Stabilization of retromer for treating alzheimer disease and other neurodegenerative disorders

Abstract

To provide stabilization of a retromer for treating Alzheimer disease and other neurodegenerative disorders.SOLUTION: Provided is a method and a composition for increasing and stabilizing a retromer in order to treat and / or prevent Alzheimer disease and other neurodegenerative disorders. Furthermore, provided is treatment of an adenovirus base for treating Alzheimer disease (AD), and other neurodegenerative states, which are Parkinson disease (PD), neuronal ceroid lipofuscinosis (NCL), and transmissible spongiform encephalopathy (TSE or prion disease), multiple system atrophy (MSA), Down syndrome, and hereditary spastic paraplegia for example, and which are progressive supranuclear palsy (PSP), frontotemporal dementia linked to chromosomes 17q21 to 22 and subtype (FTLD-17 / FTLD-Tau) thereof, Lewy body disease (LBD), amyotrophic lateral sclerosis (AES), frontotemporal degeneration (FTD), ALS-FTD and chronic traumatic encephalopathy (CTE) for example.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 943,999, filed December 5, 2019, and U.S. Provisional Patent Application No. 63 / 074,578, filed September 4, 2020, both of which are hereby incorporated by reference in their entireties.

[0002] Field The present disclosure relates to methods and compositions for elevating and stabilizing retromer to treat and / or prevent Alzheimer's disease and other neurodegenerative disorders. [Background technology]

[0003] background Alzheimer's disease (AD) has been characterized as a disease of misfolded proteins and neuroinflammation. However, AD therapeutics targeting amyloid, tau, cholinesterase inhibitors, anti-inflammatory compounds, and alternative treatments, such as memantine and nutritional supplements, have failed, and the disease remains a major cause of mortality, morbidity, and economic burden. The failure of AD clinical trials has compelled researchers to further explore the causal mechanisms of the disease. Numerous preclinical studies have examined novel AD-related genes, intracellular proteostasis pathways, and interactions between neurons and their microenvironment and glial cells. Several investigations are still ongoing.

[0004] Recent genetic and cell biological findings in Alzheimer's disease have implicated endosomal trafficking as a central role in disease pathophysiology. Current literature suggests that four classes of genes are involved in AD. These gene classes are: 1) endosomal trafficking; 2) cholesterol metabolism; 3) immune response; and 4) amyloid precursor protein (APP) processing. All four of these gene classes are directly or indirectly linked to defects in endosomal trafficking. Defects in endosomal trafficking have also been linked to other neurodegenerative diseases, such as Parkinson's disease (PD), transmissible spongiform encephalopathies (TSEs or prion diseases), and neuronal ceroid lipofuscinosis (NCL).

[0005] Retromer is a protein complex associated with endosomal organelles that regulates the transport of certain cellular cargo molecules within tubular vesicular carriers to the trans-Golgi network. Defects in this transport have been associated with various neurodegenerative diseases (Small and Petsko 2015; Anderson et al. 2014). Neurodegeneration is an umbrella term for the progressive loss of neuronal structure or function, including neuronal death. Many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease, arise as a result of neurodegenerative processes.

[0006] The use of small molecules to improve retromer complex function has been described. However, developing successful drugs that exhibit positive pharmacological kinetics in vivo is challenging and can take years. There is an urgent need for effective treatments for these neurodegenerative diseases, and currently no gene-based therapies offer long-term benefits. Summary of the Invention [Means for solving the problem]

[0007] overview The present disclosure relates to compositions and methods that can be used to treat a subject (e.g., a mammalian subject, e.g., a human subject) having or at risk of developing a neurodegenerative disease, including, but not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), Down's syndrome, hereditary spastic paraplegia (HSP) and multiple system atrophy (MSA), and tauopathies, such as progressive supranuclear palsy (PSP), frontotemporal lobar dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0008] Using the compositions and methods of the present disclosure, a subject (e.g., a mammalian subject, e.g., a human subject) having or at risk of developing the above-described disease can be administered a composition containing a transgene encoding one or more of the retromer proteins described herein. The composition can comprise a vector, e.g., a viral vector, e.g., an adeno-associated virus (AAV) vector. In some embodiments, the subject is administered a second composition containing a transgene encoding one or more of the retromer proteins described herein. The second composition can comprise a vector, e.g., a viral vector, e.g., an AAV vector. In some embodiments, the subject is administered a third composition containing a transgene encoding one or more of the retromer proteins described herein. The third composition can comprise a vector, e.g., a viral vector, e.g., an AAV vector.

[0009] In a first aspect, the disclosure features a composition containing a transgene encoding the retromer core protein VPS35 and / or VPS26a and / or VPS26b. In embodiments, the transgene encodes VPS35. In embodiments, the transgene encodes VPS26a and / or VPS26b. In embodiments, the transgene encodes VPS35 and either VPS26a or VPS26b. In embodiments, the transgene encodes VPS35 and VPS26a. In embodiments, the transgene encodes VPS35 and VPS26b. In embodiments, the transgene encodes VPS26a and VPS26b. In other aspects, the composition includes two transgenes, one of which encodes VPS35 and the other of which encodes VPS26a or VPS26b. In other aspects, the composition includes two transgenes, one of which encodes VPS35 and the other of which encodes VPS26a or VPS26b. In other aspects, the composition includes two transgenes, one of which encodes VPS26a and the other of which encodes VPS26b. In other embodiments, the composition comprises three transgenes, one transgene encoding VPS35, one encoding VPS26a, and one encoding VPS26b.

[0010] In some embodiments, the transgene encodes the retromer core protein VPS35. In some embodiments, the retromer core protein VPS35 protein is human. The retromer core protein VPS35 encoded by the transgene may have an amino acid sequence at least 85% identical to that of VPS35 (e.g., an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of VPS35). In some embodiments, the retromer core protein VPS35 encoded by the transgene has an amino acid sequence at least 90% identical to that of VPS35 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of VPS35). In some embodiments, the retromer core protein VPS35 encoded by the transgene has an amino acid sequence that is at least 95% identical to the amino acid sequence of VPS35 (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of VPS35). In some embodiments, the retromer core protein VPS35 encoded by the transgene has an amino acid sequence that differs from VPS35 by one or more amino acid substitutions, insertions and / or deletions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more amino acid substitutions, insertions and / or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions). In some embodiments, the retromer core protein VPS35 encoded by the transgene has an amino acid sequence that differs from VPS35 by one or more conservative amino acid substitutions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions).

[0011] In some embodiments, the transgene encoding the retromer core protein VPS35 comprises human VPS35 (Gene ID 55737). In some embodiments, the transgene encoding the retromer core protein VPS35 has a nucleic acid sequence at least 70% identical to a nucleic acid sequence encoding VPS35 (e.g., a nucleic acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer core protein VPS35 has a nucleic acid sequence at least 85% identical to a nucleic acid sequence encoding VPS35 (e.g., a nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer core protein VPS35 has a nucleic acid sequence at least 90% identical to a nucleic acid sequence encoding VPS35 (e.g., a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer core protein VPS35 has a nucleic acid sequence at least 95% identical to a nucleic acid sequence encoding VPS35 (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer core protein VPS35 is codon optimized.

[0012] In some embodiments, the transgene encodes the retromer core protein VPS26a. In some embodiments, the retromer core protein VPS26a protein is human. The retromer core protein VPS26a encoded by the transgene may have an amino acid sequence at least 85% identical to the amino acid sequence of VPS26a (e.g., an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of VPS26a). In some embodiments, the retromer core protein VPS26a encoded by the transgene has an amino acid sequence at least 90% identical to that of VPS26a (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to that of VPS26a). In some embodiments, the retromer core protein VPS26a encoded by the transgene has an amino acid sequence at least 95% identical to that of VPS26a (e.g., 95%, 96%, 97%, 98%, 99% or 100% identical to that of VPS26a). In some embodiments, the retromer core protein VPS26a encoded by the transgene has an amino acid sequence that differs from VPS26a by one or more amino acid substitutions, insertions and / or deletions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more amino acid substitutions, insertions and / or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions). In some embodiments, the retromer core protein VPS26a has an amino acid sequence that differs from VPS26a by one or more conservative amino acid substitutions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions).

[0013] In further embodiments, the transgene encoding the retromer core protein VPS26a comprises human VPS26a (Gene ID 9559). In some embodiments, the transgene encoding the retromer core protein VPS26a has a nucleic acid sequence at least 70% identical to a nucleic acid sequence encoding VPS26a (e.g., a nucleic acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer core protein VPS26a has a nucleic acid sequence at least 85% identical to a nucleic acid sequence encoding VPS26a (e.g., a nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer core protein VPS26a has a nucleic acid sequence at least 90% identical to a nucleic acid sequence encoding VPS26a (e.g., a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer core protein VPS26a has a nucleic acid sequence at least 95% identical to a nucleic acid sequence encoding VPS26a (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer core protein VPS26a is codon optimized.

[0014] In some embodiments, the transgene encodes the retromer core protein VPS26b. In some embodiments, the retromer core protein VPS26b protein is human. The retromer core protein VPS26b encoded by the transgene may have an amino acid sequence at least 85% identical to the amino acid sequence of VPS26b (e.g., an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of VPS26b). In some embodiments, the retromer core protein VPS26b encoded by the transgene has an amino acid sequence at least 90% identical to that of VPS26b (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to that of VPS26b). In some embodiments, the retromer core protein VPS26b encoded by the transgene has an amino acid sequence at least 95% identical to that of VPS26b (e.g., an amino acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to that of VPS26b). In some embodiments, the retromer core protein VPS26b encoded by the transgene has an amino acid sequence that differs from VPS26b by one or more amino acid substitutions, insertions and / or deletions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more amino acid substitutions, insertions and / or deletions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions).In some embodiments, the retromer core protein VPS26b encoded by the transgene has an amino acid sequence that differs from VPS26b by one or more conservative amino acid substitutions, e.g., 1 to 10, 1 to 15, 1 to 20, 1 to 25 or more conservative amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more conservative amino acid substitutions).

[0015] In further embodiments, the transgene encoding the retromer core protein VPS26b comprises human VPS26b (Gene ID 112936). In some embodiments, the transgene encoding the retromer core protein VPS26b has a nucleic acid sequence at least 70% identical to a nucleic acid sequence encoding VPS26b (e.g., a nucleic acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer core protein VPS26b has a nucleic acid sequence at least 85% identical to the nucleic acid sequence encoding VPS26b (e.g., a nucleic acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer core protein VPS26b has a nucleic acid sequence at least 90% identical to the nucleic acid sequence encoding VPS26b (e.g., a nucleic acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer core protein VPS26b has a nucleic acid sequence at least 95% identical to a nucleic acid sequence encoding VPS26b (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer VPS26b core protein is codon optimized.

[0016] In some embodiments of the foregoing aspects, the composition comprises a vector, e.g., a viral vector. The viral vector can be, for example, an AAV vector, an adenoviral vector, a lentiviral vector, a retroviral vector, a poxvirus vector, a baculovirus vector, a herpes simplex virus vector, a vaccinia virus vector, or a synthetic viral vector (e.g., a chimeric virus, a mosaic virus, or a pseudotyped virus, and / or a virus containing a foreign protein, a synthetic polymer, a nanoparticle, or a small molecule).

[0017] In some embodiments of the foregoing aspects, the viral vector is an AAV vector, for example, AAV1 (i.e., an AAV containing AAV1 inverted terminal repeats (ITRs) and AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., an AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., an AAV9 ITRs and AAV9 capsid protein), AAVrh74 (i.e., an AAV containing AAVrh74 ITRs and AAVrh74 capsid protein), AAVrh.8 (i.e., an AAV containing AAVrh.8 ITRs and AAVrh.8 capsid protein), or AAVrh.10 (i.e., an AAV containing AAVrh.10 ITRs and AAVrh.10 capsid protein).

