A method for detecting, preventing, promoting recovery, and treating nerve diseases

Abstract

Among various aspects of the present disclosure, methods are provided for detecting, preventing, ameliorating, treating, or delaying the onset of neurological disorders (e.g., adult-onset neurological disorders, Alzheimer's disease, Parkinson's disease, frontotemporal dementia).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority from U.S. Provisional Application No. 62 / 775,626, filed on December 05, 2018, which is hereby incorporated by reference in its entirety.

[0002] Description of Research or Development Sponsored by the Federal Government Not applicable

[0003] Incorporation by Reference of Materials Not applicable

[0004] The present disclosure generally relates to the treatment and detection of neurological diseases (e.g., adult - onset neurological diseases, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), etc.).

Summary of the Invention

Problems to be Solved by the Invention

[0005] Among various aspects of the present disclosure, methods are provided for detecting, preventing, treating, rehabilitating, or delaying the onset of neurological diseases (e.g., adult - onset neurological diseases, AD, PD, or FTD).

Means for Solving the Problems

[0006] One aspect of the present invention is a method of modulating the autophagy - lysosome pathway in a subject heterozygous for a lysosomal gene comprising a loss - of - function variant, the method comprising administering to the subject in need thereof a therapeutically effective amount of an autophagy - lysosome pathway modulator.

[0007] One aspect of the present invention is a method for preventing, treating, restoring, or delaying the onset of a nerve disease, disorder, or condition associated with lysosomal dysfunction in a subject, the method comprising detecting at least one lysosomal gene comprising a loss-of-function variant in a biological sample of the subject, or having detected the same, and administering a therapeutically effective amount of an autophagy-lysosome pathway regulator, wherein the subject is heterozygous for a lysosomal gene comprising a loss-of-function variant, or the subject is a carrier of a lysosomal storage disease (LSD).

[0008] One aspect of the present invention is a method for detecting at least one lysosomal gene loss-of-function variant in a subject, the method comprising providing a biological sample from the subject and detecting the presence of a lysosomal gene comprising a loss-of-function variant, and if at least one lysosomal gene comprising a loss-of-function variant is detected, determining that the subject has or is at risk of having a nerve or neurodegenerative disease, disorder, or condition associated with APP processing dysfunction. In some embodiments, the method comprises administering a therapeutically effective amount of an autophagy-lysosome pathway regulator, wherein the autophagy-lysosome pathway regulator is a treatment related to a lysosomal gene comprising a detected loss-of-function variant.

[0009] In some embodiments, the lysosomal gene comprising a loss-of-function variant is associated with a lysosomal storage disease (LSD).

[0010] In some embodiments, the subject is suspected of having or is at risk of having a nerve or neurodegenerative disease, disorder, or condition associated with lysosomal dysfunction.

[0011] In some embodiments, the subject is heterozygous for a lysosomal gene loss-of-function variant, or is a carrier of a lysosomal storage disease (LSD), or is suspected of being such.

[0012] In some embodiments, lysosomal genes including loss-of-function variants are selected from the group consisting of CTNS, MAN2B1, MFSD8, GLB1, GALNS, NAGLU, CLN3, GNPTAB, SGSH, CLN8, NPC1, TPP1, DNAJC5, MANBA, PPT1, SMPD1, GAA, HGSNAT, GNS, CTSA, HEXB, and combinations thereof.

[0013] In some embodiments, lysosomal genes including loss-of-function variants are associated with lysosomal storage diseases (LSDs) and are selected from the group consisting of AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0014] In some embodiments, lysosomal genes including loss-of-function variants are selected from the group consisting of NEU1, NAGLU, GBA, GLB1, MANBA, MAN2B1, HGSNAT, IDS, PPT1, GNS, and combinations thereof.

[0015] In some embodiments, lysosomal genes including loss-of-function variants are selected from the group consisting of GALC, ACD, and combinations thereof.

[0016] In some embodiments, the autophagy-lysosome pathway regulator includes gene therapy (GT), and GT increases or enhances the enzymatic activity associated with lysosomal genes comprising loss-of-function variants selected from the group consisting of AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0017] In some embodiments, the subject is treated with an autophagy-lysosome pathway regulator that modulates the expression of AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0018] In some embodiments, the subject is haploinsufficient for a lysosomal gene comprising a loss-of-function variant selected from the group consisting of AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof, or has a heterozygous variant in a lysosomal gene.

[0019] In some embodiments, the lysosomal gene comprising a loss-of-function variant is selected from rare functional variants in genes contributing to heparan sulfate (HS) metabolism in the group consisting of SGSH, NAGLU, HGSNAT, GNS, and combinations thereof.

[0020] In some embodiments, the loss-of-function variant is one or more variants selected from the group consisting of deletions, substitutions, or deletions of a lysosomal gene.

[0021] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with lysosomal dysfunction.

[0022] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with altered APP processing (e.g., changes in the levels of interstitial Aβ in the brain and increased amounts of Aβ plaques) or α-Syn aggregation.

[0023] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with Aβ accumulation.

[0024] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is Alzheimer's disease (AD).

[0025] In some embodiments, the autophagy-lysosome pathway modulator is an agent related to a lysosomal gene comprising a loss-of-function variant selected from the group consisting of chemical chaperone therapy (CCT), enzyme replacement therapy (ERT), gene therapy (GT), gene editing, hematopoietic stem cell transplantation (HSCT), chemical chaperone therapy (CCT), stop codon readthrough drugs, substrate reduction therapy (SRT), and combinations thereof.

[0026] In some embodiments, the method comprises supplementation with exogenous lysosomal proteins by enzyme replacement therapy (ERT), gene therapy (GT), or stem cell therapy.

[0027] In some embodiments, the autophagy-lysosome pathway modulator is a treatment related to a lysosomal gene comprising a loss-of-function variant, including cysteamine, cyclodextrin, or miglustat.

[0028] In some embodiments, compared to an untreated subject, Aβ, apoE, tau, or α-Syn aggregation is decreased in the subject, or Aβ, apoE, tau, or α-Syn clearance is enhanced.

[0029] In some embodiments, the subject has or is suspected of having dementia, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, motor neuron disease, polyglutamine disorder, Huntington's disease, familial amyloid polyneuropathy (FAP), Lewy body dementia, or multiple system atrophy.

[0030] Other objectives and features are in part obvious and in part pointed out below. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0032]

Fig. 1A-1C

Fig. 2

Fig. 3A-3B

Fig. 4A-4C

Fig. 5

Fig. 6

Fig. 7A-7E

Fig. 8A-8C

Fig. 9A-9D

Fig. 10A-10B

Fig. 12A-12D

Fig. 13A-13B

Fig. 14A-14B

Fig. 15

Fig. 16A-16B

Fig. 17A-17C

Fig. 18

Fig. 19

Fig. 20

Fig. 21A-21F

Fig. 22

Fig. 23

Fig. 24

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present disclosure is based, at least in part, on the discovery that subjects (carriers) who are heterozygous for a deleterious mutation in a lysosomal protein gene that causes a lysosomal storage disorder (LSD) (carriers of LSD, without symptoms of LSD) have an increased risk of developing Alzheimer's disease (AD). The current dogma is that heterozygous carriers of genes causing LSD, and LOF variants in many other genetic diseases, are considered normal and have no predisposition to any disease.

[0034] LSD patients are homozygous for the gene defect causing LSD (two alleles with a complete or nearly complete loss-of-function lysosomal gene variant) and survive within a range from infancy to 20 years, depending on the LSD. These LSD patients do not survive long enough to develop AD.

[0035] As described herein, the inventors have discovered that in subjects with AD, one or more variants (harmful mutations) in the genes that cause LSD are detected, enriched, and detected only in one allele (heterozygous). The inventors have discovered that these variants are loss-of-function variants in heterozygous subjects.

[0036] Currently, there are multiple treatment strategies available instead of treating lysosomal storage diseases (LSDs) (e.g., enzyme replacement therapy (ERT), gene therapy (GT), stem cell therapy (SCT) (e.g., hematopoietic stem cell transplantation), oral small molecule substrate inhibition therapy, small molecule chaperones, or pharmacological restoration of the autophagy-lysosome pathway), which can be used to treat AD patients with harmful heterozygous mutations in lysosomal proteins.

[0037] Heterozygous loss-of-function variants in lysosomal genes It is well known that subjects homozygous for loss-of-function variants (e.g., also known as harmful variants or mutations) in genes associated with LSDs cause severe lysosomal dysfunction. However, surprisingly, it has been discovered herein that the genomes of human AD patients are enriched for heterozygous complete or nearly complete loss-of-function variants in lysosomal genes (e.g., LSD carriers). Furthermore, it is shown herein that heterozygosity of harmful mutations in the lysosomal genes of mice that appear completely normal causes slight lysosomal dysfunction and directly affects normal APP processing.

[0038] A loss-of-function variant can be a complete loss-of-function variant, a near-complete loss-of-function variant, or a partial loss-of-function variant, as described herein. Thus, a loss-of-function variant is a variant in a lysosomal gene or an LSD-related gene that prevents the production or normal function of a lysosomal enzyme or other integral lysosomal protein. The variant can be a deletion, substitution, insertion, splice variant, promoter variant, a mutation that causes a change in stability, a frameshift or stop variant, a nonsense mutation, and / or any other mutation that adversely affects the normal function of the lysosomal gene or the resulting protein. For example, a complete, near-complete, or partial loss-of-function variant can cause an interference or reduction in the production or activity of the enzyme.

[0039] A complete loss-of-function (LOF) variant can be defined as a variant or variants that are expected to correlate with complete LOF of the affected transcript, e.g., a variant that results in a downstream premature stop codon or a larger deletion that removes more than 50% of the first exon or the protein-coding sequence of the affected transcript (haploinsufficiency) (MacArthur et al., 2012 Science 335, 823-828). A near-complete or partial LOF variant reduces gene activity but does not completely abolish gene activity.

[0040] As shown herein, it has been discovered that several genes, including variants associated with lysosomal storage diseases (LSDs), are strongly associated with Alzheimer's disease (AD) and Parkinson's disease (PD). This is a novel finding because while a subject needs to be homozygous for these mutations to develop an LSD, only heterozygosity is required for a subject to develop a disease associated with APP processing or mild lysosomal dysfunction, such as AD or PD.

[0041] This discovery is also important because it was previously not understood that being haploinsufficient for these genes and having reduced expression of these LSD proteins is associated with any other disease state. As noted above, it has long been believed that people (carriers) with heterozygous mutations that can cause LSD are not predisposed to any disease.

[0042] As shown herein, heterozygous deleterious mutations in lysosomal genes (e.g., genes associated with LSD or normal ALP function) are associated with AD. These genes can provide great insight into the mechanism of lysosomal dysfunction in the pathogenesis of AD, which is currently lacking and is knowledge that can lead to new therapeutic targets. The disclosed results can establish a basis for repurposing current treatment strategies that now exist in place of LSD for the potential treatment of AD. Surprisingly, the models of NAGU and PPT1 were not the ones in which genes with deleterious variants identified in AD were most significantly enriched (see, e.g., Table 13), but were shown to respond to LSD therapy. Thus, LSD treatment provided to subjects with deleterious heterozygous variants (or complete or nearly complete loss-of-function variants) for other identified LSD-causing genes that are significantly enriched in AD would be predicted to respond at least similarly, or better.

[0043] It has been discovered herein that 45 lysosomal enzyme genes with complete or nearly complete loss-of-function heterozygous variants are enriched in AD and PD patients. The following are known LSD-related genes: AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, or GRN. Assays for measuring enzyme deficiencies associated with these genes and genotyping variants of these genes are well known in the art.

[0044] A population of subjects having a deletion in an ALP-related gene may also have lysosomal dysfunction. Currently, 453 known lysosomal genes exist. Thus, LOF variants in the following genes may cause lysosomal dysfunction and may be treatable with the therapies described herein. ABCA2, ABCA3, ABCA5, ABCB9, ABCC10, ACP2, ACP5, ACPP, ADA, ADAM8, ADRB2, AGA, AHNAK, ALDOB, ANKFY1, ANKRD27, ANPEP, ANXA11, AP1B1, AP1G1, AP1M1, AP1M2, AP1S1, AP1S2, AP1S3, AP3B1, AP3B2, AP3D1, AP3M1, AP3M2, AP3S1, AP3S2, AP4B1, AP4E1, AP4M1, AP4S1, AQP2, ARF1, ARL8A, ARL8B, ARRB1, ARSA, ARSB, ARSD, ARSG, ASAH1, ASS1, ATP11A, ATP11C, ATP13A2, ATP6AP1, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD164, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, CTSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, CTSZ, CUBN, CXCR2, CYBASC3, DAGLB, DEPDC5, DKFZp761E198, DNAJC13, DNAJC5, DNAJC6, DNASE1, DNASE2, DNASE2B, DNM2, DOC2A, DPP4,DPP7, DRAM1, DRAM2, ECE1, EGF, ELANE, ENPEP, ENPP1, ENTPD4, EPDR1, FAM176A, FGFR3, FLOT1, FLOT2, FNBP1, FUCA1, FUCA2, GAA, GABARAP, GALC, GALNS, GBA, GC, GDAP2, GGA1, GGA2, GGA3, GGH, GJA1, GLA, GLB1, GM2A, GNA11, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR137, GPR137B, GPR143, GRN, GUSB, HEXA, HEXB, HGSNAT, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HPS1, HPS4, HPSE, HSPA8, HYAL1, HYAL2, HYAL3, IDS, IDUA, IFI30, IGF2R, IL4I1, ITM2C, KCNE1, KCNE2, KIAA0226, KIAA0415, KIAA1609, LAMP1, LAMP2, LAMP3, LAMTOR1, LAMTOR2, LAPTM4A, LAPTM4B, LAPTM5, LDLR, LGMN, LHCGR, LIPA, LITAF, LMBRD1, LNPEP, LOC653653, LRBA, LRP1, LRP2, M6PR, MAN2B1, MAN2B2, MANBA, 1-Mar, 2-Mar, 3-Mar, 8-Mar, 9-Mar, MCOLN1, MCOLN2, MCOLN3, MFSD1, MFSD8, MIOS, MMD, MON1B, MPO, MTOR, MYLPF, MYO7A, NAAA, NAGA, NAGLU, NAGPA, NAPA, NAPG, NAPSA, NBR1, NCSTN, NEU1, NEU4, NPC1, NPC2, NPPA, NSF, OCA2, OSTM1, P2RX4, P2RY2, PCSK9, PCYOX1, PEBP4, PGCP, PI4K2A, PLA2G15, PLA2G4E, PLA2G4F, PLBD1, PLBD2, PLD1, PLD3, PLEKHF1, PLOD1, PNPLA7, PON2, PPT1, PPT2PRCP, PRDX6, PRF1, PRTN3, PSAP, PSAPL1, PSEN1, PSEN2, PTGDS, RAB14, RAB27A, RAB2A, RAB5C, RAB7A, RAB7B, RAB9A, RAMP2, RAMP3, RDH14, RILP, RNASE1, RNASE2, RNASE6, RNASET2, RNF13, RNF152, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, SCARB1, SCARB2, SCPEP1, SELRC1, SERINC2, SFTPB, SFTPD, SGSH, SH3GL2, SIAE, SIDT2, SLC11A1, SLC11A2, SLC12A4, SLC15A3, SLC15A4, SLC17A5, SLC26A11, SLC29A3, SLC2A13, SLC2A8, SLC30A2, SLC36A1, SLC37A3, SLC44A2, SLC48A1, SMCR8, SMPD1, SMPD4, SMPDL3A, SNAP23, SNX16, SORT1, SPACA3, SPG11, SPHK2, SPNS1, SPPL2A, SRGN, STARD3, STARD3NL, STS, STX3, STX7, STXBP2, SUMF1, TCIRG1, TIAL1, TLR3, TLR7, TLR9, TM9SF1, TMBIM1, TMEM127, TMEM175, TMEM192, TMEM55A, TMEM55B, TMEM63A, TMEM74, TMEM8A, TMEM9, TMEM92, TMEM97, TOM1L1, TPCN1, TPCN2, TPP1, TRIM23, TRIP10, TSPAN1, TSPAN8, TXNDC5, TYR, UBA52, UNC13D, UNC93B1, USP4, USP5, USP6, UVRAG, VAMP4, VAMP7, VASN, VMA21, VPS11, VPS16, VPS18, VPS33A, VPS33B, VPS35, VPS36, VPS39, VPS41, VPS4B, WDR11, WDR41, WDR48, ZFYVE26, ZNRF1, or ZNRF2.,

[0045] Neurological diseases, disorders, and conditions associated with autophagy-lysosome pathway (ALP) dysfunction It has been discovered herein that a subject (carrier) heterozygous for a complete or nearly complete loss-of-function variant, typically associated with lysosomal storage disease (LSD), can cause ALP dysfunction or defects in APP processing (when the subject is homozygous for these gene variants). Harmful variants in lysosomal genes can cause recessive lysosomal dysfunction, which can, in particular, cause altered APP processing (e.g., changes in the levels of interstitial brain Aβ and increased amounts of Aβ plaques) or α-Syn aggregation in subjects with Alzheimer's disease (AD) and Parkinson's disease (PD). This is particularly evident when growing on mouse models of AD or PD with heterozygous complete or nearly complete loss-of-function mutations in lysosomal genes (NAGLU, PPT1, or Csp-α). In this case, the heterozygous complete or nearly complete loss-of-function mutation significantly exacerbates Aβ plaque formation or α-synuclein aggregation. Thus, it has been discovered herein that recessive lysosomal dysfunction can cause neurological diseases, disorders, or conditions. Other neurological diseases, disorders, or conditions associated with lysosomal dysfunction can be characterized by hereditary cerebral amyloid angiopathy, stroke, and conditions characterized by a decline in intellectual function (dementia), Creutzfeldt-Jakob disease, motor neuron diseases, polyglutamine disorders, such as Huntington's disease, and diseases of peripheral tissues, such as familial amyloid polyneuropathy (FAP), Lewy body dementia, multiple system atrophy, or frontotemporal dementia.

[0046] The present disclosure provides methods for treating a subject (e.g., a carrier) heterozygous for a gene variant associated with lysosomal storage disease (LSD) and for treating or preventing a neurological or neurodegenerative disease, disorder, or condition associated with the autophagy-lysosome pathway (ALP).

[0047] For example, Table 13 describes genes having variants found in genes that cause LSD in subjects with AD. If a subject is homozygous for these genes, they would have developed LSD. However, surprisingly, as discovered herein, heterozygosity is herein associated as predictive of future risk of being diagnosed with a neurological or neurodegenerative disease, disorder, or condition associated with the autophagy-lysosome pathway, such as AD or PD.

[0048] Accordingly, the disclosed methods of diagnosing and treating subjects having these heterozygous complete or near-complete loss-of-function lysosomal gene variants can be used to detect or treat neurological disease states associated with the autophagy-lysosome pathway. For example, the method can be used in subjects having or suspected of having a neurological disease, disorder, or condition, such as any neurological or neurodegenerative disease (e.g., AD, PD, FTD, etc.) associated with a lysosomal or autophagy dysfunction.

[0049] Other neurological or neurodegenerative diseases, disorders, or conditions in subjects heterozygous for a lysosomal storage disease (LSD)-associated or deleterious lysosomal gene having a complete or near-complete loss-of-function variant can be treated by the methods described herein.

[0050] Lysosomal storage disease (LSD) Lysosomal storage diseases (LSDs) or disorders are characterized by homozygous complete or nearly complete loss-of-function gene variants in autophagy-lysosome pathway genes that cause a decrease or complete loss of lysosomal proteins or lysosomal protein function that are essential for the degradation pathway of macromolecules in lysosomes. As described herein, it has been discovered that subject heterozygous complete or nearly complete loss-of-function lysosomal gene variants are at risk of developing a neurological disease associated with APP processing dysfunction (AD) or a neurological disease associated with α-synuclein aggregation (PD). Thus, in the newly discovered neurological lysosome-related diseases, treatment of diseases caused by heterozygous loss-of-function variants can be carried out.

[0051] LSDs are a group of at least 50 hereditary diseases characterized by the total or partial absence of one specific lysosomal protein involved in the degradation pathway of macromolecules in lysosomes. They are single genes and, in most cases, numerous mutations have been described. Some mutations cause a complete loss of protein function, while others only reduce normal function. The storage of undegraded or partially degraded substances, usually substrates of defective lysosomal enzymes, occurs within lysosomes. Conventionally, LSDs are grouped based on the chemical nature of the accumulating undegraded substrates, including mucopolysaccharidoses, lipidoses, glycogenoses, and oligosaccharidoses.

[0052] Despite the diversity of symptoms, most of these diseases are characterized by a progressive course with significant variation between different diseases and among patients with the same disease, but with an increased incidence and mortality. Generally, these diseases are multi-systemic and clinical features include organomegaly, central nervous system dysfunction, and coarse hair. Most patients are asymptomatic at birth and present onset in childhood. Their frequencies vary in different regions and populations, are rare individually, and the total estimated prevalence ranges from 1 in 4000 to 1 in 9000 live births. Interestingly, most pediatric LSDs have a significant neurological component.

[0053] Some of these diseases have not had specific therapies to date, but for some LSDs, hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), gene therapy (GT), and small molecule drugs are available or in clinical trials.

