CN118591385A - Methods and uses of BAG3 for treating inflammation - Google Patents

Abstract

Bag3 is a multifunctional protein that is expressed primarily in the heart, skeletal muscle, central nervous system and many cancers. Although BAG3 was cloned ten years ago, studies have shown that genetic variation, especially genetic variation that leads to haploid deficiency, can lead to severe left ventricular dysfunction; however, its complete mechanism is still unclear. To exclude the effect of heart failure itself on Bag3 biology, transgenic mice carrying a single allele knockout were studied at 8 to 10 weeks of age before signs of heart failure are apparent. The results of the study are surprising and instructive. First, it was found that the proteome of young Bag3 ^+/‑ mice was significantly altered despite normal phenotypes, which is characterized by altered proteins associated with metabolism and apoptosis. Consistent with this finding, a decrease in the level of key proteins responsible for maintaining mitochondrial membrane potential was also observed. Studies have also found that the balance between the extrinsic and intrinsic pathways of young mice apoptosis is altered. However, in the presence of pressure and in the absence of Bag3, the transition from equilibrium to the dominant system of the exon type (cleavage of Caspase 8) occurs. The variety of key pathways regulated by Bag3 is varied, suggesting that Bag3 is important for its role, especially during stress, and that this role may include acting as an intracellular adhesive, keeping proteins in the most efficient place, rather than letting them meet accidentally.

Description

Methods and uses of BAG3 for treating inflammation

Government support

The invention is completed with government support, and the dialing number granted by the national institutes of health is: HL091799 and HL123093. The government has certain rights in this invention.

Background

BAG3 is a universally expressed multifunctional protein, but is most prominent in heart, skeletal muscle, central nervous system and many cancers (1, 2). Multiple whole genome related studies and whole genome sequencing of the DNA of patients with hereditary and sporadic Dilated Cardiomyopathy (DCM) showed that the deletion of single allele BAG3 is a significant cause of the disease (3-6). This has been demonstrated in animal models, as homologous chromosomal deletions of BAG3 are lethal early after birth (7), whereas functional deletions of a single allele lead to zebra fish cardiomyocyte dysfunction (3), human mutant carrying mesenchymal stem cell derived cardiomyocyte dysfunction (8), mouse model point mutations and haploid deletions (9, 10), and human haploid deletions (11, 12). Studies have also shown that patients with normal genotypes with reduced heart failure and ejection fraction (HFrEF) when receiving heart transplants, the reduction in BAG3 in the ventricular myocardium is comparable to that of patients with BAG3 truncations (11, 12). Unlike the heart, overexpression of BAG3 in cancer cells leads to chemotherapy resistance and increases the propensity for metastasis and local invasion (13, 14). Although the importance of BAG3 in two major diseases in industrialized countries, heart disease and cancer, is apparent, its role in health and disease has not been fully defined.

BAG3 has a structure with many proteins and protein binding domains that enable it to influence various molecules and cellular activities. In the heart BAG3 enhances autophagy by acting as a chaperone for heat shock protein 70 (hsp/hsc 70) (15). It inhibits apoptosis by interacting with the anti-apoptotic protein Bcl-2 (16), improves excitation-contraction coupling by linking β -adrenergic receptor (b-AR) and L-type Ca ²⁺ channels (17), and maintains sarcomere integrity (18). Different functions of BAG3 benefit from multiple binding sites and a set of different binding partners. For example, the pxp domain may serve as a molecular anchor proximal to the motor-generator transport system, while two isoleucine-proline-valine (IPV) motifs may bind to the small heat shock proteins HspB6 and HspB8 and support large autophagy (1).

Recent animal model studies began to link specific heterozygous genetic variations of BAG3 to unique cellular or molecular phenotypes. For example, E455K loss-of-function mutations disrupt the interaction between BAG3 and Hsp-70, resulting in destabilization of heat shock proteins and imbalance in protein homeostasis (9). The rare P209L mutation is a dominant gain-of-function mutation that causes self-aggregation with Hsp70 individuals, resulting in arrest of the Hsp70 autophagy network, leading to restrictive cardiomyopathy (19). In contrast, P209S variation in BAG3 has been reported to be associated with late axonal fibular muscular dystrophy lesions in two patients (20). However, it is still unclear since the effect of BAG3 ^P209L mutations on the heart phenotype of mice is inconsistent (21) (22).

Disclosure of Invention

In order to better understand the cardiac biological properties of BAG3 haploid deletions, high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques were used to identify differentially expressed proteins in 8-10 week old heterozygous BAG3 knockout mice, as described herein: at this point the Left Ventricle (LV) function and heart size of the mice remained normal. Heterozygote BAG3 knockout mice were normative to the echocardiography at 8-10 weeks of age, but had significantly altered left ventricular function by 18 weeks of age.

Proteomic analysis reveals two areas of cardiac biology: mitochondrial function and apoptosis or apoptosis. Evaluation of the 8 to 10 week old BAG3 ^+/ -mouse proteome showed that, despite normal phenotypes, proteins associated with metabolism and programmed cell death or apoptosis were abnormal.

As disclosed herein, among other things, BCL 2-related immortalized gene 3 (BAG 3) has been found to be a key component of the intrinsic and extrinsic pathways of apoptosis in heart and other tissues and cell types. In particular, BAG3 regulates apoptosis via canonical and non-canonical pathways.

The application also discloses in particular that BAG3 has been found to alter the late steps of apoptosis, i.e.activate caspase 3.BAG3 interacts directly with apoptosis inhibitor protein 1/2 (cIAP 1/2 or cIAP). Under normal conditions, BAG3 protein levels are normal and bind to apoptosis inhibitor 1 (CIAP-1), promoting binding of CIAP1 to caspase 3 and inhibiting caspase 3 activation. However, in the case of a BAG3 haploid loss, a significant abnormality in mitochondrial function may occur, including a drop in membrane potential, resulting in leakage of the second Caspase activator SMAC (also known as direct IAP binding protein (DIABLO)) from the mitochondria. SMAC then migrates to the cytoplasm and binds to CIAP-1, releasing it from Caspase 3, allowing Caspase 3 to be activated. Activated caspase 3 can lyse key components of cells. Thus, BAG3 may be used or formulated to reduce, inhibit or decrease caspase-3 activation, thereby reducing inflammation or inflammatory response.

As further disclosed in the present application, it has now been found that a decrease in BAG3 levels results in a shift in the balance between the intrinsic and extrinsic pathways of caspase activation to a signal favoring caspase-8 (cleaved caspase 8) activation. Thus, BAG3 may be used or formulated to reduce, inhibit or decrease caspase-8 activation.

The present application discloses, among other things, that BAG3 has been found to interact directly with the mitochondrial import receptor subunits TOM22, ca ²⁺, the one-way transporter, mitochondrial metabolism and the production of mitochondrial membrane potential, respectively.

The present application also discloses, among other things, that a decrease in BAG3 has been found to result in an increase in poly (ADP-ribose) polymerase 1 (PARP 1). An increase in PARP1 leads to an increase in alpha-synuclein and exacerbation of parkinson's disease. Thus, BAG3 may be used or formulated to reduce, inhibit or decrease PARP1.

According to the invention BAG3 may be used or formulated as a medicament for modulating TNF signalling. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit, or reduce TNF signaling.

According to the invention BAG3 may be used or formulated as a medicament for modulating inflammation. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit, reduce, or treat inflammation.

According to the invention BAG3 may be used or formulated as a medicament for modulating inflammatory reactions. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit, reduce, or treat an inflammatory response.

The inflammation or inflammatory response may be systemic, regional or local, for example in an organ or tissue. In particular aspects, BAG3 may be used or formulated to reduce, inhibit, reduce or treat inflammatory or inflammatory reactions, non-limiting examples of which occur in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joints, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney and pancreas.

In particular aspects, non-limiting examples of inflammatory conditions or inflammatory reactions that BAG3 may be used or formulated to reduce, inhibit, reduce or treat include chronic inflammatory diseases, chronic inflammatory demyelinating polyneuropathy, primary immune thrombocytopenia, anorexia senile, intestinal inflammation, inflammatory bowel disease, ulcerative colitis, crohn's disease, lupus, rheumatoid arthritis, chronic myocarditis after Covid infection, psoriasis, psoriatic arthritis, and ankylosing spondylitis.

According to the present invention BAG3 may be used or formulated as a medicament for modulating PARP1 levels, expression or activity. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit or reduce the level, expression or activity of PARP 1. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit, reduce, or stabilize the amount of α -synuclein. In particular embodiments, BAG3 may be used or formulated to reduce, inhibit, reduce, or alleviate the exacerbation or severity of one or more symptoms of parkinson's disease.

In a specific embodiment, the BAG3 encoding nucleic acid comprises an expression vector expressing a BAG3 protein or an active BAG3 peptide thereof.

In particular embodiments, the expression vector comprises a promoter, including an inducible promoter, a constitutive promoter, a bicistronic promoter, a tissue-specific promoter, or a heart-specific promoter.

In particular embodiments, the expression vector comprises a viral vector, a heart-specific vector, a plasmid, or a yeast vector.

In particular embodiments, the viral vector or heart-specific vector comprises an adenovirus vector, an adeno-associated virus vector (AAV), a coxsackievirus vector, a cytomegalovirus vector, an epstein barr virus vector, a paravirus vector, or a hepatitis virus vector.

In particular embodiments, the AAV vector comprises a capsid protein having 90% or more sequence identity to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7mAAV, AAV9, AAV10, AAV11, or AAV 12.

In a particular embodiment, the expression vector is a pseudoviral vector.

In certain embodiments, the inflammation or inflammatory response is induced or increased by a cytokine.

In particular embodiments, the cytokine comprises Tumor Necrosis Factor (TNF).

In certain embodiments, the patient expresses a BAG3 level in a tissue or organ that is below normal or fails to detect expression or production of functional BAG3.

In particular embodiments, the inflammation or inflammatory response occurs in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney, or pancreas.

In particular embodiments, the expression vector further comprises a promoter, optionally including an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue specific promoter.

In particular embodiments, the promoter confers expression in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney or pancreas.

In particular embodiments, the expression vector further comprises an AAV Inverted Terminal Repeat (ITR).

In certain embodiments, the expression vector further comprises a polyadenylation sequence and/or a stop codon.

In particular embodiments, the patient or subject is a human.

In particular embodiments, the endogenous BAG3 polynucleotide or polypeptide of the patient, subject or human is mutated.

In particular embodiments, the expression or activity of an endogenous BAG3 polynucleotide or polypeptide is reduced in a patient, subject, or human.

In particular embodiments, the viral vector is administered or formulated at a dose of from about 0.1x10 ¹² vector genomes (vg)/patient weight (kg) (vg/kg) to about 1.0x10 ¹⁴ vg/kg.

In particular embodiments, the viral vector is administered or formulated at a dose of about 1.0x10 ¹² vg/kg to about 0.5x10 ¹⁴ vg/kg.

In particular embodiments, the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 1.0x10 ¹³ vg/kg.

In particular embodiments, the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 9.0x10 ¹² vg/kg.

In particular embodiments, the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 8.0x10 ¹² vg/kg.

In particular embodiments, the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 5.0x10 ¹² vg/kg.

Drawings

The data shown in FIGS. 1A-1B demonstrate that young mice with one allele of Bag3 deleted exhibit a unique proteome that emphasizes apoptosis and alterations in cellular metabolic pathways, despite the presence of normal left ventricular function and size. A) Scatter plots and bar charts show transthoracic echocardiography results for 8-10 week old BAG3 ^+/ -and BAG3 ^wt mice, including measurements of left ventricular Ejection Fraction (EF), diastolic left ventricular inner diameter (LVIDd), and systolic Left Ventricular Inner Diameter (LVIDs), all in millimeters (mm). B) In all BAG3 ^+/- groups BAG3 levels were reduced by about 50%.

The data shown in FIGS. 2A-2B indicate that cardiomyocyte-specific Bag3 gene Knockout (KO) disrupts the expression of mitochondrial proteins involved in cell metabolism and apoptosis. (A) Volcanic map analysis of all proteins in the left ventricle of wild-type and BAG3-KO mice from bottom-up mass spectrometry; the significance threshold is set to p=0.05; green indicates a decrease in expression level as compared with the wild type, and red indicates an increase in expression level; n=3wt, 3ko. B) The protein patterns of the BAG 3KO mice with altered expression are summarized and grouped by their major cellular compartments. C) BAG 3KO mice express altered proteins summarized in a graph of their biological functional groupings; biological function and cellular component information was obtained by the DAVID bioinformatics program (version 6.8).

