Stabilization of reverse transport complexes for treatment of alzheimer's disease and other neurodegenerative disorders

Abstract

The present disclosure relates to methods and compositions for enhancing and stabilizing reverse transport complexes for the treatment and / or prevention of Alzheimer's disease and other neurodegenerative diseases. Furthermore, the present disclosure relates to use in the treatment of Alzheimer's disease (AD) and other neurodegenerative diseases such as Parkinson's disease (PD), neuronal wax-like lipofuscia deposition (NCL) and infectious cavernous encephalopathy (TSE or prion disease), multi-system atrophy (MSA), Down's syndrome and hereditary spasmodic paraplegia, as well as tau cases such as progressive suprakaryotic paralysis (PSP), the present invention relates to adenovirus-based therapies of frontotemporal dementia (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal lobe degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE) associated with chromosome 17q21-22 and subtypes thereof.

Description

[0001] This application is a divisional application, and the application number of its parent application is 202080093935.2, the application date is December 7, 2020, and the invention title is "Stabilization of Retromer Complexes for the Treatment of Alzheimer's Disease and Other Neurodegenerative Disorders".

[0002] Cross - reference to related applications

[0003] This application claims the priority of U.S. Provisional Application No. 62 / 943,999, filed on December 5, 2019, and U.S. Provisional Application No. 63 / 074,578, filed on September 4, 2020, both of which are incorporated herein by reference in their entirety. Technical field

[0004] The present disclosure relates to methods and compositions for enhancing and stabilizing retromer complexes for the treatment and / or prevention of Alzheimer's disease and other neurodegenerative diseases. Background art

[0005] Alzheimer's disease (AD) is a disease characterized by misfolded proteins and neuroinflammation. However, AD therapies and alternative therapies targeting amyloid, tau, cholinesterase inhibitors, anti - inflammatory compounds (such as memantine and nutritional supplements) have failed, and the disease remains a major source of mortality, morbidity, and economic burden. The failure of AD clinical trials has forced researchers to further investigate the causality of the disease. Many pre - clinical studies have examined new AD - related genes, intracellular protein homeostasis pathways, the interaction of neurons with their microenvironment and with glial cells. Multiple studies are still ongoing.

[0006] Recent genetic and cell biological findings in Alzheimer's disease suggest that "endosomal trafficking" plays a central role in disease pathophysiology. Current literature posits four gene categories associated with AD. These gene categories are: 1) endosomal trafficking; 2) cholesterol metabolism; 3) immune response; and 4) amyloid - precursor protein (APP) processing. All four of these gene categories are directly or indirectly related to endosomal trafficking defects. Endosomal trafficking defects are also associated with other neurodegenerative diseases, such as Parkinson's disease (PD), transmissible spongiform encephalopathies (TSE or prion diseases), and neuronal ceroid lipofuscinosis (NCL).

[0007] The retrograde transport complex is a protein complex associated with intracellular organelles that controls the transport of certain cellular cargo molecules within tubular vesicular carriers to the trans-Golgi network. Defects in this transport are associated with various neurodegenerative diseases (Small and Petsko 2015; Anderson et al., 2014). Neurodegeneration is an umbrella term for the progressive loss of structure or function of neurons, including neuronal death. Many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD), and Huntington's disease, are the result of neurodegenerative processes.

[0008] The use of small molecules to improve the function of the retrograde transport complex has been described. However, it will be difficult to develop successful drugs that show positive pharmacokinetics in vivo and may take many years. There is an urgent need for effective treatments for these neurodegenerative diseases, and there are currently no gene-based therapies that can provide long-term benefits. SUMMARY OF THE INVENTION

[0009] The present disclosure relates to compositions and methods useful for treating subjects (e.g., mammalian subjects, such as human subjects) suffering from or at risk of developing a neurodegenerative disease, said neurodegenerative diseases including but not limited to Alzheimer's disease (AD), Parkinson's disease (PD), neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathy (TSE or prion disease), Down syndrome, hereditary spastic paraplegia (HSP), and multiple system atrophy (MSA), as well as tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0010] Using the compositions and methods of the present disclosure, a composition containing a transgene encoding one or more of the retrograde transport complex proteins described herein can be administered to a subject suffering from or at risk of developing the above diseases (e.g., mammalian subjects, such as human subjects). The composition can comprise a vector, such as a viral vector, such as an adeno-associated virus (AAV) vector. In some embodiments, a second composition is administered to the subject, said second composition comprising a transgene encoding one or more of the retrograde transport complex proteins described herein. The second composition can comprise a vector, such as a viral vector, such as an AAV vector. In some embodiments, a third composition is administered to the subject, said third composition containing a transgene encoding one or more of the retrograde transport complex proteins described herein. The third composition can comprise a vector, such as a viral vector, such as an AAV vector.

[0011] In a first aspect, the disclosure features a composition comprising a transgene encoding the retromer complex core protein VPS35 and / or VPS26a and / or VPS26b. In an embodiment, the transgene encodes VPS35. In an embodiment, the transgene encodes VPS26a and / or VPS26b. In an embodiment, the transgene encodes VPS35 and VPS26a or VPS26b. In an embodiment, the transgene encodes VPS35 and VPS26a. In an embodiment, the transgene encodes VPS35 and VPS26b. In an embodiment, the transgene encodes VPS26a and VPS26b. In other aspects, the composition comprises two transgenes, wherein one transgene encodes VPS35 and the other encodes VPS26a or VPS26b. In other aspects, the composition comprises two transgenes, wherein one transgene encodes VPS26a and the other encodes VPS26b. In other aspects, the composition comprises three transgenes, wherein one transgene encodes VPS35, another encodes VPS26a, and another encodes VPS26b.

[0012] In some embodiments, the transgene encodes the retromer complex core protein VPS35. In some embodiments, the retromer complex core protein VPS35 is human. The retromer complex core protein VPS35 encoded by the transgene can have an amino acid sequence that is at least 85% identical to the amino acid sequence 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 the amino acid sequence of VPS35). In some embodiments, the retromer complex core protein VPS35 encoded by the transgene has an amino acid sequence that is at least 90% identical to the amino acid sequence of VPS35 (e.g., an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VPS35). In some embodiments, the retromer complex 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., an amino acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of VPS35). In some embodiments, the retromer complex core protein VPS35 encoded by the transgene has an amino acid sequence that is different from the amino acid sequence of VPS35 by one or more amino acid substitutions, insertions, and / or deletions, such as by 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 complex core protein VPS35 encoded by the transgene has an amino acid sequence that is different from the amino acid sequence of VPS35 by one or more conservative amino acid substitutions, such as by 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 some embodiments, the transgene encoding the retromer complex core protein VPS35 comprises human VPS35 (Gene ID 55737). In some embodiments, the transgene encoding the retromer complex core protein VPS35 has a nucleic acid sequence that is at least 70% identical to the 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 the nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer complex core protein VPS35 has a nucleic acid sequence that is at least 85% identical to the 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 the nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer complex core protein VPS35 has a nucleic acid sequence that is at least 90% identical to the 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 the nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer complex core protein VPS35 has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence encoding VPS35 (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence encoding VPS35). In some embodiments, the transgene encoding the retromer complex core protein VPS35 is codon-optimized.

[0014] In some embodiments, the transgene encodes the retromer core protein VPS26a. In some embodiments, the retromer core protein VPS26a is human. The retromer core protein VPS26a encoded by the transgene can have an amino acid sequence that is 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 that is at least 90% identical to the amino acid sequence of VPS26a (e.g., an amino acid sequence that is 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 that is at least 95% identical to the amino acid sequence of VPS26a (e.g., an amino acid sequence that is 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 that is different from the amino acid sequence of VPS26a by one or more amino acid substitutions, insertions, and / or deletions, such as by 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 is different from the amino acid sequence of VPS26a by one or more conservative amino acid substitutions, such as by 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 additional embodiments, the transgene encoding the retromer complex core protein VPS26a comprises human VPS26a (Gene ID 9559). In some embodiments, the transgene encoding the retromer complex core protein VPS26a has a nucleic acid sequence that is at least 70% identical to the 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 the nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer complex core protein VPS26a has a nucleic acid sequence that is at least 85% identical to the 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 the nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer complex core protein VPS26a has a nucleic acid sequence that is at least 90% identical to the 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 the nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer complex core protein VPS26a has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence encoding VPS26a (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence encoding VPS26a). In some embodiments, the transgene encoding the retromer complex core protein VPS26a is codon-optimized.

[0016] In some embodiments, the transgene encodes the retromer core protein VPS26b. In some embodiments, the retromer core protein VPS26b is human. The retromer core protein VPS26b encoded by the transgene can have an amino acid sequence that is 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 that is at least 90% identical to the amino acid sequence of VPS26b (e.g., an amino acid sequence that is 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 that is at least 95% identical to the amino acid sequence of VPS26b (e.g., an amino acid sequence that is 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 that is different from the amino acid sequence of VPS26b by one or more amino acid substitutions, insertions, and / or deletions, such as by 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 is different from the amino acid sequence of VPS26b by one or more conservative amino acid substitutions, such as by 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).

[0017] In additional embodiments, the transgene encoding the retromer complex core protein VPS26b comprises human VPS26b (Gene ID 112936). In some embodiments, the transgene encoding the retromer complex core protein VPS26b has a nucleic acid sequence that is at least 70% identical to the 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 the nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer complex core protein VPS26b has a nucleic acid sequence that is 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 complex core protein VPS26b has a nucleic acid sequence that is 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 complex core protein VPS26b has a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence encoding VPS26b (e.g., a nucleic acid sequence that is 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleic acid sequence encoding VPS26b). In some embodiments, the transgene encoding the retromer complex VPS26b core protein is codon-optimized.

[0018] In some embodiments of the foregoing aspects, the composition comprises a vector, such as a viral vector. The viral vector can be, for example, an AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus 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 an exogenous protein, a synthetic polymer, a nanoparticle, or a small molecule).

[0019] In some embodiments of the foregoing aspects, the viral vector is an AAV vector, such as AAV1 (i.e., AAV containing AAV1 inverted terminal repeats (ITRs) and AAV1 capsid proteins), AAV2 (i.e., AAV containing AAV2 ITRs and AAV2 capsid proteins), AAV3 (i.e., AAV containing AAV3 ITRs and AAV3 capsid proteins), AAV4 (i.e., AAV containing AAV4 ITRs and AAV4 capsid proteins), AAV5 (i.e., AAV containing AAV5 ITRs and AAV5 capsid proteins), AAV6 (i.e., AAV containing AAV6 ITRs and AAV6 capsid proteins), AAV7 (i.e., AAV containing AAV7 ITRs and AAV7 capsid proteins), AAV8 (i.e., AAV containing AAV8 ITRs and AAV8 capsid proteins), AAV9 (i.e., AAV containing AAV9 ITRs and AAV9 capsid proteins), AAVrh74 (i.e., AAV containing AAVrh74 ITRs and AAVrh74 capsid proteins), AAVrh.8 (i.e., AAV containing AAVrh.8 ITRs and AAVrh.8 capsid proteins), or AAVrh.10 (i.e., AAV containing AAVrh.10 ITRs and AAVrh.10 capsid proteins).

[0020] In some embodiments of the foregoing aspects, the viral vector is a pseudotyped AAV vector, comprising 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).

[0021] In some embodiments of the foregoing aspects, the AAV vector contains a recombinant capsid protein, such as a capsid protein of a chimera containing one or more of the capsid proteins from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh74, AAVrh.8, or AAVrh.10. In an embodiment, the capsid is a variant AAV capsid, such as the AAV2 variant rAAV2-retro (SEQ ID NO: 44 from WO2017 / 218842, which is incorporated herein by reference).

[0022] In certain embodiments, the viral vector is AAV10. For example, the composition can comprise AAV10, which contains a nucleic acid sequence containing 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 AAV9. For example, the composition can comprise AAV9 that contains a nucleic acid sequence encoding a transgene that encodes retromer complex core protein VPS35 and / or retromer complex core protein VPS26a and / or retromer complex core protein VPS26b.

[0024] In certain embodiments, the viral vector is AAV2 / 10. For example, the composition can comprise AAV2 / 10 that contains a nucleic acid sequence encoding a transgene that encodes retromer complex core protein VPS35 and / or retromer complex core protein VPS26a and / or retromer complex core protein VPS26b.

