WO2025255576A1 - Compositions and methods for the treatment of neurodegeneration - Google Patents

Abstract

Compositions and methods for the treatment of neurodegenerative diseases and disorders are disclosed.

Description

Compositions and Methods for the Treatment of Neurodegeneration

By

Hakon Hakonarson Arthur Feldman

Cross-Reference to Related Applications

This application claims the benefit of the filing date of U.S. Provisional Application No. 63/567,649, filed June 7, 2024, the entire contends of which is incorporated by reference herein.

Grant Support Statement

This invention was made with government support under grant number HL091799 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field of the Invention

The present invention relates to the fields of macroautophagy, neuroinflammation and neurodegeneration. More specifically, the invention provides agents useful for the treatment of neurodegenerative disorders associated with aberrant protein clearing in patients in need thereof, via modulation of BAG3 and CLEC16A function and, or expression levels.

Background of the Invention

Several publications and patent documents are cited through the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

BAG3 plays multiple existential roles in the heart. By coupling the beta-1 adrenergic receptors (bl -AR) to the L-type Ca2⁺ channel BAG3 facilitates increased contractility during exercise or disease when there is a need for increased cardiac output. Binding to Bcl2 by BAG3 results in a decrease in apoptosis through the intrinsic, mitochondrial-dependent and terminal caspase-3 activation of apoptosis by in part suppressing the release of the inhibitor of caspase activation (cIAPl) from caspases and in particular caspase 3. BAG3 also serves to support autophagy by binding to the ATPase domain on both small (Hsp-B6 and Hsp-B8) and large (Hsp70) heat shock proteins thereby forming complexes with mis-folded proteins, parkin, which can significantly damage mitochondria (mitophagy). These complexes then bind with phagophores as they engulf the protein debris and organelles carried by BAG3. The phagophore then forms an autophagophore that contains the misfolded proteins, protein debris and organelles. The autophagophore then fuses with a lysozyme. The lysozyme releases the acidic material that includes digestive enzymes into the auto-phagophore which dissolves the proteins it carries. The resulting amino acids and proteinaceous debris are then released into the cytoplasm where they are used by the cell to build new proteins and protein structures.

Additionally, BAG3 binds to TOM20/TOM22 thereby regulating control of the uptake of proteins through the outer mitochondrial membrane and modifying the ability of mitochondria to manufacture proteins from small fragments of proteins that are transported into the mitochondria. BAG3 also binds to the mitochondrial uniporter which helps maintain the negative electrical potential across the mitochondrial membrane that is necessary for maintaining the transport of Ca²⁺ levels of solute transit. Through these roles BAG3 maintains the negative charge of the mitochondria which facilitates not only respiratory gas exchange but also the enzymes of the tri-carboxylic acid cycle. Lastly, BAG3 also binds to a moiety += of the TNFR1 receptor preventing its activation through binding to TNF-alpha and thus preventing inflammation.

Mutations in BAG3, such as the P209L mutation, can result in profound loss of normal skeletal muscle structure and severe myofibrillar disarray with only mild dilation seen in the heart. This mutation is a gain of function mutation which results in a large amount of proteinaceous debris being brought into the proteosome, functionally clogging it and leading to all of the adverse events associated with decreases in BAG3. Such mutations can cause paralysis of the intercostal muscles, which can result in patients having a reduced life expectancy and being wheelchair and/or respirator bound by their early teens. Alternatively, the replacement of the leucine by a serine leads to a Charcot-Marie-Tooth like phenotype whereas replacement of the leucine by a threonine is associated with a pure dilated cardiomyopathy phenotype. Mutations at amino acid 209 are quite rare and result in an extremely severe phenotype which appears in young children.

A single study in Europe has followed a cohort of patients with BAG3 variants that numbered just slightly greater than 100 patients. In that study of adults, patients with BAG3 variants had an earlier presentation and a worse course than did those with other forms of dilated cardiomyopathy. Most of the affected family members died of worsening heart failure suggesting that BAG3 insufficiency at least in that family could not be prevented by the use of an ICD. To date, no patient having homozygous loss of BAG3 has ever been reported. In fact, in animal models, homozygous deletions are lethal by approximately 8 weeks of age. Importantly, BAG3 deficiency leads to markedly increased levels of PARP whereas increased levels of BAG3 (normalization) leads to a decrease in PARP. Seminal studies by Ted and Valina Dawson reported that increased levels of PARP drive pathologic alpha synuclein accumulations in neurodegeneration.(7./T76d is implicated in multiple neurodegenerative diseases. Turning off CLEC16A in adult mice leads to, severe neurological symptoms with neuroinflammation and progressive neurodegeneration resembling spinocerebellar ataxia.

Given the large number of neurodegenerative diseases correlated with CLEC16A and BAG3 loss or dysfunction, it is clear that new treatments and therapeutic agents which ameliorate the effects of neurodegenerative disorders and disease and reduce associated symptoms, are urgently needed.

Summary of the Invention

In accordance with the present invention, methods of treating a neurodegenerative disorder in a subject are provided. In certain embodiments, the methods comprise administering an effective amount of one or more agents that elevate intracellular levels of BAG3 in neuronal cells, thereby providing therapeutic benefit to said subject. In certain embodiments the one or more agents that elevates intracellular levels of GAB3 also elevates intracellular levels of CLEC16A.

In another aspect of the invention methods treating a neurodegenerative disorder in a subject in need thereof, comprising administering an effective amount of one or more agents that elevate intracellular levels of BAG3 and one or more agents that elevate intracellular levels of CLEC16A, thereby providing therapeutic benefit to said subject, are provided.

In another aspect, methods for treating neurodegenerative disease in a subject in need thereof, the method comprising a) diagnosing the subject with the neurodegenerative disease, and b) administering an effective amount of one or more agents that elevate intracellular levels of BAG3 are provided. In certain embodiments, the one or more agents include a BAG3 polypeptide or functional fragment thereof and a CLEC16A polypeptide or functional fragment thereof In certain embodiments, the BAG3 and CLEC16A are present in an expression vector. In certain embodiments, the expression vector further comprises an expression control element, promoter or enhancer that is active in the brain or central nervous system. In certain embodiments, the expression control element, promoter or enhancer active in the brain or central nervous system is selected from an expression control element, promoter or enhancer set forth in Table 1, such as a cytomegalovirus (CMV) promoter. In certain embodiments, the expression vector crosses the blood-brain barrier.

In certain embodiments, the BAG3 polynucleotide, polypeptide or active fragment thereof is transmittable across the blood brain barrier or directly administered to the brain or central nervous system. In certain embodiments, the expression vector comprises a viral vector, plasmid, or a yeast vector. In certain embodiments, the viral vector comprises an adeno- associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a coxsackie viral vector, a cytomegalovirus vector, retroviral vector, a lipid nanoparticle, or an Epstein Barr virus vector.

In certain embodiments, the treatment delays neurodegenerative disease symptom progression when compared to an untreated control.

Still other aspects and advantages of these compositions and methods are readily apparent and described further in the following detailed description of the invention.

Brief Description of the Drawings

Figure 1. Immunofluorescent images depicting induced human pluripotent stem cells (iPSC’s) stained with the indicated antibodies. Confocal imaging shows abundant BAG3 and alpha-synuclein under baseline conditions in differentiated cells.

Figure 2. iPSCs imaged after augmenting expression of BAG3 with an AAV-BAG3 vector or silencing expression of BAG3 with using siRNA.

Figure 3. iPSCs stained with beta tubulin III to outline the cells, Clecl6A and BAG3. The lower row was also treated with AAV-BAG3. Figure 4. iPSCs stained with beta tubulin III to outline the cells, Clecl6A and BAG3. The upper row shows the cells on the incubation medium. The lower row shows the cells after 24 hours after treatment with AAV-BAG3.

Figure 5. The proximity ligation assay (PLA) technique.

Figure 6. Results of the PLA.

Figure 7. Baseline levels of a-synuclein in normal human dopamine-producing neurons derived from normal human stem cells. Parkinson’s Disease iCell DopaNeurons display increased a-synuclein accumulation. GBA N370S, LRRK2, G2019S, and SNCA A53T iCell DopaNeurons (42 DIV) were analyzed for a-synuclein accumulation using a Meso Scale Discovery assay, a-synuclein in the disease lines increased as compared to the apparently healthy normal (AHN) donor line (n=4, One-way ANOVA with Dunnett’s multiple comparisons).

Figure 8. Analysis of a-syn in mutant GBA after BAG3 or AAV-BAG3 administration.

Figure 9A-9B. Representative images of cells from Parkinsons’ s disease patients after treatment with a control or with AAV-BAG3.

Figure 10A-10C. U-Shaped Curve of AAV9 on aSyn AT53T cells.

Figure 11 A-l ID. AAV treatment in GBA mutant cell line. Figure HA shows levels of BAG3 in neurons harboring a GBA variant or an A53T variant compared to BAG3 levels in wild-type neurons. Figure 1 IB shows the effect of increasing metformin concentrations on Bag 3 levels. Representative images of the neurons in Figures 11 A and 1 IB are shown in Figures 11C and 1 ID respectively.

