death. Scale bar equals to 50 pm. FIG. 44D shows IHC staining of tumor section demonstrating upregulation of tumor immunity, apoptosis, and inhibition of proliferation. Scale bar equals to 50 pm. Fig. 44E is a graphical representation of PDX tumor volume profde. TNBC PDX xenografted in female NSG mice treated with CD276 mAb-DMl and saline following schedule of Q4Dx4 as indicated by the black arrow, n = 7. Data were presented as mean ± SEM. *P<0.05 vs. saline using ANOVA followed by Dunnett’s t-test. FIG. 44F is a graphical representation of normalized body weight.T2025-099 (069596.00097)

[0281] FIGS. 45A-D represent the synergism of combined cmLumiOpto and CD276 ADC. FIG. 45Ais a graphical representation of the cytotoxic effects of saline, cmLumiOpto, ADC and cmLumiOpto+ADC in MDA-MB-231 and MDA-MB-468 cells, n = 3. FIG. 45B is a graphical representation of the seahorse assay indicating oxygen consumption rate of TNBC MDA-MB- 231 cells post treatment, n = 3. FIGS. 45C-D are graphical representation of Multi -pl ex Luminex assay results identifying several enhanced cytokines in tumor microenvironment by synergism of cmLumiOpto and ADC. n = 4. The lung tissues with TNBC metastasis were harvested were used in this study.

[0282] FIGS. 46A-C represent in vivo anti-TNBC synergy of cmLumiOpto and CD276 ADC in immunocompetent metastatic models. Mice were treated with saline (control), CD276 ADC (low dose of 8 mg / kg-BW), cmLumiOpto (low dose of lOxlO10ptc / kg-BW), and combination of cmLumiOpto with CD276 ADC (same dose as monotherapy), n = 5. FIG. 46A shows IVIS imaging of BALB / cJ mice carrying metastatic 4Tl-FLuc. FIG. 46C shows H&E staining of harvested lung tissue from the mouse with strongest bioluminescent signal. Scale bar equals to 2 mm. FIG. 46C shows H&E staining of lung tissue with TNBC tumor. Scale bar equals to 70 pm.

[0283] FIGS. 47A-C represent anti-cancer mechanisms of combined cmLumiOpto and CD276 ADC using syngeneic models of 4T1 xenografted BALB / cJ mice. FIG. 47A shows RNA- seq demonstrating cmLumiOpto downregulated metastasis signaling of Wnt, TGF-P, IL-6 and FGFR. FIG. 47B shows upregulation of tumor immunity (T cells) and IL12. FIG. 47C shows downregulation of DNA damage response and histone lysine methylation, n = 4 / group.

[0284] FIGS. 48A-D represent in vivo anti-TNBC synergy of CD276 ADC and cmLumiOpto in PDX model. Mice were treated with low dose of combined cmLumiOpto (lOxlO10ptc / kg) and CD276 ADC (8 mg / kg-BW) and saline (control), n = 5. FIG. 48Ais a graphical representation of tumor volume profdes. FIG. 48B is a graphical representation of body weight. FIG. 48C shows H&E staining of tumor. Scale bar equals 70 pm. FIG. 48D shows H&E staining of major organs harvested including brain, heart, lungs, liver, spleen and kidneys. Scale bar equals 70 pm.

[0285] FIGS. 49A-B represent evaluation of toxicity of anti-CD276 mAb and ADC in BALB / cJ mice without TNBC tumor xenograft. FIG. 49A shows H&E staining of major organs (brain, heart, liver, kidney, lung, and spleen) at 28 days post i.v. injection of 20 mg / kg ADC via tail vein. Scale bar equals to 100 pm. n = 7. FIG. 49B is a graphical representation of wholeT2025-099 (069596.00097) blood analysis showing minimal effect of anti-CD276 mAb on blood cell count and peripheral immunity, n = 7. WBC: white blood cell; NE: neutrophils; LY: lymphocyte; RBC: red blood cell; Hb: hemoglobin; PTL: platelet.

[0286] FIGS. 50A-F represent in vivo anti-TNBC efficacy of CD276 ADC in immunocompromised models. The human TNBC MDA-MB-231-FLuc line was xenografted in female NSG mice and treated with single-payload ADC, 16 mg / kg anti-human CD276 mAb- DM1 and saline (controls), n = 4. FIG. 50A is a graphical representation of tumor volume post treatment which was started on Day 4 indicated by the black arrow. Tumor volume was measured with calipers and calculated as ellipsoid. Data were presented as mean ± SEM, Saline and ADC were i.v. injected through tail vein. FIG. 50B is a graphical representation of body weight (BW) after treatment. FIG. 50C is a graphical representation of tumor weight on the endpoint. FIG. 50D shows HE staining of tumor tissues indicated low tumor intensity and cell death. Scale bar equals to 70 pm. FIG. 50E shows IHC staining of tumor section demonstrated improvement of tumor immunity, apoptosis, and inhibition of proliferation. Scale bar equals to 70 pm. FIG. 50F shows HE staining of major organs.