[0054] Autophagy-lysosome pathway enhancer: Treatment and therapy of lysosomal storage diseases The present disclosure provides for the identification and treatment of subjects heterozygous for a complete or nearly complete loss-of-function lysosomal gene associated with an LSD disease, disorder, or condition. These heterozygous subjects are at high risk of having or developing a neurological or neurodegenerative disease, disorder, or condition associated with the autophagy-lysosome pathway (ALP). The present LSD therapies and treatments are shown herein for treating or preventing neurological or neurodegenerative diseases, disorders, or conditions associated with ALP in animal models by rescuing or enhancing autophagy-lysosome pathway (ALP) function.

[0055] Treatments for LSDs, such as gene therapy and enzyme replacement therapy, have been shown to treat LSDs in homozygous animal models of the disease and in human patients. Accordingly, it would be expected that these treatments would be even more effective and more amenable to treating the disease in the heterozygous population compared to the homozygous population. In other words, since the dysfunction associated with the heterozygous population (e.g., AD and PD) is milder compared to LSD patients, the threshold for treatment efficacy would be even lower in the heterozygous population compared to the homozygous population.

[0056] Treatments (LSD therapeutics) and treatment methods for LSDs are well known. See, for example, Ohashi 2018 Gene therapy for lysosomal storage diseases and peroxisomal diseases Journal of Human Genetics (2019) 64:139 - 143, Beck 2017 Treatment strategies for lysosomal storage disorders, Dev Med & Child Neuro, 13 - 18, Ferreira and Gahl 2017 Lysosomal storage diseases, Translational Science of Rare Diseases 2(1 - 2)1 - 71, Platt 2017 Emptying the stores: lysosomal diseases and therapeutic strategies, Nature Reviews Drug Discovery 17 133 - 150, Marques and Saftig 2019 Lysosomal storage disorders - challenges, concepts and avenues for therapy: beyond rare diseases J Cell Sci 132 jcs221739. Accordingly, unless otherwise stated herein, the treatments and treatment methods of the present disclosure can be carried out according to such processes. [Table 1]

Table 2

[0057] Since these and other therapies for LSD have already been found to be effective in homozygous subjects, it would be expected that they would also be effective in heterozygous subjects.

[0058] Substrate reduction therapy (SRT) In metabolic or genetic pathways, enzymes catalyze a series of reactions. Each enzyme is regulated or mediated by genes via its RNA and protein products. At each step in the pathway, enzyme activity catalyzes the reaction in which a precursor molecule (substrate) is converted to its next intermediate state. Failure of a metabolic pathway causes accumulation of the substrate, which can have harmful effects. Substrate reduction therapy addresses this failure by reducing the level of the substrate to a point where the remaining catabolic activity is sufficient to prevent substrate accumulation.

[0059] The theoretical basis behind substrate reduction therapy is a reduction in the formation of lysosomal material such that the remaining enzyme activity is decreased to the rate at which it can catabolize incoming lysosomal material. Examples of SRT can include miglustat (Zavesca) or eliglustat (Cerdelga).

[0060] Hematopoietic stem cell transplantation (HSCT) In HSCT, bone marrow-derived stem cells or umbilical cord blood from a healthy donor are transplanted. Its effectiveness has been shown as evidence to depend not only on the migration of donor cells into the bone marrow and the reconstitution of the blood system, but also on the migration of the subsequently engrafted cells into many disease target organs including the brain, where they replenish the population of resident enzyme deficiencies and thus serve as a local and stable source of functional enzymes. This is further enhanced by a process generally referred to as "cross-correction". Cross-correction is a process by which lysosomal enzymes can be secreted from one cell (in this case, donor hematopoietic cells) and taken up by adjacent cells (hematopoietic or non-hematopoietic-derived) by a receptor-mediated process. In many cases, sufficient enzyme is shared to completely correct the biochemical defect associated with a homozygous complete or nearly complete loss-of-function mutation. When successful, HSCT can extend the patient's lifespan, maintain neurocognition, and enhance somatic changes. Disadvantages of HSCT include significant risks associated with this procedure, such as the possibility of developing graft-versus-host disease, the difficulty of finding an HLA-compatible donor, and the occurrence of chimerism. Therefore, in many countries, its use has been postponed in favor of ERT whenever possible.

[0061] Enzyme replacement therapy (ERT) In ERT, the missing recombinant enzyme is administered to the patient by repeated intravenous injection. In this case, the recombinant enzyme is taken up by cells by a process mediated by the same receptors involved in "cross-correction". Despite being an effective and safe treatment option for various LSDs, ERT also has important limitations. These include adverse reactions presented by some patients, the high cost of treatment, the lifelong dependence on long infusions of 4 to 5 hours per week, and the limited ability to correct neurological and skeletal lesions.

[0062] Gene therapy and genome editing Gene therapy can involve inserting functional genes using viral vectors. Gene therapy for lysosomal storage diseases (LSDs) is rapidly evolving. Most LSDs are characterized by brain involvement, which promotes the development of therapies targeting the brain. There are two types of gene therapy for brain involvement in LSDs: direct transfer of therapeutic genes into brain cells and gene therapy targeting ex vivo hematopoietic stem cells. The rationale for the latter approach is that brain microglia are derived from hematopoietic cells. Thus, gene-corrected hematopoietic cells migrate to the brain and differentiate into microglial cells. These gene-corrected microglial cells cross-correct the metabolic defects associated with LSDs, reduce inflammation in LSDs, and provide clinical benefit. Gene editing technologies are also applied in this field, and clinical trials focusing on LSDs are currently underway (see, for example, de Carvalho et al. 2015 Genome Editing: Potential Treatment for Lysosomal Storage Diseases Current Stem Cell Reports 1(1)9-15). These approaches are still under investigation but have yielded very promising results. Currently, there are several approved gene therapies on the market, including AAV / LPL (Glyvera) for lipoprotein lipase deficiency, retrovirus / ADA (Strimvelis) for adenosine deaminase deficiency, and AAV / RPE65 (Luxturna) for Leber congenital amaurosis.

[0063] Recently, there has been an improved outlook for gene therapy. For example, in the first quarter of 2019, there were 372 ongoing gene therapy clinical trials (Alliance for Regenerative Medicine, 5 / 9 / 19).

[0064] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or hybrids thereof. [Table 3]

[0065] Gene therapy can enable continuous direct delivery of an enzyme to the target organ, eliminating the need for weekly injections. Also, correction of some cells may lead to the enzyme being secreted into the circulation and taken up by their neighboring cells (cross-collection), resulting in correction over a wide range of biochemical defects. Therefore, the number of cells that need to be modified with a gene transfer vector is relatively small. Furthermore, overexpression of lysosomal enzymes does not seem to be harmful, and as little as 5 - 10% of normal levels of the enzyme can be a therapeutic agent for some LSDs, so precise transcriptional regulation may not be necessary.

[0066] Genetic modification can be performed either ex vivo or in vivo. Ex vivo strategies are based on modification of cells in culture and transplantation of the modified cells into the patient. The cells most commonly considered as therapeutic targets for single-gene diseases are stem cells. Advances in the collection and isolation of these cells from various sources have promoted autologous gene therapy as a viable option for LSDs. In a mouse model of LSD, genetically modified neural stem cells encoding the enzyme gene effectively reduced lysosomal storage in the animals, decreased lesions, and extended lifespan. Mesenchymal stem cells and induced pluripotent stem cells (iPSCs) have also been used for this purpose. However, conventional gene therapy protocols may have limitations, particularly safety issues related to the immune response and, in the case of viral vectors, the potential for insertional mutagenesis, as well as low efficiency with non-viral vectors.

[0067] The use of endonucleases for targeted genome editing can address the limitations presented by conventional gene therapy protocols. These enzymes are custom molecular scissors that enable the cutting of DNA into precisely defined and fully specified segments in virtually all cell types. Furthermore, these enzymes can be delivered to cells by plasmids that transiently express the nuclease or by transcribed RNA that avoids the use of viruses.

[0068] Combination therapy Combinations of treatment approaches have been shown to be effective in LSDs. For example, the inventors have previously shown that a combination of gene therapy, HST transplantation, and small molecule substrate inhibition was most effective in the treatment of Krabbe disease. Thus, similar combination approaches are also expected to be most effective in subjects with neurological diseases associated with lysosomal dysfunction such as AD or PD. However, as disclosed earlier herein, the heterozygous population is expected to be more amenable to treatment and have a lower threshold for treatment efficacy because the dysfunction associated with the heterozygous population is expected to be much less than that of the heterozygous LSD population.

[0069] As another example, gene therapy can be combined with HSCT. Hematopoietic stem cells extracted from a patient can be transfected with a vector encoding an endonuclease designed to cut at a site near a specific mutation and a donor vector. However, the donor vector will contain regions homologous to the mutated region with the correct nucleotide sequence and will function as a template for the repair of DNA damage after double-strand break. Cells that internalize the two vectors where cleavage and homologous recombination occur will have the correct gene sequence and can be selected and transplanted back into the patient. Combining autologous HSCT with nuclease-mediated genome editing would have the advantage of a low risk of infection during the patient's treatment due to the rapid recovery of immune function. Also, since the donor and recipient are the same individual, the onset of rejection (graft-versus-host disease) will be avoided. Corrected hematopoietic stem cell (HSC) therapy may include steps where hematopoietic stem cells extracted from a patient can be transfected with a vector encoding a modified endonuclease and a donor vector for inducing homologous recombination. The corrected cells can then be selected ex vivo and transplanted back into the patient.

[0070] Personalized / Precision Medicine The discovery that heterozygosity for lysosomal gene defects causes changes in APP processing and increased Aβ plaque deposition enables a personalized therapeutic approach. Methods for detecting complete or nearly complete loss-of-function variants in lysosome-related genes can be used as a basis for treating them based on that information (see, for example, Tables 1, 2, and 3).

[0071] Here, it becomes possible to identify and treat subjects at risk of neurological diseases associated with lysosomal dysfunction based on the detected lysosomal gene variants. Defects in lysosomal genes and treatment of LSDs are well known and have been shown to be effective in rescuing the effects of neurological diseases associated with lysosomal dysfunction (e.g., AD).

[0072] Together with the evidence provided herein, the current treatment of LSD in homozygous children with LSD is expected to act on at-risk carriers (heterozygotes) as well. This is because both involve the autophagy-lysosome pathway and the efficacy threshold may be much lower for heterozygous subjects.

[0073] Autophagy-lysosome pathway (ALP) Methods are described herein for modulating the autophagy-lysosome pathway (ALP) for the treatment of neurological or neurodegenerative diseases, disorders, or conditions.

[0074] The autophagy-lysosome pathway (ALP) is a major pathway for the degradation of intracellular organelles and aggregation-prone proteins. Autophagy ("self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients via lysosomes. There is increasing evidence that lysosomal dysfunction may play a role in several neurodegenerative diseases, most notably Alzheimer's disease (AD) and Parkinson's disease (PD). Loss-of-function variants in lysosomal genes may also be associated with cases of dementia of unknown etiology.

[0075] As described herein, the human genome contains at least 430 genes related to ALP (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Individuals with heterozygous deleterious variants within autophagy-lysosome pathway-related genes are at risk of developing common adult-onset neurological diseases (e.g., Alzheimer's disease, Parkinson's disease, frontotemporal dementia, etc.). Furthermore, supplementation of exogenous lysosomal proteins by enzyme replacement therapy (ERT), gene therapy (GT), stem cell therapy, etc. can slow the progression of these diseases.

[0076] As described herein, by identifying specific defects within the ALP gene in PD and AD, multiple treatment strategies can be utilized for the potential treatment of PD and AD, and other neurological or neurodegenerative diseases and disorders, including gene therapy, enzyme replacement, oral small molecule substrate inhibition therapy, small molecule chaperones, and pharmacological restoration of the autophagy pathway. As described herein, the expression of genes related to ALP can be regulated for the treatment of neurological or neurodegenerative diseases or disorders. Protein products derived from genes related to ALP can also be supplemented by enzyme replacement therapy (ERT).

[0077] LSD-related genes can include, but are not limited to, the following. AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, or GRN. For example, LSD-related genes correlated with adult-onset neurodegenerative diseases can include, but are not limited to, the following. ASAH1, CLN3, CLN8, CSTD, CTNS, CTSA CTSF, DNAJC5, GAA, GALC, GALNS, GBA, GLA, GLB1, GNPTAB, GNS, GRN, HEXB, HGSNAT, IDS, IDUA, MAN2B1, MANBA, MFSD8, NAGLU, NEU1, NPC1, NPC2, PLD3, PPT1, SGSH, SMPD1, SORL1, and TPP1.

[0078] Genes related to ALP may include, but are not limited to, the following: ABCA2, ABCA3, ABCA5, ABCB9, ABCC10, ACP2, ACP5, ACPP, ADA, ADAM8, ADRB2, AGA, AHNAK, ALDOB, ANKFY1, ANKRD27, ANPEP, ANXA11, AP1B1, AP1G1, AP1M1, AP1M2, AP1S1, AP1S2, AP1S3, AP3B1, AP3B2, AP3D1, AP3M1, AP3M2, AP3S1, AP3S2, AP4B1, AP4E1, AP4M1, AP4S1, AQP2, ARF1, ARL8A, ARL8B, ARRB1, ARSA, ARSB, ARSD, ARSG, ASAH1, ASS1, ATP11A, ATP11C, ATP13A2, ATP6AP1, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD164, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, CTSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, CTSZ, CUBN, CXCR2, CYBASC3, DAGLB, DEPDC5, DKFZp761E198, DNAJC13, DNAJC5, DNAJC6, DNASE1, DNASE2, DNASE2B, DNM2, DOC2A, DPP4, DPP7, DRAM1, DRAM2, ECE1, EGF, ELANE, ENPEP, ENPP1, ENTPD4, EPDR1, FAM176A, FGFR3, FLOT1, FLOT2, FNBP1, FUCA1,FUCA2, GAA, GABARAP, GALC, GALNS, GBA, GC, GDAP2, GGA1, GGA2, GGA3, GGH, GJA1, GLA, GLB1, GM2A, GNA11, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR137, GPR137B, GPR143, GRN, GUSB, HEXA, HEXB, HGSNAT, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HPS1, HPS4, HPSE, HSPA8, HYAL1, HYAL2, HYAL3, IDS, IDUA, IFI30, IGF2R, IL4I1, ITM2C, KCNE1, KCNE2, KIAA0226, KIAA0415, KIAA1609, LAMP1, LAMP2, LAMP3, LAMTOR1, LAMTOR2, LAPTM4A, LAPTM4B, LAPTM5, LDLR, LGMN, LHCGR, LIPA, LITAF, LMBRD1, LNPEP, LOC653653, LRBA, LRP1, LRP2, M6PR, MAN2B1, MAN2B2, MANBA, 1-Mar, 2-Mar, 3-Mar, 8-Mar, 9-Mar, MCOLN1, MCOLN2, MCOLN3, MFSD1, MFSD8, MIOS, MMD, MON1B, MPO, MTOR, MYLPF, MYO7A, NAAA, NAGA, NAGLU, NAGPA, NAPA, NAPG, NAPSA, NBR1, NCSTN, NEU1, NEU4, NPC1, NPC2, NPPA, NSF, OCA2, OSTM1, P2RX4, P2RY2, PCSK9, PCYOX1, PEBP4, PGCP, PI4K2A, PLA2G15, PLA2G4E, PLA2G4F, PLBD1, PLBD2, PLD1, PLD3, PLEKHF1, PLOD1, PNPLA7, PON2, PPT1, PPT2, PRCP, PRDX6, PRF1, PRTN3, PSAP, PSAPL1, PSEN1, PSEN2, PTGDS, RAB14, RAB27A, RAB2A, RAB5C, RAB7A, RAB7B, RAB9A,RAMP2, RAMP3, RDH14, RILP, RNASE1, RNASE2, RNASE6, RNASET2, RNF13, RNF152, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, SCARB1, SCARB2, SCPEP1, SELRC1, SERINC2, SFTPB, SFTPD, SGSH, SH3GL2, SIAE, SIDT2, SLC11A1, SLC11A2, SLC12A4, SLC15A3, SLC15A4, SLC17A5, SLC26A11, SLC29A3, SLC2A13, SLC2A8, SLC30A2, SLC36A1, SLC37A3, SLC44A2, SLC48A1, SMCR8, SMPD1, SMPD4, SMPDL3A, SNAP23, SNX16, SORT1, SPACA3, SPG11, SPHK2, SPNS1, SPPL2A, SRGN, STARD3, STARD3NL, STS, STX3, STX7, STXBP2, SUMF1, TCIRG1, TIAL1, TLR3, TLR7, TLR9, TM9SF1, TMBIM1, TMEM127, TMEM175, TMEM192, TMEM55A, TMEM55B, TMEM63A, TMEM74, TMEM8A, TMEM9, TMEM92, TMEM97, TOM1L1, TPCN1, TPCN2, TPP1, TRIM23, TRIP10, TSPAN1, TSPAN8, TXNDC5, TYR, UBA52, UNC13D, UNC93B1, USP4, USP5, USP6, UVRAG, VAMP4, VAMP7, VASN, VMA21, VPS11, VPS16, VPS18, VPS33A, VPS33B, VPS35, VPS36, VPS39, VPS41, VPS4B, WDR11, WDR41, WDR48, ZFYVE26, ZNRF1, or ZNRF2.,

[0079] Molecular engineering The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in its practice. Unless otherwise specified, terms should be understood according to their conventional use by those skilled in the relevant art.

[0080] The terms "heterologous DNA sequence", "exogenous DNA segment" or "heterologous nucleic acid", as used herein, each refer to a sequence that is derived from a source foreign to a particular host cell or, if from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell but has been modified, for example, through the use of DNA shuffling. These terms also include multiple non-naturally occurring copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to the cell or that is homologous to the cell but is in a position within the host cell nucleic acid where the element is not normally found. An exogenous DNA segment is expressed to obtain an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence that naturally associates with the host cell into which it is introduced.

[0081] An expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid generated through human intervention having a series of specific nucleic acid elements that enable the transcription or translation of a particular nucleic acid (e.g., in a host cell), including recombinant means or direct chemical synthesis. An expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, an expression vector can contain a nucleic acid to be transcribed operably linked to a promoter.

[0082] A "promoter" is generally understood to be a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood to be a promoter that mediates the transcription of a gene operably linked in response to a specific stimulus. A promoter can include the nucleic acid sequences necessary near the transcription start site, for example, in the case of a polymerase II type promoter, the TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located several thousand base pairs from the transcription start site.

[0083] As used herein, a "transcribable nucleic acid molecule" refers to any nucleic acid molecule that can be transcribed into an RNA molecule. Methods are known for introducing constructs into cells in such a manner that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated to be expressed as a protein product. The construct can also be constructed to express an antisense RNA molecule to inhibit the translation of a particular RNA molecule of interest. Conventional compositions and methods for preparing and using constructs and host cells for the practice of the present disclosure are well known to those of skill in the art (see, for example, Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717, Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929, Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773, Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754).

[0084] The "transcription start site" or "start site" is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. All other sequences of the gene and its regulatory regions can be numbered with respect to this site. Downstream sequences (i.e., additional protein-coding sequences in the 3' direction) can be referred to as positive, while upstream sequences (most of the regulatory regions in the 5' direction) can be referred to as negative.

[0085] "Operably linked" or "functionally linked" preferably refers to the association of nucleic acid sequences to a single nucleic acid fragment such that one function is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked" or "associated" with a DNA sequence encoding an RNA or polypeptide when the two sequences are arranged such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of a promoter). The coding sequence can be operably linked to the regulatory sequence in the sense or antisense direction. The two nucleic acid molecules may be part of a single continuous nucleic acid molecule or may be adjacent. For example, a promoter is operably linked to a gene of interest within a cell when the promoter regulates or mediates the transcription of the gene of interest.

[0086] A "construct" is generally understood to be any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, that is derived from any source, is capable of genomic integration or autonomous replication, and contains nucleic acid molecules in which one or more nucleic acid molecules are operably linked.

[0087] The constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. Additionally, the construct can include, without limitation, additional regulatory nucleic acid molecules, for example, from the 3'-untranslated region (3'UTR). The construct can include, without limitation, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule that can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules can be derived from a source that is native or heterologous with respect to other elements present on the promoter construct.

[0088] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell that results in genetically stable inheritance. A host cell containing the transformed nucleic acid fragment is referred to as a "transgenic" cell, and an organism containing transgenic cells is referred to as a "transgenic organism".

[0089] "Transformed", "transgenic", and "recombinant" refer to a host cell or organism into which a heterologous nucleic acid molecule has been introduced, such as a bacterium, cyanobacterium, animal, or plant. Nucleic acid molecules are generally known in the art and, as disclosed, can be stably integrated into the genome (Sambrook 1989, Innis 1995, Gelfand 1995, Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc. The term "untransformed" refers to normal cells that have not experienced the transformation process.

[0090] "Wild type" refers to a virus or organism found in nature that has no known mutations.

