WO2016018454A2

WO2016018454A2 - Compositions and methods employing bcl2 and bcl2 family members

Info

Publication number: WO2016018454A2
Application number: PCT/US2015/000070
Authority: WO
Original assignee: Cisthera, Inc.
Priority date: 2014-06-18
Filing date: 2015-06-19
Publication date: 2016-02-04
Also published as: WO2016018454A3

Abstract

Provided are methods employing BCL-2 for the treatment of VDAC-associated disease, wherein the methods comprise administering to a mammal a Bcl-2 protein in an amount sufficient to inhibit a VDAC-associated disease. The disclosure also provides methods for identifying a Bcl- 2 protein that inhibits a VDAC-associated disease when administered to an animal, as well as methods for increasing the effectiveness of a gene, stem cell and/or vaccine therapy involving the administration of an extracellular Bcl-2 protein, a Bcl-2 family member, and/or a fragment thereof comprising a BH4 domain. The disclosure further provides methods for treating diseases involving administering a Bcl-2 protein to a cell that has been engineered to produce exosomes comprising nucleic acid constructs engineered for selective therapeutic transfection of target cell types.

Description

COMPOSITIONS AND METHODS EMPLOYING BCL2 AND

BCL2 FAMILY MEMBERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application was filed on June 18, 2015 as a PCT International patent application and claims the benefit of U.S. Provisional Patent Application Nos. 62/014,048, filed June 18, 2014 and 62/014,055, filed June 18, 2014. The disclosures of U.S. Provisional Patent Application Nos. 62/014,048 and 62/014,055 are incorporated by reference herein in their entirety.

BACKGROUND OF THE DISCLOSURE

Technical Field

[0002] The present disclosure is directed, generally, to Bcl-2 and related proteins, fragments, and variants thereof including extracellular Bcl-2 protein, Bcl-2 family members, and/or fragments comprising a BH4 domain of a Bcl-2 protein or a Bcl-2 family member. The present disclosure also concerns various therapeutic methods and other uses for Bcl-2 and related proteins, fragments, and variants thereof including extracellular Bcl-2 protein, Bcl-2 family members, and/or fragments comprising a BH4 domain of a Bcl-2 protein or a Bcl-2 family member.

Description of the Related Art

[0003] Microparticles such as exosomes constitute a mechanism of cell-cell communication that efficiently delivers micro-RNA (miRNA), mRNA, and proteins to different cell types. Exosomes, including those originating from mesencyhmal stem cells and other cell types, can exhibit therapeutic activity in animal disease models, such as models of fibrosis. An extracellular Bcl-2 family (Bel) member (Bcl-2, A l , Bcl-xL, Bcl-Xs, Bcl-w, CED- 9, Diva. Bfl- 1 , or Mcl- 1 ), or a BH4 domain region thereof, can induce changes in the quantity and/or type of microparticles, such as exosomes, released from mammalian cells. The Bcl-2 protein fami ly governs permeabilization of the mitochondrial outer membrane (MOMP) and can either serve pro-apoptotic or anti-apoptotic functions. According to one view, Bcl-2 proteins are believed to regulate apoptosis by inducing or releasing cytochrome c from the mitochondria into the cytosol. Once released into the cytosol, cytochrome c is known to activate caspases, such as caspase-3 and caspase-9, leading ultimately to apoptosis. [0004] VDAC (Voltage-Dependent Anion Channel), also known as mitochondrial porin, is a class of porin ion channel protein located in the mitochondrial outer membrane. VDAC functions to control influx and efflux of ions and molecules between the mitochondria and cytosol. So named for its ability to change between an "open" conformation at low or zero membrane potential and a "closed" conformation at potentials above 30-40 mV, VDAC forms a beta barrel structure that spans the mitochondrial outer membrane. An anion-selective open conformation facilitates a high-conductance state with high metabolite flux, while a cation- selective low conductance state decreases metabolite flow.

[0005] VDAC is involved in the transport of ATP, ADP, pyruvate, malate, and other metabolites, and, therefore, interacts extensively with various metabolic enzymes. The ATP- dependent cytosolic enzymes hexokinase, glucokinase, and glycerol kinase, as well as the mitochondrial enzyme creatine kinase, have all been found to bind to VDAC. This binding orients the proximal enzymes to ATP released from the mitochondria. In particular, the binding of VDAC to hexokinase is presumed to play a key role in coupling glycolysis to oxidative phosphorylation. Additionally, VDAC is an important regulator of Ca²⁺ transport in and out of the mitochondria. Because Ca²⁺ is a cofactor for metabolic enzymes such as pyruvate dehydrogenase and isocitrate dehydrogenase, energetic production and homeostasis are both affected by VDAC's permeability to Ca²⁺.

[0006] VDAC proteins have also been shown to play a role in apoptosis by assuming an open conformation, which increases permeability of the outer mitochondrial membrane and releases apoptogenic factors such as cytochrome c. In the cytosol, cytochrome c activates proteolytic caspases, which play a major role in cell death. Thus, VDAC activity is implicated in disease and injury contexts including, among others, ischemia/reperfusion injury, muscular dystrophy, Alzheimer's disease, cardiotoxicity and fibrosis, including cystic fibrosis. cCommis et al, Biochim. Biophys. Acta. 181 8(6): 1444- 1450 (2012) and Okada et al., J. Gen. Physiol. 124(5^:513-526 (2004).

SUMMARY OF THE DISCLOSURE

[0007] In accordance with the foregoing, the present disclosure provides Bcl-2 proteins, Bcl-2 protein family members, extracel lular domains of Bcl-2 proteins and family members, and exosomes released from mammalian cells that have been treated with a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein, an extracellular domain of a Bcl-2 protein family member, a BH4 domain region of a Bcl-2 protein, or a BH4 domain region of a Bcl-2 protein family member. Also provided herein are compositions comprising one or more Bcl-2 protein, Bcl-2 protein family member, extracellular domain of a Bcl-2 protein, extracellular domain of a Bcl-2 protein family member, BH4 domain region of a Bcl-2 protein, or BH4 domain region of a Bcl-2 protein family member.

[0008] The present disclosure also provides nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members. Also provided herein are compositions comprising one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members.

[0009] Within yet other embodiments, the present disclosure provides vectors for the delivery and expression of Bcl-2 proteins, Bcl-2 family members, or fragments thereof comprising a BH4 domain region of a Bcl-2 protein or other Bcl-2 family member and compositions comprising one or more Bcl-2 proteins, Bcl-2 family members, or fragments thereof comprising a BH4 domain region of a Bcl-2 protein or other Bcl-2 family member.

[0010] Within still further embodiments, the present disclosure provides methods for identifying Bcl-2 proteins that modulate the production of, or promote the stabilization of, microparticles from a cell, including a stem cell or a lymphoid cell, and, thereby, promoting or improving the delivery of nucleic acids, including therapeutic nucleic acids, or for enhancing or modulating an immune response. Related aspects of these embodiments involve the identification of cell types, including stem cell types and lymphoid cel l types, which are responsive to a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl- 2 protein family member, or an exosome released from a mammalian cell, and, as a consequence of contacting with a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell, are susceptible to one or more of the cellular, physiological, or therapeutic benefits that are described in greater detail herein.

[0011] Within still further embodiments, the present disclosure provides compounds and genetic constructs comprising a polynucleotide that encodes a Bcl-2 protein, a Bcl-2 protein family member, or an extracellular domain of a Bcl-2 protein family member. Within certain aspects, such compounds and genetic constructs may be used in methods for modulating, inducing, enhancing, or inhibiting one or more activity of a cell, which methods comprise contacting a cell with a compound or genetic construct comprising a polynucleotide encoding a Bcl-2 protein, a Bcl-2 protein family member, or an extracellular domain of a Bcl-2 protein family member thereby modulating, inducing, enhancing, or inhibiting the cellular activity. Within related aspects, such compounds and genetic constructs may be used in methods for the treatment of an injury, a condition, or a disease, which methods comprise administering a compound or genetic construct comprising a polynucleotide encoding a Bcl-2 protein, a Bcl-2 protein family member, or an extracellular domain of a Bcl-2 protein family member thereby modulating, inducing, enhancing, or inhibiting one or more cellular activity or phenotype that is associated with the injury, condition, or disease. Within certain aspects,

[0012] Within related aspects, such compounds and genetic constructs may be used in methods for the treatment of an injury, a condition, or a disease, which methods comprise administering a compound or genetic construct comprising a polynucleotide encoding a Bcl-2 protein, a Bcl-2 protein family member, or an extracellular domain of a Bcl-2 protein family member thereby modulating, inducing, enhancing, or inhibiting one or more cellular activity or phenotype that is associated with cellular damage, such as toxic shock damage, in a healthy tissue. Thus, for example, such compounds and genetic constructs may be used in methods for the treatment of a CAR T-cell cancer that is associated with a toxic shock response in a mammal.

[0013] Within other embodiments, the present disclosure provides compositions and methods for treating cells, including stem cells and lymphoid cells, or a disease, such as a cancer, which is associated with a particular cell, which methods comprise contacting with, or administration of, a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein family member.

[0014] Within other embodiments, the present disclosure provides compositions and methods for inducing the in vivo production of microparticles and/or vesicles, including therapeutically active microparticles and/or vesicles, such as exosomes, ectosomes, and nonvesicular microparticles, which methods comprise contacting a cell with one or more Bcl-2 proteins, Bcl-2 family members, or fragments thereof comprising a BH4 domain region of a Bcl-2 protein or other Bcl-2 family member.

[0015] Within further embodiments, the present disclosure provides compositions and methods for the treatment of diseases, such as fibrosis and other VDAC-associated diseases, which compositions comprise a Bcl-2 protein, a Bcl-2 protein family member, an extracellular Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein, a Bcl-2 protein family member, or a BH4 domain region thereof, and which methods comprise the administration of, including the in vivo administration of, a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein family member.

[0016] Within other embodiments, the present disclosure provides gene, stem cell, and vaccine therapies and related methods for enhancing the effectiveness of gene therapies, stem cell therapies, or vaccine therapies, which methods comprise the in vivo administration of a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein family member.

[0017] Certain aspects of the methods disclosed herein involve the use of a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein family member to modulate, induce, or enhance the production, release, or stabilization of exosomes that are engineered to modulate, induce, or enhance the production of CAR T-cells for cancer therapy.

[0018] Other aspects of the present methods involve the use of a Bcl-2 protein, a Bcl-2 protein family member, an extracellular domain of a Bcl-2 protein family member, or an exosome released from a mammalian cell that has been treated with an extracellular domain of a Bcl-2 protein family member to modulate, induce, or enhance the production, release, or stabilization of exosomes that are engineered to selectively modulate, induce, or enhance one or more therapeutic activities of a cel l, which cel l may be associated with one or more disease states or other conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The present disclosure will be best understood by reference to the following drawings.

[0020] FIG. 1 shows the design of a laser-induced choroidal neovascularization (CNV) mouse study from which it was discovered that Bel proteins can inhibit fibrosis. Eyes were subjected at four sites to laser-induced injury on day 0. On day 1 and day 4 CTA l (recombinant human A l protein) was administered intraperitoneally to 8 mice. As a positive control, a humanized anti-VEGF antibody was administered to another 8 mice. At days 4 and/or 7, vascular leak (a measure of neovascularization) was assessed by fluorescein fundus angiography. On day 7 or 8, mice were sacrificed and eyes processed for histology.

[0021] FIG. 2 shows results of the laser-induced CNV study presented in FIG. 1. FIG. 2A presents representative fluorescein angiogram images that show a decrease in the area of leakage in CTAl treated animals. CTAl treatment significantly reduced the mean grade of lesion. FIG. 2B shows the quantification of lesion area leakage by flat mount and further confirms that CTAl treated animals had significantly less leakage compared to control. These data demonstrate that CTAl treatment decreases neovascularization as determined by vascular leak assay.

[0022] FIG. 3 shows immunohistochemistry (IHC) data obtained with a mouse model of choroidal neovascularization. These data revealed that the in vivo administration of CTAl resulted in a decrease in vessel formation in tissue stained with an anti-CD31 antibody having vessel binding activity and demonstrated that CTAl treatment decreased neovascularization in the mouse model.

[0023] FIG. 4 shows immunohistochemistry (IHC) data obtained with the mouse model of choroidal neovascularization. These data revealed that the in vivo administration of CTA l reduced the number of macrophage/monocytic cells, which are key contributors to fibrogenesis through their production of profibrotic cytokines and growth factors (Wynn and Barron, Semin Liver Dis 30(3):245-257 (2010)), in laser-induced CNV tissue stained with an anti-CD 163 antibody.

[0024] FIG. 5 shows that CTA l treatment reduced collagen accumulation in laser-induced CNV tissue as visualized by staining with Sirius Red. These data demonstrated that CTA l treatment reduced fibrosis in the laser-induced CNV model. [0025] FIG. 6 shows that CTA 1 treatment reduces fibrosis in a glaucoma filtration surgery (GFS) model. Mice were treated with CTAl (0.8 μg, via subconjunctival injection) or PBS vehicle (n=10/group) on the day of surgery and on day 2 post-surgery. Bleb survival was determined by slit lamp biomicroscopy on day 0, -2, -7 and weekly thereafter. These data demonstrate that CTA l treatment significantly prolongs bleb survival over 30 days. Because failure of bleb survival is due to fibrosis, CTA l treatment appears to reduce fibrosis in the GFS model.

[0026] FIG. 7 shows that Bcl-2 proteins and a BH4 domain region peptide can induce a change in the quantity or type of microparticles/exosomes released from cultured mammalian cells including mesencyhmal stem cells, monocytic cells and dendritic cells. Microparticles/exosomes released from mammalian cells treated with a Bcl-2 protein or a Bcl- 2 protein fragment containing the BH4 domain region can be used to treat a disease or an injury, such as that caused by ischemia and reperfusion. Bcl-2 protein treatment of cultured mammalian cells with microparticles induced therapeutic cytoprotective activity in the culture media that was demonstrated in a mouse model of ischemic injury.

[0027] FIG. 8 is a diagram presenting an in vitro assays for developing a Bcl-2 -related protein or exosome drug and identifying Bel-induced mediators of activity. Bcl-2 family members or their BH4 domain region peptides can induce microparticles (e.g., exosomes) containing mediators of activity from cultured cells.

[0028] FIGs. 9A-9C presents exemplary in vitro assays that may be employed for developing a Bel drug candidate and for identifying Bel-induced mediators of extracellular Bel activity. Cultured media of Bcl-2 -treated cells can be processed in several assay systems for quantitative and qualitative analysis of induced mediators. For example, centrifuged and buffer exchanged culture media may be subjected to spectrophotometry to determine induced protein and R A concentrations (FIG. 9A), subjected to SDS-PAGE and protein stain to determine size profile or induced proteins (FIG. 9B), subjected to an ELISA designed to capture exosomes to determine the induction of exosomes (FIG. 9C). The exosome ELISA may use antibodies that bind to exosome membrane markers (e.g., Rab5b and CD63) in a sandwich ELISA (FIG. 9C).

[0029] FIG. 10 shows that a BH4 domain peptide binds to a mammalian cell surface receptor. A concentration range (20 nM to 1 μΜ) of n-terminal biotinylated A l BH4 peptide, scrambled A l BH4 peptide (sBH4), or a Bax BH3 peptide (BH3) were allowed to bind to dendritic cells (Jaws2) and T cells (Jurkat) for 30 minutes at 4°C. Peptide binding was determined by subsequent binding of phycoerythin labeled-strepavidin and flow cytofluorimetry. Specific and saturable binding of the A l BH4 peptide (red) indicates binding to a cell surface receptor. These data demonstrated that biotinylated-BH4 peptide is functional, maintaining its anti-apoptotic activity in the mouse hind limb I R injury model, performed as described in Iwata et al., PLoS one 5(2):e9103 (2010).

[0030] FIG. 11 is a diagrammatic representation of putative Bcl-2 BH4 binding sites or motifs from A l , Bcl-2, and their respective BH4 peptides, each of which induce microparticle mediators of therapeutic activity. The BH4 domain region peptides of Bcl-2 family members that induce microparticles/exosomes contain the BH4 domain alpha-helix and a loop structure (top; A l , M3QP; Bcl2, 1 GJH). The A l peptide binding to cells in a specific and saturable fashion indicates a specific plasma membrane receptor. The structure and homology between Bcl-2 family members reveals at least 3 potential binding motifs: ( 1 ) a conserved hydrophobic motif on one face of the amphipathic helix, (2) conserved hydrophilic residues in 3 out of 4 family members) on the other face of the helix, and (3) conserved residues in the amino terminal region of the loop region. A solvent-exposed, accessible glutamine is positioned in the amino-terminal region of the loop (blue arrows). The loop structure may allow for a large BH4 helix movement allowing receptor accessibility to any of the three potential binding sites.

[0031] FIG. 12 presents representative liver tissue sections following treatment with Bcl- 2 protein family members (Bcl-W, Bcl-A l , and Bcl-X) and demonstrates that treatment with Bcl-2 protein family members was effective in protecting mice from CC14-induced fibrosis. Moreove, Bcl-2 treatment decreased the extent of bridging fibrosis (arrows) and thickness of collagen fibers as determined by Sirius red staining. Treatment with Bcl-w and Bcl-A l (CTA 1 ) exhibited a greater effect than Bcl-x in this liver fibrosis model system, which is diagrammed in FIG. 13.

[0032] FIG. 13 is a diagram that illustrates the design of the four-week CCL4-induced liver fibrosis study presented in FIG. 12. Mice (n=5) were administered either Bcl-A l , Bcl-W, or Bcl-X ( 1 .2 μg ip/mouse) on days three and twelve. In parallel, as a negative control, mice (n=5) were administered saline. CCI4 was administered (ip) to each of the mice (i.e.. Bcl-A l , Bcl-W, Bcl-X and saline) twice per week. At the end of the fourth week, mice were sacrificed and blood and liver tissue was collected for analysis. [0033] FIG. 14 is a graph showing that treatment with the Bcl2 family members Bcl-W, Bcl2-A l , and Bcl-X was effective in decreasing the blood levels of the enzyme alanine transaminase (ALT), an indicator of liver injury. Treatment with Bel family members decreased blood levels of ALT relative to control mice. ALT was measured according to manufacturer's instructions (ThermoTrace™ transaminase kits). Each point represents the mean of triplicate measurements.

[0034] FIG. 15 is a graph showing that treatment with Bcl-2 family members decreased the blood levels of the enzyme aspartate transaminase (AST), an indicator of liver injury. Treatment with Bcl-2 family members decreased blood levels of AST relative to control mice. AST was measured according to manufacturer's instructions (ThermoTrace™ transaminase kits). Each point represents the mean of triplicate measurements.

[0035] FIG. 16 is the amino acid sequence of Homo sapiens Bcl2-A l .

[0036] FIG. 17 is the amino acid sequence of Homo sapiens Bcl2

[0037] FIG. 18 is the amino acid sequence of Homo sapiens Bcl-X.

[0038] FIG. 19 is the amino acid sequence of Homo sapiens Bcl-W.

[0039] FIG. 20 is a flow chart showing the Bcl-2 pull-down protocol presented in Example 7.

[0040] FIG. 21 is an SDS-polyacrylamide gel showing proteins pulled-down with Bcl-W BH4 peptide- A agarose (lanes 1 and 2); with Bcl2-A l -biotin agarose (lanes 3 and 4); and with NA agarose preclear (lane 5). Molecular weight markers were run in lane 6.

[0041 ] FIG. 22 is an SDS polyacrylamide gel showing proteins isolated by interaction with the BH4 domain of Bcl2 fami ly members. Analysis of a 30-40 kDa protein band identified in a pull down using a Bcl-W BH4 domain peptide revealed peptides from the voltage-dependent anion channels (VDACs) 1 , 2, and 3).

[0042] FIG. 23 presents flow cytometry data showing CTA l binding to a subpopulation of HE cells that also bind a VDAC- 1 antibody (LS-C 160510; Life Science Bio™) and demonstrates that, relative to a biotinylated BSA control, CTA l predominantly binds the same subpopulation that binds the VDAC-1 antibody but does not bind to a second subpopulation of HE that does not bind the VDAC- 1 antibody.

[0043] FIG. 24 presents flow cytometry data showing CTAl and VDAC binding for HEK cells in both isotonic and hypotonic conditions. Two gated HEK cell populations were analyzed. These data demonstrate that all VDAC1 positive HEK cells tested also bound CTA l .

[0044] FIG. 25 is a diagrammatic representation of VDAC, which is a beta-barrel composed of 19 strands and an alpha helical n-terminal sequence of 26 amino acids. The n- terminus of VDAC is shown localized within the lumen of the channel stabilizing a resting open state.

[0045] FIGs. 26-28 presents a short term in vivo assessment of CTA l activity for facilitating CTAl development, which is achieved by assaying liver enzymes as a measure of tissue damage/apoptosis, inflammatory mediators, exosome mediators/PD markers, and comparing the activity of different lots of CTAl or engineered variants (e.g., CTAl .2 and CTAl 40).

[0046] FIGs. 29A and 29B present immunohistochemistry data for PAI-1 on 6 μπι cryo- sectioned retina (laser burn area) obtained from a laser induced retinal choroidal neovascularization mouse model. Slides were incubated with primary antibodies specific for PAl- 1 and, before examination, the nuclei were counterstained with DAPI using UltraCruz™ Mounting Medium sc-24941. CTAl treatment reduced PAI-1 expression relative to control in a particular vesicle-associated PAI-1 (arrow in FIG. 29A). These images were taken at 40X magnification with an exposure time of 2000 ms.

[0047] FIGs. 30A-30C present immunohistochemistry data for VEGF on 6 μι cryo- sectioned retina (laser burn area) obtained from a laser induced choroidal neovascularization mouse model. VEGF is highly expressed at the area of laser burn in the PBS treated control (FIG. 30A). CTA l treated groups exhibited minimal VEGF expression as compared to the PBS and VEGF antibody treated groups (compare FIGs. 30B and 30C). These images were taken at 20X magnification with an exposure time of 1000 ms.

[0048] FIGs. 31A-31C present immunohistochemistry data for platelet-derived growth factor (PDGF) on 6 μιη cryo-sectioned retina (laser burn area) obtained from a laser induced choroidal neovascularization mouse model. PDGF is highly expressed in the VEGF antibody treated group whereas other groups demonstrated lower levels of PDGF expression (compare FIGs. 31B and 31C). These images were taken at 40X magnification with an exposure time of 1500 ms.

[0049] FIG. 32 is a diagram illustrating the design of a four-week CCL4-induced liver fibrosis study. At day 0 (DO), Mice (n=28) were administered either CCL4 (n=24) or a saline negative control (n=4). At D14, a test group (n=8, middle arrow set) was administered CTA 1 while an internal control group (n=16) was administered either CTA1 (n=8) or saline (positive control, n=8) and the negative control group received a saline. At D21 , the test group was administered a second dose of CTA1. All animals were sacrificed at D28 and liver tissue was collected for analysis.

[0050] FIG. 33 presents representative liver tissue sections from negative control mice (FIGs. 33A and 33A'), positive control mice (CCL4 + saline; FIGs. 33B and 33B'), and test mice (CCL4 at D14 and two doses of CTA- 1 (D14 and D21 ; FIGs. 33C and 33C') from the study described in FIG. 32. Anti-alpha-SMA antibody (FIGs. 33A-33C) was used as an indicator of fibrosis. Histology staining was performed with Sirius Red (FIGs. 33A'-33C), a marker of collagen accumulation. These data demonstrate that CTA-1 decreases collagen accumulation in a CCL4-induced liver-wound model.

[0051] FIG. 34 presents hepatic mRNA expression data (determined by standard qPCR) for known fibrosis mediators TGF-β, alpha-SMA, Col l al , and PAI-1 from the mouse study groups described in FIG. 32. Expression levels are presented as a X-fold increase relative to control. Bars (L-R) in each representative graph are as follows: (1 ) saline negative control, (2) CCL4+saline, (3) CCL4 + D14 CTA-1 , and (4) CCL4 + D14 and D21 CTA- 1. Second CTA-1 doses were administered at D21 to demonstrate efficacy in a model of repetitive injury and late therapeutic dosing. These data demonstrate that treatment with CTA- 1 decreases expression of pro-fibrogenic components Col l al (collagen), TGF-β, and PAI-1 . Expression levels of these components were significantly lower in CTA- 1 -treated mice as compared to negative control mice (p<0.05) and mice administered CCL4 alone (p<0.05). Statistical analysis was performed by ANOVA with Tukey's post-hoc test. These data demonstrate that CTA-1 is effective in reducing alpha-SMA levels in this mouse fibrosis model system.

[0052] FIG. 35 presents hepatic mRNA expression data for TGF-β, alpha-SMA. Col l al , and PAI- 1 in negative control, CCL4-dosed and CCL4+CTA-1 (2X)-dosed mice. mRNA expression for each fibrosis mediator was significantly higher in CCL4 mice than in negative control mice (p<0.05), but CCL4 +CTA- 1 mice (2X) exhibited significantly lower expression levels relative to CCL4 mice (p<0.05). Statistical analysis was performed by ANOVA with Tukey 's post-hoc test. These data demonstrate efficacy of CTA- 1 in this mouse fibrosis model system.

[0053] FIG. 36 is a diagrammatic representation of the mechanistic relationship between PAl- 1 expression and plasmin-mediated degradation of Αβ (beta-amyloid) protein in Alzheimer's disease and supports the administration of CTA-1 to reduce PAI- 1 expression and, thereby, promote the degradation of Αβ and APP.

[0054] FIG. 37 presents data demonstrating that treatment with a Bcl-2 protein family member induces a therapeutic cytoprotective activity in mammalian cells, including mesencyhmal stem cells, monocytic cells, and dendritic cells, which therapeutic cytoprotective activity is shown to be associated with the generation of microparticles.

[0055] FIG. 37A presents data obtained from various mammalian cell types, including human THP- 1 monocytic, mouse mesenchymal stem cells (MSC) and mouse JAWS II dendritic cells, which were treated with 300 ng/ml of recombinant human AI (rhAI; SEQ ID NO. 169) having a six-histidines carboxy -terminal tag. After four hours, unbound rhAI was removed by three large volume washes and after 24 hours of culture, cytoprotective activity assessed in the ischemic injury model. Cytoprotective activity was measured by TUNEL stain for apoptosis and MTT (tetrazolium dye) was measured for metabolic activity or tissue viability as was reported for the direct administration of rhAI, Bcl-2, or their respective BH4 domains in Iwata et al. , PLoS one 5(2) e9103 (2010).