[0287] FIGS. 51 A-B represent a toxicity evaluation of combined cmLumiOpto and CD276 ADC in immunocompetent metastasis models. Mice were treated with saline c(control), low- dose cmLumiOpto (10xl010ptc / kg-BW), low-dose CD276 mAb-DMl ADC (8 mg / kg-BW), and combination of cmLumiOpto with CD276 ADC (same dose as monotherapy), n = 5. FIG. 51 A is a graphical representation of mouse body weight profiles during treatment. FIG. 5 IB shows H&E staining of major organs harvested including brain, heart, liver, spleen and kidneys. Scale bar equals 70 pm.

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[0289] Additional exemplary embodiments contemplated herein are as follows: Embodiment 1. A composition comprising an expression vector, wherein the expression vector comprises: a first nucleic acid sequence encoding a channelrhodopsin fusion protein; and a second nucleic acid sequence encoding a luciferase protein, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence; and wherein the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM- MLS).T2025-099 (069596.00097)Embodiment 2. The composition of Embodiment 1 , wherein the channelrhodopsin fusion protein further comprises a channelrhodopsin ion channel domain, wherein the ion channel domain can change the mitochondrial membrane potential (ATm) when light and / or bioluminescence is present.Embodiment 3. The composition of Embodiment 1 or 2, wherein when the expression vector is expressed, the luciferase protein is localized to the cytosol.Embodiment 4. The composition of any one of Embodiments 1-3, wherein the luciferase protein does not comprise and / or lacks a targeting peptide.Embodiment 5. The composition of any one of Embodiments 1-4, wherein the luciferase protein does not comprise and / or lacks a targeting sequence.Embodiment 6. The composition of any one of Embodiments 1-5, wherein the luciferase protein is not a luciferase fusion protein.Embodiment 7. The composition of any one of Embodiments 1-6, wherein the luciferase protein comprises only a luciferase domain.Embodiment 8. The composition of any one of Embodiments 1-7, wherein the luciferase protein is not linked to an outer mitochondrial membrane-mitochondrial localization signal (OMM- MLS), such as for example OMA25 or TOM20.Embodiment 9. The composition of any one of Embodiments 1-8, wherein the luciferase protein is not localized to the outer membrane.Embodiment 10. The composition of any of Embodiments 1-9, wherein the IMM-MLS comprises a leading sequence from a mitochondrial inner membrane protein selected from ABCB10, ABCB140, Cytochrome C, and renal outer medullary potassium channel (ROMK). Embodiment 11. The composition of any of Embodiments 1-10, wherein the IMM-MLS comprises ABCB10.Embodiment 12. The composition of any of Embodiments 1-11, wherein the luciferase protein comprises hRluc, hGluc, NLuc, M23hGluc, or sbGluc.Embodiment 13, The composition of any of Embodiments 1-12, wherein the luciferase protein comprises NLuc.Embodiment 14. The composition of any of Embodiments 1-11, wherein the luciferase protein has an amino acid sequence that is no more than 200 amino acids and the luciferase protein’sT2025-099 (069596.00097) emission spectrum is matched to or overlaps effectively with the channelrhodopsin fusion protein..Embodiment 15. The composition of any of Embodiments 1-14, wherein the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are operably linked to the same expression control sequence Embodiment 16. The composition of any of Embodiments 1-15, wherein the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are separated by a self-cleavable linker.Embodiment 17. The composition of Embodiment 16, wherein the self-cleavable linker is 2A. Embodiment 18. The composition of any of Embodiments 1-17, wherein the expression control sequence comprises a cancer-specific promoter.Embodiment 19. The composition of any of Embodiments 1-18, wherein the cancer-specific promoter is cfos.Embodiment 20. The composition of any of Embodiments 1-19, wherein the expression vector comprises a viral vector.Embodiment 21. The composition of any of Embodiments 1-20, wherein the viral vector is an adeno-associated virus (AAV) vector.Embodiment 22. The