[0091] The design, generation, and testing of variant nucleotides having the required percent identity described above and retaining the required activity of the expressed protein are within the scope of the art. For example, directed evolution and rapid isolation of variants can follow methods described in the literature including, but not limited to, Link et al. (2007) Nature Reviews 5(9),680-688, Sanger et al. (1991) Gene 97(1),119-123, Ghadessy et al. (2001) Proc Natl Acad Sci USA 98(8)4552-4557. Thus, one of ordinary skill in the art can generate, for example, a number of nucleotide and / or polypeptide variants having at least 95-99% identity to the reference sequences described herein and screen for the desired phenotype according to routine methods in the art.

[0092] The percent identity (%) of a nucleotide and / or amino acid sequence is understood as the percentage of nucleotide or amino acid residues in a candidate sequence that are identical to the nucleotide or amino acid residues in a reference sequence when the two sequences are aligned. To determine percent identity, the sequences are aligned and gaps are introduced as necessary to achieve the maximum percent sequence identity. Sequence alignment procedures for determining percent identity are well known to those of skill in the art. In many cases, publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align the sequences. Those of skill in the art can determine appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment over the full length of the sequences being compared. When sequences are aligned, the percent sequence identity of a given sequence A with respect to, or with, or to a given sequence B (or can be expressed as a given sequence A having, or including, a particular percent sequence identity with respect to, or with, or to a given sequence B) can be calculated as percent sequence identity = X / Y×100, where X is the number of residues scored as exact matches by the alignment of the alignment program or algorithm of A and B, and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A with respect to B is not equal to the percent sequence identity of B with respect to A.

[0093] Generally, conservative substitutions can be made at any position as long as the required activity is retained. So-called conservative exchanges can be made in which the replaced amino acid has similar properties to the original amino acid, for example, the exchanges of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine), hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine), cyclic amino acids (e.g., proline), aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan), basic amino acids (e.g., histidine, lysine, arginine), or acidic and their amides (e.g., aspartate, glutamate, asparagine, glutamine). Deletion is the replacement of an amino acid by a direct bond. Positions for deletion include the ends of the polypeptide and the bonds between individual protein domains. Insertion is the introduction of an amino acid into the polypeptide chain in which a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known to those skilled in the art that can produce, for example, polypeptides with improved activity or altered regulation. Based on this artificially generated polypeptide sequence, the corresponding nucleic acid molecule encoding such an adjusted polypeptide can be synthesized in vitro using the specific codon usage of the desired host cell.

[0094] "Very stringent hybridization conditions" are defined as hybridization at 65 °C in 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Considering these conditions, for a given set of sequences, the melting point (T mBy calculating [[ID=]], a determination can be made as to whether hybridization occurs. If a particular double-strand has a melting point of less than 65 °C under the salt conditions of 6×SSC, the two sequences do not hybridize. On the other hand, if the melting point exceeds 65 °C under the same salt conditions, the sequences hybridize. Generally, the melting point of any hybridized DNA:DNA sequence can be determined using the following formula. T m = 81.5 °C + 16.6(log 10 [Na + ) + 0.41 (fraction G / C content) - 0.63 (% formamide content) - (600 / l). Furthermore, the T m of a DNA:DNA hybrid decreases by 1 to 1.5 °C for every 1% decrease in nucleotide identity (see, for example, Sambrook and Russel, 2006).

[0095] Host cells can be transformed using a variety of standard techniques known in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717, Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929, Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773, Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, and the like. Transfected cells can be selected and propagated to provide recombinant host cells containing an expression vector stably integrated into the host cell genome.

Number

[0096] Exemplary nucleic acids that can be introduced into a host cell include, for example, a DNA sequence or gene from another species, or a gene or sequence that is from the same species or present within the same species but is incorporated into the recipient cell by genetic engineering methods. The term "exogenous" also refers to a gene that is not normally present in the cell being transformed, or perhaps a gene that simply does not exist in the form, structure, etc. as found in the transformed DNA segment or gene, or a gene that is normally present and is desired to be expressed (e.g., overexpressed) in a manner different from its natural expression pattern. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The types of DNA included in the exogenous DNA can include DNA already present in the cell, DNA from another individual of the same type of organism, DNA from different organisms, or externally generated DNA, for example, a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified form of a gene.

[0097] Host strains developed according to the approaches described herein can be evaluated by several means known in the art (see, for example, Studier (2005) Protein Expr Purif. 41(1), 207 - 234, Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley - VCH, ISBN - 10: 3527310363, Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN - 10: 0954523253).

[0098] Methods for down-regulating or silencing genes are known in the art. For example, the activity of an expressed protein can be down-regulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), and microRNAs (miRNAs) (see, e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, which describes hammerhead ribozymes and short hairpin RNAs; Helene, C., et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36, which describes target deoxyribonucleotide sequences; Maher (1992) Bioassays 14(12):807-15; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, which describes aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326-330, which describes RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, which describes RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, which describes RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423, which describes RNAi). RNAi molecules are commercially available from a variety of sources (e.g., (Ambion, TX, Sigma Aldrich, MO, Invitrogen).Several siRNA molecule design programs that use various algorithms are known in the art (see, e.g., Cenix algorithm, Ambion, BLOCK-iT™ RNAi Designer, Invitrogen, siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits that affect in defining the optimal siRNA sequence include the G / C content at the ends of the siRNA, the Tm of specific internal domains of the siRNA, the siRNA length, the position of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0099] Genome editing Recent advances in genome editing technologies using engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system have enabled the precise modification of target sites in the genome. This technology offers hope for the treatment of many genetic diseases. Here, targeting genome editing can be used or combined with hematopoietic stem cell transplantation and other approaches used for the treatment of subjects heterozygous for loss-of-function lysosomal gene variants.

[0100] As described herein, enzyme activity can be enhanced or increased using genome editing. The process for genome editing is well known. See, e.g., Aldi 2018 Nature Communications 9(1911). Thus, unless otherwise described herein, the processes of the present disclosure can be carried out in accordance with such processes.

[0101] For example, genome editing can include CRISPR / Cas9, CRISPR-Cpf1, TALEN, or ZNF. Appropriate enhancement of enzymatic activity by genome editing can result in protection from LSD.

[0102] As an example, the clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) system is a new type of genome editing tool that targets desired genomic sites in mammalian cells. The recently published type II CRISPR / Cas system uses a Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence immediately preceding the NGG motif recognized by Cas9 (thus, the (N) 20 NGG target DNA sequence). This causes a double-strand break three nucleotides upstream of the NGG motif. Double-strand breaks can be exploited by error-prone, non-homologous end joining, which is advantageous for knocking out gene alleles with frameshift mutations, or by homologous recombination repair using exogenously introduced double-stranded or single-stranded DNA repair templates to knock in or correct mutations within the genome. Thus, for example, genome editing using the CRISPR / Cas system can be a useful tool for therapeutic applications for treating heterozygous subjects against loss-of-function lysosomal gene variants by enhancing or increasing enzyme production or activity.

[0103] For example, the methods described herein can include methods of modifying a target polynucleotide sequence in a cell, including contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeat-associated (Cas) protein.

[0104] Formulation The agents and compositions described herein can be formulated in any conventional manner using, for example, one or more pharmaceutically acceptable carriers or excipients described in Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations contain a therapeutically effective amount of the biologically active agent described herein, which may be in purified form, together with a suitable amount of carrier to provide a form suitable for proper administration to a subject.

[0105] The term "formulation" refers to the preparation of a drug in a form suitable for administration to a subject such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, such as diluents or carriers, for example, capsid proteins.

[0106] As used herein, the term "pharmaceutically acceptable" can describe a substance or component that does not cause an unacceptable loss of pharmacological activity or unacceptable side effects. Examples of pharmaceutically acceptable components can be those having monographs in the United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 ("USP / NF"), or more recent editions, and components listed in the FDA's continuously updated Inactive Ingredient Search online database. Other useful components not described in USP / NF, etc. can also be used.

[0107] As used herein, the term "pharmaceutically acceptable excipient" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents with a pharmaceutically active substance is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R.Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). The use of any conventional media or agent in a therapeutic composition is contemplated, except where it is incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

[0108] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as from about 0 °C to about 60 °C, for a commercially reasonable period, such as at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0109] Formulations must be compatible with the mode of administration. The agents used in connection with the present disclosure can be formulated by known methods for administration to a subject using several routes including, but not limited to, intrathecal (e.g., gene therapy), intracranial (e.g., gene therapy), parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implantation, intramuscular, intraperitoneal, intravenous (e.g., enzyme replacement therapy), intraventricular, subcutaneous, intranasal, epidural, intraocular, transdermal, buccal, and rectal. The individual agents can also be administered in combination with one or more additional agents or together with other biologically active or biologically inactive agents. Such biologically active or inactive agents can be in fluid or mechanical communication with the agent or can be bound to the agent by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0110] Controlled release (or sustained release) preparations may be formulated to extend the activity of a drug and reduce the dosing frequency. Controlled release preparations can also be used to effectively modify other characteristics such as the onset time of action or the blood level of the drug, and as a result, affect the occurrence of side effects. Controlled release preparations may be designed to first release an amount of the drug that provides the desired therapeutic effect and then gradually and continuously release other amounts of the drug to maintain the level of the therapeutic effect over a long period of time. To maintain a substantially constant level of the drug in the body, the drug may be released from the dosage form at a rate that replaces the amount of the drug that is metabolized or excreted from the body. The controlled release of a drug can be stimulated by various inducing factors, such as changes in pH, temperature, enzymes, water, or other physiological conditions or molecular changes.

[0111] The drugs or compositions described herein may also be used in combination with other therapeutic modalities, as further described below. Thus, in addition to the therapies described herein, it is also possible to provide other therapies known to be effective in the treatment of a disease, disorder, or condition.

[0112] Methods of treatment There is also provided a process for treating, preventing, or reversing a neurological or neurodegenerative disease, disorder, or condition in a subject in need of administration of a therapeutically effective amount of an ALP function enhancer (e.g., an LSD therapeutic agent) to substantially inhibit, slow the progression of, or suppress the onset of a neurological disease, disorder, or condition associated with an increase in Aβ or an APP processing dysfunction, or in a subject heterozygous for a loss-of-function gene variant associated with lysosomal dysfunction who has, is suspected of having, or is at risk of developing a neurological disease, disorder, or condition associated with an increase in Aβ or an APP processing dysfunction.

[0113] The methods described herein are generally performed on a subject in need thereof. A subject in need of the treatment methods described herein can be a subject having, diagnosed as having, suspected of having, or at risk of developing a neurological disease, disorder, or condition. The determination of the need for treatment is typically evaluated by a medical history and physical examination consistent with the disease or condition in question. Diagnosis of the various conditions treatable by the methods described herein is within the scope of the art. The subject can be an animal subject including mammals such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans. For example, the subject can be a human subject.

[0114] Generally, a safe and effective amount of an ALP function enhancer will be an amount that, for example, will produce the desired therapeutic effect in a subject while minimizing undesirable side effects. In various embodiments, an effective amount of the ALP function enhancer described herein can substantially inhibit, slow the progression of, or suppress the onset of a neurological disease, disorder, or condition.

[0115] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.

[0116] When used in the treatments described herein, a therapeutically effective amount of an ALP function enhancer can be used in pure form or, if such form exists, in a pharmaceutically acceptable salt form, and with or without pharmaceutically acceptable excipients. For example, the compounds of the disclosure can be administered in an amount sufficient to substantially inhibit, slow the progression of, or suppress the onset of a neurological disease, disorder, or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0117] The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single-dose administration form will vary depending on the host to be treated and the particular method of administration. It will be understood by those skilled in the art that the unit content of the agent contained in the individual dose of each administration form need not itself constitute a therapeutically effective amount, since the therapeutically effective amount can be reached by administration of several individual doses.

[0118] The toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for determining the LD 50 (lethal dose, 50% of a population) and the ED 50 (therapeutically effective dose, 50% of a population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 and a greater therapeutic index is generally considered optimal in the art.

[0119] The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors including the disorder being treated and the severity of the disorder, the activity of the specific compound being used, the specific composition being used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion of the composition being used, the duration of the treatment, drugs used in combination with or concurrently with the specific compound being used, as well as similar factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN0781748453, Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN0781741475, Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN0071375503). For example, it is well within the skill in the art to initiate administration of the composition at a level below that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dosage may be divided into multiple dosages for purposes of administration. Thus, a single dosage composition may contain such an amount or a multiple thereof to constitute a daily dosage. However, it will be understood that the total daily usage of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0120] Here again, each of the situations, diseases, disorders, and conditions described herein, as well as other situations, diseases, disorders, and conditions, can benefit from the compositions and methods described herein. Generally, treating a situation, disease, disorder, or condition means preventing clinical symptoms, promoting recovery, or delaying their onset in a mammal that may be affected by or predisposed to those situations, diseases, disorders, or conditions but has not yet experienced or manifested their clinical or subclinical symptoms. Treatment can also include inhibiting a situation, disease, disorder, or condition, for example, stopping or reducing the development of a disease or at least one of its clinical or subclinical symptoms. Further, treatment can include alleviating a disease, for example, causing at least partial recovery of a situation, disease, disorder, or condition, or one of its clinical or subclinical symptoms. The benefit to the subject being treated can be statistically significant or at least recognizable to the subject or the physician.

[0121] Administration of an ALP function enhancer can occur as a single event or over the course of a treatment. For example, the ALP function enhancer can be administered daily, weekly, bi-weekly, or monthly. In the case of gene therapy, the course of treatment is usually at least 1 day to several days. For certain treatments, such as ERT, the treatment can be extended from several days to several weeks. For example, the treatment can be extended over 1 week, 2 weeks, or 3 weeks. For more chronic conditions and long-term treatment methods, the treatment can be extended from several weeks to several months, or even 1 year or more.

[0122] Treatment according to the methods described herein can be carried out before, simultaneously with, or after conventional treatment modalities for neurological diseases, disorders, or conditions associated with loss-of-function variants in lysosomal genes.

[0123] Administration The agents and compositions described herein can be administered according to the methods described herein by various means known in the art. The agents and compositions can be used for treatment as either exogenous or endogenous materials. Exogenous agents are those produced or manufactured outside the body and administered into the body. Endogenous agents are those produced or manufactured in the body by some type of device (biological or otherwise) inside other organs of the body or for delivery to other organs of the body.

[0124] As described above, administration can be intracranial administration, intrathecal administration, parenteral administration, pulmonary administration, oral administration, topical administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, intraventricular administration, subcutaneous administration, intranasal administration, epidural administration, ophthalmic administration, oral cavity administration, or rectal administration.

[0125] The agents and compositions described herein can be administered by various methods well-known in the art. Administration can include, for example, oral ingestion, direct injection (e.g., systemic or stereotactic), transplantation of cells engineered to secrete the factor of interest, biomaterials that release drugs, polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1 - 100 μm), reservoir devices, any combination of the above, or other suitable delivery vehicles for providing a desired release profile at various rates. Other methods of controlled release delivery of the agent or composition are known to those skilled in the art and are within the scope of the present disclosure.

[0126] The delivery system may include an infusion pump that can be used to administer the drug or composition in a manner similar to that used, for example, to deliver insulin or chemotherapy to a particular organ or tumor. Typically, using such a system, the drug or composition can be administered in combination with a biodegradable biocompatible polymer implant that releases the drug at a selected site over a controlled period. Examples of polymer materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and their copolymers and combinations. In addition, the sustained release system can be placed in the vicinity of the treatment target, thereby requiring only a small fraction of the systemic dose.

[0127] The drug can be encapsulated and administered in various carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymer implants, smart polymer carriers, and liposomes (see generally Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for molecular or biomolecular drug delivery can provide intracellular delivery, can adjust the biomolecule / drug release rate, can increase the proportion of biomolecules reaching their site of action, can improve the transport of the drug to its site of action, can enable co-localized deposition with other drugs or excipients, can improve the stability of the drug in vivo, can extend the residence time of the drug at its site of action by reducing clearance, can reduce the non-specific delivery of the drug to non-target tissues, can reduce the irritation caused by the drug, can reduce the toxicity resulting from a high initial dose of the drug, can modify the immunogenicity of the drug, can reduce the dosing frequency, can improve the taste of the product, or can improve the shelf life of the product.

[0128] The compositions and methods described herein that utilize protocols of molecular biology can follow various standard techniques known in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717, Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929, Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773, Elhai, J. and Wolk, C.P. 1988. Methods in Enzymology 167, 747-754, Studier (2005) Protein Expr Purif. 41(1), 207-234, Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363, Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0129] The definitions and methods described herein are provided to better define the present disclosure and to guide one of ordinary skill in the art in practicing the invention. Unless otherwise specified, terms should be understood according to their conventional use by those of ordinary skill in the relevant art.

[0130] In some embodiments, it should be understood that the numbers expressing amounts of components, properties such as molecular weight, reaction conditions, etc. used to describe and claim particular embodiments of the present disclosure are, in some cases, modified by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the mean standard deviation for the device or method used to determine that value. In some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may include certain errors necessarily resulting from the standard deviation found in their respective test measurements. The recitation of a range of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0131] In some embodiments, the terms "a," "an," and "the," as well as similar references used in the context of describing particular embodiments (especially in the context of the following claims), may be construed to include both the singular and the plural, unless otherwise indicated to the contrary. In some embodiments, as used herein, including in the claims, the term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0132] The terms "comprise", "have", and "include" are non-limiting conjunctive verbs. Any form or tense of one or more of these verbs, such as "comprises", "comprising", "has", "having", "includes", and "including", is also non-limiting. For example, any method that "comprises", "has", or "includes" one or more steps is not limited to having only those one or more steps, and may also include other unrecited steps. Similarly, any composition or device that "comprises", "has", or "includes" one or more features is not limited to having only those one or more features, and may also include other unrecited features.

[0133] All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (e.g., "for example") provided herein with respect to particular embodiments is merely intended to better illustrate the disclosure and is not intended to limit the scope of the disclosure, unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0134] The grouping of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Members of each group can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in or deleted from the group for reasons of convenience or patentability. When such inclusion or deletion occurs, this specification is considered to contain the modified group and thus meets all written descriptions of the Markush groups used in the appended claims.

[0135] All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference were specifically and individually indicated to be incorporated by reference for all purposes. The citation of a reference in this specification should not be construed as an admission that such is prior art to the present disclosure.

[0136] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples.

Examples

[0137] The following non-limiting examples are provided to further illustrate the present disclosure. The techniques disclosed in the following examples represent approaches that the inventors have found to function well in the practice of the present disclosure and can thus be considered to constitute examples of modes for its practice. However, those skilled in the art should understand that, in light of the present disclosure, many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain the same or similar results.

[0138] Example 1: Investigate the role of NAGLU variants in the lesions of Alzheimer's disease (AD) and Parkinson's disease (PD) This example illustrates the in vitro and in vivo verification of the role of genetic variations in genes involved in the lysosomal degradation of heparan sulfate in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0139] There is compelling genetic and biochemical evidence suggesting that lysosomal dysfunction is a common pathogenic mechanism in several adult-onset neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). However, the age-dependent decline in lysosomal function and the genetic variations underlying this decline in relation to AD and PD are not fully understood. In addition, the contribution of genetic variations within the general population of each lysosomal gene to the risk of developing AD or PD, and the systematic and comprehensive evaluation of its role in disease pathogenesis have not been completed. To address this gap in current knowledge, single-variant and gene-based analyses of 45 lysosomal genes were performed in AD and PD case-control cohorts. As described herein, variants were discovered in several lysosomal enzyme genes associated with both AD and PD. These data confirm the association between GBA and PD. Of particular interest was the enrichment of AD and PD patients with rare functional variants in genes involved in heparan sulfate (HS) metabolism (GNS, NAGLU, SGSH, and HGSNAT). In addition, the transcript level of NAGLU was decreased in dopaminergic neurons from the substantia nigra of PD patients.

[0140] Interestingly, NAGLU transcript levels are also significantly higher in AD cases compared to age-matched controls and show a proportional age-dependent increase with the onset of lesions in a mouse model of AD. Heparan sulfate proteoglycan (HSPG), consisting of HS chains covalently linked to specific protein cores, is a molecule abundant on the cell surface and extracellularly that interacts with a spectrum of ligands. HSPG regulates the oligomerization, clearance, endocytosis, and transport of various pathogenic proteins, including amyloid (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (α-Syn). Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. So far, it has not been clear whether the decrease in NAGLU activity and the resulting decrease in HSPG accumulation affect APP metabolism, Aβ plaque amount, or the aggregation and diffusion of α-Syn.

[0141] As described herein, biochemical assays and cell-based assays can be used to fully characterize the functional effects of selected genetic variants in NAGLU activity. The effects of mutant NAGLU on full-length APP levels, APP transport, Aβ production in neurons, and Aβ degradation by glial cells are examined. It can be determined whether haploinsufficiency of NAGLU accelerates AD lesions present in a well-characterized mouse model of AD. It can be determined whether the binding, internalization, and aggregation of α-Syn PFF are affected in primary neurons derived from NAGLU-deficient and heterozygous mice stably expressing the selected variant. Finally, intracerebral inoculation of α-Syn PFF is performed in heterozygous or knockout (KO) NAGLU mice, and the formation of pSyn aggregates, connectivity-dependent diffusion, and their effects on disease progression and lifespan are quantified.