[0025] In certain embodiments, the viral vector is AAV2 / 9. For example, the composition can comprise AAV2 / 9 that contains a nucleic acid sequence encoding a transgene that encodes retromer complex core protein VPS35 and / or retromer complex core protein VPS26a and / or retromer complex core protein VPS26b.

[0026] In certain embodiments, the viral vector is an AAV vector and the transgene encodes the retromer complex core protein VPS35. For example, the composition can comprise recombinant AAV (rAAV), such as AAV10, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS35. For example, the composition can comprise recombinant AAV (rAAV), such as AAV9, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS35. For example, the composition can comprise recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS35.

[0027] In certain embodiments, the viral vector is an AAV vector and the transgene encodes the retromer complex core protein VPS26a. For example, the composition can comprise recombinant AAV (rAAV), such as AAV10, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS26a. For example, the composition can comprise recombinant AAV (rAAV), such as AAV9, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS26a. For example, the composition can comprise recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, that contains a nucleic acid sequence encoding a transgene that encodes a functional retromer complex core protein VPS26a.

[0028] In some embodiments, the viral vector is an AAV vector and the transgene encodes the retromer complex core protein VPS26b. For example, the composition can comprise recombinant AAV (rAAV), such as AAV10, which comprises a nucleic acid sequence containing a transgene encoding a functional VPS26b retromer complex core protein. For example, the composition can comprise recombinant AAV (rAAV), such as AAV9, which comprises a nucleic acid sequence containing a transgene encoding a functional retromer complex core protein VPS26b. For example, the composition can comprise recombinant AAV (rAAV), such as AAV2 / 9 or AAV2 / 10, which comprises a nucleic acid sequence containing a transgene encoding a functional retromer complex core protein VPS26b.

[0029] In some embodiments of any of the above aspects of the present disclosure, the composition comprises liposomes, vesicles, synthetic vesicles, exosomes, synthetic exosomes, dendrimers, or nanoparticles.

[0030] In some embodiments of any of the above aspects of the present disclosure, the transgene is operably linked to a promoter that induces transgene expression in neurons. The promoter can be, for example, the chicken β-actin promoter, the cytomegalovirus (CMV) promoter, the myosin light chain-2 promoter, the α-actin promoter, the troponin 1 promoter, the Na+ / Ca2+ exchanger promoter, the dystrophin promoter, the creatine kinase promoter, the α7 integrin promoter, the brain natriuretic peptide promoter, the αB-crystallin / small heat shock protein promoter, the α-myosin heavy chain promoter, or the atrial natriuretic factor promoter.

[0031] In some embodiments of any of the above aspects of the present disclosure, the transgene is operably linked to an enhancer that induces transgene expression in neurons. Exemplary enhancers that can be used in conjunction with the compositions and methods of the present disclosure are the CMV enhancer, the myocyte enhancer factor 2 (MEF2) enhancer, and the MyoD enhancer.

[0032] In another aspect, the present disclosure features methods of 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 foregoing embodiments. In some embodiments, the composition is administered to the subject immediately upon or immediately 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 encephalopathy (TSE or prion disease), multiple system atrophy (MSA), Down syndrome, and hereditary spastic paraplegia (HSP), and tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with 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).

[0033] In another aspect, the present disclosure features methods of treating a degenerative disease or disorder in a subject in need thereof by administering one or more compositions comprising a transgene encoding retromer complex core protein VPS35 and / or VPS26a and / or VPS26b, as described in the foregoing paragraphs. 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 the subject immediately upon or immediately 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 encephalopathy (TSE or prion disease), Down syndrome, hereditary spastic paraplegia (HSP), and multiple system atrophy (MSA), and tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with 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).

[0034] In some embodiments, the method comprises administering to a subject a therapeutically effective amount of a first composition comprising a transgene encoding retromer complex core protein VPS35 and / or VPS26a and / or VPS26b, as described in the foregoing paragraphs. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a second composition comprising a transgene encoding retromer complex core protein VPS35 and / or VPS26a and / or VPS26b, as described in the foregoing paragraphs. In an embodiment, the method comprises administering a first composition comprising a transgene encoding VPS35 and administering a second composition comprising a transgene encoding VPS26a or VPS26b. In an embodiment, the method comprises administering a first composition comprising a transgene encoding VPS26a or VPS26b and administering a second composition comprising a transgene encoding VPS35. In an embodiment, the method comprises administering a first composition comprising a transgene encoding VPS26a and administering a second composition comprising a transgene encoding VPS26b. In an embodiment, the method comprises administering a first composition comprising a transgene encoding VPS26b and administering a second composition comprising a transgene encoding VPS26a.

[0035] In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of a third composition comprising a transgene encoding retromer complex core protein VPS35 and / or VPS26a and / or VPS26b, as described in the foregoing paragraphs. In an embodiment, the first, second, and third compositions each comprise a transgene encoding VPS35, VPS26a, or VPS26b, respectively.

[0036] In some embodiments, the first and second compositions are administered to the subject simultaneously. In some embodiments, the first, second, and third compositions are administered to the subject simultaneously.

[0037] In some embodiments, the second composition is administered to the subject after the first composition has been administered to the subject. The second composition can be administered, for example, one day or more or weeks after the first composition has been administered to the subject. In some embodiments, the second composition is administered to the subject at least one month after the first composition has been administered to the subject. In some embodiments, the administration of the first composition continues while the second composition is being administered to the subject.

[0038] In some embodiments, after administering the first composition and the second composition to a subject, a third composition is administered to the subject. The third composition can be administered to the subject, for example, one day or more or weeks after administering the first composition and the second composition to the subject. In some embodiments, the third composition is administered to the subject at least one month after administering the first composition and the second composition to the subject. In some embodiments, the administration of the first composition and the second composition continues while the third composition is being administered to the subject.

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

[0040] In some embodiments, the second composition is administered to the subject by a route of administration including intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and / or oral administration.

[0041] In some embodiments, the third composition is administered to the subject by a route of administration including intravenous, intrathecal, intradermal, transdermal, parenteral, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular, inhalation, perfusion, lavage, and / or oral administration.

[0042] In another aspect, the present disclosure features a method of treating, preventing, and / or curing a neurodegenerative disease or disorder in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of one or more compositions that contain a transgene encoding retromer complex core protein VPS35 and / or retromer complex core protein VPS26a and / or retromer complex core protein VPS26b.

[0043] In a further aspect, the present 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 includes administering to the subject a therapeutically effective amount of one or more compositions that contain a transgene encoding retromer complex core protein VPS35 and / or retromer complex core protein VPS26a and / or retromer complex core protein VPS26b.

[0044] As part of the foregoing aspects, the present disclosure also provides one or more compositions as described herein for use in the methods as described herein. The present disclosure also provides the use of one or more compositions as described herein for the preparation of one or more medicaments for use in the methods as described herein. The transgene(s) may encode vacuolar protein sorting 35 (VPS35), a core protein of the retromer complex, and / or vacuolar protein sorting 26a (VPS26a), a core protein of the retromer complex, and / or vacuolar protein sorting 26b (VPS26b), a core protein of the retromer complex.

[0045] As part of the foregoing aspects, the present disclosure thus also provides a composition comprising one or more transgenes encoding vacuolar protein sorting 35 (VPS35), a core protein of the retromer complex, and / or vacuolar protein sorting 26a (VPS26a), a core protein of the retromer complex, and / or vacuolar protein sorting 26b (VPS26b), a core protein of the retromer complex, for treating, preventing, and / or curing a neurodegenerative disease or disorder. One or more compositions are also provided comprising one or more transgenes encoding vacuolar protein sorting 35 (VPS35), a core protein of the retromer complex, and / or vacuolar protein sorting 26a (VPS26a), a core protein of the retromer complex, and / or vacuolar protein sorting 26b (VPS26b), a core protein of the retromer complex, for alleviating one or more symptoms associated with a neurodegenerative disease or disorder.

[0046] As part of the foregoing aspects, the present disclosure also provides the use of one or more compositions for the preparation of one or more medicaments for treating, preventing, and / or curing a neurodegenerative disease or disorder, said compositions comprising one or more transgenes encoding vacuolar protein sorting 35 (VPS35), a core protein of the retromer complex, and / or vacuolar protein sorting 26a (VPS26a), a core protein of the retromer complex, and / or vacuolar protein sorting 26b (VPS26b), a core protein of the retromer complex. The use of one or more compositions comprising one or more transgenes encoding vacuolar protein sorting 35 (VPS35), a core protein of the retromer complex, and / or vacuolar protein sorting 26a (VPS26a), a core protein of the retromer complex, and / or vacuolar protein sorting 26b (VPS26b), a core protein of the retromer complex, in the preparation of one or more medicaments for alleviating one or more symptoms associated with a neurodegenerative disease or disorder is also provided.

[0047] In some embodiments of any of the foregoing aspects, the disease or disorder is a neurodegenerative disease or disorder. In some embodiments of any of the foregoing aspects, the disease or disorder is Alzheimer's disease (AD). In some embodiments of any of the foregoing aspects, the disease or disorder is Parkinson's disease. In some embodiments of any of the foregoing aspects, the disease or disorder is neuronal ceroid lipofuscinosis (NCL). In some embodiments of any of the foregoing aspects, the disease or disorder is transmissible spongiform encephalopathy (TSE or prion disease). In some embodiments of any of the foregoing aspects, the disease or disorder is multiple system atrophy (MSA). In some embodiments of any of the foregoing aspects, the disease or disorder is progressive supranuclear palsy (PSP). In some embodiments of any of the foregoing aspects, the disease or disorder is frontotemporal dementia associated with chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau). In some embodiments of any of the foregoing aspects, the disease or disorder is chronic traumatic encephalopathy (CTE). In some embodiments of any of the foregoing aspects, the disease or disorder is Down syndrome. In some embodiments of any of the foregoing aspects, the disease or disorder is HSP. In some embodiments of any of the foregoing aspects, the disease or disorder is LBD. In some embodiments of any of the foregoing aspects, the disease or disorder is ALS. In some embodiments of any of the foregoing aspects, the disease or disorder is FTD or ALS-FTD.

[0048] In another aspect, the present disclosure features a kit comprising the composition of any of the foregoing aspects. The kit may further comprise a package insert, such as a package insert instructing a user of the kit to administer the composition to a subject according to the methods of any of the above aspects or embodiments of the present disclosure. Brief Description of the Drawings

[0050] To illustrate the invention, certain embodiments of the invention are depicted in the drawings. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

[0051] Figure 1 A backbone rendering (PyMOL, Schroedinger, Inc.) of the three-dimensional structure of the retrograde transport complex cargo recognition core is shown, highlighting the interaction of VPS35 (orange) with VPS29 (red) and VPS26a (green). Vps26b binds to Vps35 in an almost identical manner (Collins et al., 2008; Shi et al., 2006). The atomic coordinates (PDB file 6vac) were taken from the cryoEM structure of the murine heterotrimer (Kendall et al., 2020).

[0052] Figure 2 shows that VPS35 expression alone is not sufficient to enhance the trimerization and function of the retrograde transport complex. Figure 2ARepresentative immunoblots showing retrograde transport complex and Sorl1 expression levels after AAV9-VPS35-HA. AAV9-GFP and AAV9-EV (empty vector) were used as controls. Figure 2B Bar graph showing mean levels of retrograde transport complex components and Sorl1, normalized to actin; control (left bar, dark grey, n = 23), VPS35 overexpression (right bar, red, n = 16). ***P < 0.001, ns = not significant.

[0053] Figure 3 shows the map of the plasmid used in the examples. Figure 3A Shows the map of the empty frame control plasmid. Figure 3B Shows the map of the GFP control plasmid. Figure 3C Shows the map of the VPS35 plasmid. Figure 3D Shows the map of the VPS26a plasmid. Figure 3E Shows the map of the VPS26b plasmid.