[0018] In some embodiments of the aforementioned aspects, the viral vector is a pseudotyped AAV vector containing ITRs from one AAV serotype and capsid proteins from a different AAV serotype. In some embodiments, the pseudotyped AAV is AAV2 / 9 (i.e., AAV containing AAV2 ITRs and AAV9 capsid proteins). In some embodiments, the pseudotyped AAV is AAV2 / 10 (i.e., AAV containing AAV2 ITRs and AAV10 capsid proteins).

[0019] In some embodiments of the foregoing aspects, the AAV vector contains a recombinant capsid protein, such as a capsid protein containing a chimera of one or more capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In embodiments, the capsid is a variant AAV capsid, e.g., the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, hereby incorporated by reference).

[0020] In certain embodiments, the viral vector is AAV10. For example, the composition may include an AAV10 comprising a nucleic acid sequence comprising a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0021] In certain embodiments, the viral vector is AAV9. For example, the composition may include AAV9 comprising a nucleic acid sequence comprising a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0022] In certain embodiments, the viral vector is AAV2 / 10. For example, the composition may include an AAV2 / 10 comprising a nucleic acid sequence comprising a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0023] In certain embodiments, the viral vector is AAV2 / 9. For example, the composition may include AAV2 / 9 comprising a nucleic acid sequence that includes a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0024] In certain embodiments, the viral vector is an AAV vector, and the transgene encodes a VPS35 retromer core protein. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV10, which comprises a nucleic acid sequence comprising a transgene encoding a functional VPS35 retromer core protein. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV9, which comprises a nucleic acid sequence comprising a transgene encoding a functional VPS35 retromer core protein. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, which comprises a nucleic acid sequence comprising a transgene encoding a functional VPS35 retromer core protein.

[0025] In certain embodiments, the viral vector is an AAV vector, and the transgene encodes the retromer core protein VPS26a. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV10, which comprises a nucleic acid sequence comprising a transgene encoding a functional VPS26a retromer core protein. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV9, which comprises a nucleic acid sequence comprising a transgene encoding a functional retromer core protein VPS26a. For example, the composition may comprise a recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, which comprises a nucleic acid sequence comprising a transgene encoding a functional retromer core protein VPS26a.

[0026] In certain embodiments, the viral vector is an AAV vector, and the transgene encodes the retromer core protein VPS26b. For example, the composition may include a recombinant AAV (rAAV), such as AAV10, which includes a nucleic acid sequence comprising a transgene encoding a functional VPS26b retromer core protein. For example, the composition may include a recombinant AAV (rAAV), such as AAV9, which includes a nucleic acid sequence comprising a transgene encoding a functional retromer core protein VPS26b. For example, the composition may include a recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, which includes a nucleic acid sequence comprising a transgene encoding a functional retromer core protein VPS26b.

[0027] In some embodiments of any of the above aspects of the present disclosure, the composition comprises a liposome, a vesicle, a synthetic vesicle, an exosome, a synthetic exosome, a dendrimer, or a nanoparticle.

[0028] In some embodiments of any of the above aspects of the present disclosure, the transgene is operably linked to a promoter that induces expression of the transgene in neurons. The promoter may be, for example, a chicken beta-actin promoter, a cytomegalovirus (CMV) promoter, a myosin light chain-2 promoter, an alpha-actin promoter, a troponin 1 promoter, a Na+ / Ca2+ exchanger promoter, a dystrophin promoter, a creatine kinase promoter, an alpha 7 integrin promoter, a brain natriuretic peptide promoter, an alpha B-crystallin / small heat shock protein promoter, an alpha myosin heavy chain promoter, or an atrial natriuretic factor promoter.

[0029] In some embodiments of any of the above aspects of the present disclosure, the transgene is operably linked to an enhancer that induces expression of the transgene in neurons. Exemplary enhancers that can be used in conjunction with the compositions and methods of the present disclosure include the CMV enhancer, the myocyte enhancer factor 2 (MEF2) enhancer, and the MyoD enhancer.

[0030] In another aspect, the disclosure features a method for treating a degenerative disease or disorder in a subject in need thereof by administering one or more compositions comprising one or more viral vectors according to the preceding embodiments. In some embodiments, the composition is administered to the subject upon or shortly after the subject is diagnosed with the degenerative disease or disorder. In embodiments, the degenerative disease or disorder is a neurodegenerative disease, such as Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), multiple system atrophy (MSA), Down's syndrome and hereditary spastic paraplegia (HSP), and tauopathies, such as progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0031] In another aspect, the disclosure features methods for treating a degenerative disease or disorder in a subject in need thereof by administering one or more compositions comprising transgenes encoding the retromer core proteins VPS35 and / or VPS26a and / or VPS26b described in the preceding paragraph. In embodiments, the composition comprises a transgene encoding VPS35 and a transgene encoding VPS26a. In embodiments, the composition comprises a transgene encoding VPS35 and a transgene encoding VPS26b. In embodiments, the composition comprises a transgene encoding VPS26a and a transgene encoding VPS26b. In embodiments, the composition comprises a transgene encoding VPS35, a transgene encoding VPS26a, and a transgene encoding VPS26b. In some embodiments, the composition is administered to a subject upon or shortly after the subject is diagnosed with a degenerative disease or disorder. In embodiments, the degenerative disease or disorder is a neurodegenerative disease, such as Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), Down's syndrome, hereditary spastic paraplegia (HSP) and multiple system atrophy (MSA), and tauopathies, such as progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0032] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a first composition containing a transgene encoding the retromer core protein VPS35 and / or VPS26a and / or VPS26b described in the preceding paragraph. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a second composition containing a transgene encoding the retromer core protein VPS35 and / or VPS26a and / or VPS26b described in the preceding paragraph. In embodiments, the method comprises administering a first composition containing a transgene encoding VPS35 and a second composition containing a transgene encoding VPS26a or VPS26b. In embodiments, the method comprises administering a first composition containing a transgene encoding VPS26a or VPS26b and a second composition containing a transgene encoding VPS35. In embodiments, the method comprises administering a first composition containing a transgene encoding VPS26a and a second composition containing a transgene encoding VPS26b. In embodiments, the method comprises administering a first composition comprising a transgene encoding VPS26b and administering a second composition comprising a transgene encoding VPS26a.

[0033] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a third composition containing a transgene encoding the retromer core protein VPS35 and / or VPS26a and / or VPS26b described in the preceding paragraph. In embodiments, the first, second, and third compositions comprise a transgene encoding VPS35, VPS26a, or VPS26b, respectively.

[0034] In some embodiments, the first and second compositions are administered to the subject at the same time. In some embodiments, the first, second and third compositions are administered to the subject at the same time.

[0035] In some embodiments, the second composition is administered to the subject after the first composition is administered to the subject.The second composition can be administered to the subject, for example, within one or more days or one or more weeks after the first composition is administered to the subject.In some embodiments, the second composition is administered to the subject at least one month after the first composition is administered to the subject.In some embodiments, the second composition is administered to the subject while the first composition is still being administered.

[0036] In some embodiments, the third composition is administered to the subject after the first and second compositions are administered to the subject.The third composition can be administered to the subject, for example, within one or more days or one or more weeks after the first and second compositions are administered to the subject.In some embodiments, the third composition is administered to the subject at least one month after the first and second compositions are administered to the subject.In some embodiments, the third composition is administered to the subject while the first and second compositions are still being administered.

[0037] In some embodiments, the first composition is administered to the subject by intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage and / or oral administration.

[0038] In some embodiments, the second composition is administered to the subject by intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage and / or oral administration.

[0039] In some embodiments, the third composition is administered to the subject by intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage and / or oral administration.

[0040] In a further aspect, the disclosure features a method of treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of a composition(s) containing a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0041] In a further aspect, the disclosure features a method of alleviating one or more symptoms associated with a neurodegenerative disease or disorder in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of a composition or compositions containing a transgene encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0042] As part of the above-described aspects, the present disclosure also provides one or more compositions described herein for use in the methods described herein. The present disclosure also provides use of one or more compositions described herein for the manufacture of one or more medicaments for the methods described herein. The transgene may encode retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0043] Thus, as part of the above-described aspects, the present disclosure also provides compositions containing one or more transgenes encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b for use in treating, preventing, and / or curing a neurodegenerative disease or disorder. Further provided are one or more compositions containing one or more transgenes encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b for use in alleviating one or more symptoms associated with a neurodegenerative disease or disorder.

[0044] As part of the above aspects, the present disclosure also provides the use of one or more compositions containing one or more transgenes encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b for the manufacture of one or more medicaments for treating, preventing and / or curing a neurodegenerative disease or disorder. Further provided is the use of one or more compositions containing one or more transgenes encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b for the manufacture of one or more medicaments for alleviating one or more symptoms associated with a neurodegenerative disease or disorder.

[0045] In some embodiments of any of the above aspects, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments of any of the above aspects, the disease or disorder is Alzheimer's disease (AD). In some embodiments of any of the above aspects, the disease or disorder is Parkinson's disease. In some embodiments of any of the above aspects, the disease or disorder is neuronal ceroid lipofuscinosis (NCL). In some embodiments of any of the above aspects, the disease or disorder is a transmissible spongiform encephalopathy (TSE or prion disease). In some embodiments of any of the above aspects, the disease or disorder is multiple system atrophy (MSA). In some embodiments of any of the above aspects, the disease or disorder is progressive supranuclear palsy (PSP). In some embodiments of any of the above aspects, the disease or disorder is frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau). In some embodiments of any of the above aspects, the disease or disorder is chronic traumatic encephalopathy (CTE). In some embodiments of any of the above aspects, the disease or disorder is Down's syndrome. In some embodiments of any of the above aspects, the disease or disorder is HSP. In some embodiments of any of the above aspects, the disease or disorder is LBD. In some embodiments of any of the above aspects, the disease or disorder is ALS. In some embodiments of any of the above aspects, the disease or disorder is FTD or ALS-FTD.

[0046] In another aspect, the present disclosure features a kit containing the composition of the aforementioned aspect. The kit may further contain a package insert, e.g., a package insert instructing a user of the kit to administer the composition to a subject according to the method of any of the above aspects or embodiments of the present disclosure.