Detailed Description of the Invention

While alpha-synuclein is known to play a role in neurological diseases, such as Parkinson’s Disease, the critical role of BAG3 in clearing deleterious proteins from the brain has not been previously described. Furthermore, studies have demonstrated the heightened susceptibility of individuals with loss of functional variants in BAG3. To date, abnormalities in protein quality control in the brain have not been linked to modulation of BAG3 expression levels. The data provided herein demonstrate that modulation of BAG3 expression levels can be used to advantage to ameliorate symptoms of neurological diseases, such as Parkinson’s Disease.

Definitions

For purposes of the present invention, “a” or “an” entity refers to one or more of that entity; for example, “a cDNA” refers to one or more cDNA or at least one cDNA. As such, the terms “a” or “an,” “one or more” and “at least one” can be used interchangeably herein. It is also noted that the terms “comprising,” “including,” and “having” can be used interchangeably. Furthermore, a compound “selected from the group consisting of’ refers to one or more of the compounds in the list that follows, including mixtures (i.e. combinations) of two or more of the compounds. According to the present invention, an isolated, or biologically pure molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route.

As used herein, the terms “component,” “composition,” “composition of compounds,” “compound,” “drug,” “pharmacologically active agent,” “active agent,” “therapeutic,” “therapy,” “treatment,” or “medicament” are used interchangeably herein to refer to a compound or compounds or composition of matter which, when administered to a subject (human or animal) induces a desired pharmacological and/or physiologic effect by local and/or systemic action. The terms “agent” and “test compound” denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues.

It is also contemplated that the term “compound” or “compounds” refers to the compounds discussed herein and includes precursors and derivatives of the compounds, and pharmaceutically acceptable salts of the compounds, precursors, and derivatives. The invention also includes prodrugs of the compounds, pharmaceutical compositions including the compounds and a pharmaceutically acceptable carrier, and pharmaceutical compositions including prodrugs of the compounds and a pharmaceutically acceptable carrier.

The phrase "consisting essentially of when referring to a particular nucleotide or amino acid means a sequence having the properties of a given SEQ ID NO. For example, when used in reference to an amino acid sequence, the phrase includes the sequence per se and molecular modifications that would not affect the functional and novel characteristics of the sequence.

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used herein “BAG3,” “BAG3 molecules,” “BCL2-associated athanogene 3 (BAG3) genes,” “BCL2-associated athanogene 3 (BAG3) molecules” are inclusive of all family members, mutants, cDNA sequences, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc. (HGNC (939) Entrez Gene (9531) Ensembl (ENSG00000151929) OMIM (603883) UniProtKB (095817)). Similarly, “BAG3,” “BAG3 molecules,” “BCL2-associated athanogene 3 (BAG3) molecules” also refer to BAG3 polypeptides or active fragment thereof, proteins, variants, derivatives etc. The term “molecule,” thus encompasses both the nucleic acid sequences and amino acid sequences of BAG3.

BAG3-mediated selective macroautophagy is involved in the clearance of aggregated proteins associated with age-related neurodegenerative disorders, like Alzheimer’s disease (tau- protein), Huntington’s disease (mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). BAG3 is also anti-apoptotic; therefore, BAG3 is a promising candidate in the search for therapeutics that might block neurodegeneration. BAG3 also plays a role in neuroinflammation mediated by microglia, a pathway thought to play a role in the pathogenesis of PD through the regulation of a-synuclein.

In heart cells, a reduction in BAG3 leads to a dilated cardiomyopathy, and similarly in the brain, a reduction in BAG3 also causes changes consistent with decreased autophagy resulting in neurodegeneration. BAG3 also maintains the homeostasis of the mitochondria by activating the mitochondrial pump (uniporter). In the heart, BAG3 also couples the beta- adrenergic receptor and the L type calcium channel to maintain excitation contraction coupling. Thus, an absence of BAG3 leads to many cellular perturbations and abnormal cell function.

As used herein “CLEC16A,” “CLEC16A molecules,” “C-type lectin domain containing 16A (CLEC16A) genes,” “C-type lectin domain containing 16A (CLEC16A) molecules” are inclusive of all family members, mutants, cDNA sequences, alleles, fragments, species, coding and noncoding sequences, sense and antisense polynucleotide strands, etc. (Gene ID: 23274; HGNC (29013); Ensembl (ENSG00000038532); MIM(611303)). Similarly, “CLEC16A,” “CLEC16A molecules,” “C-type lectin domain containing 16A (CLEC16A) molecules” also refer to CLEC16A polypeptides or active fragment thereof, proteins, variants, derivatives etc. The term “molecule,” thus encompasses both the nucleic acid sequences and amino acid sequences of CLEC16A.

Regional mapping of CLEC16A first identified this locus as a novel Type 1 Diabetes (T1D) susceptibility locus within a 233-kb LD block on chromosome 16p 13. CLEC16A modulation is associated with the susceptibility to nearly 20 human diseases, including Typel diabetes (T1D), multiple sclerosis (MS), primary adrenal insufficiency (PAI), systemic lupus erythematosus (SLE), Crohn’s disease (CD), selective immunoglobulin A deficiency (IgA), alopecia areata (AA), juvenile idiopathic arthritis (JIA), rheumatoid arthritis (RA), primary biliary cirrhosis (PBC) and asthma. CLEC16A encodes an E3 ubiquitin ligase which regulates mitochondrial quality control by clearing damaged or aged mitochondria through a type of selective autophagy, termed mitophagy. CLEC16A forms and stabilizes a tripartite mitophagy complex with the E3 ubiquitin ligase RNF41/Nrdpl and the deubiquitinase USP8, which together regulate mitophagic flux by controlling the activity of the mitophagy-effector Parkin (PRKN), an important gene in Parkinson disease.

CLEC16A is required for normal glucose stimulated insulin release through its effect on mitophagy. PINKl/Parkin mediated mitophagy is shown to restrain innate immunity in vivo and that dysfunction in mitophagy leads to upregulation of the stimulator of interferon genes (STING) pathway causing an inflammatory phenotype that ultimately may contribute to the loss of dopaminergic neurons. Importantly, components of innate immunity activation are also found in patients with Parkin mutations. A vicious cycle has been proposed whereby inflammation leads to more mitochondrial damage and that promotes further inflammatory processes. It may therefore be that synergy between mitochondrial dysfunction and immune responses is required for Parkinson’s disease to develop.

Incomplete mitophagy, caused by a deficiency in CLEC16A, predisposes mice to a cascade of altered immune signaling functions leading to pathogenic inflammation with hyperactive NK cells under dysregulated mitophagy settings. Additionally, dysfunctional mitochondria have been shown to contribute to human diseases by damaging proteins, lipids and DNA molecular structure and function, as a result of hyperactive inflammatory pathways. Mitophagy orchestrates the autophagic degradation of dysfunctional mitochondria preventing their pathological accumulation and contributing to cellular homeostasis.

“Adenosine monophosphate-activated protein kinase” or “Adenosine monophosphate kinase” (AMPK) refers to a cellular energy sensor that plays a role in regulating metabolic processes and energy homeostasis. When activated, AMK stimulates energy-producing pathways and inhibits energy consuming pathways.

An “AMPK activator” or “activator of AMPK” refers to any compound which increases expression of AMPK or levels of AMPK proteins in a subject, or any compound which catalyzes the interaction of AMPK ligand with any of the receptors. Exemplary AMPK activators include, for example exogenous AMPK, Metformin, AICAR, Kazinol B, Marein, Amarogentin, A 769662, PF 06409577, Metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, Nilotinib, Phenformin, Nilotinib hydrochloride monohydrate, Adenosine 5 '-monophosphate monohydrate, Hispidulin, MK 8722, Euphorbiasteroid, ASP4132, GSK621, EX229 (compound 991), Trans- feluric acid, 0-304, MK 3903, BAM 15, ligustroflavone, ETC-1002, BC1618, IMM-H007, IM156, Chikusetsusaponin IVa, Poricoic acid A, 7-Methoxyisoflavone, Urolithin B, Danthron, Demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, Malvidin-3- O-arabinoside chloride, RSVA 405, Etilefrin, COH-SR4, Buformin, Buformin hydrochloride, PT1, Bempedoic acid, 3a-Hydrocymogrol, Ampkinone, and combinations thereof. In certain embodiments, vectors encoding a functional AMPK protein may be used as an AMPK activator. A list of recently identified indirect and direct AMPK activators is provided in Steinberg et al. (Nat Rev Drug Dis 2019; 18(7): 527-55141) which is incorporated in its entirety.

The phrase “proximity ligation assay” (PLA) as used herein refers to a method which extends the capabilities of traditional immunoassays to include direct detection of proteins, protein interactions, extracellular vesicles and post translational modifications with high specificity and sensitivity. Protein targets can be readily detected and localized with single molecule resolution and objectively quantified in unmodified cells and tissues. Utilizing only a few cells, sub-cellular events, even transient or weak interactions, are revealed in situ and subpopulations of cells can be differentiated. Within hours, results from conventional coimmunoprecipitation and co-localization techniques can be confirmed.