[0054] Figure 4 shows the optimization of combined VPS35 and VPS26 expression in neuroblastoma cells. Figure 4A Representative immunoblots showing retrograde transport complex expression levels after transfection with plasmids containing VPS35, VPS26a, and VPS26b alone or double transfection of VPS35 with VPS26a or VPS26b. GFP or empty backbone plasmids were used as controls. To correctly control the amount of plasmid DNA / lipofectamine complex introduced under each condition, control plasmids (GFP or empty backbone) were included whenever only one component of the retrograde transport complex was transfected. VPS29 shows two different bands, which represent two different isoforms of this protein in these N2a cells. Figures 4B to 4E Graph of the mean levels of retrograde transport complex core proteins (VPS35, VPS26a, VPS26b, VPS29) normalized to actin in neuroblastoma cells transfected with viral vectors containing empty vector (EV) and GFP as controls (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 4EVPS29 is shown. Control (dark gray, n = 15), overexpression of VPS35 alone (n = 30), VPS26a (n = 30), and VPS26b (n = 15) (blue), VPS35+VPS26 combination (red, n = 15). ***P < 0.001, **P < 0.01,!P = 0.07, ns = not significant.

[0055] Figure 5 shows that combined VPS35 and VPS26 expression functions in concert to reverse the transport complex in neurons. Figure 5A is a representative immunoblot showing the levels of the retrograde transport complex expressed 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. The 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 properly control the amount of DNA and AAV9 introduced in each case, control AAV9 (GFP or EV) was included whenever only one component of the retrograde transport complex was transduced. Figures 5B to 5E Bar graph of the average levels of the retrograde transport complex core proteins (VPS35, VPS26a, VPS26b) normalized to actin in neuroblastoma cells transfected with one or more of five viral vectors (AAV9) containing an 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 VPS35 is shown. Figure 5C VPS29 is shown. Figure 5D VPS26a is shown. Figure 5E VPS26b is shown. Control (dark gray, n = 9), single overexpression of VPS35, VPS26a, or VPS26b (blue, n = 18), VPS35+VPS26a or VPS26b combination (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.

[0056] Figure 6 shows the combined VPS35 and VPS26 expression synergistically functioning in the retrograde transport complex in neurons. Figure 6A Is a representative immunoblot showing the 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. The 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 correctly control the amount of DNA and AAV9 introduced in each case, control AAV9 (GFP or EV) was included whenever only one component of the retrograde transport complex 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 an empty vector (EV), GFP, VPS35, VPS26a, or VPS26b. The results are shown as 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 combination (red, n = 9), VPS35+VPS26a+VPS26b (dark red, n = 9), VPS26a+VPS26b (pink, n = 9). ***P<0.001, ns = not significant. Figure 6C and 6D Is a scatter plot generated from multiple regression indicating that VPS26a (t = 5.6, p = 1.4E-7) and VPS26b (F = 7.2, p = 3.2E-11) are independently associated with Sorl1 levels. Figure 6C Shows VPS26a. Figure 6D Shows VPS26b. Summary of the Invention

[0058] Definitions

[0059] The terms used in this specification generally have their ordinary meanings in the context of the present invention and in the particular context in which each term is used. Certain terms are discussed below or elsewhere in the specification to provide additional guidance to the practitioner in describing the methods of the present invention and how to use them. In addition, it is understood that the same thing can be expressed in more than one way. Accordingly, alternative languages and synonyms may be used for any one or more of the terms discussed herein, and there is no special significance whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in the specification, including examples of any of the terms discussed herein, is illustrative only and in no way limits the scope and meaning of the present invention or any exemplary term. Similarly, the present invention is not limited to its preferred embodiments.

[0060] The term "about" or "approximately" means within an acceptable error range of a particular value as determined by a person of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measuring system, i.e., the precision required for a particular purpose (e.g., a pharmaceutical formulation). For example, in accordance with the practice in the art, "about" can mean within 1 or more standard deviations. Alternatively, "about" can represent a range of up to 20%, preferably up to 10%, more preferably up to 5%, 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 of the value, preferably within 5-fold, more preferably within 2-fold. Where a particular value is described in the application and claims, unless otherwise stated, the term "about" should be assumed to mean within the acceptable error range of the particular value.

[0061] The term "subject" as used in this application refers to an animal in need of treatment or prophylactic treatment. Subjects include mammals such as canines, felines, rodents, bovines, equines, porcines, ovines, and primates. Thus, the compositions and methods can be used in veterinary medicine, e.g., treating companion animals, farm animals, laboratory animals in zoos, and wild animals. The compositions and methods disclosed herein are particularly desirable for human medical applications.

[0062] As used herein, the term "patient" refers to a human subject. In some embodiments, the "patient" is known or suspected 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 encephalopathy (TSE or prion disease), multiple system atrophy (MSA), Down syndrome, and hereditary spastic paraplegia (HSP), and tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE). In some embodiments, the "patient" is known or suspected of having a disorder or disease associated with endosomal trafficking, such as retrograde transport complex dysfunction.

[0063] The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to effect a clinically significant improvement in a subject, or to delay, minimize, or alleviate one or more symptoms associated with a disease or disorder, or to cause a desired beneficial physiological change in a subject.

[0064] The terms "treat", "treatment", etc. refer to means for slowing, alleviating, ameliorating, or reducing at least one symptom of a disease or disorder or reversing a disease or disorder after its onset.

[0065] The terms "prevent", "prevention", etc. refer to taking action prior to the onset of an overt disease or disorder to prevent the development of a disease or disorder, or to minimize the extent of a disease or disorder, or to slow its progression.

[0066] The term "cure", etc. refers to recovery, improvement, or restoration to health, or allowing a period of time without recurrence of a disease such that the risk of recurrence is low.

[0067] The term "in need" shall be a subject known or suspected of having a neurodegenerative disease or disorder 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 encephalopathy (TSE or prion disease), multiple system atrophy (MSA), Down syndrome, and hereditary spastic paraplegia (HSP), and tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with chromosome 17q21-22 and its subtypes (FTLD-17 / FTLD-Tau), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTD), ALS-FTD, and chronic traumatic encephalopathy (CTE).

[0068] As used herein, the term "reagent" refers to a substance that produces or is capable of producing an effect, and includes, but is not limited to, carriers, chemicals, drugs, biological products, organic small molecules, antibodies, nucleic acids, peptides, and proteins.

[0069] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle used to administer a therapeutic agent, and includes 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 reagents for pharmaceutical active substances is well known in the art.

[0070] The term "pharmaceutically acceptable" refers to molecular entities and compositions that, when administered to a host, do not produce allergic or similar adverse reactions (such as gastric discomfort, dizziness, etc. when administered to humans) and are approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other recognized pharmacopeias for use in animals and especially in humans.

[0071] An "isolated nucleic acid molecule" refers to a genomic, mRNA, cDNA, or synthetically derived DNA or RNA, or some combination thereof, that is not associated with all or part of the polynucleotide with which it is associated in nature or is not linked to polynucleotides to which it is not linked in nature. For the purposes of this disclosure, it should be understood that a nucleic acid molecule "comprising" a particular nucleotide sequence does not include an entire chromosome. An isolated nucleic acid molecule "comprising" a particular nucleic acid sequence may, in addition to the particular sequence, include up to ten or even up to twenty or more other protein-encoding sequences or portions or fragments thereof, or may include regulatory sequences operably linked thereto that control the expression of the coding region of the nucleic acid sequence, and / or may include vector sequences.

[0072] The phrase "control sequence" refers to the DNA sequences necessary to express an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optionally operator sequences, and ribosome binding sites. It is known that eukaryotic cells use promoters, polyadenylation signals, and enhancers.

[0073] A nucleic acid is "operably linked" when placed into a functional relationship with another nucleic acid sequence. For example, if a DNA for a presequence or secretory leader is expressed as a preprotein participating in the secretion of a polypeptide, the DNA is operably linked to the DNA for the polypeptide; if a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is positioned to facilitate translation, it is operably linked to the coding sequence. 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. However, enhancers do not have to be contiguous. The ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adaptors are used according to conventional practice.

[0074] As used herein, the terms "cell", "cell line", and "cell culture" are used interchangeably and all such designations include progeny. Thus, the terms "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 may have identical DNA content due to either intentional or unintentional mutations. Mutant progeny that have the same functionality or biological activity as screened for in the originally transformed cell are included. Where different designations are intended, it will be clear from the context.

[0075] In some aspects, the present disclosure provides isolated adeno-associated virus vectors (AAV). As used herein with respect to AAV, the term "isolated" means an AAV that has been separated 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 capabilities such that the transgene of the rAAV will be specifically delivered to one or more predetermined tissues. The AAV capsid is an important factor in determining these tissue-specific targeting capabilities.

[0076] Methods for obtaining recombinant AAVs with desired capsid proteins have been described (see, e.g., U.S. Patent No. 7,906,111). Many different AAV capsid proteins have been described, e.g., 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 the desired packaging embodiments of the presently described constructs and methods, the recombinant AAV can 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 can be applicable to the methods and compositions of the present invention. Generally, the methods include 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 consisting 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.

[0077] The components to be cultured in the host cell to package the rAAV vector in the AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the desired components (e.g., the recombinant AAV vector, the rep sequence, the cap sequence, and / or the helper functions) can be provided by a stable host cell that has been engineered using methods known to those of skill in the art to contain one or more of the desired components. Most desirably, such a stable host cell will contain one or more of the desired components under the control of an inducible promoter. However, one or more of the desired components may be under the control of a constitutive promoter. In yet another alternative, a selected stable host cell can contain one or more selected components under the control of a constitutive promoter and other one or more selected components under the control of one or more inducible promoters. For example, a stable host cell can be generated that is derived from 293 cells (which contain E1 helper functions under the control of a constitutive promoter), but which contains the rep and / or cap proteins under the control of an inducible promoter.

[0078] Any suitable genetic element (vector) can be used to deliver the recombinant AAV vector, the rep sequence, the cap sequence, and the helper functions for producing rAAV to the packaging host cell. The selected genetic element can be delivered by any suitable method, including those described herein. See, e.g., Fisher et al., J. Virology 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0079] In some embodiments, a triple transfection method (e.g., as described in detail in U.S. Patent No. 6,001,650) can be used to generate recombinant AAV. Generally, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (containing a transgene), a recombinant AAV vector that is packaged into AAV particles, an AAV helper function vector, and a helper function vector. The AAV helper function vector encodes "AAV helper function" sequences (i.e., rep and cap), which are used in trans for productive AAV replication and encapsulation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). Non-limiting examples of suitable vectors include pHLP19 described in U.S. Patent No. 6,001,650 and pRep6cap6 vector described in U.S. Patent No. 6,156,303, the entire contents of both of which are incorporated herein by reference. The helper function vector encodes nucleotide sequences of non-AAV-derived viral and / or cellular functions (i.e., "helper functions") upon which AAV replication depends. Helper functions include those functions required for AAV replication, including but not limited to those involved in AAV gene transcriptional activation, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. The virus-based helper functions can be derived from any known helper virus, such as adenovirus, herpes virus (except herpes simplex virus type 1), and vaccinia virus.

[0080] As used herein, the terms "AAV1", "AAV2", "AAV3", "AAV4", etc. refer to AAV vectors that contain the ITRs from AAV1, AAV2, AAV3, or AAV4, respectively, and the capsid proteins from AAV1, AAV2, AAV3, or AAV4, respectively. The terms "AAV2 / 1", "AAV2 / 8", "AAV2 / 9", etc. refer to pseudotyped AAV vectors that contain the ITRs from AAV2 and the capsid proteins from AAV1, AAV8, or AAV9, respectively.

[0081] With respect to transfected host cells, the term "transfection" is used to refer to the uptake of exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced within the cell membrane. Many transfection techniques are generally known in the art. See, for example, 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 (e.g., nucleotide integration vectors and other nucleic acid molecules) into a suitable host cell.

[0082] "Host cell" means any cell that contains or is capable of containing a substance of interest. Typically, the host cell is a mammalian cell. The host cell can serve as a recipient for an AAV helper construct, an AAV minigene plasmid, a helper function vector, or other transfer DNA associated with the production of a recombinant AAV vector. The term includes the progeny of the original cell that has been transfected. Thus, "host cell" as used herein can refer to a cell that has been transfected with an exogenous DNA sequence. It should be understood that the progeny of a single parental cell may not be exactly identical to the original parent in morphology or genomic or total DNA complement due to natural, accidental or deliberate mutations.