[0142] Description of the project The goal of this study is to verify in vitro and in vivo the role of genetic variations in genes involved in the lysosomal degradation of heparan sulfate in the onset of Alzheimer's disease (AD) and Parkinson's disease (PD). The study described herein incorporates an innovative integrated framework that combines computational methods and experimental data to verify the functional impact of selected NAGLU variants both in vitro and in vivo. The experiments outlined here can reveal novel lysosomal genes associated with AD and PD and provide deeper insights into the mechanisms of lysosomal dysfunction in the onset of AD and PD.

[0143] There is compelling genetic and biochemical evidence suggesting that severe lysosomal dysfunction caused by homozygous mutations in lysosomal genes is a common pathogenic mechanism in several adult-onset neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). In case-control cohorts of AD (5712 cases / 5011 controls) and PD (821 cases / 750 controls), single-variant and gene-based analyses of 45 lysosomal genes were performed. Variants were identified in many lysosomal enzyme genes associated with both AD and PD. Importantly, this data confirms the association between GBA and PD. Of particular interest was the enrichment of rare heterozygous functional variants in genes involved in heparan sulfate (HS) metabolism (SGSH, NAGLU, HGSNAT, and GNS) in AD and PD patients. The transcript level of N-acetyl-α-glucosaminidase (NAGLU) is decreased in dopaminergic neurons from the substantia nigra (SN) of PD patients. Enrichment of predicted rare heterozygous functional variants in NAGLU and SGSH was found in AD patients.

[0144] Interestingly, NAGLU transcript levels are also significantly higher in AD cases compared to age-matched controls and show a proportional age-dependent increase associated with the co-occurrence of lesions in mouse models of AD. Analyses identified haploinsufficiency within several lysosomal enzyme genes as risk factors for AD and PD, examined the effects of altered lysosomal HS metabolism, and performed proof-of-concept experiments in well-characterized homozygous NAGLU-deficient mice. Thus, there has been no direct biological evidence to date that haploinsufficiency of NAGLU is associated with neurological diseases. Heparan sulfate proteoglycan (HSPG), consisting of HS chains covalently linked to specific protein cores, is a molecule abundant on the cell surface and extracellularly that interacts with a spectrum of ligands. HSPG regulates the oligomerization, clearance, endocytosis, and transport of various pathogenic proteins, including amyloid (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (α-Syn).

[0145] Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote clearance of pathogenic proteins and reduce their aggregation. Most HSPGs and their bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. SGSH, NAGLU, HGSNAT, and GNS enzymes are involved in the stepwise breakdown of HS in lysosomes. Loss-of-function (LoF) mutations in these genes result in the accumulation of partially degraded HS within lysosomes, causing mucopolysaccharidosis (MPS) III types A, B, C, and D. The role of altered lysosomal degradation of HSPGs in the pathogenesis of multiple adult-onset neurodegenerative diseases is mostly unclear, although homozygous NAGLU-deficient mice show intracellular accumulation of hyperphosphorylated tau, Aβ, and HSPG in the medial olfactory cortex. In addition, MPS IIIB patients show significant SN neuron loss and accumulation of phosphorylated α-Syn (pSyn) in neurons within the temporal cortex, hippocampus, and SN. These and other data strongly suggest severe lysosomal dysfunction as a common pathogenic mechanism between AD and PD. However, as described above, there is no direct biological data implicating haploinsufficiency of lysosomal proteins in adult-onset neurodegenerative diseases (except for one notable exception of glucocerebrosidase (GBA) and Parkinson's disease).

[0146] (I) Determine the functional effects of rare variants in the NAGLU gene To verify the functional effects of cells derived from NAGLU-deficient mice, cells derived from NAGLU-deficient mice are transduced with a lentiviral vector having three variants predicted to be the most deleterious. Their effects on enzyme activity and HSPG levels are measured.

[0147] (II) Determine the effects of variants in the NAGLU gene on APP metabolism, Aβ production, and Aβ degradation in vitro, and the effects of NAGLU haploinsufficiency on AD lesions in vivo Primary neurons from NAGLU-deficient and heterozygous mice that stably express the validated variant (as described in Section (I)) are examined for their effects on APP transport, APP half-life, mechanisms of APP processing, and Aβ production. Glial cells from NAGLU-deficient or heterozygous mice that stably express the selected variant are examined for Aβ uptake and degradation.

[0148] It can be determined whether NAGLU haploinsufficiency affects Aβ production, Aβ clearance, plaque deposition, synapse loss, and neuroinflammation in 5XFAD mice at early (4 months) and late (8 months of age). The most deleterious variant (from Section (I)) is expressed in the brains of neonatal heterozygous mice using the AAV2 / 9-PHP.B pseudotyped vector, and a quantitative pathological investigation is performed on the effects of NAGLU haploinsufficiency on AD-related phenotypes in 24-month-old mice in the absence of FAD mutations.

[0149] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn spreading in vivo It is hypothesized that functional variants related to PD in the NAGLU gene affect α-Syn aggregation and intercellular transmission. It can be determined whether the binding, internalization, and aggregation of α-Syn PFFs are affected in primary neurons from NAGLU-deficient and heterozygous mice that stably express the selected variant. Finally, it can be determined whether recombinant enzyme supplementation or gene therapy rescues the effects on α-Syn PFF internalization and aggregation.

[0150] Intrastriatal injection of α-Syn PFF recapitulates the accumulation of intracellular Lewy body (LB) lesions, the selective loss of SN neurons, and the impairment of coordinated movement in transgenic mice expressing wild-type and mutant A53T human α-Syn. Intrastriatal inoculation of α-Syn PFF is performed in hemizygous or NAGLU-deficient mice injected with the AAV2 / 9-PHP.B pseudotyped vector expressing the most deleterious NAGLU variant at birth. The formation of aggregates of pSyn, their connectivity-dependent spread, and their effects on disease progression and lifespan are quantified.

[0151] Significance ALP dysfunction in AD The familial form of AD is pathogenically caused by increased amyloid-β (Aβ) production and subsequent aggregation into soluble oligomers or insoluble Aβ plaques in the extracellular space (ISF, interstitial fluid), but recent studies in late-onset sporadic AD patients have shown impaired clearance of Aβ1. Thus, the balance between production and clearance determines Aβ levels and the propensity for Aβ plaque development. The autophagy-lysosome pathway (ALP) is the major pathway for the degradation of organelles and aggregation-prone proteins. Autophagy ("self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients via lysosomes. ALP "core" genes are transcriptionally downregulated during normal aging of the human brain. In contrast, there is transcriptional upregulation of ALP in the brains of AD patients. In the brains of sporadic AD, there are decreased levels of beclin 1 (a multifunctional protein essential for autophagosome formation in ALP), increased levels of rab5 and rab7 (small ras-related GTPase (rab) proteins that regulate vesicle transport along the endosome-lysosome pathway), abnormal activation of macroautophagy (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase), and massive neuronal accumulation of autophagic vacuoles (AV) and lysosomal dense bodies in degenerating neurites.

[0152] Neuropathological studies have also found that autophagy-lysosome lesions in the AD brain contribute to the onset of AD, but the underlying mechanism is not fully understood. Changes in ALP have also been found in multiple transgenic mouse AD models. Haploinsufficiency of beclin 1 in two AD mouse models caused further disruption of their lysosomes, promoted intracellular and extracellular Aβ accumulation, and worsened neurodegeneration. Homozygous deletion of lysosomal neuraminidase 1 (NEU1) worsened Aβ lesions in the AD model. In contrast, overexpression of NEU1 reduced AD lesions. These results fully suggest that changes in ALP "core" genes or lysosomal proteins accelerate AD lesions. Cellular studies suggest that the endosome-lysosome system is the main site of Aβ production. However, there is no consensus on where Aβ is actually produced. Aβ is generated after inducing macroautophagy both in vitro and in vivo. Accumulation of Aβ increases mTOR signaling, while decreasing mTOR signaling reduces Aβ levels, suggesting a negative feedback loop between ALP activation and Aβ levels. When autophagy is activated, autophagosomes become the cellular site with the highest γ-secretase activity. Presenilin 2 (PSEN2) and nicastrin (an essential γ-secretase component for catalysis) are located in lysosomes. In fact, PSEN1 regulates lysosomal pH.

[0153] Pharmacological impairment of lysosomal function in vitro causes changes in Aβ production. Changes in lysosomal pH decrease Aβ secretion. Lysosomal protease inhibitors reduce the production of amyloidogenic APP fragments. All of these studies suggest that overall lysosomal function plays an important role in normal and abnormal amyloid precursor protein (APP) processing and subsequent amyloidogenesis. All evidence from human lesions, mouse and cell models strongly suggests that defects in autophagy induction occur early in the disease, while defects in lysosomal clearance occur at a more advanced stage of the disease.

[0154] Lysosomal dysfunction in PD Critical genetic defects in endocytic transport, lysosomal integrity, and lysosomal hydrolase activity are well-established risk factors for synucleinopathies in human postmortem studies and model systems. Lysosomal dysfunction as a pathogenic mechanism in Parkinson's disease (PD) is aided by mutations in the ATP13A2 (lysosomal ATPase) and VPS35 genes (intra-lysosomal transport) in familial PD. In addition, low-frequency variants in the GBA gene (lysosomal hydrolase glucocerebrosidase) and SMPD1 gene (lysosomal acid sphingomyelinase) increase the risk of sporadic PD. Recent meta-analyses have also found that common variants in the SCARB2 (lysosomal integral membrane protein type 2), TMEM175 (transmembrane protein 175), CTSB (lysosomal cysteine protease cathepsin B), ATP6V0A1 (ATPase H+ transporting V0 subunit a1), and GALC (lysosomal galactosylceramidase) genes are also associated with PD risk. Lysosomal markers (LAMP-1, LAMP-2a, cathepsin-D, GBA, and ATP13A2) have been identified as components of Lewy bodies (LB) in patients with sporadic PD. Thus, it has been suggested that LBs and Lewy neurites (LN) may form seeds around damaged lysosomes and increase in size by continuous deposition of undegraded material derived from lysosomes as the disease progresses.

[0155] Although models based on multiple cells still leave mechanistic questions unanswered, in recent years they have converged on the importance of the potential for intercellular transfer of proteinopathic seeds in the progression of synucleinopathies. Whether specific α-Syn strains are internalized via distinct receptors or endocytosis mechanisms remains unclear. The uptake of α-Syn by macropinocytosis in immortalized cells and primary neurons appears to be mediated by HSPG. However, the role of HS in α-Syn diffusion in vivo has not been evaluated. Lysosomal processing is the main fate of α-Syn protofibrils internalized in primary neurons. Briefly, severe pharmacological disruption of lysosomal function causes abnormal intracellular processing of α-Syn protofibrils, along with an increased rate of inclusion formation due to mobilization of endogenous α-Syn. The processes governing this mobilization are still poorly understood, suggesting that pathogenic species must escape from lysosomal transport. Thus, exogenous α-Syn species have been reported to cause rupture of vesicles and lysosomal membranes by endocytosis, thereby escaping endocytic transport and lysosomal degradation. Upon entry into the cytoplasmic matrix, these α-Syn protofibrils or oligomers can interact with soluble species and initiate the mobilization of endogenous α-Syn. These results further support the idea that defects in lysosomal activity and integrity can accelerate pathological α-Syn aggregation and transmission.

[0156] Lysosomal defects are thought to contribute to the new aggregation of α-Syn and the impairment of the autophagic degradation of mature cytoplasmic aggregates. Interestingly, neuroprotection in several in vitro and in vivo α-Syn overexpression models has been reported using mammalian targets of rapamycin (mTOR)-dependent or mTOR-independent autophagy enhancers. Similarly, viral vector-mediated expression of beclin-1 reduces α-Syn aggregates and synaptic lesions in α-Syn transgenic mice. Overexpression of transcription factor EB (TFEB), a master activator of ALP, also protects against α-Syn aggregation. Overall, these studies indicate that novel therapeutics aimed at restoring lysosomal function in PD may provide a much-needed disease-modifying treatment strategy.

[0157] Heparan sulfate in AD and PD Heparan sulfate proteoglycan (HSPG), consisting of HS chains covalently linked to a specific protein core, is a molecule abundant on the cell surface and extracellularly that interacts with a spectrum of ligands. Membrane HSPGs function as endocytosis receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and transport of various pathogenic proteins, including Aβ, apoE, tau, and α-Syn. HSPGs are present in Aβ plaques and in LBs and LNs. HSPGs have been shown to bind Aβ and accelerate its oligomerization and aggregation. HS significantly stimulates α-Syn fibril formation in vitro. HS also mediates cellular Aβ uptake. HSPGs mediate the uptake of α-Syn by macropinocytosis. Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. These findings suggest that HS and HSPGs play important roles in Aβ and α-Syn metabolism and in the pathogenesis of AD and PD. NAGLU encodes N-acetyl-α-glucosaminidase, which is involved in the lysosomal degradation of heparan sulfate (HS). LoF mutations in NAGLU cause mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome B. Qualitatively increased levels of intracellular full-length APP have been reported in the brains of both NAGLU-deficient and human MPS-IIIB patients in the absence of Aβ plaques. MPS-IIIB patients show a significant 3-fold increase in the levels of soluble Aβ40 compared to normal control brains. MPS IIIB patients show significant SN neuronal loss and accumulation of phosphorylated α-Syn in neurons within the temporal cortex, hippocampus, and SN. To date, it is not clear whether the decrease in NAGLU activity and the resulting HSPG accumulation affect APP metabolism, Aβ production or clearance, or the aggregation and spread of α-Syn.

[0158] Innovation The goal of the research described in this specification is to validate genetic findings and provide more insights into lysosomal dysfunction in AD and PD. In addition, this research can facilitate the identification of PD and AD patients with genetically determined lysosomal dysfunction, and the restoration of such dysfunction can provide effective therapies. The research outlined in this specification is conceptually innovative in systematically and comprehensively evaluating the functional consequences of genetic variations associated with the NAGLU gene in AD and PD ((Chapter (I))). These studies will provide a better understanding of the effects of aging and NAGLU haploinsufficiency on Aβ production and clearance ((Chapter (II))), as well as on α-Syn aggregation and spread in vitro and in vivo ((Chapter (III))). Furthermore, these studies will carefully examine neuronal lesions and determine the consequences of genetically altering NAGLU, as well as its effects on Aβ and the effects of α-Syn lesions on clinically relevant endpoints. The research described in this specification incorporates an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of the NAGLU gene both in vitro and in vivo. Cell-based assays are complemented by biochemical data, RNAseq data from specific cell types in the mouse brain, genome-wide gene expression data in human AD and PD cases and controls, and genome-wide gene expression data from an AD mouse model correlated with Aβ plaques. The studies described in (II) and (III) address the question of how age and NAGLU haploinsufficiency in vulnerable brain regions affect APP processing and trafficking, Aβ plaque burden and Aβ40 / 42 levels, as well as α-Syn aggregation in vitro and α-Syn spread in vivo. These studies are made possible by an innovation in collaborative research involving investigators with expertise spanning neurogenetics, lysosomal biology, LSD animal models, and AD and PD pathophysiology in cell and mouse models.

[0159] Approach Here, state-of-the-art genomic tools can be used to evaluate the functional impact, both in vitro and in vivo, of NAGLU gene variants associated with the risk of AD and PD.

[0160] Data and Results Heterozygous variants in lysosomal HS-degrading genes affect the risk of developing AD. Single-variant and gene-based analyses of 45 lysosomal genes were performed in two case-control cohorts of AD. The discovery sample consisted of whole-exome sequencing (WES) data from 667 unrelated AD cases and 511 controls. As expected, the gene-specific cumulative minor allele frequencies (cMAFs) from the ExAC dataset (European, non-Finnish) were in very good agreement with the cMAFs from the in-house AD database (r 2 = 0.96). The amount of variants (cMAF) that alter rare proteins was compared to the burden observed in controls and ExAc. For most genes, there was an excess of variation when compared to controls, but only a slight association with the SGSH gene was found (p = 4.2 × 10 -3 , odds ratio (OR) = 3.7, 95% confidence interval (CI) 1.4–9.6). When compared to the cMAF of the ExAc samples, the SGSH gene (p = 7.9 × 10 -5 and the NAGLU gene (p = 4.8 × 10 -4 passed the multiple testing correction threshold of p < 1.0 × 10 -3 (0.05 / 50). Next, the Alzheimer's Disease Sequencing Project (ADSP) cohort (5045 AD cases and 4500 controls) was used to replicate these findings. In this independent sample, NAGLU was replicated (p = 3 × 10 -3 , OR = 2.3, 95% CI 1.2–5.2). Of note is the fact that the associations found in the replication sample are in the same direction and have a similar effect size.

[0161] NAGLU transcript levels associated with age, AD status, and in an AD mouse model RNAseq data from brain cell types in mice indicate that NAGLU transcripts are expressed at higher levels (about 20-fold) in microglia than in neurons. In neuropathologically normal human brain samples, there was a significant increase in NAGLU transcript levels with age (p = 0.02) (see, e.g., FIG. 1A). NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (p = 0.007) (see, e.g., FIG. 1B). NAGLU transcript levels also showed an age-dependent proportional increase with the onset of AD lesions in the cortex of an AD mouse model (APP, p.K670N / p.M671L / PSEN1, p.M146V, heterozygous [HET] or homozygous [HO], see, e.g., FIG. 1C) compared to levels in wild-type mice (see, e.g., the black line in FIG. 1C) (see, e.g., the right panel of FIG. 1C).

[0162] Heterozygous variants in lysosomal HS degradation genes affect the risk of developing PD. The discovery sample consisted of WES data from 331 unrelated PD cases from the PPMI cohort. Gene-specific cMAFs from the NFE ExAC dataset were highly concordant with cMAFs from the in-house PD database (PPMI r 2 = 0.92, in-house r 2 = 0.96). The burden of variants (cMAF) that alter rare proteins was compared to the burden observed in controls and ExAc. When compared to the cMAF of the ExAc sample, seven genes, including GBA (p = 6.1×10 -6 , OR = 2.1, CI = 1.3 - 3.3), GNS (p = 2.5×10 -5 , OR = 2.4, CI = 1.4 - 4.6), and NAGLU (p = 1.0×10 -4 , OR = 4.7, CI = 1.5 - 8.3), passed the multiple testing correction threshold of p < 1.0×10 -3 . There was also a trend for HGSNAT (p = 8.1×10 -3, OR = 1.8, CI = 1.1 - 2.8). Next, using an additional PD cohort (WUSTL), these findings were replicated including 490 PD cases for which data were obtained using a human exome chip. In particular, the associations found in the replication samples were in the same direction and the effect sizes were similar for NAGLU (p = 3.6×10 -7 , OR = 3.6, CI = 2.8 - 8.3) and HGSNAT (p = 9.7×10 -4 , OR = 1.9, CI = 1.4 - 3.2).

Table 4

[0163] NAGLU transcript levels in the context of PD SN lesions and LB accumulation have been reported in MPS IIIB patients with mutations in the NAGLU gene. Additionally, it has been found that the transcript levels of the NAGLU gene are decreased in dopaminergic (DA) neurons from the substantia nigra of PD patients compared to controls (see, for example, Figure 2). The preparation and use of α-Syn PFFs in neuronal cultures have been previously optimized. α-Syn PFFs were added to primary cortical neurons from wild-type mice at 7 days in vitro (DIV). Seven days after treatment, the neurons were fixed and stained with a pSyn-specific antibody. The PFFs were abnormal, phosphorylated, and induced the recruitment of endogenous expressed α-Syn into insoluble aggregates (see, for example, Figures 3A and 3B). α-Syn aggregates initially appeared as small punctate inclusions in presynaptic terminals and axons (see, for example, the lower right panel, Figure 3A). The aggregates grew and appeared more elongated and serpentine in appearance, resembling Lewy neurites (see, for example, the lower left panel, Figure 3A). Figure 3B shows that PBS-treated control neurons showed a band just above 15 kDa corresponding to monomeric α-Syn. Several bands with higher molecular weights appeared in neurons treated with PFFs. These additional bands are likely to correspond to α-Syn oligomers. This is a tractable in vitro system for studying the effects of lysosomal dysfunction on α-Syn aggregation.

[0164] Diffusion of pSyn lesions in NAGLU-deficient mice Intrastriatal inoculation of α-Syn PFF or PBS (control) was performed in 6 NAGLU-deficient mice and 6 wild-type littermates. All mice survived this injection and are currently aging. Consistent with published data, animals treated with PBS at 30 days post-injection (dpi) showed no pSyn lesions (see, e.g., Fig. 4A). In contrast, wild-type mice injected with PFF showed numerous ipsilateral pSyn lesions and very few contralateral pSyn lesions (see, e.g., Fig. 4B). At 90 dpi, there was a gradient of pSyn lesions with greater intensity ipsilateral than contralateral in wild-type mice injected with PFF. This gradient was very prominent in the motor cortex and SN. The amygdala and somatosensory cortex had more symmetric lesions. Surprisingly, NAGLU-deficient mice treated with α-Syn PFF showed α-Syn lesions in both the ipsilateral and contralateral prefrontal and olfactory cortex to the injection site at 30 dpi (see, e.g., Fig. 4C). NAGLU-deficient mice appear to have more symmetric pSyn lesions, which may indicate greater spread to the contralateral side than that observed in WT mice. To further characterize the effect of NAGLU deficiency on the regional and temporal spread of pSyn lesions and to extend these initial results suggesting an increase in the spread of pSyn lesions in NAGLU-deficient mice, more α-Syn PFF-treated mice are currently being analyzed.