[0047] For the purpose of illustrating the invention, there are shown in the drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of one or more viral vectors comprising a transgene encoding a retromer core protein VPS35 and at least one transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 2) 1. A method of treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more compositions comprising at least one viral vector comprising a transgene encoding a retromer core protein VPS35 and at least one transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 3) A method of treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more compositions comprising a nucleic acid encoding the retromer core protein VPS35 and at least one nucleic acid encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 4) 1. A method for alleviating one or more symptoms of a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of one or more viral vectors comprising a transgene encoding the retromer core protein VPS35 and at least one transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 5) 1. A method of alleviating one or more symptoms of a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more compositions comprising at least one viral vector comprising a transgene encoding a retromer core protein VPS35 and at least one transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 6) A method for alleviating one or more symptoms of a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of one or more compositions comprising a nucleic acid encoding a retromer core protein VPS35 and at least a nucleic acid encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 7) 6. The method of any of items 2 and 5, wherein a therapeutically effective amount of a first composition comprising at least one viral vector comprising a transgene encoding a retromer core protein VPS35 is administered to the subject, and a therapeutically effective amount of a second composition comprising at least one viral vector comprising a transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof is administered to the subject. (Item 8) 8. The method of claim 7, wherein a therapeutically effective amount of a third composition comprising at least one viral vector comprising a transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof is administered to the subject. (Item 9) 8. The method of claim 7, wherein the first and second compositions are administered sequentially. (Item 10) 8. The method of claim 7, wherein the first and second compositions are administered simultaneously. (Item 11) 9. The method of claim 8, wherein the first, second and third compositions are administered sequentially. (Item 12) 9. The method of claim 8, wherein the first, second and third compositions are administered simultaneously. (Item 13) 7. The method of any of items 2, 3, 5 and 6, wherein the composition further comprises a pharmaceutical carrier. (Item 14) 14. The method of any of items 1 to 13, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE). (Item 15) A viral vector encoding the retromer core protein VPS35, comprising at least one transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 16) 16. The vector of item 15, wherein the retromer core protein VPS35 encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS35 (SEQ ID NO: 1). (Item 17) Item 16. The vector of Item 15, wherein the retromer core protein VPS35 encoded by the transgene has the amino acid sequence of VPS35 (SEQ ID NO: 1). (Item 18) 16. The vector of item 15, wherein the transgene encoding the retromer core protein Vps35 comprises human Vps35 (SEQ ID NO: 1). (Item 19) 16. The vector of item 15, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS35 (SEQ ID NO: 1). (Item 20) 16. The vector of item 15, wherein the transgene has the nucleic acid sequence of SEQ ID NO: 10. (Item 21) 16. The vector of item 15, wherein the retromer core protein VPS26a encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS26a (SEQ ID NO: 2). (Item 22) Item 16. The vector of Item 15, wherein the retromer core protein Vps26a encoded by the transgene has the amino acid sequence of VPS26a (SEQ ID NO: 2). (Item 23) 16. The vector of item 15, wherein the transgene encoding the retromer core protein Vps35 comprises human VPS26a (SEQ ID NO: 2). (Item 24) 16. The vector of item 15, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS26a (SEQ ID NO: 2). (Item 25) 16. The vector of item 15, wherein the retromer core protein VPS26b encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS26b (SEQ ID NO: 3). (Item 26) Item 16. The vector of Item 15, wherein the retromer core protein VPS26b encoded by the transgene has the amino acid sequence of VPS26b (SEQ ID NO: 3). (Item 27) 16. The vector of item 15, wherein the transgene encoding the retromer core protein VPS35 comprises human VPS26b (SEQ ID NO: 3). (Item 28) 16. The vector of item 15, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS26b (SEQ ID NO: 3). (Item 29) 29. The vector of any one of items 1 to 28, wherein the vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus. (Item 30) 30. The vector of item 29, wherein the viral vector is AAV. (Item 31) Item 32. The vector according to Item 30, wherein the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVrh10. 31. The vector of item 30, wherein the AAV is selected from the group consisting of AAV9 and AAV2 / 9. (Item 33) 33. The vector of any one of items 1 to 32, wherein the transgene is operably linked to a promoter that directs expression of the transgene in neuronal cells. (Item 34) 34. The vector of any one of items 1 to 33, wherein the transgene is operably linked to an enhancer that directs expression of the transgene in neuronal cells. (Item 35) A composition comprising at least one viral vector comprising a transgene encoding a retromer core protein VPS35 and a transgene encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b and combinations thereof. (Item 36) 36. The composition of claim 35, wherein the retromer core protein VPS35 encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS35 (SEQ ID NO: 1). (Item 37) 36. The composition of claim 35, wherein the retromer core protein VPS35 encoded by the transgene has the amino acid sequence of VPS35 (SEQ ID NO: 1). (Item 38) 36. The composition of claim 35, wherein the transgene encoding the retromer core protein VPS35 comprises human VPS35 (SEQ ID NO: 1). (Item 39) 36. The composition of item 35, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS35 (SEQ ID NO: 1). (Item 40) 36. The composition of item 35, wherein the transgene has the nucleic acid sequence of SEQ ID NO: 10. (Item 41) 36. The composition of claim 35, wherein the retromer core protein VPS26a encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS26a (SEQ ID NO: 2). (Item 42) 36. The composition of claim 35, wherein the retromer core protein Vps26a encoded by the transgene has the amino acid sequence of VPS26a (SEQ ID NO: 2). (Item 43) 36. The composition of claim 35, wherein the transgene encoding the retromer core protein Vps35 comprises human VPS26a (SEQ ID NO: 2). (Item 44) 36. The composition of claim 35, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS26a (SEQ ID NO: 2). (Item 45) 36. The composition of claim 35, wherein the retromer core protein VPS26b encoded by the transgene has an amino acid sequence that is at least 85% identical to the amino acid sequence of VPS26b (SEQ ID NO: 3). (Item 46) 36. The composition of claim 35, wherein the retromer core protein VPS26b encoded by the transgene has the amino acid sequence of VPS26b (SEQ ID NO: 3). (Item 47) 36. The composition of claim 35, wherein the transgene encoding the retromer core protein VPS35 comprises human VPS26b (SEQ ID NO: 3). (Item 48) 36. The composition of claim 35, wherein the transgene has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence encoding VPS26b (SEQ ID NO: 3). (Item 49) Item 50. The composition of item 35, wherein the vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus. 50. The composition of item 49, wherein the viral vector is AAV. (Item 51) 51. The composition of item 50, wherein the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrhlO. (Item 52) 51. The composition of item 50, wherein the AAV is selected from the group consisting of AAV9 and AAV2 / 9. (Item 53) 53. The composition of any one of items 35 to 52, wherein the transgene is operably linked to a promoter that directs expression of the transgene in neuronal cells. (Item 54) 53. The composition of any one of items 35 to 52, wherein the transgene is operably linked to an enhancer that directs expression of the transgene in neuronal cells. (Item 55) 36. The composition of claim 35, further comprising a pharmaceutical carrier. (Item 56) A composition comprising at least one nucleic acid encoding VPS35 and a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 57) 57. The composition of item 56, comprising a first nucleic acid encoding VPS35 and a second nucleic acid encoding a retromer core protein selected from the group consisting of VPS26a, VPS26b, and combinations thereof. (Item 58) 58. A kit comprising the composition according to any of items 35 to 57. [Brief explanation of the drawings]

[0048] [Figure 1] Figure 1 shows a backbone rendering (PyMOL, Schroedinger, Inc.) of the three-dimensional structure of the cargo recognition core of retromer, highlighting the interactions of VPS35 (orange) with VPS29 (red) and VPS26a (green). VPS26b binds to Vps35 in a virtually identical manner (Collins et al. 2008; Shi et al. 2006). Atomic coordinates taken from the cryoEM structure of the mouse heterotrimer (PDB file 6vac) (Kendall et al. 2020).

[0049] [Figure 2-1] Figure 2 shows that VPS35 expression alone is insufficient to increase retromer trimer and function. Figure 2A is a representative immunoblot showing retromer and Sorl1 expression levels after AAV9-VPS35-HA. AAV9-GFP and AAV9-EV (empty vector) were used as controls. Figure 2B is a bar graph showing the average levels of retromer components and Sorl1 normalized to actin; control (left bar, dark gray, n = 23), VPS35 overexpression (right bar, red, n = 16). ***P < 0.001, ns = not significant. [Figure 2-2] Same as above.

[0050] [Figure 3-1] Figure 3 shows maps of the plasmids used in the examples. Figure 3A shows the empty chassis control plasmid map. Figure 3B shows the GFP control plasmid map. Figure 3C shows the VPS35 plasmid map. Figure 3D shows the VPS26a plasmid map. Figure 3E shows the VPS26b plasmid map. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above.

[0051] [Figure 4-1] Figure 4 shows the optimization of combined VPS35 and VPS26 expression in neuroblastoma cells. Figure 4A shows representative immunoblots showing retromer expression levels after transfection with plasmids containing VPS35 alone, VPS26a alone, and VPS26b alone, or dual transfection of VPS35 together with VPS26a or VPS26b. GFP or empty backbone plasmids were used as controls. To appropriately control for the amount of plasmid DNA / lipofectamine complex introduced in each condition, control plasmids (GFP or empty backbone) were included when only one component of the retromer was transfected. VPS29 shows two distinct bands representing two different isoforms of this protein in these N2a cells. Figures 4B-4E are graphs of the average levels of retromer core proteins (VPS35, VPS26a, VPS26b, and VPS29) normalized to actin in neuroblastoma cells transfected with an empty vector (EV) as a control, a viral vector containing GFP (EV + GFP), VPS35 alone, VPS26a alone, VPS26b alone, VPS35 vector + VPS26a vector, and VPS35 vector + VPS26b vector. Figure 4B shows VPS35. Figure 4C shows VPS26a. Figure 4D shows VPS26b. Figure 4E shows VPS29. Control (dark gray, n = 15), VPS35 (n = 30), VPS26a (n = 30), and VPS26b (n = 15) overexpression alone (blue), and the VPS35 + VPS26 combinatorial (red, n = 15). ***P<0.001, **P<0.01, !P=0.07, ns=not significant. [Figure 4-2] Same as above. [Figure 4-3] Same as above.

[0052] [Figure 5-1]Figure 5 shows that combined expression of VPS35 and VPS26 confer synergy on retromer function in neurons. Figure 5A shows a representative immunoblot showing retromer expression levels after transduction with AAV9 vectors containing VPS35, VPS26a, and VPS26b. AAV9-GFP and AAV9-EV (AAV9 containing an empty backbone plasmid) were used as controls. Experimental AAV9 vectors were expressed in neurons in all possible combinations: single protein expression (VPS35 alone, VPS26a alone, VPS26b alone); dual protein expression (VPS35 + VPS26a, VPS35 + VPS26b, VPS26b + VPS26a); and triple protein expression (VPS35 + VPS26a + VPS26b). To appropriately control for the amount of DNA and AAV9 introduced in each condition, control AAV9 (GFP or EV) was included when only one component of the retromer was transduced. Figures 5B-5E are bar graphs of the average levels of retromer core proteins (VPS35, VPS26a, VPS26b) normalized to actin in neuroblastoma cells transfected with one or more of five viral vectors (AAV9) containing empty vector (EV), GFP, VPS35, VPS26a, or VPS26b. Results are shown for empty vector (EV) + GFP as a control, VPS35 vector alone, VPS26a vector alone, VPS26b vector alone, VPS35 vector + VPS26a vector, VPS35 vector + VPS26b vector, VPS35 vector + VPS26a vector + VPS26b vector, and VPS26a vector + VPS26b vector. Figure 5B shows VPS35. Figure 5C shows VPS29. Figure 5D shows VPS26a. Figure 5E shows VPS26b. Control (dark gray, n=9), single overexpression of VPS35, VPS26a, or VPS26b (blue, n=18), VPS35+VPS26a or VPS26b combinatorial (red, n=9), VPS35+VPS26a+VPS26b (dark red, n=9), VPS26a+VPS26b (pink, n=9). ***P<0.001, **P<0.01, *P<0.05, ns=not significant. [Figure 5-2] Same as above. [Figure 5-3] Same as above.

