BAG3 and protein quality control in the brain

Abstract

BAG3 proteins are used in the treatment of, for example, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body disease, vascular dementia, mixed dementia, and traumatic brain injury, for example, complicated by chronic traumatic encephalopathy (CTE). Therapeutically effective BAG3 compositions, uses of BAG3, and methods of treating BAG3 are described.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 337,914, filed May 3, 2022. The entire contents of the above application, including all text, tables, sequence listings, and figures, are hereby incorporated by reference.

[0002] Government support This invention was made with Government support under Grant No. HL91799 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] FIELD OF THEINVENTION Aspects of the present invention are directed to protein quality control of BAG3 in diseases or disorders of the brain, central nervous system, and nervous system, including traumatic injury. [Background technology]

[0004] background BAG3 plays a key role in the heart by controlling protein quality control through enhanced autophagy and reduced apoptosis. Most recently, we identified a group of functional variants in BAG3 found exclusively in individuals of African ancestry that conferred a nearly two-fold increased risk of either death or worsening heart failure requiring hospitalization. Summary of the Invention

[0005] overview Aspects of the present invention are directed to protein quality control of BAG3 in diseases or disorders of the brain, central nervous system, and nervous system, including traumatic injury. Examples include, but are not limited to, Parkinson's disease, Alzheimer's disease, and traumatic brain injury combined with chronic traumatic encephalopathy.

[0006] Aspects of the invention may be practiced without relying on the theoretical aspects presented. Moreover, theoretical aspects are presented with the understanding that applicants do not intend to be bound by the theory presented.

[0007] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable.Thus, this term includes, but is not limited to, genes and gene products from human and mouse.When a gene or gene product from a particular species is disclosed, it is understood that this disclosure is intended to be exemplary only and should not be interpreted as limiting unless the context in which it appears clearly indicates otherwise.Thus, for example, the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals, including but not limited to other mammals, fish, amphibians, reptiles, and birds.In a preferred embodiment, the gene or nucleic acid sequence is from human. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] definition The terminology used herein is intended to describe specific embodiments only and is not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context indicates otherwise. Furthermore, when the terms "including", "includes", "having", "has", "with" or variations thereof are used in any of the detailed description and / or claims, such terms are intended to be as inclusive as the term "comprising".

[0009] As used herein, "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) gene", "BCL2-associated athanogene 3 (BAG3) molecule" includes all family members, variants, cDNA sequences, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide strands, and the like (HGNC (939) Entrez Gene (9531) Ensembl (ENSG00000151929) OMIM (603883) UniProtKB (095817)). Similarly, "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) molecule" also refers to BAG3 polypeptides or active fragments, proteins, variants, derivatives, and the like thereof. Thus, the term "molecule" encompasses both the nucleic acid and amino acid sequences of BAG3.

[0010] As used herein, the terms "comprising," "comprise," or "comprised," and variations thereof, with respect to defined or described elements of an item, composition, apparatus, method, process, system, etc., are intended to be inclusive or open-ended and allow for additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements - or their equivalents, as appropriate - and that other elements may also be included and still be included within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0011] 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, which depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within 1 standard deviation or more than 1 standard deviation, according to practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% from a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a value. When a particular value is described in this application and claims, unless otherwise specified, the term "about" should be assumed to mean within an acceptable error range for that particular value.

[0012] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, and thus the description includes instances where the circumstance occurs and instances where it does not occur.

[0013] The term "expression vector" as used herein refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, such as in the production of antisense molecules, siRNA, and ribozymes, these sequences are not translated. Expression vectors can contain various control sequences. Control sequences refer to nucleic acid sequences necessary to transcribe and possibly translate operably linked coding sequences in a particular host organism. In addition to the control sequences that govern transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that perform other functions.

[0014] "Recombinant viral vector" refers to a viral vector that contains one or more heterologous gene products or sequences. Because many viral vectors exhibit size limitations related to packaging, heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses may become replication-deficient and require the missing functions to be provided during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line that has gene products necessary for replication and / or encapsidation). Modified viral vectors in which the polynucleotide to be delivered is carried outside the viral particle have also been described (see, e.g., Curiel, DT, et al., PNAS 88: 8850-8854, 1991).

[0015] The term "modulate" means, for example, to increase, enhance, increase, stimulate (act as an agonist), promote, decrease, reduce, inhibit, hinder, or antagonize (act as an antagonist) any of the aforementioned activities of the compounds embodied herein. Modulation can increase activity, for example, 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or more, compared to baseline value. Modulation can also decrease activity below baseline value.

[0016] As used herein, the term "agent" is intended to encompass any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent, or biological agent capable of preventing, improving, or treating disease or other medical conditions. This term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides, organic or inorganic molecules, and natural or synthetic compounds, etc. Agents can be assayed according to the method of the present invention during clinical trials, during pre-clinical testing, or at any stage after FDA approval.