[0165] Study design and methods (I) Determine the functional impact of variants in the NAGLU gene Evaluate the impact on the protein product Evaluating all variants of the NAGLU gene associated with a specific AD or PD is beyond the scope of the studies described herein. Thus, this study focused on the top 3 - 5 variants identified in the NAGLU gene. The top variants were defined based on their frequency in AD / PD patients, the predicted effects on proteins by SIFT and Polyphen2, and the GERP conservation score. These genes were selected based on the strength of the data from both the discovery and replication samples. The effects of the selected variants in the NAGLU gene on enzyme activity, protein level, and lysosome function can be determined using the 3 variants listed in Table 4, which is the focused area. Very stringent criteria were used to select potential functional variants in NAGLU (one has previously been identified as a pathogenic variant). However, it is important to characterize their effects on protein level and enzyme activity. Cells have already been immortalized from NAGLU - deficient mice. The variants outlined in Table 4 can be transduced and their effects on enzyme activity and protein level determined. The effects on the lysosome function and accumulation of HSPG can also be determined. The selected variants are manipulated using site - directed mutagenesis and sub - cloned into a lentiviral vector as described above. The lentiviral vector is produced, handled, and disposed of in a BSL2 facility according to Section III - E - 1 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. To determine the effect of the variant on enzyme activity, a fluorescence assay is used for NAGLU activity as described above. The variant is determined to affect the function of the lysosome as described above. The level of HSPG is quantified by ELISA. To ensure rigor and reproducibility, the quantification is performed in at least 3 independent experiments, each measured 3 times by double - blind observers.

[0166] Expected results It is expected that the NAGLU variant will cause partial loss of function, increase partially degraded HS in lysosomes, and alter ALP function. It is expected that 5 - 20% residual NAGLU activity will be detected. If the selected variant cannot reduce activity compared to wild - type levels, evaluate their effects on intracellular localization and misfolding. Additional variants associated with AD or PD can be selected and tested for their effects on enzyme activity.

[0167] (II)(a) Determine the functional effects of NAGLU on APP metabolism, Aβ production, and Aβ degradation in vitro Evaluate the effects on APP transport, endocytosis, and intracellular localization Multiple studies have shown that APP endocytosis is essential for the co-localization of APP with β- and γ-secretases in endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impairment of endosomal flux following lysosomal dysfunction increases the transport time within this organelle, increases the tendency for β-cleavage and γ-cleavage, and thus increases the tendency for Aβ production. In the absence of Aβ plaques, increased levels of intracellular full-length APP have been reported in the brains of both NAGLU-deficient patients and Sanfilippo B patients. To determine whether selected variants in the NAGLU gene affect the steady-state levels of APP, APP endocytosis, or the enhancement of APP flux to lysosomes for degradation, the dynamics of intracellular APP appearance and the levels of APP on the cell surface can be determined using the cell surface biotinylation assay as previously published. The effect on the full-length APP half-life is measured by Western blot at 0, 5, 10, 30 minutes under treatment with the protein synthesis inhibitor cycloheximide. The effects on APP and SorL1 intracellular localization are studied using co-localization techniques. The protein and transcript levels of the APP processing machinery, including α-secretases (ADAM10 and ADAM17), β-secretase 1 (BACE1), and the γ-secretase complex (PSEN1 and Nicastrin), are measured by Western blot and RT-qPCR, respectively.

[0168] Evaluate the effect on Aβ production A significant proportion of APP targets lysosomes, and intracellular APP levels rapidly accumulate in the presence of lysosomal acidification inhibitors, suggesting that lysosomal degradation causes APP proteolysis and eliminates the formation of Aβ peptides. Sanfilippo B patients show a significant increase (three-fold) in the levels of soluble Aβ compared to normal control brains. A significant increase in Aβ oligomer levels has been reported in the brains of NAGLU-deficient mice. These findings suggest that the accumulation of HS and lysosomal dysfunction in both NAGLU-deficient mice and humans cause abnormal APP processing that is dependent on γ-secretase. Therefore, the selected variants can be evaluated for whether they affect Aβ production in cell culture. Primary neuron cultures are performed as described above. Primary neurons are transduced from both NAGLU-deficient and hemizygous mice using lentiviral vectors carrying the selected variants and wild type under a neuron-specific promoter (synapsin). Different volumes of concentrated lentivirus are used to determine appropriate expression levels in neurons. NAGLU levels are measured by RT-qPCR and fluorescence assays. Aβ species in cell lysates and cell-derived media are detected by sandwich ELISA as previously published. Briefly, Aβx-40 and Aβx-42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). HJ5.1 (anti-Aβ13-28), a biotinylated antibody targeting the central domain, or HJ3.5 targeting the N-terminal amino acid is used as the detection antibody, followed by streptavidin-poly-HRP-40. APP-derived proteolytic fragments such as α-CTF and β-CTF, and sAPPα and sAPPβ are measured by Western blot. As described above, the levels of full-length APP are monitored by Western blot.

[0169] Evaluate the effect on Aβ degradation Microglia proliferate around Aβ plaques and phagocytose Aβ substances, but subsequent degradation is impaired, contributing to the progressive Aβ accumulation in AD. It is not clear why microglial cells can take up fibrillar Aβ but cannot degrade it. However, microglial cells derived from AD patients show a decrease in beclin-1 and subsequent ALP dysfunction. In addition, insoluble fibrillar Aβ affects the transport of the chloride channel CIC-7 to lysosomes in primary microglia, impairing lysosomal degradation. However, when lysosomal acidification is restored, Aβ degradation is enhanced. Collectively, this evidence suggests that ALP deficiency in microglial cells may contribute to the development of AD. The data mining effort presented here revealed that NAGLU is expressed at a higher level in microglial cells than in neurons. Therefore, primary microglial cells derived from NAGLU-deficient and heterozygous mice transduced with the selected variant can be evaluated for their ability to take up and degrade exogenous Aβ. The evaluation of Aβ uptake and degradation is performed as previously published.

[0170] Evaluate the effect on ALP function Increased levels of beclin 1, p62, and LC3-II in heart and brain tissues from NAGLU-deficient mice suggest abnormal activation of the lysosomal autophagy system with accumulation of autophagosomes. Neurons from heterozygous NAGLU mice can be evaluated for whether they exhibit ALP dysfunction and whether these changes are increased by the selected variants. Western blots of LC3 and p62 are used as an indirect indicator of macroautophagy activation. Autophagic flux is evaluated, as previously published, by the amount of LC3-II present in cells in the absence or presence of autophagy activators (rapamycin and Torin 1), autophagy inhibitors (bafilomycin A1), lysosome-directed agents (chloroquine, ammonium chloride), and E64 / pepstatin. This is complemented by live cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion can be further evaluated by co-localization of LC3 and LAMP1. Lysotracker is used to quantify the number of acidic compartments per cell. Activation of TFEB is evaluated by its nuclear localization. RT-qPCR is used to measure changes in transcript levels of TFEB-regulated mRNA transcripts (SQSTM1 / p62, MAP1LC3B, and LAMP2). To ensure rigor and reproducibility, the researchers are blinded to the genotype during the quantification and analysis steps. For each experiment, the data obtained are averaged within each of the groups described. The experiments are performed using at least two independently generated preparations per genotype, measured three times each. Statistical significance is tested using two-way ANOVA and appropriate post hoc tests to determine whether each marker or functional analysis is associated with NAGLU relative to the control.

[0171] Generation of the AAV2 / 9-PHP.B vector The NAGLU wild type and the most deleterious variants are subcloned into the AAV2 / 9-PHP.B vector. AAV2 / 9-PHP.B transfers genes across the entire central nervous system (CNS) with at least 40-fold greater efficiency than AAV9, transducing the majority of astrocytes and neurons across multiple CNS regions. High-titer AAV2 / 9 vector stocks were obtained from the UNC Viral Vector Core facility. The AAV vector stocks were diluted to 10 12 vg / ml in lactated Ringer's solution for all experiments outlined in this project.

[0172] Expected Results It is expected that there will be a gene dosing effect of NAGLU on APP trafficking, APP metabolism, Aβ production, or Aβ degradation. In addition, it is expected that the experiments outlined herein will enable the evaluation of the effect of selected variants in the NAGLU gene on the survival of neurons and microglial cells. Alternatively, primary neurons derived from 5XFAD transgenic mice or N2A695 cells could be used, transduced with selected variants in the NAGLU gene, and the effect on Aβ production could be evaluated. The next step after identifying variants in the NAGLU gene that affect both the risk and onset of AD in vitro is to generate direct induced pluripotent stem cells (iPScs) from human fibroblasts and utilize advances in genome editing methods. Using iPSc-derived neurons or glial cells from AD patients carrying variants in the NAGLU gene, the effect of such variants on APP metabolism can be compared to CRISPr-corrected cells with the same genetic background. Combining the results from this example with the availability of a fluorescence assay for NAGLU activity would enable the screening of cerebrospinal fluid (CSF), plasma, serum, or brain tissue from AD cases and controls to detect specific defects that could be used as biomarkers for AD.

[0173] (II)(b) Determine the functional impact of NAGLU haploinsufficiency on the development of AD lesions in aged mice The neurodegenerative consequences of complete loss of NAGLU function have been characterized in mice and humans, but little is known about the long-term consequences of a single copy (haploinsufficiency) of this gene. Hemizygous mice and humans have always been assumed to be normal. However, recently, haploinsufficiency in lysosomal genes has been shown to cause significant metabolic abnormalities in humans and mice. Here, the hypothesis is put forward that AD lesions develop from a mild form of hereditary ALP dysfunction and that the appearance of these may require additional age-related ALP impairment. The primary endpoints are Aβ levels measured at 4 months of age (before plaque deposition) in the presence of mutations that cause FAD in mice, and plaque burden at 8 months of age. The effect on Aβ levels is the primary endpoint in 24-month-old NAGLU heterozygous mice expressing the most deleterious NAGLU variant associated with AD.

[0174] Effect on a mouse model of AD lesions Complete loss of NAGLU protein function in human patients with Sanfilippo B disease causes a significant three-fold increase in the level of soluble Aβ40 compared to normal control brains. NAGLU transcript levels showed an age-dependent proportional increase with the onset of AD lesions in the cortex of the AD mouse model (see, e.g., FIG. 1C). Similar to AD transgenic mice, cognitive decline in humans does not correlate with Aβ plaque load but correlates with soluble Aβ species. Considering data from human Sanfilippo B patients and NAGLU-deficient mice that support the role of these genes in intracellular Aβ production, it is possible to determine whether mild lysosomal impairment (hemizygosity in NAGLU) accelerates Aβ production in a well-characterized mouse model of AD carrying a familial Alzheimer's disease (FAD) mutation favorable for Aβ production. NAGLU-deficient mice show highly sulfated HS brain accumulation, neuroinflammation, increased lysosomes in both neurons and microglia, and a decrease in synaptic proteins at about 4 months, followed by changes in the circadian rhythm, auditory and visual deficits, and in older mice (over 8 months), Purkinje cell loss and impaired coordinated movement. The intermediate lifespan of NAGLU-deficient mice is about 12 months of age. The 5XFAD model is a very aggressive Aβ deposition model in which intracellular Aβ42 develops at 1.5 months of age, plaques develop at 2 months, loss of synaptic markers and memory deficits develop at 4 months, and neuronal loss develops at 9 months. The development of plaques is accompanied by reactive gliosis. To further confirm whether NAGLU haploinsufficiency worsens the existing amyloidogenic process, NAGLU mice are crossed with 5XFAD transgenic mice. NAGLU and 5XFAD mice have the same genetic background relative to the C57Bl / 6 background. The effect of the most deleterious NAGLU variant (described in section (I)) and the gene dosage of NAGLU on Aβ plaque load is determined by histology and Aβ40 / Aβ42 levels by sandwich ELISA at 4 and 8 months as described above. APP metabolism is evaluated by measuring APP-CTF by Western blot.Four months is an early time point for the deposition of Aβ plaques in 5XFAD mice to detect whether Aβ accumulation starts early, while eight months represents a late stage with abundant Aβ plaques.

[0175] Randomization, biological variables, and sample size Calculation of the sample size indicates that at 80% power, with a 40% increase in plaque burden (SD = 20%, α = 5%), and to detect detergent-soluble and -insoluble Aβ40 and Aβ42, at least n = 10 mice / group are required. Twenty NAGLU-deficient mice and 20 NAGLU heterozygous mice crossed with 5XFAD are injected with the most deleterious NAGLU variant using AAV2 / 9-PHP.B at birth. In addition, 20 NAGLU-deficient mice, 20 NAGLU heterozygous mice crossed with 5XFAD, and 20 additional 5XFAD mice (to control for genetic background) at 4 and 8 months of age are collected for histological and biochemical studies. Since females generally have greater Aβ accumulation than males, each group consists of 10 males and 10 littermate females (n = 20). When experimental animals are generated for each experiment, independent members of the laboratory randomly assign a number to each animal. Thus, the researchers most closely associated with this study are blinded to the genotype and treatment regimen to ensure an unbiased experiment. Samples for biochemical and histological analyses retain the same randomly assigned numbers. Biological variation is minimized by using animals with the same genetic background and the same batch of reagents within one experiment.

[0176] Effect of NAGLU haploinsufficiency on aged mice AD lesions (e.g., Aβ plaques) are typically age-dependent. However, published studies do not address the interaction between age and ALP dysfunction. Most studies evaluating the role of ALP in AD in vivo use pharmacological approaches or complete absence of the ALP gene and short-term endpoints. An increase in Aβ oligomers has been reported in the brains of 10-month-old NAGLU-deficient mice. As described herein, a genetic approach can be used due to the decrease in endogenous levels of NAGLU, and a quantitative pathological investigation of the effect of hereditary chronic lysosomal disorders on AD-related phenotypes with Aβ can be performed. The results show that in normal human brain samples, NAGLU transcript levels increase very significantly with age (see, for example, FIG. 1A). In addition, NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (see, for example, FIG. 1B). These results suggest that a compensatory response to aggregated proteins derived from NAGLU may be part of the normal aging process. The abnormal increase found in the AD model suggests an attempt to control abnormal levels of Aβ. Therefore, haploinsufficiency in NAGLU may exacerbate AD-related phenotypes in aged mice.

[0177] Sample size Calculation of the sample size indicates that at 80% power, at least n = 15 mice / group are required to detect a 20% increase in detergent-soluble and insoluble Aβ40 and Aβ42. Fifteen NAGLU-deficient mice, 15 NAGLU heterozygous mice, and 15 wild-type mice are injected with the most deleterious NAGLU variant using AAV2 / 9-PHP.B 1-2 days after birth. The mice can recover and survive for at least 12 months of age. Dying mice are anesthetized and euthanized, and brain biochemical studies such as detergent-soluble and insoluble Aβ40 and Aβ42 levels are collected.

[0178] Quantification of Aβ plaques and Aβ production Fixed frozen brain sections (50 μm) are stained in a subcohort of mice with X-34, immunostained with the HJ3.4 (anti-Aβ) antibody, and the plaque burden is quantified (expressed as % area). Aβ levels in brain tissue homogenates from the contralateral hemisphere are fractionated into soluble (PBS) and insoluble (5 M guanidine) fractions and quantified using ELISA. The effect on the APP processing machinery is evaluated.

[0179] Synaptic marker Synaptic loss is a common finding in humans and AD mouse models. Antibodies against the following presynaptic markers can be used to evaluate by Western blot whether NAGLU haploinsufficiency accelerates synaptic loss in 5XFAD mice. As previously published, SNAP-25, vesicle-associated membrane protein 2, syntaxin 1, and synaptophysin.

[0180] In vivo Aβ microdialysis Aβ has a relatively short half-life in the brain, approximately 1-2 hours in the interstitial fluid (ISF) of mice and approximately 8 hours in human cerebrospinal fluid (CSF). To investigate the effects of NAGLU on Aβ production and clearance, in vivo microdialysis was used to dynamically evaluate ISF Aβ metabolism in the hippocampus of 5XFAD / NAGLU (+ / -), 5XFAD / NAGLU (- / -) mice and 5XFAD littermates injected with AVV2 / 9 or PBS at 3-4 months of age. To evaluate the ISF Aβ levels over time in the awake hippocampus, in vivo microdialysis of freely moving mice was performed as described above. Briefly, under isoflurane anesthesia, a guide cannula was implanted stereotaxically on the hippocampus (1.2 mm under the dura at 3.1 mm posterior to bregma, 2.5 mm lateral to the midline, at an angle of 12°). A microdialysis probe was inserted into the brain through the guide cannula. Artificial CSF was used as the microdialysis perfusion buffer. Microdialysis samples were collected every 60-90 minutes and evaluated for Aβ40 or Aβ42 by ELISA. The average concentration of Aβ over 6 hours was defined as the basal concentration of ISF Aβ. For each animal, all Aβ concentrations were normalized to the basal Aβ concentration of that mouse. After the basal concentration was determined, the mice were administered a blood-brain permeable γ-secretase inhibitor (LY411575, 3 mg / kg subcutaneous) to rapidly block Aβ production. Microdialysis samples were collected every 60 minutes for 6 hours and then assayed for Aβ40 by ELISA. The half-life of ISF Aβ was calculated based on the slope of the semi-logarithmic plot of the percent change in Aβ over time. Only continuously decreasing Aβ values were included in the half-life analysis. Based on the power analysis, n = 10 mice / group was determined to detect a 30% decrease in ISF Aβ levels and clearance rate. (5 groups × 10 = 50 mice, equal numbers of males and females).

[0181] ALP dysfunction Brain sections from NAGLU-deficient, NAGLU heterozygous, and NAGLU heterozygous mice crossed with 5XFAD mice are immunostained with anti-LAMP1, LC3, and p62 antibodies as described above.

[0182] Effects on neuronal dystrophy and reactive gliosis Previous studies by the inventors have shown that NAGLU-deficient mice exhibit an increase in astrogliosis. Therefore, in parallel studies, brain sections are stained with anti-CD11b and anti-GFAP antibodies to investigate the effect of a single copy of one of the selected genes on reactive gliosis. Fixed frozen brain sections are immunostained with the reticulon-3 (RTN-3) antibody (RTN-3 selectively accumulates in degenerating neurites), and degenerating neurites are quantified as previously done.

[0183] Expected results It is expected that mice heterozygous for the NAGLU gene will accelerate and exacerbate the amount of Aβ plaques in 5XFAD mice. It is expected that NAGLU haploinsufficiency will affect APP metabolism and Aβ production in aged mice, and subsequently increase synaptic loss and reactive gliosis without the occurrence of Aβ plaque formation. If changes in APP metabolism and Aβ production are not found in the brains of heterozygous mice, the NAGLU transcript can be knocked down in neonatal 5XFAD transgenic mice by injecting an AAV2 / 9 vector carrying the shRNA / RNAi validated against them. Using CRISPr technology, knock-in mice for variants in the selected genes with the most potent effects on in vitro assays can be generated. To extend the findings in NAGLU mice, the same approach can be applied to other lysosomal enzymes that degrade HS in available mouse models (e.g., N-sulphoglucosamine sulphonohydrolase [SGSH (Jax 003780)]).

[0184] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn spreading in vivo Determine the functional effect of NAGLU on the uptake, transport, aggregation, and clearance of α-synuclein in vitro The uptake of α-Syn by macropinocytosis in immortalized cells and primary neurons appears to be mediated by HSPG. In addition, HS significantly stimulates the formation of α-Syn protofibrils in vitro. In cultured neurons, a well-characterized model of α-Syn aggregation has been developed. In this model, PFFs generated from recombinant α-Syn are added directly to primary neurons and endocytosed by the neurons. These PFFs are abnormal, phosphorylated, and induce the mobilization of endogenous expressed α-Syn into insoluble and ubiquitinated aggregates. The formation of these aggregates from endogenous α-Syn in primary neurons from wild-type non-transgenic mice in vitro occurs after a 2- to 3-day induction period, followed by formation in axons by day 4-7 and spreading to the cell body dendritic compartment by day 7-10, leading to neuronal death approximately 14 days after PFF addition. The effect of NAGLU on the uptake, transport, aggregation, and clearance of α-Syn protofibrils can be determined using this well-characterized model. It can also be determined whether there are any changes in the clearance of endogenous α-Syn in primary hippocampal neurons derived from NAGLU-deficient and heterozygous mice transduced with the most deleterious NAGLU variants. The rate and level of α-Syn aggregates (inclusions) (see, for example, Figure 2) in primary neurons derived from NAGLU-deficient mice after treatment with α-Syn PFFs can be determined. Finally, it can be determined whether recombinant enzyme supplementation can rescue α-Syn PFFs in neurons derived from NAGLU-deficient mice.

[0185] Generation of α-Syn PFFs Recombinant monomeric α-Syn is prepared from bacteria and sequentially purified by size exclusion and ion exchange chromatography according to a previously established protocol. The protofibril form of α-Syn is prepared by size selection using a centrifugal filter device with specific molecular weight cut-off parameters after stirring the recombinant monomer at 37 °C for about 72 - 120 hours. The conditions for PFF generation have already been optimized. PFF is diluted with Tris-buffered NaCl and added to primary neurons cultured after 5 - 10 DIV. PFF transduction and seeding are confirmed by immunofluorescence or sequential extraction and immunoblotting 4 - 7 days after exposure. Abnormal α-Syn aggregates derived from endogenous α-Syn are detected by immunofluorescence using an anti-pSyn(Ser129) antibody, clone 81A (Biolegend, MMS-5091), and also by Western blot. α-Syn PFF is produced, handled, and disposed of in a BSL2 facility.