composition of any of Embodiments 1-21, further comprising a nanoparticle.Embodiment 23. The composition of Embodiment 22, further comprising one or more antibodies disposed on the nanoparticle surface.Embodiment 24. The composition of Embodiment 23, wherein the antibody is specific for a tumor antigen.Embodiment 25. The composition of Embodiment 23, wherein the one or more antibodies disposed on the nanoparticle surface target CD276, EGFR, or a combination thereof. Embodiment 26. The composition of Embodiment 23, wherein the one or more antibodies disposed on the nanoparticle surface target NK-1R, MET, HER2, Trop-2, Nectin-4, SSTR2, CD 19, CD20, CD22, CD33, CD47, or combinations thereof.Embodiment 27. The composition of Embodiment 23, wherein the one or more antibodies disposed on the nanoparticle surface target NK-1R, MET, HER2, Trop-2, Nectin-4, CD 19, CD20, CD22, CD33, CD47, or combinations thereof.T2025-099 (069596.00097)Embodiment 28. The composition of Embodiment 21, wherein the one or more antibodies disposed on the nanoparticle surface target NK-1R, MET, HER2, Trop-2, Nectin-4, or combinations thereof.Embodiment 29. The composition of any of Embodiments 23-28, wherein the one or more antibodies is attached to the surface of the nanoparticle through a linker.Embodiment 30. The composition of any of Embodiments 23-29, wherein the linker comprises DMPE-PEG or DSPE-PEG.Embodiment 31. The composition of any of Embodiments 23-29, wherein a stabilizer is disposed on the surface of the nanoparticle.Embodiment 32. The composition of Embodiment 31, wherein the stabilizer comprises DMPE, DSPE, PEG or combinations thereof.Embodiment 33. The composition of Embodiment 31 or 32, wherein the stabilizer comprises DMPE-PEG or DSPE-PEG.Embodiment 34. The composition of any of Embodiments 22-33, wherein the nanoparticle comprises an adenovirus.Embodiment 35. The composition of Embodiment 34, wherein the nanoparticle has an AAV packing rate of at least 80 vg per particle.Embodiment 36. The composition of any of Embodiments 22-33, wherein the nanoparticle comprises an extracellular vesicle.Embodiment 37. The composition of Embodiment 36, wherein the extracellular vesicle comprises an exosome.Embodiment 38. The composition of Embodiment 37, wherein the exosome has an average particle size of 150-250 nm, such as for example, 200 nm.Embodiment 39. The composition of any of Embodiments 22-33, wherein the nanoparticle comprises a liposome.Embodiment 40. A method of treating a disease in a patient in need thereof, wherein the method comprises: administering to a patient a therapeutically effective amount of the composition of any of Embodiments 1-39.Embodiment 41. The method of Embodiment 40, wherein the disease is cancer.Embodiment 42. The method of Embodiment 41, wherein the cancer comprises a heterogeneous cancer and / or metastatic cancer.T2025-099 (069596.00097)Embodiment 43. The method of Embodiment 41 or 42, wherein the cancer is a heterogeneous cancer.Embodiment 44. The method of Embodiment 41 or 42, wherein the cancer is a metastatic cancer. Embodiment 45. The method of any of Embodiments 41-44, wherein the composition of any of Embodiments 1-39 reduces the heterogeneity of the cancer.Embodiment 46. The method of any of Embodiments 40-45, wherein the cancer comprises lung cancer, including non-small cell lung cancer, glioma, including glioblastoma multiforme (GBM) or breast cancer, including triple-negative breast cancer (TNBC).Embodiment 47. The method of Embodiment 46, wherein the cancer is recurrent GBM.Embodiment 48. A method of upregulating tumoral immunity in a patient in need thereof, wherein the method comprises: administering to the patient the composition of any of Embodiments 1-39.Embodiment 49. The method of Embodiment 48, wherein tumoral immunity is measured by one or more of single-cell RNA sequencing, flow cytometry, immunohistochemistry, and multiplex Luminex.Embodiment 50. The method of Embodiment 49, wherein tumoral immunity is improved by at least about 20%.Embodiment 51. A method of reducing the mass of cancer stem cells in a patient in need thereof, wherein the method comprises administering to the patient the composition of any of Embodiments 1-39.Embodiment 