[0056] FIG. 37B presents data demonstrating an increase in microparticle production in response to rhAI treatment as assessed by measuring an increase in protein production and RNA levels by optical density (O.D.) in buffer exchanged rhAI treated cell culture (n=5) media using a 100 to 300 kD molecular weight filter to remove proteins and other relatively small molecules.

[0057] FIGs. 37C and 37D are bar graphs of tissue MTT assay data that was obtained from a model of skeletal muscle ischemic injury, which data demonstrate a therapeutically-effective cytoprotective activity in culture media from rhAl-induced THP- 1 monocytic cells and rhAI- induced mesencyhmal stem cells (MSC). [0058) FIGs. 37E and 37F are bar graphs of data that demonstrates a cytoprotective activity that is associated with induced microparticles/exosomes that were recovered in a pellet fraction following ultracentrifugation. Exosomes may also be purified by Exoquick (Systems Biosciences. CA).

[0059] FIG. 37G presents transmission electron microscopy images at Ι Ο,ΟΟΟΧ magnification. These data demonstrate that rhAl treatment of mammalian cells specifically induces the production of microparticles having diameters of 40- 100 nm, which size range is consistent with the formatyion of exosome vesicles (indicated by the arrows).

[0060] FIG. 37H is a graph of data obtained from a mouse model of hind leg ischemic injury, which data demonstrate that a Bcl -2 fragment containing a BH4 domain (SEQ ID NO. 173) induced Jaws II cellular expression of an extracellular mediator of cytoprotective activity.

[0061] FIG. 38 presents flow cytometry data demonstrating that treatment with a Bel protein (rhA l ) promotes the intercellular transfer of a microRNA (miRNA) between two distinct cell types. A fluorescently labeled marker miRNA (PAM-labeled pre-miR; Ambion Inc) was introduced into human monocytic cells (THP- 1 ) using lipofectamine 2000 (Invitrogen Inc). At 2 hours post-transfection, the THP- 1 cells were treated with 300 ng/ml of rhA l (SEQ ID NO. 169) or saline. At 6 hours post-transfection, THP- 1 cells were subjected to 3 large volume washes and the culture continued. At 12 hours post-transfection, microparticles were prepared as described herein and transferred to a culture of HE cells for 10 hours of exposure. Prior to flow cytofluorimetry, the HEK cells were trypsinized to remove remaining extracellular exosomes. Percent specific fluorescence is indicated in the upper right quadrant of each scatter plot. rhA l treatment resulted in an approximate 3-fold increase in miRNA transfer relative to the saline control (after mock background subtract).

[0062] FIG. 39 is a graph showing survival following radiation exposure wherein a single dose of Bcl -2 A l protein was administered either prior to or following exposure to radiation (see, Example 21 ). These data demonstrate that pre-treatment with Bc l -2 A l resulted in increased survival (as compared with saline) while post-treatment with Bc l -2 A 1 had no effect.

[0063] FIG. 40 is a diagrammatic representation of certain methods for the in vitro production of exosomes with a CAR vector and a T-cell ligand, as well as methods that employ the administration of exosomes to T-cells, whereby CAR T-cells are generated by integration of the CAR vector, which methods may optionally include the expression of CAR and stimulated T-cell proliferation, in vivo administration of the CAR T-cells to a patient, and targeted killing of cells by the CAR T-cells.

DETAILED DESCRIPTION

[0064] The present disclosure is based upon the discovery that Bcl-2, Bcl-2 family members, fragments comprising a BH4 domain region of Bcl-2 and Bcl-2 family members, and exosomes/microparticles produced in response to Bcl-2, a Bcl-2 family member, a Bcl-2 BH4 domain region, or a Bcl-2 family member BH4 domain region may be advantageously employed in methods for the treatment of conditions, injuries, or diseases, including VDAC- associated diseases such as fibrosis.

[0065] As disclosed herein, Bcl-2 proteins, Bcl-2 family member proteins, and fragments comprising a BH4 domain region of a Bcl-2 protein or Bcl-2 family member protein may be employed to induce a qualitative or quantitative change in microparticles/exosomes, which are released in vivo and deliver mediators of therapeutic activity to different target cells. Moreover, it is disclosed herein that microparticle-dependent cellular communication can be modulated by an extracellular Bcl-2 protein, an extracellular Bcl-2 family member, and fragments comprising a BH4 domain region of Bcl-2 or other Bcl-2 family members thereby enhancing, inhibiting, or modulating one or more cellular activities, including one or more therapeutic activities, such as nucleic acid transfer, and may be employed in methods for generating stem cells or vaccines.

Bcl-2, Bcl-2 Family Members, and BH4 Domain Resigns Thereof

[0066] The present disclosure provides Bcl-2 proteins, Bcl-2 family member proteins, and fragments comprising a BH4 domain region of Bcl-2 protein or other Bcl-2 family member and compositions comprising such proteins and fragments for use in the methods of the present disclosure. Also provided are nucleic acids encoding Bcl-2 proteins, Bcl-2 fami ly member proteins, and fragments comprising a BH4 domain region of Bcl-2 proteins or other Bcl-2 family members and vectors for the delivery of Bcl-2 proteins, Bcl-2 family member proteins, and fragments comprising a BH4 domain region of Bcl-2 proteins or other Bcl-2 family members as well as vectors for the expression and delivery of nucleic acids encoding Bcl-2 proteins. Bcl-2 family member proteins, and fragments comprising a BH4 domain region of Bcl-2 proteins or other Bcl-2 family members. [0067] The various Bcl-2 proteins, Bcl-2 family member proteins, and fragments comprising a BH4 domain region of Bcl-2 proteins or other Bcl-2 family members and corresponding nucleic acids and vectors may be advantageously employed for use in the various methods disclosed herein, including methods for inducing the production and release of exosomes having desired therapeutic properties, for the production of CAR T-cells for cancer therapy, for the production of other recombinant cell types, and for facilitating gene transfer between cells, each of which will find application in the methods disclosed herein for the treatment of a tissue injury, a disease, or a condition that is susceptible to such treament. It was discovered in connection with the present disclosure that Bcl-2 protein may, for example, be used to inhibit, prevent, or mitigate toxic shock that is associated with certain injuries, conditions, diseases, or therapies, such as CAR T-cell therapy.

[0068] The Bcl-2 proteins and Bcl-2 family proteins disclosed herein, or identified by the presently disclosed methodologies, may be advantageiously employed in the methods of this disclosure to: ( 1 ) inhibit fibrosis; (2) increase the effectiveness of microparticle mediated nucleic acid, stem cell, or vaccine therapy in a mammal; (3) induce production or stabilization of exosomes from a cell engineered to produce certain exosomes; and (4) prevent or mitigate cytotoxic shock. Bcl-2 proteins, including Bcl-2-A l proteins, Bcl-2-X proteins, and Bcl-2- W proteins, are intracellular cytoplasmic proteins, which are known to inhibit apoptosis. See, e.g., Adams and Cory, Science 281 : 1322- 1326 ( 1998); Cory et ai, Oncogene 22:8590-8607 (2003); Karsan et ai , Blood 87(8):3089-3096 (1996); Choi et ai, Mammalian Genome 8:781 -782 ( 1997); Boise et al, Cell 74(4):597-608 ( 1993); and Gibson et ai, Oncogene 13(4):665-675 (1996). Bcl-2 proteins and Bcl-2 family proteins include members of the following Groups (a) through (g):

[0069] Group (a) proteins include those Bcl-2 proteins having an amino acid sequence that is at least 50% identical to, and retaining one or more of the functional activities of, the amino acid sequence set forth in SEQ ID NO: 169, which is the amino acid sequence of Bcl-2-A l having a deletion of 23 carboxy-terminal amino acids. Group (a) proteins also include those Bcl-2 proteins that are at least 55%, or at least 60%, or at least 65%, or at least 70%. or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%. or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 169, wherein each such Group (a) protein retains one or more of the functional activities of the Bcl-2 protein having the amino acid sequence of SEQ I D NO: 169. TDCEFGYIYRLAQDYLQCVLQIPQPGSGPS TSRVLQN

VAFSVQ EVE NLKSCLDNVNVVSVDTARTLFNQVME EFEDGIINWGRIVTIFAFEGIL1 LLRQQIAPDVDTYKE 1SYFVAEFIMNNTGEWIRQNGGWENGFVKKFEP S (SEQ ID NO: 169).

[0070] Group (a) proteins include those Bcl-2 proteins having an amino acid sequence that is at least 50% identical to, and retaining one or more of the functional activities of, the amino acid sequence set forth in SEQ ID NO: 170, which is the amino acid sequence of Bcl-2 having a deletion of 28 carboxy-terminal amino acids. Group (a) proteins also include those Bcl-2 proteins that are at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 170, wherein each such Group (a) protein retains one or more of the functional activities of the Bcl-2 protein having the amino acid sequence of SEQ ID NO: 170.

MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGA

APPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAP

GAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSS

QLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGV

MCVESVNREMSPLVDNIALW TEYLNRHLHTWIQDNGG

WDAFVELYGPSMRPLFD (SEQ ID NO: 170).

[0071] Group (a) proteins include those Bcl-2 proteins having an amino acid sequence that is at least 50% identical to, and retaining one or more of the functional activities of, the amino acid sequence set forth in SEQ ID NO: 171 , which is the amino acid sequence of Bcl-2 having a deletion of 22 carboxy-terminal amino acids. Group (a) proteins also include those Bcl-2 proteins that are at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 171 , wherein each such Group (a) protein retains one or more of the functional activities of the Bcl-2 protein having the amino acid sequence of SEQ ID NO: 171.

MSQSNRELVVDFLSY LSQKGYSWSQFSDVEENRTEAPE GTESE ETPSAINGNPSWHLADSPAVNGATAHSSSLDARE VIPMAAV QALREAGDEFELRYRRAFSDLTSQLH1TPGTA

YQSFEQVVNELFPvDGVNWGRIVAFFSFGGALCVESVD E MQVLVSR1AAWMATYLNDHLEPWIQENGGWDTFVELYG NNAAAESRKGQERF^{^}N (SEQ ID NO: 171 ).

[0072] Group (a) proteins include those Bcl-2 proteins having an amino acid sequence that is at least 50% identical to, and retaining one or more of the functional activities of, the amino acid sequence set forth in SEQ ID NO: 172, which is the amino acid sequence of Bcl-2-W having a deletion of 29 carboxy-terminal amino acids. Group (a) proteins also include those Bcl-2 proteins that are at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 172, wherein each such Group (a) protein retains one or more of the functional activities of the Bcl-2 protein having the amino acid sequence of SEQ ID NO: 172.

MATPASAPDTRALVADFVGYKLRQKGYVCGAGPGEGPAA DPLHQA RAAGDEFETRFRRTFSDLAAQLHVTPGSAQQRF TQVSDELFQGGPNWGRLVAFFVFGAALCAESVNKE EPL VGQVQEWMVAYLETRLADWIHSSGGWAEFTALYGDGAL EEARRLRE (SEQ ID NO: 172).

[0073] Group (b) proteins include those Bcl-2 proteins having an amino acid sequence of at least four amino acids, which is at least 50% identical to, and retaining one or more of the functional activities of, a corresponding fragment of the amino acid sequence set forth in SEQ ID NO: 170, which is the amino acid sequence of Bcl-2 having a deletion of 28 carboxy- terminal amino acids.

[0074] Group (b) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 174, wherein each such Group (b) proteins retain one or more of the functional activities of the Bcl-2 protein having the amino acid sequence of SEQ ID NO: 174 (TGYDNREIV KYIHYKLSQRGYEWD). (0075] Group (c) proteins include those Bcl-2 proteins having an amino acid sequence of at least four amino acids, which is at least 50% identical to. and retaining one or more of the functional activities of, a corresponding fragment of the amino acid sequence set forth in SEQ ID NO: 169, which is the amino acid sequence of Bcl-2-Al having a deletion of 23 carboxy- terminal amino acids.

[0076] Group (c) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%), or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the segment of a Bcl-2-Al protein, which is referred to as Bfl-1, wherein the Al protein consists of the amino acid sequence set forth in SEQ ID NO: 169.

[0077] Group (c) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the segment of a Bcl-2-A l protein, wherein the Al protein consists of the amino acid sequence set forth in SEQ ID NO: 173 (FGYIYRLAQDYLQCVLQIPQPGSGP).

[0078] Group (d) proteins include those Bcl-2 proteins having an amino acid sequence of at least four amino acids, which is at least 50% identical to, and retaining one or more of the functional activities of, a corresponding fragment of the amino acid sequence of Bcl-2-X set forth in SEQ ID NO: 171.

[0079] Group (d) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%>, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%o, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the segment of Bcl-2-X having the amino acid sequence set forth in SEQ ID NO: 175 (MSQSNRELVVDFLSYKLSQKG YS WSQF).

[0080] Group (e) proteins include those Bcl-2 proteins having an amino acid sequence of at least four amino acids, which is at least 50% identical to. and retaining one or more of the functional activities of, a corresponding fragment of the amino acid sequence of the segment of a Bcl-2- W protein set forth in SEQ ID NO: 172. [0081] Group (e) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%>, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the segment of a Bcl-2-W having the amino acid sequence set forth in SEQ ID NO: 172.

[0082] Group (e) proteins also include those Bcl-2 proteins that are at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%), or at least 75%, or at least 80%, or at least 85%>, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the segment of a Bcl-2-W having the amino acid sequence set forth in SEQ ID NO: 176 (SAPDTRALVADFVGY LRQKG YVC).

[0083] As used herein, the term "sequence identity" is expressed as "percent identity" between two proteins and refers to the number of amino acid residues in the two sequences that are the same when the two sequences are aligned for maximum correspondence over a specified comparison window. For example, if two 100 amino acid protein sequences are compared and 75 of the amino acids in the first sequence are the same as, and align with, 75 of the amino acids in the second sequence, then the percent identity between those two proteins is 75%.

[0084] Sequence identity values provided herein refer to the value obtained using GAP (e.g., GCG programs (Accelrys, Inc., San Diego, Calif.) version 10) using GAP Weight of 50 and Length Weight of 3. GAP uses the algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443-53 (1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.

[0085] An "equivalent method" to GAP may be used where the term "equivalent method" refers to any sequence comparison method, such as a sequence comparison program, that, for any two sequences in question, generates an alignment having identical amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP.

[0086] As used herein, the term "sequence similarity" refers to a statistical measure of the degree of relatedness of two compared protein sequences. "Percent similarity" is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g.. whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair.

[0087] Calculations of "sequence similarity" are made after a best fit alignment of the two sequences has been made empirically by iterative comparison of all possible alignments, (see, e.g., Henikoff and Henikoff, Proc. Nat'lAcad. Sci. USA 89: 10915- 10919, 1992). For example, "sequence similarity" can be determined using the ClustalW alignment program for full alignment, single CPU mode, using the GONNET matrix, a gap opening penalty of 100, a gap closing penalty of - 1 , a gap extending penalty of .2 and a gap separation penalty of 4. In the aligned sequences, similarity is defined as two amino acids being identical or having conserved substitutions or having semi-conserved substitutions. The ClustalW alignment program is available, for example, on the Internet at the web page of the European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CBIO 1 SD, U.K.

[0088] Bcl-2 proteins include, for example, naturally-occurring Bcl-2 proteins, synthetic Bcl-2 proteins that may incorporate non-natural amino acids, modified peptides such as "stapled" peptides or cyclic peptides and Bcl-2 fusion proteins in which a protein, peptide, amino acid sequence, or other chemical structure, is attached to a portion (e.g., N-terminal or C-terminal) of a Bcl-2 protein. Representative examples of proteins or chemical structures that can be fused to a Bcl-2 protein include: human serum albumin, an immunoglobulin, polyethylene glycol, or other protein or chemical structure that, for example, increases the serum half-life of the Bcl-2 protein, or increases the efficacy of the Bcl-2 protein, or reduces the immunogenicity of the Bcl-2 protein.

[0089] Representative, non-limiting, amino acid sequences of exemplary Bcl-2 proteins, which may be used in the compositions and methods of the present disclosure, are set forth in the protein database accessible through the Entrez search tool of the National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, Maryland 20894, and are exemplified by the amino acid sequences of those Bcl-2 and Bcl-2 family member proteins presented in Table 1 . TABLE 1

Amino Acid Sequences of Bcl-2 Proteins and Bcl-2 Family Member Proteins

SEQUENCE IDENTIFIER

GENBANK ACCESSION LITERATURE

O. AMINO ACID SEQUENCE

CITATION

DESCRIPTION

Bak protein [Mus 121 swgr vallg fgyrlalyvy qrgltgflgq vtcfladiil hhyiarwiaq rggwvaalnl 44(2), 195-200 musculus] 181 rrdpiltvmv ifgvvllgqf vvhrffrs (1997)

SEQUENCE ID NO: 110 1 mdgsgeqprg ggptsseqim ktgalllqgf iqdragrmgg etpelpleqv pqdastkkls Yazawa,

BAC53619.1 '61 eclkrigdel dsnmelqrmi aavdtdspre vffrvaaemf sdgnfnwgrv valfyfaskl Submitted (21-

Bax [Canis lupus 121 vlkalctkvp elirtimgwt ldflrerllg wiqdqggwdg llsyfgtptw qtvtifvagv FEB-2002) familiaris] 181 ltasltiwkk mg Contact : Mitsuh iro Yazawa The University of Tokyo, Tokyo, 113-8657, Japan

SEQUENCE ID NO: 111 1 maapsgggdt gsgndqildl gaallnnfvy ervrrhgdrd aevtrsqlgg velcdpshkr Strausberg,

AAH55592.1 61 laqclqqigd eldgnaqlqs mlnnsnlqpt qdvfirvare ifsdgkfnwg rvvalfyfac PNAS U.S.A.

Bcl2-associated X 121 rlvikaistr vpdiirtiis wtmsyiqehv inwireqggw dgirsyfgtp twqtvgvfla 99(26), 16899- protein, a [Danio 181 gvittalvir km 16903 (2002) rerio]

SEQUENCE ID NO: 112 1 maaskkavlg plvgavdqgt sstrflvfns rtaellshhq veikqefpre gwveqdpkei Strausberg,

AAH66960.1 61 lhsvyeciek tceklgqlni gisnikaigv snqrettvvw dkitgeplyn avvwldlrtq PNAS U.S.A.

G 3P protein [Homo 121 stveslskri pgnnnfvksk tglplstyfs avklrwlldn vrkvqkavee kralfgtids 99(26), 16899- sapiens] 181 wliwsltggv nggvhctdvt nasrtmlfni hslewdkqlc effgipmeil phvrssseiy 16903 (2002)

241 glmkagaleg vpisgclgdq saalvgqmcf qigqakntyg tgcfllcntg hkcvfsdhgl

301 lttvayklgr dkpvyyaleg svaiagavir wlrdnlgiik tseeieklak evgtsygcyf

361 vpafsglyap ywepsargii cgltqftnkc hiafaaleav cfqtreilda mnrdcgipls

421 hlqvdggmts nkilmqlqad ilyipvvkpl mpettalgaa maagaaegvd vwslepedls

481 avtmkrfepq inaeeseiry stwkkavmks mgwvttqspe ggdpsvfcsl plgffivssm

541 amligaryis g^jp

SEQUENCE ID NO: 113 1 mdgsgeqprg ggptsseqim ktgalllqgf iqdragrmgg etpelgleqv pqdastkkls Reyes,

AAC48806.1 61 eclkrigdel dsnmelqrmi aavdtdspre vffrvaaemf sdgnfnwgrv valfyfaskl Virology bax-alpha 121 vlkalctkvp elirtimgwt ldflrerllg wiqdqggwdg llsyfgtptw qtvtifvagv 242(1), 184- taurus] 181 ltasltiwkk mg 192 (1998)

SEQUENCE ID NO: 114 1 mdgsgeqprg ggptsseqim ktgalllqgf iqdragrmgg eapelaldpv pqdastkkls Schmitt,

AAF71267.1 61 eclkrigdel dsnmelqrmi aavdtdspre vffrvaadmf sdgnfnwgrv valfyfaskl Biochem.

bax-sigma [Homo 121 vlkalctkvp elirtimgwt ldflrerllg wiqdqggwtv tifvagvlta sltiwkkmg Biophys. Res. sapiens] Commun .

270(3), 868- 879 (2000)

SEQUENCE IDENTIFIER

GENBANK ACCESSION LITERATURE

AMINO ACID SEQUENCE NO. CITATION

DESCRIPTION

SEQUENCE ID NO: 122 1 mdgsgeqprg ggptsseqim ktgalllqgf iqdragrmgg eapelaldpv pqdastkkls Strausberg,

AAH14175.1 61 eclkrigdel dsnmelqrmi aavdtdspre vffrvaadmf sdgnfnwgrv valfyfaskl PNAS U.S.A.

BCL2-associated 121 vlkalctkvp elirtimgwt ldflrerllg wiqdqggwdg llsyfgtptw qtvtif agv 99(26), 16899- protein [Homo 181 ltasltiwkk mg 16903 (2002) sapiens]

SEQUENCE ID NO: 123 1 mdgsgeqprg ggptsseqim ktgalllqgf iqdragrmgg eapelaldpv pqdastkkls Oltvai, Cell

AAA03619.1 61 eclkrigdel dsnmelqrmi aavdtdspre vffrvaadmf sdgnfnwgrv valfyfaskl 74(4), 609-619

Bax alpha [Homo 121 vlkalctkvp elirtimgwt ldflrerllg wiqdqggwdg llsyfgtptw qtvtifvagv (1993)

sapiens] 181 ltasltiwkk mg

SEQUENCE ID NO: 124 1 gfiqdrdgrm ggetpelgle qvpqdastkk lseclkrigd eldsnmelqr miaavdtdsp Murray,

AAF98242.1 61 revffrvaae mfsdgnfnwg rvvalfyfas klvlkalctk vpelirtimg wtldflrerl Unpublished

Bcl2-associated 121 lgwiqdqggw dgllsyfgtp twqtvtifv

protein Bax,

partial [Ovis

aries]

SEQUENCE ID NO: 125 1 masgqgpgpp rqecgepalp saseeqvaqd teevfrsyvf yrhqqpsstm gqvgrqlaii Ma, Submitted

AAM74949.1 61 gddinrryds efqtmlqhlq ptaenayeyf tkiatslfes ginwgrvval lgfgyrlalh (07-JUN-2002) pro-apoptotic 121 vyqhgltgfl gqvtrfvvdf mlhhciarwi aqrggwvaal nlgngpilnv Ivvlgvvllg School of Life protein BAKM 181 qfvvrrffks Science, USTC, variant [Homo Hefei, Anhui sapiens] 230027, China

SEQUENCE ID NO: 126 1 mktgalllqg fiqdragrmg geapelaldp vpqdastkkl seclkrigde ldsnmelqrm Cartron, Hum.

CAD10744.1 61 iaavdtdspr evffrvaadm fsdgnfnwgr vvalfyfask lvlkalctkv pelirtimgw Mol . Genet. bax isoform psi 121 tldflrerll gwiqdqggwd gllsyfgtpt wqtvtifvag vltasltiwk kmg 11(6), 675-687 [Homo sapiens] (2002)

SEQUENCE ID NO: 127 1 masgqgpgpp kgdcdealsa seqqvaqdte evfrsyvfyl hqqeqetqga aapanpemdn Itoh, J.

AAF71760.1 61 lslepnsvlg qvgrqlalig ddinrrydte fqnlleqlqp tagnayelft kiasslfksg Neurochem.

BA protein [Rattus 121 iswgrvvall gfgyrlalyv yqrgltgflg qvtcfladii Ihhyiarwia qrggwvaals 85(6), 1500- norvegicus] 181 lrrdpilsvv vifgvvllgq fvvhrffrs 1512 (2003)

SEQUENCE ID NO: 128 1 mfglrrnavi glnlycggas lgagggspag trlaaeeaka rregggeaal lpgarvvarp Leo,

AAD13295.1 61 ppvgaedpdv tasaerrllk spgllavppe emaasaaaim speeeldgce pevlskrpav Endocrinology

Mcl-1 protein 121 Ipllervsea akssgadgsl pstppppeee ddelyhqsle iisrylreqa tgskdakplg 140(12), 5469- [Rattus norvegicus] 181 eagaagrral etlrrvgdgv qrnhetafqg mlrkldikne ddvksfsrvm thvfkdgvtn 5477 (1999)

241 wgrivtlisf gafvakhlks inqesciepl aesitdvlvr tkrdwlvkqr gwdgfveffh

301 v dleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 129 1 mfglrrnavi glnlycggas lgagggspag trlaaeeaka rregggeaal lpgarvvarp

SEQUENCE IDENTIFIER

GENBANK ACCESSION LITERATURE

AMINO ACID SEQUENCE

O. CITATION

DESCRIPTION

myeloid cell 121 vvtlisfgaf vakhlksinq ekcitslagi itdalvsskr ewlmsqggwe gfvdffrved Lee, Genes leukemia protein 181 lessirnvlm afagvaglga slaymirkwr s Dev. 13(6), MCL-1, partial 718-728 (1999) [Gallus gallus]

SEQUENCE ID NO: 137 1 mktgafllqg fiqdraerma getpeltleq ppqdastkkl seclrrigde ldnnmelqrm Jin, Redwood

AAF36411.1 61 iadvdtdspr evffrvaadm fadgnfnwgr vvalfyfask lvlkalctkv pelirtimgw Blvd., Novato,

Bax protein splice 121 tldflrerll vwiqdqggwd gllsyfgtpt wqtvtifvag vltasltiwk kmg CA 94945, USA variant k [Rattus

norvegicus]

SEQUENCE ID NO: 138 1 mdgsgeqlgg ggptsseqfm ktgafllqgf iqdraermag etpeltleqp pqdastkkls Han, Genes

AAC26327.1 61 eclrrigdel dnnmelqrmi advdtdspre vffrvaadmf adgnfnwgrv valfyfaskl Dev. 10(4), rBax alpha [Rattus 121 vlkalctkvp elirtimgwt Idflrerllv wiqdqggweg llsyfgtptw qtvtifvagv 461-477 (1996) norvegicus] 181 ltasltiwkk mg

SEQUENCE ID NO: 139 1 mktgafllqg fiqdragrma getpeltleq ppqdastkkl seclrrigde ldsnmelqrm Chartrand,

AAM34436.1 61 iadvdtdspr evffrvaadm fadgnfnwgr vvalfyfask lvlkalctkv pelirtimgw Unpublished

Bcl2-associated X 121 tldflrerll vwiqdqggwe gllsyfgtpt wqtvtifvag vltasltiwk kmg

protein kappa

isoform [Mus

musculus]

SEQUENCE ID NO: 140 1 fiqdragrmg getpelgleq vpqdastkkl seclkrigde ldsnmelqrm iaavdtdspr Hopwood,

CAE54428.1 61 evffrvaaem fadgnfnwgr vvalfyfask lvlkalctrv pelirtimgw tldflrdrll Unpublished

Bax-alpha protein, 121 gwiqdqggwd gllsyfgtpt wqtvtifvag vltasltiwk kmgc

partial [Sus

scrofa]

SEQUENCE ID NO: 141 1 mfglrrnavi glnlycggas lgagggspag arlvaeeaka rregggeaal lpgarvvarp Strausberg,

AAH03839.1 61 ppvgaedpdv tasaerrlhk spgllavppe emaasaaaai vspeeeldgc epeaigkrpa PNAS U.S.A.