[0053] [Figure 6-1]Figure 6 shows that combined expression of VPS35 and VPS26 confer synergy on retromer function in neurons. Figure 6A shows a representative immunoblot showing Sorl1 expression levels after transduction with AAV9 vectors containing VPS35, VPS26a, and VPS26b. AAV9-GFP and AAV9-EV (AAV9 containing an empty backbone plasmid) were used as controls. Experimental AAV9 vectors were expressed in neurons in all possible combinations: single protein expression (VPS35 alone, VPS26a alone, VPS26b alone); dual protein expression (VPS35 + VPS26a, VPS35 + VPS26b, VPS26b + VPS26a); and triple protein expression (VPS35 + VPS26a + VPS26b). To appropriately control for the amount of DNA and AAV9 introduced in each condition, control AAV9 (GFP or EV) was included when only one component of the retromer was transduced. Figure 6B is a bar graph of Sorl1 levels normalized to actin in neurons transfected with one or more of five viral vectors (AAV9) containing empty vector (EV), GFP, VPS35, VPS26a, or VPS26b. Results are shown for empty vector (EV) + GFP as a control, VPS35 vector alone, VPS26a vector alone, VPS26b vector alone, VPS35 vector + VPS26a vector, VPS35 vector + VPS26b vector, VPS35 vector + VPS26a vector + VPS26b vector, and VPS26a vector + VPS26b vector. Control (dark gray, n = 9), single overexpression of VPS35, VPS26a, or VPS26b (blue, n = 18), VPS35 + VPS26a or VPS26b combinatorial (red, n = 9), VPS35 + VPS26a + VPS26b (dark red, n = 9), VPS26a + VPS26b (pink, n = 9). ***P < 0.001, ns = not significant. Figures 6C and 6D are scatter plots generated from multivariate regression demonstrating that VPS26a (t = 5.6, p = 1.4E-7) and VPS26b (F = 7.2, p = 3.2E-11) are independently correlated with Sorl1 levels.Figure 6C shows VPS 26a, and Figure 6D shows VPS 26b. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description definition The terms used herein generally have their ordinary meaning in the art within the context of the present invention and the specific context in which each term is used. Certain terms are discussed below or elsewhere herein to provide practitioners with further guidance in describing the method of the present invention and how to use it. It is further understood that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, and no particular emphasis is placed on whether a term is detailed or discussed herein. Synonyms are provided for certain terms. The detailed description of one or more synonyms does not preclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is merely illustrative and does not in any way limit the scope and meaning of the present invention or any exemplified term. Similarly, the present invention is not limited to its preferred embodiments.

[0055] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the constraints of the measurement system, i.e., the precision required for a particular purpose, such as pharmaceutical formulation. For example, "about" can mean within one standard deviation or within more than one standard deviation, in accordance with practice in the art. Alternatively, "about" can mean within a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably 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. When particular values ​​are described in this application and claims, unless otherwise specified, the term "about" meaning within an acceptable error range for the particular value should be assumed.

[0056] The term "subject" as used herein refers to an animal that requires therapeutic or preventive treatment. Subjects include mammals, such as dogs, cats, rodents, cattle, horses, pigs, sheep, and primates. Thus, the compositions and methods can be used in veterinary medicine to treat, for example, companion animals, livestock, zoo laboratory animals, and wild animals. The compositions and methods disclosed herein are particularly desirable for human medical applications.

[0057] The term "patient," as used herein, refers to a human subject. In some embodiments, a "patient" is known to have or suspected to have a neurodegenerative disease or disorder, including, but not limited to, Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), multiple system atrophy, Down's syndrome and hereditary spastic paraplegia (HSP), (MSA), and tauopathies, such as progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE). In some embodiments, a "patient" is known to have or suspected to have a disorder or disease associated with endosomal trafficking, e.g., retromer dysfunction.

[0058] The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in a subject, or an amount that delays, minimizes, or alleviates one or more symptoms associated with a disease or disorder, or an amount that produces a desired beneficial change in physiology in a subject.

[0059] The terms "treat," "treatment," and the like refer to slowing, relieving, ameliorating, or alleviating at least one symptom of a disease or disorder, or a measure for reversing a disease or disorder after its onset.

[0060] The terms "prevent," "prevention," and the like refer to acting prior to the onset of overt disease or disorder, to prevent the disease or disorder from occurring or to minimize the extent of the disease or disorder, or to slow the course of its development.

[0061] The term "cure" and the like means to cure, improve, or restore to good health, or to allow a period of time without recurrence of a disease so that the risk of recurrence is reduced.

[0062] The term "in need thereof" refers to a subject who is known to have, suspected to have, or at risk of having a neurodegenerative disease or disorder, including, but not limited to, Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), multiple system atrophy, Down's syndrome and hereditary spastic paraplegia (HSP), (MSA), and tauopathies, such as progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0063] The term "agent," as used herein, means a substance that produces an effect or is capable of producing an effect, including, but not limited to, vectors, chemicals, pharmaceuticals, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

[0064] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered, including any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0065] The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a host, e.g., hypertension, dizziness, etc. when administered to humans, and that have been approved by a federal or state government regulatory agency or are listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, humans.

[0066] An "isolated nucleic acid molecule" means DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or some combination thereof, where the isolated polynucleotide is not associated with all or a portion of a polynucleotide found in nature, or is linked to a polynucleotide with which it is not naturally linked. For purposes of this disclosure, a "nucleic acid molecule comprising" a particular nucleotide sequence should be understood to not encompass an intact chromosome. An isolated nucleic acid molecule "comprising" a specified nucleic acid sequence may, in addition to the specified sequence, include coding sequences for up to 10 or even up to 20 or more other proteins or portions or fragments thereof, or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences.

[0067] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

[0068] A nucleic acid is "operably linked" when it is functionally related to another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0069] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where a separate designation is intended, it will be clear from the context.

[0070] In some aspects, the present disclosure provides an isolated adeno-associated virus vector (AAV). As used herein, the term "isolated" in relation to AAV refers to AAV that is isolated from its natural environment (e.g., from a host cell, tissue, or subject) or artificially produced. Isolated AAV can be produced using recombinant methods. Such AAV is referred to herein as "recombinant AAV." Recombinant AAV (rAAV) preferably has tissue-specific targeting ability, so that the transgene of rAAV is specifically delivered to one or more predetermined tissues. AAV capsid is an important element in determining these tissue-specific targeting abilities.

[0071] Methods for obtaining recombinant AAVs with desired capsid proteins have been described (see, for example, U.S. Patent No. 7,906,111). Several different AAV capsid proteins have been described, such as those disclosed in Gao, et al., J. Virology 78(12):6381-6388 (June 2004); Gao, et al., Proc Natl Acad Sci USA 100(10):6081-6086 (May 13, 2003); and U.S. Patent No. 7,906,111; U.S. Patent No. 8,999,678. In embodiments of the desired packaging of the constructs and methods described herein, the recombinant AAV may be an AAV9 or AAV10 vector and capsid. However, it should be noted that other suitable AAVs, such as rAAVrh.8 and rAAVrh.10 or other similar vectors, may be adapted for use in the methods and compositions of the present invention. Typically, the method involves culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein.

[0072] The components cultured in the host cell to package the rAAV vector into an AAV capsid can be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequence, cap sequence, and / or helper functions) can be provided by a stable host cell engineered to contain one or more of the required components using methods known to those skilled in the art. Most suitably, such a stable host cell contains the required components under the control of an inducible promoter. However, the required components may also be under the control of a constitutive promoter. In yet another alternative, the selected stable host cell can contain selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, stable host cells can be generated that are derived from 293 cells (containing E1 helper functions under the control of a constitutive promoter) but contain rep and / or cap proteins under the control of an inducible promoter.

[0073] The recombinant AAV vector, rep sequence, cap sequence, and helper function for producing rAAV can be delivered to the packaging host cell using any suitable genetic element (vector). The selected genetic element can be delivered by any suitable method, including those described herein. For example, Fisher et al., J. See Virology 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0074] In some embodiments, recombinant AAV can be produced using a triple transfection method (e.g., as described in detail in U.S. Pat. No. 6,001,650). Typically, recombinant AAV is produced by transfecting host cells with a recombinant AAV vector (including a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap) that function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without producing detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use include the pHLP19 vector described in U.S. Pat. No. 6,001,650 and the pRep6cap6 vector described in U.S. Pat. No. 6,156,303, both of which are hereby incorporated by reference in their entireties. Accessory function vectors encode nucleotide sequences for non-AAV-derived viral and / or cellular functions (i.e., "accessory functions") on which AAV depends for replication. Accessory functions include functions required for AAV replication, including, but not limited to, moieties involved in activation of AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, cap expression product synthesis, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus.

[0075] As used herein, the terms "AAV1," "AAV2," "AAV3," "AAV4," etc. refer to AAV vectors containing ITRs from AAV1, AAV2, AAV3, or AAV4, respectively, and capsid proteins from AAV1, AAV2, AAV3, or AAV4, respectively. The terms "AAV2 / 1," "AAV2 / 8," "AAV2 / 9," etc. refer to pseudotyped AAV vectors containing ITRs from AAV2 and capsid proteins from AAV1, AAV8, or AAV9, respectively.

[0076] With respect to transfected host cells, the term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell is "transfected" when exogenous DNA is introduced inside the cell membrane. Several transfection techniques are generally known in the art. For example, see Graham et al., Virology 52:456 (1973); Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratories, New York (1989), Davis et al., Basic Methods in Molecular Biology, Elsevier (1986), and Chu et al., Gene 13:197 (1981). Such techniques can be used to introduce one or more exogenous nucleic acids, such as nucleotide integration vectors and other nucleic acid molecules, into a suitable host cell.

[0077] "Host cell" refers to any cell that contains or is capable of containing a substance of interest. Host cells are often mammalian cells. Host cells can be used as recipients of AAV helper constructs, AAV minigene plasmids, accessory function vectors, or other transfer DNAs involved in the production of recombinant AAV vectors. This term includes the progeny of the original transfected cell. Thus, as used herein, "host cell" can refer to a cell transfected with an exogenous DNA sequence. It is understood that the progeny of a single parent cell may not necessarily be completely identical to the original parent in morphology or in genomic or total DNA complement due to natural, accidental, or deliberate mutations.

[0078] With respect to cells, the term "isolated" refers to cells that have been isolated from their natural environment (e.g., from a tissue or subject). The term "cell line" refers to a population of cells capable of continuous or long-term growth and division in vitro. Often, a cell line is a clonal population derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in the karyotype during storage or transfer of such clonal populations. Thus, cells derived from the referenced cell line may not be exactly identical to the ancestral cell or culture, and the referenced cell line includes such variants. As used herein, the term "recombinant cell" refers to a cell into which an exogenous DNA segment, for example, a DNA segment that results in the transcription of a biologically active polypeptide or the production of a biologically active nucleic acid such as RNA, has been introduced.

[0079] The term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, or virion, that can replicate and transfer gene sequences between cells when associated with appropriate control elements. Thus, the term includes cloning and expression vehicles and viral vectors. In some embodiments, a useful vector is intended to be a vector in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery required to initiate the specific transcription of a gene. The phrases "operably positioned," "operably linked," "under control," or "under transcriptional control" mean that the promoter is in the correct position and orientation relative to the nucleic acid so as to control RNA polymerase initiation and gene expression.

[0080] The term "expression vector" or "expression construct" or "construct" refers to any type of genetic construct containing a nucleic acid from which some or all of a nucleic acid coding sequence can be transcribed. In some embodiments, expression includes transcription of a nucleic acid, for example, to produce a biologically active polypeptide product or inhibitory RNA from the transcribed gene.

[0081] Standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis, "Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1982 & 1989 2nd Edition, 2001 3rd Edition); Sambrook and Russell, "Molecular Cloning," 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); and Wu, "Recombinant DNA," Vol. 217, Academic Press, San Diego, CA (1993). Standard methods also appear in Ausbel, et al., "Current Protocols in Molecular Biology," Vols. 1-4, John Wiley and Sons, Inc. New York, NY (2001). Retromer and Neurodegenerative Diseases

[0082] The genetics, cytopathology, and cell biology of Alzheimer's disease (AD) have focused on endosomal trafficking as a key defect in AD pathogenesis. Three lines of evidence implicate retromer dysfunction in AD. First, genetic and gene expression studies have identified an increasing number of retromer-associated molecules associated with AD, including bona fide loss-of-function mutations. Second, retromer dysfunction recapitulates the cytopathology of AD, characterized by dilated, dysfunctional endosomes in which amyloid precursor protein (APP) fragments accumulate. Third, retromer dysfunction mistransports several AD-associated molecules, including APP in neurons and phagocytic receptors in microglia.

[0083] Retromer is a multiprotein complex that is the "master conductor" of endosomal trafficking. The retromer core is a trimer of three different proteins, technically a heterotrimer. All of these proteins are members of the "Vacuolar Protein Sorting" (VPS) family of proteins. VPS35 is the central protein of the trimer core, to which VPS29 and VPS26 bind. VPS26 is the only core protein with two paralogs, designated VPS26a and VPS26b. Thus, neurons have two distinct retromer cores: VPS29-VPS35-VPS26a and VPS29-VPS35-VPS26b. See Figure 1.