Figures 3A-3E show that Bag3 ^+/- mice had more TUNEL positive cells under hypoxic/reoxygenation conditions, but lower mitochondrial membrane potential. A) And B) TUNEL staining. Adult mouse cardiomyocytes isolated from WT and BAG3 ^+/- mice were placed under hypoxic/reoxygenation (H/R) and normoxic (Norm) control conditions. Cells were stained with nonylacridine orange (NAO), TMR red and DAPI, and then imaged with Zeiss LSM 900 confocal microscopy. Statistical significance was analyzed using one-way anova and multiple sub-comparisons were corrected using Bonferroni. * And < 0.0001, "ns" indicates no obvious. n=10 images per group. Scale bar = 50 μm. C) There was no difference in Bcl-2 levels in the BAG3 ^+/- LV myocardium compared to the WT control group. D) Mitosox staining. Adult mouse cardiomyocytes isolated from WT and Bag3 ^-/- mice were stained with MitoSOX and imaged with Zeiss LSM 900 confocal microscopy. Mitosox fluorescence was quantified with FijiImage J and data plotted using GRAPHPAD PRISM software. The statistical significance is determined by t-test, and "ns" indicates no significance. n=75 to 182 cells per group. Scale bar = 20 μm. E) TMRM staining. Adult mouse cardiomyocytes isolated from WT and Bag3 ^-/- mice were stained with TMRM and imaged with Zeiss LSM 900 confocal microscopy. TMRM fluorescence was quantified using Fiji Image J and the data plotted in GRAPHPAD PRISM software. Mitochondrial content was quantified as a single mitochondrial area using Mito-Morphology mjciacro in Image J. Statistical significance was determined using t-test, with x representing < 0.001 and "ns" representing no significant. n=136 to 162 cells per group. Scale bar = 20 μm.

Figures 4A-4F show western blot analysis of proteins involved in mitochondrial dependent or mitochondrial independent apoptosis signaling in young and old Bag3 ^+/- and Bag3 ^WT mice. The data shown in the bar graph are from the accompanying western blot. The "n" of each study group was equal to 5, except for the samples obtained from the older (18 week) mice in fig. 3B, the sample size of each study group was 3. Each study was repeated at least once and samples were taken from the same mice or mice of the same age. A) The total caspase 3 level in BAG3 ^+/- mice was significantly higher than that in BAG3 ^WT mice (p < 0.01). B) The ratio of cleaved caspase 3 to pro caspase 3 from BAG3 ^WT mouse tissue was higher than in BAG3 ^+/- mice, also in 18 week old mice: although the LV EF was significantly reduced in the aged mice, LV distention was significant (see FIG. 1A). C) The TNFa levels were significantly elevated in BAG3 ^+/- mice, but the IL-6 levels were unchanged, indicating that the cytokine action was highly specific. D) In contrast to Caspase 3, there was a significant increase in the level of cleaved Caspase 8 divided by total Caspase 8 (p < 0.01), indicating that Caspase 8 was physiologically increased in mice without significant heart failure. E) A significant decrease in TOM22 levels was observed (p < 0.01), TOM22 being a member of the TOM (outer membrane translocator) family of proteins that carry amino acid, small peptide sequences and membrane segments across the mitochondrial membrane and subsequently incorporate into larger proteins produced in the mitochondrial matrix. F) Shown is an immunoprecipitation study that attempts to identify the partners for BAG3 in order to better understand the biological properties of BAG3 haploid defects. BAG3 binds to TOM22 and cIAP, but not to homologous XIAP or SMAC.

The data shown in FIGS. 5A-5C indicate that there is no difference in the expression of SMAC and cIAP in BAG3 haplotype deficient mice; SMAC, however, requires Bag3 for transport from the mitochondrial outer membrane (OMM) to the cytoplasm. A) Neonatal mouse ventricular cardiomyocytes (NMVM) were isolated and cultured under normoxic conditions, one hour hypoxic and two hours normoxic conditions, normoxic conditions but in the presence of SiRNA for BAG3, and normoxic experimental conditions of hypoxia followed by reoxygenation. TOM22 (mitochondria), MCU (mitochondria), and GAPDH (cytoplasm) were used as controls to demonstrate that mitochondrial isolation has been completed. BAG3 is expressed more in the cytoplasm of H/R stressed cardiomyocytes, but is not or hardly expressed in cells where one allele of BAG3 is subtracted. A) SMACs are present in both cytoplasm and mitochondria; however, SMAC is not apparent in the cytoplasm when BAG3 is subtracted with siRNA, and TOM22 is also present only in mitochondria. B) And C), the levels of both endo-enzyme G and cIAP1 are unchanged by the biological properties of BAG3 and its interactions with cells. SMAC is not seen in the cytoplasm of the cells during stress.

The data shown in fig. 6A-6E demonstrate that protein levels known to be altered in heart failure animal models and in failing human hearts are not alternately regulated early in the Bag3 gene-deleted heart failure model. A) To C) shows western blots for analysis of total content and phosphorylated form of JNK, JUN and ERK 1/2. Each blot represents data from one study, n=5. The data is shown in block diagram form and p values are listed as appropriate. Data are expressed as phosphorylated form of protein divided by the total amount of each protein. As can be seen from the block diagram attached to each western blot, there was no significant statistical difference in the levels of these proteins in BAG3 haplotype deleted mouse tissue compared to wild type mice. D) And E), in contrast, western blot analysis showed significantly higher levels of HuR and C PARP 1/GAPDH.

Figures 7A-7H show the effect of Bag3 haploid loss on mitochondrial membrane potential and Ca ²⁺ uptake in isolated cardiomyocytes and intact structures of Bag3 ^+/- and Bag3 ^+/+ mouse mitochondrial inner membranes and matrices (mitoplast). left ventricular cardiomyocytes were isolated from BAG3 ^+/- and WT mice, exposed to hypoxic conditions for 1 hour, and then re-inhaled for 2 hours as described in "methods". Cells were infiltrated with digoxin and succinic acid was supplemented. A) A ratio indicator JC-1 was added as indicated by the downward arrow to monitor the membrane potential (Δψ _m). At the second arrow, mitochondrial uncoupling agent CCCP (2 mM) was added. B) Summary of Δψ _m after Ca ²⁺ addition but before CCCP addition (n=4 per group). C) After the addition of the ratio dye Fura FF at 0 seconds, a pulse of Ca ²⁺ (10 mM) was subsequently added and extra-mitochondrial Ca ²⁺ was measured in the individual cardiomyocyte groups, as indicated by (arrow). The cell membrane Ca ²⁺ clearance was then measured as arbitrary units of fluorescence after the first Ca ²⁺ pulse. D) Summary of cell membrane Ca ²⁺ clearance. * p < 0.05, p < 0.01, p < 0.001 (n=4 per group). Δψ _m is produced by Ca ²⁺ flux through Ca ²⁺ one-way transporter, which consists of five proteins: MICU1, MICU2, MCUb, MCU and EMRE. In E) and F), the BAG3 haploid deletion resulted in a significant decrease in the relative level of MICU1 (p < 0.05), with a decreasing trend in MICU2, resulting in an increase in membrane function potential (disadvantage). In G) and H), the current of the intact structure of the cardiac mitochondrial inner membrane and matrix (cardiac mitoplasts) (I _MCU) was recorded before and after the application of 5mM Ca ²⁺ in the water bath. In G), the current is measured during the voltage jump shown. Representative single I _MCU recordings of intact structures of WT-GFP, BAG3 ^+/- -GFP, and BAG3 ^+/- -BAG3 mitochondrial inner membranes and matrices are shown. h) The mean Means ± SEM of I _MCU (pA/pF) of the intact structure of the mitochondrial inner membrane and matrix of WT-GFP (n=5), BAG3 ^+/- -GFP (n=4) and BAG3 ^+/- -BAG3 (n=5). * p < 0.05).

Figures 8A-8F show the human Bag3 proteome: bag3 and Bag3 related protein levels in heart failure and non-heart failure humans. Tissue was taken from the left ventricular free wall of a human heart with non-ischemic dilated cardiomyopathy at heart transplant (IDC) and compared to non-heart failure (NF) tissue taken from a transplant donor whose heart was not available for transplantation. a-F represents western blots for each indicated protein and the data are summarized in the cumulative plot to the right of each blot. Each study was repeated at least once with comparable results. * p is less than 0.05; * P < 0.01.

Fig. 9 is a diagram of mitochondrial and extramitochondrial pathways that maintain mitochondrial balance by modulating the activity of the extrinsic and intrinsic pathways of apoptosis, mitochondrial function, and the role of BAG3 in these pathways. Individual proteins, including Bcl2, bid, tBid, BAX, BAK-Bcl-2 family members, have the function of inhibiting and stimulating apoptosis; cIAP-1-apoptosis inhibitor-1 (cIAP-2 consists of the same gene); OMM member-mitochondrial outer membrane; IMM-mitochondrial inner membrane; MCU-mitochondrial unidirectional transport protein; SMAC-a second caspase activator derived from mitochondria expressed by the DIABLO gene that activates caspases by blocking inhibition of caspase activation by cIAP, thereby promoting apoptosis; TNFR 1-tumor necrosis factor-alpha receptor; VDAC-voltage dependent anion channels, VDAC is a gatekeeper for the passage of metabolites, nucleotides and ions, and plays a role in regulating apoptosis by interacting with Bcl-2 protein family members and hexokinase. While not wanting to be bound by any theory, when cIAP is linked to caspases and to BAG 3-BAG 3 stabilizes the cIAP-caspase dimer, thereby preventing SMAC from activating (cleaving) the caspases. When BAG3 binds to ciIAP coupled to the TNFR receptor, it is speculated to stabilize the receptor complex, prevent tnfα from binding to the receptor, or down regulate the receptor, thereby disabling normal activation of the receptor, followed by caspase 8.

Detailed Description

The following description of certain embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. Embodiments of the invention may be practiced without the theoretical aspects described. Furthermore, in introducing any theoretical aspect, applicants do not seek to be bound by the theory introduced.

In certain embodiments, a method or formulation for treating a patient suffering from or at risk of suffering from inflammation comprises administering to the patient a therapeutically effective amount of the formulation, wherein the formulation modulates the expression or amount of a BCL 2-related immortal gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby treating inflammation.

Inflammation includes, but is not limited to, abnormal or adverse inflammatory reactions, autoimmune reactions, disorders, and diseases. These reactions, disorders and diseases may be antibody or cell mediated, or a combination of antibody and cell mediated. These reactions include T cell or B cell reactions.

An inflammatory response refers to any immune response, activity or function that is beyond an expected or physiological normal response, activity or function, including an acute or chronic response, activity or function. Such inflammatory responses are generally described as adverse or abnormal increases in the immune system or inappropriate responses, activities or functions. However, the adverse inflammatory response, function or activity may also be a normal response, function or activity. Thus, normal inflammation or inflammatory response is included in the meaning of these terms, as long as it is considered undesirable, if not considered abnormal. Abnormal (abnormal) inflammatory reactions, functions or activities deviate from normal.

Inflammation and inflammatory response manifest as a number of different physiological adverse symptoms or complications, which may be humoral, cell-mediated, or a combination thereof. Inflammation, inflammatory responses, disorders, and diseases that may be treated according to embodiments herein include, but are not limited to, those that directly or indirectly result in or cause damage to cells or tissues/organs of a patient. At a systemic, regional or local level, the inflammation or inflammatory response may manifest as swelling, pain, headache, fever, nausea, bone joint stiffness, fluid accumulation, lack of mobility, rash, redness or other discoloration. At the cellular level, inflammation may manifest itself as one or more of T cell activation and/or differentiation, regional cell infiltration, antibody production, cytokines, lymphokines, chemokines, interferons and interleukins, cell growth and maturation factors (such as proliferation and differentiation factors), cell aggregation or migration, and cell, tissue or organ damage. Thus, methods, uses and formulations include treating and ameliorating any such physiological symptoms or cellular or biological responses that are characteristic of inflammation or inflammatory responses.

In certain embodiments, a method, use, or formulation according to embodiments herein may reduce, decrease, inhibit, suppress, limit, or control inflammation or inflammatory response in a patient. In other particular embodiments, a method, use, or formulation may reduce, decrease, inhibit, suppress, limit, or control an undesirable symptom of inflammation or inflammatory response.