52. A method of inducing differentiation of cancer stem cells in a patient in need thereof, wherein the method comprises administering to the patient the composition of any of Embodiments 1-39.Embodiment 53. The method of Embodiment 51 or 52, wherein the cancer stem cells comprise non-small cell lung cancer stem cells, GBM stem cells, or TNBC stem cells.Embodiment 54. The method of any of Embodiments 40-53, wherein the method further comprises administering a luciferase substrate.Embodiment 55. The method of Embodiment 54, wherein luciferase substrate is coelenterazine (CTZ) or a CTZ analogueEmbodiment 56. The method of Embodiment 54 wherein the CTZ analogue has the structure:T2025-099 (069596.00097)Embodiment 57. The method of any of Embodiments 54-56, wherein the composition of any of Embodiments 1-39 and the luciferase substrate are administered simultaneously or sequentially. Embodiment 58. The method of any of Embodiments 40-57, wherein administration of the composition of any of Embodiments 1-39 is intravenous.Embodiment 59. The method of any of Embodiments 54-57, wherein administration of the composition of any of Embodiments 1-39 is intravenous and administration of the luciferase substrate is parenteral or transdermal.Embodiment 60. The method of any of Embodiments 54-57, wherein administration of the composition of any of Embodiments 1-39 is intravenous and administration of the luciferase substrate is intramuscular, subcutaneous, or transdermal.Embodiment 61. The method of any of Embodiments 40-57, wherein administration is intracerebroventricular.Embodiment 62. The method of any of Embodiments 40-61, further comprising administering a chemotherapeutic.Embodiment 63. The method of Embodiment 62, wherein the chemotherapeutic has a low response rate in monotherapy.Embodiment 64. The method of Embodiment 62, wherein the chemotherapeutic is an antibodydrug conjugate.Embodiment 65. The method of Embodiment 62, wherein the chemotherapeutic comprises a poly (ADP-ribose) polymerase inhibitor (PARPi).Embodiment 66. The method of Embodiment 65, wherein the PARPi is selected from the group consisting of pamiparib (BGB-290), veliparib (ABT-888), olaparib (Lynparza®), rucaparib (Rubraca®), CEP 9722, niraparib (Zejula®), rucaparib, talazoparib (Talzenna®, BMN-673), 4- amino-l,8-naphthalimide, and E7016.Embodiment 67. The method of Embodiment 66, wherein the PARPi is olaparib.T2025-099 (069596.00097)Embodiment 68. The method of any of Embodiments 62-67, wherein the composition of any of Embodiments 1-39 and the chemotherapeutic are administered simultaneously or sequentially. Embodiment 69. A kit comprising (a) the composition of any of Embodiments 1-39; (b) a luciferase substrate; and (c) instructions for treating a cancer.Embodiment 70. The kit of Embodiment 69, wherein the composition and the luciferase substrate are co-packaged.Embodiment 71. The kit of Embodiment 69, wherein the composition and the luciferase substrate are co-formulated.Embodiment 72. The kit of Embodiment 69, further comprising a plurality of dosage forms, the plurality of dosage forms comprising one or more doses; wherein each dose comprises a therapeutically effective amount of the composition of any of Embodiments 1-39; or the luciferase substrate.Embodiment 73. The kit of Embodiment 69, wherein the dosage form for the composition of any of Embodiments 1-39 is formulated for intravenous administration and the dosage form for the luciferase substrate is formulated for intravenous administration.Embodiment 74. The kit of any of Embodiments 66-72, further comprising (d) at least one chemotherapeutic.Embodiment 75. The kit of Embodiment 74, wherein the chemotherapeutic is an antibody-drug conjugate.Embodiment 76. The kit of Embodiment 74, wherein the chemotherapeutic comprises a poly (ADP -ribose) polymerase inhibitor (PARPi).Embodiment 77. The method of Embodiment 76, wherein the PARPi is selected from the group consisting of pamiparib (BGB-290), veliparib (ABT-888), olaparib (Lynparza®), rucaparib (Rubraca®), CEP 9722, niraparib (Zejula®), rucaparib, talazoparib (Talzenna®, BMN-673), 4- amino-l,8-naphthalimide, and E7016.Embodiment 78. The method of Embodiment 77 wherein the PARPi is olaparib.