[0084] When overexpressed, these core proteins can bind to endogenous retromer components, resulting in increased retromer function. These proteins are very tightly self-regulated inside the cell. To overcome this barrier, two retromer proteins are co-expressed at the same time, as described herein.

[0085] Increasing VPS35 levels with pharmacological chaperones or via viral vectors increases retromer function. Compared to either VPS35 or VPS26, VPS29 protein may be present in excess. As shown herein, co-expression of both VPS35 and VPS26a or VPS35 and VPS26b has a synergistic effect on the cellular levels of VPS35 and VPS26a or VPS35 and VPS26b, respectively.

[0086] Evidence for retromer function is also provided by an approximately 34% increase in Sorl1 levels, an effect size that mirrors the degree of Sorl1 deficiency observed in Alzheimer's disease (Sager et al. 2007; Scherzer et al. 204; Dodson et al. 2006). Broader comparison of Sorl1 effects across these studies provides information about retromer functionality. In the first neuronal study, overexpression of VPS35 alone robustly elevated two of the three retromer core proteins but had no effect on retromer function. In the second neuronal study, synergy resulted in robust elevation of all three trimeric proteins, leading to an increase in retromer function. This result provides the first empirical evidence that co-elevation of all three retromer core proteins is necessary to upregulate overall retromer function. The studies herein demonstrate that by exploiting retromer stoichiometry and protein-protein interactions, exogenous expression of all three proteins is not required. Exogenously expressing VPS35 and VPS26 is sufficient to also upregulate levels of VPS29 and increase the endosomal cargo recycling function of retromer.

[0087] Importantly, it is also shown herein that the levels of VPS26a and VPS26b are independent of each other, and thus appropriate combinatorial selection allows for the selective increase of one retromer heterotrimer over the other.

[0088] Described herein is a biologically based method for increasing retromer levels and function in vivo: overexpression of retromer by using recombinant AAV (adeno-associated adenovirus) technology. Establishing a novel retromer-AAV tool for use in retromer-based therapeutics has a significant impact because this viral delivery system, recently approved for clinical application, can circumvent the obstacles encountered by small molecules in living organisms (i.e., low absorption rate, degradation, toxicity, lack of target / organ specificity, blood-brain barrier permeability). Compositions containing AAV vectors and retromer transgenes offer many advantages, including increased expression of therapeutic agents, bypassing strict protein autoregulation, the potential for long-term expression of stabilized proteins, and increased half-life of stabilized proteins. Methods of treating, preventing and / or curing neurodegenerative diseases

[0089] Patients who would benefit from administration of the described gene therapies include those diagnosed with neurodegenerative diseases or disorders involving defects in endosomal trafficking, including, but not limited to, Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathies (TSEs or prion diseases), multiple system atrophy (MSA), Down's syndrome and hereditary spastic paraplegia (HSP), and tauopathies, e.g., progressive supranuclear palsy (PSP), frontotemporal lobe dementia linked to chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0090] In these patients, compositions containing nucleic acids encoding one or more of the retromer core proteins (e.g., viral vectors, e.g., AAV vectors, containing such nucleic acids) can be administered to the patient. These compositions can be administered alone or in combination with other agents for the treatment of neurodegenerative diseases or disorders.

[0091] In some embodiments, the present disclosure provides methods for treating, preventing, curing, and / or reducing the severity or extent of a neurodegenerative disease or disorder by administering to a subject in need thereof a therapeutically effective amount of a composition(s), e.g., a viral vector (e.g., AAV), comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26 and / or retromer core protein VPS26b. In some embodiments, the viral vector is AAV, e.g., rAAV2-retro, AAV10, AAV2 / 10, AAV9, or AAV2 / 9. In some embodiments, a composition(s) (e.g., a viral vector, e.g., AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b is administered as soon as a neurodegenerative disease or disorder is diagnosed or suspected. In embodiments, the composition administered comprises a nucleic acid encoding VPS35 and either VPS26a or VPS26b. In embodiments, the methods comprise administering, simultaneously or sequentially, one or more compositions comprising a nucleic acid encoding VPS35 and a nucleic acid encoding either VPS26a or VPS26b. In embodiments, the methods comprise administering, simultaneously or sequentially, one or more compositions comprising a nucleic acid encoding VPS35, a nucleic acid encoding VPS26a, and a nucleic acid encoding VPS26b. In embodiments, the methods comprise administering, simultaneously or sequentially, one or more compositions comprising a nucleic acid encoding VPS26a and a nucleic acid encoding VPS26b.

[0092] In some embodiments, the amount of AAV vector containing a transgene administered is about 4.2 x 10 11 or 4.2 x 10 10 The genome or vector or vector copy of the vector.

[0093] The present disclosure also provides methods for treating, preventing, curing, and / or reducing the severity or extent of a neurodegenerative disease or disorder by administering to a subject in need thereof a therapeutically effective amount of a first composition (e.g., a viral vector, e.g., AAV) containing a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, the method further comprising the step of administering to the subject a therapeutically effective amount of a second composition (e.g., a viral vector, e.g., AAV) containing a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In embodiments, the method comprises administering a therapeutically effective amount of a first composition containing a nucleic acid encoding retromer core protein VPS35 and a therapeutically effective amount of a second composition containing a nucleic acid encoding retromer core protein VPS26a or VPS26b. In embodiments, the method comprises administering a therapeutically effective amount of a first composition containing a nucleic acid encoding the retromer core protein VPS26a or VPS26b and a therapeutically effective amount of a second composition containing a nucleic acid encoding the retromer core protein VPS35.

[0094] The present disclosure also provides methods for treating, preventing, curing, and / or reducing the severity or extent of a neurodegenerative disease or disorder by administering to a subject in need thereof a therapeutically effective amount of a first composition (e.g., a viral vector, e.g., AAV) containing a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, the method further comprising the step of administering to the subject a therapeutically effective amount of a second composition (e.g., a viral vector, e.g., AAV) containing a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, and the method further comprising the step of administering to the subject a therapeutically effective amount of a third composition (e.g., a viral vector, e.g., AAV) containing a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In embodiments, the first, second and third compositions each comprise a nucleic acid encoding the retromer core protein VPS35, VPS26a or VPS26b.

[0095] In some embodiments, the first, second and third AAV vectors are each independently AAV9 vectors encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or VPS26b retromer core protein.In some embodiments, the first AAV vector, the second AAV vector and the third AAV vector are administered at the same time.In some embodiments, the first AAV vector is administered before the second AAV vector.In some embodiments, the second AAV vector is administered before the third AAV vector.

[0096] In some embodiments, the first composition (e.g., the AAV vector) is administered as soon as a neurodegenerative disease or disorder is diagnosed or suspected, and the second composition (e.g., the AAV vector) is administered at a time later than the first composition. In some embodiments, the second composition (e.g., the AAV vector) is administered within a few hours of the first composition (e.g., the AAV vector). In some embodiments, the second composition (e.g., the AAV vector) is administered within a few days of the first composition (e.g., the AAV vector). In some embodiments, the second composition (e.g., the AAV vector) is administered several weeks after the first composition (e.g., the AAV vector). In some embodiments, the first composition (e.g., the AAV vector) and the second composition (e.g., the AAV vector) are administered simultaneously at any given time.

[0097] In some embodiments, the third composition (e.g., AAV vector) is administered at a time later than the second composition. In some embodiments, the third composition (e.g., AAV vector) is administered within a few hours of the second composition (e.g., AAV vector). In some embodiments, the third composition (e.g., AAV vector) is administered within a few days of the second composition (e.g., AAV vector). In some embodiments, the third composition (e.g., AAV vector) is administered several weeks after the second composition (e.g., AAV vector).

[0098] In some embodiments, the first composition (e.g., AAV vector), the second composition (e.g., AAV vector), and the third composition (e.g., AAV vector) are administered simultaneously at any given time, including at the time a neurodegenerative disease or disorder is diagnosed or suspected, or after a neurodegenerative disease or disorder is diagnosed or suspected. In some embodiments, the three compositions (e.g., AAV vectors) are present within the same larger composition; in some embodiments, the three are separate compositions.

[0099] In embodiments of the methods described herein, one or more compositions comprising one or more nucleic acids encoding VPS35 and VPS26b (preferentially expressed in the cortex) are administered to a subject having or at risk of developing a disorder in which endosomal trafficking defects occur primarily in the cortex and in which VPS35 is not affected. Examples of cortical endosomal trafficking disorders in which VPS35 is not affected include biomarker-negative sporadic AD, AD patients with SORL1 mutations, FTD, prion disease, and Down's syndrome.

[0100] In other embodiments of the methods described herein, one or more compositions comprising one or more nucleic acids encoding VPS35 and VPS26a (which are preferentially expressed in the subcortical region) are administered to a subject having or at risk of developing a disorder in which defects in endosomal trafficking occur predominantly in the subcortical region, such as biomarker-negative sporadic PD, HSP, prion disease, and NCL.

[0101] In other embodiments of the methods described herein, one or more compositions comprising one or more nucleic acids encoding VPS35, VPS26a, and VPS26b are administered to subjects having or at risk of developing endosomal trafficking neurological disorders where the disease is more diffuse, such as Lewy Body Disease (LBD), prion diseases, and ALS-FTD.

[0102] In addition to treating, preventing, curing, and / or reducing the severity or extent of neurodegenerative diseases or disorders, in embodiments, the methods and compositions described herein are used to treat, prevent, cure, and / or reduce the severity of other disorders or diseases associated with endosomal trafficking and retromer dysfunction. Recombinant AAV vectors

[0103] A "recombinant AAV (rAAV) vector" as described herein generally comprises a transgene (e.g., encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b). The transgene is flanked by 5'-ITRs and 3'-ITRs, and the transgene may be operably linked to one or more regulatory elements in a manner that allows transcription, translation, and / or expression of the transgene in cells of the target tissue. Such regulatory elements may include promoters or enhancers, such as the chicken beta-actin promoter or cytomegalovirus enhancer, among others described herein. The recombinant AAV genome is generally encapsidated by capsid proteins (e.g., from the same AAV serotype from which the ITRs are derived, or from an AAV serotype different from that from which the ITRs are derived). The AAV vector is then delivered to the selected target cell type or tissue. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence that encodes one or more of VPS35, VPS26a, and / or VPS26b. Exemplary AAV vector components that can be used in conjunction with the compositions and methods of the present disclosure are described herein.

[0104] Any AAV serotype or combination of AAV serotypes can be used in the method and composition of the present disclosure.Because the method and composition of the present disclosure are for treating and curing neurodegenerative disease or disorder, the AAV serotype that at least targets central nervous system can be used in some embodiments, including but not limited to AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10.

[0105] In some embodiments, the broad tropism AAV9 serotype is used, hi some embodiments, AAV2 / 9 is used. AAV vector components

[0106] The AAV vectors described herein can contain cis-acting 5'ITR and 3'ITR (see, e.g., Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are typically about 145 bp in length. Preferably, substantially the entire ITR-encoding sequence is used in the molecule, although some minor modifications of these sequences are acceptable (see, for example, texts such as Sambrook et al., (1989) and Fisher et al., (1996)). An example of such a molecule is a "cis-acting" plasmid containing a transgene, in which the selected transgene sequence and associated regulatory elements are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequences can be obtained from any known AAV, including the mammalian AAV types identified herein.

[0107] In addition to the elements identified above for recombinant AAV vectors, the vector may also contain conventional control elements operably linked to the transgene in a manner that allows its transcription, translation, and / or expression in cells transfected with a plasmid vector or infected with a virus. As used herein, "operably linked" sequences include both expression control sequences contiguous with the gene of interest and expression control sequences that act in trans or remotely to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing signals and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and can be utilized.