Bcl-2 related immortalized gene-3 (BAG 3), also known as BCL 2-related immortalized gene 3, MFM6, bcl-2-binding protein BIS, CAIR-1, docking protein CAIR-1, BAG family chaperone modulator 3, BAG-3, BCL 2-binding immortalized gene 3, or BIS, is a cytoprotective polypeptide that competes with Hip-1 for binding to HSP 70. The NCBI reference amino acid sequence of BAG3 can be found in Genbank under accession No. np_004272.2; common GI:14043024.genbank accession No. np_004272.2, common GI:14043024 are referred to herein as SEQ ID NOs: 1. the NCBI reference nucleic acid sequence of BAG3 can be found in Genbank under accession No. nm_004281.3GI:62530382.genbank accession No. nm_004281.3GI:62530382 is referred to as SEQ ID NO:2. other BAG3 amino acid sequences include, for example, but are not limited to ,095817.3GI：12643665(SEQ ID NO：3);EAW49383.1GI：119569768(SEQ ID NO：4);EAW49382.1GI：119569767(SEQ ID NO：5); and CAE55998.1GI:38502170 (SEQ ID NO: 6). The BAG3 polypeptides of the invention may be variants of the polypeptides described herein, but must retain their functionality.

The term "formulation" as used herein is meant to include any molecule, chemical entity, composition, drug, therapeutic agent, or biological agent capable of preventing, ameliorating, or treating a disease or other condition. The term includes small molecule compounds, antisense agents, siRNA, agents, antibodies, enzymes, peptide organic or inorganic molecules, natural or synthetic compounds, and the like. The formulations may be tested according to the methods of the invention at any stage of a clinical trial, pre-trial test or FDA approval.

The terms "polypeptide", "protein" and "peptide" are used interchangeably herein. "polypeptides", "proteins" and "peptides" encoded by a "polynucleotide sequence" include full-length native sequences (e.g., naturally occurring proteins), and functional subsequences, modified forms or sequence variants, provided that the subsequences, modified forms or variants retain some degree of function of the full-length native protein. Such polypeptides, proteins and peptides encoded by the polynucleotide sequences may be, but are not required to be, identical to endogenous proteins of the patient being treated.

The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, and antisense DNA, as well as spliced or non-spliced mRNA, rRNA tRNA, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microrna (miRNA), small or short interfering (si) RNA, trans-spliced RNA, or antisense RNA).

Nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. The nucleic acid may be single-stranded, double-stranded or triple-stranded, linear or circular, and is not limited in length. In discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein in accordance with the convention of providing sequences in the 5 'to 3' direction.

"Heterologous" polynucleotide or nucleic acid sequence refers to a polynucleotide inserted into a plasmid or vector for the purpose of vector-mediated transfer/delivery of the polynucleotide into a cell. Heterologous nucleic acid sequences are distinct from viral nucleic acids, i.e., are non-native relative to viral nucleic acids. Once transferred/delivered into the cell, the heterologous nucleic acid sequence contained in the vector can be expressed (e.g., transcribed and translated). Or a heterologous polynucleotide transferred/delivered into a cell, contained in a vector, without expression. Although the term "heterologous" is not always used herein in reference to nucleic acid sequences and polynucleotides, reference to nucleic acid sequences or polynucleotides is intended to include heterologous nucleic acid sequences and polynucleotides even if the term "heterologous" is absent.

The term "expression vector" as used herein refers to a vector comprising a nucleic acid sequence (e.g.BAG3) encoding a gene product that is at least partially capable of being transcribed. In some cases, the RNA molecule will be translated into a protein, polypeptide or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, etc. Expression vectors may contain a variety of control sequences, which refer to nucleic acid sequences necessary for transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may contain nucleic acid sequences that have other functions.

As used herein, a "promoter" may refer to a DNA sequence that is typically adjacent to a nucleic acid sequence (e.g., BAG 3). The promoter generally increases the expression level of a nucleic acid sequence (e.g., BAG 3) compared to the expression level in the absence of the promoter.

As used herein, an "enhancer" may refer to a sequence adjacent to a nucleic acid sequence (e.g., BAG 3). Enhancer elements are typically located upstream of promoter elements, but also functional, and may be located downstream or within a nucleic acid sequence (e.g. BAG 3). Thus, the enhancer element may be located 100 base pairs, 200 base pairs or 300 or more base pairs upstream or downstream of the nucleic acid sequence (e.g. BAG 3). Enhancer elements generally increase the expression level of a nucleic acid sequence (e.g., BAG 3) over that of a promoter element.

Examples of expression regulatory elements or expression control elements that can be used in the methods according to the invention include, for example and without limitation, the Cytomegalovirus (CMV) direct early promoter/enhancer, the Rous Sarcoma Virus (RSV) promoter/enhancer, the SV40 promoter, the dihydrofolate reductase (DHFR) promoter, the chicken β -actin (CBA) promoter, the phosphoglycerate kinase (PGK) promoter, and the elongation factor-1 a (EF 1-a) promoter.

In certain embodiments, viral vectors may be used in the methods and formulations of the present invention including, by way of non-limiting example, AVV particles. In certain embodiments, viral vectors useful in the present invention include, for example, but are not limited to, retrovirus, adenovirus, helper-dependent adenovirus, hybrid adenovirus, herpes simplex virus, lentivirus, poxvirus, epstein-Barr virus (Epstein-Barr virus), vaccine virus, and human cytomegalovirus vectors, including recombinant versions thereof.

The term "recombinant" as a modifier to a viral vector (e.g., recombinant AAV (rAAV) vector), and to a sequence of a recombinant polynucleotide and polypeptide, refers to manipulation (i.e., engineering) of a composition in a manner that does not normally occur in nature. Thus, a "recombinant viral vector" refers to a viral vector comprising one or more heterologous gene products or sequences.

Because many viral vectors are subject to packaging-related size limitations, heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may suffer from replication defects, requiring trans (i.e., a "helper" function) to provide the deleted function during viral replication and encapsulation (e.g., by using helper viruses or encapsulated cell lines carrying the gene products required for replication and/or encapsulation, such as AAVrep, AAVcap, human adenovirus E4, and adenovirus VARNA). In addition, improved viral vectors carrying the polynucleotide to be delivered outside of the viral particle are described (see, e.g., curiel, D T, et al, PNAS 88:8850-8854, 1991).

A specific example of an AAV vector is the insertion of nucleic acids (heterologous polynucleotides) in the viral genome that are not normally present in the wild-type AAV genome. One example of this is the cloning of a nucleic acid (e.g., a gene) encoding a therapeutic protein or polynucleotide sequence into a vector, whether or not there are 5', 3' and/or intron regions of the gene that are normally present in the AAV genome in the vector. Although the term "recombinant" is not always used herein to refer to sequences such as AAV vectors and polynucleotides, recombinant forms including AAV vectors, polynucleotides, etc. are expressly included, although any such omissions are made.

"RAAV vectors" are derived from a wild-type AAV genome, for example, by removal of all or a portion of the wild-type AAV genome using molecular methods, and substitution with a non-native (heterologous) nucleic acid, such as a nucleic acid encoding a therapeutic protein or polynucleotide sequence. Typically, a rAAV vector will retain one or both Inverted Terminal Repeats (ITRs) of the AAV genome. rAAV differs from AAV genomes in that all or part of the AAV genome has been replaced with a non-native sequence of AAV genomic nucleic acid, e.g., with a heterologous nucleic acid encoding a therapeutic protein or polynucleotide sequence. Thus, the addition of non-native (heterologous) sequences defines AAV as a "recombinant" AAV vector, which may be referred to as a "rAAV vector.

Recombinant AAV vector sequences (or genomes) can be packaged-referred to herein as "particles" for subsequent infection (transduction) of cells in vitro, in vivo, or in vivo. When the recombinant vector sequence is packaged or packaged into an AAV particle, the particle can also be referred to as a "rAAV", "rAAV particle", and/or "rAAV virus". Such rAAV, rAAV particles, and rAAV viruses include proteins that encapsulate or package the vector genome. In the case of AAV, specific examples include capsid proteins.

"Vector genome" may be abbreviated "vg" and refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsulated to form a rAAV particle. In the case of recombinant AAV vectors constructed or manufactured using recombinant plasmids, the AAV vector genome does not include a "plasmid" portion that is inconsistent with the vector genomic sequence of the recombinant plasmid. This portion of the non-vector genome of the recombinant plasmid, referred to as the "plasmid backbone", is important for cloning and amplification of the plasmid (the process required for propagation and recombinant AAV vector production), but is not itself packaged or encapsulated into rAAV particles. Thus, a "vector genome" refers to a nucleic acid packaged or encapsulated by a rAAV.

The term "serotype" as used herein refers to an AAV vector that is serologically distinct from other AAV serotypes. Serological differences are determined by the lack of cross-reactivity between antibodies from one AAV and antibodies from another AAV. Cross-reactive differences are typically due to differences in capsid protein sequences/antigenic determinants (e.g., due to VP1, VP2, and/or VP3 sequence differences of AAV serotypes). Antibodies to AAV may cross-react with one or more other AAV serotypes due to homology of capsid protein sequences.

By serotype is meant that the relevant virus has passed neutralization activity tests with the specific sera of all existing and characteristic serotypes, and no antibodies were found that could neutralize the relevant virus, according to conventional definitions. As more naturally occurring viral isolates are discovered and/or capsid mutants are generated, there may or may not be serological differences from any of the serotypes currently present. Thus, in the case where there is no serological difference in a new virus (e.g., AAV), the new virus (e.g., AAV) will be a subset or variant of the corresponding serotype. In many cases, serological tests for neutralizing activity have not been performed on mutant viruses with capsid sequence modifications to determine whether they belong to another serotype based on the traditional definition of serotypes. Thus, for convenience and to avoid duplication, the term "serotype" refers broadly to serologically distinct viruses (e.g., AAV) as well as serologically distinct viruses (e.g., AAV) that may belong to a subset or variety of a serotype.

RAAV vectors include any viral strain or serotype. For example, the rAAV vector genome or particle (capsid, e.g., VP1, VP2, and/or VP 3) can be based on any AAV serotype, e.g., AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, AAV3B, or AAV-2i8. These vectors may be based on the same strain or serotype (or subgroup or variant) or may be different from each other. For example, a rAAV plasmid or vector genome or particle (capsid) based on one serotype genome may be identical to one or more capsid proteins of the packaging vector, but is not limited thereto. Furthermore, the rAAV plasmid or vector genome may be based on an AAV serotype genome that is different from one or more phage proteins packaging the vector genome, in which case at least one of the three phage proteins may be a different AAV serotype, e.g., AAV1, AAV2, AAV3B, AAV-2i8 (AAV 2/AAV8 chimera), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof. More specifically, the rAAV2 vector genome may comprise AAV2 ITRs, but the capsids are from different serotypes, e.g., AAV1, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, or variants thereof. Thus, rAAV vectors include gene/protein sequences that are identical to a particular serotype signature gene/protein sequence, as well as "mixed" serotypes, which may also be referred to as "pseudoserotypes".

In certain embodiments, the rAAV vector comprises or consists of at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3B, AAV-2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins (VP 1, VP2, and/or VP3 sequences). In certain embodiments, a rAAV vector comprises or consists of a sequence that is at least 70% or more (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 ITRs.

In certain embodiments, rAAV vectors include variants (e.g., ITR and capsid variants such as amino acid insertions, additions, substitutions, and deletions) of AAV1, AAV2, AAV3B, AAV-2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12, e.g., as described in WO 2013/158879 (international application PCT/US 2013/037170), WO 2015/01353 (international application PCT/US 2014/047670), and US 2013/0059732 (US application No. 13/594,773).

RAAV, such as AAV1, AAV2, AAV3B, AAV-2i8, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and variants, hybrids and chimeric sequences, can be constructed using recombinant techniques known to the skilled artisan to include one or more heterologous polynucleotide sequences (transgenes) and one or more functional AAV ITR sequences. Such AAV vectors typically retain at least one functional flanking ITR sequence, which is necessary for rescue, replication and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome will include cis sequences (e.g., functional ITR sequences) that are required for replication and packaging.

In certain embodiments, the lentiviruses used in the present invention may be human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian Immunodeficiency Virus (SIV), feline Immunodeficiency Virus (FIV), bovine Immunodeficiency Virus (BIV), jem bar's disease virus (JDV), equine Infectious Anemia Virus (EIAV), or Caprine Arthritis Encephalitis Virus (CAEV). Lentiviral vectors are capable of efficiently delivering, integrating and long-term expressing heterologous polynucleotide sequences into non-dividing cells in vitro and in vivo. A variety of lentiviral vectors are known in the art, see Naldini et al (Proc. Natl. Sci. USA,93:11382-11388 (1996); science,272:263-267 (1996)), zufferey et al (Nat. Biotechnol.,15:871-875, 1997), dull et al (JVirol. 1998nov;72 (11): 8463-71, 1998), U.S. Pat. Nos. 6,013,516 and 5,994,136, any of which may be suitable viral vectors for use in the present invention.