[0083] With respect to cells, the term "isolated" means a cell that has been separated from its natural environment (e.g., from tissue or a subject). The term "cell line" means a population of cells capable of growing and dividing continuously or for a long period of time in vitro. Typically, a cell line is a clonal population derived from a single progenitor cell. It is also known in the art that spontaneous or induced changes in karyotype may occur during the storage or transfer of such clonal populations. Thus, cells derived from the cell line mentioned may not be exactly identical to the ancestral cells or culture, and the cell line mentioned includes such variants. As used herein, the term "recombinant cell" means a cell into which an exogenous DNA segment (e.g., a DNA segment that results in the transcription of a bioactive polypeptide or the production of a bioactive nucleic acid such as RNA) has been introduced.

[0084] The term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus or virion, which is capable of replicating when associated with appropriate control elements and can transfer a gene sequence between cells. Thus, the term includes cloning and expression vectors, as well as viral vectors. In some embodiments, the vectors of interest are those in which the nucleic acid segment to be transcribed is under the transcriptional control of a promoter. A "promoter" is a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery and is 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 to control the initiation of RNA polymerase and the expression of the gene.

[0085] The term "expression vector" or "expression construct" or "construct" refers to any type of genetic construct that contains nucleic acid, where part or all of the nucleic acid coding sequence is capable of being transcribed. In some embodiments, expression includes the transcription of nucleic acid, e.g., to produce a bioactive polypeptide product or inhibitory RNA from the transcribed gene.

[0086] 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 Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); 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).

[0087] Retrotranslocation Complexes and Neurodegenerative Diseases

[0088] The genetics, cytopathology, and cell biology of Alzheimer's disease (AD) have identified endosomal trafficking as a key defect in the pathogenesis of AD. Three lines of evidence suggest a dysfunction of the retromer complex in AD. First, genetic and gene expression studies have identified an increasing number of retromer complex-related molecules associated with AD, including true loss-of-function mutations. Second, retromer complex dysfunction recapitulates the cytopathology of AD, which is characterized by enlarged and dysfunctional endosomes that accumulate amyloid precursor protein (APP) fragments. Third, retromer complex dysfunction mis-traffics many AD-related molecules, including APP in neurons and phagocytic receptors in microglia.

[0089] The retromer complex is a multiprotein complex that is the "master conductor" of endosomal trafficking. The core of the retromer complex is a trimer of three different proteins, making it technically a heterotrimer. These proteins are all members of the "vacuolar protein sorting" (VPS) protein family. VPS35 is the central protein of the trimeric core to which VPS29 and VPS26 bind. VPS26 is the only core protein with two paralogs called VPS26a and VPS26b. Thus, neurons have two distinct retromer complex cores: VPS29-VPS35-VPS26a and VPS29-VPS35-VPS26b. See Figure 1 。

[0090] These core proteins bind to endogenous retromer complex components when overexpressed and can lead to increased retromer complex function. These proteins are very tightly autoregulated within the cell. To overcome this hurdle, as described herein, two retromer complex proteins are co-expressed.

[0091] Increasing VPS35 levels by pharmacological chaperones or by viral vectors increases retromer complex function. VPS29 protein may be present in excess compared to VPS35 or VPS26. As shown herein, co-expression of both VPS35 and VPS26a or both VPS35 and VPS26b has a synergistic effect on the cellular levels of VPS35 and VPS26a or VPS35 and VPS26b, respectively.

[0092] Evidence of retromer complex function was also shown by an approximately 34% increase in Sorl1 levels, an effect size that reflects the degree of Sorl1 deficiency observed in Alzheimer's disease (Sager et al., 2007; Scherzer et al., 2004; Dodson et al., 2006). A more extensive comparison of the effects of Sorl1 in the study provided information on retromer complex functionality. In the first neuronal study, when VPS35 was overexpressed alone, two of the three retromer complex core proteins were significantly elevated, but there was no effect on retromer complex function. In the second neuronal study, when all three trimeric proteins were significantly elevated due to synergy, this led to an increase in retromer complex function. This result provides the primary empirical evidence that all three retromer complex core proteins are required to be co - elevated to upregulate the overall function of the retromer complex. The studies herein show that by leveraging the stoichiometry and protein - protein interactions of the retromer complex, it is not necessary to exogenously express all three proteins. Exogenous expression of VPS35 and VPS26 is also sufficient to upregulate the level of VPS29 and increase the endosomal cargo recycling function of the retromer complex.

[0093] Importantly, the present study also shows that the levels of VPS26a and VPS26b are independent of each other, so appropriate combinatorial selection allows for the selective increase of one retromer complex trimer relative to another.

[0094] The present invention describes a biology - based method for increasing retromer complex levels and function in vivo: overexpressing the retromer complex by using recombinant AAV (adeno - associated virus) technology. Establishing a novel retromer - AAV tool for retromer - based therapies will have a significant impact because this viral delivery system (recently approved for clinical use) can bypass the obstacles that small molecules will encounter in the body (i.e., low absorption rate, degradation, toxicity, lack of target / organ specificity, blood - brain barrier permeability). Compositions containing an AAV vector and one or more retromer complex transgenes have many advantages, including increased therapeutic agent expression, bypassing of stringent protein autoregulation, the potential for long - term expression of stable proteins, and increased half - life of stable proteins.

[0095] Methods for treating, preventing, and / or curing neurodegenerative diseases

[0096] Patients who would benefit from the administration of the described gene therapy include those diagnosed with neurodegenerative diseases or disorders involving defects in endosomal trafficking, said neurodegenerative diseases or disorders including but not limited to Alzheimer's disease (AD), Parkinson's disease, neuronal ceroid lipofuscinosis (NCL), transmissible spongiform encephalopathy (TSE or prion disease), multiple system atrophy (MSA), Down syndrome, and hereditary spastic paraplegia (HSP), as well as tauopathies such as progressive supranuclear palsy (PSP), frontotemporal dementia associated with 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).

[0097] In these patients, a composition (e.g., a viral vector containing such nucleic acid, such as an AAV vector) containing nucleic acid encoding one or more core proteins of the retrograde transport complex can be administered to the patient. These compositions can be administered alone or in combination with other agents to treat the neurodegenerative disease or disorder.

[0098] In some embodiments, the present disclosure provides a method of treating, preventing, curing a neurodegenerative disease or disorder and / or reducing the severity or degree of a neurodegenerative disease or disorder by administering to a subject in need a therapeutically effective amount of one or more compositions such as viral vectors (e.g., AAV) containing nucleic acid encoding the core protein VPS35 of the retrograde transport complex and / or the core protein VPS26 of the retrograde transport complex and / or the core protein VPS26b of the retrograde transport complex. In some embodiments, the viral vector is AAV, such as rAAV2-retro, AAV10, AAV2 / 10, AAV9, or AAV2 / 9. In some embodiments, once a neurodegenerative disease or disorder is diagnosed or suspected, one or more compositions (e.g., viral vectors, such as AAV) containing nucleic acid encoding the core protein VPS35 of the retrograde transport complex and / or the core protein VPS26a of the retrograde transport complex and / or the core protein VPS26b of the retrograde transport complex are administered. In an embodiment, the administered composition contains nucleic acid encoding VPS35 and VPS26a or VPS26b. In an embodiment, the method includes administering simultaneously or sequentially one or more compositions containing nucleic acid encoding VPS35 and nucleic acid encoding VPS26a or VPS26b. In an embodiment, the method includes administering simultaneously or sequentially one or more compositions containing nucleic acid encoding VPS35, nucleic acid encoding VPS26a, and nucleic acid encoding VPS26b. In an embodiment, the method includes administering simultaneously or sequentially one or more compositions containing nucleic acid encoding VPS26a and nucleic acid encoding VPS26b.

[0099] In some embodiments, the amount of the AAV vector comprising the transgene administered is about 4.2×10 11 or 4.2×10 10 genomes or vector or vector copies.

[0100] The present disclosure also provides a method for treating, preventing, curing a neurodegenerative disease or disorder and / or reducing the severity or degree 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, such as AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, and further comprising administering to the subject a therapeutically effective amount of a second composition (e.g., a viral vector, such as AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In an embodiment, the method comprises administering a therapeutically effective amount of a first composition comprising a nucleic acid encoding retromer core protein VPS35 and a therapeutically effective amount of a second composition comprising a nucleic acid encoding retromer core protein VPS26a or VPS26b. In an embodiment, the method comprises administering a therapeutically effective amount of a first composition comprising a nucleic acid encoding retromer core protein VPS26a or VPS26b and a therapeutically effective amount of a second composition comprising a nucleic acid encoding retromer core protein VPS35.

[0101] The present disclosure also provides a method for treating, preventing, curing a neurodegenerative disease or disorder and / or reducing the severity or degree 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, such as AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, and further comprising administering to the subject a therapeutically effective amount of a second composition (e.g., a viral vector, such as AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b, and further comprising administering to the subject a therapeutically effective amount of a third composition (e.g., a viral vector, such as AAV) comprising a nucleic acid encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In an embodiment, the first composition, the second composition, and the third composition each comprise a nucleic acid encoding retromer core protein VPS35, VPS26a, or VPS26b, respectively.

[0102] In some embodiments, the first, second, and third AAV vectors are each independently an AAV9 vector encoding retromer core protein VPS35 and / or retromer core protein VPS26a and / or retromer core protein VPS26b. In some embodiments, the first AAV vector, the second AAV vector, and the third AAV vector are administered simultaneously. 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.

[0103] In some embodiments, the first composition (e.g., AAV vector) is administered once a neurodegenerative disease or disorder is diagnosed or suspected, and the second composition (e.g., AAV vector) is administered at a time point after the first composition. In some embodiments, the second composition (e.g., AAV vector) is administered within hours of the first composition (e.g., AAV vector). In some embodiments, the second composition (e.g., AAV vector) is administered within days of the first composition (e.g., AAV vector). In some embodiments, the second composition (e.g., AAV vector) is administered weeks after the first composition (e.g., AAV vector). In some embodiments, the first composition (e.g., AAV vector) and the second composition (e.g., AAV vector) are administered simultaneously at any given time point.

[0104] In some embodiments, the third composition (e.g., AAV vector) is administered at a time point after the second composition. In some embodiments, the third composition (e.g., AAV vector) is administered within hours of the second composition (e.g., AAV vector). In some embodiments, the third composition (e.g., AAV vector) is administered within days of the second composition (e.g., AAV vector). In some embodiments, the third composition (e.g., AAV vector) is administered weeks after the second composition (e.g., AAV vector).

[0105] 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 point (including at or after the time of diagnosis or suspicion of a neurodegenerative disease or disorder). In some embodiments, the three compositions (e.g., AAV vectors) are present in the same larger composition, and in some embodiments, the three are separate compositions.

[0106] In embodiments of the methods described herein, one or more compositions comprising one or more nucleic acids encoding VPS35 and VPS26b, which are preferably expressed in the cortex, are administered to a subject having a disorder in which endosomal trafficking defects occur primarily in the cortex and in which VPS35 is unaffected or at risk of developing a disorder in which endosomal trafficking defects occur primarily in the cortex and in which VPS35 is unaffected. Examples of cortical endosomal trafficking disorders in which VPS35 is unaffected include biomarker-negative sporadic AD, AD patients with SORL1 mutations, FTD, prion disease, and Down syndrome.

[0107] In other embodiments of the methods described herein, one or more compositions comprising one or more nucleic acids encoding VPS35 and VPS26a, which are preferably expressed in the subcortex, are administered to a subject having a disorder in which endosomal trafficking defects occur primarily in the subcortical region or at risk of developing a disorder in which endosomal trafficking defects occur primarily in the subcortical region (e.g., biomarker-negative sporadic PD, HSP, prion disease, and NCL).

[0108] 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 a subject having a more diffuse endosomal trafficking neurological disorder or at risk of developing a more diffuse endosomal trafficking neurological disorder (e.g., Lewy body disease (LBD), prion disease, and ALS-FTD).

[0109] In addition to treating, preventing, curing a neurodegenerative disease or disorder and / or reducing the severity or extent of a neurodegenerative disease or disorder, in embodiments, the methods and compositions described herein are also used to treat, prevent, cure other diseases or disorders associated with endosomal trafficking and retrograde transport complex dysfunction and / or reduce the severity of other diseases or disorders associated with endosomal trafficking and retrograde transport complex dysfunction.

