Quality control of BAG3 and proteins in brain

Abstract

The BAG3 proteins are useful in the treatment of Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, Lewy body disease, vascular dementia, mixed dementia and traumatic brain injury, such as chronic traumatic encephalopathy (CTE). A therapeutic BAG3 composition, use of BAG3, and a method of treatment of BAG3 are described.

Description

Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 337,914, filed on May 3, 2022. The entire contents of the foregoing application are incorporated herein by reference, including all text, tables, sequence listings, and drawings. Government funding

[0002] 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. Technology Neighborhood

[0003] An embodiment of the present invention is directed to quality control of BAG3 protein in diseases or disorders of the brain, central nervous system, nervous system, including traumatic injury. Background Art

[0004] BAG3 plays a critical role in the heart by regulating protein quality control by enhancing autophagy and reducing apoptosis. Recently, the applicants discovered a group of functional mutations in BAG3 found only in individuals of African ancestry that resulted in a nearly 2-fold increased risk of death or worsening heart failure requiring hospitalization. Summary of the invention

[0005] Embodiments of the present invention are directed to quality control of BAG3 proteins in brain, central nervous system, nervous system diseases or disorders, including traumatic injury. For example, traumatic brain injury including but not limited to Parkinson's disease, Alzheimer's disease, and concurrent chronic traumatic encephalopathy.

[0006] Embodiments of the present invention can be practiced without presenting theoretical aspects. Furthermore, when presenting theoretical aspects, applicants do not seek to be bound by the presented theory.

[0007] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs of any species to which the compositions and methods disclosed herein are applicable. Therefore, these terms include, but are not limited to, genes and gene products from humans and mice. It goes without saying that when a gene or gene product of a particular species is disclosed, the disclosure is exemplary only and should not be construed as limiting unless explicitly stated in the context. Therefore, for example, the genes disclosed herein, in certain embodiments, relate to nucleic acid and amino acid sequences of mammals, with the intent to include 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 human. definition

[0008] The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. As used herein, the singular forms "a", "an", and "the" also include the plural forms, unless the context clearly indicates otherwise. In addition, when the terms "include", "comprise", "contain", "have", "have", "with", or variations thereof are used in the detailed description and / or claims, these terms are intended to be included in a manner similar to the term "comprising".

[0009] "BAG3", "BAG3 molecule", "BCL2-associated immortality gene 3 (BAG3) gene", "BCL2-associated immortality gene 3 (BAG3) molecule" used herein include all family members, mutants, cDNA sequences, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide chains, etc. (HGNC (939) Entrez Gene (9531) Ensembl (ENSG00000151929) OMIM (603883) UniProtKB (095817)). Similarly, "BAG3", "BAG3 molecule", "BCL2-associated immortality gene 3 (BAG3) molecule" also refers to BAG3 polypeptides or their active fragments, proteins, variants, derivatives, etc. Therefore, the term "molecule" includes the nucleic acid sequence and amino acid sequence of BAG3.

[0010] As used herein, the terms "include," "comprising," or "containing," and variations thereof, when referring to elements of a definition or description of an item, composition, apparatus, method, process, system, etc., are intended to be inclusive or open-ended, allowing for the inclusion of other elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes the specified elements - or their equivalents as appropriate - and may include other elements and still belong to and fall within the scope / definition of the item, composition, apparatus, method, process, system, etc.

[0011] The term "approximately" or "approximately" means within an acceptable error range of a specific numerical value determined by a person of ordinary skill in the art, which error range depends on the measurement or determination method of the numerical value, i.e., the limitations of the measurement system. For example, according to the practice in the art, "approximately" can refer to 1 or more standard deviations. Alternatively, "approximately" can also mean within 20% of a given value, preferably within 10%, more preferably within 5%, and more preferably within 1%. Alternatively, particularly in terms of biological systems or processes, the term can refer to within an order of magnitude, preferably within 5 times, and more preferably within 2 times of a numerical value. In the case of describing specific values ​​in the present application and claims, unless otherwise stated, it should be assumed that the term "approximately" means within an acceptable error range of a specific value.