The dose of recombinant viral vector may be formulated, administered or delivered in any suitable dosage. Generally, the dosage range is at least 1x10 ⁸ per kilogram, or more, for example 1x10 ⁹、1x10¹⁰、1x10¹¹、1x10¹²、1x10¹³ or 1x10 ¹⁴ vector genomes (vg/kg) per kilogram of patient body weight, or more, to achieve the effect. AAV dose range of mice is 1x10 ¹⁰-1x10¹¹ vg/kg, and rAAV dose range of dogs is 1x10 ¹²-1x10¹³ vg/kg, all effective. More specifically, the dosage is from about 1x10 ¹¹ vg/kg to about 5x10 ¹⁴ vg/kg, or from about 5x10 ¹¹ vg/kg to about 1x10 ¹⁴ vg/kg, Or from about 5x10 ¹¹ vg/kg to about 5x10 ¹³ vg/kg, inclusive, or from about 5x10 ¹¹ vg/kg to about 1x10 ¹³ vg/kg, inclusive, Or from about 5x10 ¹¹ vg/kg or about 5x10 ¹² vg/kg, inclusive, or from about 5x10 ¹¹ vg/kg to about 1x10 ¹² vg/kg, inclusive. For example, the dose may be a dose of about 5x10 ¹⁴ vg/kg, or less than about 5x10 ¹⁴ vg/kg, such as about 2x10 ¹¹ to about 2x10 ¹⁴ vg/kg, In particular, for example, about 2X10 ¹² vg/kg, about 6X10 ¹² vg/kg or about 2X10 ¹³ vg/kg.

An "effective amount," "sufficient amount," or "therapeutically effective amount" refers to an amount that provides a detectable response, expected or desired result, or any measurable or detectable degree or benefit of any duration (e.g., minutes, hours, days, months, years, or cure) to a patient, in a single dose or multiple doses, alone or in combination with one or more other compositions, methods of treatment, regimens, or treatment regimen formulations. An "effective amount" or "sufficient amount" of a therapeutic "or" sufficient amount "dose (e.g., to improve or provide a therapeutic benefit or relief) is generally effective to address one or more or all of the adverse symptoms, consequences, or complications of the disease, one or more of the adverse symptoms, disorders, diseases, pathologies, or complications, e.g., caused by or associated with the disease, to a measurable extent, although a reduction, decrease, inhibition, suppression, limitation, or control of the progression or worsening of the disease is a satisfactory result.

An effective amount or quantity may be, but need not be, provided in a single formulation or mode of administration, may require multiple administrations, and may be, but need not be, administered alone or in combination with another composition (e.g., formulation), treatment, regimen, or regimen of treatment. For example, the amount may be scaled up according to the needs of the patient, the type, state and severity of the disease being treated, or the side effects of the treatment (if any). Furthermore, an effective amount or amount is not necessarily an effective amount or amount administered in a single or multiple dose without a second composition (e.g., another drug or formulation), method of treatment, regimen, or regimen of treatment, as additional doses, amounts, or durations beyond those doses, or additional compositions (e.g., drugs or formulations), methods of treatment, regimens, or regimens may be included in order to be considered effective or sufficient for a given patient. Dosages considered to be effective also include dosages that result in a reduction in the use of another treatment method, regimen or regimen.

An effective amount or quantity is not necessarily effective for every patient treated, nor is it necessarily effective for most patients treated in a given population or population. An effective amount or amount refers to an amount or amount effective for a particular patient, and not an amount or amount effective for a population or general population. As is typical of such methods, some patients will exhibit greater responsiveness, or less responsiveness or no responsiveness to a given treatment regimen or use.

Thus, the methods, uses and formulations of the invention include providing a detectable or measurable benefit to a patient, or any objective or subjective transient or temporary or longer term improvement (e.g., cure) of inflammation or inflammatory response. Thus, a satisfactory clinical endpoint is achieved when the patient's disease is progressively improved, or one or more associated adverse symptoms or complications of the condition, disorder, or disease are partially reduced in severity, frequency of occurrence, duration, or extent of exacerbation, or one or more physiological, biochemical, or cellular manifestations or characteristics of inflammation or inflammatory response are inhibited, reduced, eliminated, prevented, or reversed. Thus, a therapeutic benefit or improvement ("improvement" is synonymous) is not necessarily a complete elimination of any or all adverse symptoms or complications associated with inflammation or inflammatory response, but rather any measurable or detectable, objective or subjective, meaningful improvement in inflammation or inflammatory response. For example, inhibiting the worsening or progression of inflammation or inflammatory response or related symptoms (e.g., slowing the progression or stabilizing one or more symptoms, complications, or physiological or psychological effects or responses), even if only for days, weeks, or months, even if failing to completely diminish inflammation or inflammatory response or related adverse symptoms, is considered a beneficial effect.

"Treatment" is an intervention aimed at preventing the development of a disorder, altering a pathology or symptom, or delaying the development or progression of a disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic measures. "treatment" may also be designated as palliative treatment.

"Preventing" and grammatical variations thereof refers to a method according to the present invention wherein the condition, disorder or disease (or associated symptom or physiological or psychological response) is contacted, administered or administered in vivo prior to the manifestation or onset of the condition, disorder or disease, such that the probability, susceptibility, onset or frequency of the condition, disorder or disease or associated symptom can be eliminated, prevented, inhibited, reduced or decreased. As described herein, the target patient for prophylaxis may be suffering from a certain inflammation or inflammatory response or related symptoms, or recurrence of inflammation or inflammatory response or related symptoms previously diagnosed.

The person in need of treatment includes those already suffering from a disorder and those in need of prevention of a disorder. Thus, "treating" or "treatment" a disorder or condition includes: (1) Preventing or delaying the onset of clinical symptoms of the condition, disorder or disease in a human or other mammal that may be suffering from or susceptible to the condition, disorder or disease but has not yet developed or exhibited clinical or subclinical symptoms of the condition or disorder; (2) Suppression of symptoms or disorders, i.e., preventing, alleviating or delaying the development or recurrence of symptoms or disorders (in the case of maintenance therapy) or at least one clinical or sub-clinical symptom; or (3) relief of the condition, i.e., resolution of the symptom or disorder or at least one clinical or sub-clinical symptom. The benefit to the patient receiving the treatment is statistically significant, or at least perceptible to the patient or physician.

The term "improvement" refers to a detectable or measurable improvement in a patient's disease or its symptoms or potential cellular response. Detectable or measurable improvements include subjective or objective reduction, decrease, inhibition, suppression, limitation, or control of the disease, or the occurrence, frequency, severity, progression, or duration of complications arising from or associated with the disease, or improvement of a symptom or underlying cause or outcome of the disease, or reversal of the disease.

The formulation may be administered one or more times per day; once every other day; once or more times per week; once or more monthly; once or more times per year; or administered 1-2 times during the life of the patient. The skilled artisan will appreciate that certain factors will affect the dosage and time required to treat a patient, including but not limited to the severity of the disorder or disease, the desired outcome, previous treatments, the general health and/or age of the patient, and other diseases present. Furthermore, treating a patient with a therapeutically effective amount according to the present invention may include a single treatment or multiple treatments, such as a series of treatments.

The formulations, compositions and pharmaceutical compositions of the invention include compositions in which the active agent is present in an amount effective to achieve the intended therapeutic purpose. The skilled medical practitioner is fully capable of determining an effective dose using techniques and guidelines known in the art, and using the teachings provided herein.

Formulations, such as pharmaceutical compositions, may be delivered to a patient to allow transcription of nucleic acids and translation of encoded proteins. In certain embodiments, the formulation (e.g., pharmaceutical composition) comprises sufficient genetic material to produce a therapeutically effective amount of BAG3 in a patient, e.g., to modulate TNF signaling.

The term "modulate" refers to any of the activities of the compounds embodied herein, such as increasing, enhancing, elevating, agonizing (as an agonist), promoting, decreasing, reducing, inhibiting, blocking, or antagonizing (as an antagonist). The modulator may decrease or reduce its activity below a baseline value, e.g., by a factor of 1 to 5, 1 to 10, 5 to 10, 10 to 20, 20 to 30, 40 to 50, etc., or at least 1, 2,3, 5, 10, 20, 50, 100, etc. Modulation may also increase or enhance activity based on baseline values, e.g., 1 to 5-fold, 1 to 10-fold, 5 to 10-fold, 10 to 20-fold, 20 to 30-fold, 40 to 50-fold, etc., or at least 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, etc.

The formulations, compositions, methods and uses of the invention are useful in primates (e.g., humans) and veterinary medicine. Thus, suitable patients include mammals, such as humans, as well as non-human mammals.

The terms "patient" and "subject" refer to animals, typically mammals, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (birds such as chickens and ducks, horses, cattle, goats, sheep, pigs) and laboratory animals (mice, rats, rabbits, guinea pigs). Human patients include fetal, neonatal, infant, juvenile, and adult subjects. Patients also include animal disease models, such as mice and other animal models of BAG3 dysfunction.

The compositions and formulations may be sterile and the methods and uses may employ sterile compositions and formulations. The composition may be formulated with or administered in any biocompatible pharmaceutical carrier, including, but not limited to, physiological saline, buffered physiological saline, dextrose, and water. The compositions may be formulated or administered to a patient alone or in combination with other agents which affect the amount, frequency and/or efficacy of the administration.

The formulations, methods and uses of the invention include systemic, regional or local (e.g., specific regions, tissues, organs or cells) administration, or administration by any route, such as injection or infusion. Administration or delivery of the compositions, formulations and pharmaceutical compositions in vivo can generally be accomplished by injection using conventional syringes, but other methods of administration, such as convection enhanced delivery, can also be employed (see U.S. patent No. 5,720,720). For example, the formulations and compositions may be administered subcutaneously, epidermically, transdermally, intrathecally, intraorbitally, intramuscularly, intraperitoneally, intravenously, intrapleurally, intraarterially, orally, intrahepatic, or intramuscularly. The optimal route of administration may be determined by a clinician specifically treating the patient based on a number of criteria including, but not limited to, the patient's condition and the therapeutic purpose (e.g., modulating TNF signaling, reducing TNF signaling, treating inflammation, reducing inflammatory response, etc.).

Also according to the invention, nucleic acids, expression vectors including viral vectors and viral particles may be encapsulated or complexed with liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles or extracellular vesicles.

"Lipid nanoparticle" or "LNP" refers to lipid-based vesicles useful for the administration or delivery of nucleic acids, expression vectors (including viral vectors), and having a size on the order of nanometers, i.e., from about 10nm to about 1000nm, or from about 50nm to about 500nm, or from about 75nm to about 127nm. Without being bound by theory, LNP is believed to partially or completely shield the immune system from the effects of nucleic acids, expression vectors, or recombinant viral vectors. Shielding can deliver the nucleic acid, expression vector, or viral vector to a tissue or cell while avoiding eliciting a substantial immune response against the nucleic acid, expression vector, or viral vector in vivo. Shielding may also allow for repeated administration without eliciting a substantial immune response. Shielding may also improve or enhance in vivo delivery efficiency, duration of efficacy, and/or efficacy.

AAV surfaces carry a slight negative charge. Thus, it may be beneficial for the LNP to comprise cationic lipids (e.g., amino lipids). Exemplary amino lipids are described in U.S. Pat. Nos. No.9,352,042、9,220,683、9,186,325、9,139,554、9,126,966、9,018,187、8,999,351、8,722,082、8,642,076、8,569,256、8,466,122 and 7,745,651 and U.S. Pat. publication Nos. 2016/0213785、2016/0199485、2015/0265708、2014/0288146、2013/0123338、2013/0116307、2013/0064894、2012/0172411 and 2010/017125.

The terms "cationic lipid" and "amino lipid" are used interchangeably herein to include lipids having one, two, three or more fatty acid chains or fatty alkyl chains, and an amino group (e.g., alkylamino or dialkylamino) that can adjust the pH, and salts thereof. At pH values below the pKa of the cationic lipid, the cationic lipid is typically protonated (i.e., positively charged), whereas at pH values above the pKa, the cationic lipid is substantially neutral. The cationic lipid may also be a titratable cationic lipid. In certain embodiments, the cationic lipid comprises: a protonatable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain independently has from 0 to 3 (e.g., 0, 1,2, or 3) double bonds; and ether, ester or ketal linkages between head groups and alkyl chains.