The phase “neurodegenerative diseases” or “neurodegenerative disorders” refers to a disease which progressively affects the functioning of the nervous system. Generally, the functioning of the nerve cells, and more particularly of the neurons, becomes altered. These diseases develop at different rates and are often irreversible. More particularly, neurodegenerative diseases refers to the following diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies, Pick's disease, progressive supranuclear palsy, multiple sclerosis, Alpers' disease, batten disease, Benson's syndrome, Cerebro-oculo-facio-skeletal (COFS) syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, dementias, Friedreich's ataxia, Gerstmann-Strussler-Scheinker disease, Lewy body syndrome, Leigh's disease, monomelic amyotrophy, motor neuron diseases, multiple system atrophy, opsoclonus myoclonus, progressive multifocal leukoencephalopathy, Parkinson's disease, primary progressive aphasia, progressive supranuclear palsy, spinocerebellar ataxia, spinal muscular atrophy, kuru, and Shy-Drager syndrome human or animal prion diseases for example such as bovine spongiform encephalopathy, and myopathy.

In certain embodiments, the neurodegenerative disease is Parkinson’s Disease (PD). Experiments described herein were carried out in whole animals including mice with a heterozygous knock-out of BAG3, the data recently obtained from the CLEC16A knock-out mouse and from human stem cells obtained from patients with PD who harbor one of three heterozygous mutations that have been linked to PD: the LRRK variant, the GBA variant or a transformed neuronal cell line in which a variant was inserted at aa 53 where a T replaces an A53T.

Autosomal dominant mutations in the Leucine-Rich-Repeat Kinase 2 (LRRK) gene are recognized as one of the most common genetic contributors to the development of PD with various ethnic populations affecting the outcome. However, sporadic occurrence of LRRK variants have also been identified. LRRK2 is a 2527 amino acid protein with multiple independently acting domains and its expression is increased following inflammation. Much like BAG3, LRRK serves multiple roles in the cell including support of vesicular trafficking, autophagy and immune response through genetic variants at multiple sites. Also, mutations in the LRRK2 gene have been associated with dysregulation of endocytic pathways in iPS derived from alterations of the dopaminergic (DAergic) system neurons carrying a mutation in the LRRK2 gene. However, all of the pathogenic LRRK2 mutations are within the GPTase and kinase domains and affect kinase activity.

A second genetic variant that has been associated with PD are those that affect lysosomal function, the most common being an inherited deficiency of the enzyme glucocerebrosidase (GBA) which when inherited in an autosomal recessive pattern leads to Gaucher’s disease. A portion of patients presenting with GD have clinical features of PD and GD-associated heterozygous mutations are a strong risk factor for idiopathic PD. A mouse model of GBA showed features of synucleinopathies including progressive accumulation of proteinase K resistant a-synuclein accumulation and cognitive defects. These abnormalities could be reduced by administering a vector containing recombinant GBA. Animal models have shown that manipulation of presumptive pathways including providing the GBA substrate and studies in embryonic fibroblasts of GBA KO mice showed findings consistent with a role for GBA mutations in PD. Consistent with the results in animal models, it was notable that all of the GBA cases that have been examined post-mortem harbored a-synuclein pathology.

“Sample” or “patient sample” or “biological sample” generally refers to a sample which may be tested for a particular molecule, such as a marker described hereinbelow. Samples may include but are not limited to cells, body fluids, including blood, serum, plasma, cerebral spinal fluid, urine, saliva, tears, pleural fluid and the like.

The terms “agent” and “compound” are used interchangeably herein and denote a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Biological macromolecules include siRNA, shRNA, antisense oligonucleotides, peptides, and peptide/DNA complexes. Agents and compounds may also be referred to as “test agents” or “test compounds” which are evaluated for potential biological activity by inclusion in screening assays described herein below.

The term "delivery" as used herein refers to the introduction of foreign molecule (i.e., miRNA containing nanoparticle) into cells. The term "administration" as used herein means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term "delivery".

METHODS OF TREATMENT

The invention described herein includes methods for treating neurodegenerative diseases and disorders. Neurodegenerative diseases and disorders include, without limitation, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies, Pick's disease, progressive supranuclear palsy, multiple sclerosis, Alpers' disease, batten disease, Benson's syndrome, Cerebro-oculo-facio-skeletal (COFS) syndrome, corticobasal degeneration, Creutzfeldt-Jakob disease, dementias, Friedreich's ataxia, Gerstmann- Strussler-Scheinker disease, Lewy body syndrome, Leigh's disease, monomelic amyotrophy, motor neuron diseases, multiple system atrophy, opsoclonus myoclonus, progressive multifocal leukoencephalopathy, Parkinson's disease, primary progressive aphasia, progressive supranuclear palsy, spinocerebellar ataxia, spinal muscular atrophy, kuru, and Shy-Drager syndrome human or animal prion diseases for example such as bovine spongiform encephalopathy, and myopathy. In certain embodiments, the neurodegenerative disease is Parkinson’s disease (PD).

The phrase “in need of treatment” as used herein refers to a judgment made by a caregiver (e.g., physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care givers expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.

By “treatment” and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, or stabilize, a pathological condition or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. It is understood that treatment, while intended to cure, ameliorate, or stabilize, a disease, pathological condition, or disorder, need not actually result in the cure, ameliorization, and stabilization. The effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms. Thus, for example, characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.

As used herein, the terms “modulate”, “modulation” or “modulation” refer to changing the rate at which a particular process occurs, inhibiting a particular process, reversing a particular process, and/or preventing the initiation of a particular process. In certain embodiments, the term “modulate” as used herein refers to increasing/promoting or decreasing/inhibiting a particular cellular, biological or signaling function associated with the normal activities of BAG3 or CLEC16A molecules described herein. For example, the term modulate refers to the ability of a test compound or test agent to increase intracellular levels of one or more of BAG3 and CLEC16A, thereby treating the neurodegenerative disease.

As used herein, the phrase “effective amount” of a compound or pharmaceutical composition refers to an amount sufficient to modulate symptoms associated with neurodegenerative diseases or disorders in an animal, especially a human, including without limitation mitigation of the negative health consequences associated with mitochondrial damage and or reducing such consequences by prophylactic administration prior to the onset of symptoms.

Determination of an effective amount of the compound(s) for a particular use and mode of administration is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in animals may be formulated to achieve a specific circulating blood or serum concentration of the metabolite active compound. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via the desired route of administration is well within the capabilities of skilled artisans. Initial dosages of compound can also be estimated from in vivo data, such as animal models. Animal models useful for testing the efficacy of the active metabolites to treat or prevent the various diseases described above are well-known in the art. Animal models suitable for testing the bioavailability and/or metabolism of compounds into active metabolites are also well- known. Ordinarily skilled artisans can routinely adapt such information to determine dosages of particular compounds suitable for human administration.

Dosage amounts will typically be in the range of from about 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active metabolite compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and/or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.

Generally, the initial therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the compound or a pharmaceutically acceptable salt thereof. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the compound or a pharmaceutically acceptable salt thereof. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the compound or a pharmaceutically acceptable salt thereof.

An exemplary method for treating a neurodegenerative disease or disorder entails administering a pharmaceutically effective amount of an agent that increases intracellular levels of one or more of BAG3 and CLEC16A to a subject in need thereof. In certain embodiments, BAG3 and/or CLEC16A are administered as the agent. In certain embodiments, the agents are present in a vector, as described below. In certain embodiments, treatment can also include administration of an AMPK activator.

In certain embodiments, treatment can also include administration of an auxiliary therapeutic agent. In certain embodiments, the auxiliary therapeutic agent is LRRK2 kinase inhibitor. Exemplary LRRK2 kinase inhibitors include, without limitation, MLi-2, PF-06447475, PF-06685360, GNE-0877, GNE-7915, GSK2578215A, HG-10-102-1, LRRK2-IN-1, and those described in WO 2019/074810 Al and Wojewska DN, et al. LRRK2 Targeting Strategies as Potential Treatment of Parkinson's Disease. Biomolecules. 2021 lul 26; 11(8): 1101. doi: 10.3390/bioml 1081101. PMID: 34439767; PMCID: PMC8392603, each of which is incorporated herein by reference.

In certain embodiments the method of treatment prevents advancement or the disease, delays progression, or causes regression of a disease, or which is capable of reducing symptoms caused by the disease. Symptoms can vary according to the type of neurodegenerative disease or disorder. In certain embodiments, symptoms include without limitation, confusion, memory loss, trouble thinking or concentrating, behavior changes, numbness, pain, muscle spasms, weakness and paralysis, coordination issues, fatigue, slowed movements, shaking and tremors, balance problems, shuffling steps, hunched posture, weakness, paralysis, severe neurological symptoms, neuroinflammation, and progressive neurodegeneration resembling spinocerebellar ataxia. In certain embodiments the method of treatment prevents the degradation of the CNS and neuronal cells.