[0108] As used herein, a nucleic acid sequence (e.g., a coding sequence) and a regulatory sequence are said to be operably linked when they are covalently linked in such a way that the expression or transcription of the nucleic acid sequence is under the influence or control of the regulatory sequence. When it is desired that the nucleic acid sequence be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences (1) does not result in the introduction of frame-shift mutations, (2) does not interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) does not interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably linked to a nucleic acid sequence if it is capable of effecting transcription of that DNA sequence so that the resulting transcript can be translated into a desired protein or polypeptide. Similarly, two or more coding regions are operably linked if they are linked in such a way that transcription from a common promoter results in the expression of two or more proteins. In some embodiments, operably linked coding sequences result in a fusion protein. In some embodiments, the operably linked coding sequences produce a functional RNA (e.g., shRNA, miRNA). In some embodiments, the operably linked coding sequences produce two or more separate functional proteins (e.g., VPS35 and VPS26a or VPS26b).

[0109] For nucleic acids encoding proteins, a polyadenylation sequence is generally inserted after the transgene sequence and before the 3'AAV ITR sequence. The rAAV construct of the present disclosure can also include an intron, which is preferably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is called the SV-40T intron sequence.

[0110] Another vector element that can be used is an internal ribosome entry site (IRES).IRES sequences are used to produce more than one polypeptide or protein from a single transcript.For example, IRES elements can be used to express VPS35 and VPS26a, VPS35 and VPS26b, or VPS26a and VPS26b from the same AAV vector.

[0111] The exact nature of regulatory sequences required for gene expression in host cells may vary between species, tissues, or cell types, but generally will include 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like, as needed. In particular, such 5' non-transcribed regulatory sequences include promoter regions containing promoter sequences for transcriptional control of operably linked genes. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired. Vectors may also include 5' leader or signal sequences, as needed.

[0112] Examples of constitutive promoters include, but are not limited to, the chicken beta-actin promoter, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the human elongation factor-la (EFla) promoter (Invitrogen).

[0113] Inducible promoters allow for the regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or specific physiological conditions such as the presence of an acute phase, a specific differentiation state of cells, or only in replicating cells. Inducible promoters and inducible systems are available from a variety of commercial sources, including, but not limited to, Invitrogen, Clontech, and Ariad. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al. al., Proc. Natl. Acad. Sci. USA 93:3346-3351 (1996)), tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)), tetracycline-inducible system (Gossen et al., Science 268:1766-1769 (1995), RU486-inducible systems (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)), and rapamycin-inducible systems (Magari et al., J. Clin. Invest. 100:2865-2872 (1997)). Still other types of inducible promoters that may be useful in this regard are inducible promoters that are regulated by specific physiological conditions, such as temperature, acute phase, a specific differentiation state of the cell, or only in replicating cells.

[0114] In another embodiment, the native promoter or its fragment for the transgene can be used.When it is desired that the expression of the transgene should mimic native expression, the native promoter can be preferred.The native promoter can be used when the expression of the transgene needs to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to a specific transcription stimulus.In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0115] In some embodiments, the regulatory sequence confers tissue-specific gene expression. In some cases, the tissue-specific regulatory sequence binds to a tissue-specific transcription factor that induces transcription in a tissue-specific manner.

[0116] In some embodiments, one or more binding sites for one or more miRNAs are incorporated into the transgene of an rAAV vector to inhibit transgene expression in one or more tissues of a subject carrying the transgene. The miRNA target site in the mRNA can be in the 5'-UTR, 3'-UTR, or coding region. Typically, the target site is in the 3'UTR of the mRNA. Furthermore, the transgene can be designed so that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites can result in the cooperative action of multiple RISCs, providing highly efficient inhibition of expression. The target site sequence can contain a total of 5 to 100, 10 to 60, or more nucleotides. The target site sequence can include at least 5 nucleotides of the target gene binding site sequence.

[0117] For example, a 3'-UTR region can be incorporated into the transgene to inhibit expression in the liver. This is beneficial for transgenes encoding therapeutic proteins that are toxic to the liver, since the majority of administered viruses (approximately 60-90%) are ultimately found in the liver. Therefore, suppressing therapeutic gene expression in the liver relieves the burden on liver cells.

[0118] In some embodiments, the AAV vector is modified to be a self-complementary AAV. The self-complementary AAV has a complementary sequence of the transgene (i.e., a double copy of the transgene). Self-complementarity makes the gene more stable after entering the cell. Transgene coding sequence

[0119] The nucleic acid sequence of the transgene described herein can be designed based on knowledge of the particular composition (e.g., viral vector) in which the transgene will be expressed. For example, one type of transgene sequence includes a reporter sequence that produces a detectable signal upon expression. In another example, the transgene encodes a therapeutic protein or a therapeutic functional RNA. In another example, the transgene encodes a protein or functional RNA intended to be used for research purposes, for example, to create a somatic transgenic animal model carrying the transgene, for example, to study the function of the transgene product. In another example, the transgene encodes a protein or functional RNA intended to be used to create an animal model of a disease. Suitable transgene coding sequences will be apparent to those skilled in the art.

[0120] In embodiments, the transgene encodes a functional protein, including but not limited to, retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In embodiments, the transgene encodes either VPS35 and VPS26a or VPS26b. In embodiments, the transgene encodes VPS35, VPS26a and VPS26b. In embodiments, the transgene encodes VPS26a and VPS26b. In embodiments, the transgene encodes only one of VPS35, VPS26a and VPS26b.

[0121] Amino acid sequence information is available from the National Center for Biotechnology Information (NCBI) and is shown below.

[0122] The gene encoding the human retromer core protein VPS35 (Gene ID: 55737) was found to encode the functional retromer core protein VPS35 (SEQ ID NO: 1): [ka] [ka] can be used to obtain a transgene encoding

[0123] The gene encoding the human retromer core protein VPS26a (Gene ID: 9559) was found to encode the functional retromer core protein VPS26a (SEQ ID NO: 2): [ka] can be used to obtain a transgene encoding

[0124] The gene encoding the human retromer core protein VPS26b (Gene ID: 112936) was found to encode the functional retromer core protein VPS26b (SEQ ID NO: 3): [ka] can be used to obtain a transgene encoding

[0125] The wild-type mouse mRNA sequence (ie, the coding sequence) for the retromer core protein was obtained from the National Center for Biotechnology Information (NCBI) and is shown below.

[0126] mVPS35 (SEQ ID NO: 4) [ka] [ka]

[0127] mVPS26a isoform A (SEQ ID NO: 5) [ka] [ka]

[0128] mVPS26b (SEQ ID NO: 6) [ka] [ka] Codon optimization of transgene coding sequences

[0129] Codon optimization of a transgene coding sequence can increase the efficiency of gene therapy. Thus, in some embodiments, a nucleic acid that is at least 70% identical to the coding sequence of a transgene encoding a therapeutic protein (e.g., a nucleic acid sequence that is 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence) is used.

[0130] Codon optimization tools are known in the art.

[0131] Exemplary codon-optimized nucleic acids are as follows:

[0132] Codon-optimized mVPS35 (SEQ ID NO: 7) [ka] [ka]

[0133] Codon-optimized mVPS26a, isoform A (SEQ ID NO: 8) [ka] [ka]

[0134] Codon-optimized mVPS26b (SEQ ID NO: 9) [ka] [ka]

[0135] Human VPS35 coding sequence (with certain codons modified to remove restriction sites) (SEQ ID NO: 10) [ka] [ka] Route of administration and administration

[0136] The present disclosure provides rAAV vectors for use in methods of treating, preventing, and / or curing a neurodegenerative disease or disorder and / or alleviating at least one symptom associated with a neurodegenerative disease and / or disorder in a subject. In some embodiments, the methods comprise administering to a subject an rAAV vector encoding one or more therapeutic polypeptides or proteins in a pharmaceutically acceptable carrier in an amount and for a period of time sufficient to treat, prevent, and / or cure the neurodegenerative disease or disorder in a subject having or suspected of having such a neurodegenerative disease or disorder.

[0137] The rAAV vector can be delivered to a subject in a composition according to any suitable method known in the art. The rAAV vector, preferably suspended in a physiologically compatible carrier (e.g., a composition), can be administered to a subject. In certain embodiments, the composition can include the rAAV vector alone or in combination with one or more other vectors (e.g., a second rAAV vector carrying one or more different transgenes). In one embodiment, the composition can include an rAAV9 vector containing a nucleic acid sequence including a transgene encoding a functional protein, including, but not limited to, retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In one embodiment, the composition can include an rAAV2 / 9 vector containing a nucleic acid sequence including a transgene encoding a functional protein, including, but not limited to, retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In one embodiment, the composition may comprise an rAAV10 or rAAV2 / 10 vector comprising a nucleic acid sequence comprising a transgene encoding a functional protein, including, but not limited to, retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b.

[0138] Suitable carriers can be easily selected by those skilled in the art, taking into consideration the indications for which rAAV is intended.For example, one suitable carrier includes saline, which can be formulated with various buffer solutions (e.g., phosphate-buffered saline).Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil and water.The selection of carriers is not a limitation of the present disclosure.

[0139] If necessary, the compositions disclosed herein may contain other conventional pharmaceutical ingredients, such as preservatives or chemical stabilizers, in addition to rAAV and carriers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0140] In some embodiments, the rAAV composition may contain, in particular, a high rAAV concentration (e.g., about 10 13 The composition is formulated to reduce aggregation of AAV particles in the composition when rAAV is present in the presence of 0.1 GC / ml or higher. Methods for reducing rAAV aggregation are well known in the art and include, for example, the addition of detergents, pH adjustment, and salt concentration adjustment (see, e.g., Wright, et al., Molecular Therapy 12:171-178 (2005)).

[0141] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms. In many cases, the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0142] For administration of injectable aqueous solutions, for example, the solution may be appropriately buffered if necessary, and the liquid diluent may first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those skilled in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion therapy fluid, or injected at the proposed injection site. Some variation in dosage will inevitably occur depending on the condition of the host. In any case, the person responsible for administration will determine the appropriate dose for each individual host.

[0143] Sterile injectable solution is prepared by incorporating the active rAAV in the required amount in a suitable solvent together with various other components listed herein, and then optionally sterilizing by filtration.Generally, dispersion is prepared by incorporating various sterilized active ingredients into a sterile vehicle that contains basic dispersion medium and other components listed above that are required.For the preparation of sterile injectable solution, the preferred method of preparing sterile powder is vacuum drying and freeze-drying, which obtains the powder of active ingredient plus any other desired components from the solution that has been previously sterilized and filtered.

[0144] In addition to the above delivery methods, the following techniques are also contemplated as alternative methods for delivering rAAV compositions to a host. Sonophoresis (i.e., ultrasound) is used as a device to enhance the rate and effectiveness of drug penetration into and through the circulatory system and is described in U.S. Patent No. 5,656,016. Other contemplated drug delivery alternatives are intraosseous injection (U.S. Patent No. 5,779,708), microchip devices (U.S. Patent No. 5,797,898), ophthalmic formulations, transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208), and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0145] The rAAV is administered by an administration route and in an amount sufficient to transfect cells in the desired tissue and provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional pharmaceutically acceptable administration routes include, but are not limited to, direct delivery to the selected tissue (e.g., intracerebral administration, intrathecal administration), intravenous, oral, inhalation (including nasal and intratracheal delivery), intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. Administration routes can be combined as desired. The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic composition, the level of symptoms, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic composition to cause improvement in the target disease state while simultaneously minimizing undesirable side effects. Thus, the amount of biologic delivered depends in part on the specific therapeutic composition and the severity of the condition being treated.