Myeloid cell 121 vlpllervse aakssgadgs Ipstppppee eeddlyrqsl eiisrylreq atgskdskpl 99(26), 16899- leukemia sequence 1 181 geagaagrra letlrrvgdg vqrnhetafq gmlrkldikn egdvksfsrv mvhvfkdgvt 16903 (2002) (Mus musculus] 241 nwgrivtlis fgafvakhlk svnqesfiep laetitdvlv rtkrdwlvkq rgwdgfveff

301 hvqdleggir nyllafagva gvgaglayli r

SEQUENCE ID NO: 142 1 mfglrrnavi glnlycggas lgagggspag arlvaeeaka rregggeaal lpgarvvarp Strausberg,

AAH21638.1 61 ppvgaedpdv tasaerrlhk spgllavppe emaasaaaai vspeeeldgc epeaigkrpa PNAS U.S.A.

301' hvqdleggir nyllafagva gvgaglayli

SEQUENCE IDENTIFIER

GENBANK ACCESSION LITERATURE

AMINO ACID SEQUENCE NO. CITATION

DESCRIPTION

SEQUENCE ID NO: 143 1 mfglrrnavi glnlycggas lgagggspag arlvaeeaka rregggeaal lpgarvvarp Strausberg,

AAH05427.1 61 ppvgaedpdv tasaerrlhk spgllavppe emaasaaaai vspeeeldgc epeaigkrpa PNAS U.S.A.

Myeloid cell 121 vlpllervse aakssgadgs Ipstppppee eeddlyrqsl eiisrylreq atgskdskpl 99(26), 16899- leukemia sequence 1 181 geagaagrra letlrrvgdg vqrnhetafq gmlrkldikn egdvksfsrv mvhvfkdgvt 16903 (2002) [Mus musculus] 241 nwgrivtlis fgafvakhlk svnqesfiep laetitdvlv rtkrdwlvkq rgwdgfveff

301 hvqdleggir nyllafagva gvgaglayli

SEQUENCE ID NO: 144 1 mfglrrnavi glnlycggas lgagggspag arlvaeeaka rregggeaal lpgarvvarp Okita,

AAC31790.1 61 ppvgaedpdv tasaerrlhk spgllavppe emaasaaaai vspeeeldgc epeaigkrpa Biochim.

EAT/MCL-1 [Mus 121 vlpllervse aakssgadgs Ipstppppee eeddlyrqsl eiisrylreq atgskdskpl Biophys. Acta musculus ] 181 geagaagrra letlrrvgdg vqrnhetafq gmlrkldikn egdvksfsrv mvhvfkdgvt 1398(3), 335-

241 nwgrivtlis fgafvakhlk svnqesfiep laetitdvlv rtkrdwlvkq rgwdgfveff 341 (1998)

301 hvqdleggir nvllafagva gvgaglayli r

SEQUENCE ID NO: 145 1 mfglkrnavi glnlycggag laagsggass sggrlvavgk eatarrevgg geagaviggs Nagafuchi,

BAC77771.1 61 agasppatla pdarrvarps pigaegpdvt atppkllffa atrcasppee megpaadaim Published Only

Mcl-1 [Felis catus] 121 speeeldgye peplgkrpav lpllelvgea ssgpgtdgsl pstpppaeee edelfrqsle in Database

181 iisrylreqa tgakdakplg gsgaasrkal etlrrvgdgv qrnhetafqg mlrkldikne (2003)

241 ndvkslsrvm vhvfsdgvtn wgrivtlisf gafvakhlks inqesciepl aesitdvlvr

301 tkrdwlvkqr gwdgfveffh vedleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 146 1 masgqgpgpp rqecgepalp saseeqvaqd teevfrsyvf yhhqqeqeae gaaapadpem Kiefer, Nature

AAA74467.1 61 vtlplqpsst mgqvgrqlai igddinrryd sefqtmlqhl qptaenayey ftkiasslfe 374 (6524) ,

Bak-2 protein [Homo 121 sginwgrvva llgfsyrlal hiyqrgltgf lgqvtrfvvd fmlhhciarw iaqrggwvaa 736-739 (1995) sapiens] 181 lnlgngpiln vlvvlgvvll gqfvvrrffk

SEQUENCE ID NO: 147 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Bingle, J.

AAF64255.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Biol. Chem.

Mcl-1 [Homo 121 speeeldgye peplgkrpav lpllelvges gnntstdgsl pstpppaeee eddlyrqsle 275 (29) , sapiens] 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne 22136-22146

241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr (2000)

301 tkrdwlvkqr gwdgfveffh vedleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 148 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Kalnine,

AAP36208.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Unpublished

Homo sapiens 121 speeeldgye peplgkrpav lpllelvges gnntstdgsl pstpppaeee edelyrqsle

myeloid cell 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne

leukemia sequence 1 241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr

(BCL2-related) , 301 tkrdwlvkqr gwdgfveffh vedleggirn vllafagvag vgaglaylir 1

SEQUENCE IDENTIFIER

GENBANK ACCESSION LITERATURE

AMINO ACID SEQUENCE NO. CITATION

DESCRIPTION

partial [synthetic

construct]

SEQUENCE ID NO: 149 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Kalnine,

AAP35286.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Unpublished

Myeloid cell 121 speeeldgye peplgkrpav Ipllelvges gnntstdgsl pstpppaeee edelyrqsle

leukemia sequence 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne

(BCL2-related) 241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr

(Homo sapiens] 301 tkrdwlvkqr gwdgfveffh vedleggirn vllafagvag vgaglaylir

SEQUENCE ID NO: 150 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Strausberg,

AAH71897.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim PNAS U.S.A.

Myeloid cell 121 speeeldgye peplgkrpav Ipllelvges gnntstdgsl pstpppaeee edelyrqsle 99(26), 16899- leukemia sequence 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne 16903 (2002) (BCL2-related) 241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr

[Homo sapiens] 301 tkrdwlvkqr gwdgfveffh vedleggirn vllafagvag vgaglaylir

SEQUENCE ID NO: 151 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Strausberg,

AAH17197.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim PNAS U.S.A.

[Homo sapiens] 301 tkrdwlvkqr gwdgfveffh vedleggirn vllafagvag vgaglaylir

SEQUENCE ID NO: 152 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Akgul, Cell.

AAF74821.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Mol . Life Sci . myeloid cell 121 speeeldgye peplgkrpav Ipllelvges gnntstdgsl pstpppaeee edelyrqsle 57(4), 684-691 differentiation 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne (2000) protein [Homo 241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr

sapiens] 301 tkrdwlvkqr gwdgfveffh vedleggirn vllafagvag vgaglaylir

SEQUENCE ID NO: 153 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Umezawa, Cell

AAD13299.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Struct. Funct. myeloid cell 121 speeeldgye peplgkrpav Ipllelvges gnntstdgsl pstpppaeee edelyrqsle 21(2), 143-150 leukemia sequence 1 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne (1996)

[Homo sapiens] 241 ddvkslsrvm ihvfsdgvtn wgrivtlisf gafvakhlkt inqesciepl aesitdvlvr

301 tkrdwlvkqr gwdgfveffh vedleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 154 1 mfglkrnavi glnlycggag lgagsggatr pggrllatek easarreigg geagaviggs Bae, J. Biol.

AAG00896.1 61 agasppstlt pdsrrvarpp pigaevpdvt atparllffa ptrraaplee meapaadaim Chem. 275(33),

SEQUENCE IDENTIFIER

GENBAN ACCESSION LITERATURE

AMINO ACID SEQUENCE

O. CITATION

DESCRIPTION

Myeloid cell 121 speeeldgye peplgkrpav lpllelvges gnntstdgsl pstpppaeee edelyrqsle 25255-25261 leukemia protein 1 181 iisrylreqa tgakdtkpmg rsgatsrkal etlrrvgdgv qrnhetafqg mlrkldikne (2000)

301 tkrdwlvkqr gwdgfveffh vedleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 155 1 mevlrrssvf aaeimdafdr sptdkelvaq akalgreyvh arllraglsw saperaspap Strausberg,

AAH78871.1 61 ggrlaevctv llrlgdeleq irpsvyrnva rqlhiplqse pvvtdaflav aghifsagit PNAS U.S.A.

BCL2-related 121 wgkvvslysv aaglavdcvr qaqpamvhal vdclgefvrk tlatwlrrrg gwtdvlkcvv 99(26), 16899- ovarian killer 181 stdpgfrshw lvatlcsfgr flkaafflll per 16903 (2002) [Rattus norvegicus]

SEQUENCE ID NO: 156 1 mevlrrssvf aaeimdafdr sptdkelvaq akalgreyvh arllraglsw saperaspap Strausberg,

AAH30069.1 61 ggrlaevctv llrlgdeleq irpsvyrnva rqlhiplqse pvvtdaflav aghifsagit PNAS U.S.A.

BCL2-related 121 wgkvvslysv aaglavdcvr qaqpamvhal vdclgefvrk tlatwlrrrg gwtdvlkcvv 99(26), 16899- ovarian killer 181 stdpgfrshw lvatlcsfgr flkaafflll per 16903 (2002) protein [Mus

musculus]

SEQUENCE ID NO: 157 NO GENBANK RECORD AVAILABLE

AAAC53582.1

NO GENBANK RECORD AVAILABLE

SEQUENCE ID NO: 158 1 mevlrrssvf aaeimdafdr sptdkelvaq akalgreyvh arllraglsw saperaspap Hsu, PNAS

AAB87418.1 61 ggrlaevctv llrlgdeleq irpsvyrnva rqlhiplqse pvvtdaflav aghifsagit U.S.A. 94(23),

Bcl-2-related 121 wgkvvslysv aaglavdcvr qaqpamvhal vdclgefvrk tlatwlrrrg gwtdvlkcvv 12401-12406 ovarian killer 181 stdpgfrshw lvatlcsfgr flkaafflll per (1997) protein [Rattus

norvegicus]

SEQUENCE ID NO: 159 1 mfglkrnavi rtqlycggag lgagsggass sggrllasgr eattrreggg geagaviggs Sano, Res.

BAC21258.1 61 agasppttla pdarrvarps pigaegpnvs atpprlllla ppcrasppee megpaadaim Vet. Sci.

Mcl-1 [Canis lupus 121 speeeldgye peplgkrpav lpllelvgea ssgpgmdgsl pstpppaeee edelyrqsle 78(2), 183 familiaris] 181 iisrylreqa tgakdakplg gsraasrkal etlqrvgdgv qrnhetafqg mlrkldikne (2005)

241 ddvkslsrvi vhvfsdgvtn wgrivtlisf gafvakhlks inqesciepl aesitdvlvr

301 tkrdwlvkqr gwdgfveffh vedleggirn yllafagvag vgaglaylir

SEQUENCE ID NO: 160 1 mevlrrssvf aaeimdafdr wptdkelvaq akalgreyvh arllraglsw saperaspap Hsu,

AAF09129.1 61 ggrlaevctv llrlgdeleq irpsvyrnva rqlhiplqse pvvtdaflav aghifsagit Unpublished

Bcl-2 related 121 wgkvvslysa aaglavdcvr qaqpamvhal vdclgefvrk tlatwlrrrg gwtdvlkcvv

ovarian killer 181 stkpgfrshw lvatlcsfgr flkaafflll per

[Homo sapiens]

Methods for Identifying Bcl-2, Bcl-2 Family Members, and BH4 Domain Resigns Thereof

[0090] The present disclosure provides methods for identifying a Bcl-2 peptide, modified Bcl- 2 peptide, Bcl-2 domain-containing peptide, or Bcl-2 mimetic that induces a change in the quantity and/or type of microparticles/exosomes released when administered to mammalian cells ("Bcl-2- induced exosomes") wherein the methods of this aspect of the invention each include the step of screening a plurality of proteins or peptides to identify one or more Bcl-2 proteins or peptide, including one or more BH4 peptides, and/or one or more Bcl-2 mimetics, including one or more BH4 mimetics, which induces changes in the quantity and/or type of microparticles/exosomes released.

[0091] In another aspect, the present disclosure provides methods for identifying a small molecule that induces a change in the quantity and/or type of microparticles/exosomes released when administered to mammalian cells wherein the methods of this aspect of the invention each include the step of screening a small molecule library for compounds that affect the binding of a Bcl-2 protein or peptide to its mammalian cell receptor and induces a change in the quantity and/or type of microparticles/exosomes released from a mammalian cell.

[0092] In another aspect, the present disclosure provides methods for identifying a mammalian cell surface receptor for one or more Bcl-2 proteins or peptides, including one or more BH4 peptides, which are involved in Bcl-2-induced microparticle/exosome release wherein the methods of this disclosure each include a step of binding one or more Bcl-2 proteins or peptides, including one or more BH4 peptides, to the receptor to isolate and identify the receptor from a mixture of cell proteins.

[0093] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein, domain-containing peptide, or mimetic thereof that induces microparticles when administered to a mammal or to cells ex vivo, wherein the methods of this aspect of the disclosure each include screening a plurality of proteins to identify a Bel protein that induce or increases microparticles or microparticle dependent nucleic acid delivery in a mammal or to mammalian cells ex vivo.

[0094] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein, domain-containing peptide, or mimetic thereof that induces microparticles when administered to a mammal or to cells ex vivo, wherein the methods of this aspect of the disclosure each include screening a plurality of proteins to identify a Bcl-2 protein that induces or increases stem cell microparticles with therapeutic activity in vivo.

[0095] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein, domain-containing peptide, or mimetic thereof that induces microparticles when administered to a mammal or to cells ex vivo, wherein the methods of this aspect of the disclosure each include screening a plurality of proteins to identify a Bcl-2 protein that induces or increases microparticles capable of stimulating or suppressing an immune response.

[0096] In another aspect, the present disclosure provides methods for identifying a mammalian cell type that responds to treatment with a Bcl-2 protein, domain-containing peptide, or mimetic thereof by production of microparticles that mediate the delivery of a therapeutic nucleic acid.

[0097] In another aspect, the present disclosure provides methods for identifying a mammalian stem cell type that responds to treatment with a Bcl-2 protein, domain-containing peptide, or mimetic thereof by production of microparticles that mediate therapeutic activity.

[0098] In another aspect, the present disclosure provides methods for identifying a mammalian lymphoid cell type that responds to treatment with a Bcl-2, domain-containing peptide, or mimetic thereof protein by production of microparticles that regulate an immune response.

[0099] In the practice of this aspect of the disclosure, at least two proteins are screened to identify a Bcl-2 protein that increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal. Thus, for example, between two and 1 00 proteins may be screened to identify a Bcl-2 protein that increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal; or, for example, from 100 to 500 proteins may be screened to identity a Bcl-2 protein increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal; or, for example, from 100 to 1000 proteins may be screened to identity a Bcl-2 protein that increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal; or, for example, more than 1000 proteins may be screened to identify a Bcl-2 protein that increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal. [00100] Any useful assay can be used to identify a protein that increases the effectiveness of microparticle mediated nucleic acid delivery when administered to a mammal. For example, a useful assay can be an in vitro assay, or an in vivo assay, or an assay that includes an in vitro component and an in vivo component. Representative examples of useful assays include the assays described supra for assessing the ability of a Bcl-2 protein to increase microparticle production or the effectiveness of a nucleic acid delivery.

[00101] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein that increases the effectiveness of a nucleic acid therapy when administered to a mammal. The methods of this aspect of the disclosure each include analyzing data obtained from an experiment wherein a plurality of proteins are screened to identify a Bcl-2 protein that increases the effectiveness of a nucleic acid therapy when administered to a mammal. The analysis can include comparing the effect(s) of candidate Bcl-2 proteins to increase microparticle mediated nucleic acid delivery in vivo and/or in vitro, and comparing the effect(s) of the candidate proteins to the effects on microparticle mediated nucleic acid delivery of a non-Bcl-2 protein, or a Bcl-2 protein that has been modified (e.g., by site-directed mutagenesis) to be biologically inactive, or to some other control treatment. A statistically significant increase in microparticle mediated nucleic acid delivery caused by the candidate Bcl-2 protein, compared to the amount of microparticle mediated nucleic acid delivery caused by the control treatment, indicates that the candidate Bcl-2 protein increases microparticle mediated nucleic acid delivery. If desired, the candidate Bcl-2 protein may be subjected to further study.

[00102] Any of the methods disclosed herein for screening a plurality of Bcl-2 proteins to identify a Bcl-2 protein that increases microparticle mediated nucleic acid delivery can be used in this aspect of the disclosure.

[00103] In the practice of this aspect of the disclosure, the analyzed data are obtained from an experiment wherein a plurality of proteins is screened to identify a Bcl-2 protein that increases microparticle mediated nucleic acid delivery when administered to a mammal. For example, between two and 100 proteins may be screened to identify a Bcl-2 protein that increases microparticle mediated nucleic acid delivery when administered to a mammal; or, for example, from 100 to 500 proteins may be screened to identity a Bcl-2 protein that increases microparticle mediated nucleic acid delivery when administered to a mammal; or, for example, from 100 to 1000 proteins may be screened to identify a Bcl-2 protein that increases microparticle mediated nucleic acid delivery when administered to a mammal; or, for example, more than 1000 proteins may be screened to identity a Bcl-2 protein that increases microparticle mediated nucleic acid delivery when administered to a mammal.

[00104] Exemplary in vitro assays that may be employed for developing a Bel drug candidate and for identifying Bel-induced mediators of extracellular Bel activity are presented in FIGs. 9A- 9C. Cultured media of Bcl-2-treated cells can be processed in several assay systems for quantitative and qualitative analysis of induced mediators. For example, centrifuged and buffer exchanged culture media may be subjected to spectrophotometry to determine induced protein and RNA concentrations (FIG. 9A), subjected to SDS-PAGE and protein stain to determine size profile or induced proteins (FIG. 9B), subjected to an ELISA designed to capture exosomes to determine the induction of exosomes (FIG. 9C). The exosome ELISA may use antibodies that bind to exosome membrane markers (e.g., Rab5b and CD63) in a sandwich ELISA (FIG. 9C).

[00105] Other assay systems or methods may be used to analyze Bcl-2 -induced vesicles or mediators including, but not limited to, nanoparticle tracking analysis (NTA), cytofluorimetry (e.g., following the capture of exosomes on beads), light scattering, RNA arrays (e.g., mRNA, miRNA), PCR, qPCR and proteomic profiling methods. Functional assays may be used to characterize the activity Bcl-2 -induced exosomes or mediators. Appropriate functional systems related to extracellular Bcl-2 activities, includes protection from hypoxia, chemical or mechanical injuries and in particular, protection from ER stress or the unfolded protein response (UPR). Negative controls for Bcl-2 induction of exosomes or mediators from cultured cells may include saline, PBS, BH3 domain peptides or other non-Bel proteins.

[00106] These in vitro bioanalytical assays using media, processed media from Bel treated cultured cells may be used for: determination of minimal Bcl-2 peptide size, active Bcl-2 peptide modifications, identification of active Bcl-2 peptidomimetics, identification of active exosomes, small molecules that induce exosome release, Bcl-2 or Bcl-2 peptide lot release, determination of Bcl-2 protein potency such as 50% effective concentration (EC50), to determine the kinetics of mediator induction, to optimize the exosome ELISA (e.g., different pairs of capture and detection antibodies and incubation time), to compare protein, RNA & exosome production induction in THP- 1 , MSC or other cell types, to identify induced proteins and RNAs (mediators of mechanism) via quantitative proteomic methods (such as iTRAQ labeling and LC-MS MS) and RNA array methods (for example miRXplore microarray), to facilitate identification of an in vitro functional assay for Bel- induced exosomes.

[00107] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein, BH4 peptide or mimetic that inhibits fibrosis when administered to a mammal. The methods of this aspect of the invention each include the step of screening a plurality of proteins to identify a Bcl-2 protein that inhibits fibrosis, when administered to a mammal.

[00108] In the practice of this aspect of the invention, at least two proteins are screened to identify a Bcl-2 protein that inhibits fibrosis when administered to a mammal. Thus, for example, between two and 100 proteins may be screened to identify a Bcl-2 protein that inhibits fibrosis when administered to a mammal; or, for example, from 100 to 500 proteins may be screened to identify a Bcl-2 protein increases that inhibits fibrosis when administered to a mammal; or, for example, from 100 to 1000 proteins may be screened to identify a Bcl-2 protein that inhibits fibrosis when administered to a mammal; or, for example, more than 1000 proteins may be screened to identify a Bcl-2 protein that inhibits fibrosis when administered to a mammal.

[00109] Any useful assay can be used to identify a protein that inhibits fibrosis when administered to a mammal. For example, a useful assay can be an in vitro assay, or an in vivo assay, or an assay that includes an in vitro component and an in vivo component. Representative examples of useful assays include the assays described supra for assessing the ability of a Bcl-2 protein to induce a change in the quantity and/or type of microparticles/exosomes released from mammalian cells or inhibit fibrosis.

[00110] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein that inhibits fibrosis when administered to a mammal. The methods of this aspect of the invention each include the step of analyzing data obtained from an experiment wherein a plurality of proteins are screened to identify a Bcl-2 protein that inhibits fibrosis when administered to a mammal. The analysis can include comparing the effect(s) of candidate Bcl-2 proteins on fibrosis in vivo and/or in vitro, and comparing the effect(s) of the candidate proteins to the effects on fibrosis of a non-Bcl-2 protein, or a Bcl-2 protein that has been modified (e.g., by site-directed mutagenesis) to be biologically inactive, or to some other control treatment. A statistically significant increase in the amount of inhibition of fibrosis caused by the candidate Bcl-2 protein, compared to the amount of inhibition of fibrosis caused by the control treatment, indicates that the candidate Bcl-2 protein inhibits fibrosis. If desired, the candidate Bcl-2 protein may be subjected to further study. Any of the methods disclosed herein for screening a plurality of Bcl-2 proteins to identify a Bel protein that inhibits fibrosis can be used in this aspect of the invention.

Vectors for the Delivery and Expression of Nucleic Acids Encoding Bcl-2, Bcl-2 Family

Members, and BH4 Domain Regions Thereof

[0001] The present disclosure provides vectors comprising and for the delivery or expression of a nucleic acid encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof. Suitable nucleic acid delivery or expression vectors include those that are non-specific c with respect to the target cell type to which the nucleic acids are delivered or may be configured for target cell-specific delivery of one or more nucleic acids to achieve target cell specificity and, consequently, regulate, promote, normalize, restore, inhibit, or modulate a desired cellular activity or phenotype within the targeted cell.

[0002] Within certain aspects, the present disclosure provides vectors for the expression of a a nucleic acid encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof within a target cell, wherein the expression vectors comprise: (1) a transcriptional promoter that is activated in response to one or more factors each of which is produced within a target cell and (2) nucleotide sequences encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof, which are operably linked to and under regulatory control of the transcriptional promoter, wherein the one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof can regulate, promote, normalize, restore, inhibit, or modulate a desired cellular phenotype within the targeted cell.

[0003] Within related aspects, the transcriptional promoter can be activated in a target cell that is associated with a disease or condition or a target cell, such as a stem cell {e.g., an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), or a mesenchymal stem cell (MSC)) that is to be induced to produce microsomes or exosomes (as is described in further detail herein). Transcriptional activation can be achieved by the action of one or more factors that are produced in the target cell, such as a cancer cell, a precancerous cell, a dysplastic cell, or a cell that is infected with an infectious agent. Target cells may also include a hematopoietic cell, an adipose cell, an eye cell, a brain cell, a liver cell, a colon cell, a lung cell, a pancreas cell, a breast cell, a prostate cell, a colorectal cell, or a heart cell.

[0004] Suitable transcriptional promoters that may be employed in vectors for the expression of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof include, for example, the p2 l ^c|P^{| /wafl} promoter, the p27^kipl promoter, the p57^k,p2 promoter, the TdT promoter, the Rag-1 promoter, the B29 promoter, the Blk promoter, the CD 19 promoter, the BLNK promoter, and the λ5 promoter, which transcriptional promoter is responsive to activation by one or more transcription factors such as an EBF3, O/E-l , Pax-5, E2A, p53, VP16, MLL, HSF 1 , NF-IL6, NFAT1 , AP-1 , AP-2, HOX, E2F3, and/or NF-κΒ transcription factor, and which transcriptional activation induces the expression of a nucleic acid that encodes a Bcl-2, Bcl-2 family member, BH4 domain region, or mimetic thereof as described in further detail herein.

[0005] Within other aspects of these embodiments, the vector may be configured to non- specifically deliver a nucleic acid encoding a Bcl-2, Bcl-2 family member, BH4 domain region, or mimetic thereof to a target cell as well as a non-target cell, wherein the vector comprises a transcriptional promoter that is responsive to a transcription factor that is specifically or preferentially expressed in the target cell (e.g., a cell that is associated with a disease or other condition or a cell that is in a particular stage of differentiation), but is not expressed in the non- target cell and wherein the vector comprises a nucleic acid that encodes a Bcl-2, Bcl-2 family member, BH4 domain region, or mimetic thereof that can regulate, promote, normalize, restore, inhibit, or modulate a desired cellular phenotype within the targeted cell.

[0006] Thus, certain aspects, the present disclosure provides vectors that are configured for the expression of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof within a target cell, such as a cell that is associated with a disease, a condition, or a stage of differentiation, wherein the vectors comprise: (a) a transcriptional promoter that is activated in response to one or more factors that are produced within a target cell and (b) a nucleic acid that is operably linked to and under regulatory control of the transcriptional promoter, wherein the nucleic acid encodes one or more a Bcl-2, Bcl-2 family member, BH4 domain region, or mimetic thereof that can regulate. promote, normalize, restore, inhibit, or modulate a desired cellular phenotype within the targeted cell.