In certain embodiments, the cationic lipid can be present in an amount from about 10 wt% of the LNP to about 85 wt% of the lipid nanoparticle, or from about 50 wt% of the LNP to about 75 wt% of the LNP.

LNP may include neutral lipids. Neutral lipids can include any lipid species that exists as an uncharged or neutral oligomer at physiological pH. Such lipids include, but are not limited to, diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. In selecting neutral lipids, particle size and necessary stability are typically considered. In certain embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacyl phosphatidylcholine and diacyl phosphatidylethanolamine).

In certain embodiments, the neutral lipid may be present in an amount from about 0.1% by weight of the lipid nanoparticle to about 75% by weight of the LNP, or from about 5% by weight of the LNP to about 15% by weight of the LNP.

Biological samples are typically obtained from or produced by biological organisms. For example, patient biological samples that may be analyzed include, but are not limited to, whole blood, serum, plasma, and the like, and combinations thereof. Other biological samples from a patient include, but are not limited to, cerebrospinal fluid or spinal fluid. The biological sample may be free of cells, and may include cells (e.g., erythrocytes, platelets, and/or lymphocytes).

The compositions, such as kits, provided herein include packaging materials and one or more components thereof. Kits typically include a label or package insert including instructions for the composition or instructions for use of the composition in vitro, in vivo or in vitro. The kit may comprise a collection of such components, e.g., nucleic acids, recombinant vectors, viral (e.g., AAV, lentiviral) vectors, or viral particles.

Kits refer to the physical structure that houses one or more components of the kit. The packaging material may maintain the sterility of the assembly and may be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoule, vial, test tube, etc.).

The label or insert may include identification information of one or more of the components, dosage, clinical pharmacology of the active ingredient (including mechanism of action, pharmacokinetics, and pharmacodynamics). The label or instructions may include identification information for the manufacturer, lot number, production location and date, expiration date, and the like. The label or instructions may include manufacturer information, lot number, production location, and date. The label or insert may include information about the disease for which the kit assembly may be used to treat. The label or insert may include instructions for the clinician or patient to use one or more kit components in a method, use, treatment regimen, or treatment regimen. Instructions may include amounts, frequencies, or durations, instructions for performing any of the methods, uses, treatment regimens, or prevention or treatment regimens described herein.

The label or instructions may include information of any benefit that the ingredient may provide, such as a prophylactic or therapeutic benefit. The label or instructions may include information regarding potential adverse side effects, complications or reactions, such as alerting the patient or clinician that the particular component is not suitable for use. Adverse side effects or complications may also occur when a patient has, is about to, or is taking one or more other medications that may be incompatible with the composition, or when a patient has, is about to, or is receiving another treatment regimen or treatment regimen that is incompatible with the composition, and thus, the instructions may include information regarding such incompatibility.

The label or insert includes "printed matter" such as paper or cardboard, either alone or adhered to the component, kit or packaging material (e.g., a package), or attached to an ampoule, tube or vial containing the kit component.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

All patents, patent applications, publications and other references, genBank references, and ATCC references cited herein are incorporated by reference in their entirety. In case of conflict, the specification, including definitions, will control.

All of the features disclosed herein may be combined in any combination. Each feature disclosed in the description may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed features are one example of a generic series of equivalent or similar features.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, a "nucleic acid" includes a plurality of nucleic acids, a "vector" includes a plurality of vectors, and a "virus" or "particle" includes a plurality of viruses/particles.

The terms "comprises," "comprising," or "consists of," and variations thereof, as used herein, when referring to defined or described elements of an item, composition, formulation, method, process, system, etc., are meant to be inclusive or open ended, allowing the inclusion of additional elements, thereby indicating that the defined or described item, composition, formulation, method, process, system, etc., includes the specified elements-or equivalents thereof as appropriate-and may include additional elements as well, yet remain within the scope/definition of the defined item, composition, formulation, method, process, system, etc.

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, depending in part on the manner in which the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may refer to 1 or more standard deviations, as is conventional in the art. Or "about" may mean up to 20%, or up to 10%, or up to 5% of the range of a given value. In addition, particularly in terms of biological systems or processes, the term may refer to within an order of magnitude, for example within 5, 4, 3, or 2 times the given value. In describing particular values in the application and claims, unless otherwise indicated, the word "about" should be assumed to mean within an acceptable error range for the particular value.

Unless the context clearly indicates otherwise, all numbers or ranges of numbers include integers within those ranges as well as fractions of numbers or integers within the range. Thus, for example, 95% or greater reduction includes 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc. Thus, it is also possible to exemplify that the numerical ranges of "1-4" and the like include 2,3, and 1.1, 1.2, 1.3, 1.4, and the like. For example, "1 to 4 weeks" includes 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days.

Further, reference to numerical ranges such as "0.01 to 10" includes 0.011, 0.012, 0.013, and the like, as well as 9.5, 9.6, 9.7, 9.8, 9.9, and the like. For example, a dose of about "0.01 mg/kg to about 10 mg/kg" of a patient's body weight includes 0.011mg/kg, 0.012mg/kg, 0.013mg/kg, 0.014mg/kg, 0.015mg/kg, etc., and 9.5mg/kg, 9.6mg/kg, 9.7mg/kg, 9.8mg/kg, 9.9mg/kg, etc., and the like.

Integers more (greater) or less than include any number greater or less than a reference number, respectively. Thus, for example, reference to greater than 2 includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., and so forth. For example, a "2 or more times" of administration of a recombinant viral vector, proteasome, and/or glycosidase includes 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more times.

Furthermore, reference to numerical ranges such as "1 to 90" includes 1.1, 1.2, 1.3, 1.4, 1.5, etc., and 81, 82, 83, 84, 85, etc., etc. For example, "about 1 minute to about 90 days" includes 1.1 minute, 1.2 minutes, 1.3 minutes, 1.4 minutes, 1.5 minutes, etc., as well as1 day, 2 days, 3 days, 4 days, 5 days. 81 days, 82 days, 83 days, 84 days, 85 days, etc., and so forth.

"Optional" or "optionally" means that the subsequently described event may or may not occur, such that the description includes instances where the event occurs and instances where the event does not.

The present invention generally uses affirmative language to describe the many embodiments of the invention herein. The invention also specifically includes embodiments such as compositions or formulations, uses, method steps and conditions, procedures or programs that exclude particular subject-matter in whole or in part. For example, in certain embodiments of the invention, the composition and/or method steps are excluded. Accordingly, even though the invention is not generally described herein as not comprising the invention, aspects of the invention not explicitly excluded are still disclosed herein.

Several embodiments of the present invention have been described. However, various changes and modifications may be made to the invention by one skilled in the art to adapt it to various usages and conditions without departing from the spirit and scope of the invention. Accordingly, the following examples are intended to illustrate the invention, but not to limit the scope of the invention in any way.

Examples

Example 1: materials and methods

Animal, animal model, surgical procedure and human tissue

Mice with one or both alleles of BAG3 deleted were generated by crossing mice bearing the deletion BAG3 (BAG 3 ^flox/flox) with Cre mice bearing the alpha-myosin heavy chain (alpha-MHC) (both C57B1/6 background), as described in (10) above. BAG3 ^flox/flox mice (BAG 3 (hepd0556_7_b06)) were from MRC HARWELL (international mouse phenotype association member), which represents european mouse mutant archive (www.infrafrontier.eu) for the generation and distribution of transgenic mice. The phenotypes of the Bag3 ^+/- and Bag3 ^-/- mice were as described above (10). Heart failure and non-heart failure human heart samples were from heart tissue banks from university of pittsburgh and university of cororado, healthy branch of academic or vocational study, as previously described (23, 24).

Mass spectrometry-Bag 3 ^+/+ (WT) and Bag3 ^+/- mice

The left ventricular tissue of three wild-type mice and three Bag3-KO mice was homogenized in lysis buffer containing 9M urea and then subjected to brief sonication. The supernatant containing the dissolved proteins was then collected by centrifugation at 10000RCF for 10 minutes. Protein concentration was determined by BCA method (Pierce). About 300. Mu.g of protein per sample was used for proteomic analysis.

Raw mass spectral data was imported Peaks into bioinformatics software and amino-methylated cysteines were used as fixed modifications, phosphorylated to variable modifications, and searched against a mouse (Mus musculus) database. The data were analyzed using a built-in Label Free Quantification (LFQ) option and normalized with the total ion flow (TIC) for each sample. The analysis of the pathways was performed using the DAVID bioinformatics program (version 6.8) to further investigate the relevant proteins identified by this method and thereby determine the function and cellular compartment characteristics of the proteins.

TUNEL staining for cell death

Isolated adult mouse cardiomyocytes were transplanted into 35mm ² dishes, which were covered with a 10mm ² diameter glass coverslip No. 1.5 (MatTek Corporation Cat #P35G-1.5-10-C) coated with fibronectin. Cells were hypoxized (1 hr) and reoxygenated (2 hr) prior to staining with 100nM nonylacridine orange (Molecular Probes Cat #A1372) in Table fluid. The cells are then imaged with a confocal or fluorescence microscope, as described previously.

TMRM staining method for detecting mitochondrial membrane potential and mitochondrial content

Isolated adult mouse cardiomyocytes were transplanted into cells as described in TUNEL staining section. Cells were stained with 100nM tetramethylrhodamine methyl ester (TMRM) (Thermo FISHER SCIENTIFIC CAT #T668) in Table fluid. Imaging was performed by confocal microscopy as previously described. Mitosox staining for mitochondrial ROS content

Isolated adult mouse cardiomyocytes were transplanted into cells as described in TUNEL staining section. Cells were stained with 5 μm MitoSOX ^TM red mitochondrial superoxide indicator (Thermo FISHER SCIENTIFIC CAT #m36008) and confocal images were quantified with Fiji Image J.

Preparation of primary Neonatal Mouse Ventricular Cardiomyocytes (NMVC)

Neonatal mouse ventricular cardiomyocytes were isolated from FVB mice 1 to 3 days old as described previously (27) using the Pierce primary cardiomyocyte isolation kit (Cat No.88281, thermo Scientific, rockford IL) following the manufacturer's instructions. .

Isolation and culture of adult cardiomyocytes

Bulk cardiomyocytes were isolated from the ventricular septum and left ventricular free wall of Bag3 ^+/+(WT)、Bag3^-/- and Bag3 ^+/- mice, placed on a glass coverslip coated with laminin, then treated as described for the first time by Zhou et al (28), and subsequently modified by members of the study group (29) (30). Detailed descriptions of experimental techniques have been presented in previous articles.

Bag3 Gene knockout

As previously described, lipofectamine 3000 system (Thermo Scientific, waltham, mass.) and Bag3 specific siRNA and lipofectamine RNAimax (ThermoFisher) were used and transfected at NMVC at a fusion level of 60% -70% according to the manufacturer's instructions.

Hypoxia/reoxygenation (H/R)

H/R was performed on NMVC as previously described (31). Briefly, NMVC was exposed to humidified 5% co ₂：95％N₂ for 16 hours at 37 ℃ and cultured in glucose-free medium. Then in a medium containing glucose with 5% co ₂: reoxygenation of 95% humidified air for 4 hours.

Cell collection and protein extraction

The cultured cells were rinsed with 1XPBS, lysed with lysis buffer supplemented with mammalian protease inhibitor cocktail, and then scraped from the petri dish. The cells were vortexed and centrifuged at 13,000Xg for 5 minutes at low temperature. The supernatant was collected for protein analysis (27).

Protein separation

The heart was excised, the left ventricle isolated, flash frozen in liquid nitrogen and stored at-80 ℃ until use. Membrane proteins were prepared as described previously (32) using Bullet Blender (Next Advance, AVERILL PARK, NY).

Cytoplasmic and mitochondrial isolation

Mitochondrial and cytoplasmic proteins were isolated using a mitochondrial culture cell isolation kit (Thermofisher, # 89874) according to the manufacturer's instructions. The isolated proteins were quantified using Braford assay (Bio-Rad, USA). The protein was then isolated by western blot analysis.

Immunoprecipitation

NVCM or AC16 cardiomyocytes were transplanted into 10cm dishes and treated as described above. The cells were washed rapidly with cold PBS and then placed in IP lysis buffer (Thermofisher) with phosphate and arrestin inhibitors added and homogenized with beads in a stirrer. The protein lysates were then incubated with Magna magnetic beads (A/G) (Millipore, sigma) for one hour and the amount of lysates was quantified as described previously.