In certain embodiments, the subject treated by the methods disclosed herein has a mutation in the enzyme glucocerebrosidase (GBA). In certain embodiments, the mutation is an A53T mutation. In certain embodiments, the methods comprise detecting a mutation in the subject.

Genetic alterations containing DNA, RNA, or fragments thereof may be used as probes to detect the presence of and/or expression of genetic alteration specific markers. Methods in which these markers’ nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR). The term "probe" as used herein refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single stranded or double stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide probe typically contains 15 25 or more nucleotides, although it may contain fewer nucleotides. The probes herein are selected to be complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to "specifically hybridize" or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non-complementary nucleotide fragment may be attached to the 5' or 3' end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically.

Further, assays for detecting genetic alterations may be conducted on any type of biological sample. Clearly, genetic alteration-containing nucleic acids, vectors expressing the same, genetic alteration-containing marker proteins and anti-genetic alteration specific marker antibodies of the invention can be used to detect genetic alterations in the subject, cells, or fluid, and alter genetic alteration-containing marker protein expression for purposes of assessing the genetic and protein interactions involved in BAG3 or CLEC16A production.

In most embodiments for screening for genetic alterations, the genetic alterationcontaining nucleic acid in the sample will initially be amplified, e.g. using PCR, to increase the amount of the templates as compared to other sequences present in the sample. This allows the target sequences to be detected with a high degree of sensitivity if they are present in the sample. This initial step may be avoided by using highly sensitive array techniques that are important in the art.

Alternatively, new detection technologies can overcome this limitation and enable analysis of small samples containing as little as Ipg of total RNA. Using Resonance Light Scattering (RLS) technology, as opposed to traditional fluorescence techniques, multiple reads can detect low quantities of mRNAs using biotin labeled hybridized targets and anti-biotin antibodies. Another alternative to PCR amplification involves planar wave guide technology (PWG) to increase signal-to-noise ratios and reduce background interference. Both techniques are commercially available from Qiagen Inc. (USA).

Any of the aforementioned techniques may be used to detect or quantify genetic alteration marker expression and accordingly, identify subject and cells of subject that would benefit from the methods of treatment described herein.

THERAPEUTIC VECTORS

The elucidation of the roles played by the BAG3 and CLEC16A facilitates the development of pharmaceutical compositions useful for treatment and diagnosis neurodegenerative diseases. These compositions may comprise, in addition to one of the therapeutics, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

In certain embodiments, the pharmaceutical compositions comprise nucleic acids, polypeptides, or functional fragments thereof that encodes BAG3 and/or CLEC16A. In certain embodiments, the nucleic acids or functional fragments thereof are present in a vector. In certain embodiments, the vector increases expression of BAG3 and/or CLEC16A.

For purposes of the invention, “nucleic acid”, “nucleotide sequence” or a “nucleic acid molecule” as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5’ to 3’ direction. With reference to nucleic acids of the invention, the term “isolated nucleic acid” is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an “isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism. Alternatively, this term may refer to a DNA that has been sufficiently separated from (e.g., substantially free of) other cellular components with which it would naturally be associated. “Isolated” is not meant to exclude artificial or synthetic mixtures with other compounds or materials, or the presence of impurities that do not interfere with the fundamental activity, and that may be present, for example, due to incomplete purification. When applied to RNA, the term “isolated nucleic acid” refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.

As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and "polypeptide" are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

The term "identity" as used herein and as known in the art, is the relationship between two or more oligo sequences, and is determined by comparing the sequences. Identity also means the degree of sequence relatedness between oligo sequences, as determined by the match between strings of such sequences. Identity can be readily calculated (see, e.g., Computation Molecular Biology, Lesk, A. M., eds., Oxford University Press, New York (1998), and Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993), both of which are incorporated by reference herein). While a number of methods to measure identity between two polynucleotide sequences are available, the term is well known to skilled artisans (see, e.g., Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); and Sequence Analysis Primer, Gribskovm, M. and Devereux, J., eds., M. Stockton Press, New York (1991)). Methods commonly employed to determine identity between oligo sequences include, for example, those disclosed in Carillo, H., and Lipman, D., Siam J. Applied Math. (1988) 48: 1073. In certain embodiments, the present invention may have 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity with the SEQ ID NOs disclosed herein.

A “derivative” of a polypeptide, polynucleotide or fragments thereof means a sequence modified by varying the sequence of the construct, e.g. by manipulation of the nucleic acid encoding the protein or by altering the protein itself. “Derivatives” of a gene or nucleotide sequence refers to any isolated nucleic acid molecule that contains significant sequence similarity to the gene or nucleotide sequence or a part thereof. In addition, “derivatives” include such isolated nucleic acids containing modified nucleotides or mimetics of naturally-occurring nucleotides.

The term "functional" as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

According to the present invention, an isolated or biologically pure molecule or cell is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified. An isolated compound of the present invention can be obtained from its natural source, can be produced using laboratory synthetic techniques or can be produced by any such chemical synthetic route. The terms "increased", "increase", "enhance", or "activate" are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms "increased", "increase", "enhance", or "activate" can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an "increase" is a statistically significant increase in such level.

In one embodiment, "increased expression" refers to an increase in the level or in another embodiment, activity of target gene product relative to the level or activity of target gene product in a standard. In another embodiment, increased expression refers to between a 10 to about a 250% increase in mRNA levels, or in another embodiment, in protein levels. In another embodiment, increased expression refers to changes in gene expression at the mRNA or protein level, in terms of its pattern of expression in particular examples, such as, for example, and in one embodiment, increased expression in CNS, but not in the blood. In one embodiment, increased expression is synonymous with overexpression, or stimulated expression. In another embodiment, increased expression is a relative determination, wherein expression is greater than the standard, or in cases where expression is absent in the standard, this despite expression being barely detectable in the subject. It is to be understood that any such circumstance described hereinabove, represents increased expression for the methods of this invention.

In one embodiment, "compared to a standard", refers to relative changes in expression where the standard is derived from a single individual, or is derived from pooled subjects. In another embodiment, a standard can be derived from a single subject following about 1 to about 5 years of having undergone successful treatment. In another embodiment, the standard is derived from a healthy subject.

The polynucleotides described herein may be delivered through any known method, such as through one or more vectors, to a host cell. In certain embodiments, the vector allows the agents to cross the blood brain barrier (BBB). Exemplary methods for formulating the above described agents to enhance its penetration across the blood brain barrier are described in Yeini et al., Advanced Therapeutics, DOI: 10.1002/adtp.202000124.

A “vector” as used herein is a biological or chemical moiety comprising a nucleic acid sequence which can be introduced into an appropriate host cell for replication or expression of said nucleic acid sequence.

As used herein, an “expression cassette” refers to a nucleic acid molecule which comprises a biologically useful nucleic acid sequence (e.g., a gene cDNA encoding a protein, enzyme or other useful gene product, mRNA, etc.) and regulatory sequences operably linked thereto which direct or modulate transcription, translation, and/or expression of the nucleic acid sequence and its gene product.

As used herein, “operably linked” sequences include both regulatory sequences that are contiguous with the nucleic acid sequence and regulatory sequences that act in trans or at a distance to control the sequence. Such regulatory sequences typically include, e.g., one or more of a promoter, an enhancer, an intron, a Kozak sequence, a polyadenylation sequence, and a TATA signal. The expression cassette may contain regulatory sequences upstream (5’ to) of the gene sequence, e.g., one or more of a promoter, an enhancer, an intron, etc., and one or more of an enhancer, or regulatory sequences downstream (3’ to) a gene sequence, e.g., 3’ untranslated region comprising a polyadenylation site, among other elements. In other embodiments, the term “transgene” refers to one or more DNA sequences from an exogenous source which are inserted into a target cell. Typically, such an expression cassette for generating a viral vector contains the coding sequence for the gene product described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein. In certain embodiments, a vector genome may contain two or more expression cassettes.

In addition to the coding sequence, in certain embodiments the vector includes regulatory sequences which direct expression in a host cell. In certain embodiments, the regulatory elements include a promoter.

The term “promoter” or “promoter polynucleotide” is understood to mean a regulatory sequence/ element or control sequence/element that is capable of binding/recruiting an RNA polymerase and initiating transcription of sequence downstream or in a 3’ direction from the promoter. A promoter can be, for example, constitutively active, or always on, or inducible in which the promoter is active or inactive in the presence of an external stimulus. In certain embodiments, where the vector may be designed such that the protein of interest is expressed under the control of a CNS-specific promoter. Exemplary promoters that drive expression to the

CNS are provided in Table 1: In certain embodiments, the promoter is an inducible promotor, such as a doxycycline- inducible expression control system. In certain embodiments, the vector comprising an inducible promoter prevents over-production of the protein of interest by allowing using an exogenous protein to turn on or off expression of the protein of interest. In one embodiment, the doxycycline-inducible expression control system is a tetracycline-Controlled Operator system (Tet-On system). The Tet-On system employs nucleic acid encoding a reverse tetracycline transactivator (rtTA) protein, which is a fusion of the tetracycline repressor (TetR) protein mutated at four amino acid positions to reverse the response to tetracycline/doxycycline, and the activation domain of VP 16. In the absence of tetracycline (or a derivative thereof such as doxycycline) rtTA does not bind to TetO operator sequences and the polypeptide is not expressed. In the presence of tetracycline/doxycycline, rtTA binds to TetO sequences in the TRE and activates transcription of the nucleic acid downstream of the promoter. The Tet-On system is used herein activates specific proteins that increase expression of BAG3 or CLEC16A.