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

[0013] As used herein, the term "expression vector" refers to a vector containing a nucleic acid sequence encoding 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, ribozymes, etc., these sequences are not translated. Expression vectors can contain various control sequences, which refer to nucleic acid sequences necessary for transcription and possible translation of an operably linked coding sequence in a specific host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences with other functions.

[0014] A "recombinant viral vector" refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors have size limitations associated with packaging, heterologous gene products or sequences are usually introduced by replacing one or more parts of the viral genome. Such viruses may be replication-defective and require the deleted functions to be provided transiently (in trans) during viral replication and packaging (e.g., by using a helper virus or a packaging cell line carrying gene products necessary for replication and / or packaging). Modified viral vectors that carry the polynucleotide to be delivered on the outside of the viral particle have also been described (see, e.g., Curiel, DT, et al., PNAS 88:8850-8854, 1991).

[0015] The term "modulate" refers to any reference to an activity of a compound described herein, such as increasing, enhancing, increasing, activating (as an agonist), promoting, decreasing, reducing, inhibiting, blocking, or antagonizing (as an antagonist). Modulation can increase an activity by 1, 2, 3, 5, 10, 100 times, etc., compared to a baseline value. Modulation can also reduce an activity below a baseline value.

[0016] As used herein, the term "agent" refers to any molecule, chemical entity, composition, drug, therapeutic agent, chemotherapeutic agent or biological agent that can prevent, improve or treat a disease or other condition. The term includes small molecule compounds, antisense agents, siRNA agents, antibodies, enzymes, peptides, organic or inorganic molecules, natural or synthetic compounds, etc. The agent can be tested according to the method of the present invention during clinical trials, pre-clinical testing, or at any stage after FDA approval.

[0017] As defined herein, the "therapeutically effective" amount (i.e., effective dose) of a compound or formulation refers to an amount sufficient to produce a desired effect of treatment (e.g., clinical). The composition is administered in a manner from once or more per day to once or more per week (including administration every other day). It is understood by those skilled in the art that certain factors may affect the dose and time required for effective treatment of the subject, including but not limited to the severity of the disease or disorder, previous treatment, the general health and / or age of the subject, and other diseases present. In addition, treatment of subjects with a therapeutically effective amount of the compound of the present invention may include a single treatment or a series of treatments.

[0018] "Treatment" is an intervention aimed at preventing the development of a disease or altering a pathology or symptom. Thus, "treatment" refers to both curative treatment and prophylactic or preventive measures. "Treatment" may also specifically refer to palliative care. Those in need of treatment include those already suffering from the disease as well as those in need of prevention of the disease. Thus, "treating" or "treating" a condition, disorder, or condition includes: (1) preventing or delaying the onset of clinical symptoms of the condition, disorder, or condition in a human or other mammal that may be suffering from or susceptible to the condition, disorder, or condition but does not yet develop or show clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, or (3) ameliorating the disease, i.e., causing regression of the condition, disorder, or condition or at least one clinical or subclinical symptom thereof. The benefit to the individual receiving the treatment is statistically significant or at least perceptible to the patient or physician. DETAILED DESCRIPTION

[0019] It is well known that tau and alpha-synuclein play a role in disease, but before the present invention, the possibility that BAG3 plays a key role in clearing these two proteins from the brain had not been demonstrated. In addition, it has not been previously demonstrated that individuals with BAG3 loss-of-function variants would be at high risk for brain diseases associated with abnormal quality control of proteins. Finally, the use of BAG3 as a therapy in Parkinson's disease, Alzheimer's disease and CTE has not been previously involved.

[0020] In certain embodiments, the agent modulates the expression or amount of a BCL2-associated immortality gene 3 (BAG3) molecule, protein, or peptide thereof in a target cell or tissue as compared to a normal control, and the agent comprises an expression vector, 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 a peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or natural compound.