[0017] As defined herein, a "therapeutically effective" amount (i.e., effective dosage) of a compound or agent means an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The composition may be administered from one or more times per day to one or more times per week (including once every other day). Those skilled in the art will understand that several factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a compound of the present invention may include a single treatment or a series of treatments.

[0018] "Treatment" is an intervention intended to prevent the onset of a disease or to modify the pathology or symptoms of a disease. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be designated as palliative care. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. Thus, "treating" or "treatment" of a condition, disorder, or pathology includes (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder, or pathology in a human or other mammal that may be afflicted with or susceptible to the condition, disorder, or pathology, but that has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or pathology; (2) inhibiting the condition, disorder, or pathology, i.e., preventing, alleviating, or delaying the onset or recurrence of the disease (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) palliating the disease, i.e., causing a reduction in the condition, disorder, or pathology, or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0019] Detailed Description Although it is known that tau and α-synuclein play some role in disease, the possibility that BAG3 plays a crucial role in removing them from the brain has not been revealed before the present invention.Furthermore, the concept that individuals with loss-of-function variants of BAG3 are at higher risk of brain diseases associated with abnormalities in protein quality control has also not been shown.Finally, the use of BAG3 as a therapeutic agent for Parkinson's disease, Alzheimer's disease, and CTE has not been addressed before.

[0020] In certain embodiments, the agent modulates the expression or amount of BCL2-associated athanogene 3 (BAG3) molecule, its protein or peptide in a target cell or tissue compared to a normal control, and includes an expression vector, an oligonucleotide, or a combination thereof that expresses a BAG3 protein or an active fragment thereof.

[0021] In certain embodiments, the agent induces expression of BAG3. In certain embodiments, the agent comprises a protein or peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or natural compound.

[0022] Bcl-2-associated athanogene 3 (BAG3), also known as BCL2-associated athanogene 3; MFM6; Bcl-2-binding protein Bis; CAIR-1; docking protein CAIR-1; BAG family molecular chaperone regulator 3; BAG-3; BCL2-associated athanogene 3; or BIS, is a cytoprotective polypeptide that competes with Hip-1 for binding to HSP70. The NCBI reference amino acid sequence of BAG3 can be found in Genbank under accession number NP_004272.2; Public GI:14043024. The NCBI reference nucleic acid sequence of BAG3 can be found in Genbank under accession number NM_004281.3 GI:62530382. Other BAG3 amino acid sequences include, but are not limited to, e.g., 095817.3 GI:12643665; EAW49383.1 GI:119569768; EAW49382.1 GI:119569767; and CAE55998.1 GI:38502170. The BAG3 polypeptides of the present invention can be variants of the polypeptides described herein, provided they retain functionality.

[0023] Table 1. Exemplary gene promoters that drive expression in the central nervous system (CNS) 1 TIFF2025514489000001.tif2111551. Harris J. et al. Anatomical characterization of Cre driver mice for neural circuit mapping and manipulation. Front Neural Circuits. 2014;8. doi:10.3389 / fncir.2014.00076. EXAMPLES

[0024] Abnormalities in protein quality control are thought to be important in the pathobiology of Alzheimer's disease, traumatic brain injury combined with chronic traumatic encephalopathy, and Parkinson's disease. We hypothesized whether abnormalities in BAG3 may contribute to the pathobiology of these CNS diseases and potentially serve as a therapeutic target. We also hypothesized whether the development of these CNS diseases may be more prevalent in individuals with mutations in BAG3. It is usually assumed that BAG3 is primarily present in the heart and skeletal muscles, and its role in the brain has received little attention.

[0025] The experimental results supported the hypothesis that BAG3 is involved in neurodegenerative diseases of the brain and may serve as an important therapeutic target: (1) in contrast to previous reports, BAG3 levels are indeed highly expressed in mouse brain, with brain levels comparable to those found in the heart; (2) BAG3 binds tightly to both tau and α-synuclein, peptides that play important roles in Alzheimer's disease / CTE and Parkinson's disease, respectively; and (3) common (>1%) loss-of-function gene variants found in individuals of African ancestry are unable to bind these disease-causing peptides.

Claims

1. A method of treating a neurodegenerative disease of the brain or central nervous system comprising administering a Bcl2-associated athanogene 3 (BAG3) polynucleotide, polypeptide, or active fragment thereof to a subject having or at risk of having a neurodegenerative disease of the brain or central nervous system to effect expression of a BAG3 polypeptide or an active fragment thereof in the brain or central nervous system, thereby treating or reducing the risk of the neurodegenerative disease of the brain or central nervous system.

2. 2. The method of claim 1, wherein the neurodegenerative disease of the brain or central nervous system is Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body disease, vascular dementia, or mixed dementia.