Tet-On systems are described in Das et al., Curr Gene Ther. (2016)16(3): 156-67 (hereby incorporated by reference in its entirety), and include systems using optimized rtTA variants such as the Tet-On Advanced system (which uses the rtTA variant protein rtTA2.sup.s-M2) and Tet-On 3G system.

The Tet-On Advanced systems are also described in Urlinger et al. Proc. Natl. Acad. Sci. U.S.A. (2000) 97( 14): 7963 -8 (hereby incorporated by reference in entirety), and Kallunki T, et al. How to Choose the Right Inducible Gene Expression System for Mammalian Studies? Cells. 2019; 8(8):796. Tet-On 3G is described in Zhou et al., Gene Ther. 13(19): 1382-1390 (hereby incorporated by reference in entirety).

In addition to a promoter, the vector may contain one or more appropriate “regulatory elements” or “regulatory sequences”, which comprise but are not limited to an enhancer; transcription factor; transcription terminator; efficient RNA processing signals such as splicing and polyadenylation signals (poly A); sequences that stabilize cytoplasmic mRNA, for example Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE); sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. Examples of suitable polyA sequences include, e.g., SV40, bovine growth hormone (bGH), and TK polyA. Examples of suitable enhancers include, e.g., the alpha fetoprotein enhancer, the TTR minimal promoter/enhancer, LSP (TH-binding globulin promoter/alphal-microglobulin/bikunin enhancer), amongst others. These control sequences or the regulatory sequences are operably linked to the nuclease coding sequence or transgene coding sequence.

In some aspects, the invention further provides cells produced with said vectors, and organisms or cells comprising or produced from such cells. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory compounds to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11 :211-217 (1993); Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTECH 11 :167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);

Kremer & Perricaudet, British Medical Bulletin 51(1):31 -44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bihm (eds) (1995); and Yu et al., Gene Therapy 1 : 13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, poly cation or lipidmucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

The preparation of lipidmucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66: 1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700). In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94: 1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenovirus, and \|/2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producing a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also be infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

The compounds described herein can be formulated for enteral, parenteral, topical, or systemic administration. The compounds can be combined with one or more pharmaceutically acceptable carriers and/or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions. The carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. Typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the compounds are known by those skilled in the art. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like.

Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers, for example to a diluent, adjuvant, excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

A pharmaceutical composition of the present invention can be administered by any suitable route, for example, by injection, by oral, pulmonary, nasal or other forms of administration. In general, pharmaceutical compositions contemplated to be within the scope of the invention, comprise, inter alia, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions can include diluents of various buffer content (e.g., Tris HC1, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical composition of the present invention. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435 1712 which are herein incorporated by reference. A pharmaceutical composition of the present invention can be prepared, for example, in liquid form, or can be in dried powder, such as lyophilized form. Particular methods of administering such compositions are described infra.

In certain embodiments, the compositions described herein can be injected into the heart through the coronary sinus. Methods for administering pharmaceutical compositions into the heart through the coronary sinus are known by those skilled in the art. For example, Myers, Valerie D., et al. "Cardiac transduction in mini-pigs after low-dose retrograde coronary sinus infusion of AAV9-BAG3 : a pilot study." Basic to Translational Science 7.9 (2022): 951-953, which is incorporated herein by reference in its entirety, provides an exemplary method.

In yet another embodiment, a pharmaceutical composition of the present invention can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In a particular embodiment, a pump may be used [see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321 :574 (1989)]. In another embodiment, polymeric materials can be used [see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71 :105 (1989)]. In yet another embodiment, a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose [see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115 138 (1984)]. Other controlled release systems are discussed in the review by Langer [Science 249: 1527 1533 (1990)].

In certain embodiments, the gene editing system is a Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR) system that edits the sequence of a gene involved in production of BAG3 or CLEC16A. In one embodiment, provided herein is a CRISPR/Cas dual vector system (see, e.g. WO 2016/176191, which is incorporated herein by reference). Alternatively, in certain embodiments, a suitable gene editing system includes a zinc-finger nuclease (ZFN) to induce DNA double-strand breaks, which may or may not be in conjunction with delivery of an exogenous DNA donor substrate (See, e.g., Ellis et al, Gene Therapy (epub January 2012) 20:35-42 which is incorporated herein by reference). In other embodiments, a suitable gene editing system includes a meganuclease (see, e.g., in US Patent 8,445,251; US 9,340,777; US 9,434,931; US 9,683,257, and WO 2018/195449, each of which is incorporated herein by reference) or transcription activator-like (TAL) effector nucleases (TALENs).

In certain embodiments, a suitable CRISPR gene editing system includes, at a minimum, a Cas enzyme, such as a Cas9 enzyme, and an sgRNA specific for a target site in the sequence of a gene involved in BAG3 or CLEC16A production. Accordingly, in one embodiment, the gene editing vector comprises a Cas9 gene as the editing enzyme and an sgRNA which is at least 20 nucleotides in length and specifically binds to a selected site in a gene involved in BAG3 or CLEC16A production, 5 ' to a protospacer- adjacent motif (PAM) that is specifically recognized by the Cas9. In certain embodiments, the expression cassette or vector genome includes a nucleic acid sequence encoding the sgRNA molecule and a nucleic acid sequence encoding a Cas9 enzyme. In certain embodiments, the gene editing system also includes a donor or repair template. The expression cassette providing the donor template may be the same as the expression cassettes encoding the sgRNA and Cas9, or a different expression cassette. Thus, in certain embodiments, a dual-vector system (as described for example in WO 2016/176191) is provided, wherein the gene editing system includes an expression cassette comprising a Cas9 gene under control of regulatory sequences which direct its expression and a second expression cassette comprising a sgRNA and a donor template.

“Cas9” (CRISPR associated protein 9) refers to family of RNA-guided DNA endonucleases which is characterized by two signature nuclease domains, RuvC (cleaves noncoding strand) and HNH (coding strand). Suitable bacterial sources of Cas9 include Staphylococcus aureus (SaCas9), Stapylococcus pyogenes (SpCas9), and Neisseria meningitides (KM Estelt et al, Nat Meth, 10: 1116-21 (2013)). The wild-type coding sequences may be utilized in the constructs described herein. Alternatively, bacterial codons are optimized for expression in humans, e.g. using any of a variety of known human codon optimizing algorithms. Other endonucleases with similar properties may optionally be substituted. See, e.g., the public CRISPR database (db) accessible at crispr.u-psud.fr/crispr. CRISPR/Cas9 gene targeting requires a single guide RNA (sgRNA) that contains a targeting sequence (crRNA sequence) and a Cas9 nuclease-recruiting sequence (tracrRNA). The crRNA region is a 20-nucleotide sequence that is homologous to a target site and will direct Cas9 nuclease activity. Strategies for identifying suitable target sites in the genome while also eliminating off target effects are known to those of skill in the art (see, e.g., ChopChop available online at chopchop.cbu.uib.no/).

In another embodiment, the CRISPR nuclease may be Cpfl (CRISPR from Prevotella and Francisella). Cpfl's preferred PAM is 5 '-TTN; this contrasts with that of SpCas9 (5'-NGG) and SaCas9 (5 '-NNGRRT; N=any nucleotide; R=adenine or guanine) in both genomic location and GC-content. While at least 16 Cpfl nucleases have been identified, two humanized nucleases (AsCpfl and LbCpfl) are particularly useful. See, www.addgene.Org/69982/sequences/#depositor-full (AsCpfl sequences; and www.addgene.Org/69988/sequences/#depositor-full (LbCpfl sequences), which are incorporated herein by reference. Further, Cpfl 1 does not require a tracrRNA; allowing use of shorter guide RNAs (about 42 nucleotides) as compared to Cas9. Plasmids may be obtained from Addgene, a public plasmid database. As described herein, a gene editing system is utilized to introduce a mutation in a gene involved in BAG3 or CLEC16A production in target cell. In certain embodiments, this gene editing system increases production of BAG3 or CLEC16A. In some embodiments, the target polynucleotide sequence is cleaved such that a double-strand break results. In some embodiments, the target polynucleotide sequence is cleaved such that a single-strand break results. In certain embodiments, the alteration is an insertion or deletion (indel), which can result in random insertion/deletion mutations at the site of junction as a result of non-homologous end joining. Indel mutations occurring within the coding region of a gene can result in frame-shift and a premature stop codon, and disrupt transcription.