[0110] Recombinant AAV vector

[0111] The "recombinant AAV (rAAV) vector" 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'- and 3'-ITRs and can be operably linked to one or more regulatory elements in a manner that permits transcription, translation, and / or expression of the transgene in the cells of a target tissue. Such regulatory elements can include a promoter or enhancer, such as the chicken β-actin promoter or the cytomegalovirus enhancer, and other elements described herein. The recombinant AAV genome is generally encapsulated by a capsid protein (e.g., from the same AAV serotype from which the ITR is derived or from an AAV serotype different from the AAV serotype from which the ITR is derived). The AAV vector can then be delivered to a selected target cell type or tissue. In some embodiments, the transgene is a nucleic acid sequence heterologous to the vector sequence and encodes one or more of VPS35, VPS26a, and / or VPS26b. Components of exemplary AAV vectors that can be used in conjunction with the compositions and methods of the present disclosure are described herein.

[0112] Any AAV serotype or combination of AAV serotypes can be used in the methods and compositions of the present disclosure. Because the methods and compositions of the present disclosure are for treating and curing neurodegenerative diseases or disorders, in some embodiments, an AAV serotype that targets at least the central nervous system can be used and includes, but is not limited to, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

[0113] In some embodiments, the AAV9 serotype with broad tropism is used. In some embodiments, AAV2 / 9 is used.

[0114] Components of the AAV vector

[0115] The AAV vectors described herein may contain cis - acting 5' and 3' ITRs (see, e.g., Carter, "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 - 168 (1990)). The length of the ITR sequences is typically about 145 bp. Preferably, substantially complete sequences encoding the ITRs are used in the molecule, although some minor modifications to these sequences are allowed. (See, e.g., 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, wherein 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 currently identified mammalian AAV types.

[0116] In addition to the elements identified above for recombinant AAV vectors, the vector may also include conventional control elements that are operably linked to the transgene in a manner that permits its transcription, translation, and / or expression in cells transfected with plasmid vectors or infected with virus. As used herein, "operably linked" sequences include expression control sequences adjacent to the gene of interest and expression control sequences that act in trans or at a distance to control the expression of the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing 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, when desired, sequences that enhance the secretion of the encoded product. A wide variety of expression control sequences (including natural, constitutive, inducible, and / or tissue - specific promoters) are known in the art and can be used.

[0117] As used herein, nucleic acid sequences (e.g., coding sequences) and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequence. If a nucleic acid sequence is to be translated into a functional protein, then two DNA sequences are considered to be operably linked if induction of a promoter in a 5' regulatory sequence results in transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region will be operably linked to a nucleic acid sequence if the promoter region is capable of affecting the transcription of that DNA sequence such that the resulting transcript may be translated into the desired protein or polypeptide. Similarly, when two or more coding regions are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins, they are operably linked. In some embodiments, operably linked coding sequences produce a fusion protein. In some embodiments, operably linked coding sequences produce a functional RNA (e.g., shRNA, miRNA). In some embodiments, operably linked coding sequences produce two or more separate functional proteins (e.g., VPS35 and VPS26a or VPS26b).

[0118] For nucleic acids encoding a protein, a polyadenylation sequence is typically inserted after the transgene sequence and before the 3' AAV ITR sequence. The rAAV constructs of the present disclosure may also contain an intron, desirably located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40 and is referred to as the SV-40T intron sequence.

[0119] 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, an IRES element can be used to express VPS35 and VPS26a, VPS35 and VPS26b, or VPS26a and VPS26b from the same AAV vector.

[0120] The exact nature of the regulatory sequences required for gene expression in a host cell may vary between species, tissues, or cell types, but generally should include 5' non-transcribed and 5' transcribed sequences that are involved in the initiation of transcription and translation, respectively, such as TATA box, capping sequence, CAAT sequence, enhancer elements, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region that contains a promoter sequence for transcriptional control of the operably linked gene. Optionally, the regulatory sequences may also include enhancer sequences or upstream activation sequences. The vector may optionally include a 5' leader or signal sequence.

[0121] Examples of constitutive promoters include, but are not limited to, the chicken β-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 phosphoglycerate kinase (PGK) promoter, and the human elongation factor-1α (EF1α) promoter (Invitrogen).

[0122] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors (such as temperature), or specific physiological states such as the presence of an acute phase, a specific differentiation state of a cell, or regulation 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 metallothionein (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA 93:3346-3351 (1996)), the tetracycline repressor system (Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science 268:1766-1769 (1995)), the RU486-inducible system (Wang et al., Nat. Biotech. 15:239-243 (1997) and Wang et al., Gene Ther. 4:432-441 (1997)), and the rapamycin-inducible system (Magari et al., J. Clin. Invest. 100:2865-2872 (1997)). Other types of inducible promoters that can be used in this context are those that are regulated by specific physiological states (such as temperature, acute phase, a specific differentiation state of a cell, or regulation only in replicating cells).

[0123] In another embodiment, the native promoter of the transgene or a fragment thereof will be used. The native promoter may be preferred when it is desired that the expression of the transgene should mimic native expression. Native promoters can be used when the expression of the transgene must be regulated temporally or developmentally or in a tissue-specific manner or in response to a specific transcriptional stimulus. In another embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites, or Kozak consensus sequences, can also be used to mimic native expression.

[0124] In some embodiments, the regulatory sequence confers the ability of 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.

[0125] In some embodiments, one or more binding sites for one or more miRNAs are incorporated into the transgene of the rAAV vector to inhibit the expression of the transgene 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. Generally, the target site is located in the 3'UTR of the mRNA. In addition, the transgene can be designed such that multiple miRNAs regulate the mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may lead to the synergistic action of multiple RISCs and provide efficient expression inhibition. The target site sequence can comprise a total of 5-100, 10-60, or more nucleotides. The target site sequence can comprise at least 5 nucleotides of the target gene binding site sequence.

[0126] For example, a 3'-UTR site that inhibits transgene expression in the liver can be integrated into the transgene. This is beneficial for transgenes encoding therapeutic proteins that are toxic to the liver because most of the administered virus (about 60 to 90%) is ultimately found in the liver. Thus, inhibiting the expression of the therapeutic gene in the liver can relieve the burden on hepatocytes.

[0127] In some embodiments, the AAV vector will be modified to be self-complementary AAV. The self-complementary AAV carries the complementary sequence of the transgene (i.e., a double copy of the transgene). Self-complementarity makes the gene more stable after entering the cell.

[0128] Transgene coding sequence

[0129] The nucleic acid sequence of the transgene described herein can be designed based on the knowledge of a particular composition (e.g., a viral vector) that expresses the transgene. 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 a functional RNA intended for research purposes, e.g., to create a somatic transgenic animal model containing the transgene, e.g., to study the function of the transgene product. In another example, the transgene encodes a protein or a functional RNA intended for creating a disease animal model. Suitable transgene coding sequences will be apparent to those skilled in the art.

[0130] In embodiments, the transgene encodes a functional protein, including but not limited to vacuolar protein sorting 35 (VPS35) and / or vacuolar protein sorting 26a (VPS26a) and / or vacuolar protein sorting 26b (VPS26b). In embodiments, the transgene encodes 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.

[0131] Amino acid sequence information is available from the National Center for Biotechnology Information (NCBI) and is listed below.

[0132] The gene encoding human vacuolar protein sorting 35 (VPS35, Gene ID: 55737) can be used to obtain a transgene encoding a functional vacuolar protein sorting 35 (SEQ ID NO: 1):

[0133] MPTTQQSPQDEQEKLLDEAIQAVKVQSFQMKRCLDKNKLMDALKHASNMLGELRTSMLSPKSYYELYMAISDELHYLEVYLTDEFAKGRKVADLYELVQYAGNIIPRLYLLITVGVVYVKSFPQSRKDILKDLVEMCRGVQHPLRGLFLRNYLLQCTRNILPDEGEPTDEETTGDISDSMDFVLLNFAEMNKLWVRMQHQGHSRDREKRERERQELRILVGTNLVRLSQLEGVNVERYKQIVLTGILEQVVNCRDALAQEYLMECIIQVFPDEFHLQTLNPFLRACAELHQNVNVKNIIIALIDRLALFAHREDGPGIPADIKLFDIFSQQVATVIQSRQDMPSEDVVSLQVSLINLAMKCYPDRVDYVDKVLETTVEIFNKLNLEHIATSSAVSKELTRLLKIPVDTYNNILTVLKLKHFHPLFEYFDYESRKSMSCYVLSNVLDYNTEIVSQDQVDSIMNLVSTLIQDQPDQPVEDPDPEDFADEQSLVGRFIHLLRSEDPDQQYLILNTARKHFGAGGNQRIRFTLPPLVFAAYQLAFRYKENSKVDDKWEKKCQKIFSFAHQTISALIKAELAELPLRLFLQGALAAGEIGFENHETVAYEFMSQAFSLYEDEISDSKAQLAAITLIIGTFERMKCFSEENHEPLRTQCALAASKLLKKPDQGRAVSTCAHLFWSGRNTDKNGEELHGGKRVMECLKKALKIANQCMDPSLQVQLFIEILNRYIYFYEKENDAVTIQVLNQLIQKIREDLPNLESSEETEQINKHFHNTLEHLRLRRESPESEGPIYEGLIL

[0134] The gene encoding the core protein VPS26a of the human retromer complex (Gene ID: 9559) can be used to obtain a transgenic encoding a functional core protein VPS26a of the retromer complex (SEQ ID NO: 2):

[0135] MSFLGGFFGPICEIDIVLNDGETRKMAEMKTEDGKVEKHYLFYDGESVSGKVNLAFKQPGKRLEHQGIRIEFVGQIELFNDKSNTHEFVNLVKELALPGELTQSRSYDFEFMQVEKPYESYIGANVRLRYFLKVTIVRRLTDLVKEYDLIVHQLATYPDVNNSIKMEVGIEDCLHIEFEYNKSKYHLKDVIVGKIYFLLVRIKIQHMELQLIKKEITGIGPSTTTETETIAKYEIMDGAPVKGESIPIRLFLAGYDPTPTMRDVNKKFSVRYFLNLVLVDEEDRRYFKQQEIILWRKAPEKLRKQRTNFHQRFESPESQASAEQPEM

[0136] The gene encoding the human retromer complex core protein VPS26b (Gene ID: 112936) can be used to obtain a transgenic encoding the functional retromer complex core protein VPS26b (SEQ ID NO: 3):

[0137] MSFFGFGQSVEVEILLNDAESRKRAEHKTEDGKKEKYFLFYDGETVSGKVSLALKNPNKRLEHQGIKIEFIGQIELYYDRGNHHEFVSLVKDLARPGEITQSQAFDFEFTHVEKPYESYTGQNVKLRYFLRATISRRLNDVVKEMDIVVHTLSTYPELNSSIKMEVGIEDCLHIEFEYNKSKYHLKDVIVGKIYFLLVRIKIKHMEIDIIKRETTGTGPNVYHENDTIAKYEIMDGAPVRGESIPIRLFLAGYELTPTMRDINKKFSVRYYLNLVLIDEEERRYFKQQEVVLWRKGDIVRKSMSHQAAIASQRFEGTTSLGEVRTPSQLSDNNCRQ

[0138] The mouse mRNA sequence (i.e., the coding sequence) of the wild-type mouse of the retromer complex core protein was obtained from the National Center for Biotechnology Information (NCBI) and is shown below.