Claims

T2025-099 (069596.00097)CLAIMS1. A composition comprising an expression vector, wherein the expression vector comprises: a first nucleic acid sequence encoding a channelrhodopsin fusion protein; and a second nucleic acid sequence encoding a luciferase protein, wherein the first nucleic acid sequence and the second nucleic acid sequence are operably linked to an expression control sequence; and wherein the channelrhodopsin fusion protein comprises a channelrhodopsin protein linked to an inner mitochondrial membrane-mitochondrial localization signal (IMM-MLS).

2. The composition of claim 1, wherein when the expression vector is expressed, the luciferase protein is localized to the cytosol.

3. The composition of claim 1 or claim 2, wherein the IMM-MLS comprises a leading sequence from a mitochondrial inner membrane protein selected from ABCB10, ABCB140, Cytochrome C, and renal outer medullary potassium channel (ROMK).

4. The composition of claim 3, wherein the IMM-MLS comprises ABCB10.

5. The composition of any of claims 1-4, wherein the luciferase protein comprises hRluc, hGluc, NLuc, M23hGluc, or sbGluc.

6. The composition of claim 5, wherein the luciferase protein comprises NLuc.

7. The composition of any of claims 1-6, wherein the first nucleic acid sequence encoding a channelrhodopsin fusion protein and the second nucleic acid sequence encoding a luciferase protein are operably linked to the same expression control sequence and are separated by a self- cleavable linker.

8. The composition of any of claims 1-7, wherein the self-cleavable linker is 2A.T2025-099 (069596.00097)9. The composition any of claims 1-8, wherein the expression control sequence comprises a cancer-specific promoter.

10. The composition of claim 9, wherein the cancer-specific promoter is cfos.

11. The composition of any of claims 1-10, wherein the expression vector comprises a viral vector.

12. The composition of claim 11, wherein the viral vector is an adeno-associated virus (AAV) vector.

13. The composition of any one of claims 1-12 comprising a nanoparticle.

14. The composition of claim 13, further comprising an antibody disposed on the nanoparticle surface.

15. The composition of claim 14, wherein the antibody is specific for a tumor antigen.

16. The composition of claim 15, wherein the tumor antigen comprises CD276, EGFR, or a combination thereof.

17. The composition of any of claims 14-16, wherein the antibody is attached to the surface of the nanoparticle via a linker.

18. The composition of claim 17 wherein the linker comprises DMPE-PEG or DSPE-PEG.

19. The composition of any of claims 13-18, wherein the nanoparticle comprises an adenovirus.T2025-099 (069596.00097)20. The composition of any of claims 13-18, wherein the nanoparticle comprises an extracellular vesicle.

21. The composition of claim 20, wherein the extracellular vesicle comprises an exosome.

22. The composition of any of claims 13-18, wherein the nanoparticle comprises a liposome.

23. A method of treating a disease in a patient in need thereof, wherein the method comprises: administering to a patient a therapeutically effective amount of the composition of any of claims 1-22.

24. The method of claim 23, wherein the disease is cancer.

25. The method of claim 24, wherein the cancer comprises a heterogeneous cancer and / or metastatic cancer.

26. The method of claim 24 or claim 25, wherein the cancer comprises non-small cell lung cancer, a glioblastoma multiforme (GBM) or triple-negative breast cancer (TNBC).

27. A method of upregulating tumoral immunity in a patient in need thereof, wherein the method comprises: administering to a patient the composition of any of claims 1-22.

28. A method of reducing the mass of cancer stem cells and / or inducing differentiation of cancer stem cells in a patient in need thereof, wherein the method comprises: administering to a patient the composition of any of claims 1-22.

29. The method of claim 28, wherein the cancer stem cells comprise non-small cell lung cancer stem cells, GBM stem cells, or TNBC stem cells.T2025-099 (069596.00097)30. The method of any of claims 23-29, further comprising administering a luciferase substrate.

31. The method of claim 30, wherein the composition of any one of claims 1-22 and the luciferase substrate are administered simultaneously or sequentially.

32. The method of any of claims 23-29, wherein administration of the composition is intravenous.

33. The method of any of claims 23-29, wherein administration of the composition is intracerebroventricular.

34. The method of any one of claims 23-33, further comprising administering a chemotherapeutic.

35. The method of claim 34, wherein the composition of any one of claims 1-22 and the chemotherapeutic are administered simultaneously or sequentially.

36. A kit comprising (a) the composition of any one of claims 1-22; (b) a luciferase substrate; and (c) instructions for treating a cancer.

37. The kit of claim 36, wherein the composition and the luciferase substrate are copackaged.

38. The kit of claim 36, wherein the composition and the luciferase substrate are coformulated.

39. The kit of claim 36, further comprising a plurality of dosage forms, the plurality of dosage forms comprising one or more doses; wherein each dose comprises a therapeutically effective amount of the composition or the luciferase substrate.T2025-099 (069596.00097)40. The kit of claim 39, wherein the dosage form for the composition is formulated for intravenous administration and the dosage form for the luciferase substrate is formulated for parenteral administration.

41. The kit of any one of claims 36-40 further comprising (d) at least one chemotherapeutic.

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