[0146] The present disclosure provides stable pharmaceutical compositions comprising rAAV virions that remain stable and active even when subjected to freeze / thaw cycles and when stored in containers made of various materials, including glass.

[0147] The appropriate dose will depend on, among other factors, the subject being treated (e.g., a human or non-human primate or other mammal), the age and general condition of the subject being treated, the severity of the condition being treated, and the mode of administration of the rAAV virions. An appropriate effective amount can be readily determined by one of ordinary skill in the art.

[0148] The dose of rAAV virions required to achieve a desired effect or "therapeutic effect," e.g., a dose unit of vector genome per kilogram of body weight (vg / kg), varies based on several factors, including, but not limited to, the route of administration of the rAAV; the level of gene or RNA expression required to achieve a therapeutic effect; the particular disease or disorder being treated; and the stability of the gene or RNA product. Those skilled in the art can easily determine the rAAV virion dose range for treating a subject with a particular disease or disorder based on the factors described above, as well as other factors well known in the art. An effective amount of rAAV is generally about 10 per subject. 9 ~10 16 The volume of solution used typically depends on, among other things, the size of the subject, the dose of rAAV, and the route of administration. For example, for intrathecal or intracerebral administration, volumes in the range of 1 μl to 10 μl or 10 μl to 100 μl may be used. For intravenous administration, volumes in the range of 10 μl to 100 μl, 100 μl to 1 ml, 1 ml to 10 ml, or more may be used. In some cases, about 10 volumes per subject may be used. 10 ~10 12 In certain embodiments, a dosage of between 10 and 100 rAAV genome copies per subject is appropriate. 12 In some embodiments, 10 rAAV genome copies per subject are effective for targeting the desired tissue. 10 , 10 11 , 10 12 , 10 13 , 10 14 or 10 15 In some embodiments, the rAAV is administered at a dose of 10 genome copies. 10 , 10 11 , 10 12 , 10 13 or 10 14 It is administered at a dose of genome copies / kg.

[0149] Thus, a "therapeutically effective amount" falls in a relatively broad range that can be determined through clinical trials. For example, for in vivo injection, i.e., direct injection into a subject, a therapeutically effective dose is about 10 5 ~10 16 rAAV virions, more preferably 10 8 ~10 14 For in vitro transduction, the effective amount of rAAV virions delivered to cells can be on the order of 10 5 ~10 13 , preferably 10 8 ~10 13 When the composition includes transduced cells to be delivered back to a subject, the amount of transduced cells in the pharmaceutical composition can be on the order of about 10 4 ~10 10 cells, more preferably 10 5 ~10 8 The dosage may be in the form of a single cell. The dosage will, of course, depend on the transduction efficiency, the promoter strength, and the stability of the message and the protein encoded thereby. Effective dosages can be readily established by those skilled in the art through routine testing to establish dose-response curves.

[0150] The dosage treatment can be a single dose schedule or a multiple dose schedule that ultimately delivers the amount specified above.In addition, the subject can be administered multiple doses as needed.Thus, the subject can be administered, for example, a single dose, or, for example, 10 doses. 5 ~10 16 at two, three, four, five, six or more doses that collectively result in delivery of 10 rAAV virions. 5 ~10 16 A person skilled in the art can readily determine the appropriate number of doses to administer.

[0151] Therefore, the pharmaceutical composition contains sufficient genetic material to produce a therapeutically effective amount of the protein of interest, i.e., an amount sufficient to reduce or ameliorate the symptoms of the disease state in question, or an amount sufficient to provide the desired benefit.Therefore, the rAAV virion is present in the subject composition in an amount sufficient to provide a therapeutic effect when given in one or more doses.The rAAV virion can be provided as a lyophilized preparation and can be diluted in a virion-stabilizing composition for immediate or future use.Alternatively, the rAAV virion can be provided immediately after production and stored for future use.

[0152] Pharmaceutical compositions also contain pharmaceutically acceptable excipients or carriers. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that can be administered without undue toxicity. Pharmaceutically acceptable excipients include liquids such as water, saline, glycerol, and ethanol, but are not limited to these. Pharmaceutically acceptable salts, such as salts of mineral acids, e.g., hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and salts of organic acids, e.g., acetates, propionates, malonates, benzoates, and the like, may be included therein. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0153] Formulations of the therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients or stabilizers, for example, in the form of a lyophilized powder, a slurry, an aqueous solution or a suspension.

[0154] The toxicity and therapeutic efficacy of a therapeutic composition administered alone or in combination with another agent can be measured, for example, by the LD 50(a dose lethal to 50% of the population) and ED 50 The dose that is therapeutically effective in 50% of the population can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is known as the therapeutic index (LD 50 / ED 50 In certain embodiments, therapeutic compositions that exhibit high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds preferably is such that the ED 50 The dosage may vary within this range depending upon the dosage form used and the route of administration.

[0155] The determination of appropriate dosage is made by clinicians, for example, by using parameters or factors known or suspected in the art to affect treatment.Dosage can be started at a slightly lower amount than the optimal dose, and then can be increased by small increments until desired or optimal effect is achieved, compared with any negative side effects.Important diagnostic measures include, for example, the measure of inflammatory symptoms or the level of inflammatory cytokines produced.Generally, it is desirable that the biological agent used is derived from the same species as the animal that is targeted for treatment, thereby minimizing any immune response to the reagent.

[0156] The preferred route of administration of AAV is intravenous. Other routes of administration of the rAAV vectors described herein include intracranial, intraparenchymal, and intraspinal.

[0157] A preferred dose is about 1 x 10 10 ~Approx. 8×10 11 , about 2×10 10 ~about 6×10 11 , about 4×10 10 ~Approx. 4×10 11 The total administration of genomic or viral copies (vc) of rAAV ranges from approximately 4 x 10 11is the total dose of genome or viral copies (vc).

[0158] If more than one rAAV is used, the preferred total dose of vector is approximately 1 x 10 10 ~Approx. 6×10 11 , about 2×10 10 ~Approx. 5×10 11 , about 1×10 10 ~Approx. 4×10 11 The total dose of vector is in the range of about 3 x 10 genome or viral copies (vc). 11 The AAVs can be administered in equal amounts, for example, in a 50 / 50 ratio, or in about 5 / 95, 10 / 90, 15 / 85, 20 / 80, 25 / 75, 30 / 70, 35 / 65, 40 / 60, 45 / 55, 55 / 45, 60 / 40, 65 / 35, 70 / 30, 75 / 25, 80 / 20, 85 / 15, 90 / 10, and 95 / 5 or ratios thereof.

[0159] Dosage can be adjusted to optimize the effect in the subject.In addition, before increasing dosage, the subject can be monitored for the improvement of their condition.The response of the subject to the therapeutic administration of rAAV can be monitored by observing the changes in the subject's muscle strength and control, mobility, and height and weight.If one or more of these parameters increase after administration, treatment can be continued.If one or more of these parameters remain the same or decrease, dosage can be increased. kit

[0160] The present disclosure also provides a kit that includes the components of the combination disclosed herein in kit form.The kit of the present disclosure includes one or more components, including but not limited to the viral vector (for example, AAV vector) described herein.The kit can further include a pharmaceutically acceptable carrier as discussed herein.The viral vector can be formulated as a pure composition or in pharmaceutical composition in combination with a pharmaceutically acceptable carrier.

[0161] In some embodiments, the kit comprises an AAV vector containing a transgene described herein in one container (e.g., a sterile glass or plastic vial).

[0162] In some embodiments, the kit includes an AAV vector containing a transgene described herein in one container (e.g., a sterile glass or plastic vial), a second AAV vector encoding a transgene described herein in another container (e.g., a sterile glass or plastic vial), and a third AAV vector encoding a transgene described herein in another container (e.g., a sterile glass or plastic vial).

[0163] In some embodiments, the kit comprises an AAV vector encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, or a pharmaceutical composition thereof, in one or more containers (e.g., sterile glass or plastic vials).

[0164] If the kit includes one or more pharmaceutical compositions for parenteral administration to a subject, the kit may include a device for performing such administration. For example, the kit may include one or more hypodermic needles or other injection devices, as discussed above.

[0165] The kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information will assist patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information about the combination may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, appropriate dosage and administration, how it is provided, appropriate storage conditions, references, manufacturer / distributor information, and patent information. [Example]

[0166] The present invention may be better understood with reference to the following non-limiting examples, which are presented in order to more fully illustrate preferred embodiments of the invention, and should in no way be construed as limiting the broad scope of the invention. Example 1 material and method Plasmid production

[0167] The mRNA sequences for VPS35, VPS26a, and VPS26b were obtained from the National Center for Biotechnology Information (NCBI). The sequences were codon-optimized and then de novo synthesized. The synthesized constructs were subcloned into an AAV transfer plasmid containing AAV2 inverted terminal repeats (ITRs) and the ubiquitous chicken beta-actin wild-type promoter. The transgene was followed by a bovine growth hormone polyadenylation signal. An empty chassis vector control was generated by deleting the VPS35 sequence. A GFP control was designed to mimic the rational design of the targeted VPS construct. It contained enhanced green fluorescent protein, the same bovine growth hormone polyA (BgH) as the VPS construct, and the chicken beta-actin wild-type promoter. The resulting plasmid is shown in Figure 3. AAV9 production

[0168] Each of the above constructs was individually packaged into recombinant adeno-associated virus vector 9 (AAV9) using MeiraGTx capsid and helper plasmid DNA. Briefly, the transfer plasmid, rep-cap plasmid, and helper plasmid were co-transfected into HEK 293 cells. The collected suspension containing virus and cell debris was filtered through a millipore SHC XL150 filter (140 cm). 2The suspension was clarified using a 20 mL column of AVB Sepharose. The clarified suspension was then purified using an AVB Sepharose, 20 mL column, elution in 3 column volumes. Concentration and diafiltration were performed using a 100 kD mPES hollow fiber (Spectrum MicroKros Cat. No. C02-E100-05-S). Further concentration was performed using an Amicon Ultra-4 Centrifugal Filter 30 kD (Cat. No. UFC8030). N2A culture

[0169] Mouse neuroblastoma (N2a) cells were cultured in 50% DMEM (high glucose) and 50% Opti-MEM + 10% FBS and glutamine (2 mM) containing penicillin and streptomycin to prevent microbial contamination. Transfection

[0170] The Lipofectamine transfection protocol was used with some modifications. Briefly, Vps35 and Vps26 (Vps26a or Vps26b) plasmids were cotransfected into neuronal-like cells, Neuro2a (N2a), in a 6-well format using Lipofectamine LTX. 100k cells were plated into each well containing medium along with the DNA-Lipofectamine complex. Empty chassis and GFP were used as control plasmids. The amount of transfected DNA copies per well was 2.81E+11. Cells were harvested 48 hours post-transfection using RIPA buffer as previously described (Qureshi et al. 2019). Neuronal culture and transduction

[0171] Primary mouse cortical and hippocampal neuron cultures were prepared as previously described (Bhalla et al., 2014). (Eds. et al. 2012). Neurons (450k cells per well) were transduced with retromer AAV9 (2.27E+10 vector genomes per well per condition) 7 days after plating in 12-well plates. Empty chassis AAV9 and GFP AAV9 were used as controls. Cultures were maintained for 3 weeks post-transduction (4 weeks in total). On day 28, neurons were lysed using RIPA buffer containing protease and phosphatase inhibitors. Western blot

[0172] Cells from N2A and neuronal cultures were lysed in RIPA and proteins were isolated as previously described (Qureshi et al. 2019; Kirby et al. 2015). Lysates from samples were run on NuPAGE® Bis-Tris 4-12% gels, transferred to nitrocellulose membranes using an iBlot, and probed with antibodies.