[0139] mVPS35 (SEQ ID NO: 4)

[0140]

[0141] mVPS26a isoform A (SEQ ID NO:5)

[0142] ATGAGTTTTCTTGGAGGCTTTTTTGGTCCCATTTGTGAGATTGATGTTGCCCTTAATGATGGGGAAACCAGGAAAATGGCAGAAATGAAAACTGAGGATGGCAAAGTAGAAAAACACTATCTCTTCTATGATGGCGAGTCTGTCTCAGGAAAGGTAAACCTAGCCTTTAAGCAGCCTGGAAAGAGGCTAGAGCATCAAGGAATTAGAATTGAATTTGTAGGTCAAATTGAGCTTTTCAATGACAAGAGTAATACTCATGAATTTGTAAACCTAGTGAAGGAACTAGCCTTGCCTGGAGAACTGACTCAGAGCAGAAGCTATGACTTTGAATTTATGCAAGTTGAAAAGCCATATGAGTCATACATCGGTGCCAATGTCCGCCTGAGGTATTTTCTTAAGGTGACAATTGTGAGAAGATTGACAGACTTAGTGAAAGAGTACGATCTTATTGTTCATCAGCTAGCCACCTATCCTGATGTCAACAACTCTATTAAAATGGAAGTGGGCATTGAAGACTGTCTGCACATAGAGTTTGAATATAATAAGTCCAAGTATCATTTAAAGGATGTAATTGTTGGAAAAATTTACTTCTTATTAGTAAGAATAAAAATACAACACATGGAATTACAGCTGATCAAGAAAGAGATCACAGGAATTGGACCCAGCACCACAACAGAGACAGAAACAATCGCTAAGTATGAAATAATGGATGGGGCGCCAGTAAAAGGAGAATCTATTCCGATAAGATTGTTCTTAGCAGGGTATGACCCAACCCCCACGATGAGAGATGTGAACAAGAAGTTTTCAGTAAGGTACTTTCTAAACCTCGTGCTTGTTGATGAGGAGGACCGAAGGTACTTCAAGCAGCAGGAGATCATCCTGTGGAGAAAAGCACCCGAGAAACTGAGAAAACAGAGGACGAACTTTCACCAGCGGTTTGAATCTCCAGACTCGCAGGCCTCTGCGGAGCAGCCTGAGATGTAA

[0143] mVPS26b (SEQ ID NO:6)

[0144]

[0145] Codon Optimization of Transgenic Coding Sequences

[0146] Codon optimization of transgenic coding sequences can improve the efficiency of gene therapy. Thus, in some embodiments, a nucleic acid that is at least 70% identical to the coding sequence of a transgenic encoding a therapeutic protein is used (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).

[0147] Codon optimization tools are known in the art.

[0148] Exemplary codon-optimized nucleic acids are as follows.

[0149] Codon-optimized mVPS35 (SEQ ID NO:7)

[0150]

[0151] Codon-optimized mVPS26a isoform A (SEQ ID NO:8)

[0152] ATGAGCTTCCTGGGCGGCTTCTTCGGCCCCATCTGTGAGATCGACGTGGCCCTGAACGACGGCGAGACCAGAAAGATGGCCGAGATGAAGACAGAGGATGGCAAGGTGGAGAAGCACTACCTGTTCTACGACGGAGAGTCTGTGTCCGGCAAGGTGAACCTGGCCTTCAAGCAGCCTGGGAAGAGGCTGGAGCACCAGGGCATCAGAATCGAGTTCGTGGGCCAGATCGAGCTGTTCAACGACAAGAGCAACACCCACGAGTTTGTGAACCTGGTGAAGGAGCTGGCTCTGCCTGGCGAGCTGACCCAGAGCAGAAGCTACGACTTCGAGTTCATGCAGGTGGAGAAGCCTTACGAGAGCTACATCGGCGCCAACGTGAGACTGAGATACTTCCTGAAGGTGACCATCGTGAGGAGACTGACCGACCTGGTGAAGGAGTATGACCTGATCGTGCACCAGCTGGCCACCTACCCTGACGTGAACAACAGCATCAAGATGGAGGTGGGCATCGAGGACTGCCTGCACATCGAGTTCGAGTACAACAAGTCCAAGTACCACCTGAAGGACGTGATCGTGGGCAAGATCTACTTCCTGCTGGTGAGGATCAAGATCCAGCACATGGAGCTGCAGCTGATCAAGAAGGAGATCACCGGCATCGGCCCTTCCACAACCACCGAGACAGAGACAATCGCCAAGTACGAGATCATGGACGGCGCCCCTGTGAAGGGCGAGAGCATCCCTATCAGGCTGTTCCTGGCCGGCTACGACCCTACCCCTACCATGAGAGACGTGAACAAGAAGTTCAGCGTGAGGTACTTCCTGAACCTGGTGCTGGTGGACGAGGAGGACAGAAGATACTTCAAGCAGCAGGAGATCATCCTGTGGAGGAAGGCCCCTGAGAAGCTGAGGAAGCAGAGGACCAACTTCCACCAGAGATTCGAGTCCCCTGACAGCCAGGCCAGCGCCGAGCAGCCAGAGATGTGA

[0153] Codon-optimized mVPS26b (SEQ ID NO:9)

[0154]

[0155] Human VPS35 coding sequence (certain codons are modified to remove restriction sites) (SEQ ID NO:10)

[0156]

[0157] Route of administration and dosage

[0158] The present disclosure provides rAAV vectors for methods of treating, preventing, and / or curing a neurodegenerative disease or disorder and / or alleviating at least one of the symptoms associated with a neurodegenerative disease and / or disorder in a subject. In some embodiments, the method comprises 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 such neurodegenerative disease or disorder in a subject having or suspected of having a neurodegenerative disease or disorder.

[0159] The rAAV vector can be delivered to the 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., in a composition)) can be administered to the subject. In certain embodiments, the composition can comprise the rAAV vector alone or in combination with one or more other vectors (e.g., a second rAAV vector having one or more different transgenes). In one embodiment, the composition can comprise an rAAV9 vector containing a nucleic acid sequence comprising a transgene encoding a functional protein, the functional protein including but not limited to the retrograde transport complex core protein VPS35 and / or the retrograde transport complex core protein VPS26a and / or the retrograde transport complex core protein VPS26b. In one embodiment, the composition can comprise an rAAV2 / 9 vector containing a nucleic acid sequence comprising a transgene encoding a functional protein, the functional protein including but not limited to the retrograde transport complex core protein VPS35 and / or the retrograde transport complex core protein VPS26a and / or the retrograde transport complex core protein VPS26b. In one embodiment, the composition can comprise an rAAV10 or rAAV2 / 10 vector comprising a nucleic acid sequence comprising a transgene encoding a functional protein, the functional protein including but not limited to the retrograde transport complex core protein VPS35 and / or the retrograde transport complex core protein VPS26a and / or the retrograde transport complex core protein VPS26b.

[0160] One of skill in the art can readily select a suitable carrier depending on the indication for which the rAAV is targeted. For example, one suitable carrier includes saline, which can be formulated with a variety of 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 choice of carrier is not a limitation of the present disclosure.

[0161] Optionally, in addition to the rAAV and one or more carriers, the compositions disclosed herein may further comprise other conventional pharmaceutical components, such as preservatives or chemical stabilizers. 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.

[0162] In some embodiments, the rAAV composition is formulated to reduce aggregation of AAV particles in the composition, particularly in the presence of high rAAV concentrations (e.g., about 10 13 GC / ml or higher). Methods for reducing rAAV aggregation are well known in the art and include, for example, addition of surfactants, pH adjustment, and salt concentration adjustment (see, e.g., Wright et al., Molecular Therapy 12:171-178 (2005)).

[0163] Pharmaceutical forms suitable for injection 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 their mixtures and 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, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and / or vegetable oils. Proper fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. The action of microorganisms can be prevented by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents, such as sugars or sodium chloride. The absorption of injectable compositions can be prolonged by the use of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0164] For example, for the administration of injectable aqueous solutions, the solution can be buffered appropriately if desired, and the liquid diluent is made isotonic first 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 dose can be dissolved in 1 ml of isotonic NaCl solution and then added to 1000 ml of subcutaneous injection fluid or injected at the proposed infusion site. Some variation in dosage will necessarily occur depending on the host. In any case, the person responsible for administration will determine the appropriate dose for the individual host.

[0165] Sterile injectable solutions are prepared by incorporating the required amount of the active rAAV with the various other components enumerated herein (as required) into a suitable solvent and then filtering sterilization. Generally, dispersions are prepared by incorporating the various sterilized active components into a sterile vehicle which contains the basic dispersion medium and the required other components from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active ingredient plus any other desired components from its previously sterile-filtered solution.

[0166] In addition to the above delivery methods, the following techniques are also considered as alternative methods for delivering the rAAV composition to the host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and efficacy of drug penetration into and through the circulatory system. Other alternative drug delivery methods considered 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. Patents Nos. 5,770,219 and 5,783,208), and feedback control delivery (U.S. Patent No. 5,697,899).

[0167] rAAVs are administered in an amount sufficient via the administration route to transfect the cells of the desired tissue and provide a sufficient level of gene transfer and expression without undue side effects. Conventional and 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 intranasal and intratracheal administration), intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral administration routes. If desired, administration routes can be combined. The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic composition, the symptom level, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers a sufficient amount of the therapeutic composition to effect an improvement in the target disease state while minimizing undesired side effects. Thus, the amount of the biological agent delivered depends in part on the particular therapeutic composition and the severity of the condition being treated.

[0168] The present disclosure provides a stable pharmaceutical composition comprising an rAAV virion. The composition remains stable and active even when subjected to freeze - thaw cycles and stored in containers made of various materials including glass.

[0169] A suitable dose will depend on the subject being treated (e.g., a human, non - human primate, or other mammal), the age and general condition of the subject being treated, the severity of the condition being treated, the mode of administration of the rAAV virion, and other factors. A person skilled in the art can readily determine a suitable effective amount.

[0170] The dose of rAAV virion required to achieve a desired effect or “therapeutic effect” (e.g., dose units in the vector genome per kilogram body weight (vg / kg)) will vary according to several factors including, but not limited to: the rAAV administration route; 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. Based on the above factors and other factors well known in the art, a person skilled in the art can readily determine the dose range of rAAV virion to treat a subject suffering from a particular disease or disorder. The effective amount of rAAV is typically in the range of about 10 μl to about 100 ml of solution containing about 10 9 to 10 16 genomic copies per subject. Other volumes of solution can be used. The volume used will generally depend on factors such as the size of the subject, the dose of rAAV, and the administration route. For example, for intrathecal or intracerebral administration, volumes in the range of 1 μl to 10 μl or 10 μl to 100 μl can be used. For intravenous administration, volumes of 10 μl to 100 μl, 100 μl to 1 ml, 1 ml to 10 ml, or more can be used. In some cases, a dose of about 10 10 to 10 12 rAAV genomic copies per subject is suitable. In certain embodiments, a dose of 10 12 rAAV genomic copies per subject is effective for targeting the desired tissue. In some embodiments, rAAV is administered at a dose of 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 genomic copies per subject. In some embodiments, rAAV is at 10 10 , 10 11 , 10 12 , 10 13 , or 10 14administered at a dose of genomic copies / kg.

[0171] Thus, a "therapeutically effective amount" will fall within a relatively broad range that can be determined by clinical trials. For example, for in vivo injection, i.e., direct injection into a subject, a therapeutically effective dose can be about 10 5 to 10 16 rAAV virions, more preferably 10 8 to 10 14 rAAV virions. For ex vivo transduction, the effective amount of rAAV virions to be delivered to cells can be about 10 5 to 10 13 virions, preferably 10 8 to 10 13 rAAV virions. If the composition contains transduced cells to be delivered back to the subject, the amount of transduced cells in the pharmaceutical composition can be about 10 4 to 10 10 cells, more preferably 10 5 to 10 8 cells. Of course, the dose depends on transduction efficiency, promoter strength, stability of the message, and the protein encoded thereby. A person of ordinary skill in the art can readily establish an effective dose by routine tests that establish a dose-response curve.

[0172] Dose therapy can be a single-dose regimen or a multi-dose regimen to ultimately deliver the above-specified amount. In addition, as many doses as appropriate can be administered to the subject. Thus, the subject can be given, for example, 10 5 to 10 16 rAAV virions in a single dose, or the subject can be given two, three, four, five, six or more doses that together result in the delivery of, for example, 10 5 to 10 16 rAAV virions. A person skilled in the art can readily determine the appropriate number of doses to administer.

[0173] Thus, the pharmaceutical composition will contain 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 under discussion or an amount sufficient to confer the desired benefit. Thus, when administered in one or more doses, the rAAV virions will be present in the subject composition in an amount sufficient to provide a therapeutic effect. The rAAV virions can be provided as a lyophilized preparation and diluted in a virion-stabilizing composition for immediate or future use. Alternatively, the rAAV virions can be provided immediately after production and stored for future use.