[0186] Transport, endocytosis, and intracellular localization of α-Syn PFF Lysosomal processing is the main fate of α-Syn protofibrils endocytosed in primary neurons. PFF-treated neurons are co-stained with presynaptic (CSPα), endocytosis (EEA1), autophagosome (Rab7), and lysosome (LAMP1) markers to determine whether there are changes in the transport of α-Syn aggregates in the lysosomal pathway.

[0187] Evaluate the impact on ALP function α-Syn aggregates impair overall macroautophagy by reducing autophagosome clearance. Therefore, pharmacological modulation of the autophagy pathway can determine whether α-Syn clearance can be improved.

[0188] Evaluate the impact on chaperone-mediated autophagy (CMA) α-Syn is degraded by chaperone-mediated autophagy (CMA), and α-Syn variants that are prone to aggregation block CMA. Therefore, examine the levels of LAMP2A and HSP70 proteins and see if they are affected in PFF-treated cells. Treat PFF-treated neurons with a CMA activator and determine AR7 (retinoic acid receptor alpha-specific antagonist) and α-Syn levels in cell lysates and conditioned media.

[0189] Effects on endocytosis and lysosomal membrane integrity α-Syn PFF induces the rupture of vesicles and lysosomes after endocytosis. These ruptured vesicles are positive for EEA1, LC3, and galectin-3. It is possible to determine whether α-Syn PFF affects the integrity of the lysosomal membrane by Gal-3 and LC3 staining.

[0190] Rescue experiments by enzyme supplementation Expose recombinant NAGLU to α-Syn PFF before, during, and after addition in the presence or absence of an uptake / binding inhibitor (mannose-6-phosphate: M3655, Sigma) and test its effect on α-Syn PFF aggregation. There is already sufficient recombinant NAGLU present to add in vitro to the deficient neurons.

[0191] Expected results The gene dosing effect of NAGLU on the uptake, transport, and aggregation of α-Syn PFF in vitro is expected, and it is predicted that the recombinant enzyme can rescue those effects. NAGLU-deficient cells accumulate HS and HSPG in the cell membrane and endosomal system. Therefore, an increase in the uptake of α-Syn PFF is expected, followed by disruption of vesicles and lysosomal membranes by endocytosis, increasing the cytoplasmic levels and mobilization of endogenous α-Syn. Lentiviral vectors can be generated to overexpress aggregation-prone α-Syn mutants in primary neurons derived from NAGLU-deficient and heterozygous mice, and the rates of aggregation and disassembly were evaluated. Alternatively, primary neurons from transgenic mice overexpressing human A53T α-Syn can be used, and those neurons can be transduced with selected variants in the NAGLU gene, and then the effects on α-Syn can be tested. iPSC-derived neurons from PD patients carrying variants in the NAGLU gene can also be used to compare the effects of such variants on α-Syn processing compared to CRISPr-corrected cells with the same genetic background.

[0192] Determine the effect of NAGLU on α-Syn spreading in vivo The accumulation experimental data indicate that the intercellular transmission of α-Syn follows a seeding principle similar to that observed for prion proteins. Intracerebral injection of brain extracts containing aggregated α-Syn (brain extracts derived from examinations of cases of Lewy body disease) into young mice (about 3 - 4 months old) overexpressing human A53T α-Syn stimulated the formation of pSyn lesions in the host, which were initially observed up to 30 days post-injection (dpi), and by about 90 dpi, pSyn had spread and was abundant in anatomically relevant regions of the brain, suggesting a spread comparable to the apparent spread of α-Syn deposits observed in human PD cases. At about 100 dpi, these mice developed motor dysfunction and died prematurely (about 126 dpi) compared to non-injected mice. Intracerebral injection of synthetic (human or mouse) α-Syn PFFs also induced LB-like lesions and neuronal degeneration in non-transgenic (wild-type) host mice (see, for example, Figure 4B). Approximately 50% of wild-type mice injected with insoluble pSyn from demented brains with LB developed pSyn lesions. In contrast, the induction efficiencies of pSyn lesions by human and mouse α-Syn PFFs were 90% and 100%, respectively. At 30 dpi, pSyn-positive LB-like accumulations were completely ipsilateral to the injection site (see, for example, Figures 4B and 4C). Not only in the LB / LN lesions in the affected ipsilateral region, but also in the contralateral neocortex, a marked increase in pSyn immunoreactivity was shown in mice examined at 90 and 180 dpi. α-Syn lesions in the substantia nigra pars compacta (SNpc) developed progressively after PFF injection, starting with faint cytoplasmic accumulations at 30 dpi and evolving into high-density perinuclear LB-like inclusions, particularly in the ventromedial SNpc population, at 90 and 180 dpi. At 90 and 180 dpi, SNpc dopaminergic (DA) neurons decreased by 15% and 35%, respectively, suggesting that LB / LN formation precedes SNpc DA neuron loss. Thus, the proliferation of LB / LN is connectivity-dependent, and the pathological accumulation of α-Syn appears to be upstream of and directly related to SNpc DA neuron loss.This in vivo model of intrastriatal injection of α-Syn PFF recapitulates the accumulation of intracellular LB / LN lesions, the selective loss of SNpc DA neurons, and the impairment of coordinated movement. Both LB and LN contain HSPG. However, the role of HS in α-Syn diffusion in vivo has not been evaluated. So far, it is not clear whether the decrease in NAGLU activity and the resulting HSPG accumulation affect the aggregation and diffusion of α-Syn. As described herein, NAGLU-deficient and heterozygous mice expressing the most deleterious NAGLU variants associated with PD can be used to test whether the accumulation of HS and HSPG affects α-Syn aggregation, diffusion, and accelerates the disease in vivo.

[0193] Intrastriatal inoculation of α-Syn PFF α-Syn PFF is prepared as described above. Intrastriatal inoculation of α-Syn PFF is performed in NAGLU-deficient and heterozygous mice injected with the most deleterious NAGLU variant using AAV2 / 9-PHP.B at birth, and NAGLU-deficient mice, heterozygous mice, and 12 wild-type mice. The diffusion of pSyn aggregates is quantified to evaluate whether α-Syn PFF affects the lifespan of NAGLU mice. Wild-type, NAGLU-deficient or heterozygous mice at 3-4 months of age are unilaterally injected into the ventral striatum (0.2 mm A / P, 2.0 mm M / L relative to bregma, 3.2 mm below the skull surface) by single inoculation of mouse αSyn PFF (or PBS or αSyn monomer as a control). The mice are allowed to recover and aged until 30, 90, or 180 days after injection, at which point the brains are harvested and processed with an anti-pSyn (Ser129) antibody for immunohistochemistry. pSyn co-localization staining is performed using anti-ubiquitin and HSP90. Based on the output analysis, n = 12 mice / group are required to detect a 30% increase in pSyn levels. (5 groups × 12 mice = 60 mice).

[0194] Quantification of αSyn and pSyn The cortex, hippocampus, striatum, and brainstem were excised from the hemisphere of each animal, and insoluble αSyn was isolated by ELISA and Western blot after sequential detergent extraction as previously published, using anti-synuclein-1 / Clone 42 (BD Biosciences) and anti-pSyn 81A (biolegend) as capture antibodies.

[0195] Immunoblot analysis of the ipsilateral and contralateral striatum from animals treated with PFF and PBS was performed using antibodies against tyrosine hydroxylase (TH) and dopamine transporter (DAT) as previously published. Brain atrophy and neuron loss in the SN were evaluated, and immunohistochemistry for markers of injury and inflammation (e.g., GFAP, Iba-1) was performed as described above.

[0196] The lifespan is compared between NAGLU-deficient animals treated with PFF and those treated with PBS. The rotarod and wire hang tests are known to be most sensitive for detecting motor deficits in wild-type mice 6 months after α-Syn PFF injection. The use of the rotarod and wire hang behavioral tests has been previously published in mouse models, and appropriate experimental design and statistical tools (ANOVA with post hoc analysis for multiple group comparisons, and Student's t-test for between-pair comparisons) are known. Gait analysis is performed in animals treated with PFF and PBS. It has been previously shown that gait analysis is highly sensitive for capturing "Parkinson-like" symptoms in a mouse model of LSD.

[0197] Regarding performance in the rotor rod and wire hang assays, for comparing five groups at once, if normal animals perform in 60 s, NAGLU-deficient animals perform in 0 s (40 weeks), and the standard deviation is 30 s, the effect size is 0.89. If the effect size is 0.89, α = 0.05, and power = 0.95, six animals are needed to detect a significant difference between groups. Regarding lifespan, for a comparison between five groups including normal animals, hemizygous animals with an intermediate lifespan of about 730 days, and NAGLU-deficient animals of about 322 days, with a standard deviation of 20 days, the effect size is 5.51. If the effect size is 5.51, α = 0.05, and power = 0.95, only two to three animals are needed to detect a significant difference between groups. Since 10 - 12 mice / group are used, the studies of behavior and lifespan are well-powered.

[0198] Expected results It is expected that α-Syn PFF injection accelerates the phenotypes of NAGLU-deficient mice that increase gliosis, neurodegeneration, worsen motor impairment, and shorten lifespan. Hemizygous NAGLU mice exacerbate the propagation of pSyn lesions. Alternatively, an AAV2 / 9 vector carrying the α-Syn mutation can be generated and injected stereotaxically into the striatum of young NAGLU-deficient mice, or mice overexpressing human A53T α-Syn can be crossed with NAGLU-deficient mice to evaluate pSyn lesions, disease progression, and changes in lifespan. The same approach can be applied to SGSH-deficient mice.

[0199] Example 2: Investigate age-dependency in the autophagy-lysosome pathway (ALP) and genetic variations underlying the decline associated with Alzheimer's disease (AD) This example describes the identification of genetic variations underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the onset of Alzheimer's disease (AD).

[0200] Multiple in vitro and in vivo studies have suggested that autophagy-lysosome pathway (ALP) dysfunction contributes to the development of AD, but the age-dependence of ALP function and the genetic variations underlying the AD-related decline are not well understood. Rare functional variants in genes causing AD and multiple AD risk genes cause ALP dysfunction. Studies on a single ALP gene in isolated populations support the genetic overlap between AD and lysosomal storage diseases (LSDs). However, a systematic and comprehensive assessment of the contribution of genetic variations within each gene in ALP to the risk of developing AD and its role in AD pathogenesis has not been completed. To address this gap in current knowledge, the studies described herein can identify and prioritize rare functional variants with large effect sizes in ALP genes associated with the risk of developing AD. As described herein, an integrated framework combining computational methods and experimental data can be used to validate the functional impact of selected ALP genes in both in vitro and in vivo settings. First, the amount of rare functional variants in each gene of ALP from 33,350 non-Finnish European controls is compared to 2,000 AD cases and 3,000 controls. The results from an additional independent sample including 2,000 AD cases and 2,000 controls are replicated with 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, cell-based assays are used to examine the effect of selected variants in candidate ALP genes associated with AD risk on enzyme activity, protein stability, and / or mRNA levels, and its effect on lysosome function. The effect of validated functional variants on amyloidogenesis and Aβ degradation is examined. Finally, quantitative and qualitative pathological investigations of the spontaneous occurrence of AD lesions are performed in hemizygous or knockout models of candidate ALP genes associated with AD risk. The effect of gene dosage of candidate ALP genes on the amount of Aβ plaques is also measured in well-characterized mouse models of AD.The research outlined in this specification can identify novel ALP genes associated with AD. These experiments can provide deeper insights into the mechanisms of ALP dysfunction in the onset of AD and establish a basis for repurposing existing treatment strategies for lysosomal storage diseases for potential treatment of AD.

[0201] The goal of the research described in this specification is to identify genetic variations underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the onset of Alzheimer's disease (AD). These studies incorporate an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. The experiments outlined in this specification can identify novel ALP genes associated with AD. These experiments can provide deeper insights into the mechanisms of ALP dysfunction in the onset of AD and establish a basis for repurposing existing treatment strategies for lysosomal storage diseases for potential treatment of AD.

[0202] Age is the greatest risk factor for the onset and progression of Alzheimer's disease (AD). At the cellular level, aging reduces the degradative capacity of the autophagy-lysosome pathway (ALP). Multiple in vitro and in vivo studies have suggested that defects in the clearance of aggregated proteins due to ALP dysfunction contribute to the onset of AD, but the age-dependence of ALP function and the genetic variations underlying its AD-related decline are not fully understood. Rare functional variants in genes that cause AD and multiple AD risk genes cause ALP dysfunction. Studies on single ALP genes in isolated populations support the genetic overlap between AD and lysosomal storage diseases (LSDs). However, a systematic and comprehensive assessment of the contribution of genetic variations within each ALP gene in the general population to the risk of developing AD and its role in AD pathogenesis has not been completed.

[0203] To address this gap in current knowledge, the study described herein identifies and prioritizes rare functional variants with large effect sizes in genes of ALP associated with the risk of developing AD. Using an integrated framework that combines the computational methods and experimental data described herein, the functional impact of selected ALP genes can be verified both in vitro and in vivo. The feasibility of the study described herein is supported by an unbiased approach with previously identified rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that account for a large proportion in both familial and sporadic AD. Thus, analysis of all ALP genes is expected to reveal an enrichment of rare functional variants in AD patients compared to the variation found in the general population. The study described herein overcomes the current false associations resulting from stratified populations present in studies on isolated populations and provides complementary functional characterization at both the variant and gene levels in vitro and in vivo.

[0204] (I) Identify ALP genes enriched with rare functional variants in AD The hypothesis has been proposed that rare, untested functional variants in the ALP gene affect the risk of developing AD. To identify these risk variants, whole-exome sequencing (WES) data from 33,350 controls [Exome Aggregation Consortium (ExAC) database, non-Finnish European] were analyzed to determine baseline genetic variation in each ALP gene from European individuals. Next, gene-based analysis of the ALP gene was performed in 2,000 AD cases and 3,000 in-house controls by combining exome chip and WES data. These results were replicated in an independent sample including 2,000 AD cases and 2,000 controls, along with 10,000 publicly available samples of WES data from the Alzheimer's Disease Sequencing Project (ADSP). After collapsing rare variants, enrichment of predicted functional variants with large effect sizes (OR>2.5) was identified in at least 12 lysosomal genes (see results).

[0205] (II) Determine the functional impact of selected candidate genes of ALP on AD pathogenesis in vitro The hypothesis has been proposed that novel ALP genes associated with AD risk play a role in AD pathogenesis in vitro. Using cell-based assays, the effects of selected variants in candidate ALP genes associated with AD risk on enzyme activity, protein stability and / or mRNA levels, and their effects on lysosomal function are examined. The effects of validated functional variants on amyloidogenesis and Aβ degradation are examined. A novel role for the protein CSPα encoded by the DNAJC5 gene as a functional lysosome-related protein has been previously discovered. In addition, compelling data have been collected showing that CSPα plays a role in APP processing and amyloidogenesis (see results).

[0206] (III) Determine the functional impact of selected candidate genes of ALP on AD lesions in vivo The hypothesis has been put forward that novel ALP genes associated with AD risk play a role in the development of AD in vivo. Quantitative and qualitative pathological investigations of the spontaneous occurrence of AD lesions are performed in hemizygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice at three time points defined by the time of onset of the endogenous lesions. The effect of the gene dosage of the NAGLU, NPC1, and DNAJC5 genes on the amount of Aβ plaques is measured in well-characterized mouse models of AD.

[0207] The experiments described herein can identify novel ALP genes associated with AD. These can provide deeper insights into the mechanisms of ALP dysfunction in the development of AD, the currently lacking knowledge, and the promise of obtaining novel therapeutic targets. The studies described herein can establish a basis for repurposing existing therapeutic strategies for LSD for potential treatment of AD.

[0208] Significance There are at least 430 genes in the human genome associated with the autophagy-lysosome pathway (ALP) (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosome genes, and 167 lysosome regulatory genes). Mutations in 38% of all ALP genes (157 genes) cause Mendelian genetic diseases (OMIM), among which the most studied are classical lysosomal storage diseases (LSDs). There are at least 50 different LSDs, which together occur at a frequency of approximately 1 in 7,700 live births. LSDs are generally regarded as pediatric disorders and are typically caused by complete loss-of-function (LoF) mutations. However, adult-onset forms of LSDs carrying hypomorphic variants have been reported. Although LSDs are single-gene disorders, they can exhibit complex clinical features. In fact, approximately 75% of LSDs have a clinically significant neurological component.

[0209] The significantly high co-occurrence rate of complex diseases and Mendelian genetic diseases indicates that genes and pathways disrupted in Mendelian genetic disorders also play a role in the etiology of the corresponding complex diseases. In summary, nearly 20% of the genes involved in Mendelian phenotypes either contain variants that cause genome-wide association study (GWAS) signals for complex traits or are closest to those variants. In contrast, about 15% of all genes underlie Mendelian phenotypes overall, suggesting that genes involved in Mendelian phenotypes are enriched in GWAS signals. Approximately 35% of the ALP genomic region is associated with GWAS traits (GWAS Catalog). In fact, 18.5% of the genes within the ALP pathway play a role in both Mendelian genetic diseases and common diseases. Approximately 22% are genes that cause LSD. Evidence from multiple lines (in vitro and in vivo) suggests that AD shares molecular mechanisms with LSD, but the genetic variations underlying AD-related dysfunction in ALP are not fully understood.

[0210] The major hypotheses regarding the cause of AD are derived from genetic studies of age-related and early-onset diseases, both of which are associated with increased production and aggregation of amyloid-β (Aβ) peptide. Complementary hypotheses suggest that defects in the clearance of pathogenic proteins, including Aβ, can cause AD in patients without mutations in Mendelian AD genes. Dysregulation of endosome-lysosome and autophagy occurs in AD patients and AD mouse models. Neuronal accumulation of autophagic vacuoles (AV) has been found in the brains of AD patients and mice treated with lysosome inhibitors or cathepsin-deficient mice. Lysosomal hydrolases are also strongly upregulated in neurons of AD patients. In addition, lysosomes play an important role in both normal and abnormal APP processing and subsequent amyloidogenesis. Impairment of lysosomal function in vitro causes changes in Aβ production. Exposure to ammonium chloride or bafilomycin A1 reduces Aβ secretion. Treatment with lysosomal protease inhibitors decreases the production of amyloidogenic APP fragments within lysosomes. Lysosomal enzyme and lysosome-related protein levels change in the cerebrospinal fluid (CSF) of AD patients. All of these findings support the hypothesis that cumulative "hits" at multiple sites within the ALP during AD cause a selective failure that impairs the clearance of pathogenic proteins.

[0211] Rare variants in genes that cause LSDs such as CSTD, NPC1, and NPC2 increase the risk of AD in selected populations. In addition, studies in mice lacking genes that cause LSDs have revealed the unique roles of each gene in APP processing and amyloidogenesis. Mice lacking the NPC1, CLN3, and HEXB genes increase both the levels of α-CTF / β-CTF and Aβ40 / 42. Mice lacking the IDUA, SGSH, GBA, and TPP1 genes increase intracellular APP / Aβ levels without having Aβ plaques. Mice lacking the IDUA and SGSH genes cause a three-fold increase in Aβ40 compared to controls that do not have detectable levels of Aβ42. Mice lacking the ASAH1 and PPT1 genes decrease intracellular APP / Aβ without having plaques. However, a systematic and comprehensive assessment of the contribution of genetic variation within each gene of ALP to the risk of developing AD and its role in AD pathogenesis has not been completed (see, for example, FIG. 5).

[0212] Newborn screening studies have shown that there are 10-fold differences in the levels of lysosomal enzyme activity reported in healthy humans. Heterozygous carriers of disease-causing variants in the GBA, NPC1, GALC, GAA, GLA, and IDUA genes show significantly lower levels of enzyme activity than controls. In addition, heterozygous carriers of disease-causing variants in NPC1 show significant metabolic abnormalities downstream of the primary pathway affected in Niemann-Pick patients. So far, there do not appear to be systematic studies that have focused on such metabolic changes and the long-term consequences of ALP dysfunction. However, there are few studies suggesting that heterozygous carriers of disease-causing variants are at high risk of neurodegenerative diseases.

[0213] The research described in this specification can address multiple gaps in current knowledge. First, using whole exome sequencing (WES) data from large databases, the actual baseline gene variations in each gene of ALP for European ancestry (EA) individuals are determined. Second, the cumulative allele frequencies of rare functional variants in each gene of ALP are analyzed in AD cases to identify candidate genes that influence the risk of developing AD. Finally, the roles of these candidate genes are verified in the development of AD in vitro and in vivo. By identifying specific defects in the ALP genes in AD, multiple existing treatment strategies for LSDs, including gene therapy, enzyme replacement, oral small molecule substrate inhibition therapy, small molecule chaperones, and pharmacological restoration of the autophagy pathway, can be utilized for potential treatment of AD.

[0214] Innovation The experiments outlined in this specification are innovative in their design to define gene variations associated with the well-known dysfunction in ALP related to AD and to understand the impact of rare functional variants in the ALP genes related to AD in vitro and in vivo.