Western blot analysis

Protein lysates (90 μl) were mixed with reducing agent (ThermoFisher), proteins were separated using NuPAGE Gel (ThermoFisher), and transferred to nitrocellulose membrane (LiCor, lincoln, NE) using a wet transfer system as described previously (17). The membranes were washed rapidly, blocked with LicorOdyssey blocking buffer (LiCor), incubated with secondary antibodies, and images captured with a LiCor imaging system.

Immunofluorescent staining

Neonatal or adult cardiomyocytes were fixed with 4% paraformaldehyde in PBS for 15 min and washed; infiltration with 0.5% Triton X-100 in PBS for 10 min and washing; blocking with Licor blocking buffer S containing 5% Bovine Serum Albumin (BSA) and 0.1% Triton X-100 for 1 hour; all operations were performed at room temperature. Cells were incubated overnight at 4 ℃ with rabbit anti-protein-related antibodies diluted in blocking solution. The cells were then rinsed with PBS and incubated with the appropriate secondary antibody and DAPI (4', 6-diamidino-2 phenylindole) diluted with blocking solution for 45 minutes at room temperature.

Confocal microscope

Confocal microscopy was used to detect adult cardiomyocytes as described previously (17). Briefly, NMVC was isolated and cultured on mucin-coated 4-well chamber slides (Lab-tek., rochester, NY). Bag3 (28, 29) was identified using primary anti-rabbit antibody (1:200;Proteintech Group Inc,Chicago IL). The total laser intensity and photomultiplier gain were constant for all experimental groups, and the data were verified by two independent observers, who were blind to the experimental group. At least three coverslips were used for each experimental group, and at least three cell images were acquired per coverslip.

Measurement of mitochondrial Membrane potential (Aψm) and mitochondrial Ca ²⁺ uptake

The measurement method is as described above (35). Briefly, LV cardiomyocytes isolated from WT and Bag3 ^+/- hearts were exposed to 21% o ₂-5％CO₂ (normoxic) or 1%O ₂-5％CO₂ (hypoxic) conditions for 30 minutes, then reoxygenated for 30 minutes (33). Fura-FF (0.5. Mu.M) was added at 0 seconds, JC-1 (800nM;Molecular Probes) was added at 20 seconds, and extra-mitochondrial Ca ²⁺ and Δψm were measured, respectively. The fluorescence signal was monitored by a multi-wavelength excitation and dual wavelength emission spectrofluorimeter (Delta RAM, photon Technology International) and the calibration method for the ratiometric dye Fura-FF was as described previously (30). 10. Mu.M Ca ²⁺ pulses were added at the indicated times while monitoring Δψm and extra mitochondrial Ca ²⁺. Δψm is calculated from the fluorescence ratio of JC-1 oligomer to monomer form. Cytoplasmic Ca ²⁺ clearance represents mitochondrial Ca ²⁺ uptake.

Echocardiography (UGV)

All mice were evaluated for global LV function after mild sedation (2% isoflurane) using VisualSonics Vevo imaging system 770 and 707 scan head (miam, FL), as previously described (11), left Ventricular Ejection Fraction (LVEF) calculated as EF% = [ (LVEDV-LVESV)/LVEDV ] ×100; wherein LVEDV and LVESV are left ventricular end diastole volume and left ventricular end systole volume, respectively.

Measurement of mitochondrial Ca ²⁺ one-way transporter (MCU) current (I _MCU)

Cardiomyocytes were isolated from LV and ventricular septum of 8 to 12 week old WT or BAG3 ^+/- mice (29), then infected with Adv-GFP or Adv-BAG3 (7X 10 ⁶ pfu/ml) and cultured for 24 months for complete structural separation of mitochondrial inner membrane and matrix (34). The complete structural patch clamp recordings of the mitochondrial inner membrane and matrix were performed at 30℃as detailed in the previous description (35-37). I _MCU was recorded using a computer controlled Axon200B patch clamp amplifier and Digidata 1320A acquisition board (pClamp 10.0 software; axon Instruments). The intact structure of the mitochondrial inner membrane and matrix was immersed in a physiological solution, and after forming the gΩ seal, the intact structure of the mitochondrial inner membrane and matrix was ruptured and capacitance was measured. After capacitance compensation, the intact structure of the mitochondrial inner membrane and matrix was maintained at 0mV, and I _MCU was excited by voltage ramp (from-160 mV to +80mV,120 mV/s) before and after 5mM Ca addition.

Statistical analysis

Data for continuous variables are expressed as mean ± SEM. Variance analysis with Bonferroni multiple comparison adjustment was used to evaluate differences between study groups. For western blot analysis, p values less than 0.05 were significant. The control group (e.g., ad-GFP or normal hypoxia) for each experiment was set to 1.0. Each experiment showed a complete blot for assessing protein levels, including loading levels. All blots were normalized to the appropriate standard for the same gel as the blots. The individual elements on each plot represent different biological measurements in one sample. The results of this study were, in part, without technical repetition unless noted. The original print of each graph may be solicited from a verifiable scientist. In measuring the levels of the various components of the unidirectional conveyor, each experiment was repeated five times because of the smaller protein yield per experiment.

Example 2: results

Deletion of one allele of mouse BAG3 results in age-related LV dysfunction

Bag3 single allele knockout mice are ideal models for studying BAag3 biology because they reflect deleted or truncated molecular biology (8-10,27,38). As shown in fig. 1A, 8 to 10 week old mice deleted for one allele of BAG3 (BAG 3 ^+/-) were examined by echocardiography for normal LV phenotype. However, by 18 weeks of age, the LV function of the Bag3 ^+/- mice significantly decreased and LV increased. Without this, the levels of Bag3 were continuously reduced by about 50% in all Bag3 ^+/- groups (FIG. 1B). Thus, young Bag3 ^+/- mice are ideal models for studying the effects of Bag3 deficiency on cardiac cells and molecular biology.

Mouse central myocyte restricted Bag3 KO was associated with changes in mitochondrial protein expression.

About 50% decrease in Bag3 expression in the heart is associated with human heart failure (11, 12), while progressive left ventricular dysfunction resulting from mouse cardiomyocyte-restricted Bag3 haploid loss is consistent with the human phenotype (10). To determine the impact of Bag3 reduction on the heart and to gain insight into which specific pathways are deregulated, we analyzed the proteome of cardiomyocyte-specific Bag3 knockout mice with age-matched wild-type controls (9, 10) using unbiased mass spectrometry.

The protein of the left ventricle of the mouse was digested with trypsin, and the resulting peptide was subjected to high pressure liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The results obtained were then searched against a museulus database and analyzed using the Label Free Quantification (LFQ) method normalized to total ion flow. LFQ analysis determined that 86 proteins were significantly altered in BAG3 KO mice (p < 0.05 compared to WT, fig. 2A and table 1 below).

Notably, pathway analysis of these proteins showed that the largest proportion (36%) of the proteins were localized mainly to mitochondria (fig. 2B), suggesting that disruption of mitochondrial function may be one of the causes of heart dysfunction due to reduced Bag3 expression. Analysis of the biological functions of 86 proteins revealed that their main functions include regulation of mitochondrial metabolism and mitochondrial-dependent apoptosis (fig. 2C). In neonatal rat ventricular cardiomyocytes, bag3 is associated with mitochondrial function and Bag3 gene knockout is associated with reduced mitosis (39). However, this was the first demonstration that reducing Bag3 severely alters mitochondrial protein expression and further demonstrates the role of Bag3 in the mitochondrial-mediated cell survival pathway and cardiac non-mitochondrial inflammation.

Hypoxia-reoxygenation stress: apoptosis in BAG3 ^+/- mice

To determine how BAG3 haploid loss affects apoptosis in a BAG 3-deleted mouse model, adult cardiomyocytes were harvested from 8 to 10 week old WT and BAG3 ^+/- mice and stained for nuclear DNA (DAPI), viable mitochondria (NAO), and damaged DNA (TMR Red-TUNEL), and the resulting confocal images were analyzed using one-way analysis of variance (fig. 3A) and sub-analyzed using Bonferroni correction (fig. 3b; p < 0.0125 has significant statistical significance). In the absence of stress, the increase in apoptosis in Bag3 ^+/- mice was very small and insignificant (p= 0.1130). TUNEL positive cells were significantly increased (p < 0.0001) after H/R injury when WT cardiomyocytes were stressed with one hour hypoxia followed by two hours reoxygenation (H/R) compared to WT hypoxic cells. Likewise, H/R resulted in a significant increase in TUNEL-positive Bag3 ^+/- cardiomyocytes (p < 0.0001) compared to Bag3 ^+/- hypoxic cells. More interestingly, H/R resulted in a significant increase in TUNEL positive cells in Bag3 ^+/- cardiomyocytes compared to WT-H/R cardiomyocytes (p=0.0076); this suggests that BAG3 haploid loss exacerbates H/R injury.

BAG3 haploid loss alters mitochondrial membrane potential (Δψ _m)

Another set of studies uses confocal microscopy to measure the effect of Bag3 loss on mitochondrial area (size) and Reactive Oxygen Species (ROS) levels. Since pilot experiments showed that heterozygote BAG3 deletion had no effect on mitochondrial reactive oxygen species, these experiments used cells isolated from BAG3 ^-/- mice, but were scheduled for subsequent positive studies. As shown in fig. 3D, homologous gene deletion had no effect on mitochondrial reactive oxygen species nor on mitochondrial content. In contrast, when these cells were stained with the membrane potential marker, mitochondrial membrane potential (Δψ _m) was significantly reduced (p < 0.01) in Bag3 ^-/- mice compared to WT mice (fig. 3E).

Comparative analysis of the levels of the related proteins of the mice of Bag3 ^+/- and Bag3 ^WT

To confirm the results of proteomic studies, protein levels critical for cardiomyocyte homeostasis and protein homeostasis in the left ventricular myocardium of Bag3 ^WT and Bag3 ^+/- mice were assayed, with emphasis on proteins associated with apoptosis. The intrinsic pathway of apoptosis is activated by mitochondrial signaling.

As shown in FIG. 4A, the level of caspase3 was significantly increased (p < 0.01) by the major executive (effector) proteins of apoptosis. However, there was no increase in the ratio of cleaved caspase3 protein/total caspase3 protein in the ventricular myocardium of Bag3 ^+/ -mice compared to proteins isolated from WT mouse hearts, suggesting that Bag3 haploid loss would result in only moderate or slight apoptosis at the early stages of the disease. While not wanting to be bound by any theory or hypothesis, in early disease, caspase3 activation may be observed when cardiac remodeling is absent, whereas in aged mice with cardiac remodeling and hypofunction, caspase3 activation may be present. However, in aged mice of 10-12 weeks of age or 18-22 weeks of age, the levels of activated caspase-3 were significantly increased, but the ratio of cleaved caspase 3/total caspase3 protein was still not increased (compared to WT), indicating that only severe stress plus aged mice would activate caspase 3-mediated apoptosis in the heart (fig. 4B).

BAG3 lacks an exogenous pathway to apoptosis

Tumor necrosis factor-alpha (TNFa) activates tumor necrosis factor-alpha (TNFa) by binding to TNFR1 receptor and subsequently cleaves and activates caspase 8 (40), thereby assessing an exogenous/mitochondrial-independent pathway. Cleaved caspase 8 can then activate caspase 3, either directly leading to apoptosis, or bind to Bcl-2 family member bid, then interact with tBid, and start releasing cytochrome c after transfer of tBid into mitochondria. Cytochrome c then binds to apoptotic bodies, which in turn activates caspase 9

The exogenous pathway was significantly activated in the Bag3 ^+/- mice, as the tnfα (58.0±2.7, n= 5;p =0.014) levels were significantly higher in Bag3 ^+/- mice compared to WT littermate control mice (36.742.7; n=5) (fig. 4C). These effects were limited to TNF only, as no change in the levels of the pro-inflammatory cytokine IL-6 was detected (fig. 4C). The ratio of cleaved caspase 8 to total caspase 8 in the ventricular myocardium of Bag3 ^+/- (0.94±0.09, n=5) was significantly increased (p < 0.003) compared to tissues from WT mice, further confirming the role of exogenous signals in Bag3 ^+/ -mouse apoptosis. (0.54±0.03, n=5, fig. 4D).

The significant increase in the content of poly (ADP-ribose) polymerase-1 (PARP-1) in the heart (fig. 4E) demonstrates that a decrease in Bag3 levels is associated with an increase in cellular inflammation, poly (ADP-ribose) polymerase-1 (PARP-1) being a protein that transfers ADP ribose to Apoptosis Inducing Factor (AIF) and that is capable of transporting ADP ribose from mitochondria to the nucleus where cell death is initiated by signaling DNA breaks (41, 42). Thus, in summary, these studies of the protein level of BAG3 one allele deleted mouse cardiomyocytes indicate that typical TNF receptor signaling is characteristic of BAG3 haploid allele deletion, which leads to activation of PARP1 and AIF, and in turn to aseptic inflammation and DNA fragmentation (41, 42).