[0174] The pharmaceutical composition will also contain a pharmaceutically acceptable excipient or carrier. Such excipients include any 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, but are not limited to, liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts may be included therein, such as inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic acid salts, such as acetate, propionate, malonate, benzoate, etc. Additionally, auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances, etc. may be present in these carriers. A comprehensive discussion of pharmaceutically acceptable excipients is provided in Remington's Pharmaceutical Sciences and U.S.Pharmacopeia:National Formulary,Mack Publishing Company,Easton,PA(1984).

[0175] Preparations of therapeutic and diagnostic agents can be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, or suspensions.

[0176] The toxicity and therapeutic efficacy of a therapeutic composition administered alone or in combination with another agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as for determining the LD 50 (dose lethal to 50% of the population) and ED 50 (dose therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index (LD 50 / ED 50 ). In certain aspects, a therapeutic composition exhibiting a high therapeutic index is desirable. Data obtained from these cell culture assays and animal studies can be used to formulate a range of doses for humans. The dose of such compounds is preferably within the range that exhibits little or no toxicity and includes the circulating concentration of ED 50 . The dose can vary within this range depending on the dosage form used and the route of administration.

[0177] Determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected to affect treatment in the art. The dose can be started at an amount slightly below the optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic metrics include, for example, symptoms of inflammation or the level of inflammatory cytokines produced. Generally, it is desirable that the biological agent to be used be derived from the same species as the animal being targeted for treatment, thereby minimizing any immune response to the reagent.

[0178] The preferred route of administration of AAV is intravenous. Other routes of administration of the rAAV vectors described herein include intracranial, intrathecal, and intraspinal.

[0179] The preferred dose range is from about 1x10 10 to about 8x10 11 、about 2x10 10 to about 6x10 11 、about 4x10 10 to about 4x10 11 total administered genomes or viral copies (vc). The preferred dose is about 4x10 11 total administered genomes or viral copies (vc) of rAAV.

[0180] If more than one rAAV is used, the preferred total dose range of the vectors is from about 1x10 10 to about 6x10 11 、about 2x10 10 to about 5x10 11 、about 1x10 10 to about 4x10 11 total administered genomes or viral copies (vc). The preferred dose of the total vector is about 3x10 11 . AAV can be administered in equal amounts, for example, at a ratio of 50 / 50, or at ratios of 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.

[0181] The dose can be adjusted to optimize the effect in the subject. In addition, the improvement of the subject's condition can be monitored before increasing the dose. The response of the subject to the rAAV treatment administration can be monitored by observing changes in the subject's muscle strength and control, mobility, and height and weight. If one or more of these parameters increase after administration, the treatment can be continued. If one or more of these parameters remain unchanged or decrease, the dose can be increased.

[0182] Kit

[0183] The present disclosure also provides a kit comprising the components of the combination disclosed herein in kit form. The kits of the present disclosure include one or more components, including but not limited to the viral vectors (e.g., AAV vectors) described herein. As discussed herein, the kit may also include a pharmaceutically acceptable carrier. The viral vector can be formulated as a pure composition or combined with a pharmaceutically acceptable carrier in a pharmaceutical composition.

[0184] In some embodiments, the kit comprises an AAV vector in a container (e.g., in a sterile glass or plastic vial), the AAV vector containing the transgene described herein.

[0185] In some embodiments, the kit comprises an AAV vector containing the transgene described herein in a first container (e.g., in a sterile glass or plastic vial), a second AAV vector encoding the transgene described herein in a second container (e.g., in a sterile glass or plastic vial), and a third AAV vector encoding the transgene described herein in a third container (e.g., in a sterile glass or plastic vial).

[0186] In some embodiments, the kit contains an AAV vector encoding the retromer core protein VPS35 and / or the retromer core protein VPS26a and / or the retromer core protein VPS26b in one or more containers (e.g., in a sterile glass or plastic vial), or a pharmaceutical composition thereof.

[0187] If the kit comprises one or more pharmaceutical compositions for parenteral administration to a subject, the kit may include a device for such administration. For example, the kit may include one or more subcutaneous injection needles or other injection devices as discussed above.

[0188] The kit may include a package insert that includes information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in the effective and safe use of the accompanying pharmaceutical compositions and dosage forms. For example, the following information about the combination may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdose, appropriate dosage and administration, how supplied, appropriate storage conditions, references, manufacturer / distributor information, and patent information. Examples

[0189] The present invention may be better understood with reference to the following non-limiting examples, which are provided to more fully illustrate the preferred embodiments of the present invention. They should in no way be construed as limiting the broad scope of the present invention.

[0190] Example 1 - Materials and Methods

[0191] Plasmid Production

[0192] The mRNA sequences of VPS35, VPS26a, and VPS26b were obtained from the National Center for Biotechnology Information (NCBI). The sequences were codon-optimized and then synthesized de novo. The synthesized constructs were subcloned into an AAV transfer plasmid with AAV2 inverted terminal repeats (ITRs) and the ubiquitous chicken β-actin wild-type promoter. The transgene was followed by the bovine growth hormone polyadenylation signal. An empty-frame vector control was generated by deleting the VPS35 sequence. The GFP control was designed to mimic the rationale of the target VPS construct. It contains enhanced green fluorescent protein, the same bovine growth hormone polyA (BgH) as the VPS construct, and the chicken β-actin wild-type promoter. The resulting plasmids are shown in Figure 3.

[0193] AAV9 Production

[0194] Each of the above constructs was individually packaged into recombinant adeno-associated virus vector 9 (AAV9) using the capsid and helper plasmid DNA from MeiraGTx. Briefly, the transfer plasmid, rep-cap plasmid, and helper plasmid were co-transfected into HEK293 cells. The harvested suspension containing virus and cell debris was clarified using a millipore SHC XL150 filter (140 cm 2 ). The clarified suspension was then purified using an AVB Sepharose, 20 mL column and eluted with 3 column volumes. Concentration and diafiltration were performed using a 100 kD mPES hollow fiber (Spectrum MicroKros cat#C02-E100-05-S). Further concentration was performed using an AmiconUltra-4 centrifugal filter 30 kD (cat#UFC8030).

[0195] N2A Cultivation

[0196] 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.

[0197] Transfection

[0198] Use a modified Lipofectamine transfection protocol. Briefly, co-transfect Vps35 and Vps26 (Vps26a or Vps26b) plasmids into neuronal-like cells Neuro2a (N2a) in 6-well format using lipofectamine LTX. Plate 100k cells into each well that already contains medium with the DNA-lipofectamine complex. Empty frame and GFP are used as control plasmids. The amount of DNA copies introduced per well is 2.81E+11. As previously described (Qureshi et al., 2019), harvest cells using RIPA buffer 48 hours after transfection.

[0199] Neuron culture and transduction

[0200] Perform primary mouse cortical and hippocampal neuron culture as previously described (Bhalla et al., 2012). Seven days after plating in 12-well plates, transduce neurons (450k cells per well) with the retrograde transport complex AAV9 (2.27E+10 vector genomes / condition / well). Empty frame AAV9 and GFP AAV9 are used as controls. Maintain the cultures for 3 weeks after transduction (4 weeks in total). On day 28, lyse neurons using RIPA buffer containing protease and phosphatase inhibitors.

[0201] Western blotting

[0202] Cells from N2A and neuron cultures are lysed in RIPA and proteins are separated as previously described (Qureshi et al., 2019; Kirby et al., 2015). Lysates from samples are run on Bis-Tris 4-12% gels, transferred to nitrocellulose membranes using iblot and probed with antibodies.

[0203] Primary antibodies against the following proteins are 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). The 800 or 680 antibodies (LI-COR) were used as secondary antibodies, with a dilution of 1:10k for 800CW, 1:15k for 680RD, and 1:25k for the 680LT antibody. As described previously (Eaton et al., 2013), the Western blots were scanned using an Odyssey imaging system.

[0204] 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 the blots were scanned on a Fujifilm LAS-3000 Imager.

[0205] Statistics

[0206] Statistical analyses were performed using Microsoft Excel and SPSS. Unless otherwise stated, all experiments used an independent two-sample Student's t-test, assuming equal variances, with a two-tailed distribution. All data are presented as mean values, and error bars represent the standard error of the mean. All bar graphs were created in GraphPad Prism8. Scatter plots were created in SPSS.

[0207] Example 2 - Overexpression of VPS35 alone is insufficient to increase the trimerization and function of the retrograde transport complex

[0208] To determine the effect of exogenous VPS35 overexpression on the core proteins of the retrograde transport complex and on the function of the retrograde transport complex in a non-defective state, cultured wild-type mouse neurons were transduced with AAV9-VPS35-HA and AAV9-GFP or AAV9-empty vector (EV) as control conditions and harvested after 3 weeks.

[0209] The levels of all core proteins of the retrograde transport complex were determined by immunoblotting ( Figure 2A ). Compared with the control group, 90% VPS35-HA overexpression led to a significant increase of 67% in endogenous VPS29 (p = 9E-09), a small increase of 22% in VPS26a (p = 2E-08), while there was no increase in VPS26b (p = 0.62) ( Figure 2B ).

[0210] Sorl1 levels were also determined by immunoblotting. Compared with the control group, overexpression of VPS35 alone showed a slight increase (11%) in Sorl1 and was not statistically reliable (p = 0.06) ( Figure 2B ).

[0211] By showing that overexpression of VPS35 results in significant overexpression of VPS29, but no or little increase in VPS26 paralogs and no obvious effect on the function of the retrotransporter complex, these results justify the study of the role of co-expression of VPS35 and VPS26.

[0212] Example 3 - Results using neuroblastoma cells and plasmids and AAV9 constructs

[0213] Neuroblastoma (N2A) cells were transfected with plasmids expressing a single protein (VPS35, VPS26a or VPS26b) or protein combinations (VPS35+VPS26a or VPS35+VPS26b). A plasmid expressing GFP or an empty plasmid was used as a control.

[0214] Single-protein conditions led to overexpression above control levels for each protein: VPS35 alone (80%, p = 3.4E-09), VPS26a alone (550%; p = 2.2E-06), and VPS26b alone (362%; p = 0.0002). Compared to single-protein conditions, VPS35+VPS26a expression led to significant increases in VPS35 (31%; p = 0.0003), VPS29 (17%; p = 0.0007), and VPS26a (52%; p = 0.015), but minimal change in VPS26b. VPS35+VPS26b expression led to non-significant increases 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.

[0215] Example 4 - Results using neurons and AAV9 constructs

[0216] To test the role of the VPS35 and VPS26 combination in cultured neurons, five experimental AAV9 vectors were generated, expressing mouse VPS35, VPS26a, VPS26b, and two control AAV9 vectors, one expressing 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, VPS26b alone); dual-protein expression (VPS35+VPS26a, VPS35+VPS26b, VPS26b+VPS26a); and triple-protein expression (VPS35+VPS26a+VPS26b).

[0217] In exploratory studies, the dose of each viral vector was optimized, and when used in the final combination study, the average AAV9-VPS35 overexpression was 11% (range: 1% to 23%), the average AAV9-VPS26a was 218% (range: 154% to 354%), and the average AAV9-VPS26b was 80% (range: 50% to 107%) ( Figure 5B , blue bars). This configuration has proven particularly useful for testing interactions.

[0218] To test whether there is a VPS35-VPS26 interaction on the expression of the retromer core proteins, the levels detected under single protein conditions were compared with those detected in combination experiments. Compared with single protein expression, VPS35+VPS26a expression led to significant increases in VPS35 (70%; p = 4.4E-18), VPS26a (53%; p = 2.1E-05), VPS29 (∼42%; p < 1.22E-05) but not in VPS26b. VPS35+VPS26b expression led to significant increases in VPS35 (64%; p = 3.6E-09), VPS26b (15%; p = 0.003), VPS29 (∼18%; p < 0.013) but not in VPS26a.

[0219] Finally, compared with the control group (EV+GFP), VPS35+VPS26a+VPS26b expression led to significant increases in all four retromer components: 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).

[0220] See Figure 5.

[0221] These results again demonstrate the co-expression of VPS35+VPS26 on VPS35 expression and the synergistic effect on VPS26a and VPS26b.

[0222] Although the main purpose of this series of comprehensive experiments was to test the synergistic interaction, the fact that VPS35+VPS26a has no effect on VPS26b and VPS35+VPS26b has no effect on VPS26a indicates that in neurons, each VPS26 paralog exists as a biochemically distinct trimer.