[0173] Primary antibodies targeting the following proteins were used: VPS35 (ab57632, Abcam, 1:1k); VPS26a (ab211530, Abcam, 1:500); VPS26b (NBP1-92575, Novus, 1:500 or 15915-1-AP, Proteintech, 1:500); VPS29 (sab2501105, Sigma-Aldrich, 1:500); Sorl1 (611861, BD-biosciences, 1:2k and 79322, Cell Signaling, 1:500); and β-actin (ab6276, Abcam, 1:5k). IRDye® 800 or 680 antibodies (LI-COR) were used as secondary antibodies at dilutions of 1:10k for 800CW, 1:15k for 680RD, and 1:25k for 680LT. Western blots were scanned using an Odyssey imaging system as previously described (Eaton et al. 2013).

[0174] For Sorl1 (BD-611861), Peroxidase AffiniPure Donkey Anti-Mouse IgG (H+L) was used as the secondary antibody (Jackson Immuno Research labs, 1:2k), and blots were scanned with a Fujifilm LAS-3000 Imager. statistics

[0175] Statistical analysis was performed using Microsoft Excel and SPSS. An independent two-sample Student's t-test, assuming equal variances and with a two-tailed distribution, was used for all experiments unless otherwise stated. All data are presented as means, and error bars indicate the standard error of the mean. All bar graphs were generated in GraphPad Prism 8. Scatter plots were generated in SPSS. Example 2 VPS35 expression alone is insufficient to increase retromer trimer and function

[0176] To determine the effect that exogenous VPS35 overexpression has on the non-deficient retromer core protein and retromer function, cultured wild-type mouse neurons were transduced with AAV9-VPS35-HA and either AAV9-GFP or AAV9-empty vector (EV) were used as control conditions and harvested 3 weeks later.

[0177] Levels of all retromer core proteins were determined by immunoblotting (Figure 2A). Compared to controls, 90% VPS35-HA overexpression resulted in a robust 67% increase in endogenous VPS29 (p=9E-09), a smaller 22% increase in VPS26a (p=2E-08), and no increase in VPS26b (p=0.62) (Figure 2B).

[0178] Sorl1 levels were also determined by immunoblotting. Compared to controls, overexpression of VPS35 alone had a small (11%), statistically insignificant (p=0.06) increase in Sorl1 (Figure 2B).

[0179] By showing that VPS35 overexpression leads to robust overexpression of VPS29 but no or modest increase in the VPS26 paralog and has no apparent effect on retromer function, these results warrant investigation of the effects of VPS35 and VPS26 coexpression. Example 3 Results using neuroblastoma cells and plasmids and AAV9 constructs

[0180] Neuroblastoma (N2A) cells were transfected with plasmids expressing single proteins (VPS35, VPS26a, or VPS26b) or protein combinations (VPS35 + VPS26a or VPS35 + VPS26b). A GFP-expressing plasmid or an empty plasmid served as controls.

[0181] The single-protein condition resulted in overexpression of each protein above control levels: VPS35 alone (80%, p=3.4E-09), VPS26a alone (550%; p=2.2E-06), and VPS26b alone (362%; p=0.0002). Compared to the single-protein condition, VPS35 + VPS26a expression resulted in significant increases in VPS35 (31%; p=0.0003), VPS29 (17%; p=0.0007), and VPS26a (52%; p=0.015), but minimal changes in VPS26b. VPS35 + VPS26b expression resulted in a non-significant increase in VPS35 (15%; p=0.07) and VPS26b (56%; p=0.14), a significant increase in VPS29 (22%; p=0.0005), but no increase in VPS26a (see Figure 4). Example 4 Results using neurons and AAV9 constructs

[0182] To test the effects of VPS35 and VPS26 combinations in cultured neurons, we generated five experimental AAV9 vectors expressing mouse VPS35, VPS26a, and VPS26b, as well as two control AAV9 vectors, one of which expressed GFP and the other an empty vector. The experimental vectors were expressed in neurons in all possible combinations: single protein expression (VPS35 alone, VPS26a alone, and VPS26b alone); dual protein expression (VPS35 + VPS26a, VPS35 + VPS26b, and VPS26b + VPS26a); and triple protein expression (VPS35 + VPS26a + VPS26b).

[0183] When the dose of each viral vector was optimized in preliminary studies and used in the final combinatorial study, the average AAV9-VPS35 overexpression was 11% (range: 1%-23%), the average AAV9-VPS26a was 218% (range: 154%-354%), and the average AAV9-VPS26b was 80% (range: 50%-107%) (Figure 5B, blue bars). This profile proved particularly useful for testing interactions.

[0184] We first tested whether there was a VPS35-VPS26 interaction on retromer core protein expression by comparing the levels detected in single-protein conditions with those detected in combinatorial experiments. Compared to single-protein expression, VPS35 + VPS26a expression resulted in a significant increase in VPS35 (70%; p = 4.4E-18), VPS26a (53%; p = 2.1E-05), and VPS29 (approximately 42%; p < 1.22E-05), but not VPS26b. VPS35 + VPS26b expression resulted in a significant increase in VPS35 (64%; p = 3.6E-09), VPS26b (15%; p = 0.003), and VPS29 (approximately 18%; p < 0.013), but not VPS26a.

[0185] Finally, VPS35+VPS26a+VPS26b expression resulted in a significant increase in all four retromer proteins compared to the control (EV+GFP) - VPS35 (81%; p=5.6E-15), VPS29 (51%; p<1.8E-07), VPS26a (220%; p=1.7E-08) and VPS26b (51%; p=9.8E-10).

[0186] Please refer to Figure 5.

[0187] These results again demonstrated the synergistic effect of co-expression of VPS35+VPS26 on VPS35 expression, as well as on VPS26a and VPS26b.

[0188] Although the primary goal of this comprehensive series of experiments was to test for synergistic interactions, the fact that VPS35 + VPS26a had no effect on VPS26b and VPS35 + VPS26b had no effect on VPS26a suggests that in neurons, each VPS26 paralog exists in a biochemically distinct trimer. Example 5 Combined VPS35 and VPS26 expression synergizes retromer function in neurons

[0189] Because loss-of-function mutations in SORL1 cause Alzheimer's disease (Holstege et al. 2017) and an approximately 30% reduction in Sorl1 protein is found even in early stages of sporadic disease (Sager et al. 2007; Scherzer et al. 2004; Dodson et al. 2006), we next tested whether the VPS35 and VPS26 combinatorial have a synergistic effect on retromer function by comparing the levels of Sorl1 measured across all conditions.

[0190] A univariate ANOVA was used, including the control, single, and combinatorial conditions as fixed factors and Sorl1 as the dependent variable. Results revealed a group effect (F = 19.3, p = 9.3E-8), and simple comparisons indicated that there were no differences between the control and single conditions (contrast estimate = 0.1, p = 0.9), but there were significant differences between the single and combined conditions (contrast estimate = 0.4, p = 2.2E-7). Post-hoc comparisons revealed that each combined condition was significantly different from the single conditions (Figure 6B).

[0191] Interestingly, a significant effect of VPS26a + VPS26b overexpression on Sorl1 (34%; p = 0.006) (Figure 6B) was also observed, although this combination did not increase the levels of VPS35 or VPS29 (Figure 5B). Sorl1 has been found to interact with the retromer core via VPS26 (Suzuki et al. (2019)), suggesting that both paralogs may interact with Sorl1 independently.

[0192] We used a large-scale dataset generated across over 140 experimental or control conditions, measuring four retromer core proteins and Sorl1 across a wide dynamic range. We used a multiple linear regression model, including Sorl1 levels as the dependent variable and VPS35, VPS26, VPS26a, and VPS26b levels simultaneously entered as independent variables. A significant association with Sorl1 levels was found (F = 19.5, p = 1.1E-12), with only the VPS26 paralog contributing significantly to the model. Therefore, we trimmed the model to include both paralogs, confirming that both VPS26a (t = 5.6, p = 1.4E-7) and VPS26b (F = 7.2, p = 3.2E-11) were independently correlated with Sorl1 levels (Figures 6C and 6D). This result was consistent with the interpretation that neurons possess two trimers (VPS26b-VPS35-VPS29 and VPS26a-VPS35-VPS29) that are not only biochemically distinct but also functionally distinct.

[0193] References

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Claims

1. 1. A composition for increasing retromer function in a neuronal cell, the composition comprising: (i) a vector comprising a transgene encoding the retromer core protein VPS35 and a transgene encoding the retromer core protein VPS26a; or (ii) a vector containing a transgene encoding the retromer core protein VPS35 and a vector containing a transgene encoding the retromer core protein VPS26a; whereby, when the neuronal cells are contacted with the composition, (a) increasing the level of VPS35 and VPS26a in the neuronal cell compared to the level of VPS35 and VPS26a when only a transgene encoding either VPS35 or VPS26a is introduced into the neuronal cell, thereby increasing retromer function; and / or (b) A composition that increases the level of Sorl1 in the neuronal cell compared to the level of Sorl1 when only a transgene encoding either VPS35 or VPS26a is introduced into the neuronal cell, thereby increasing retromer function.

2. The composition of claim 1, wherein the retromer core protein VPS35 comprises the amino acid sequence of SEQ ID NO:

1.

3. The composition of claim 1, wherein the retromer core protein VPS26a comprises the amino acid sequence of SEQ ID NO:

2.

4. The composition of claim 2, wherein the retromer core protein VPS26a comprises the amino acid sequence of SEQ ID NO:

2.

5. 2. The composition of claim 1, wherein the transgene encoding the retromer core protein VPS35 comprises the nucleic acid sequence of SEQ ID NO:

10.

6. The composition of claim 1 , wherein the vector is a viral vector.

7. 7. The composition of any one of claims 1 to 6, wherein the vector is selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic viruses.

8. The composition of claim 7, wherein the vector is an AAV vector.

9. 9. The composition of claim 8, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrhlO.

10. The composition of claim 7, wherein the vector is an AAV2 / 9 vector.

11. The composition of any one of claims 1 to 6, wherein the transgene is operably linked to a promoter that directs expression of the transgene in neuronal cells.

12. 1. A composition for increasing retromer function in a neuronal cell, the composition comprising: (i) a vector comprising a transgene encoding the retromer core protein VPS35 and a transgene encoding the retromer core protein VPS26b; or (ii) a vector containing a transgene encoding the retromer core protein VPS35 and a vector containing a transgene encoding the retromer core protein VPS26b; whereby, when the neuronal cells are contacted with the composition, (a) increasing the level of VPS35 and VPS26b in the neuronal cell compared to the level of VPS35 and VPS26b when only a transgene encoding either VPS35 or VPS26b is introduced into the neuronal cell, thereby increasing retromer function; and / or (b) A composition that increases the level of Sorl1 in the neuronal cell compared to the level of Sorl1 when only a transgene encoding either VPS35 or VPS26b is introduced into the neuronal cell, thereby increasing retromer function.

13. The composition of claim 12, wherein the retromer core protein VPS35 comprises the amino acid sequence of SEQ ID NO:

1.

14. The composition of claim 12, wherein the retromer core protein VPS26b comprises the amino acid sequence of SEQ ID NO:

3.

15. 14. The composition of claim 13, wherein the retromer core protein VPS26b comprises the amino acid sequence of SEQ ID NO:

3.

16. 13. The composition of claim 12, wherein the transgene encoding the retromer core protein VPS35 comprises the nucleic acid sequence of SEQ ID NO:

10.

17. The composition of claim 12 , wherein the vector is a viral vector.

18. 18. The composition of any one of claims 12 to 17, wherein the vector is selected from the group consisting of AAV, adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus and synthetic viruses.

19. 19. The composition of claim 18, wherein the vector is an AAV vector.

20. 20. The composition of claim 19, wherein the AAV vector is selected from the group consisting of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAVrhlO.

21. 19. The composition of claim 18, wherein the vector is an AAV2 / 9 vector.

22. The composition of any one of claims 12 to 17, wherein the transgene is operably linked to a promoter that directs expression of the transgene in neuronal cells.

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