[0007] In other aspects, the present disclosure provides target cells comprising one or more vectors as described herein, which vectors are configured for the expression of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof within the target cell, such as a cell that is associated with a disease, a condition, or a stage of differentiation, wherein the target cells are susceptible to regulation, promotion, normalization, restoration, inhibition, or modulation of a desired cellular phenotype in response to a nucleic acid encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof when delivered to the target cell and expressed by the one or vectors disclosed herein and may be used in methods for the treatment of tissue injuries, diseases, or other conditions as well as in methods for inducing the in vivo or ex vivo production of microparticles or exosomes as described in further detail herein.

[0008] Thus, the present disclosure provides vectors effectuating one or more cellular activity of a broad range of cells, including those that are associated with a disease or other condition, which vectors comprise an expression construct comprising (a) a target cell specific transcriptional promoter and (b) a nucleic acid encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof.

[0009] Within certain embodiments, provided herein are vectors for effectuating the growth, survival, or differentiation of target cells, which vectors comprise an expression construct that includes: (a) a transcriptional promoter, which transcriptional promoter may, optionally, be activated in a target cell but not in non-target cells, and (b) a nucleic acid that is under the control of the transcriptional promoter, which nucleic acid encodes one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof.

|0010] In certain aspects of these embodiments wherein the human target cell is a cancer cell, such as a brain cancer cell, a prostate cancer cell, a lung cancer cell, a colorectal cancer cell, a breast cancer cell, a liver cancer cell, a hematologic cancer cell, and a bone cancer cell, the transcriptional promoter can include at least a transcription factor binding site (i.e., a response element), which transcriptional promoter is responsive to activation by one or more transcription factors and which transcriptional activation induces the expression of a nucleic acid that encodes one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof that can regulate, promote, normalize, restore, inhibit, or modulate a desired cellular activity or phenotype within the targeted cell.

[0011] As used herein, the term "transcriptional promoter" refers to a promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 3' region of the anti- sense strand, also called template strand and non-coding strand). Promoters can be about 100— 1000 base pairs long. For the transcription to take place, the enzyme that synthesizes RNA, known as RNA polymerase, must attach to the DNA near a gene. Promoters contain specific DNA sequences and response elements that provide a secure initial binding site for RNA polymerase and for proteins called transcription factors that recruit RNA polymerase. These transcription factors have specific activator or repressor sequences of corresponding nucleotides that attach to specific promoters and regulate gene expressions. The process is more complicated, and at least seven different factors are necessary for the binding of an RNA polymerase II to the promoter. Promoters represent critical elements that can work in concert with other regulatory regions (enhancers, silencers, boundary elements/insulators) to direct the level of transcription of a given gene.

[0012] Eucaryotic transcriptional promoters are often categorized according to the following classes: ( 1 ) AT-based class, (2) CG-based class, (3) ATCG-compact class, (4) ATCG-balanced class, (5) ATCG-middle class, (6) ATCG-less class, (7) AT-less class, (8) CG-spike class, (9) CG- less class, and ( 10) ATspike class (Gagniuc and Ionescu-Tirgoviste, BMC Genomics 13:512 (2012)) and comprise a number of essential elements, which collectively constitute a core promoter (i.e.. the minimal portion of a promoter that is required to initiate transcription).

[0013] The elements that are essential to a Eucaryotic transcriptional promoter include one or more of the following: (1 ) a transcription start site (TSS), (2) an RNA polymerase binding site (in particular an RNA polymerase II binding site in a promoter for a gene encoding a messenger RNA), (3) a general transcription factor binding site (e.g., a TATA box having a consensus sequence TATAAA, which is a binding site for a TATA-binding protein (TBP)). (4) a B recognition element (BRE), (5) a proximal promoter of approximately 250 bp that contains regulatory elements, (6) transcription factor binding sites (e.g., an E-box having the sequence CACGTF, which is a binding site for basic helix-loop-helix (bHLH) transcription factors including BMAL 1 1 -Clock nad cMyc), and (7) a distal promoter containing additional regulatory elements. As used herein, the term "transcriptional promoter" is distinct from the term "enhancer," which refers to a regulatory element that is distant from the transcriptional start site.

[0014] Eucaryotic promoters can be "unidirectional" or "bidirectional." Unidirectional promoters regulate the transcription of a single gene and are characterized by the presence of a TATA box. Bidirectional promoters are short (<1 kbp), intergenic regions of DNA between the 5' ends of genes in a bidirectional gene pair (i.e., two adjacent genes coded on opposite strands having 5' ends oriented toward one another. Bidirectional genes are often functionaly related and because they share a single promoter, can be co-regulated and co-expressed. Unlike unidirectional promoters, bidirectional promoters do not contain a TATA box but do contain GpC islands and exhibit symmetry around a midpoint of dominant Cs and As on one side and Gs and Ts on the other. CCAAT boxes are common in bidirectional promoters as are NRF-1 , GABPA, YY1 , and ACTACAnnTCCC motifs.

[0015] Transcriptional promoters often contain two or more transcription factor binding sites. Thus, the efficient expression of a nucleic acid that is downstream of a promoter having multiple transcription factor binding sites typically requires the cooperative action of multiple transcription factors. Accordingly, the specificity of transcriptional regulation, and hence expression of an associated nucleic acid, can be increased by employing transcriptional promoters having two or more transcription factor binding sites.

[0016] As used herein, the term "transcription factor" refers to sequence-specific DNA- binding factors that bind to specific sequences within a transcriptional promoter thereby regulating the transcription of a nucleic acid that is in operable proximity to and downstream of the promoter. Transcription factors include activators, which promote transcription, and repressors, which block transcription by preventing the recruitment or binding of an RNA polymerase. Transcription factors typically contain ( 1 ) one or more DNA-binding domains (DBDs), which facilitate sequence specific binding to a cognate transcription factor binding site (a/k/a response element) within a transcriptional promoter; (2) one or more signal-sensing domains (SSDs). which includes ligand binding domains that are responsive to external signals; and (3) one or more transactivation domains (TADs), which contain binding sites for other proteins, including transcription coregulators. The term "transcription factor" refers to those factors having one or more DBDs and is not intended to include other regulatory proteins such as coactivators, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases, which no not contain DBDs.

[0017] Suitable vectors may comprise a transposon that contains at least a cell-type specific promoter sequence and a coding region for a CAR. The cell-type specific promoter sequence may be any promoter sequence with an activity that is restricted to T-cells, such as, but not limited to, CD2, CD3, CD4, CD8 and CD28 and other T-cell specific genes.

[0018] A wide variety of both non-viral and viral nucleic acid delivery vectors are well known and readily available in the art and may be adapted for use for the non-specific cellular delivery of the expression constructs disclosed herein. See, for example, Elsabahy et al, Current Drug Delivery 8(31:235-244 (201 1) for a general description of viral and non-viral nucleic acid delivery methodologies. The successful delivery of a nucleic acid into mammalian cells relies on the use of efficient delivery vectors. Viral vectors exhibit desirable levels of delivery efficiency, but often also exhibit undesirable immunogenicity, inflammatory reactions, and problems associated with scale-up, all of which can limit their clinical use. The ideal vectors for the delivery of a nucleic acid are safe, yet ensure nucleic acid stability and the efficient transfer of the nucleic acid to the appropriate cellular compartments.

[0019] Non-limiting examples of suitable non-viral and viral nucleic acid delivery vectors are described in the scientific and patent literature and include liposomal vectors, viral vectors, nanoparticles, polyplexesm dendrimers, each of which has been developed for the non-specific delivery of nucleic acids and can be adapted for the non-specific delivery of the presently disclosed nucleic acids encoding one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof, and can be modified to incorporate one or more agents for promoting the targeted delivery of a system to a target cell of interest thereby enhancing the target cell specificity of the presently disclosed systems.

[0020] An expression cassette may be incorporated within and/or associated with a lipid membrane, a lipid bi-layer, and/or a lipid complex such as, for example, a liposome, a vesicle, a micelle and/or a microsphere. Suitable methodology for preparing lipid-based delivery systems that may be employed with the expression constructs of the present disclosure are described in Metselaar et al, Mini Rev. Med. Chem. 2{4}:319-29 (2002); O'Hagen et al. Expert Rev. Vaccines 2(2):269-83 (2003); O'Hagan, Curr. Durg Targets Infect. Disord. Ι£3):273-86 (2001); Zho et al., Biosci Rep. 22(2):355-69 (2002); Chikh et al., Biosci Rep. 22(2):339-53 (2002); Bungener et al., Biosci. Rep. 22(2):323-38 (2002); Park, Biosci Rep. 22(2):267-81 (2002); Ulrich, Biosci. Rep. 22(2): 129-50: Lofthouse, Adv. Drug Deliv. Rev. 54(6):863-70 (2002); Zhou et al, J. Immunother.

25(4):289-303 (2002); Singh et al., Pharm Res. 19(6):715-28 (2002); Wong et al, Curr. Med. Chem. 8(9): 1 123-36 (2001); and Zhou et al, Immunomethods 4 3):229-35 (1994). Midoux et al, British J. Pharmacol 157: 166-178 (2009) describe chemical vectors for the delivery of nucleic acids including polymers, peptides and lipids. Sioud and Sorensen, Biochem Biophys Res Commun 3 12(4): 1220-5 (2003) describe cationic liposomes for the delivery of nucleic acids.

[0021] Due to their positive charge, cationic lipids have been employed for condensing negatively charged DNA molecules and to facilitate the encapsulation of DNA into liposomes. Cationic lipids also provide a high degree of stability to liposomes. Cationic liposomes interact with a cell membrane and are taken up by a cell through the process of endocytosis. Endosomes formed as the results of endocytosis, are broken down in the cytoplasm thereby releasing the cargo nucleic acid. Because of the inherent stability of cationic liposomes, however, transfection efficiencies can be low as a result of lysosomal degradation of the cargo nucleic acid.

[0022] Helper lipids (such as the electroneutral lipid DOPE and L-a-dioleoyl phosphatidyl choline (DOPC)) can be employed in combination with cationic lipids to form liposomes having decreased stability and, therefore, that exhibit improved transfection efficiencies. These electroneutral lipids are referred to as fusogenic lipids. See, Gruner et al, Biochemistry 27(8):2853-66 (1988) and Farhood et al, Biochim Biophys Acta 1235(2):289-95 (1995). DOPE forms an HI1 phase structure that induces supramolecular arrangements leading to the fusion of a lipid bilayer at a temperature greater than 5°C to 10°C. The incorporation of DOPE into liposomes also helps the formation of HII phases that destabilize endosomal membranes.

[0023] Cholesterol can be employed in combination with DOPE liposomes for applications in which a liposomal vector is administered intravenously. Sakurai et al, Eur J Pharm Biopharm 52(2): 165-72 (2001 ). The presence of one unsaturation in the acyl chain of DOPE is a crucial factor for membrane fusion activity. Talbot et al., Biochemistry 36(19):5827-36 (1997).

[0024] Fluorinated helper lipids having saturated chains, such as DF4C 1 1 PE (rac-2,3-Di[ l 1 - (F-butyl)undecanoyl) glycero-l -phosphoethanolamine) also enhance the transfection efficiency of lipopolyamine liposomes. Boussif et al., J Gene Med 3(2): 109- 14 (2001 ); Gaucheron et al., Bioconj Chem 12(6):949-63 (2001); and Gaucheron et al., J Gene Med 3 (4):338-44 (2001).

[0025] The helper lipid l ,2-dioleoyl-3-trimethylammonium-propane (DOTAP) enhances efficient of in vitro cell transfection as compared to DOPE Hpoplexes. Prata et al., Chem Commun 13: 1566-8 (2008). Replacement of the double bond of the oleic chains of DOPE with a triple bond as in Distear-4-ynoyl L-a-phosphatidylethanolamine [DS(9-yne)PE] has also been shown to produce more stable Hpoplexes. Fletcher et al., Org Biomol Chem 4(2): 196-9 (2006).

[0026] Amphiphilic anionic peptides that are derived from the N-terminal segment of the HA- 2 subunit of influenza virus haemagglutinin, such as the IFN7 (GLFEAIEGFIENGWEGMIDGWYG) and E5CA (GLFEAIAEFIEGGWEGLIEGCA) peptides, can be used to increase the transfection efficiency of liposomes by several orders of magnitude. Wagner et al., Proc Natl Acad Sci U.S.A. 89(17):7934-8 (1992); Midoux et al., Nucl Acids Res. 21 (4):871 -8 (1993); Kichler et al., Bioconjug Chem 8(2):213-21 (1997); Wagner, Adv Drug Deliv Rev 38(3):279-289 (1999); Zhang et al., J Gene £?c/ 316):560-8 (2001 ). Some artificial peptides such as GALA have been also used as fusogenic peptides. See, for example, Li et al., Adv Drug Deliv Rev 56(7):967-85 (2004) and Sasaki et al, Anal Bioanal Chem 391 (8):2717-27 (2008). The fusogenic peptide of the glycoprotein H from herpes simplex virus improves the endosomal release of DNA/Lipofectamine Hpoplexes and transgene expression in human cell (Tu and Kim, J Gene e</ 10(6):646-54 (2008).

[0027] PCT Patent Publication No. WO 2002/044206 describes a class of proteins derived from the family Reoviridae that promote membrane fusion. These proteins are exemplified by the p i 4 protein from reptilian reovirus and the p i 6 protein from aquareovirus. PCT Patent Publication No. WO 2012/040825 describes recombinant polypeptides for facilitating membrane fusion, which polypepides have at least 80% sequence identity with the ectodomain of p 14 fusion- associated small transmembrane (FAST) protein and having a functional myristoylation motif, a transmembrane domain from a FAST protein and a sequence with at least 80% sequence identity with the endodomain of p i 5 FAST protein. The ^:825 PCT further describes the addition of a targeting ligand to the recombinant polypeptide for selective fusion. The recombinant polypeptides presented in the '825 PCT can be incorporated within the membrane of a liposome to facilitate the delivery of nucleic acids. Fusogenix liposomes for delivering therapeutic compounds, including nucleic acids, to the cytoplasm of a mammalian cell, which reduce liposome disruption and consequent systemic dispersion of the cargo nucleic acid and/or uptake into endosomes and resulting nucleic acid destruction are available commercially from Innovascreen Inc. (Halifax, Nova Scotia, CA).

[0028] A wide variety of inorganic nanoparticles, including gold, silica, iron oxide, titanium, hydrogels, and calcium phosphates have been described for the delivery of nucleic acids and can be adapted for the delivery of the expression constructs described herein. See, for example Wagner and Bhaduri, Tissue Engineering J_8 JQ: 1 - 14 (2012) (describing inorganic nanoparticles for delivery of nucleic acid sequences); Ding ei al., Mol Ther e-pub (2014) (describing gold nanoparticles for nucleic acid delivery); Zhang et al., Langmuir 30(3):839-45 (2014) (describing titanium dioxide nanoparticles for delivery of DNA oligonucleotides); Xie et al., Curr Pharm Biotechnol 14(10):918-25 (2014) (describing biodegradable calcium phosphate nanoparticles fro gene delivery); Sizovs et al., J Am Chem Soc 136(0:234-40 (2014) (describing sub-30 monodisperse oligonucleotide nanoparticles).

[0029] Among the advantages of inorganic vectors are their storage stability, low immunogenicity, and resistance to microbial attack. Nanoparticles of less than 100 nm can efficiently trap nucleic acids and allows its escape from endosomes without degradation. Inorganic nanoparties exhibit improved in vitro transfection for attached cell lines due to their high density and preferential location on the base of the culture dish. Quantum dots have been described that permit the coupling of nucleic acid delivery with stable fluorescence markers.

[0030] Hydrogel nanoparticles of defined dimensions and compositions, can be prepared via a particle molding process referred to as PRINT (Particle Replication in Non-wetting Templates), and can be used as delivery vectors for the expression constructs disclosed herein. Nucleic acids can be encapsulated in particles through electrostatic association and physical entrapment. To prevent the disassociation of cargo nucleic acids from nanoparticles following systemic administration, a polymerizable conjugate with a degradable, disulfide linkage can be employed.

[0031] The PRINT technique permits the generation of engineered nanoparticles having precisely controlled properties including size, shape, modulus, chemical composition and surface functionality for enhancing the targeting of the expression cassette to a target cell. See, e.g., Wang et al, J Am Chem Soc 132: 1 1306-1 1313 (2010); Enlow et al., Nano Lett _Π :808-813 (201 1); Gratton et al., Proc Natl Acad Sci USA 105: 1 1613- 1 1618 (2008); Kelly, J Am Chem Soc 130:5438-5439 (2008); Merkel et al. Proc Natl Acad Sci USA 108:586-591 (201 1). PRINT is also amenable to continuous roll-to-roll fabrication techniques that permit the scale-up of particle fabrication under good manufacturing practice (GMP) conditions.

[0032] Nanoparticles can be encapsulated with a lipid coating to improveoral bioavailability, minimize enzymatic degradation and cross blood brain barrier. The nanoparticle surface can also be PEGylated to improve water solubility, circulation in vivo, and stealth properties.

[0033] A wide variety of viral vectors are well known by and readily available to those of skill in the art, including, for example, herpes simplex viral vectors lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, which viral vectors can be adapted for use in the systems disclosed herein for the delivery of nucleic acids, in particular nucleic acids comprising an expression cassette for the target cell specific expression of a therapeutic protein.

[0034] The tropisms of natural or engineered viruses towards specific receptors are the foundations for constructing viral vectors for delivery of nucleic acids. The attachment of these vectors to a target cell is contingent upon the recognition of specific receptors on a cell surface by a ligand on the viral vector. Viruses presenting very specific ligands on their surfaces anchor onto the specific receptors on a cell. Viruses can be engineered to display ligands for receptors presented on the surface of a target cell of interest. The interactions between cell receptors and viral ligands are modulated in vivo by toll like receptors.

[0035] The entry of a viral vector into a cell, whether via receptor mediated endocytosis or membrane fusion, requires a specific set of domains that permit the escape of the viral vector from endosomal and/or lysosomal pathways. Other domains facilitate entry into nuclei. Replication, assembly, and latency determine the dynamics of interactions between the vector and the cell and are important considerations in the choice of a viral vector, as well as in engineering therapeutic cargo carrying cells, in designing cancer suicide gene therapies.

[0036] Herpes simplex virus (HSV) belongs to a family of herpesviridae, which are enveloped DNA viruses. HSV binds to cell receptors through orthologs of their three main Iigand glycoproteins: gB, gH, and gL, and sometimes employ accessory proteins. These ligands play decisive roles in the primary routes of virus entry into oral, ocular, and genital forms of the disease. HSV possesses high tropism towards cell receptors of the nervous system, which can be utilized for engineering recombinant viruses for the delivery of expression cassettes to target cells, including senescent cells, cancer cells, and cells infected with an infectious agent. Therapeutic bystander effects are enhanced by inclusion of connexin coding sequences into the constructs. Herpes Simplex Virus vectors for the delivery of nucleic acids to target cells have been reviewed in Anesti and Coffin, Expert Opin Biol Ther 10(1 ):89-103 (2010); Marconi et al., Adv Exp Med Biol 655: 1 18-44 (2009); and Kasai and Saeki, Curr Gene Ther 6(3}:303-14 (2006).

[0037] Lentivirus belongs to a family of retroviridae, which are enveloped, single stranded RNA retroviruses and include the Human immunodeficiency virus (HIV). HIV envelope protein binds CD4, which is present on the cells of the human immune system such as CD4+ T cells, macrophages, and dendritic cells. Upon entry into a cell, the viral RNA genome is reverse transcribed into double-stranded DNA, which is imported into the cell nucleus and integrated into the cellular DNA. HIV vectors have been used to deliver the therapeutic genes to leukemia cells. Recombinant lentiviruses have been described for mucin-mediated delivery of nucleic acids into pancreatic cancer cells, to epithelial ovarian carcinoma cells, and to glioma cells, without substantial non-specific delivery to normal cells. Lentiviral vectors for the delivery of nucleic acids to target cells have been reviewed in Primo et al., Exp Dermatol 21 (3): 162-70 (2012); Staunstrup and Mikkelsen, Curr Gene Ther 1 1 (5):350-62 (201 1 ); and Dreyer, Mol Biotechnol 47(2): 169-87 (201 1 ).

[0038] Adenovirus is a non-enveloped virus having a double-stranded, linear DNA genome and a capsid. Naturally, adenovirus resides in adenoids and may be a cause of upper respiratory tract infections. Adenovirus utilizes a cell's coxsackievirus and adenovirus receptor (CAR) for the adenoviral fiber protein for entry into nasal, tracheal, and pulmonary epithelia. Recombinant adenovirus can be generated that are capable of nucleic acid deliver to target cells. Replication- competent adenovirus-mediated suicide gene therapy (ReCAP) is in the clinical trials for newly- diagnosed prostate cancer. Adenovirus vectors for the delivery of nucleic acids to target cells have been reviewed in Huang and Kamihira, Biotechnol Adv. 31 2):208-23 (2013); Alemany, Adv Cancer Res 1 15:93-1 14 (2012); Kaufmann and Nettelbeck, Trends Mol Med 18(7):365-76 (2012); and Mowa et al. , Expert Opin Drug Deliv 7Π 2): 1373-85 (2010).

[0039] Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. Vectors using AAV can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV a very attractive candidate for creating viral vectors for use in the systems of the present disclosure. Adeno-associated virus (AAV) vectors for the delivery of nucleic acids to target cells have been reviewed in Li et al., J. Control Release 172(2):589-600

(2013) ; Hajitou, Adv Genet 69:65-82 (2010); McCarty, Mol Ther 16(10): 1648-56 (2008); and Grimm et al, Methods Enzymol 392:381 -405 (2005).

[0040] Polyplexes are complexes of polymers with DNA. Polyplexes consist of cationic polymers and their fabrication is based on self-assembly by ionic interactions. One important difference between the methods of action of polyplexes and liposomes and lipoplexes is that polyplexes cannot directly release their nucleic acid cargo into the cytoplasm of a target cell. As a result co-transfection with endosome-lytic agents such as inactivated adenovirus is required to facilitate escape from the endocytic vesicle made during particle uptake. Better understanding of the mechanisms by which DNA can escape from endolysosomal pathway {i.e., the proton sponge effect) has triggered new polymer synthesis strategies such as the incorporation of protonable residues in polymer backbone and has revitalized research on polycation-based systems. See, e.g. , Parhamifar et al., Methods e-pub (2014); Rychgak and ilbanov, Adv Drug Deliv Rev e-pub

(2014) ; Jafari et al. , Curr Med Chem 19(2): 197-208 (2012).

[0041] Due to their low toxicity, high loading capacity, and ease of fabrication, polycationic nanocarriers exhibit substantial advantages over viral vectors, which show high immunogenicity and potential carcinogenicity and lipid-based vectors which cause dose dependent toxicity. Polyethyleneimine, chitosan, poly(beta-amino esters), and polyphosphoramidate have been described for the delivery of nucleic acids. See, e.g., Buschmann et al., Adv Drug Deliv Rev 65(9 : 1234-70 (2013). The size, shape, and surface chemistry of these polymeric nano-carriers can be easily manipulated.

[0042] Dendrimers are highly branched macromolecules having a spherical shape. The surface of dendrimer particles may be functionalized such as, for example, with positive surface charges (cationic dendrimers), which may be employed for the delivery of nucleic acids. Dendrimer-nucleic acid complexes are taken into a cell via endocytosis. Dendrimers offer robust covalent construction and extreme control over molecule structure and size. Dendrimers are available commercially from Dendritic Nanotechnologies Inc. (Priostar; Mt Pleasant, MI), who produce dendrimers using kinetically driven chemistry, which can be adapted fro the delivery of nucleic acids and can transfect cells at a high efficiency with low toxicity.

[0043] It will be understood that, while targeted delivery of a vector may not be required for use in certain methods of the present disclosure and that the targeted reduction, prevention, and/or elimination in the growth and/or survival of a target cell may be achieved by exploiting the intracellular transcriptional machinery of a target cell that is unique to the target cell, it may be desirable, depending upon the precise application contemplated, to incorporate into an otherwise non-specific delivery vector one or more components that facilitate the targeted delivery to a subset of cells at least some of which include a target cell to which a nucleic acid that encodes one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof is to be delivered.

[0044] Vectors can be administered to a human patient by themselves or in pharmaceutical compositions where they are mixed with suitable carriers or excipient(s) at doses to treat or ameliorate a disease or condition as described herein. Mixtures of these systems can also be administered to the patient as a simple mixture or in pharmaceutical compositions.

Methods for the Production and Release of Microparticles and Exosomes

[00111] Within certain aspects, the present disclosure provides methods for inducing the production and release of microparticles or exosomes, which methods include contacting ex vivo a mammalian cell, such as a mesenchymal or other stem cell, or administering to a mammal in vivo, one or more Bcl-2 proteins, Bcl-2 family member proteins, fragments comprising a BH4 domain region of a Bcl-2 protein or other Bcl-2 family member protein, including one or more mimetics thereof or delivery vectors comprising such proteins, in an amount sufficient to modulate, induce, promote, enhance, or inhibit one or more cellular activities or phenotypes of the mammalian cell.

[00112] Within related aspects, the present disclosure provides methods for inducing the production and release of microparticles or exosomes, which methods include contacting ex vivo a mammalian cell, such as a mesenchymal or other stem cell, or administering to a mammal in vivo, one or more nucleic acids, or one or more delivery or expression vectors comprising one or more such nucleic acids, encoding a Bcl-2 protein, Bcl-2 family member protein, fragment comprising a BH4 domain region of a Bcl-2 protein or other Bcl-2 family member protein, or a mimetic thereof in an amount sufficient to modulate, induce, promote, enhance, or inhibit one or more cellular activities or phenotypes of the mammalian cell.

[00113] The ability of a Bel protein to enable or increase microparticle-mediated nucleic acid delivery can be assessed, for example, using a mammalian cell or mammal. For example, one or more of the following markers and/or assays may be used to assess the ability of a Bcl-2 protein to enable or increase microparticle-mediated nucleic acid delivery:

(1 ) An increase in microparticlesin the culture media of a mammalian cell following treatment with a Bcl-2 protein as measured by ELISA, flow cytometric analysis, electron microscopy, light scattering, optical density or another technique used to measure microparticles (see, e.g. Examples 3 and 19 herein and Logozzi, PLos ONE 4(4):5219 (2009); Orozco, Cytometry 6:502-14 (2010); and Van Der Pol et. al, Journal of Thrombosis and Haemostasis 8:2596-2607 (2010). Prior to measurement microparticles may be purified by, e.g., differential ultracentrifugation, chromatography or membrane filtration methods.