In certain embodiments, the gene editing system one or more elements of an RNA- targeting CRISPR system, such as a member of the Cast 3 enzyme family and/or crRNA construct. The diverse Cast 3 family contains at least four known subtypes, including Casl3a (formerly C2c2), Casl3b, Casl3c, and Casl3d. The Casl3 family is the only family of class 2 Cas enzymes known to exclusively target single-stranded RNA. Cast 3 enzymes and systems are known in the art, see, e.g., US Patent No. 10,362,616, Abudayyeh, et al, C2c2 is a singlecomponent programmable RNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573 (2016); S. Shmakov, et al, Discovery and functional characterization of diverse class 2 CRISPR- Cas systems. Mol. Cell 60, 385-397 (2015); S. Shmakov, et al, Diversity and evolution of class 2 CRISPR-Cas systems. Nat. Rev. Microbiol. 15, 169-182 (2017). A. A. Smargon, et al, Casl3b is a type VI-B CRISPR-associated RNA-guided RNase differentially regulated by accessory proteins Csx27 and Csx28. Mol. Cell 65, 618-630.e7 (2017); J. S. Gootenberg, et al, Nucleic acid detection with CRISPR-Cas 13 a/C2c2. Science 356, 438-442 (2017); O. O. Abudayyeh, et al, RNA targeting with CRISPR-Casl3. Nature 550, 280-284 (2017). Each of these documents is incorporated herein.

A Casl3 protein uses a short crRNA that interacts with the Casl3 molecule through a stem loop and facilitates target binding and cleavage through a series of conformational changes in the Casl3 molecule. In certain embodiments, the Casl3 protein is Casl3a, Casl3b, Casl3c, or Casl3d. In one embodiment, the Casl3 comprises one or more mutations the HEPN domain(s).

The Casl3d protein is a Class 2, Type VI CRISPR effector guided by a crRNA. Two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains have been found in the Casl3d, flanking a helical domain. See, for example, WO 2019/010384 Al, US 2019/0169595 Al , Zhang C, et al. (2018). Structural Basis for the RNA-Guided Ribonuclease Activity of CRISPR-Casl3d. Cell 175, 212-223. e217, golden.com/wiki/CRISPR-Casl3d, and zlab.bio/casl3, which publication is incorporated herein by reference in its entirety. While the term Class 2, Type VI is a broader genus, of which Casl3d is exemplary, throughout the Specification, one of skill in the art would appreciate that the use of the terms “Cast 3d” or “Casl3d and a variant thereof’ also encompass other Class 2, Type VI proteins, and the terms can be interchangeable. Casl3d and a variant thereof includes, e.g., a wild type or naturally occurring Casl3d protein, an ortholog of a Casl3d, a functional variant thereof, or another modified variant as disclosed.

Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. In some embodiments, the Casl3d is selected from a /? rCas l 3d from Ruminococcus flavefaciens strain XPD3002, an AdmCasl3d from Anaerobic digester metagenome 15706, //.sCasl 3d from Eubacterium siraeum DSM15702, P / Cas l3d from Gut metagenome assembly PlE0-k21, /7'Cas l 3d from Uncultured Ruminoccocus sp., /(//Cas l 3d from Ruminoccocus flavefaciens FD1, and Ak/Casl 3d from Ruminoccocus albus. In one embodiment, the Casl3d protein is a A rCasl3d or a variant thereof. The amino acid sequences of the Cast 3d orthologs are publicly available. In one embodiment, the Casl3d has an amino acid sequence as provided by a Protein Data Bank (PDB) accession number 60AW B or 60AW A or 6E9F A or 6E9E A or 6IV9 A, or an amino acid sequence as provided by the UniProtKB identifier B0MS50 (B0MS50 9FIRM) or A0A1C5SD84 (AOA1C5SD84_9FIRM). Each of the sequences of these references is incorporated by reference herein in its entirety.

The term “target RNA” refers to an RNA polynucleotide being or comprising the target sequence, including coding and non-coding transcripts. In other words, the target RNA may be an RNA polynucleotide or a part of a RNA polynucleotide to which a part of a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) is designed to have complementarity and to which the effector function mediated by the complex comprising CRISPR enzyme and a guide RNA (gRNA) is to be directed.

In certain embodiments, a viral vector is used to deliver one more elements of a gene editing system. It will be understood that a different, partially or wholly integrating vector or virus may be used in the system in place of the gene editing vector and/or the vector carrying template. See, e.g., Jinek, M.; Chilynksi, K.; Fonfara, I.,; Hauer, M.„ Doudna, J.,; Charpentier, E., (August 17, 2012). “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity”. Science. 337 (6069): 816-821. Bibcode:2012 Sci..337..816J. doi: 10.1126/science.1225829. PMID 22745249; US Patent 8,697,359; US 9,909,122, US 2017/0051312; US 2017/0137801; US 2017/0166893; US2017/0360048; US 2018/0002682, which are incorporated by reference in their entirety.

In certain embodiments, a viral vector delivers one or more components of a genome editing system, such as CRISPR/Cas9 or CRISPR/Casl3. In another embodiment, a combination or dual AAV vector system is provided to deliver the components of the CRISPR system when co-administered to a subject (see, e.g. WO 2016/176191, which is incorporated by reference herein in its entirety). The vectors may be formulated together or separately and delivered essentially simultaneously, preferably by the same route.

The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

Example I

Abnormalities in protein quality control are thought to be important in the pathobiology of various neurodegenerative diseases including Alzheimer’s Disease, Chronic Traumatic Encephalopathy complicating traumatic brain injury and Parkinson’s Disease. Abnormalities in BAG3 were analyzed herein to determine whether they contribute to the pathobiology of these central nervous system diseases and potentially serve as a therapeutic target. In addition, we determined whether the development of these central nervous system diseases might be more common in individuals harboring mutations in BAG3. It is generally assumed that BAG3 is found predominantly in the heart and the skeletal muscle and little attention has been paid to its role in the brain.

Provided herein are differentiated human stem cells that, when stained with appropriate antibodies and imaged with confocal imaging, show there is abundant BAG3 and a-synuclein under baseline conditions in differentiated human stem cells. (Fig 1) By contrast, as seen in Figure 2, when BAG3 was knocked down using an siRNA, there was a significant decrease in BAG3 levels in the IPSCs and an increase a-synuclein indicating that BAG3 protected the cell from over-expression of a-synuclein and the accompanying changes we now know characterize cells in which a-synuclein are over-expressed.

Next, differentiated human iPSCs that expressed Beta Tubulin III, a brain cell marker were treated. We found that normal stem cell-derived dopamine+ neurons harvested from patients with Parkinson’s Disease secondary to GD-associated genetic variants show changes in the levels of alpha-synuclein that are consistent with earlier studies. Therefore, we hypothesized thatBAG3 augmentation in human dopamine-producing neuronal cells plays a role in modulating the function of those cells. To test this hypothesis, we took both normal human dopamine- producing neuronal cells and cells with a PD causing variant in LRRK2 and human cells with a PD causing variant in GBA, and cells that expressed the A53T variant and exposed them to AAV9-BAG3. As seen below in Figure 3, AAV9-BAG3 significantly lowered a-synuclein levels in GBA neurons and to a lesser extent in normal neurons but has no effect on a-synuclein levels in LRRK2 neuron. The effects of AAV9-BAG3 were biphasic in that lower levels of expression were associated with beneficial effects whereas higher concentrations were associated with adverse effects.

We next analyzed the role of CLEC in maintaining homeostasis in normal cells as well as cells with a background of high levels of a-synuclein. iPSCs cells were stained with beta tubulin III antibody to outline the cells, along with antibodies which bind Clecl6A and BAG3. Treatment of cells with AAV-BAG3 (lower Fig. 3- AAV-BAG3) substantially increased BAG3 levels in cells as shown in lane 1 and in lane 4while there was only a modest-minimal increase in CLEC16A. (Fig. 3)

The ability of AAV-BAG3 to alter levels of CLEC16A and BAG3 in human iPSCs were further analyzed. When AAV-BAG3 was added to the incubation medium and the cells imaged 24 hours later, there was a small but non-significant change in the housekeeping gene beta tubulin III. However, there was a significant increase in BAG3 as well as CLEC16A. (Fig. 4)

To determine whether the two proteins were working in unison to modulate intracellular processes, we analyzed whether BAG3 and CLEC16A co-localized in the cell. Accordingly, we performed a proximity ligation assay (PLA) to assess the position of the two proteins within neuronal cells obtained from maturation of normal human stem cells. The technique is described in Figure 5 and the results are seen in Figure 6. There was clear co-localization of BAG3 and CLEC16A in neuronal cells that produced dopamine and these results specifically indicate that the proteins are no more than 50 A⁰ apart in the cell. Since both CLEC16A and BAG3 have been shown to independently play an important role in cellular homeostasis, the finding that they are located together in the neuron indicates that they are synergistic in their activities and that levels of both proteins is important for normal neuron function. Also, since CLEC6A effects are only seen when there is a complete loss of protein, BAG3 might play a pivotal role as untoward effects of BAG3 loss are seen in heterozygotes whereas homozygous loss of BAG3 is lethal.