[0215] This data provides compelling evidence to support the feasibility of the research described in this specification. Enrichment of predicted rare functional variants has been identified in several candidate genes, including the NAGLU, NPC1, PPT1, GLB1, and DNAJC5 genes (described below). These results include novel ALP genes associated with AD risk, opening new avenues into the mechanisms of ALP dysfunction in AD pathogenesis. For example, based on this gene analysis, a novel role as a functional lysosome-related protein has been found for the protein encoded by the DNAJC5 gene CSPα. Additionally, compelling evidence is provided that CSPα plays a role in APP processing and amyloidogenesis.

[0216] The current state of knowledge regarding gene variations in the ALP gene in AD is dominated by limited studies reporting spurious associations in isolated populations with very low replication rates. The study described herein can overcome that limitation because its design allows for a systematic and comprehensive assessment of the contribution of gene variations in each ALP gene to the risk of developing AD in a very large sample representing the general population. Thus, data were used from two large publicly available databases of WES data, in addition to in-house WES and exome chip data, for a total of AD cases (n ~ 4000) and controls (n ~ 5000), from the Exome Aggregation Consortium (ExAC) (n ~ 61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n ~ 10,000). Using these multiple datasets, a series of analyses were designed to resolve the genetic architecture of the well-known dysfunction in ALP associated with AD.

[0217] There is a general assumption that ALP dysfunction is associated with AD. However, there is no consensus on the role of the ALP gene in AD pathogenesis. This is due in part to the fact that most studies have focused on a few genes (e.g., cathepsin D) using classical expression systems (neuron-like cell lines) that may not be the appropriate cell type. In addition, these studies do not evaluate the appropriate AD pathway (APP processing vs. tau aggregation). The study described herein incorporates an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. The cell-based assays complement biochemical data, live cell assays, RNAseq data from specific brain cell types in mice, genome-wide gene expression data in human AD cases and controls, genome-wide gene expression data from human AD cases at different stages, genome-wide gene expression data from humans of different ages, and genome-wide gene expression data from AD mouse models correlated with Aβ plaque and neurofibrillary tangle burden.

[0218] Most of the studies using ALP gene knockout mice attempt to understand the role of the ALP gene in the development of AD by focusing on AD lesions. However, the lesions resulting from the defective gene are rapid and more closely related to LSDs. This complicates the interpretation of these results and, in most cases, there is no clear rationale. The study described herein can evaluate vulnerable brain regions for AD lesions, including the amount of Aβ plaques at different time points, in hemizygous mice from selected ALP genes based on the results of genetic analysis supported by cell-based assays.

[0219] Experimental approach Overview of the project Multiple in vitro and in vivo studies suggest that ALP dysfunction contributes to the development of AD. However, the genetic architecture underlying the AD-related decline in ALP function is not fully understood. To address this issue, an innovative approach that combines the analysis of predicted rare functional variants in the genes of ALP in large datasets (both in-house databases and publicly available ones) is used to prioritize candidate genes in ALP that have evidence of involvement in AD risk (see, for example, Chapter (I)). The main result expected from the analysis in Chapter (I) is the identification of genes that are not single variants related to AD. Next, the functional impact of the selected variants is verified in their respective encoded proteins. In Chapter (II), the impact of partial loss of function of the selected genes related to AD risk can be determined in cell-based assays. This process is integrated with a wide-ranging data mining process from both human diseases and mouse models, collecting biochemical, pathological, and cell functional evidence to support the role of the selected candidate ALP genes in AD pathogenesis. In this interconnected process, computational data defines and refines experimental data and vice versa (see, for example, Chapter (II)). Finally, it can be determined whether the selected ALP genes are associated with in vivo AD lesions (see, for example, Chapter (III)). The experiments outlined here are based not only on genetic and cell-based findings but also on the availability of mouse models. In addition, it can be determined whether partial loss of function of the selected ALP genes related to AD modifies AD lesions (Aβ plaques in vulnerable brain regions). Both homozygous (- / -) and heterozygous (+ / -) mice for the selected genes are examined to distinguish whether the AD lesions are the result of endogenous lesions in these mouse models. In addition, the onset of AD lesions is examined in aged heterozygous (+ / -) mice for the selected genes. The effect of the gene dosage of the selected ALP genes on the amount of Aβ plaques is also measured in well-characterized mouse models of AD.The expected results from these experiments are the confirmation of the contribution of the single ALP gene to AD lesions in vivo.

[0220] Data The list of autophagy lysosome gene sets (about 430 genes) has been manually curated and derived by mining existing annotations in public databases (e.g., see bioinformatics analysis) and the literature. The most well-studied model of ALP dysfunction is LSD. LSD is a genetically heterogeneous group of Mendelian genetic diseases caused by LoF homozygous, compound heterozygous mutations or copy number variations (CNVs). The incidence of LSD in different populations (mostly isolated populations) is very low. Therefore, the expected frequency of variants causing LSD in the general population is extremely low. The ExAC database containing over 60,000 sequenced exomes was used to estimate the frequency of variants causing LSD in a wide-ranging sampling of multiple ethnic groups. Only three genes (GLA, IDS, and LAMP2) causing X-linked LSD are loss-of-function (LoF) intolerant (pLI ≥ 0.9). The number of LoF mutations observed in 43 additional genes causing LSD is less than the number expected under the neutral model. Genes causing LSD show variants that change a wide range of proteins, from 32 in the NPC2 gene to 423 in the GAA gene. Many of these variants are predicted LoFs and likely behave as low-penetrance variants, probably in the heterozygous situation. This large number of protein-changing variants may explain the wide range of levels of lysosomal enzyme activity reported in humans. Interestingly, the GAA gene shows the largest number of protein-changing variants in ExAc and exhibits more extensive variability in enzyme activity.

[0221] Most of the AD samples are of European origin. Therefore, this analysis focused on genes (n = 46) causing LSD in non-Finnish samples (about 33,000 individuals). In the NCBI ClinVar database, about 2,740 variants causing LSD have been reported. There are 288 variants causing LSD annotated in the ExAC sample, 76% of which are missense variants, 10% affect alternative splicing, and 12% are nonsense mutations. Most of the variants causing LSD are predicted to be harmful (87%) by SIFT and predicted to be damaged (84%) by polyphen2. Most of the variants causing LSD (73%) are located within highly conserved nucleotides (GREP score > 4). This underestimated the frequency of variants causing LSD diseases in the EA population because CNVs were not included. CNVs are a common cause of LSD. In addition, some LoF variants reported in ExAC were found not to be classified as variants causing LSD in the NCBI ClinVar database. Variants causing LSD were found in each gene causing LSD, but the number of variants causing LSD varies from 1 found in the HYAL1 gene to 20 found in the ARSA gene. The cumulative minor allele frequency (cMAF) (number of heterozygous carriers) of these variants per gene ranges from 1.50E -05 to 0.003 in the NPC2 gene. Therefore, variants with a higher frequency than this in the general population are not expected to be variants causing high-penetrance LSD, so a cMAF threshold of 1×10 -3 was applied as a conservative upper limit.

[0222] The discovery sample was composed of WES data from 523 unrelated AD cases and 386 controls. Table 5 shows the top genes causing LSD associated with AD.

Table 5

[0223] These were analyzed using inclusion and exclusion criteria defined based on the features that define the variants causing LSDs in the ExAc samples. As expected, the gene-specific cMAFs from the ExAC dataset (European, non-Finnish) were highly concordant with the cMAFs from the in-house AD database (European) (see, for example, Figure 6). Variants in each gene causing LSDs that met the inclusion criteria (n = 46) were collated gene by gene. 82% were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The number of variants included in the analysis varied from 5 in the ARSB gene to 21 in the NPC1 gene per gene. The amount of variant (cMAF) changing rare proteins was compared with the amounts observed in controls and ExAC. For most of these genes, there was excessive variation in cases compared to controls, but only weak associations were found with the SGSH gene (p = 4.2×10 -3 , OR = 3.7, 95% CI 1.4 - 9.6) and the CLN8 gene (p = 1.0×10 -2 , OR = 8.9, 95% CI 1.1 - 68.1) (see, for example, Table 5). When compared with the cMAF of the ExAC samples, 14 genes passed a very stringent multiple testing correction threshold of p < 1.0×10 -4 (0.05 / 450), and genes causing 13 LSDs passed a gene-level significance threshold of p < 2.4×10 -6 (0.05 / 20,000) (see, for example, Table 5). Next, two additional AD cohorts were used to replicate the findings listed in Table 5, including 1722 AD cases, and the data were obtained using human exome chip and WES data from 1394 familial AD (FAD) cases (see, for example, Table 6).

Table 6

[0224] As expected, only six genes were replicated in the samples using exome chip data, but most of the associations were replicated in the FAD samples (see, for example, Table 6). The most likely explanation for this discrepancy is the coverage depth of the data. The best example is the results for the NAGLU gene, from which the variant did not meet any of the inclusion criteria using data from the exome chip (see, for example, Table 6). Of note is the fact that the associations found in the replication samples are in the same direction and the effect sizes are the same.

[0225] One of the ALP genes replicated in three samples is the DNAJC5 gene encoding cysteine string protein α (CSPα), and its mutation causes adult-onset LSD. CSPα was localized to the plasma membrane within neurites. CSPα had a diffuse cytoplasmic localization in the neuron-like cell type (N2A), but also had a portion of endogenous CSPα co-localized with LAMP2 in the cell body (see, e.g., FIG. 7A). Subcellular fractionation showed that a significant proportion of CSPα co-precipitated with another lysosome marker (LAMP1) (see, e.g., FIG. 7B). These results suggest that endogenous CSPα is a lysosome-related protein. As expected, significantly higher levels of LysoTracker signal were present in cells expressing the mutation (p.L115R) that causes LSD compared to the empty vector. In contrast, CSPα-WT transduced cells had a significantly reduced LysoTracker signal compared to cells expressing CSPα-p.L115R or the empty vector (see, e.g., FIG. 7C), suggesting that CSPα may be involved in the regulation of lysosomal pH. Expression of the mutation (p.L115R) that causes LSD resulted in a significant increase in intracellular and secreted lysosomal enzymes compared to the empty vector (see, e.g., FIGS. 7D and 7E). In contrast, overexpression of CSPα-WT resulted in a significant decrease in intracellular and secreted lysosomal enzymes compared to cells transduced with the empty vector or CSPα-p.L115R (see, e.g., FIGS. 7D and 7E), suggesting that lysosomal trafficking and exocytosis are affected by CSPα.

[0226] The DNAJC5 transcript is highly expressed in neurons and in the brain regions most vulnerable to AD lesions. A decrease in DNAJC5 transcript levels with age was found in neuropathologically normal brain samples from the prefrontal cortex region of young (less than 40 years old), middle-aged (40 - 70 years old), and normal elderly adults (70 - 94 years old) (p = 0.0003, GEO database, Series GDS5204) (see, for example, Figure 8A). The DNAJC5 transcript levels were significantly lower in AD cases compared to age-matched controls in laser capture microdissected, tangle-free neurons from AD and controls (p < 0.0001, see, for example, the left graph in Figure 8B) (GEO database, Series GSE5281). This finding was replicated in a different study (GEO database, Series GSE15222) (see, for example, the right graph in Figure 8B). Also, compared to the levels in wild-type mice (black line in Figure 8C), in the cortex of two AD mouse models (TAU, p.P301L, blue line in Figure 8C) and (APP, p.K670N / p.M671L, red line in Figure 8C), the DNAJC5 transcript levels showed an age-dependent decrease and were found to be inversely proportional to the onset of AD lesions (right graph, see, for example, Figure 8C). All of these results suggest that CSPα is likely to be involved in the pathogenesis of AD.

[0227] The brains of patients with the variant (p.L115R) that causes LSD in CSPα do not show Aβ plaques or neurofibrillary tangles. However, histological analysis revealed significant intracellular accumulation of APP / Aβ (antibody 4G8) in cortical neurons (see human LSD, e.g., Figure 9A). Therefore, the role of CSPα in amyloidogenesis was tested in vitro. APP / Aβ immunoreactivity co-localized with lysosomal markers in N2A695 cells (see empty vector, e.g., Figure 9B). Knockdown of CSPα expression in N2A695 cells decreased Aβ / APP levels and their co-localization with Lamp-1 (see shRNA, e.g., Figure 9B). N2A695 cells stably expressing the variant that causes LSD in CSPα showed an intracellular accumulation mechanism of APP / Aβ (see p.L115R, e.g., Figure 9B). N2A695 cells expressing hCSPα-p.L115R released significantly higher levels of Aβ40 and Aβ42 into the medium than cells transfected with the empty vector (see the left graph in Figure 9C, for example). Conversely, N2A695 cells expressing specific shRNA-CSPα secreted significantly lower extracellular levels of Aβ40 and Aβ42 (see the left graph in Figure 9C, for example). N2A695 cells expressing hCSPα-p.L115R accumulated more Aβ40 intracellularly than when transfected with the empty vector (see the right graph in Figure 9C, for example). There was no difference in the levels of Aβ42 across different groups (see the right graph in Figure 9C, for example). Cells transfected with shRNA-CSPα showed a decrease in the levels of full-length APP, α-CTF / β-CTF, CSPα, and sAPPα (see the graph in Figure 9D, for example). In hCSPα-p.L115R, there was an increase in full-length APP and α-CTF / β-CTF, and no change in the level of sAPPα (see the graph in Figure 9D, for example). These results suggest that a novel and unexpected role of CSPα in amyloidogenesis and perhaps in the pathogenesis of AD has been revealed.

[0228] Research Design and Methods (I) Identify the ALP gene enriched with rare functional variants in AD The hypothesis has been proposed that untested rare functional variants in the ALP gene affect the risk of developing AD. As described herein, an innovative approach that combines the analysis of predicted rare functional variants in the ALP gene in large datasets (both in-house databases and publicly available ones) can be used to prioritize candidate genes in ALP with evidence of involvement in AD risk.

[0229] Define the allele frequency threshold for rare variants Using information obtained from the analysis of variants causing LSD, including the maximum MAF, SIFT, Polyphen2, or GERP score of variants causing LSD, the following inclusion criteria were defined. 1) Call rate in AD cases > 98%, 2) Maf per variant < 0.01%, 3) Only variants likely to change the protein in the designated canonical transcript annotated as missense by ExAc or Ensemble, 4) Frameshift, 5) Nonsense, 6) Variants affecting splice donor and acceptor regions, 7) Presence of evidence of pathogenicity in the NCBI ClinVar database and whether it is located in the 3' or 5' UTR region. Variants not found in ExAC, or not present in the NCBI ClinVar database, or their synonymous intronic 3' or 5' UTR variants with Maf > 0.01%, and miscalls in ExAC and ClinVar were excluded. Individuals were selected based on the results of principal components to ensure that population-specific variants had no confounding effect on this analysis.

[0230] Study population WES was obtained from 2,000 individuals. Access to exome chip data is available from a total of 3,000 individuals from Knight-ADRC. Access to GWAS data that has been cleaned and imputed for the ADNI and Knight-ADRC samples is also available. DNA is available for the Alzheimer’s disease Neuroimaging Initiative (ADNI, 600 cases and 200 controls), NIA-LOAD (867 unrelated cases and 645 unrelated controls), Knight Alzheimer’s Disease Research Center (Knight-ADRC, 779 cases, 555 controls), and Spanish dataset (167 cases and 534 controls). Descriptions of these datasets have been published previously. Each case received a diagnosis of Alzheimer's dementia using criteria equivalent to those of the National Institute of Neurological and Communication Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association for probable AD. Controls received the same evaluations as cases but were cognitively normal. All individuals were of European ancestry and written consent was obtained from all participants. Data were downloaded from ExAC (version 0.3.1, March 2015). Only genes containing a high proportion of coding regions extending to a median sequence depth of over 30-fold, and only high-quality (PASS filter) variants were included in this analysis. Data were downloaded from ADSP. The discovery stage dataset included WGS data for 584 subjects from 113 families, additional pedigree data for 853 (682 cases [510 non-Hispanic, 172 Hispanic]), and 171 Hispanic control subjects from families with multiple individuals affected by AD.

[0231] Whole exome sequencing data WES exists from 2,000 individuals. Exome enrichment is performed using the SureSelect 52Mb Target Enrichment System (Agilent). DNA is sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling are performed using Novoalign and SAM tools. These methods have good specificity and sensitivity for genotype calling and have been used previously. GWAS data are used for quality control of sequence calls. In this study, a concordance rate of over 98% was observed between exome sequencing calls and GWAS data.

[0232] Human exome chip data Access exists to exome chip data from a total of 3,000 individuals from Knight-ADRC. Illumina and Affymetrix developed an inexpensive off-the-shelf genotyping chip called "exome chip" that contains variants within exons that have been reported at least twice in the exome variant server database. Most of the coding variants included in the exome chip are very rare variants with MAF < 0.01. These arrays provide a rapid and simple method for analyzing low-frequency variants. However, these arrays do not include all coding variants.

[0233] Quality control (QC) of exome chip Genotype calls were made using best practices for calling Illumina exome chip data described elsewhere. QC of the exome chip is similar to the QC steps used for GWAS, but variants were not removed due to low MAF. Raw data exist for the exome chip, and clusters are examined for any significant associations at the single variant or gene level.

[0234] Mutation quantity test To address the impact of rare variants with moderate effect sizes, validated statistical methods have been developed to analyze the association with rare variants. Briefly, gene-based methods collapse rare variants within a region into a single value and then test the association between the rare variants within a region and the trait of interest. The Sequence Kernel Association Test (SKAT) is used to test the association between the context within a gene region and rare variants. The advantage of SKAT over other gene-based methods is that SKAT can account for variants that affect in different directions within the same gene and adjust for confounding covariates. Odds ratios with 95% confidence intervals are calculated for alternative alleles compared to the most common allele. In allele tests, if an association is detected, further analysis determines which of the additive or dominant models is a better fit. Independent case-control samples are used to replicate findings from the discovery dataset. Analyses of each different dataset are performed separately. Joint analysis is done to combine p-values and ORs. This method has been successfully used in previous studies to identify novel genes for AD.

[0235] GWAS data Genome-wide genotyping has been previously generated for most (over 90%) of the samples. Genotyping was performed on various arrays including the NeuroX-chip (WU and PPMI). Prior to association analysis or imputation, all samples and genotypes undergo stringent quality control (QC). Genotype data are cleaned by applying a minimum call rate (98%) for SNPs and individuals and a minimum minor allele frequency (MAF = 0.02). SNPs not in Hardy-Weinberg equilibrium (P < 1×10 -6 ) are excluded. Tests for unexpected duplications and cryptic relatedness are performed using pairwise genome-wide estimates of the proportion of familial identity.

[0236] Imputation Using the 1000 Genomes project data (phase 3, released in November 2014) and Impute2 software, up to 6 million SNPs are imputed. R 2 <0.5, minor allele frequency (MAF) < 0.02, out of Hardy-Weinberg equilibrium (p < 1 × 10 -6 )), call rate < 95%, or SNPs with Gprobs score < 0.90 are removed. In previous GWAS and imputation processes, a total of 6,815,690 SNPs passed the QC process.

[0237] Population structure Considering the availability of GWAS data, Eigenstrat is used as an anchor on the samples together with HapMap samples to confirm self-reported race / ethnicity. Three first principal component factors (PCs) from the population stratification analysis are included in this analysis as covariates.

[0238] Data storage and management Up to 150 GB of processed data is generated for each exome, and it takes 3 days to align the sequences and call SNPs. Therefore, it is necessary to have a large-scale and secure data storage system. All generated data is stored on a Linux server with 3 terabytes (TB) of space. When the data is processed and the sequence variants detected by exome sequencing are confirmed by genotyping, efficient and effective methods for data management, quality control, cleaning, annotation, and analysis developed and used for other exome sequencing projects are applied.

[0239] Bioinformatics analysis For complementary analysis, the following publicly available databases are used: Online Mendelian Inheritance in Man (OMIM), Exome Variant Server ExAC, GWAS Catalog, GERP4, ClinVar database, and Human Autophagy Database.

[0240] Output analysis To determine the output for detecting genetic variants related to age at onset, the analysis was performed using Proc Power in SAS. The analysis was carried out using a minor allele frequency in the range of 0.05 - 0.50, an OR in the range of 1.2 - 3.6, and a sample size in the range of 4,000 - 7,000. α was adjusted to 5×10 -8 in single variant analysis and 5×10 -6 in gene-based analysis. Based on these results, there is approximately 80% output to detect an effect with OR > 1.19 (or < 0.84).

[0241] Expected results The feasibility of the study described herein is supported by an unbiased approach that has identified rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that account for a large proportion in both familial and sporadic AD. The objective of (I) is to identify genes related to AD risk among approximately 430 genes in the human genome belonging to ALP. Studies focusing on genes causing LSD (n = 46) have been able to identify at least 12 additional novel genes in ALP that are related to AD risk and have a large effect size (see, for example, Table 5), indicating that these can be replicated in additional samples with appropriate coverage depth (see, for example, Table 6). These results support the hypothesis that variations in ALP genes are higher in AD patients than those found in the general population. Therefore, it is expected that new associations between AD risk and some of the remaining approximately 384 ALP genes included in the analysis will be revealed.