(2) An increase in microparticie mediated transfer of a labeled (e.g. enzyme, epitope, isotope, biotin or fluorescent conjugate) or unlabeled nucleic acid between mammalian cells can be determined by measuring an increased level of the label present in the recipient cells or tissues {see, e.g., Paganin-Gioanni, PNAS 108(26): 10443-7 (201 1 ) or by measuring the transferred nucleic acid by in situ hybridization, quantitative PCR or the phenotypic change mediated by the delivered nucleic acid (see, e.g., Li, Int J Nanomedicine 7:2473-81 (2012)); (3) An increase in microparticles in the blood, tissues, saliva or urine of a mammal following administration of a Bcl-2 protein {see, e.g., Mitchell et ai, Journal of Translational Medicine 7:4 (2009) and Palanisamy el ai, PLos ONE 5: 1 (2010)); and

(4) An increase in the cell type or tissue distribution of a nucleic acid using methods described supra. Such assays may be used to identify Bcl-2 proteins, relatively small Bcl-2 peptides or a Bcl-2 peptidometic capable of inducing microparticle induction, determining the potency of a Bci-2 protein, peptide or mimetic, and identifying cell types that efficiently produce relatively large quantities of microparticles in response to treatment with a Bcl-2 protein.

[00114] In further aspects, the present disclosure provides methods for increasing the efficiency of gene therapy wherein the methods each include contacting a mammalian cell with an extracellular Bcl-2 protein, Bcl-2 domain-containing peptide, or a mimetic thereof in an amount sufficient to induce the formation of microparticles that mediate the delivery of therapeutic nucleic acid including a DNA, a micro-RNA (miRNA), a short interfering RNA (siRNA), and/or a messenger RNA (mRNA).

[00115] For example, treating mammalian cells are contacted in vitro or ex vivo, such as in tissue culture, with a Bcl-2 protein, Bcl-2 domain-containing peptide, or a mimetic thereof, or administering the Bcl-2 molecule, prior, simultaneously to or following treatment with a therapeutic nucleic acid, can induce microparticles that mediate delivery of the therapeutic nucleic acid to various cell types and/or tissues. The Bcl-2 protein may be used alone or it may be conjugated to the therapeutic nucleic acid. An active fragment of a Bc l -2 protein, such as the BH4 domain, may be used to stimulate production of therapeutic microparticles in vitro, ex vivo, and/or in vivo. The treatment may address technical limitations of gene delivery vehicles such as low efficiency of gene transfer, induction of an immune or an inflammatory response and inappropriate tissue distribution.

[00116] In yet another aspect, the present disclosure provides methods of using Bcl-2 proteins, Bcl-2 domain-containing peptides, or mimetics thereof, to generate exosomes or other microparticles in vitro that can be administered to a cancer patient to generate tumoricidal CAR T- cells in vivo. The methods of this aspect involve generation of a cell line that is (a) engineered to produce exosomes that contain a unique vector for CAR (chimeric antigen receptor) expression and a T-cell specific ligand and (b) may overexpress a T-cell ligand, such as, but not limited to, anti-CD2, anti-CD3, anti-CD4, anti-CD8 or anti-CD22 single-chain variable fragment (scFv). CARs typically consist of an immunoglobulin or immunoglobulin-derived domain that can recognize specific cancer cells; a hinge and transmembrane domain that tethers the immunoglobulin to the cancer cell; and costimulatory and essential activity domains, which together signal the T-cell to divide, thereby producing additional T-cells to bind and kill the cancer cells.

[00117] Administration of a Bcl-2 protein, domain-containing peptide, or mimetic induces and/or enhances generation and/or stabilization of exosomes or other microparticles containing the unique CAR vector. Upon exit from the engineered cells, the plasma membrane of the vector- containing exosome or other microparticle may contain one or more of the aforementioned T-cell ligands to enhance binding of the exosomal surface T-cell ligand.

[00118] The ability of a Bel protein or peptide to induce a change in the type of microparticle/exosome produced from a mammalian cell may be measured by, for example, proteomic methods, miRNA arrays, gene arrays, qPCR, SDS-PAGE, ELISA or another method. Prior to measurement, microparticles may be purified by, e.g., differential ultracentrifugation, chromatography, HPLC, commercial products described to precipitate or purify exosomes such as Exoquick (Systems Biosciences) or membrane filtration methods.

[00119] An increase in microparticles in the blood, tissues, saliva or urine of a mammal following administration of a Bel protein can be measured as described (see, e.g., Mitchell el al., Journal ofTranslational Medicine 7:4 (2009) and Palanisamy et al. , PLos ONE 5 : 1 (2010)). Such assays may be used to identify Bel proteins, relatively small Bel peptides or a Bel peptidometic capable of inducing a change in the quantity and/or type of microparticles/exosomes released from a mammalian cell, determining the potency of a Bcl-2 protein, peptide or mimetic, and identifying cell types that efficiently produce relatively large quantities of microparticles/exosomes in response to treatment with a Bcl-2 protein.

[00120] Such exosomes or other microparticles may be purified by known methods such as by PEG precipitation. The exosomes or other microparticles may advantageously be stored for long periods of time. A further advantage of the exosomes or other microparticles is that they possess low immunogenicity, and therefore may be administered to any patient. This advantageously allows for more efficient production of CAR T-cells relative to known methods, which methods involve time-consuming ex vivo generation of patient-specific CAR T-cells {See, Jacobson and Ritz, Blood 1 18 (18):4761 -4762 (201 1 )).

[00121] In conjunction with the present disclosure, various mammalian cell types, including human monocytic cells, rat dendritic cells, mouse bone marrow derived macrophages, and mouse mesenchymal stem cells (MSC) were treated with CTA 1 (recombinant human A l (rhA l ) with a c-terminal his- tag) at 300 ng/ml for 4 hours. After 4 hours, unbound CTA 1 was removed by three large volume washes. After 24 hours, the culture media may be processed by low speed centrifugation to remove any cells and buffer exchange and concentration (e.g., by centrifugation filtration; Amicon with 1 -300 kDa filter). Administration of the processed media to mice protects them from ischemic injury as determined by MTT (tetrazolium dye) assay for metabolic activity or tissue viability as described in Iwata et al, PLoS one 5(2}:e9103 (2010) (FIGS. 7B-7E). The cytoprotective activity in the processed media can be depleted by 20nm filtration, a step that would remove exosomes which are 40- 100nm in diameter (FIG. 7F), or pelleted by ultracentrifugation under conditions (100K for 70 minutes) that pellet exosomes and microvesicles (FIG. 7G). Transmission electron microscopy demonstrates that relative to control treatments, CTA 1 treatment of mammalian cells appears to specifically induce production/release of microparticles 40- 100 nm in diameter consistent with exosome vesicles (FIG 71; arrows). A Bcl-2 peptide containing the BH4 domain region induced cytoprotective activity from Jaws II cells (FIG. 7H). Jaws II cells were treated as described above with a peptide containing the BH4 domain peptide to induce an extracellular mediator cytoprotective response in the mouse model of hind leg ischemic injury.

[00122] In another aspect, the present disclosure provides methods for identifying a Bcl-2 protein that increases the effectiveness of microparticle mediated nucleic acid therapy when administered to a mammal. The methods of this aspect of the disclosure each include screening a plurality of proteins to identity a Bcl-2 protein that increases the tissue concentration of an administered nucleic acid or inhibits disease, when administered to a mammal.

[00123] The ability of a Bcl-2 protein to induce a change in the quantity and/or type of microparticles/exosomes released in the culture media of a mammalian cell following treatment with a Bel protein may be measured by e.g. ELISA, use of a flow cytometric instrument such as FACS, electron microscopy, light scattering, optical density, nanoparticle tracking (NTA) eg using Nanosight or another technique used to measure microparticles (see, e.g.. Examples 3 and 19 and Logozzi, PLos ONE 4(4):5219 (2009); Orozco, Cytometry 6:502-14 (2010), and Van Der Pol et. al.. Journal of Thrombosis and Haemostasis 8:2596-2607 (2010)).

Methods for Altering a Cellular Activity or Phenotype

[00124] Within certain embodiments, the present disclosure provides methods for using the (Bcl-2, Bcl-2 family members, or BH4 domain regions thereof described herein. Within certain aspects of those embodiments, disclosed herein are methods for regulating, promoting, normalizing, restoring, inhibiting, or modulating a desired cellular phenotype including, for example, differentiation, de-differentiation, proliferation, growth, cell death, contact inhibition by expressing one or more Bcl-2, Bcl-2 family members, or BH4 domain regions as presented herein or as identified through the methodology described in this disclosure.

[00125] Thus, the present disclosure provides in various embodiments, as exemplified herein, in vitro, ex vivo, and in vivo methods that employ one or more Bcl-2 protein, Bcl-2 protein family member, extracellular domain of a Bcl-2 protein, extracellular domain of a Bcl-2 protein family member, BH4 domain region of a Bcl-2 protein, or BH4 domain region of a Bcl-2 protein family member either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activity or phenotype, including cellular activities or phenotypes that are associated with a condition, disease, or injury that can be advantageously treated by contacting a cell ex vivo with, or by the in vivo administration of, a Bcl-2 protein, Bcl- 2 protein family member, extracellular domain of a Bcl-2 protein, extracellular domain of a Bcl-2 protein family member, BH4 domain region of a Bcl-2 protein, or BH4 domain region of a Bcl-2 protein family member.

[00126] The present disclosure also provides, and exemplifies, in vitro, ex vivo, and in vivo methods that employ one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activity or phenotype, including cellular activities or phenotypes that are associated with a condition, disease, or injury that can be advantageously treated by contacting a cell ex vivo with, or by the in vivo administration of, one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of a Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members.

[00127] Further provided, and exemplified herein, are methods that employ one or more vectors for the delivery or expression of nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activity or phenotype, including cellular activities or phenotypes that are associated with a condition, disease, or injury that can be advantageously treated by contacting a cell ex vivo with, or by the in vivo administration of, one or more vectors for the delivery or expression of nucleic acids encoding one or more Bcl- 2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members.

Methods for the Treatment of Tissue Injuries, Diseases, and Other Conditions

[00128] The present disclosure provides methods for the treatment of tissue injuries, diseases, and other conditions, which methods include the in vivo administration to a mammal, including a human, of one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activities, symptoms, or phenotypes associated with a tissue injury, disease, or other condition thereby treating the tissue injury, disease, or other condition.

[00129] Also provided are methods for the treatment of tissue injuries, diseases, and other conditions, which methods include the in vivo administration to a mammal, including a human, of one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activities, symptoms, or phenotypes associated with a tissue injury, disease, or other condition thereby treating the tissue injury, disease, or other condition.

[00130] Within related aspects, the present disclosure provides methods for the treatment of a VDAC-associated disease, such as fibrosis, or other VDAC-associated disease, including a VDAC-associated disease that affects a mammal's retina, liver, kidney, lung, skin, nervous system, digestive tract, or other body system or tissue, which methods include the in vivo administration to a mammal, including a human, of one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activities, symptoms, or phenotypes associated with the VDAC-associated disease.

[00131] Within related aspects, the present disclosure provides methods for the treatment of a VDAC-associated disease, such as fibrosis, or other VDAC-associated disease, including a VDAC-associated disease that affects a mammal's retina, liver, kidney, lung, skin, nervous system, digestive tract, or other body system or tissue, which methods include the in vivo administration to a mammal, including a human, of one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members either alone or in combination with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activities, symptoms, or phenotypes associated with the VDAC-associated disease.

[00132] Within other aspects, the present disclosure provides methods for the treatment of a condition, disease, or tissue injury, which methods include contacting ex vivo a target cell with one or more microparticles/exosomes, such as mesencyhmal stem cell exosomes, isolated from a mammalian cell contacted either in vivo or ex vivo with one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, BH4 domain regions of Bcl-2 protein family members, or mimetics thereof in an amount sufficient to modulate, induce, promote, enhance, or inhibit one or more cellular activities or phenotypes of the target cell. These methods may advantageously employ the introduction of the target cell into a patient following the ex vivo contact of the target cell with the one ore more microparticles or exosomes.

[00133] Within related aspects, the present disclosure provides methods for the treatment of a condition, disease, or tissue injury, which methods include contacting ex vivo a target cell with one or more microparticles/exosomes, such as mesencyhmal stem cell exosomes, isolated from a mammalian cell contacted either in vivo or ex vivo with one or more nucleic acids encoding one or more Bcl-2 proteins, Bcl-2 protein family members, extracellular domains of Bcl-2 proteins, extracellular domains of Bcl-2 protein family members, BH4 domain regions of Bcl-2 proteins, or BH4 domain regions of Bcl-2 protein family members or mimetics thereof in an amount sufficient to modulate, induce, promote, enhance, or inhibit one or more cellular activities or phenotypes of the target cell. These methods may advantageously employ a vector for delivering the one or more nucleic acids to, or for expressing the one or more nucleic acids within, the mammalian cell and may advantageously employ the introduction of the target cell into a patient following the ex vivo contact of the target cell with the one ore more microparticles or exosomes.

[00134] These methods may further include contacting ex vivo the target cell with one or more compounds, including one or more small molecules, proteins, or nucleic acids, which can, either alone or in combination, modulate, induce, promote, enhance, or inhibit one or more cellular activities or phenotypes of the target cell.

[00135] Within other aspects, disclosed herein are methods for the treatment of a tissue injury, a disease, or a condition that associated with the expression of one or more gene or the production of one or more protein, wherein one or more aspect of the disease or condition is reduced in severity following the administration of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof.

[00136] Within further embodiments, the present disclosure provides methods for reducing, preventing, and/or eliminating the growth of a target cell, which methods comprise contacting a target cell with a vector system for the targeted production of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof wherein the vector comprises: (a) a transcriptional promoter that is activated in response to one or more factors each of which factors is produced within a target cell and (b) a nucleic acid that is operably linked to and under regulatory control of the transcriptional promoter, wherein the nucleic acid encodes one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof and wherein production of the one or more Bcl-2, Bcl- 2 family members, or BH4 domain regions thereof affects an activity or phenotype of the target cell.

[00137] Within still further embodiments, the present disclosure provides methods for the treatment of a human that is afflicted with a disease or another condition, wherein the disease, or other condition is associated with a target cell within the human, the methods comprising administering to the human a vector for the production of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof wherein the vector comprises an expression construct for the targeted production of one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof wherein the vector comprises: (a) a transcriptional promoter that is activated in response to one or more factors each of which factors is produced within a target cell and (b) one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof and wherein the nucleic acid is operably linked to and under regulatory control of the transcriptional promoter, wherein the one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof reduces, prevents, and/or eliminates growth and/or survival of the target cell thereby slowing, reversing, and/or eliminating the disease or condition in the human.

[00138] Examples of conditions, diseases, or injuries that may be treated by Bcl-2-induced microparticles/exosomes include conditions, diseases, or injuries such as liver fibrosis, kidney injury, and myocardial ischemic injury. Li et al., 2013, Exosomes derived from human umbilical cord mesencyhmal stem cells alleviate liver fibrosis, Stem Cells Dev., 22 (6); 845-854 ), (Zhou, et al., 2013, J. Stem Cell Res. & Ther. 4:34), (Lai 2010, Stem Cell Research, May; 4(3):214-22 and Lai et al, 2012 Int. J. Proteomics, Arslan et al., 2013, Stem Cell Res. May; 10(3):301 - 12). As therapeutic activity of mesencyhmal stem cells can be associated with the exosomes they release (Lai, et al., Stem Cell Res. 4(3): 214-22, 2010, Bruno, et al., J. Am. Soc. Nephrol. 20(5): 1053-67, 2009, Gatti, et.al., Nephrol. Dial. Transplant 26(5): 1474-83, 201 1 ), further examples of diseases and injuries that may benefit from treatment with Bel-induced microparticles/exosomes may include those being treated with mesencyhmal stem cells in clinical trials including, Cirrhosis, Pulmonary Fibrosis, Ulcerative Colitis, Crohn's Disease, Diabetes, Transplantation, Multiple Sclerosis, Acute Myocardial Infarction, Stroke, Osteoarthritis, Parkinson's Disease, Critical Limb Ischemia, Ophthalmic diseases, and Spinal Cord Injury (Clinical Trials.gov). As microparticles produced from monocytic or dendritic cells can enhance or suppress an immune response (Record et al., Biochemical Pharmacology, 81 , 1 171 - 1 1 82, 201 1 ; Camussi et al., Kidney International, 1 , 2010) additional examples of disease that may be treated with Bel-induced microparticles/exosomes include: asthma, allergies, autoimmune diseases and infectious diseases.

[00139] A schematic of a proposed mechanism by which a Bel protein induces or increases a physiologic response to limit injury or disease (e.g., fibrosis) progression and facilitate repair is presented herein. Without being limited by mechanistic theory, it is-believed that CTA 1 released from injured tissue/cells binds to a cell surface receptor involved in production and/or release of microparticles/exosomes that deliver associated mediators of activity (miRNA, proteins, mRNA, lipids) to various target cells. The anti-fibrotic activity of Bcl-2 proteins (see, e.g., the data presented in FIG. 5 and FIG. 6) may include exosome associated proteins and miRNA that can decrease macrophage accumulation (FIG. 3) through one or more mechanisms and hence macrophage production of profibrotic cytokines. The activity of CTA/rhA l is distinguished from "alarmins" that bind receptors, including TLR2 and TLR4. to activate and recruit cells of the innate immune system (Iwata 2010). Exosomes, but not rhA l , can directly bind TLR2 (Chalmin 2010, Anand 2010, unpublished).

[00140] Suitable target cells may be isolated from a patient afflicted with or exhibiting one or more symptoms of the medical condition or may be isolated from a suitable donor. The target cell may, for example, be a mesencyhmal stem cell, a monocytic cell, a dendritic cell, an endothelial cell, an epithelial cell, or another cell type. The target cell may be a primary cell or a cell line.

[00141] In yet another aspect of the present disclosure, a Bcl-2 protein, a Bcl-2 protein-domain peptide, such as BH4, or a mimetic thereof, maybe used for the treatment of Alzheimer's Disease (AD). In this regard, AD is characterized in part by the accumulation, aggregation and deposition of beta-amyloid plaques (Αβ) in the brain. Αβ is a neurotoxic, synaptotoxic peptide product of β- site proteolytic processing of the amyloid precursor protein (APP). In AD, Αβ peptides improperly accumulate, aggregate, and deposit to form plaques as a result of decreased catabolism and clearance. Recent animal and clinical studies using an antibody to Αβ support a central role for accumulation of Αβ for AD pathogenesis. PAI- 1 is believed to promote Αβ accumulation by preventing plasmin-directed degradation of Αβ and APP. More specifically, Αβ and APP are typically degraded via a protease cascade triggered by the plasminogen activators uPA and tPA. PAI- 1 has been shown to inhibit this protease cascade by directly binding to and antagonizing both uPA and tPA.

[00142] Work by Liu et a/ has shown that PAI- 1 is increased in the brains of AD patients (cite), and an active role for PAI- 1 is supported by small-molecule inhibition of PAI- 1 decreasing Αβ and reversing cognitive deficit in AD-model mice (Jacobsen), as well as the observation that PAI- 1 knockout decreases Αβ deposition in the brains of APP/PS 1 (AD model) mice. Thus, downregulation of PAI- 1 mRNA expression by Bcl-2-derived peptides, as shown in the present Example, supports an alternative mechanism for treating AD. In this aspect, "treatment" refers to decreasing, or slowing the increase of expression levels of PAI- 1 , APP or tau mRNA or protein, peptide, or of a mimetic variant thereof, decreasing, or slowing the increase of the presence of one or more biomarkers correlated with the presence or progression of AD, effecting an increase or improvement in the presence of one or more biomarkers correlated with the absence or decline of AD. slowing the progression or spread of AD, decreasing the size and/or number of Αβ peptides, oligomers, aggregates, deposits, fibrils or plaques corresponding to AD, preventing the onset of Αβ, such as by prophylactic introduction, or improving cognition, neurological activity, neurological function, memory, or locomotion of a mammal diagnosed with or believed to be at risk for AD or another neurodegenerative disease.

[00143] The present disclosure also provides methods for treating cancer. In various representative embodiments, the cancer is breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, oral cancer, brain cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, thyroid cancer, renal cancer, bladder cancer, leukemia, or melanoma. In other embodiments, the cancer is a different type of cancer.

[00144] The methods of this aspect of the disclosure can be used for example to treat various mammalian diseases and injuries. Examples include, genetic disorders (immunodeficiencies, metabolic disorders, muscular dystrophies, lysosomal storage diseases), and disorders of bone, joint, gastrointestinal, hematologic, ophthalmic, neurological, urogenital, pulmonary and hearing.

[00145] In another aspect, the present disclosure provides methods for enabling or increasing the efficiency of stem (embryonic or mesencyhmal) cell therapy wherein the methods each include treating mammalian cells with an extracellular Bcl-2 protein, domain-containing peptide, or mimetic thereof in an amount sufficient to induce or increase stem cell microparticles that mediate or contribute to stem cell therapeutic activity. Examples of diseases and injuries that may benefit from extracellular Bel are those being treated with mesencyhmal stem cells in clinical trials including, Ulcerative Colitis, Crohn's Disease. Diabetes, Transplantation, Multiple Sclerosis, Cirrhosis, Acute Myocardial Infarction, Stroke, Osteoarthritis, Parkinson's Disease, Critical Limb Ischemia, ophthalmic diseases, and Spinal Cord Injury (see Clinical Trials.gov).

[00146] In another aspect, the present disclosure provides methods enabling or increasing the efficiency of a vaccine wherein the methods include treating mammalian cells with an extracellular Bcl-2 protein, domain-containing peptide, or mimetic thereof in an amount sufficient to induce or increase dendritic or monocytic cell microparticles that mediate or contribute to an immune response or suppression of an immune response. Examples of diseases that may benefit from extracellular Bcl-2 treatment are inflammatory diseases such as rheumatoid arthritis, Crohn's disease, Ulcerative colitis, ophthalmic diseases, and pelvic inflammatory disease, and autoimmune diseases such as Rheumatoid arthritis, Sjogren syndrome, Type 1 diabetes, Myasthenia gravis, Systemic lupus erythematous. Multiple Sclerosis, Addison's disease, Graves disease, and pernicious anemia.

[00147] In another aspect, the present disclosure provides methods for treating mammalian cells ex vivo with a Bcl-2 protein and therapeutic nucleic acid to prepare the mammalian cell for increased nucleic acid delivery following administration in a mammal.

[00148] In another aspect, the present disclosure provides methods for treating mammalian stem cells ex vivo with a Bcl-2 protein to prepare the stem cell for increased therapeutic activity following administration in a mammal.

[00149] In another aspect, the present disclosure provides methods for treating mammalian lymphoid cells ex vivo with a Bcl-2 protein to prepare the lymphoid cell for increased immune activation or suppression activity following administration in a mammal.

[00150] In a further aspect, the present disclosure provides methods for treating a mammal or mammalian cells with a Bcl-2 protein in conjunction with ultrasound to increase and/or direct microparticle-mediated delivery of a nucleic acid to mammalian cells or mammalian tissues. For example, increasing Bcl-2 protein induction of microparticles with ultrasound applied to a mammalian cell or mammalian tissue may enhance the localized effectiveness of nucleic acid delivery.

[00151] Inhibition of a disease or disease progression as a result of a Bcl-2 protein increasing nucleic acid delivery can also be assessed in a mammalian disease or mammalian disease model that is treatable by administering a nucleic acid (see e.g., CA. Pacak et ah, Circ. Res. 18;99(4), (2006), A. V. Sauer, Front. Immunol. 3:265 (2012)). Inhibition of disease, as a result of administration of a Bel protein is indicated by changes in disease symptoms or biomarkers used to determine disease progression or regression.

[00152] Administration of the Bcl-2 proteins is accomplished by any effective route, e.g., topical, local injection or systemic injection. Bcl-2 proteins may be administered together with suitable pharmaceutically acceptable carriers including excipients and other compounds that facilitate administration of the Bcl-2 proteins to a mammalian subject. Further details on techniques for formulation and administration may be found, for example, in the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co., Easton, PA).

[00153] Bcl-2 proteins for parenteral administration include aqueous solutions of one or more Bcl-2 proteins. For injection, Bcl-2 proteins may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of Bcl-2 proteins may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[00154] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are typically used in the formulation. Such penetrants are generally known in the art (see, e.g. B. G. Short, Toxicologic Pathology 36:49-62 (2008)).

[00155] Bcl-2 proteins may be prepared in a form suitable for administration to a mammal by art-recognized techniques e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or Iyophilizing processes). The Bcl-2 proteins may also be modified to provide appropriate release characteristics, e.g., sustained release or targeted release, by conventional means {e.g., coating).

[00156] The Bcl-2 proteins may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. After such Bcl-2 proteins formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for use.

[00157] Bcl-2 protein may be introduced in association with another molecule, such as a lipid, to protect the protein from enzymatic degradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 1 1 : 139, 1990). Many polymer systems have been reported for protein delivery (Bae, Y.H., et al., J Controlled Release 9:271 , 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I., et al., J Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J Controlled Release 10: 195, 1989; Asano, M., et al., J Controlled Release 9: 1 1 1 , 1989; Rosenblatt, J., et al., J Controlled Release 9: 195, 1989; Makino, ., J Controlled Release 2:235, 1990; Takakura, Y., et al., J Pharm. Sci. 78: 1 17, 1989; Takakura, Y., et al., J Pharm. Sci. 78:219, 1989.)

[00158] The amount actually administered will be dependent upon the individual to which treatment is to be applied, and will preferably be an optimized amount such that the desired effect is achieved without significant side-effects. The determination of an effective dose is well within the capability of those skilled in the art. Of course, the skilled person will realize that divided and partial doses are also within the scope of the disclosure.

[00159] For any Bcl-2 protein, the effective dose can be estimated initially in any appropriate animal model (e.g., primate, rats and guinea pigs and other laboratory animals). The animal model is also typically used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans or other mammals.

[00160] Therapeutic efficacy and possible toxicity of Bcl-2 proteins can be determined by standard pharmaceutical procedures in experimental animals {e.g., ED50, the dose therapeutically effective in 50% of the population; and LD50, the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio ED50/LD50. Bcl-2 proteins, which exhibit large therapeutic indices, are preferred. The data obtained from animal studies is used in formulating a range of dosage for use in humans or other mammals. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED so with little or no toxicity. The dosage typically varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[00161] Exemplary Bcl-2 dosages include administration of at least 50 ng/kg/day, such as from 50 ng/kg/day to 50 mg/kg/day, or such as from 0.5 mg/kg/day to 50 mg/kg/day, for a period of time sufficient to inhibit disease in the mammal. Typically, the Bcl-2 protein is administered to the mammal on multiple occasions (e.g., daily). For example, a Bcl-2 protein can be administered to a mammal at least once per day for a period of from 1 day to 20 days, or from 1 day to 40 days, or from 1 day to 60 days. Bcl-2 protein can be administered indefinitely to a mammalian subject to treat a chronic medical condition (e.g., at least once per day each day during the remaining lifetime of the recipient). The abbreviation "ng" is an abbreviation for nanogram, or nanograms, as appropriate. The abbreviation "mg" is an abbreviation for milligram, or milligrams, as appropriate. The abbreviation "kg" is an abbreviation for kilogram, or kilograms, as appropriate.