While the homozygous knock-out model of CLEC16A allowed us to dissect the molecular mechanism whereby we observed autoimmune, inflammatory and neurodegenerative disorders, homozygous deletions of CLEC16A have not been found in humans or in the animal kingdom - presumably because such a mutation would be lethal. This led us to hypothesize that BAG3 would be an appropriate partner. Having shown co-localization in the PLA experiments and knowing that both had profound effects on either the same or complementary pathways, an accurate model of Parkinson’s Disease (PD) was needed. We used human stem cells from patients with specific genetic mutations that cause the disease by increasing a-synuclein, the common pathologic pathway of many neurodegenerative diseases.

We obtained transformed cell lines from individuals with a homozygous variant in BAG3 that is focused on identifying pathological and physiological evidence of mitochondrial dysfunction in all major neurodegenerative diseases. Neurons are especially vulnerable to injury and death from dysfunctional mitochondria as they cannot switch to glycolysis if oxidative phosphorylation is impaired. At the simplest level, mitochondrial ATP production stands at the center of cellular function in all cells including neurons.

We obtained two types of stem cells: the GBA variant stem cells and the LRRK2 variant stem cells. Pooled cells from normal donors were used as controls. The GBA variant encodes a variant form of [3-Glucocerebrosidase - a lysosomal enzyme that is the cause of PD in 5 -15% or patients. GBA mutations are the greatest risk identified to date for the development of idiopathic PD. GBA mutations are also associated with Gaucher disease. The LRRK2 variant encodes a variant form of dardarin protein-kinase which is seen in 0.5% - 2.0% sporadic PD and 5% genetic PD (Dachsel). LRRK2 is also associated with Crohn’s disease. In these experiments, cells were plated and exposed to either a control or to AAV-BAG3. Levels of a-synuclein were then measured in the cells. (FIG. 7)

Next the a-synuclein levels in healthy donor cells, GBA cells and LRRK2 cells were analyzed after administration of BAG3 or AAV-BAG3. a-synuclein (total intensity) was unchanged by the addition of BAG3 to the cells in controls, but BAG3 was increased significantly, but not substantially in GBA cells and in LRRK2 cells (FIG. 8, Blue). However, the addition of AAV-BAG3 to the culture medium resulted in a significant increase in levels of BAG3 in both the GBA and the normal cells but not in the LRRK2 cells. By contrast, the addition of BAG3 resulted in a small but significant decrease in a-synuclein in the normal cells, a large and highly significant fall in a-synuclein in the GBA cells, and no change in a-synuclein levels in the LRRK2 cells. (Fig. 8) Accordingly, this data indicates that AAV-BAG3 represents an effective treatment strategy for patients with PD caused by at least one common genetic variant - GBA. This therapy represents the first successful gene therapy approach for Parkinson’s disease.

These results were confirmed using cells from patients with Parkinsons’ s disease that were transformed to adult dopamine producing cells and treating them with AAV-BAG3. (FIG. 9) This therapy was able to lower a-synuclein levels in the iPSCs. The fibers of a-synuclein can be readily seen in FIG. 9 as these were stained for using this technique.

Results from these experiments indicate that BAG3 plays a role in both neuroinflammation and neurodegenerative diseases of the brain and can serve as an important therapeutic target. The data show 1) BAG3 levels are expressed at lows levels in the brain of mice that are comparable to those found in the heart, by contrast with earlier reports which indicate that the levels are much lower than those found in the heart 2) BAG3 couples tightly with a-synuclein, peptides that play an important role in Alzheimer’ s/CTE and Parkinson’s Disease respectively; and 3) Common (>1%) loss of function genetic variants found in individuals of African ancestry fail to bind to these disease causing peptides.

BAG3 also works with CLEC16A in a cooperative synergistic fashion, thereby providing new therapeutic avenues for effective treatment of PD. References

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Example II The Effects of Metformin on BAG3

Metformin, a drug used world-wide for the treatment of diabetes, is associated with activation of the energy regulator adenosine monophosphate-activated protein kinase, diminished neuroinflammation, inhibition of the mammalian target of rapamycin signaling, and augmented autophagy in the brain. In addition, Metformin removed protein aggregates in transgenic BAG3^P2009L-expressing fish and human myoblasts from patients with a BAG3^P209L genotype as well as rescuing the fiber disintegration and swimming deficit in BAG3' ' fish. (9) In this focused example, we sought to determine whether metformin could increase BAG3 levels in the brain by assessing the effects of metformin on BAG3 levels in human dopamine-producing neurons derived from induced pluripotent stem cells isolated from patients with Parkinson’s disease (PD) as compared to stem cells isolated from donors without known neurologic disease as well as assessing the effects of increased levels of BAG3 on neuronal levels of alpha- synuclein (a-SYN).

Methods

Dopamine producing neurons (iCell Dopa Neurons) generated from induced pluripotent stem cells (iPSCs) isolated from the sera of patients with genetic forms of Parkinson’s disease (N370S [GBA]; A53T) or from donors who did not have a history of neurological disease (AHN) were purchased from FUJIFILM Cellular Dynamics. The patients from whom cells were extracted were enrolled in the landmark Parkinson’s Progression Markers Initiative (PPI) funded by the Michael J. Fox Foundation for Parkinson’s Research’s. Dopamine producing neurons were derived from neurons that had been genetically modified by exchanging a threonine for an asparagine at amino acid 53 in the a-synuclein gene (A53T) or from patients having a GBA^N370S mutation in the glucocerebrosidase gene that is associated with Gaucher’s disease, a lysosomal storage disease. FUJIFILM used technology licensed from the laboratory of Dr. Lorenz Studer at Memorial Sloan Kettering to generate the respective adult neurons.

Cells were seeded ontol2 mm diameter round coverslips pre-coated with 10 mg/mL of fresh laminin at a density of 55,000 cells/cm² in maintenance media provided by FUJIFILM and placed in 24 well plates. Reactions were carried out as described in detail in the directions provided by FUJIFILM. In brief, cells received no treatment for the first two days after which the media was changed every 48-72 hours. Adeno-associated vectors (AAV2/9) carrying a cytomegaloviral promoter driving either BAG3 or an empty vector were added to the incubation media at the first media change. At the conclusion of the 17-day treatment period samples were either washed and fixed for confocal imaging as described in detail previously using a Zeiss LSM 800 microscope at 20x magnification or samples were washed and incubated with primary antibodies overnight and then incubated with secondary antibodies for western blot analysis as described previously. We used tubulin III as a control to account for variations in the amount of protein in each reaction.

Results

To assure that our experimental conditions produced cells having the morphologic features of adult neurons and were responsive to AAV9-BAG3, we added AAV2/9-cmv-BAG3 or AAV2/9-cmv-null to GBAN370S neurons after two days in culture and then continued the cultures for 16 days. The cover-slips were then stained for BAG3 or for a-SYN and imaged with confocal microscopy. As seen in the representative images in Figure 10A and the cumulative quantification in Figures 10D and 10C, there was a significant increase in the levels of BAG3 (<0.0001) and a significant decrease in the levels of a-SYN (p=0.018) in neurons harboring a BGA variant after exposure to AAV2/9-BAG3 when compared to cells treated with AAV2/9- null. We next sought to determine whether baseline levels of BAG3 were the same or different in neurons harboring a GBA variant or a A53T variant when compared with normal controls (AHN). As seen in Figure 11 A, BAAG3 levels were significantly lower (p<0.02) in neurons harboring a GBA variant or an A53T variant when compared with BAG3 levels in wild-type dopamine producing neurons (AHN). As seen in Figure 1 IB, the addition of increasing concentrations of metformin did not change the levels of BAG3 in the wild-type cells (AHN); however, there was an upside-down horseshoe shaped effect on BAG3 levels in both the GBA and the A53T cells as the levels of BAG3 initially increased and then subsequently decreased with increasing levels of metformin. The increase appeared to be greater in the A53T cells when compared to the GBA cells.

Next, we analyzed whether the effects of the metformin were attributable to the increase in BAG3 or whether they could be explained by a different effect of metformin. Therefore, we again increased BAG3 levels in the neurons by administering increasing amounts of AAV2/9- CMV-BAG3 as described above. Increasing amounts of AAV9-BAG3 resulted in increased levels of BAG3 in the A53T neurons after 18 days in culture.(p=0.018) Most interesting; however, was the finding that lower levels of BAG3 were associated with a decrease in the levels of a-SLN; however, higher levels of BAG3 were associated with significantly higher levels of a- SYN (p=0.024).