Caspases, SMAC and control of cIAP-apoptosis signals

To better understand the regulatory mechanisms of Bag3 on apoptosis and mitochondrial balance, proteins in the Bag3 proteome were studied. As previously described, caspase 3 is one of the executing caspases located at the ends of the apoptotic signaling cascade, whose activity is dependent on signals involving mitochondria, including the release of cytochrome c and endoenzyme G from the mitochondrial matrix. Thus, it is considered to be a "mitochondrial dependent" caspase. In the absence of stress, the apoptosis-inhibiting factor (cIAP) binds to receptors on caspase 3 (and all other caspases), inhibiting its ability to be cleaved into active molecules. However, under stress (e.g., ischemia or toxins), a second, mitochondrially derived caspase activator (SMAC) is released from the Outer Mitochondrial Membrane (OMM) (as well as other caspases). Caspase 3 is then cleaved and begins to affect apoptosis of heart cells.

The data of young Bag3 ^+/- mice presents a problem: why does the adult cardiomyocyte apoptosis of the Bag3 ^+/- mice significantly increased, but the cleaved caspase3/pro ratio did not change? One possible explanation for this apparent difference is the potential change in SMAC function. SMAC is a protein transcribed from the DIABLO gene that is located in the mitochondrial space. As shown in FIG. 4F, cIAP was first co-immunoprecipitated with Bag3 and caspase-3 (co-IP) instead of SMAC; bag3 co-immunoprecipitated with cIAP (co-IP) rather than SMAC or caspase3 (co-IP). The relationship between Bag3 and cIAP is selective in that Bag3 is not co-immunoprecipitated with highly homologous XIAP (x-linked IAP) (fig. 4F).

The second important factor regulating mitochondrial balance is the protein import system known as outer membrane Translocase (TOM) and inner membrane Translocase (TIM). TOM and TIM proteins are key elements necessary for transport into and out of mitochondria. TOM22 (along with TOM 20) is an accessory unit of the outer membrane Transporter (TOM), a well in OMM that transports short chain proteins into the mitochondrial matrix. Interestingly, bag3 also co-precipitated with TOM22 protein (fig. 4F).

In the absence of Bag3, the SMAC is stuck in the OMM

While not wanting to be bound by any theory or mechanism of action, there is a hypothesis that caspase-3 is not activated in the event of a Bag3 haploid loss, since SMACs stagnate on the mitochondrial membrane and cannot relocate to the cytoplasm. To understand this hypothesis, neonatal cardiomyocytes of wild-type mice were isolated and cultured. The cells are then exposed to 1) normal control conditions; 2) Hypoxia/reoxygenation; 3) siRNA of Bag 3; or 4) hypoxia/reoxygenation plus Bag3 siRNA.

As shown in fig. 5A, SMAC was found predominantly in mitochondria with a small amount of protein in the cytoplasm under control culture conditions. The addition of H/R stress does not itself alter this localization of SMACs. However, in the absence of Bag3 (siRNA Bag 3), there was no significant SMAC in the cytoplasm. Likewise, when cells lacking Bag3 (siBag) were exposed to H/R stress, SMAC was also not seen in the cytoplasm. TOM22 serves as a control because it is present only in mitochondria. Four study conditions indicate that Bag3 is always required for SMAC transport in mitochondria.

BAG3 ^+/- precordial dyspareunia proteome

To more broadly understand how Bag3 loss affects apoptosis, other apoptosis signaling proteins associated with heart failure were evaluated. FIG. 5 shows that in 8 week old Bag3 ^+/- mice, the effect of Bag3 deficiency was very different from the change in protein levels, which was a sign of late LV dysfunction in the disease. For example, no changes in cIAP or total-P39, a MAP kinase, associated with pathological changes during inflammation and apoptosis (43), were observed (fig. 5B). As shown in fig. 5A and 5C, none of JNK, which up-regulates and activates various pro-apoptotic signaling pathways in heart failure, or Jun, which produced very similar effects on cells by releasing SMAC in mitochondria (differently regulated in HFrEF), was observed. Finally, the expression of phospho-ERK 1/2 and total-ERK was evaluated, but no changes attributable to BAG3 were found.

One observation from proteomic screening was unexpected, but consistent with the overall hypothesis that abnormal BAG3 levels resulted in increased cardiac inflammatory corpuscles. In comparison to the levels of WT control mice (1.07±0.11, n=9, p=0.03), the RNA-binding protein human antigen R (HuR: ELAV-1) was overexpressed in the Bag3 ^+/- heart (1.51±0.16, n=8) (fig. 5D). Given that Bag3 haploid loss is associated with increased inflammatory body activity, this may not be surprising, as knockout of HuR may reduce the inflammatory response and may be a therapeutic target for pathologic cardiac hypertrophy (44, 45). Thus, in general, these studies of the protein levels of the cardiomyocytes in mice with a single allele deletion of Bag3 strongly indicate that typical TNF receptor signaling is a feature of the deficiency of the Bag3 haploid allele and leads to inflammation and DNA fragmentation. TOM22 is a protein that regulates the absorption of proteins and other nutrients by the mitochondrial matrix, and its level is also significantly reduced (fig. 4E).

Deficiency of Bag3 haploid leads to abnormal mitochondrial Ca ²⁺ balance

As shown in fig. 2A-2C and table 1, the proteomic studies disclosed herein demonstrate that the primary effect of Bag3 haploinsufficiency is to alter the number of specific mitochondrial proteins that play a role in cellular metabolism and energy production. Specifically, knocking out one allele of BAG3 results in reduced expression of enzymes associated with mitochondrial function, including isocitrate dehydrogenase, pyruvate dehydrogenase, and alpha-ketoglutarate dehydrogenase. Thus, three studies were used to evaluate the hypothesis that insufficient haploid form Bag3 may be a causative factor in impaired Bag3 ^+/- cardiac function, as it may lead to abnormal mitochondrial Ca ²⁺ homeostasis.

1) Bag3 haplotype deficiency hinders the ability of the heart to maintain Mitochondrial Membrane Potential (MMP)

In the first set of studies adult cardiomyocytes were extracted from WT and Bag3 ^+/- mice. Cardiomyocytes were then exposed to H/R, mitochondrial membrane potential (Δψm) was assessed using the ratio indicator JC-1, mitochondrial Ca ²⁺ uptake was assessed using the ratio dye Fura-FF. Fluorescence is measured using a dual wavelength emission spectrofluorimeter as previously described (35).

As shown in fig. 7A and 7B, cardiomyocytes Δψm of normoxic WT hearts were significantly reduced (p < 0.001) compared to those of WT hearts exposed to H/R stress. However, a significant decrease in Δψm of BAG3 +/-cardiomyocytes (p < 0.001) also occurred compared to H/R-exposed WT cardiomyocytes, indicating that H/R stress aggravated the potential effect of Bag3 haploid loss on mitochondrial function. Similar phenomena were also observed when studying the effect of one allele deletion of Bag3 on calcium homeostasis (fig. 7C and 7D). When adult cardiomyocytes of WT mice were compared to cells isolated from Bag3 ^+/- mice, the [ Ca ²⁺]_m uptake (1/τ) of Bag3 ^+/- mitochondria was significantly reduced (p < 0.001) compared to WT mouse mitochondria. Taken together, these results support the hypothesis that a Bag3 deficiency results in a significant change in Ca ²⁺ steady state, especially in stressed cells.

2) Heterozygous deletions of Bag3 alter the function of the Ca ²⁺ one-way transporter.

The one-way transporter consists of two pore-forming subunits (MCU and MCUb) and three regulatory subunits (MICU 1, MICU2 and EMRE) that together maintain a negative potential of the mitochondrial outer membrane (OMM) (46). The negative potential across the outer mitochondrial membrane is responsible for transporting valuable resources into mitochondria. In the resting state, MICU1 and MICU2 dimerize and act as gatekeepers for the MCU. When the cytoplasm [ Ca ²⁺ ] starts to release to mitochondria, the inhibition of the movement of Ca ²⁺ by MICU-2 is blocked, and the conformation of the protein complex is changed. MICU1 activates the channel and stimulates transport of Ca ²⁺ into mitochondria. EMRE stabilize the MCU-MICU1 complex, thereby fine tuning the Ca ²⁺ level into the mitochondria.

As shown in FIGS. 7E and 7F, there was a trend of decreasing MICU2 levels in the Bag3 ^+/- mice compared to the Bag3-WT mice, but this trend did not reach statistical significance. However, the mice with Bag3 ^+/- had significantly reduced MICU1 levels (p < 0.05) compared to the WT control group, further demonstrating that Bag3 haploid loss was directly related to and resulted in the occurrence of mitochondrial Ca ^{2 +} homeostasis abnormalities.

3) The adenovirus over-expresses Bag3 to normalize MCU function

To confirm that the decrease in mitochondrial calcium uptake was due to a decrease in MCU activity and was directly related to the haploid deficiency of Bag3, cardiac mitogens were isolated from WT cardiomyocytes and Bag3 ^+/- cardiomyocytes (both overexpressing GFP as a control). The current of the voltage clamp across the intact structure of the mitochondrial inner membrane and matrix was measured before and after the addition of 5mM Ca ²⁺ (I _MCU). These measurements, while technically challenging, can compare MCU activity of different sets of mitochondria by tightly controlling membrane potential, ca ²⁺ and H ⁺ gradient conditions. As shown, I _MCU is recorded during the voltage ramp.

As shown in fig. 7G and 7H, the peak of I _MCU for the intact structure of the WT-GFP mitochondrial inner membrane and matrix was significantly higher (p=0.018) than the intact structure of the Bag3 ^+/- GFP mitochondrial inner membrane and matrix (n=5). Importantly, adenovirus-mediated overexpression of WT Bag3 in Bag3 ^+/- cardiomyocytes restored the peak of I _MCU (p=0.028 compared to Bag3 ^+/-). These data indicate that adequate levels of BAG3 are a necessary condition to maintain MCU expression and/or activity.

Human proteome and inflammatory body reflect the situation of the Bag3 ^+/- mice

As shown in FIG. 8A, the level of Bag3 in the heart failure human tissue LV myocardium was reduced by about 50% compared to the non-heart failure control group. In addition, it was found that the inflammatory body of the human heart was similar to, but not exactly identical to, that of haploid Bag3 deficient mice: cPARP and cleaved caspase 8 levels were elevated, whereas cleaved caspase 3 levels were not (FIGS. 8D and 8E). An increase in cPARP was also observed (fig. 8D). No significant change in HuR was observed compared to mice (fig. 8G).

Example 3: theory of passing

Just ten years ago Selcen and colleagues reported for the first time a sporadic Single Nucleotide Polymorphism (SNP) in BAG3, which resulted in the substitution of leucine at amino acid 209 with proline, leading to the appearance of the phenotypes of giant axons, severe skeletal muscle confusion, and mild cardiac hypertrophy in children. Subsequent whole genome association studies (GWAS) and Whole Exome (WES) and Whole Genome (WGS) sequencing found that truncations and SNPs of BAG3 can lead to a variety of cardiac phenotypes, especially DCM (3-6). Although BAG3 was found little from a scientific knowledge perspective, BAG3 has a relatively long history from a biological perspective, and the discovery of BAG3 homologs in plants is demonstrated (47). To date, bag3 genomic abnormalities are associated with reduced autophagy, increased apoptosis, abnormal excitation-contraction coupling, and abnormal sarcomere function at the protein and molecular level. However, the full range of Bag3 functions is not yet known.

Young mice deleted BAG3 haplotype alleles but not yet showing a drop in ejection fraction were studied by using high pressure liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) and proteomics methods to determine the previously unrecognized consequences of BAG3 haploinsufficiency. As shown by the proteomic studies and subsequent investigation disclosed herein, BAG3 also plays a role in the extrinsic pathway of altered apoptosis, while also acting as an inflammatory regulator by activating the cardiac inflammatory group, particularly the TNFR1 signaling cascade. As disclosed for the first time herein, bag3 plays an equally important role in supporting transport of Ca ²⁺ into and out of mitochondria. This transport of Ca ²⁺ maintains the mitochondrial membrane potential required for Ca ²⁺ flux, while Ca ²⁺ flux is a biological activity that provides the required energy for tricarboxylic acid (TCA) cycle enzymes.