[00162] Once administered to a cancer patient (for example, by intravenous injection), the vector-containing exosomes or other microparticles may fuse with the T-cell in a receptor- dependent or receptor-independent process. The internalized transposon may then express transposase using the T-cell-specific promoter. The transposase mediates the cut-and-paste insertion of the CAR encoding transposon into the T-cell DNA. In this way, T-cells expressing CAR can be stimulated to proliferate through contact with tumor cells bearing a tumor antigen that is recognized by the given CAR. Further, Bcl-2 proteins, domain-containing peptides or mimetics thereof may be administered to patients with the engineered exosomes to enhance the survival of CAR T cells (See Charo et al., "Bcl-2 overexpression enhances tumor-specific T-cell survival" Cancer Res. 65(5): 2001 -8 (2005) and Kalbasi et al., "Prevention of interleukin-2 withdrawal- induced apoptosis in lymphocytes retrovirally cotransduced with genes encoding an antitumor T- cell receptor and an anti-apoptotic protein" J. lmmunother. 33(7): 672-83 (2010).

[00163] In a related aspect, administration of Bcl-2 may protect normal or healthy tissues from undesirable chemokine toxicity that may occur with CAR T-cell treatment of cancer. Excessive activation of CAR T-cells in immunosuppressed and/or immunocompromised patients has been associated with a toxic so-called "chemokine release syndrome" or "cytokine storm" (Maher, J. "Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells" ISRN Oncology, online, Dec. 9, 2012). Accordingly, it is believed that Bcl-2 treatment may protect normal tissues from damage related to excessive chemokine release associated with CAR T-cell treatment.

[00164] In another related aspect, Bcl-2-induced or Bcl-2-stabilized exosomes/microparticles, in conjunction with cell-type specific transposase expression, may be used to treat other diseases. The gene or genes of interest may be associated with a transposon and/or another expression system with cell-type restriction and may be used to treat a variety of diseases, including diseases related to gain-of-function or loss-of-function of a gene. For example, in certain embodiments a loss-of-function-associated disease such as diabetes may be treated by correction by Bcl-2- and exosome-mediated transposition of the wild-type gene sequence or a functionally equivalent DNA sequence into affected cells. Alternatively, the exosome may deliver to a diseased cell one or more miRNAs, shRNAs, or other nucleic acids to selectively inhibit gene expression of, for example, a mutated and/or overexpressed gene. In other embodiments, the method may be utilized as a treatment for an infectious disease.

[00165] In some embodiments, the therapeutic method may involve administration of a Bcl-2 protein, domain-containing-peptide, or mimetic, and an expression vector containing a therapeutic gene. The expression construct may be internalized in a specific cell type, such as, for example, a liver cell or a spleen cell. The Bcl-2 protein, domain-containing-peptide, or mimetic may support exosome/microparticle production enabling the transfer of the therapeutic gene to other tissues and cell types to achieve a therapeutic effect.In one aspect, the present disclosure provides methods for inhibiting VDAC-associated disease in a mammal. Each method includes the step of administering to a mammal a Bel protein or BH4 peptide in an amount sufficient to inhibit VDAC-associated disease activity. In one embodiment, the VDAC-associated disease is fibrosis.

[00166] In another aspect, the present disclosure provides methods for treatment of medical conditions that can be treated with exosomes wherein the methods each include a step of administering exosomes released from mammalian cells that have been treated with a Bel protein ex vivo, in an amount sufficient to inhibit disease or inhibit tissue damage due to an injury and/or facilitate tissue repair. The mammalian cell may be a mesencyhmal stem cell, monocytic cell, dendritic cell, endothelial cell, epithelial cell or other cell type. The mammalian cell may be a primary cell or a cell line. The mammalian cell may be an engineered cell over expressing a mediator of activity such as a miR A, protein, mR A or lipid. Examples of diseases or injuries that may be treated by Bel-induced microparticles/exosomes include those that may be treated with mesencyhmal stem cell exosomes such as liver fibrosis, kidney injury, and myocardial ischemic injury. (Li, T. et al.. 2013, "Exosomes derived from human umbilical cord mesencyhmal stem cells alleviate liver fibrosis," Stem Cells Dev. 22 (6); 845-854 ), (Zhou et al., 2013, Stem Cell Res. & Ther. 4:(34), (Lai 2010, Stem Cell Res., May;4(3):214-22 and Lai et al, 2012, Int. J. Proteomics, Arslan et al., 2013, Stem Cell Res. May; 10(3):301 - 12). As therapeutic activity of mesencyhmal stem cells can be associated with the exosomes they release (Lai et al., Stem Cell Res. 4(3): 214- 22, 2010, Bruno, et al., J. Am. Soc. Nephrol. 20(5): 1053-67, 2009, Gatti et al., Nephrol. Dial. Transplant 26(5): 1474-83, 201 1 ), further examples of diseases and injuries that may benefit from treatment with Bel-induced microparticles/exosomes may include those being treated with mesencyhmal stem cells in clinical trials including, Cirrhosis, Pulmonary Fibrosis, Ulcerative Colitis, Crohn's Disease, Diabetes, Transplantation, Multiple Sclerosis, Acute Myocardial Infarction, Stroke, Osteoarthritis, Parkinson's Disease, Critical Limb Ischemia, Ophthalmic diseases, and Spinal Cord Injury (Clinical Trials.gov). As microparticles produced from monocytic or dendritic cells can enhance or suppress an immune response (Record, et al., Pharm Biol, 81 , 1 171 -1 182, 201 1 ; Camussi et al., Kidney Int., 1, 2010) additional examples of disease that may be treated with Bel-induced microparticles/exosomes include: asthma, allergies, autoimmune diseases and infectious diseases.

[00167] Inhibition of VDAC-associated disease, such as fibrosis, in a mammal encompasses complete or partial inhibition of VDAC-associated disease in a mammal. Inhibition of diseases that can be treated with mammalian exosomes in a mammal encompasses complete or partial inhibition of diseases that can be treated with mammalian exosomes in a mammal.

[00168] In the practice of the present disclosure one or more types of Bcl-2 proteins or Bcl-2- induced exosomes can be administered to a mammal suffering from fibrosis (e.g., suffering from a disease that causes fibrosis, or undergoing a medical treatment that causes fibrosis, or suffering from an injury that causes fibrosis). Examples of diseases, medical treatments, or injuries that cause fibrosis include non-alcoholic steatohepatitis (NASH), hepatitis C virus, alcoholic steatohepatitis, diabetic nephopathy, hypertension, glomerulonephritis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, heart failure, myelofibrosis, scleroderma/systemic fibrosis, arthrofibrosis, keloid, hypertrophic scar, cystic fibrosis, cirrhosis, organ transplantation, mesenteric, retinal, and peripheral vascular disease, amyotrophic lateral sclerosis, radiation- induced fibrosis and traumatic injuries.

[00169] Additionally, in the practice of the present disclosure, one or more Bel proteins or Bcl- induced exosomes can be administered to a mammal that is not suffering from a fibrotic disease. For example, one or more types of Bcl-2 proteins or Bcl-2-induced exosomes can be administered prophylactically to a mammal to prevent, or decrease the likelihood of, the onset of fibrosis or to reduce the severity of fibrosis that may subsequently occur. The mammal may be suffering from a disease that can cause fibrosis and the Bcl-2 protein is administered to prevent, or decrease the likelihood of, the onset of fibrosis, or to reduce the severity of fibrosis that may subsequently occur. For example, the following categories of human patients may benefit from administration of one or more Bcl-2 proteins or Bcl-2-induced exosomes to prevent, or decrease the likelihood of, the onset of fibrosis: patients undergoing surgeries, percutaneous intervention, organ transplant, radiation treatment.

[00170] Such methods of the present disclosure can be practiced on any patient with a medical condition that can be treated with exosomes. Examples of diseases or injuries that may be treated by exosomes include those that may be treated with mesencyhmal stem cell exosomes such as liver fibrosis, kidney injury, and myocardial ischemic injury. (Li, T. et al 2013, Exosomes derived from human umbilical cord mesencyhmal stem cells alleviate liver fibrosis, stem cells and development, 22 (6); 845-854 ), (Zhou, et al., 2013, Stem Cell Research & Therapy 4:34), (Lai 2010, Stem Cell Research,^' May;4(3):214-22 and Lai et al, 2012 Int J of Proteomics, Arslan et al 2013, Stem Cell Research May; 10(3):301 -12). The therapeutic activity of mesencyhmal stem cells can be associated with the exosomes they release (Lai, et.al. Stem Cell Res 4(3): 214-22, 2010, Bruno, et.al., J Am Soc Nephrol 20(5): 1053-67, 2009, Gatti, et.al., Nephol Dial Transplant 26(5): 1474- 83, 201 1 ). Thus further examples of diseases and injuries that may benefit from treatment with a Bel protein or peptide include those being treated with mesencyhmal stem cells in clinical trials including, Cirrhosis, Pulmonary Fibrosis, Ulcerative Colitis, Crohn's Disease, Diabetes, Transplantation, Multiple Sclerosis, Acute Myocardial Infarction, Stroke, Osteoarthritis, Parkinson's Disease, Critical Limb Ischemia, Ophthalmic diseases, and Spinal Cord Injury (Clinical Trials.gov).

[00171] In another aspect, the present disclosure provides methods for enabling or increasing the efficiency of nucleic acid delivery. Each of the methods includes the treating mammalian cells or administering to a mammal a Bcl-2 protein or Bcl-2 protein-nucleic acid conjugate in an amount sufficient to induce and/or increase microparticle mediated nucleic acid delivery. Subsequent to treating a mammalian cell with a Bcl-2 protein and nucleic acid or Bcl-2 protein-nucleic acid conjugate the mammalian cell may be administered to a mammal. [00172] In a further aspect, the present disclosure provides methods for treating mammalian cells or administering to a mammal a Bcl-2 protein to enable or increase the efficiency of stem cell therapy. Treatment of mesenchymal stem cells with an extracellular recombinant Bcl-2 protein potently stimulates therapeutic cytoprotective activity. Microparticles from stem cells can mediate the therapeutic activity of the stem cell. For example, exosomes from mesenteric stem cells (MSC) confer the therapeutic cytoprotective activity in ischemic injury models (see, Lai et al, Stem Cell Res 4(3): 214-22 (2010); Bruno et al, J. Am. Soc. Nephrol. 20(5): 1053-67 (2009); and Gatti et al, Nephrol. Dial. Transplant 26(5): 1474-83 (201 1 )). Each of the methods includes treating mammalian cells or administering to a mammal a Bcl-2 protein in an amount sufficient to induce and/or increase stem cell microparticle mediated therapeutic activity.

[00173] In another aspect, the present disclosure provides for regulation of an immune response. Treatment of monocytic or dendritic cells with an extracellular recombinant Bcl-2 protein stimulates therapeutic cytoprotective activity associated with microparticles (FIG. 37). Microparticles produced from monocytic or dendritic cells can enhance or suppress an immune response (see, Record et al, Biochemical Pharmacology 81 : 1 171 -1 182 (201 1 ) and Camussi et al. Kidney International (2010)).

[00174] Induction of microparticle-mediated nucleic acid delivery between mammalian cells or in a mammal encompasses complete or partial induction of microparticle-mediated therapeutic nucleic acid delivery between mammalian cells or in a mammal. Nucleic acid delivery between mammalian cells or in a mammal by a Bcl-2 protein-nucleic acid conjugate encompasses complete or partial nucleic acid delivery between mammalian cells or in a mammal, such as the mammals described previously.

[00175] One or more Bcl-2 proteins can be used to treat mammalian cell types ex vivo prior, simultaneously or following treatment with a therapeutic nucleic acid. Such treated mammalian cells may be administered to a mammal to deliver a therapeutic nucleic acid to treat an injury or disease. Additionally, one or more Bcl-2 proteins can be used in conjunction with therapeutic nucleic acids and ultrasound to treat a mammal suffering from various diseases. Ultrasound may be used to prime mammalian cells or tissues for Bcl-2 protein induced microparticle production and/or uptake. Ultrasound may induce mild tissue damage and release of endogenous Bcl-2 proteins that may enhance the activity of an administered Bcl-2 protein in induction of microparticle formation in a more tissue specific manner (see, e.g., F. Prat et al. Out, 35:395-400, 1994). Ultrasound also be used to prime mammalian cells or tissues for receiving microparticles induced by a recombinant extracellular Bcl-2 protein (see eg., Polat et al. Expert Opin Drug Deliv., 7: 12: 1415-32 (2010) and Sheikh, J Clin Exp Dent 3 :3:e228-34 (201 1 )).

[00176] Additionally, in the practice of the present disclosure one or more Bcl-2 proteins can be administered to a mammal that is not suffering from an injury or disease. For example, one or more types of recombinant extracellular Bcl-2 proteins can be administered prophylactically to a mammal to prevent, or decrease the likelihood of, a disease, or to reduce the severity of a disease that may subsequently occur.

[00177] The amount actually administered will be dependent upon the individual to which treatment is to be applied, and will preferably be an optimized amount such that the desired effect is achieved without significant side-effects. The determination of an effective dose is well within the capability of those skilled in the art. Of course, the skilled person will realize that divided and partial doses are also within the scope of the invention.

[00178] For any Bcl-2 protein, Bcl-2 domain-containing peptide such as BH4, or Bcl-2-induced exosomes, the effective dose can be estimated initially in any appropriate animal model (e.g., primate, rats and guinea pigs and other laboratory animals). The animal model is also typically used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans or other mammals.

[00179] Therapeutic efficacy and possible toxicity of Bel proteins, Bel domain-containing peptides or Bcl-2-induced exosomes can be determined by standard pharmaceutical procedures in experimental animals (e.g., ED50, the dose therapeutically effective in 50% of the population; and LD50. the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio EDso/LDso. Bcl-2 proteins, Bcl-2 domain-containing peptides or Bcl-2-induced exosomes which exhibit large therapeutic indices, are preferred. The data obtained from animal studies is used in formulating a range of dosage for use in humans or other mammals. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage typically varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

[00180] Exemplary Bcl-2 protein, -domain, or -induced exosome dosages include administration of at least 50 ng/kg/day, such as from 50 ng/kg/day to 50 mg/kg/day, or such as from 0.5 mg/kg day to 50 mg kg/day, for a period of time sufficient to inhibit disease in the mammal. Typically, the Bcl-2 protein is administered to the mammal on multiple occasions (e.g., daily). For example, a Bcl-2 protein can be administered to a mammal at least once per day for a period of from 1 day to 20 days, or from 1 day to 40 days, or from 1 day to 60 days. Bcl-2 protein can be administered indefinitely to a mammalian subject to treat a chronic medical condition (e.g., at least once per day each day during the remaining lifetime of the recipient). The abbreviation "ng" is an abbreviation for nanogram, or nanograms, as appropriate. The abbreviation "mg" is an abbreviation for milligram, or milligrams, as appropriate. The abbreviation "kg" is an abbreviation for kilogram, or kilograms, as appropriate.

[00181] Administration of the Bcl-2 proteins or BcI-2-induced exosomes is accomplished by any effective route, e.g., topical, local injection or systemic injection. Bcl-2 proteins may be administered together with suitable pharmaceutically acceptable carriers including excipients and other compounds that facilitate administration of the Bcl-2 proteins to a mammalian subject. Further details on techniques for formulation and administration may be found, for example, in the latest edition of "Remington's Pharmaceutical Sciences" (Maack Publishing Co, Easton PA).

[0045] Compositions within the scope of this disclosure include compositions comprising one or more Bcl-2 proteins, Bcl-2 family member proteins, or BH4 domain regions thereof as well as compositions comprising one or more nucleic acids that encode one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof. Compositions may also include vectors for the delivery or such proteins and nucleic acids. Compositions may also include one or more additional active compounds, which compounds include nucleic acids, proteins, and small molecules that further promote the desired cellular activity or phenotype of a cell that is contacted with the Bcl-2, Bcl-2 family member, or BH4 domain region proteins or nucleic acids or a cell that is associated with an injury, disease, or condition in a patient to which the Bcl-2, Bcl-2 family member, or BH4 domain region proteins or nucleic acids are administered. [0046] Determination of optimal ranges of effective amounts of each component of such compositions is within the skill of the art. The effective dose is a function of a number of factors, including the specific system, the presence of one or more additional therapeutic agent within the composition or given concurrently with the system, the frequency of treatment, and the patient's clinical status, age, health, and weight.

[0047] Compositions include a therapeutically effective amount of a Bcl-2, Bcl-2 family member, or BH4 domain region protein or nucleic acid and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skimmed milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0048] These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such compositions will contain a therapeutically effective amount of the inhibitor, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. [0049] Compositions can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0050] The vectors disclosed herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0051] Bcl-2 proteins may be prepared in a form suitable for administration to a mammal by art-recognized techniques (e.g., by means of conventional mixing, dissolving, granulating, dragee- making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes). The Bcl-2 proteins may also be modified to provide appropriate release characteristics, e.g., sustained release or targeted release, by conventional means (e.g., coating).

[0052] The present disclosure provides compounds comprising a Bcl-2 polypeptide, or a mimetic thereof, and an immunoglobulin, which compounds exhibit an improved in vivo half-life over native or otherwise unmodified or unconjugated Bcl-2 protein. In certain embodiments, the Bcl-2 polypeptide or mimetic thereof are fused to the immunoglobulin. In certain embodiments, the immunoglobulin is an immunoglobulin fragment chain (Fc). In some embodiments, the immunoglobulin fragment chain is an IgG Fc. In some embodiments, the Bcl-2 polypeptide is Bcl-2 protein or a mimetic thereof. In other embodiments, the Bcl-2 polypeptide is a Bcl-2 peptide. In some embodiments, the Bcl-2 polypeptide is a BH4 peptide or a mimetic thereof. [0053] In the practice of the present disclosure, one or more types of Bcl-2 proteins can be used in conjunction with therapeutic nucleic acids to treat mammalian cells or administered to a mammal suffering from a disease that may be treated by a nucleic acid. Examples include medical needs that would be served by effective gene therapy such as genetic disorders (immunodeficiencies, metabolic disorders, muscular dystrophies, lysosomal storage diseases), and disorders of bone and joint, endocrine, gastrointestinal, hematologic, ophthalmic, neurological, urogenital, pulmonary and hearing. See, for example, Jain, "Textbook of Personalized Medicine," pp 529-549 (Springer, 2015).

[0054] The Bcl-2 proteins may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. After such Bcl-2 proteins formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for use.

[0055] Bcl-2 protein may be associated with another molecule, such as a lipid, to protect the protein from enzymatic degradation. For example, the covalent attachment of polymers, especially polyethylene glycol (PEG), has been used to protect certain proteins from enzymatic hydrolysis in the body and thus prolong half-life (Fuertges, F., et al., J. Controlled Release 77 : 139, 1990). Many polymer systems have been reported for protein delivery (Bae, Y.H., et al., J. Controlled Release 9:271 , 1989; Hori, R., et al., Pharm. Res. 6:813, 1989; Yamakawa, I., et al., J. Pharm. Sci. 79:505, 1990; Yoshihiro, I., et al., J. Controlled Release 70: 195, 1989; Asano, M., et al., J. Controlled Release 9: 1 1 1 , 1989; Rosenblatt, J., et al., J. Controlled Release 9: 195, 1989; Makino, ., J. Controlled Release 2:235, 1990; Takakura, Y., et al., J. Pharm. Sci. 78: \ 1 7, 1989; Takakura, Y., et al., J. Pharm. Sci. 75:219, 1989.)

[0056] In various embodiments of the present disclosure, Bcl-2 proteins may be covalently or non-covalently conjugated to a therapeutic nucleic acid using established methods. For example, a Bcl-2 protein may be fused using standard molecular biology methods to protamine to create a delivery vehicle. Protamine is a highly basic protein that condenses nucleic acids. It has been successfully fused to other proteins to direct genes to target cells (Li (2001 ) and Song (2005)). Other well established chemical crosslinkers may be used to conjugate a Bel protein to a therapeutic nucleic acid including plasmid DNA, linear DNA, miRNA, siRNA, shRNA or mRNA (see e.g. Y Singh, et. al., Chem. Soc. Rev., 39:2054-2070, 2010).

[0057] The amount of the one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof that will be effective in the treatment, inhibition, and/or prevention of cancer, infectious disease, or other disease or condition can be determined by standard clinical techniques. In vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0058] The compositions of the present disclosure can be tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of a system on a cell line or a patient tissue sample. The effect of the system or pharmaceutical composition thereof on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to proliferation and apoptosis assays. In accordance with the present disclosure, in vitro assays that can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

[0059] Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The Bcl-2, Bcl-2 family members, or BH4 domain regions thereof may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings {e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the inhibitors or compositions into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, for example, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0060] Compositions may be administered parenterally. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion. Alternatively, or concurrently, administration may be orally. Compositions may, for example, be administered intravenously via an intravenous push or bolus. Alternatively, compositions may be administered via an intravenous infusion.

[0061] Bcl-2 proteins for parenteral administration include aqueous solutions of one or more Bcl-2 proteins. For injection, Bcl-2 proteins may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of Bcl-2 proteins may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

[0062] It may be desirable to administer the Bcl-2, Bcl-2 family members, or BH4 domain regions thereof or miRNAs locally to the area in need of treatment; this may be achieved by, for example, local infusion during surgery, topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are typically used in the formulation. Such penetrants are generally known in the art (see e.g. B. G. Short, Toxicologic Pathology 36:49-62 2008). [0063] The Bcl-2, Bcl-2 family members, or BH4 domain regions thereof or miR As can be delivered in a controlled release system placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release 2: 1 15- 138 ( 1984)).

[0064] Intravenous infusion of a compositions comprising a system may be continuous for a duration of at least about one day, or at least about three days, or at least about seven days, or at least about 14 days, or at least about 21 days, or at least about 28 days, or at least about 42 days, or at least about 56 days, or at least about 84 days, or at least about 1 12 days.

[0065] Continuous intravenous infusion of a composition comprising a system may be for a specified duration, followed by a rest period of another duration. For example, a continuous infusion duration may be from about 1 day, to about 7 days, to about 14 days, to about 21 days, to about 28 days, to about 42 days, to about 56 days, to about 84 days, or to about 1 12 days. The continuous infusion may then be followed by a rest period of from about 1 day, to about 2 days to about 3 days, to about 7 days, to about 14 days, or to about 28 days. Continuous infusion may then be repeated, as above, and followed by another rest period.

[0066] Regardless of the precise infusion protocol adopted, it will be understood that continuous infusion of a composition comprising one or more Bcl-2, Bcl-2 family members, or BH4 domain regions thereof will continue until either desired efficacy is achieved or an unacceptable level of toxicity becomes evident.

* * * * *

[0067] It will be understood that, unless indicated to the contrary, terms intended to be "open" (e.g., the term "including" should be interpreted as "including but not limited to," the term "having" should be interpreted as "having at least," the term "includes" should be interpreted as "includes but is not limited to," etc.). Phrases such as "at least one," and "one or more," and terms such as "a" or "an" include both the singular and the plural.

[0068] It will be further understood that where features or aspects of the disclosure are described in terms of Markush groups, the disclosure is also intended to be described in terms of any individual member or subgroup of members of the Markush group. Similarly, all ranges disclosed herein also encompass all possible sub-ranges and combinations of sub-ranges and that language such as "between," "up to," "at least," "greater than," "less than," and the like include the number recited in the range and includes each individual member.

[0069] All references cited herein, whether supra or infra, including, but not limited to, patents, patent applications, and patent publications, whether U.S., PCT, or non-U. S. foreign, and all technical and/or scientific publications are hereby incorporated by reference in their entirety.

[0070] While various embodiments have been disclosed herein, other embodiments will be apparent to those skilled in the art. The various embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the claims.

EXAMPLES

Example 1

Bcl-2 Proteins Are Effective in Treating Retinal Disease

[00182] This Example discloses the use of Bcl-2 proteins to treat retinal disease in a mouse model system for laser-induced choroidal neovascularization (CNV) - an art accepted animal model system for age-related macular degeneration (AMD). The data presented herein demonstrate that CTA l treatment is effective in decreasing fibrosis, inflammation, and neovascularization associated with retinal disease.

[00183] In this study (FIG. 1) each eye is subjected at four sites to laser-induced injury on day 0. On day 1 and 4 CTA l (recombinant human A l ) is administered intraperitoneally to 8 mice. As a positive control a humanized anti- VEGF antibody is administered to another 8 mice. At days 4 and 7 vascular leak (a measure of neovascularization) is determined by fluorescein fundus angiography. On day 7 or 8 mice are sacrificed and eyes are processed for histology.

[00184] Results of the laser induced CNV study are shown in FIGs. 2-5. FIG. 2A includes representative fluorescein angiogram images of decreased area of leakage in CTA l treated animals. CTA l treatment significantly reduced the mean grade of lesion. Quantification of lesions by flat mount further confirms that CTA l treated animals had significantly less leakage compared to control (FIG. 2B). Thus CTA l treatment decreases neovascularization as determined by vascular leak. Immunohistochemistry (1HC) using an antibody (anti-CD31 ) that binds to vessels also indicates decreased vessel formation in CTA l treated mice (FIG. 3). CTA l treatment also reduced the number of macrophage/monocytic cells in laser-induced CNV tissue as determined by 1HC using a CD163 antibody (FIG. 4). Macrophage and monocytic cells are key contributors to a fibrogenesis through their production of profibrotic cytokines and growth factors (Wynn and Barron, Semin Liver Dis. 30(3):245-257 (2010)). CTA l treatment reduces collagen accumulation in laser-induced CNV tissue as determined by staining with Sirius Red to visualize collagen (FIG. 5). Thus CTA l treatment decreases fibrosis in the mouse laser-induced CNV model. The reduction of fibrosis may involve a reduction in macrophages, inflammatory mediators and neovascularization. Example 2

[00185] This Example describes the use of Bcl-2 proteins to inhibit fibrosis in a mouse model of glaucoma filtration surgery.