3. A method of treating traumatic brain injury comprising administering to a subject having or at risk of having traumatic brain injury a Bcl2-associated athanogene 3 (BAG3) polynucleotide, polypeptide, or active fragment thereof to effect expression of a BAG3 polypeptide or an active fragment thereof in the brain or central nervous system, thereby treating or reducing the risk of having traumatic brain injury.

4. The method of claim 3, wherein the traumatic brain injury is traumatic brain injury complicated with chronic traumatic encephalopathy (CTE).

5. The method of any one of claims 1 to 3, wherein the expression or amount of a BAG3 polynucleotide, polypeptide, or active fragment thereof is increased in cells or tissues of the brain or central nervous system compared to a normal control.

6. The method of any one of claims 1 to 3, wherein the BAG3 polynucleotide comprises an expression vector for expression of a BAG3 polypeptide or an active fragment thereof.

7. The method of claim 6, wherein the expression vector further comprises an expression control element.

8. The method of claim 7, wherein the expression control element comprises a promoter or enhancer.

9. The method of claim 8, wherein the promoter or enhancer comprises an inducible promoter or enhancer, a constitutive promoter or enhancer, a bicistronic promoter or enhancer, or a tissue-specific promoter or enhancer.

10. 9. The method of claim 7 or 8, wherein the expression control element, promoter, or enhancer is active in the brain or central nervous system.

11. The method of claim 10, wherein the expression control element, promoter, or enhancer that is active in the brain or central nervous system is selected from the expression control elements, promoters, or enhancers shown in Table 1.

12. The method of claim 7, wherein the expression control element comprises a cytomegalovirus (CMV) promoter.

13. The method of claim 6, wherein the expression vector crosses the blood-brain barrier.

14. The method of any one of claims 1 to 3, wherein the BAG3 polynucleotide, polypeptide, or active fragment thereof can be delivered across the blood-brain barrier or is administered directly to the brain or central nervous system.

15. The method of claim 6 , wherein the expression vector comprises a viral vector, a plasmid, or a yeast vector.

16. 16. The method of claim 15, wherein the viral vector comprises an adeno-associated viral (AAV) vector, an adenoviral vector, a lentiviral vector, a coxsackievirus vector, a cytomegalovirus vector, a retroviral vector, or an Epstein-Barr virus vector.

17. The method of claim 16, wherein the AAV vector comprises a recombinant AAV (rAAV) particle comprising an AAV capsid protein and a vector genome comprising a polynucleotide encoding a BAG3 polypeptide or an active fragment thereof.

18. The method of claim 17, wherein the vector genome further comprises one or more AAV inverted terminal repeats (ITRs), introns, stop codons, or polyA sequences.

19. The AAV capsid protein or the AAV ITRs (i) a capsid sequence that has 70% or more than 70% identity to the capsid sequence of AAV1, AAV2, AAV3, AAV-3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, or AAV-F VP1, and / or (ii) an ITR sequence having 70% or more than 70% identity to the AAV ITR sequences of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, or AAV-2i8; 19. The method of claim 17 or 18, comprising:

20. The method of claim 18 or 19, wherein the AAV ITRs are adjacent to the 5' or 3' end of the polynucleotide encoding a BAG3 polypeptide or an active fragment thereof.

21. The method of any one of claims 1 to 20, wherein the BAG3 polynucleotide, the BAG3 polypeptide, an active fragment thereof, an expression vector comprising a BAG3 polynucleotide, or a viral vector comprising a BAG3 polynucleotide is comprised in a pharmaceutical composition.

22. The method of any one of claims 1 to 20, wherein the BAG3 polynucleotide, the BAG3 polypeptide, an active fragment thereof, an expression vector comprising a BAG3 polynucleotide, or a viral vector comprising a BAG3 polynucleotide is contained in a microvesicle, nanovesicle, or nanoparticle.

23. The method of any one of claims 6 to 20, wherein the expression vector or viral vector is targeted to the cerebral cortex.

24. A composition comprising a Bcl2-associated athanogene 3 (BAG3) polynucleotide, polypeptide, and / or an agent that induces BAG3.

25. The composition of claim 24, wherein the agent modulates the expression or amount of a BCL2-associated athanogene 3 (BAG3) molecule, its protein or peptide in a target cell or tissue compared to a normal control.

26. 25. The composition of claim 24, wherein the agent comprises an expression vector expressing a BAG3 protein or an active fragment thereof, an oligonucleotide, or a combination thereof.

27. 27. The composition of claim 26, wherein the expression vector further comprises a promoter, the promoter comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue-specific promoter.

28. 27. The composition of claim 26, wherein the expression vector comprises a viral vector, a plasmid, or a yeast vector.

29. The composition of claim 26, wherein the vector crosses the blood-brain barrier.

30. The composition of claim 24, wherein BAG3 or an agent that induces BAG3 can be delivered across the blood-brain barrier or is administered directly.

31. 25. The composition of claim 24, wherein the agent comprises a protein or a peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or natural compound.

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