Discussion

That lower levels of BAG3 are a potential risk factor for PD was recently shown in a study from the UK Biobank cohort including over 52,000 PD-free participants and 811 newly diagnosed Pd cases.(Jiao X, Lu, Y, Huang Y, Parkinsonism and related disorders). Therefore, our results demonstrating that metformin can increase the levels of BAG3 in isolated human dopamine producing neurons would be of potential importance in view of the fact that BAG3 plays a critical role in modulating the clearance of mis-folded proteins by the proteasome.(Chen Y, 2013) The fact that the effect is biphasic raises concerns about the usefulness of traditional gene therapy for raising BAG3 levels in the brain. This is consistent with recent studies which showed that correction of cardiac dysfunction secondary to a BAG3 P209L mutation of human induced pluripotent stem cell lines from patients with myofribillar myopathy required the generation of two isogenic control lines. ( Filippi K, Wiemann M, Fleischmann BK, Hesse M.Stem Cell Res. 2025 Feb;82: 103627. doi: 10.1016/j.scr.2024.103627. Epub 2024 Dec 7.PMID: 39662463). Thus, a more effective and safer method for increasing levels of BAG3 may simply be to use a lower dose of metformin, although it will be important to gain a better understanding of the role that genetic variants play in regulating metformin levels to assure that adverse effects do not occur that result in increased levels of a-SYN or of other unwanted byproducts.

References

1. Kirk JA, Cheung JY, Feldman AM. Therapeutic targeting of BAG3: considering its complexity in cancer and heart disease. J Clin Invest 2021; 131.

2. Rosati a. BAG3: a multifaaceted protein that regulates major cell paathays. Cell Death and Disease 2011.

3. Selcen D, Muntoni F, Burton BK et al. Mutation in BAG3 causes severe dominant childhood muscular dystrophy. Ann Neurol 2009;65:83-9.

4. myers vd, tomar, d., madesh, m. Haplo-insufficiency of Bcl2-associated athanogene. journal of cellular physiology 2018.

5. Santoro A, Nicolin V, Florenzano F, Rosati A, Capunzo M, Nori SL. BAG3 is involved in neuronal differentiation and migration. Cell Tissue Res 2017;368:249-258.

6. Limanaqi F, Biagioni F, Gambardella S, Familiari P, Frati A, Fornai F. Promiscuous Roles of Autophagy and Proteasome in Neurodegenerative Proteinopathies. Int J Mol Sci 2020;21.

7. Sweeney N, Kim TY, Morrison CT et al. Neuronal BAG3 attenuates tau hyperphosphorylation, synaptic dysfunction, and cognitive deficits induced by traumatic brain injury via the regulation of autophagy-lysosome pathway. Acta Neuropathol 2024;148:52.

8. Sheehan PW, Nadarajah CJ, Kanan MF et al. An astrocyte BMAL1-BAG3 axis protects against alpha-synuclein and tau pathology. Neuron 2023;111:2383-2398 e7.

9. Ruparelia aa, McKaige, E.A., Williams, C. Metformin rescues muscle function in BAG3 Myofibrillar Myopathy Models, autophagy 20221.

Example III

Test and Treat Method for Treating Neurodegenerative Disorders

The information herein above can be applied clinically to patients for therapeutic intervention, particularly for the treatment of symptoms associated with a neurodegenerative disease or disorder. A preferred embodiment of the invention comprises clinical application of the information described herein to a patient. In some embodiments, the neurodegenerative disease or disorder is assessed, monitored, or diagnosed by a method comprising: (i) measuring one or more clinical symptoms or signs of a neurodegenerative disease or disorder in a subject, (ii) combining the measurements obtained into a single composite measurement, and (iii) assessing the overall severity of, or change in, the neurodegenerative disease or disorder in the subject by comparing the composite measurement to a reference value or another composite measurement in the same subject. The (i) one or more composite measurements are employed to measure the clinical effect on the subject of a diagnostic, therapeutic or other type of medical intervention; (ii) for each of the measurements tested, the subject is classified as: (a) a responder or a non-responder, (b) a member of a clinical category, or (c) a member of a metric range, based on the change in said one or more clinical symptoms as measured using the particular clinical symptom or metabolic pathway assessed; and (iii) the measurements obtained are combined into a single composite measurement, by either: (a) separately assessing the change in each measurement obtained from each assay conducted prior to combining each measurement into a single composite measurement, or (b) combining measurements obtained from a first time point and generating a single composite measurement for said first time point and then comparing the single composite measurement for the first time point to a single composite measurement generated from the same assay for a second time point.

Important clinical assessments for neurodegenerative disease or disorder include confusion, memory loss, trouble thinking or concentrating, behavior changes, numbness, pain, muscle spasms, weakness and paralysis, coordination issues, fatigue, slowed movements, shaking and tremors, balance problems, shuffling steps, hunched posture, weakness, paralysis, severe neurological symptoms, neuroinflammation, and progressive neurodegeneration resembling spinocerebellar ataxia.

The derived therapeutic dose of vectors and agents described herein for human could be by those skilled in the art based on response rate. The agent, or pharmaceutically acceptable composition comprising said agent, can be administered at a dose of 0.0001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active metabolite compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, discussed above.

Generally, the initial therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of the compound or a pharmaceutically acceptable salt thereof. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of the compound or a pharmaceutically acceptable salt thereof. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of the compound or a pharmaceutically acceptable salt thereof. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of the compound or a pharmaceutically acceptable salt thereof. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of the compound or a pharmaceutically acceptable salt thereof.

Treatment can occur after a patient arrives in the clinic and presents with symptoms of neurodegenerative disease or disorder. The agents and vectors described herein, have been shown to be well tolerated and the symptoms were assessed using clinical scores criteria.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A method for treating a neurodegenerative disorder in a subject in need thereof, comprising administering an effective amount of one or more agents that elevate intracellular levels of BAG3 in neuronal cells, thereby providing therapeutic benefit to said subject.

2. The method of claim 1, wherein the one or more agents that elevate intracellular levels of BAG3 also elevate intracellular levels of CLEC16A.

3. A method for treating a neurodegenerative disorder in a subject in need thereof, comprising administering an effective amount of one or more agents that elevate intracellular levels of BAG3 and one or more agents that elevate intracellular levels of CLEC16A, thereby providing therapeutic benefit to said subject

4. The method of anyone of claims 1-3, wherein the one or more agents include a BAG3 polypeptide or functional fragment thereof and a CLEC16A polypeptide or functional fragment thereof.

5. The method of claim 4, wherein the BAG3 and CLEC16A are present in an expression vector.

6. The method of claim 5, wherein said expression vector further comprises an expression control element, promoter or enhancer that is active in the brain or central nervous system.

7. The method of claim 6, wherein the expression control element, promoter or enhancer active in the brain or central nervous system is selected from an expression control element, promoter or enhancer set forth in Table 1.

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

9. The method of any one of claims 5-8, wherein the expression vector crosses the bloodbrain barrier.

10. The method of any one of claims 5-9, wherein the BAG3 polynucleotide, polypeptide or active fragment thereof is transmittable across the blood brain barrier or directly administered to the brain or central nervous system.

11. The method of any one of claims 5-10, wherein the expression vector comprises a viral vector, plasmid, or a yeast vector.

12. The method of claim 11, wherein the viral vector comprises an adeno-associated virus (AAV) vector, an adenoviral vector, a lentiviral vector, a coxsackie viral vector, a cytomegalovirus vector, retroviral vector, a lipid nanoparticle, or an Epstein Barr virus vector.

13. A method for treating neurodegenerative disease in a subject in need thereof, the method comprising a) diagnosing the subject with the neurodegenerative disease, and b) administering an effective amount of one or more agents that elevate intracellular levels of BAG3.

14. The method of any one of the preceding claims, wherein the treatment delays neurodegenerative disease symptom progression when compared to an untreated control.

15. The method of any one of the preceding claims, further comprising administering at least one of an activator of adenosine monophosphate-activated protein kinase (AMPK).

16. The method of claim 15, wherein said activator of AMPK is exogenous AMPK, Metformin,

AICAR, Kazinol B, Marein, Amarogentin, A 769662, PF 06409577, Metformin hydrochloride, ZLN 024, ZLN 024 hydrochloride, Nilotinib, Phenformin, Nilotinib hydrochloride monohydrate, Adenosine 5 '-monophosphate monohydrate, Hispidulin, MK 8722, Euphorbi asteroid, ASP4132, GSK621, EX229 (compound 991), Trans-feluric acid, 0-304, MK 3903, BAM 15, ligustroflavone, ETC-1002, BC1618, IMM-H007, IM156, Chikusetsusaponin IVa, Poricoic acid A, 7-Methoxyisoflavone, Urolithin B, Danthron, Demethyleneberberine, AMPK activator 1, AMPK activator 2, AMPK activator 4, Malvidin-3-O-arabinoside chloride, RSVA 405, Etilefrin, COH-SR4, Buformin, Buformin hydrochloride, PT1, Bempedoic acid, 3a-Hydrocymogrol, Ampkinone, and combinations thereof.

17. The method of claim 15, wherein said activator of AMPK is metformin.

18. The method of any one of the preceding claims, wherein the patient has a A53T mutation in glucocerebrosidase.