[00186] In the mouse model of glaucoma filtration surgery (GFS) the conjunctiva is surgically dissected to expose the underlying sclera, after which an incision is made through the sclera into the anterior chamber of the eye to create a fistula. This surgically-created channel facilitates the outflow of aqueous humor from the anterior chamber into the subconjunctival space, and thereby effectively reduces intraocular pressure. Filtration of fluid is obvious as an elevated conjunctival bleb which is readily observed by slit lamp microscopy (Seet et al., PlosOne 5 (2010) and Seet et al, Mol Med 17 '(5-6)557 '-567 (201 1)). Survival of the bleb is limited by fibrosis.

[00187] In the GFS study, mice were treated with CTAl (0.8 ug, via subconjunctival injection) or PBS vehicle (n=10/group) on the day of surgery and on day 2 post-surgery. Bleb survival was determined by slit lamp biomicroscopy on day 0, -2, -7 and weekly thereafter. FIGURE 6 shows that CTA l treatment significantly prolongs bleb survival over 30 days (Kaplan-Meier survival plot; P=0.029). Because failure of bleb survival is generally due to fibrosis, CTA l treatment appears to reduce fibrosis in this glaucoma filtration surgery model.

Example 3

[00188] This Example discloses the use of Bcl-2 proteins in an in vitro system that can be used to screen for active for Bel peptides, modified Bcl-2 peptides and mimetics that induce the release of microparticles/exosomes. This example also discloses an in vitro system that can be used to screen for biomarkers of Bcl-2 protein activity.

[00189] Bcl-2 family members or their BH4 domain region peptides can induce microparticles/exosomes from cultured cells (FIG. 7). The cultured media of Bcl-2-treated cells can be processed in several assay systems for quantitative and qualitative analysis of induced mediators. For example, centrifuged and buffer exchanged culture media (FIG. 7) may be subjected to spectrophotometry to determine Bcl-2-induced induced protein and RNA concentrations (FIG. 7A). [00190] The presence of RNA supports Bcl-2- induction of a microparticle/exosome. The processed media may be subjected to SDS-PAGE and protein stain to determine size profile of Bel-induced proteins (FIG. 7B). These data demonstrate that Bcl-2 induces the release of microparticles/exosomes as the media was processed by 3 buffer exchanges using a centrifugal filter with a nominal molecular weight limit of either 100 or 300 d yet proteins under this size were induced, apparently as a microparticle/exosome. The media may be subjected to an ELISA designed to capture exosomes (or other mediators) to determine the level of Bcl-2-induction of exosomes.

[00191] The exosome ELISA may use antibodies that bind to exosome membrane markers (eg Rab5b and CD63) in a sandwich ELISA (FIG. 7C). These data demonstrate the Bcl-2-induction of exosomes relative to saline or BIM control treatments. Other assay systems or methods may be used to analyze Bcl-2-induced vesicles or mediators including, but not limited to, nanoparticle tracking analysis (NTA), cytofluorimetry (e.g., following the capture of exosomes on beads), light scattering, RNA arrays (e.g., mRNA, miRNA), and proteomic profiling methods. Functional assays may be used to characterize the activity Bcl-2-induced exosomes or mediators, Appropriate systems related to extracellular Bcl-2 activities includes protection from hypoxia, chemical or mechanical injuries. In particular, protection from ER stress or the unfolded protein response (UPR), would be relevant. Negative controls for Bcl-2 induction of exosomes or mediators from cultured cells may include saline, PBS, BH3 domain peptides or other non- Bcl-2 proteins.

[00192] These in vitro bioanalytical assays (See FIG. 9) using media from Bcl-2- treated cultured cells may be used for: (a) determination of minimal Bcl-2 peptide size containing a functional binding motif (FIG. 11); (b) active Bcl-2 peptide modifications; (c) identification of active Bcl-2 peptidomimetics; (d) identification of active exosome or mediator inducing small molecules: (e) Bcl-2 or Bcl-2 peptide lot release; (f) determination of Bcl-2 potency such as 50% effective concentration (EC50); (g) determination of the kinetics of mediator induction; (h) optimization of the exosome ELISA (e.g., different pairs of capture and detection antibodies and incubation time); (i) comparison of protein, RNA, and exosome production/induction in THP- 1 , SC or other cell types, to identify induced proteins and RNAs (indicators of mechanism) via quantitative proteomic methods (such as iTRAQ labeling and LC-MS/MS) and RNA array methods (for example miRXpIore microarray), to facilitate identification of an in vitro functional assay for CTA 1 -induced exosomes.

Example 4

[00193] This Example demonstrates the use of a Bcl-2 protein or BH4 peptide to identify a cell surface receptor involved in Bcl-2-induced microparticle/exosome release and presents a system that may be used to screen for a small molecule that can compete Bcl-2 protein binding and induce release of microparticles/exosomes from mammalian cells.

[00194] FIG. 10 shows that a BH4 domain peptide binds to a mammalian cell surface receptor. A concentration range (20 nM to 1 μΜ) of biotinylated Bcl-2-A l BH4 peptide, scrambled Bcl-2- A l BH4 peptide (sBH4), or a Bax BH3 peptide (BH3) were allowed to bind to dendritic cells (Jaws2) and T cells (Jurkat) for 30 minutes at 4C. Peptide binding was determined by subsequent binding of phycoerythin labeled-strepavidin and flow cytofiuorimetry. Specific and saturable binding of the A l BH peptide (red) indicates binding to a cell surface receptor. Note: It has been determined that biotinylated-BH4 peptide maintains its anti-apoptotic activity in the mouse hind limb I/R injury model performed as described (Iwata 2010).

[00195] The binding of the Bcl-2 peptide to a specific cell surface receptor indicates that the Bcl-2 peptide may be used to isolate and identify the receptor. The Bcl-2 peptide may be conjugated to a substrate such as sepharose beads which then may be used in standard affinity chromatography to isolate the receptor from solubilized cell membrane proteins. The receptor may also be isolated by a "pull down" method using the Bcl-2peptide conjugated beads. The isolated receptor may be visualized by SDS-PAGE and protein staining. The isolated receptor may be identified by nano-LC-MS/MS. Candidate plasma membrane receptors for Bcl-2proteins include proteins or related proteins that have been described to interact intracellularly with Bcl-2 BH4 domain including Bax inhibitor- 1 (BI- 1 ) family members, BI- 1 , LFG and Ghitm or VDAC (Lee 2010, Reimers 2006, Reimers 2007, Akanda 2008).

Example 5

[00196] This Example demonstrates the use of Bcl-2 proteins to treat fibrosis using a mouse model of liver fibrosis. [00197] In this study treatment with Bcl-2 proteins decreases fibrosis and blood levels of transaminases. Efficacy is demonstrated in mice subjected to CC14 twice a week, a model often used for development of treatments for liver fibrosis. In this study (FIG. 12) CCI4 is administered intraperitoneally to each mouse two times a week beginning on day 1 . On days 3 and 12 Bel proteins A l , Bcl-W, and Bcl-X, or saline was administered intraperitoneally at 1.2 micrograms/mouse (5 mice per treatment group). After 4 weeks the mice were sacrificed and blood and liver tissue was collected for analysis.

[00198] Results of the CC14-induced liver fibrosis study are shown in FIGs. 12-15. FIG. 12 includes representative liver tissue sections stained with Sirius red to determine the quantity and pattern of collagen accumulation. Liver sections of the vehicle (saline) control mice show a large accumulation of collagen and bridging fibrosis (vessel to vessel) consistent with later stages of disease. Treatment with Bcl-2 family proteins decreases the extent of bridging fibrosis and thickness of collagen fibers. Bcl-W and Bcl-A l appeared to have greater anti-fibrotic activity relative to Bcl-X. Treatment with Bcl-2 family members also decreased blood levels of alanine transaminase (ALT) and aspartate transaminase (AST), indicators of liver injury (FIGs. 14 and 15). Relative to control mice treatment with Bcl-A l , Bcl-W and Bcl-X decreased the blood level of both ALT and AST at the end of week 4. Thus treatment with Bcl-2 family members protects mice from CC14-induced liver fibrosis and injury decreasing fibrosis, ALT and AST levels.

Example 6

Bcl-2 Protein Identification

This Example discloses methodology for the identification of Bcl-2 proteins and Bcl-2 family member proteins.

Sample Preparation and Digestion In-solution

[00199] Samples were precipitated using a ProteoExtract Protein Precipitation Kit following the protocol provided by the manufacturer (CalBiochem; http://www.emdmillipore.com/life- science-research/proteoextract-protein-precipitation-kit/E D BIO-

5391 80/p_xdCb.sl ORVMAAAEjpxp9.zLX). The resulting protein pellet was solubilized in Ι ΟΟμί of 6M urea in 50mM ammonium bicarbonate (A BIC). 200m of dithiothreitol (DTT) was added to a final concentration of 5mM and samples were incubated for 30min at 37°C. Next, 20m iodoacetamide (I AA) was added to a final concentration of 15mM and incubated for 30min at room temperature, followed by the addition of 20 μL· DTT to quench the IAA reaction. Lys- C/trypsin (Promega) was next added in a 1 :25 ratio (enzyme:protein) and incubated at 37°C for four hours. Samples were then diluted to <1 M urea by the addition of 50mM AMBIC and digested overnight at 37°C. The following day, samples were desalted using C I 8 Macro Spin columns (Nest Group) and dried down by vacuum centrifugation.

In-gel Digestion

[00200] Gel pieces were cut into ~l mm³ pieces, washed three times with 50mM ammonium bicarbonate (AmBic), pH 8, and then chemically dried twice with 100% acetonitrile (ACN). Gel pieces were then reduced in 15mM dithiothreitol for 30 minutes at 56°C, chemically dried twice with 100% ACN, and then alkylated with 20mM iodoacetamide for 20 minutes in the dark. The gel pieces were washed twice more with 50mM AmBic, chemically dried twice with 100% ACN, and then mechanically dried using vacuum centrifugation. Trypsin (Promega) in 50mM AmBic was added in a 1 :30 ratio and digested overnight at 37°C. Peptide extraction proceeded the next day by collecting the supernatant and adding 60% ACN in 0.1 % trifluoroacetic acid to the gel pieces, sonicating for 10 minutes, and centrifuging for 5 minutes. The supernatant was collected and added to the supernatant of the previously collected supernatant. The supernatant was then vacuum-centrifuged.

LC-MS MS Analysis

[00201] LC separation was done on a Waters Nano Acquity UHPLC (Waters Corporation) with a Proxeon nanospray source. The digested peptides were reconstituted in 2% acetonitrile /0.1 % trifluoroacetic acid and roughly 3μg of each sample was loaded onto a 100 micron x 25 mm Magic C I 8 l OOA 5U reverse phase trap where they were desalted online before being separated on a 75 micron x 150 mm Magic C I 8 200A 3U reverse phase column. Peptides were eluted using a gradient of 0.1 % formic acid (A) and 100% acetonitrile (B) with a flow rate of 300nL/min. A 60 minute gradient was ran with 5% to 35% B over 50 minutes, 35% to 80% B over 2 minutes, 80% B for 1 minute, 80% to 5% B over 1 minute, and finally held at 5% B for 6 minutes. Each of the gradients was followed by a 30 minute column wash.

[00202] Mass spectra was collected on an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) in a data-dependent mode with one MS precursor scan followed by 15 MS/MS scans. A dynamic exclusion of 15 seconds was used. MS spectra were acquired with a resolution of 70,000 and a target of 1 * 10⁶ ions or a maximum injection time of 30ms. MS/MS spectra were acquired with a resolution of 17,500 and a target of 5 * 10⁴ ions or a maximum injection time of 50ms. Peptide fragmentation was performed using higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) value of 27. Unassigned charge states as well as +1 and ions >+5 were excluded from MS/MS fragmentation.

Example 7

Identification of Cell Surface Receptors for Anti-apoptotic Bcl2 Family Members (Bel)

[00203] This Example discloses the identification of cell surface receptors for anti-apoptotic Bcl2 family members (Bcl-2) by receptor pull down and LC-MS/MS.

Summary of Pull-down Protocol

[00204] A pull down experiment was conducted to isolate the plasma membrane receptor(s) for Bcl-2 protein family members. Plasma membrane proteins from THP-1 cells were isolated using Abeam Plasma membrane protein extraction kit (ab65400). In brief, 9 x 10⁸ THP-1 cells were washed in ice-cold buffer, re-suspended in homogenization buffer mix and lysed by dounce homogenization. Supematants from homogenates following a 10 min 700 x g centrifugation were spun at 10,000 x g for 30 min. The pellet containing proteins from both plasma membrane and cellular organelle membrane was further processed to isolate plasma membrane proteins. This involved re-suspending the pellet and extracting plasma proteins from the upper phase solution after multiple centrifugations at 1000 x g. Finally the upper phase was diluted with water and centrifuged at 16,000 x g to pellet plasma membrane proteins.

[00205] For pull downs Bcl-2A 1 (CTA 1 ) was biotinylated using Pierce Sulfo-NHS-LC- Biotinylation kit #21435 and coupled to Neutravidin (NA) agarose (Thermo Scientific 29200). In addition, the BH4 domain peptides of Bcl-W, Bcl-2 and Bcl-XL were synthesized with an n- terminal biotin for coupling to neutravidin agarose. See, FIG. 20.

[00206] Plasma Membrane Proteins were solubilized in 1 % NP-40 homogenization buffer containing Pierce Protease and Phosphatase inhibitors (#88668). The homogenate was pre-cleared with NA agarose, prior to pull down with Bcl-2 A l -biotin-NA agarose. After 3 washes with homogenization buffer, bound proteins were eluted with SDS-PAGE protein loading buffer containing 5% DTT. PAGE gels were subsequently stained with Imperial protein stain (Pierce 24615) to visualize proteins. See, FIG. 21. Gel slices (samples S 1 -S5) were sent to the Proteomics Core Lab at UC Davis for protease digestion and protein identification by LC-MS/MS.

Example 8

Identification of Membrane Proteins Isolated by Bel Protein Pull Down

In-gel protease Digestion

[00207] Gel pieces were cut into ~1 mm³ pieces, washed three times with 50mM ammonium bicarbonate (AmBic), pH 8, and then chemically dried twice with 100% acetonitrile (ACN). Gel pieces were then reduced in 15mM dithiothreitol for 30 minutes at 56°C, chemically dried twice with 100% ACN, and then alkylated with 20mM iodoacetamide for 20 minutes in the dark. The gel pieces were washed twice more with 50mM AmBic, chemically dried twice with 100% ACN, and then mechanically dried using vacuum centrifugation. Trypsin (Promega) in 50mM AmBic was added in a 1 :30 ratio and digested overnight at 37°C. Peptide extraction proceeded the next day by collecting the supernatant and adding 60% ACN in 0.1 % trifluoroacetic acid to the gel pieces, sonicating for 10 minutes, and centrifuging for 5 minutes. The supernatant was collected and added to the supernatant of the previously collected supernatant. The supernatant was then vacuum-centrifuged.

LC-MS/MS Analysis

[00208] LC separation was done on a Waters Nano Acquity UHPLC (Waters Corporation) with a Proxeon nanospray source. The digested peptides were reconstituted in 2% acetonitrile /0.1 % trifluoroacetic acid and roughly 3μg of each sample was loaded onto a 100 micron x 25 mm Magic C I 8 l OOA 5U reverse phase trap where they were desalted online before being separated on a 75 micron x 150 mm Magic C I 8 200A 3U reverse phase column. Peptides were eluted using a gradient of 0.1 % formic acid (A) and 100% acetonitrile (B) with a flow rate of 300nL/min. A 60 minute gradient was ran with 5% to 35% B over 50 minutes, 35% to 80% B over 2 minutes, 80% B for 1 minute, 80% to 5% B over 1 minute, and finally held at 5% B for 6 minutes. Each of the gradients was followed by a 30 minute column wash.

[00209] Mass spectra was collected on an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) in a data-dependent mode with one MS precursor scan followed by 15 MS/MS scans. A dynamic exclusion of 15 seconds was used. MS spectra were acquired with a resolution of 70,000 and a target of 1 ^χ 10⁶ ions or a maximum injection time of 30ms. MS/MS spectra were acquired with a resolution of 17,500 and a target of 5 * 10⁴ ions or a maximum injection time of 50ms. Peptide fragmentation was performed using higher-energy collision dissociation (HCD) with a normalized collision energy (NCE) value of 27. Unassigned charge states as well as +1 and ions >+5 were excluded from MS/MS fragmentation.

Bel cell surface receptor candidates

[00210] To identify cell surface receptor(s) for anti-apoptotic Bcl-2 family members ("Bcl-2") a "pull down" experiment was conducted as described in Protocols. SDS-PAGE protein bands that appeared specific relative to controls were excised and processed and subjected to peptide identification by LC MS/MS. Plasma membrane proteins that were identified includes: voltage dependent anion channels (VDAC 1 -3) and HLA class 1 proteins. See, Table 2. The vast majority of the proteins identified are not exposed at the cell surface but many are known to be associated with VDAC, such as ADP/ATP translocase, tubulin, and annexin, or function in vesicle generation, such as many Rab proteins and the vesicle fusing ATPase.

Table 2

Membrane Proteins Identified by Bel Protein Pull Down

[00211] Proteins isolated by interaction with the BH4 domain of Bcl-2 family members are of particular interest as this domain has been determined to suffice for therapeutic activity in animal studies (Iwata 2010). A protein band between 30 & 40 kD was analyzed from the pull down using Bcl-W BH4 domain peptide (FIG. 22). This gel slice contained peptides from voltage dependent anion channel; VDAC 1 , 2 and 3.

VDAC 1. 2. and 3

[00212] The binding of Bcl-W BH4 domain to VDAC has not previously been reported. However, certain Bcl-2 family members, Bcl-XL and Bcl-2, have been reported to bind intracellular VDAC or VDAC peptides in particular an amino terminal peptide (Monaco, Cell Mol Life Sci 70: 1 171 -1 1 83 (2013); Haung (2013); Malia (2007); Hiller (2008); and Arbel (2010)). Moreover in addition to the outer leaflet of the mitochondrial membrane, VDAC has been reported to be present in the plasma membrane (De Pinto, FEBS Letter 584: 1793-1799 (2010); Bathori (2000); and Thinnes (1989)).

[00213] There is evidence that the BH4 domain of Bcl-2 and Bcl-XL can close mitchondrial VDAC and prevent apoptotic mitochondrial changes (Shimizu, PNAS V97(7):3100 (2000)). Bcl- XL BH4 binding has been implicated in binding plasma membrane VDAC (Thinnes 2014 Molecular Genetics and Metabolism). It is not clear that all cell surface VDACs bind all anti- apoptotic Bcl-2 family members (Bcl-2, A l , Bcl-W, Bcl-X, Mcl- 1 ). Consistent with treatment with extracellular Bcl-2 proteins, antibody binding to plasma membrane VDAC demonstartes anti- apoptotic activity (Elinder, Cell Death and Differentiation 12, 1 134- 1 140 (2005) and Akanda (2008). VDAC antibodies and Bcl-x BH4 peptide have been reported to increase cell volume in hypotonic solution (Thinnes, Molecular Genetics and Metabolism (2014). VDAC ligand- mediated induction, production, or stabilization of extracellular vesicles, in particular therapeutically effective vesicles, has not been reported, for conditions such as AMD, sepsis, ischemic injuries, inflammatory diseases or fibrosis.

[00214] Several proteins were identified in pull downs that may function in VDAC and vesicle forming activities. These may form a signaling complex involved in the generation or release of vesicles. Overall VDACs appear to serve as one receptor for Bcl-2 family members. Cytofluorimetry data suggests that there may be more than one cell surface receptor for Bcl-2 family members.

Polymerase Delta Interacting Protein-2

[00215] Another protein identified in the pull down experiment is polymerase delta interacting protein-2. This protein has been associated with functions relevant to bcl activity in disease models. Polymerase delta interacting protein-2 functions in signaling pathway activating myofibroblasts which are predominant producers of matrix proteins leading to fibrosis (Manickam et al., Renal Physiolosv 307: 159 (2014)). It has been reported to function in post-ischemic injury neovascularization, supporting cell motility, MMP-2 and -9 activity, and decreasing apoptosis (Amanso et al., 34(7): 1548-55 (2014)). Polymerase delta interacting protein-2 also appears to functions in autophagy (Brown et al., Plos One 9(5) (2014)).

Heat Shock Protein 27 (HSP 27/Beta- l )

[00216] HSP27 Provides cytoprotection, and support of cell survival under stress conditions by modulating reactive oxygen species and to raise glutathione levels. Hsp27 is involved in apoptotic signaling, interfering with the activation of cytochrome c/Apaf-l/dATP complex and therefore inhibiting the activation of procaspase-9. In vitro it acts as a chaperone by inhibiting protein aggregation and by stabilizing partially denatured proteins, which ensures refolding by the Hsp70-complex. Hsp27 activates the proteasome, accelerating the degradation of irreversibly denatured proteins. HSP27 protects actin filaments from fragmentation and preserves the focal contacts fixed at the cell membrane. Hsp27 might function in cell differentiation and may play a crucial role in the termination of growth.

Example 9

Supportive Data for VP AC as a Cell Surface Receptor for Bcl 2 Family Members

CTA 1 Binds to a Subset of HE Cells that also Bind VDAC- 1 Antibody

[00217] CTA 1 binds to a subpopulation of HEK cells that also bind a VDAC- 1 antibody (LS- C 160510; Life Science Bio). This polyclonal VDAC-1 antibody binds to the n-terminal 30 amino acid region of VDAC- 1 . HEK cells were exposed to osmotic pressure; a hypotonic (75mM salt) solution for 30 minutes on ice prior to the binding of biotinylated CTA 1 and VDAC- 1 antibody, secondary fluorescent conjugates(streptavidin-PE and anti-rabbit FITC) and flow cytofluorimetry. The results show that relative to a biotinylated BSA control, CTA l predominantly binds the same subpopulation that binds the VDAC- 1 antibody. However, there is evidence that CTA l can bind a second subpopulation of HEK that does not appear to bind the VDAC- 1 antibody. This may be due to CTA 1 binding to a VDAC conformation or different VDAC that does not bind the VDAC 1 antibody. It might be due to competition between CTA l and VDAC-1 antibody binding to an overlapping site (VDAC n-terminus). Alternatively, this may be due to a second CTA l receptor. See, FIG. 23.

[00218] In the experiment described in FIG. 24, CTA l and VDAC binding was determined for HEK cells in both isotonic and hypotonic conditions. Two gated HEK cell populations were analyzed. All VDAC 1 positive HEK cells also bound CTAl . Both CTA l and VDAC 1 antibody binding increased approximately 10% following hypotonic treatment. Dual staining HEK cells also increased 10% (approximately 2 times greater) following hypotonic treatment. In contrast, CTA l binding to the population of HEK cells that does not bind the VDAC 1 antibody remained essentially constant following hypotonic treatment at around 20%.

[00219] These data are consistent with CTA l binding to a VDAC 1 conformation recognized by the VDAC 1 n-terminal antibody which increases with hypotonic stress. An antibody that binds the N-terminus of VDAC may selectively bind VDAC in a closed conformation. CTAl also appears to bind a second receptor on approximately 20% of HEK cells whose cell surface expression is not altered by hypotonic stress. CTA l may induce mediators of therapeutic activity subsequent to binding either or both populations; VDAC antibody binding and nonbinding.

[00220] VDAC is a beta-barrel composed of 19 strands and an alpha helical n-terminal sequence of 26 amino acids. The N-terminus of VDAC has been proposed to predominately locate within the lumen of the channel stabilizing a resting open state. Under conditions of lateral pressure induced by changes in membrane osmotic pressure or in the lipid environment the n-terminus may undergo a conformational change with movement toward the exterior of the lumen. The channel may then partially collapse into an elliptical state with conductance identical to the closed channel (Zachariae, Structure 20: 1 540 (2012)). The N-terminus thus appears to serve as a mechanosensor gating switch. [00221] Antibodies binding to the N-terminus can block apoptosis. Bcl-2 can bind to the n- terminus of VDAC- 1 . See, FIG. 25. Bcl-2 proteins may, therefore, antagonize plasma membrane VDAC by binding its n-terminus in a manner that interferes with its open state conductance and VDAC dependent apoptosis. In addition, or alternatively, Bcl-2 proteins may interfere with the role of VDAC oligomers in apoptosis. VDAC oligomers have been proposed to be essential to apoptosis. VDAC oligomers have been proposed to form a large central pore that allows the passage of cytochrome C leading to apoptosis. Bel proteins can inhibit the formation of VDAC oligomers. Thus, the binding of Bcl-2 proteins to plasma membrane VDAC may also interfere with transport of larger molecules leading to apoptosis. This function of plasma membrane VDAC is not well characterized.

Example 10

Identification of Bel-induced Proteins

In Solution Protease Digestion of Bel-induced Mediators

[00222] To identify potential Bcl-2 family mediators of activity and pharmacodynamic markers, culture media was prepared from cultured cells treated with Bcl-2 protein family members or saline (control). Bcl-2 mediators were induced by treating cultured cells such as THP-

1 monocytic cells with A l for 4 hours. Cells were then washed 3 times with serum-free culture media and then cultured overnight. Culture media was then collected, concentrated and buffer exchanged using a centrifuge spin column filter with a 100 D molecular exclusion. In some experiments exosome proteins were identified by precipitating exosomes from the culture media or concentrated buffer-exchanged media using Exoquick or PEG 8000. Samples were then precipitated using the ProteoExtract Protein Precipitation Kit following the protocol described by the manufacturer (CalBiochem; http://www.emdmillipore.com/life-science- research/proteoextract-protein-precipitation-kit/EMD BIO-

5391 80/p_xdCb.s 1 ORVM AAAEjpxp9.zLX).

[00223] The resulting protein pellet was solubilized in 1 ΟΟ ί of 6M urea in 50mM ammonium bicarbonate (AMBIC). 200mM of dithiothreitol (DTT) was added to a final concentration of 5mM and samples were incubated for 30min at 37°C. Next. 20mM iodoacetamide (IAA) was added to a final concentration of 15mM and incubated for 30min at room temperature, followed by the addition of 20 \iL DTT to quench the IAA reaction. Lys-C/trypsin (Promega) was next added in a 1 :25 ratio (enzyme:protein) and incubated at 37°C for four hours. Samples were then diluted to <1 M urea by the addition of 50 mM AMBIC and digested overnight at 37°C. The following day, samples were desalted using C I 8 Macro Spin columns (Nest Group) and dried down by vacuum centrifugation.