[0223] Example 5 - Combined VPS35 and VPS26 Expression Synergistically Affects Retromer Function in Neurons

[0224] Next, to test whether the VPS35 and VPS26 combination has a synergistic effect on retromer complex function by comparing Sorl1 levels measured under all conditions, as loss-of-function mutations in SORL1 are the cause of Alzheimer's disease (Holstege et al., 2017), and a reduction of approximately 30% in Sorl1 protein has been found even in the early stages of sporadic disease (Sager et al., 2007; Scherzer et al., 2004; Dodson et al., 2006).

[0225] Univariate ANOVA was used, including control conditions, single conditions, and combination conditions as fixed factors, and including Sorl1 as the dependent variable. The results showed a population effect (F = 19.3, p = 9.3E-8), where simple comparisons showed that there was no difference between the control and single conditions (contrast estimate = 0.1, p = 0.9), but there was a significant difference between the single and combination conditions (contrast estimate = 0.4, p = 2.2E-7). Post hoc comparisons showed that each combination condition was significantly different from the single condition ( Figure 6B ).

[0226] Interestingly, a significant effect of VPS26a + VPS26b overexpression on Sorl1 was also observed (34%; p = 0.006)( Figure 6B ), even though this combination condition did not increase the levels of VPS35 or VPS29 ( Figure 5B ). Sorl1 has been found to interact with the retromer complex core through VPS26 (Suzuki et al., (2019)), and this observation suggests that the two paralogs may interact with Sorl1 independently.

[0227] The generated large-scale dataset was used, which had more than 140 experimental or control conditions, and in which four retromer complex core proteins and Sorl1 were measured over a wide dynamic range. A multiple linear regression model was used, including the Sorl1 level as the dependent variable and the VPS35, VPS26, VPS26a, and VPS26b levels as independent variables input simultaneously. A significant relationship with the Sorl1 level was found (F = 19.5, p = 1.1E-12), where only the VPS26 paralogs made a significant contribution to the model. Therefore, the model was pruned to include the two paralogs, confirming that VPS26a (t = 5.6, p = 1.4E-7) and VPS26b (F = 7.2, p = 3.2E-11) were independently correlated with the Sorl1 level ( Figure 6C and 6D)。This result is consistent with the explanation that neurons have two trimers (VPS26b-VPS35-VPS29 and VPS26a-VPS35-VPS29) that are not only biochemically different but also functionally different.

[0228] References

[0229] Andersen et al., Securing the future of drug discovery for central nervous system disorders. Nature reviews. Drug discovery. 2014;13(12):871-872.

[0230] Bhalla et al., The location and trafficking routes of the neuronal retromer and its role in amyloid precursor protein transport. Neurobiol Dis. 2012;47(1):126-34.

[0231] Collins et al., Structure of Vps26B and mapping of its interaction with the retromer protein complex. Traffic 2008;9(3):366-79.

[0232] Dodson et al., LR11 / SorLA expression is reduced in sporadic Alzheimer disease but not in familial Alzheimer disease. J Neuropathol Exp Neurol, 2006;65(9):866-72.

[0233] Eaton et al., Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting. PloS one. 2013;8(8):e72457.

[0234] Holstege et al., Characterization of pathogenic SORL1 genetic variants for association with Alzheimer's disease: a clinical interpretation strategy. Eur J Hum Genet. 2017.

[0235] Kendall et al., Mammalian Retromer Is an Adaptable Scaffold for Cargo Sorting from Endosomes. Structure. 2020;28(4):393 - 405.e4.

[0236] Kirby et al., Adult hippocampal neural stem and progenitor cells regulate the neurogenic niche by secreting VEGF. PNAS USA. 2015;112(13):4128 - 33. Epub 2015 / 03 / 17.

[0237] Qureshi et al., Retromer repletion with AAV9 - VPS35 restores endosomal function in the mouse hippocampus. bioRxiv. 2019:618496.

[0238] Sager et al., Neuronal LR11 / sorLA expression is reduced in mild cognitive impairment. Ann Neurol. 2007;62(6):640 - 7.

[0239] Scherzer et al., Loss of apolipoprotein E receptor LR11 in Alzheimer disease. Arch Neurol. 2004;61(8):1200 - 5.

[0240] Shi et al., The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain. Nat Struct Mol Biol. 2006;13(6):540-8.

[0241] Small and Petsko Retromer in Alzheimer disease, Parkinson disease and other neurological disorders. Nature reviews. Neuroscience. 2015;16(3):126-132.

[0242] Suzuki et al., A bipartite sorting signal ensures specificity of retromer complex in membrane protein recycling. J Cell Biol. 2019;218(9):2876-2886.

Claims

1. A method of increasing the function of the retrograde transport complex in neuronal cells, which comprises: introducing a nucleic acid encoding the retrograde transport complex core protein VPS35 and a nucleic acid encoding the retrograde transport complex core protein VPS26a into neuronal cells, whereby the levels of VPS35 and VPS26a in the neuronal cells are increased as compared to the levels of VPS35 and VPS26a when only the nucleic acid encoding VPS26a or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex; and / or whereby the level of Sorl1 in the neuronal cells is increased as compared to the level of Sorl1 when only the nucleic acid encoding VPS26a or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

2. The method of claim 1, wherein the levels of VPS35 and VPS26a in the neuronal cells are increased as compared to the levels of VPS35 and VPS26a when only the nucleic acid encoding VPS26a or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

3. The method of claim 1, wherein the level of Sorl1 in the neuronal cells is increased as compared to the level of Sorl1 when only the nucleic acid encoding VPS26a or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

4. A method of increasing the function of the retrograde transport complex in neuronal cells, which comprises: introducing a nucleic acid encoding the retrograde transport complex core protein VPS35 and a nucleic acid encoding the retrograde transport complex core protein VPS26b into neuronal cells, whereby the levels of VPS35, VPS26b and VPS29 in the neuronal cells are increased as compared to the levels of VPS35, VPS26b and VPS29 when only the nucleic acid encoding VPS26b or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex; and / or whereby the level of Sorl1 in the neuronal cells is increased as compared to the level of Sorl1 when only the nucleic acid encoding VPS26a or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

5. The method of claim 4, wherein the levels of VPS35 and VPS26b in the neuronal cells are increased as compared to the levels of VPS35 and VPS26b when only the nucleic acid encoding VPS26b or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

6. The method of claim 4, wherein the level of Sorl1 in the neuronal cells is increased as compared to the level of Sorl1 when only the nucleic acid encoding VPS26b or VPS35 is introduced into the neuronal cells, thereby increasing the function of the retrograde transport complex.

7. The method of any one of claims 1-6, wherein the nucleic acid encoding the retrograde transport complex core protein VPS35 and the nucleic acid encoding the retrograde transport complex core protein VPS26a or the nucleic acid encoding the retrograde transport complex core protein VPS26b are provided in one or more viral vectors.

8. The method of claim 7, wherein the nucleic acid encoding the retromer complex core protein VPS35 and the nucleic acid encoding the retromer complex core protein VPS26a or the nucleic acid encoding the retromer complex core protein VPS26b are provided in a viral vector.

9. The method of claim 7, wherein a viral vector comprising a nucleic acid encoding the retromer complex core protein VPS35 and a viral vector comprising a nucleic acid encoding the retromer complex core protein VPS26a or the nucleic acid encoding the retromer complex core protein VPS26b are administered sequentially.

10. The method of claim 7, wherein a viral vector comprising a nucleic acid encoding the retromer complex core protein VPS35 and a viral vector comprising a nucleic acid encoding the retromer complex core protein VPS26a or the nucleic acid encoding the retromer complex core protein VPS26b are administered simultaneously.

11. A viral vector, which comprises at least one transgene encoding the retromer complex core protein VPS35 and a retromer complex core protein selected from VPS26a, VPS26b, and combinations thereof.

12. The vector of claim 11, wherein the retromer complex 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).

13. The vector of claim 11, wherein the retromer complex core protein VPS35 encoded by the transgene has the amino acid sequence of VPS35 (SEQ ID NO: 1).

14. The vector of claim 11, wherein the transgene encoding the retromer complex core protein Vps35 comprises a human Vps35 nucleic acid sequence.

15. The vector of claim 11, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS35 (SEQ ID NO: 1).

16. The vector of claim 11, wherein the transgene has the nucleic acid sequence of SEQ ID NO:

10.

17. The vector of claim 11, wherein the retromer complex 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).

18. The vector of claim 11, wherein the retromer complex core protein Vps26a encoded by the transgene has the amino acid sequence of VPS26a (SEQ ID NO: 2).

19. The vector of claim 11, wherein the transgene encoding the retromer complex core protein Vps35 comprises a human VPS26a nucleic acid sequence.

20. The vector of claim 11, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS26a (SEQ ID NO: 2).

21. The vector of claim 11, wherein the retromer complex 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).

22. The vector of claim 11, wherein the retromer complex core protein VPS26b encoded by the transgene has the amino acid sequence of VPS26b (SEQ ID NO: 3).

23. The vector of claim 11, wherein the transgene encoding the retromer complex core protein VPS35 comprises a human VPS26b nucleic acid sequence.

24. The vector of claim 11, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS26b (SEQ ID NO: 3).

25. The vector of any one of claims 11-24, wherein the vector is selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus.

26. The vector of claim 25, wherein the viral vector is AAV.

27. The vector of claim 26, wherein the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrh10.

28. The vector of claim 26, wherein the AAV is selected from AAV9 and AAV2 / 9.

29. The vector of any one of claims 11-24, wherein the transgene is operably linked to a promoter or enhancer that induces expression of the transgene in nerve cells.

30. A composition comprising at least one viral vector, the viral vector comprising a transgene encoding a retromer complex core protein VPS35 and a transgene encoding a retromer complex core protein selected from VPS26a, VPS26b, and combinations thereof.

31. The composition of claim 30, wherein the retromer complex 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).

32. The composition of claim 30, wherein the retromer complex core protein VPS35 encoded by the transgene has the amino acid sequence of VPS35 (SEQ ID NO: 1).

33. The composition of claim 30, wherein the transgene encoding the retromer complex core protein VPS35 comprises a human VPS35 nucleic acid sequence.

34. The composition of claim 30, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS35 (SEQ ID NO: 1).

35. The composition of claim 30, wherein the transgene has the nucleic acid sequence of SEQ ID NO:

10.

36. The composition of claim 30, wherein the retromer complex 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).

37. The composition of claim 30, wherein the retromer complex core protein VPS26a encoded by the transgene has the amino acid sequence of VPS26a (SEQ ID NO: 2).

38. The composition of claim 30, wherein the transgene encoding the core protein VPS35 of the retromer complex comprises a human VPS26a nucleic acid sequence.

39. The composition of claim 30, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS26a (SEQ ID NO: 2).

40. The composition of claim 30, wherein the core protein VPS26b of the retromer complex 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).

41. The composition of claim 30, wherein the core protein VPS26b of the retromer complex encoded by the transgene has the amino acid sequence of VPS26b (SEQ ID NO: 3).

42. The composition of claim 30, wherein the transgene encoding the core protein VPS35 of the retromer complex comprises a human VPS26b nucleic acid sequence.

43. The composition of claim 30, wherein the transgene has a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence encoding VPS26b (SEQ ID NO: 3).

44. The composition of claim 30, wherein the vector is selected from adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, poxvirus, baculovirus, herpes simplex virus, vaccinia virus, and synthetic virus.

45. The composition of claim 44, wherein the viral vector is AAV.

46. The composition of claim 45, wherein the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVrh10.

47. The composition of claim 45, wherein the AAV is selected from AAV9 and AAV2 / 9.

48. The composition of any one of claims 30-47, wherein the transgene is operably linked to a promoter or enhancer that induces expression of the transgene in nerve cells.

49. The composition of claim 30, which further comprises a pharmaceutical carrier.

50. A composition comprising at least one nucleic acid encoding VPS35 and a core protein of the retromer complex selected from VPS26a, VPS26b, and combinations thereof.

51. The composition of claim 50, which comprises a first nucleic acid encoding VPS35 and a second nucleic acid encoding a core protein of the retromer complex selected from VPS26a, VPS26b, and combinations thereof.

52. A kit comprising the composition of any one of claims 30-51.

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