The cellular mechanism that enables Bag3 to clear damaged or diseased organelles and cells from tissue without the collateral damage that occurs when the cells die suddenly is very complex, involving multiple regulatory pathways. When programmed cells such as apoptosis die, the cell membrane remains intact throughout the process, so that internal enzymes and toxic substances are degraded before the cell eventually ends up as a functional organelle. As reported more than ten years ago, there are two typical pathways that regulate cardiac apoptosis: type I or type II pathway (48). The type 1 (extrinsic or mitochondrial independent) pathway consists of a series of events, first activating death domain receptors such as tumor necrosis factor receptor-1 (TNFR-1), and finally activating caspase-7 and-3 in the executor. In contrast, in the type II (intrinsic or mitochondrial dependent) pathway, apoptosis is activated by mitochondrial release of pro-apoptotic signals (including cytochrome c and endoenzyme g) and subsequent activation of caspase-9 and-3.

It has been suggested that apoptosis is not necessarily a direct consequence of activation of the cell death pathway, but rather, due to TNF signaling, results in cell death following depletion of one or more anti-apoptotic proteins, whereas Bcl-2 overexpression is sufficient to partially attenuate this pathway (42). However, BAG3 was only just discovered since this model was proposed more than ten years ago, and its importance to cells and whole organisms is completely unknown. Indeed, it is thought to regulate apoptosis by binding to Bcl2 and linking actin filaments to the Z disc. Indeed, an early paper erroneously believes that BAG3 haploid deficiency does not lead to a high frequency phenotype. However, it is now known that the lack of a full complement of strong anti-apoptotic Bag3 protein results in a phenotype very similar to the Bag3 haplodeficiency model, i.e. an increase in caspase 3 total activity and caspase 8 activity. The Bag3 haplodeficiency model is unique in that TNF levels increase and mitochondrial membrane potential is adversely altered (decreased) independent of exogenous TNF. Furthermore, the effect of cIAP is diminished because it cannot be complemented by intact Bag3, which may be a contributor to Bag3 deficiency pathobiology. Thus, an imbalance between apoptosis and survival can have serious consequences for the myocardium.

In a typical pathway of apoptosis, the first type of pathway is down-regulated by an apoptosis Inhibitor (IAP) (49). In contrast, the class 2 pathway is inhibited by the binding of Bag3 to Bcl-2 (50), bcl-2 being an initiating member of a large Bcl-2 protein family, including pro-apoptotic (BIM, BID, BAD, BAX/BAK) and anti-apoptotic (Bcl-2, bcl-XL, MCL-1) members (50). As disclosed herein, the deletion of a single Bag3 allele and the resulting 50% decrease in Bag3 levels is associated with an increase in caspase3, but is not associated with a change in the ratio of cleaved caspase 3/total caspase-3. In contrast, caspase-8 and cleaved caspase 8 were significantly increased, and the ratio of cleaved caspase 8 to total caspase 8 was increased, which was associated with increased apoptosis. Once activated, caspase 8 causes apoptosis under induction of death receptors Fas, TNFR1 and DR 3. Indeed, in previous studies, deletion of caspase-8 and RIPK3 prevented abnormal cell death, reduced inflammation and prolonged survival in mice (49). The role of caspase-8 in cancer has been studied extensively, but less in the heart (49, 51, 52). However, these findings indicate that caspase-8 plays a greater role in the heart than one might imagine.

An interesting and novel finding in the studies herein is co-immunoprecipitation of cIAP with Bag 3. This link between Bag3 and cIAP is selective in that it was found that Bag3 does not precipitate with the X-linked homolog XIAP, an inhibitor that directly neutralizes caspase-9 and effector caspases-3 and-7, in large numbers in many different types of advanced cancers (53). Previous studies on inflammatory bowel disease and cancer have shown that cIAP and its antagonists can regulate spontaneous and TNF-induced production of pro-inflammatory cytokines and chemokines (54). Since BAG3 can immunoprecipitate with cIAP, which has previously been demonstrated to couple with TNFR1 at the critical junction of TRAF2/TRAF5, LUBAC and RIPK1, the presence or absence of BAG3 is likely to play an important role in TNF signaling and thus in the inflammatory body of cardiac myocytes (55).

Surprisingly, bag3 did not co-precipitate with either caspase-3 or SMAC. When SMAC translocates to the cytoplasm, it competes with cIAP, displacing it, thereby activating caspase-3. Although studies herein indicate that this transport does not occur in the absence of Bag3, further studies may elucidate the mechanism by which Bag3 regulates SMAC transport, particularly considering the lack of physical association between Bag3 and SMAC.

Mitochondrial Ca ²⁺(mCa²⁺) single channel is the main pathway for mitochondrial uptake of Ca ²⁺, playing an important role in the heart, as it is responsible for ATP production through the tricarboxylic acid cycle, and in particular Ca ²⁺ regulates enzyme activity in the tricarboxylic acid cycle (56). In fact, proteomic studies in mice with Bag3 ^+/- showed that Bag3 was associated with an increase in Ca ²⁺ -dependent tricarboxylic enzymes, including pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase and isocitrate dehydrogenase (57). An abnormal level mCa ²⁺ will result in an increase in workload and thus increase cell pressure, while when mCa ²⁺ levels are lower, the opposite occurs, minimizing pressure (58). MCU is the main pathway for mitochondrial uptake of Ca ²⁺, while mitochondrial permeability transition pore is the site of excess Ca ²⁺ loss.

While reduction of MCU levels and activity in the Bag3 ^+/- heart was considered to lead to an attractive HFrEF phenotype, the biological properties of mCa ²⁺ were complex and not undisputed. In contrast, studies with zebra fish (61), diabetic mouse heart (62) and guinea pig heart failure model (58) indicate that restoring normal or abnormal levels of MCU can have a beneficial effect on cardiac function. There has also been controversy about the molecular and cellular biological mechanisms of mitochondria, particularly its relationship to Ca ²⁺ equilibrium (58, 63), (64). In view of the debate, it should be carefully interpreted that restoring normal levels of Bag3 improves mitochondrial Ca ²⁺ uptake, which in turn enhances cellular bioenergy, thereby bringing benefits to the cells.

Bag3 has many protein binding domains and is well suited to play a multi-tasking role. However, unlike intracellular proteins that reside in specific areas of the cell, such as receptors in myosin or contractile elements in sarcomere, BAG3 is ubiquitous (fig. 9). However, it appears to create a discrete intracellular microenvironment depending on its specific responsibilities and locations. For example, it links the contractile element to the Z disc in the myocardial sarcomere, links the b-adrenergic receptor to the L-type Ca ²⁺ channel in sarcoplasmic, links the motor protein to the nucleus Zhou Ningji, and co-synthesizes the autophagic protein component in the proteasome domain.

The results disclosed in the present application indicate that BAG3 is also co-located with TOM22 in mitochondria. Thus BAG3 is not just a regulatory or structural protein, it appears to be a universal glue, selectively localizing proteins to specific cellular domains where it can interact with and co-localize to partner proteins. Examples of such proteins that serve multiple functions are not uncommon: multifunctional protein 4.1R is a good example (65).

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Claims (33)

1. A method or formulation for reducing, inhibiting or reducing TNF signaling comprising: administering to the patient an amount of a BCL 2-associated immortalized gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby reducing, inhibiting, or reducing TNF signaling.

2. A method or formulation for treating a patient suffering from or at risk of developing an inflammation, comprising: administering to the patient an amount of an agent that modulates expression or amount of a BCL 2-associated immortal gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide to treat inflammation.

3. A method or formulation for reducing, inhibiting or lowering inflammation or inflammatory response comprising: administering to the patient an amount of a BCL 2-associated immortal gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby reducing, inhibiting, or reducing inflammation or inflammatory response.

4. The method or formulation of claim 1, wherein the TNF signaling occurs in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney, or pancreas.

5. The method or formulation of claim 2 or 3, wherein the inflammation or inflammatory response affects the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney, or pancreas.

6. A method or formulation according to claim 2 or 3, wherein the inflammation or inflammatory response comprises chronic inflammatory disease, chronic inflammatory demyelinating polyneuropathy, primary immune thrombocytopenia, senile anorexia, intestinal inflammation, inflammatory bowel disease, ulcerative colitis, crohn's disease, lupus, rheumatoid arthritis, chronic myocarditis after Covid infection, psoriasis, psoriatic arthritis or ankylosing spondylitis.

7. A method or formulation for modulating PARP1 levels, expression or activity comprising: administering to the patient an amount of a BCL 2-associated immortalized gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby modulating PARP1 levels.

8. A method or formulation for reducing, inhibiting or reducing PARP1 levels, expression or activity comprising: administering to the patient an amount of a BCL 2-associated immortalized gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby reducing, inhibiting, or reducing PARP1 levels, expression, or activity.

9. A method or formulation for reducing, inhibiting, reducing, or stabilizing the amount of α -synuclein, comprising: administering to the patient an amount of a BCL 2-associated immortalizing gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby reducing, inhibiting, reducing, or stabilizing the amount, expression, or activity of the α -synuclein.

10. A method or formulation for reducing, inhibiting, reducing, or attenuating the exacerbation or severity of one or more symptoms of parkinson's disease, comprising: administering to the patient an amount of a BCL 2-associated immortal gene 3 (BAG 3) encoding nucleic acid, BAG3 protein, or BAG3 peptide, thereby reducing, inhibiting, or reducing the exacerbation or severity of one or more symptoms of parkinson's disease.

11. A method or formulation according to any one of claims 1 to 10, wherein the BAG3 encoding nucleic acid comprises an expression vector expressing a BAG3 protein or an active BAG3 peptide thereof.

12. The method or formulation of claim 11, wherein the expression vector further comprises a promoter comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, a tissue-specific promoter, or a heart-specific promoter.

13. The method or formulation of claim 11 or 12, wherein the expression vector comprises a viral vector, a heart-specific vector, a plasmid, or a yeast vector.

14. The method or formulation of claim 13, wherein the viral vector or cardiac-specific vector comprises an adenovirus vector, an adeno-associated virus vector (AAV), a coxsackievirus vector, a cytomegalovirus vector, an epstein barr virus vector, a paraviral vector, or a hepatitis viral vector.

15. The method or formulation of claim 14, wherein the AAV vector comprises a capsid protein having 90% or more sequence identity to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 maav 8, AAV9, AAV10, AAV11, or AAV 12.

16. The method or formulation of claim 12 or 13, wherein the expression vector is a pseudoviral vector.

17. The method or formulation of any one of claims 2-16, wherein the inflammation or inflammatory response is induced or increased by a cytokine.

18. The method or formulation of claim 17, wherein the cytokine comprises Tumor Necrosis Factor (TNF).

19. The method or formulation of any one of claims 1-18, wherein the patient expresses a sub-normal level of BAG3 in a tissue or organ, or is unable to detect expression or production of functional BAG3.

20. The method or formulation of any one of claims 1-19, wherein the inflammation or inflammatory response occurs in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney, or pancreas.

21. The method or formulation of any one of claims 11-20, wherein the expression vector further comprises a promoter, optionally comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue specific promoter.

22. The method or formulation of claim 21, wherein the promoter confers expression in the pulmonary system, lung, cardiovascular system, central nervous system, bone, skeletal joint, skeletal muscle, gastrointestinal system, stomach, small intestine, large intestine, liver, kidney, or pancreas.

23. The method or formulation of any one of claims 11-23, wherein the expression vector further comprises an AAV Inverted Terminal Repeat (ITR).

24. The method or formulation of any one of claims 11-23, wherein the expression vector further comprises a polyadenylation sequence and/or a stop codon.

25. The method or formulation of any one of claims 1-24, wherein the patient is a human.

26. The method or formulation of any one of claims 1-25, wherein the patient or human has a mutation in its endogenous BAG3 polynucleotide or polypeptide.

27. The method or formulation of any one of claims 1-26, wherein the patient or human has reduced expression or activity of an endogenous BAG3 polynucleotide or polypeptide.

28. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 0.1x10 ¹² vector genomes (vg) per patient weight (kg) (vg/kg) to about 1.0x10 ¹⁴ vg/kg.

29. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 1.0x10 ¹² vg/kg to about 0.5x10 ¹⁴ vg/kg.

30. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 1.0x10 ¹³ vg/kg.

31. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 9.0x10 ¹² vg/kg.

32. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 8.0x10 ¹² vg/kg.

33. The method or formulation of any one of claims 13-27, wherein the viral vector is administered or formulated at a dose of about 3.0x10 ¹² vg/kg to about 5.0x10 ¹² vg/kg.