Proteins Induced by Bcl-2 Treatment of Cultured Cells

[00224] A subset of the proteins identified to be increased by CTA 1 treatment relative to saline treatment of HS-5 MSC are presented in Table 3. These proteins, which were identified by LC- MS MS in buffer exchanged (100 kDa filter) THP-1 culture media, are potential markers for Al activity in vitro and ex vivo.

Table 3

Proteins Selectively Induced by CTA1 Treatment of THP-1 Monocytic Cells

Example 1 1

Acute 24 Hour CCl4-induced Liver Injury Study

[00225] This Example discloses a short term in vivo assessment of CTA 1 activity for facilitating CTA 1 development, which is achieved by assaying liver enzymes as a measure of tissue damage/apoptosis, inflammatory mediators, exosome mediators/PD markers, and comparing the activity of different lots of CTA 1 or engineered variants (e.g., CTA 1 .2 and CTA 140). See, FIGs. 26-28.

Example 12

CTA J as a Treatment for Sepsis

[00226] This Example discloses the rationale and advantages of employing CTA 1 as a treatment for sepsis.

[00227] CTA 1 is efficacious in a severe/lethal model (CLP) of sepsis, decreasing apoptosis in multiple organs within 24 hours of administration and significantly increasing survival. CTA 1 is also efficacious in models of ischemic injury and acute chemical injury (CC14), which have in common with sepsis acute apoptotic tissue injury and release of DAMPs that trigger a cytokine release syndrome. CTA 1 provides significant tissue protection in ischemic injury models when administered post-injury. Data from sepsis, other animal disease models and mechanism studies indicates that CTA 1 may target multiple components of sepsis pathogenesis; apoptosis and inflammation (e.g., innate immune response, iNOS). Previous sepsis trials predominantly targeted a single inflammatory mediator.

Example 13

CTA1 Treatment Reduces PAI-1 Expression in a Murine CNV Model

[00228] FIGs. 29A and 29B present immunohistochemistry data for PAI- 1 on 6 μηι cryo- sectioned retina (laser burn area) obtained from a laser induced retinal choroidal neovascularization mouse model. Slides were incubated with primary antibodies specific for PAI- 1 and, before examination, the nuclei were counterstained with DAP1 using UltraCruz Mounting Medium sc-24941 . CTA1 treatment appears to reduce PAI- 1 expression relative to control in a particular vesicle-associated PAI- 1 (arrow in FIG. 29A). These images were taken at x40 magnification with an exposure time of 2000 ms.

Example 14

CTA 1 Treatment Reduces VEGF in a Choroidal Neovascularization Model

[00229] FIGs. 30A-30C present immunohistochemistry data for VEGF on 6 μηι cryo-sectioned retina (laser burn area) obtained from a laser induced choroidal neovascularization mouse model. VEGF is highly expressed at the area of laser burn in the PBS treated control (FIG. 30A). CTA 1 treated groups showed relatively minimal VEGF expression compared to the PBS and VEGF antibody treated groups (compare FIGs. 30A-30C). These images were taken at x20 magnification with an exposure time of 1000 ms.

Example 15

CTAl Treatment Reduces PDGF Expression in the CNV Model Relative to

Control and Anti-VEGF Treatment

[00230] FIGs. 31A-31C present immunohistochemistry data for PDGF on 6 μιη cryo-sectioned retina (laser burn area) obtained from a laser induced choroidal neovascularization mouse model.

PDGF is highly expressed in the VEGF antibody treated group whereas other groups demonstrated lower levels of PDGF expression (compare FIGs. 31B and 31C). These images were taken at x40 magnification with an exposure time of 1500 ms.

Example 16

CTAl Treatment Reduces Collagen Accumulation and Decreases mRNA Expression of Fibrosis

Mediators in a CCL4-Induced Liver Injury Model

[00231] FIGs. 32-35 present a therapeutic dosing study design for demonstrating CTA-1 efficacy in treating fibrosis in a liver injury model and related data. Hepatic injury was induced by administration of CCL4, a known hepatotoxin. Administration of CTA l (2 doses) showed decreased levels of fibrosis modulator alpha-SMA and collagen formation (FIG. 33). Hepatic mRNA expression levels of four known fibrosis components (TGB-β, alpha-SMA, Col lal , PAI-

1 ) were compared for control, CCL4 mice, and CCL4 + (l x or 2x) CTA l mice. CTA l treatment showed statistically significant decreases in expression of TGFp, Col l al , and PAI- 1 , with a decreasing trend in alpha-SMA expression (FIGs. 34-35). This study indicates that therapeutic dosing of CTA l is efficacious in the CCL4 liver fibrosis model. A second CTA l dose improves efficacy.

[00232] These data, as well as data from other disease models, demonstrate that CTA 1 treatment decreases fibrosis in heart failure, age-related macular degeneration (CNV), chronic liver disease (CCL4-induced) and glaucoma. These observations are consistent with tissue-independent mechanism(s) underlying fibrogenesis. Collectively, the data demonstrates anti-fibrotic activity of CTA 1 and strongly suggests that all members of this Bcl-2 peptide family will demonstrate anti- fibrotic activity in a variety of tissues. [00233] These data also suggest that CTA 1 may possess anti-proliferative properties. Liver histology of CTA 1 -treated mice shows spaces or "holes" in the tissue that may be attributed to decreased cell proliferation. This observation, in conjunction with the inhibition of PAI- 1 mR A expression, suggests an application for CTA 1 or Bcl-2 protein or a Bcl-2 protein-domain peptide, such as BH4, in the treatment of cancer. Thus, in another aspect of the present disclosure, Bcl-2, or a Bcl-2 protein-domain peptide, such as BH4, or a mimetic thereof, may be used for the treatment of cancer.

[00234] In this regard, PAI-1 has been demonstrated in numerous clinical and animal studies to be a marker of poor prognosis in many different cancer types, including, but not limited to: breast cancer, ovarian cancer, endometrial cancer, cervical cancer, prostate cancer, oral cancer, brain cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, laryngeal cancer, lung cancer, thyroid cancer, renal cancer, bladder cancer, leukemia, and melanoma. (Mekkawy (2014)).

[00235] Mechanistic studies have characterized multiple PAI- 1 pro-cancer activities. (Delias, (2005), Andreasen (2005), Bajou (2004), Zhu (2014), Bajou (2001), Bajou (2008), Paugh (2008), Samarakoon (2009), Wilkins-Port (2007), Hjortland (2007), Bourcier (2000), , Wilkins (2007), Romer (2008), Ramer (2010), Lupu-Meiri (2012), Illemann (2004), Andreasen (2007), McMillin (2013), and Deryugina (2012)), and it has been demonstrated that host cell PAI- 1 production is critical to tumor growth. In accordance, it has been shown that PAI-1 inhibition decreases tumor growth through multiple mechanisms in multiple animal cancer studies, including studies of lung and gastric cancers. (Masuda (2013), Maillard (2008), Bajou ( 1998), Tsuchiya (1997), Bajou (2001 ), Mutoh (2008), Leik (2006), Zhu (2014), Fang (2012), Yan (2014), Yu (2014), Brooks (2000), Bryan (2008), and Bajou (2004)).

[00236] As used throughout this Specification and in the accompanying claims, the term "treatment" refers to the lessening, amelioration, or elimination of one or more cancer symptoms, a decrease, or a slowed increase, in the presence of one or more biomarkers correlated with the presence or progression of a cancer, an increase or improvement in the presence of one or more biomarkers correlated with the absence or decline of a cancer, slowing the progression or spread of a cancer, decreasing the size and/or number of tumors associated with a cancer, preventing the onset of a cancer, such as by prophylactic introduction, or improving the overall health or sense of well-being of mammal diagnosed with or believed to be at risk for a cancer.

Example 17

Bcl-2 Induction of Microparticles

[00237] This Example demonstrates that Bcl-2 proteins are effective in inducing the formation og microparticles. Treatment of cultured cells with a Bcl-2 protein induces the production of microparticles as determined by electron microscopy, increase levels of culture media protein and

RNA and by induced microparticle dependent therapeutic activity.

[00238] Cultured cells, including human monocytic cells (THP-1 ), mouse mesencyhmal stem cells (MSC) and mouse dendritic cells (Jaws II), may be treated with a Bcl-2 protein such as rhA l in a concentration sufficient to induce microparticles such as 300 nanograms/mi. The Bel protein may be transient with removal of unbound Bcl-2 protein achieved by three large volume washes after four hours of treatment. Twenty-four hours following treatment an increase in microparticles and microparticle dependent activity relative to controls treated with saline or a Bcl-2 protein lacking a BH4 domain (e.g., Bim), can be demonstrated in the culture media of treated cells.

[00239] Results of these experiments are shown in FIG. 37. The culture media treated with rhA l contains an increase in microparticles with a diameter of 40 to 100 nm relative to negative control culture media as determined by transmission electron microscopy. Microparticles around 40 to 100 nm are expected to be exosomes. The culture media of rhA l treated cells following buffer exchange with a 100 or 300 molecular weight filter to remove moderate to small molecules, contain an increase of protein and RNA, relative to control media, as determined by optical density. Microparticles are composed of proteins, miRNA and mRNA that are transferred to various cell types (e.g., Camussi, Kidney International 78(9): 838-848 (2010) and Record, Biochemical Pharmacology 8J.: 1 171 - 1 182 (201 1 )). The injection of rhA 1 or the BH4 domain of rhA 1 has been previously shown to possess cytoprotective activity in rodent models of ischemic injury and sepsis (Iwata, PIOS 2010 and Iwata PIOS 201 1 ).

[00240] The culture media of rhA l treated cells contain microparticle dependent cytoprotective activity when administered to rodents subjected to ischemic injury. Microparticle dependent cytoprotective activity can be removed by filtration using filters with a 20nm size pore or particle size cut off. Microparticle dependent cytoprotective activity can be recovered in the pellet of ultracentrifuged media under standard conditions that pellet microparticles (e.g., Wang, Nucleic Acids Res. 38£20);7248-59 (2010). Thus treatment of mammalian cells with a Bel protein can induce the generation of microparticles as determined by several methods.

[00241] Data presented in FIG. 37 also show that cultured cells treated with the amino terminal region of a Bcl-2 protein, containing the BH4 domain, is sufficient to induce cytoprotective activity in a rodent model of ischemic injury. Thus treatment of mammalian cells with the BH4 domain of a Bcl-2 protein may be sufficient to induce microparticle generation.

Example 18

Bcl-2-induced Nucleic Acid Transfer or Delivery

[00242] This Example describes the Bcl-2 protein-induced, microparticle-dependent increase in nucleic acid transfer and/or delivery.

[00243] A fluorescently labeled marker miRNA (FAM-labeled pre-miR; Ambion Inc) was introduced into human monocytic cells (THP-I) using lipofectamine 2000 (Invitrogen Inc). At 2 hours post-transfection THPI were treated with rhAl (300ng/ml) or saline. At 6 hours post- transfection THP-I were subjected to 3 large volume washes and the culture continued. At 12 hours post-transfection microparticles were prepared as described in FIG. 37 and one-third transferred to a culture of HE cells for 10 hours of exposure. Prior to flow cytofluorimetry HEK cells were trypsinized to suspend cells and remove exosomes that may have not internalized. The percent specific fluorescence is indicated in the upper right quadrant of each scatter plot. rhAI treatment resulted in an approximate 3-fold increase in miRNA transfer relative to the saline control following mock background subtract. In all studies the results from negative control protein or salinetreatments appear identical. Thus treatment with rhAI can be used to increase microparticle mediated transfer/delivery of a nucleic acid such as a miRNA, from one cell type to another. Example 19

Methods for Determining the Effect of Bcl-2 Adminsitration on Mortality

[PROPHETIC]

[00244] Materials. A solution of A l drug may be used without further modification or diluted with saline solution to the desirable concentration. The A l dosing solution may be prepared so that a single administered dose will contain 1 ug of drug per animal.

[00245] Animals. Male Swiss- Webster mice may be obtained from Charles River Breeding Laboratory (Raleigh, NC). Animals may be provided food and water ad libitum during acclimation. The light cycle will be 12 hour light/12 hour dark and relative humidity and temperature maintained at 50+ IS% and 22 + 2°C. All animal use protocols will be approved by the Institutional Animal Care and Use Committee at Battelle, Pacific Northwest Division and studies will be performed according to the "Guide for the Care and Use of Laboratory Animals" (National Research Council, Washington DC, 1996). The animal facility at Battelle, Pacific Northwest Division is AAALAC accredited.

[00246] Dosing regimen. Male Swiss- Webster mice (total 90 mice) will be randomly assigned to the following groups: radiation control group (radiation only), pre-treatment group (1 ug Al per animal+ radiation in I h), and post-treatment group (radiation+ 1 ug A l per animal in 1 h). For this 30 day mortality study, each group will have n=20. All animals will be acclimated for at least 5 days in our AAALAC-accredited facility prior to whole body irradiation at 7 Gy dose level using Therapax X-RAD 320 system equipped with 320 kV high stability X-ray generator, metal ceramic X-ray tube, variable X-ray beam collimator and #8 filter (Precision X-ray Incorporated, East Haven, CT). Radiation dose of 7 Gy was previously estimated to be LD50/30 for this mouse model under the experimental conditions of our laboratory.

[00247] Animals in the treatment groups will receive an IP injection of the radioprotectant material 1 h prior to irradiation (pre-treatment group) or 1 h post irradiation (posttreatment group). Body weight will be measured daily. Any animals remaining at 30 days post X-ray exposure would be sacrificed, and selected tissues will be collected, weighed, and formalin fixed for the possible histopathology examination. Thus, at the end of the study information on the following end points will be gathered: 30 day Mortality; Mortality profile (% survival versus time); Daily body weight data; and 30 day post exposure tissue to body weight ratios. Example 20

In vitro Production of Exosomes with CAR Vector and T-cell Ligand

[PROPHETIC]

[00248] The ability of exosomes to function as favorable delivery vehicles permits a range of cell- and/or gene- therapy mechanisms. As showin in FIG. 40, a cell line may be engineered to produce exosomes comprising a unique vector.

[00249] The vector may comprise a transposable nucleic acid sequence of interest under the control of a cell specific promoter. In this example, the nucleic acid sequence encodes for a chimeric antigen receptor (CAR) and the promoter sequence is specific to T cells. In vitro application of a Bcl-2 protein, peptide, or mimetic to said engineered cells enhances exosome stabilization and/or production. The cell line may also be engineered to express target-cell-specific ligands on plasma membrane. In this Example, T-cell ligands (anti- T-cell specific single chain variable fragments such as CD2, CD3, CD4, or CD8) are incorporated into the exosome plasma membrane from the cell upon exosome release.

[00250] The isolated exosomes may be purified by standard means such as PEG. The exosomes possess low immunogenicity. To create cancer-therapeutic CAR T-cells, the exosomes are administered intravenously into a patient (in vivo), where the T-cell ligand preferentially binds T- cells. The exosomes may be fused/incorporated into the T-cell by receptor-dependent and/or independent means. Once fused, the T-cell specific transposase integrates the CAR and T-cell promoter construct into the T-cell DNA. Proliferation of transformed T-cells is stimulated by introduction of tumor-cell specific ligands recognized by the CAR. CAR T-cells can then target and kill tumor cells.

Example 23

[PROPHETIC]

[00251] The methods described in the preceding Example may be employed for the treatment of a variety of diseases. The core concepts are: engineereing a cell line to produce exosomes carrying a therapeutic vector or desired therapeutic nucleic acid; stimulating production and/or stabilization of said exosomes by administering to the cells a Bcl-2 protein, domain-containing peptide, or mimietic; and administering cells and/or exosomes from said cell line to a patient in need thereof. In one aspect, the exosomes may include a vector comprising a therapeutic nucleic acid sequence under the control of a transposable element and a target-cell-specific promoter. Further, the engineered cell line may overexpress a target cell ligand to be incorporated by the exosomes upon release and to thereby facilitate exosome binding and uptake into said target cells. Transformation of the target cells permits antigen-recognizing host cells with augmented immune- response capabilities.

[00252] Alternatively or in conjunction, the cell line may be engineered to produce exosomes comprising a therapeutic nucleic acid, such as a cDNA, a DNA, an mRNA, an miR A, an shRNA, or a functionally equivalent nucleic acid sequence. The therapeutic nucleic acid may be used, for example, to facilitate gene silencing, to preferentially regulate expression of one or more genes, or to restore gene function to an optimal or improved level in a loss-of-function scenario.

* * * * *

[00253] While certain embodiments of the present disclosure have been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the subject matter disclosed and claimed. All scientific literature, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

CLAIMS What is claimed is:

1 . A method for the treatment of a VDAC-associated disease in a mammal, said method comprising administering to said mammal a Bel protein or peptide in an amount sufficient to reduce the severity of said VDAC-associated disease selected from the group consisting of a retinal disease, a liver disease, a kidney disease, a lung disease, a skin disease, a fibrotic disease.

2. A method for treating a disease or injury in a mammal, the method comprising the step of administering to a mammal exosomes released from mammalian cells that have been treated with a Bel protein ex vivo, in an amount sufficient to inhibit a disease or inhibit tissue damage due to an injury and/or facilitate repair.

3. The method of Claims 1 -2, wherein the Bcl-2 protein or exosome is administered intravenously, subcutaneously, orally, transdermally, or intravitreally.

4. The method of Claims 1 -2, wherein the Bcl-2 protein is selected from the group consisting of:

(a) a protein comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 169;

(b) a protein comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 170;

(c) a protein comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 171 ;

(d) a protein comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 1 72;

(e) a protein comprising at least 4 amino acids, wherein the protein is at least 75% similar to a segment of Bcl-2protein, wherein the Bcl-2 protein consists of the amino acid sequence set forth in SEQ ID NO: 173;

(f) a protein comprising at least 4 amino acids, wherein the protein is at least 75% similar to a segment of Bcl-2 protein, wherein the Bcl-2 protein consists of the amino acid sequence set forth in SEQ ID NO: 174; (g) a protein comprising at least 4 amino acids, wherein the protein is at least 75% similar to a segment of Bcl-2 protein, wherein the Bcl-2 protein consists of the amino acid sequence set forth in SEQ ID NO: 175;

(h) a protein comprising at least 4 amino acids, wherein the protein is at least 75% similar to a segment of Bcl-2 protein, wherein the Bcl-2 protein consists of the amino acid sequence set forth in SEQ ID NO: 176;

5. A method for identifying a Bcl-2 protein, peptide or modified peptide that inhibits said VDAC-associated disease when administered to a mammal, the method comprising the step of screening a plurality of proteins to identify a Bcl-2 protein, peptide, or modified peptide that induces a change in the quantity and/or type of microparticles/exosomes released from mammalian cells.

6. A method for identifying a Bcl-2 protein that inhibits a VDAC-associated disease when administered to a mammal, the method comprising analyzing data obtained from at least one experiment wherein a plurality of proteins are screened to identify a Bcl-2protein, peptide or modified peptide that inhibits a VDAC-associated disease in a mammal.

7. A method for decreasing PAI-1 expression in a mammal, the method comprising administering to a mammal a Bcl-2 protein in an amount sufficient to decrease expression of PAI-1 .

8. The method of Claim 7 wherein said mammal has been diagnosed with a cancer or a neurodegenerative disease.

9. The method of Claim 8 wherein said neurodegenerative disease is Alzheimer's Disease.

10. A method for treating cancer in a mammal, comprising administering to said mammal a Bcl-2 protein or an exosome induced by ex vivo administration of a Bcl-2 protein, peptide or mimetic.

1 1 . A composition comprising a Bcl-2 protein and an immunoglobulin or immunoglobulin fragment.

12. The composition of claim 1 1 wherein said Bcl-2 polypeptide comprises a BH4 domain.

13. A method for delivering a nucleic acid molecule to a subject comprising administering to said subject a Bcl-2 protein or a cell treated with a Bcl-2 protein in an amount sufficient to induce microparticle-mediated nucleic acid delivery.

14. The method of Claim 13, wherein said nucleic acid is a DNA, RNA , mRNA, miRNA, siR A, or an shRNA.

15. The method of Claim 14 wherein said Bcl-2 protein is conjugated to said nucleic acid.

16. The method of Claim 15 wherein said nucleic acid is delivered by a delivery vehicle selected from the group comprising an attenuated virus, a cationic liposome, a protein, a carbohydrate, a cholesterol and a lipid.

17. The method of Claim 16 wherein said protein is a cell penetrating peptide.

18. The method of Claim 17 wherein said attenuated virus is a lentivirus.

19. The method of Claim 13 wherein said cell is a mesenchymal stem cell.

20. The method of Claim 13 wherein said subject is a mammal and wherein said cell is an autologous cell.

21 . The method of Claim 13, further comprising the administration of one or more ultrasound waves to a tissue of said subject to which said delivery of said nucleic acid is desired.

22. A method for enabling and/or improving a stem cell therapy in a subject, comprising administering a Bcl-2 protein or a cell treated with a Bcl-2 protein in an amount sufficient to enable or improve said stem cell therapy to said subject, prior to, concurrently with or subsequent to said subject undergoing a stem cell therapy.

23. The method of Claim 22 wherein said subject is a mammal and wherein said cell is a mammalian mesenchymal stem cell.

24. A method for enabling or improving a vaccine therapy in a subject, comprising administering a Bcl-2 protein or a cell treated with a Bcl-2 protein in an amount sufficient to enable or improve an immunological response to an immunogen or a tolerogen.

25. The method of claim 24 wherein said subject is a mammal and wherein said cell is a mammalian cell.

26. The method of Claim 13 or 24 wherein said Bcl-2 protein is selected from the group consisting of:

(a) a protein comprising an amino acid sequence that is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 1 ;

(b) a protein comprising at least 12 amino acids, wherein the protein is at least 50% similar to a segment of a Bcl-2 protein, wherein the Bcl-2 protein consists of the amino acid sequence set forth in SEQ ID NO: 50;

(c) a protein comprising at least 12 amino acids, wherein the protein is at least 50% similar to a segment of an AI protein, wherein the AI protein consists of the amino acid sequence set forth in SEQ ID NO: 98;

(d) a protein comprising at least 12 amino acids, wherein the protein is at least 50% similar to a segment of a Bcl-X protein, wherein the Bcl-X protein consists of the amino acid sequence set forth in SEQ ID NO: 175;

(e) a protein comprising at least 12 amino acids, wherein the protein is at least 50% similar to a segment of a Bcl-W protein, wherein the Bcl-W protein consists of the amino acid sequence set forth in SEQ ID NO: 79;

(f) a protein comprising at least 12 amino acids, wherein the protein is at least 50% similar to a segment of an Mcl-1 protein, wherein the Mcl-I protein consists of the amino acid sequence set forth in SEQ ID NO: 147; and

(g) a protein that is at least 50% similar to a BH4 domain consisting of the amino acid sequence set forth in SEQ ID NO: 1 78.

27. The method of Claim 26 wherein said Bcl-2 protein is administered to said mammal mammalian subject in an amount from 0.5 μg/kg/day to 50 μ^£>Λ_^.

28. A method for identifying a Bcl-2 protein for delivering a nucleic acid to a mammal, the method comprising screening a plurality of proteins to identify a Bcl-2protein that enables or improves delivery of a nucleic acid when administered to said mammal.

29. A method for identifying a Bcl-2 protein for delivering a nucleic acid to a mammal, the method comprising screening a plurality of proteins to identify a Bcl-2 protein that enables or improves delivery of a nucleic acid when administered to a mammal.

30. A method for identifying a Bcl-2protein for modifying a therapeutic activity of a stem cell when administered to a mammal in need of or undergoing a stem cell therapy, the method comprising screening a plurality of proteins to identify a Bcl-2 protein that increases therapeutic mesenteric stem cell activity when administered to said mammal.

31. A method for identifying a Bcl-2 protein for regulating an immune response of a mammal, the method comprising screening a plurality of proteins to identify a Bel protein that, when administered to said mammal, increases or suppresses an immune response of said mammal to an immunogen.

32. A method for producing a microparticle for a cell therapy, comprising: administering a Bcl-2 protein, domain-containing peptide or mimetic ex vivo to a cell that has been engineered to produce exosomes or microparticles containing a vector comprising a promoter sequence with an activity specific to a target cell type, a nucleic acid sequence, and a transposase sequence.

33. A method for producing a cell for the treatment of a disease, comprising administering an exosome or a microparticle produced by the method of claims 61 or 62 to one of said target cells in vitro under conditions sufficient to facilitate fusion and/or uptake of the exosome or microparticle by the one or more target cells.

34. A gene therapy method, comprising administering a target cell produced by the method of Claim 33 to a mammal in vivo under conditions sufficient to facilitate proliferation of said target cell and/or nucleic acid transfer between said cell and a cell of said mammal.

35. The method of Claim 34 wherein the activity of said promoter sequence is specific to a T-cell, wherein said nucleic acid sequence encodes a chimeric antigen receptor (CAR) sequence, and wherein said ligand is a T-cell ligand.

36. The method of Claim 35 wherein said T-cell ligand is selected from the group consisting of an anti-CD2 single-fragment variable chain, an anti-CD3 single-fragment variable chain, an anti-CD4 single-fragment variable chain, or an anti-CD22 single- fragment variable chain.

37. A composition comprising an exosome or a microparticle produced by the method of claim 66, wherein a plasma membrane of said exosome or microparticle contains a T-cell ligand selected from the group consisting of an anti-CD2 single-fragment variable chain, an anti-CD3 single-fragment variable chain, an anti-CD4 single-fragment variable chain, or an anti-CD22 single-fragment variable chain.

38. A method of producing a CAR T-cell, comprising administering an exosome or a microparticle produced by the method of Claim 34 to a T-cell in vitro under conditions sufficient to facilitate fusion and/or uptake of the exosome or microparticle by said T-cell [what about transposition of the CAR element.

39. A method of treating a cancer in a mammal, comprising administering a CAR T-cell produced by the method of Claim 38 in vivo to a mammal in need thereof.

40. A method of treating a disease in a mammal, comprising administering a cell produced by the method of Claim 38 to a mammal in need thereof.

41 . The method of claim 40 wherein said nucleic acid sequence encodes a gene or a functional equivalent thereof.

42. The method of Claim 41 wherein said disease is associated with a loss-of- function of a gene.

43. A composition comprising a cell that has been engineered to produce exosomes or microparticles containing a vector comprising a promoter sequence with an activity specific to a target cell type, a nucleic acid sequence, and a transposase sequence.

44. A method for preventing or mitigating a cytotoxic shock associated with a disease therapy, comprising administering a Bcl-2 protein, domain-containing fragment or mimetic, or a Bcl-2-induced exosome or microparticle to a subject prior to, contemporaneously with, or immediately following application of said disease therapy to said subject.