[0022] Bcl-2-associated immortality gene-3 (BAG3), also known as BCL2-associated immortality gene 3, MFM6, Bcl-2 binding protein Bis, CAIR-1, docking protein CAIR-1, BAG family chaperone regulator 3, BAG-3, BCL2 binding immortality gene 3, or BIS, is a cytoprotective polypeptide that competes with Hip-1 for binding to HSP70. The NCBI reference amino acid sequence of BAG3 has a Genbank accession number of NP_004272.2 and a public GI of 14043024. The NCBI reference nucleic acid sequence of BAG3 has a Genbank accession number of NM_004281.3 and a GI of 62530382. Other BAG3 amino acid sequences include, but are not limited to: 095817.3 GI: 12643665, EAW49383.1 GI: 119569768, EAW49382.1 GI: 119569767, and CAE55998.1 GI: 38502170. The BAG3 polypeptides of the present invention may be variants of the polypeptides described herein, but must retain their functionality. Table 1 | Exemplary gene promoters driving expression in the central nervous system (CNS) 1 1. Table adapted from 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.2014.00076.00076 Example

[0023] Abnormalities in protein quality control are thought to be important pathobiological factors in Alzheimer's disease, chronic traumatic encephalopathy complicated by traumatic brain injury, and Parkinson's disease. It has been hypothesized whether abnormalities in BAG3 contribute to the pathobiology of these central nervous system diseases and may be a therapeutic target. In addition, whether the incidence of these central nervous system diseases is higher in individuals carrying mutations in the BAG3 gene. It is generally believed that BAG3 is mainly present in the heart and skeletal muscle, and little attention has been paid to its role in the brain.

[0024] The results support the hypothesis that BAG3 plays a role in brain neurodegenerative diseases and could serve as an important therapeutic target: 1) BAG3 is expressed at high levels in the brain of mice; in fact, in contrast to previous reports, the levels of BAG3 in the brain are comparable to those in the heart; 2) BAG3 is tightly coupled to tau and α-synuclein, peptides that play important roles in Alzheimer's / CTE and Parkinson's, respectively; and 3) common (>1%) loss-of-function genetic mutants found in individuals of African descent are unable to bind to these pathogenic peptides.

Claims

1. A method for treating a neurodegenerative disease of the brain or central nervous system, comprising administering a Bcl2-associated immortality gene 3 (BAG3) polynucleotide, polypeptide or an active fragment thereof to a subject suffering from or at risk of a neurodegenerative disease of the brain or central nervous system to provide expression of the BAG3 polypeptide or an active fragment thereof to the brain or central nervous system, thereby treating or reducing the risk of the neurodegenerative disease of the brain or central nervous system.

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 for treating traumatic brain injury, comprising administering a Bcl2-associated immortality gene 3 (BAG3) polynucleotide, polypeptide or active fragment thereof to a subject suffering from or at risk of traumatic brain injury to provide expression of the BAG3 polypeptide or active fragment thereof to the brain or central nervous system, thereby treating or reducing the risk of traumatic brain injury.

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

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

6. The method according to any one of claims 1-3, wherein the BAG3 polynucleotide comprises an expression vector for expressing the BAG3 polypeptide or an active fragment thereof. The method according to 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 an 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. 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 according to claim 10, wherein the expression control element, promoter or enhancer active in the brain or central nervous system is selected from the expression control elements, promoters or enhancers listed 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 according to any one of claims 1-3, wherein the BAG3 polynucleotide, polypeptide or active fragment thereof can be delivered across the blood-brain barrier or directly administered to the brain or central nervous system.

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

16. The method of claim 15, wherein the viral vector comprises an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a coxsackievirus vector, a cytomegalovirus vector, a retrovirus 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 the 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 poly A sequences.

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

20. The method of claim 18 or 19, wherein the AAV ITR flanks the 5' or 3' end of the polynucleotide encoding the BAG3 polypeptide or an active fragment thereof.

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

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

23. The method of any one of claims 6-20, wherein the expression vector or viral vector targets the cerebral cortex.

24. A composition comprising Bcl2-associated immortality gene 3 (BAG3) polynucleotides, polypeptides and / or agents that induce BAG3.

25. The composition of claim 24, wherein the agent modulates the expression or amount of BCL2-associated immortality gene 3 (BAG3) molecules, proteins or peptides thereof in target cells or tissues compared to normal controls.

26. The composition of claim 24, wherein the formulation comprises an expression vector, an oligonucleotide or a combination thereof that expresses the BAG3 protein or an active fragment thereof.

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

28. 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 the BAG3 or an agent inducing the BAG3 can be delivered across the blood-brain barrier or directly administered.

31. 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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