[0242] This calculation of results and outputs suggests that there is sufficient output in gene-based analysis to detect an average odds ratio greater than 2.7. If the association of the ALP gene fails to replicate in a case-control design, an endophenotype design can be used. Thus, the impact of variants in the ALP gene on CSF biomarker levels for AD can be determined by performing single variant and gene-based analyses for each of the CSF biomarkers, the LSD gene, and t-tau, p-tau, and Aβ42. To evaluate whether the regulatory genomic region of the ALP gene may be involved in the risk of developing AD, the association of the ALP gene can be analyzed in data from a previously published GWAS by the International Genomics of Alzheimer’s Project (I-GAP) consisting of a total of 25,580 AD cases and 48,466 controls. A complementary approach would be to examine whether the ALP gene affects the age at onset (AAO). Thus, data from previously published GWAS that investigate genetic variants associated with the age at onset of AD can be examined.

[0243] Using whole exome sequencing, it is predicted that most variants that change proteins can be identified. However, it has been shown that complementing WES data with RNAseq data can identify more potential functional variants in specific variants that affect splice donor and acceptor regions. RNAseq data are currently being generated from 500 brain tissue samples of AD cases and controls for which WES data are already available, enabling analysis of the impact of variants that affect splicing in the ALP gene in AD pathogenesis. Whole genome sequencing of 100 individuals has also been generated to perform a more detailed analysis of the role of the non-coding genomic regions of the genome.

[0244] (II) Determine the functional impact of selected candidate genes of ALP on AD pathogenesis in vivo The hypothesis has been proposed that novel ALP genes associated with AD risk play a role in AD pathogenesis in vitro. As described herein, cell-based assays can be used to examine the effects of selected variants in candidate ALP genes associated with AD risk on enzyme activity, protein stability, and / or mRNA levels, as well as their effects on lysosomal function. The effects of validated functional variants on amyloidogenesis, APP processing, and Aβ degradation can be examined as described below.

[0245] Methodology and analysis Evaluating all variants of each ALP gene associated with AD identified in (I) is beyond the scope of the experiments described herein. Therefore, the top 3-5 variants identified in (I) are prioritized in genes that meet the following criteria: 1) There are available (commercially available or through collaborators) mouse models that can be obtained and used to perform the in vivo experiments outlined in (III) on primary neurons / microglial cells. 2) There are available biochemical and / or cell-based assays to detect changes in their function. 3) There are collaborators at Washington University in St. Louis with sufficient expertise to assist in the analysis of the data. Therefore, these three genes, NAGLU, NPC1, and DNAJC5, have been selected, taking into account the strength of the data from both discovery and replication samples and all of the above criteria. Expertise and appropriate collaboration are available to perform all of the experiments described herein.

[0246] Cloning of selected ALP genes It is important to characterize the effects of wild-type and mutant candidate genes on protein expression and normal function. cDNA clones are purchased from candidate genes from Origene or Invitrogen. The selected variants are engineered using a site-directed mutagenesis kit (QuikChange II (Agilent Technology, Santa Clara, CA, USA)).

[0247] Lentivirus production Wild-type and mutant cDNAs are subcloned into the pLenti-III-PGK Vector (Applied Biological Materials Inc, Richmond, Canada) carrying the puromycin resistance gene. The resulting lentiviral vectors are co-transfected into HEK-293T packaging cells as described above along with plasmids encoding VSV-G, Gag-Pol, and Rev. Viral supernatants are collected according to a previously published protocol. N2A695 cells are cultured for 24 hours with the unconcentrated viral supernatant and the cells are selected with 5 μg / ml puromycin for 4 weeks. Knockdown models using lentiviral vectors with specific shRNAs against the NAGLU, NPC1, and DNAJC5 genes are also generated in N2A695 cells.

[0248] Cell-based assays The following cell lines are used. Human fetal kidney cells (HEK293-T), N2A, and N2A695 (mouse neuroblastoma cells stably expressing human APP695WT (referred to as N2A695), routinely used to study APP processing) (see, for example, FIG. 7A). Primary neurons or microglial cells from NAGLU, NPC1 and DNAJC5 hemizygous or knockout mouse models are also transduced with the variants identified in (I). Primary neuron cultures are performed as described above.

[0249] Effects on mRNA and protein levels Quantitative real-time PCR is performed using specific primers to test the effect of the selected variant on mRNA levels and splicing. Western blot is performed using antibodies such as anti-CSPα (ADI-VAP-SV003-E, ENZO Life Sciences), anti-NAGLU (ab137685, Abcam) and anti-NPC1. Fluorescence assays are performed as described above for NAGLU activity (see, for example, FIGS. 7D and 7E). Briefly, 4-methylumbelliferyl-N-acetyl-α-glucosaminide cleavage is measured at 448 nm emission and 365 nm excitation in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi, Pleasanton, CA) using a standard curve of 4-methylumbelliferone (Sigma, St. Louis, MO) in the range of 0.02-5 mM. The NPC-1 specific assay is performed as previously published.

[0250] Lysosomal function Lysotracker is used to quantify the number of acidic compartments per cell using flow cytometry (see, for example, FIG. 7C). Lysosomal pH is measured by LysoSensor Yellow / Blue dextran (DND-160). The integrity of the lysosomal membrane is monitored by acridine orange. Intracellular fractionation is performed using the Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Scientific), and LAMP1, Rab7 and EEA1 are used as lysosomal, late and early endosomal markers, respectively (see, for example, FIG. 7B). This assay is complemented by confocal images of immunofluorescence to co-localize the selected proteins within lysosomal (LAMP-1 or -2), early endosomal (EEA1), late endosomal (Rab7), plasma membrane (flotillin) markers (see, for example, FIG. 7A).

[0251] Lysosomal enzyme activity based on cell lysates Intracellular and extracellular secondary increases in lysosomal enzyme activity are performed by fluorescence assays for PPT-1, β-gluc, and β-Hexa (see, for example, FIGS. 7D and 7E).

[0252] Pharmacological regulation of macroautophagy Transduced cells are treated with autophagy system activators and inhibitors (rapamycin, Torin1, Torin2, methylamine, brefeldin A and spautin-1), as well as chaperone-mediated autophagy (AR7). Western blots of LC3 and p62 are used as an indirect indicator of macroautophagy activation. As positive controls, lysosomal agents such as chloroquine, ammonium chloride (NH4Cl), and leupeptin are used.

[0253] Effects on APP processing and turnover N2A695 cells are treated with the protein synthesis inhibitor cycloheximide to quantify APP half-life. Protein and transcript levels of the APP processing machinery (PSEN1, Nicastrin, ADAM10, ADAM17 and BACE1) are measured by Western blot and RT-qPCR, respectively. To measure the dynamic effects of selected ALP genes on cell surface APP, APP at the plasma membrane is labeled using a non-membrane-permeable cleavable biotin derivative (sulfo-NHS-SS-biotin) at 4°C. Cell surface APP is measured by streptavidin IP and APP immunoblotting as previously published.

[0254] Aβ40 / Aβ42 levels Cell lysates and Aβ species in the media derived from the transfected N2A695 cells are detected by sandwich ELISA as previously published (see, e.g., FIG. 9C). Aβx-40 and Aβx-42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). HJ5.1 (anti-Aβ13-28), a biotinylated antibody targeting the central domain, or HJ3.5 (anti-Aβ1-13) targeting the N-terminal amino acids, is used as the detection antibody, followed by streptavidin-poly-HRP-40 (Fitzgerald Industries).

[0255] α / β-secretase processing of APP APP cleavage products (sAPPβ and sAPPα) in cell supernatants and intracellular fragments (α and β-CTF) are measured by Western blot. The following antibodies 22C11 and CT695 are used to characterize the full length, as well as the α and β-CTF fragments (see, e.g., FIG. 9D).

[0256] Uptake / degradation of Aβ Primary microglial cells transfected with the selected variant or specific shRNA are treated with 250 nM synthetic Aβ42 for 2 hours. To measure Aβ uptake, cells treated with Aβ42 are washed, trypsinized to remove surface-bound Aβ, lysed, and intracellular Aβ42 is measured by sandwich ELISA (see, e.g., FIG. 9C). The Aβ uptake rate is measured by varying the time the cells are exposed to Aβ42 to 0, 5, 10, 30 minutes, and 1, 2, 4, 8, 12, 24 hours. To measure Aβ degradation, cells are treated with 250 nM synthetic Aβ42 for 2 hours, washed thoroughly, and incubated in fresh media. The cells are then washed, trypsinized, lysed, and intracellular Aβ42 is measured at 0, 2, 4, 8, 12, 24 hours. The intracellular Aβ half-life is calculated assuming first-order kinetics as previously published.

[0257] Bioinformatics analysis For complementary analysis, the following publicly available databases are used: Gene Expression Omnibus, Brain RNA-seq2, gene expression data from Mouse Dementia Network (Mouse DemNet), PolyPhen2, SIFT, Human Splicing Finder, and Mouse Genome Informatics.

[0258] Expected results Based on the specific designs described herein to detect rare functional variants predicted for DNAJC5 and confirmation results, it is expected that the ALP gene risk variants identified in (I) cause partial loss of function and alter lysosomal function. In the functions of these proteins, 5-20% residual activity is expected to be detected. It is also expected that variants that alter overexpression, downregulation, or splicing of the ALP gene will affect APP metabolism and amyloidogenesis. Using computational resources and mining data from the literature, cell types and functional assays including Aβ or tau metabolism assays for each ALP gene can be carefully selected. In addition, the experiments described herein are expected to enable the evaluation of the effects of selected variants in the ALP gene on the survival of neurons and microglial cells.

[0259] If no stable differences are observed between the WT and risk variants in the NAGLU, NPC1, and DNAJC5 genes, this could be due to overexpression masking subtle changes in function. Thus, AD risk variants that can manifest over a lifetime but cause small changes that can be a challenge to detect in terms of the number of days of cell culture. Thus, APP mutations can be overexpressed or the ALP function can be pharmacologically altered. Primary neurons from AD mouse models such as 5XFAD transgenic mice (34840-JAX) can be used and transduced with selected variants in the NAGLU, NPC1, and DNAJC5 genes to test their effects on APP processing and tau aggregation. iPSC-derived neurons from AD patients carrying variants in the NAGLU, NPC1, and DNAJC5 genes can be used to compare the effects of such variants on APP metabolism to controls. Genome editing methods such as CRISPr technology can be used to introduce selected variants into the ALP gene and perform the functional assays described above. N2A695 cells, or primary neurons or glial cells stably expressing selected variants in the NAGLU, NPC1, and DNAJC5 genes can be treated with sub-lethal doses of lysosome-directed agents of autophagy inhibitors to measure their effects on APP processing.

[0260] The next step after identifying variants in NAGLU, NPC1, and DNAJC5 that affect both AD risk and onset in vitro is to take advantage of advances in LSD therapies. Therapeutic strategies such as enzyme replacement, substrate inhibition, molecular chaperones, or pharmacological modulation of ALP can be utilized and tested for their effects on AD onset assays in vitro.

[0261] (III) Determine the functional effects of selected candidate genes of ALP on AD lesions in vivo The hypothesis has been proposed that novel ALP genes associated with AD risk play a role in the in vivo development of AD. As described herein, quantitative and qualitative pathological investigations of the spontaneous development of AD lesions can be performed in hemizygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice at three time points defined by the time of onset of the endogenous lesions. The effect of the gene dosage of the NAGLU, NPC1, and DNAJC5 genes on the amount of Aβ plaques can also be measured in a well-characterized mouse model of early-onset familial AD, the 5XFAD transgenic mouse (34840-JAX).

[0262] Methodology and analysis Evaluating each ALP gene associated with AD identified in (I) is beyond the scope of the studies described herein. Accordingly, the following inclusion criteria for the selection of mouse models for follow-up in vivo studies have been defined. There must be available (either commercially or through collaborators), a functional assay, stable brain lesions, stable behavioral changes, an ideally short lifespan, and in vitro validation of the effect on amyloidogenesis.

[0263] Mouse groups For this experiment, three groups (18 mice / group) of NAGLU, NPC1, and DNAJC5 mice are generated. At three different time points, 1) normal littermates, 2) heterozygous (+ / −) mice, and 3) deficient (− / −) mice. The median lifespan of NAGLU KO mice is 12 months. However, brain lesions are apparent as early as 3 months. Accordingly, AD lesions are investigated in NAGLU mice at 2 months, 4 months, and 8 months. The median lifespan of DNAJC5 KO mice is 60 days, and brain lesions are apparent as early as 30 days. Accordingly, AD lesions are investigated at 21 days, 30 days, and 40 days. Since the median lifespan of NPC1 KO mice is 75 days, AD lesions are investigated in NPC1 KO mice at 30 days, 50 days, and 70 days.

[0264] The AD mouse model 5XFAD is also used. 5XFAD mice accumulate Aβ-42 in neurons at 6 months of age. Amyloid deposits in the hippocampus appear at 2 months and grow throughout the brain in older mice. To further confirm whether NAGLU, NPC1, and DNAJC5 deficiencies exacerbate the existing amyloidogenic process, NAGLU, NPC1, and Cspα mice are crossed with 5XFAD transgenic mice. For this experiment, three groups (27 mice / group) are generated. The effect of gene dosage on Aβ plaque amount is determined by measuring Aβ-42 brain levels at 3 weeks, and amyloid deposits at 1 and 2 months, in 5XFAD / NAGLU(+ / -), 5XFAD / NPC-1(+ / -), and 5XFAD / DNAJC5(+ / -) mice. For this experiment, three groups (27 mice / group) are generated. The effect of complete absence of the selected gene in 5XFAD lesions is evaluated by analyzing 5XFAD / NAGLU(- / -), 5XFAD / NPC-1(- / -), and 5XFAD / DNAJC5(- / -) mice at the same time points as described for evaluating the endogenous lesions of each KO mouse. The experimenter is blinded to the genotype and age of the animals during histological analysis. Each mouse is assigned a random ID number.

[0265] Quantification of Amyloid Plaques Vibratome brain sections with a thickness of 50 micrometers are collected every 300 μM from the rostral anterior commissure to the caudal hippocampus. For plaque imaging, the sections are stained with ThioS or immunostained with the HJ3.4 anti-Aβ antibody. High-resolution digital images of the stained brain sections are obtained with a NanoZoomer Digital Scanner (Hamamatsu Photonics). The total area of plaque coverage is measured using NIH ImageJ in the area of the hippocampus or piriform cortex and expressed as a percentage of the total area for each section. The results from N = 4 sections are averaged to represent each animal.

[0266] Aβ40 / Aβ42 Levels To detect total Aβ in the hippocampus of young mice, the excised tissue is sequentially homogenized in PBS followed by RIPA buffer to obtain detergent-soluble Aβ at an age where plaques are not observed, and samples are pooled for analysis. In aged mice (when plaques are abundant), the hippocampal tissue is sequentially homogenized in PBS followed by 5 M guanidine in TBS (pH 8.0) (to extract fibrillar and membrane-bound Aβ). Aβ40 / Aβ42 levels are quantified by ELISA as described in (II).

[0267] Expected results Based on the large effect sizes reported by gene analysis, it is expected that in mice lacking candidate ALP genes that have been found to be associated with AD risk in hemizygotes and verified in in vitro assays, more AD lesions will be found than in their age-matched controls. It is also expected that the effect of gene dosage on AD lesions will be determined. Hemizygous and KO mouse NAGLU, NPC1, and DNAJC5 genes should accelerate and exacerbate the amount of Aβ plaques in 5XFAD mice.

[0268] If AD lesions are not found in the brains of hemizygous and knockout mice, an AAV2 / 9 vector with the most significant variant verified in (II) is generated and stereotaxically injected into the hippocampus of hemizygous mice, and the presence of AD lesions is re-evaluated. If AD lesions are found, a rescue experiment can also be attempted by injecting an AAV2 / 9 vector with a wild-type copy of the missing gene and retesting for AD lesions. Another way to test the role of NAGLU, NPC1, and DNAJC5 genes in AD lesions is to inject an AAV2 / 9 vector with verified shRNA / RNAi, knockdown in 5XFAD transgenic mice, and test for the presence of an effect on AD lesions.

[0269] Therapies for LSD, such as enzyme replacement, substrate inhibition, molecular chaperones, and pharmacological modulation of ALP, can be tested for their effects on in vivo AD pathogenesis assays. Combining the results from the studies described herein with the availability of fluorescence assays for NAGLU activity and mass spectrometry assays for NPC1 biomarkers enables screening of CSF, plasma, serum, or dried blood spots from large cohorts of AD cases and controls to detect specific defects that can be used as biomarkers for AD. To determine whether NAGLU, NPC1, and DNAJC5 deficiencies can exacerbate tau lesions, NAGLU, NPC1, and DNAJC5 mice are mated with mice expressing p.P301L (015815-JAX) which is tau. p.P301L mice which are tau develop tangles in the cortex by 4 months of age. Thus, tau levels are measured at 1 month of age in mice mated with NAGLU, NPC1, and DNAJC5 mice. Conditions for measuring the amount of hyperphosphorylated tau in mouse brains have been optimized. Quantification of tau brain levels by ELISA has also been optimized previously. Genome editing methods such as CRISPr technology are used to generate knock-in mice for variants in the ALP gene that have the most potent effects on in vitro assays. These knock-in mice are also mated with AD mouse models including mice expressing 5XFAD and p.P301L which is tau.

[0270] Example 3: Identifying genetic variations underlying dysfunction of the autophagy-lysosome pathway (ALP) in Alzheimer's disease (AD) This example describes the role of rare functional variants in genes of the autophagy-lysosome pathway in Alzheimer's disease.

[0271] Multiple in vitro and in vivo studies have suggested that autophagic lysosomal pathway (ALP) dysfunction contributes to the pathogenesis of AD, but the age-dependence of ALP function and the genetic variations underlying the AD-associated decline are not fully understood. Rare functional variants in genes causing AD and multiple AD risk genes cause ALP dysfunction. However, a systematic and comprehensive assessment of the contribution of genetic variations within each gene in the general population of ALP to the risk of developing AD and its role in AD pathogenesis has not been completed. To address this gap in current knowledge, a powerful approach for identifying and prioritizing rare functional heterozygous variants in genes of ALP associated with the risk of developing AD is described herein. An innovative integrated framework that combines computational methods and experimental data can be used to validate the functional impact of selected ALP genes both in vitro and in vivo. First, the amount of rare functional variants in each gene of ALP from 33,350 non-Finnish European controls is compared to 2,000 AD cases and 3,000 controls. This result is replicated in an additional independent sample that includes 2,000 AD cases and 2,000 controls, along with 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, biochemical assays and cell-based assays are used to fully characterize the functional impact of selected genetic variants in candidate ALP proteins associated with AD. Examine the effect of the mutated protein on ALP function, and the results on full-length APP levels, APP trafficking, Aβ production in neurons and Aβ degradation by glial cells. Finally, it can be determined whether haploinsufficiency of the NAGLU, NPC1, and DNAJC5 genes accelerates AD lesions present in a well-characterized mouse model of AD. A quantitative pathological investigation of the effect of hereditary chronic lysosomal disorders on AD-related phenotypes with Aβ is performed. The experiments described herein can reveal novel ALP genes associated with AD. This can provide deeper insights into the mechanisms of ALP dysfunction in AD pathogenesis.

[0272] The goal of the research described in this specification is to identify genetic variations underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the onset of Alzheimer's disease (AD). The research described in this specification incorporates an innovative integrated framework that combines computational methods and experimental data to verify the functional impact of selected ALP genes both in vitro and in vivo. The experiments outlined in this specification can reveal novel ALP genes associated with AD. This can provide deeper insights into the mechanism of ALP dysfunction in AD onset.

[0273] Role of rare functional variants in genes of the autophagy-lysosome pathway in Alzheimer's disease Age is the greatest risk factor for the onset and progression of Alzheimer's disease (AD). Aging also reduces the degradative capacity of the autophagy-lysosome pathway (ALP). Multiple in vitro and in vivo studies have suggested that ALP dysfunction contributes to the onset of AD, but the age-dependence of ALP function and the genetic variations underlying the AD-related decline are not fully understood. The main hypothesis is that a mild form of hereditary ALP dysfunction, exacerbated by further age-related ALP impairment, contributes to the onset of AD lesions. Thus, hereditary ALP impairment in neurons may increase amyloidogenesis, while ALP impairment in glial cells may reduce their ability to degrade amyloid plaques. Thus, the balance between production and clearance determines Aβ levels and the propensity for amyloid plaque formation.

[0274] Small sample size studies focusing on single coding variants in a few ALP genes (e.g., cathepsin D) and intronic "hits" from large-scale genome-wide association analyses (GWAS) (e.g., SQSTM1) in isolated populations support the role of genetic variants in ALP genes in the risk of AD. In addition, rare mutations in genes causing AD (e.g., presenilin) and functional coding variants in AD risk genes (e.g., SORL1) cause ALP dysfunction. The human genome encodes at least 430 genes related to ALP. However, a systematic and comprehensive assessment of the contribution of coding variants in each ALP gene to the risk of developing AD has not been completed. As described herein, Chapter (I) can address this gap.

[0275] Combining whole exome sequencing (WES) data with a large database of AD cases and controls in which rare variants in the putative ALP gene (phospholipase D family, member 3, PLD3) have previously been shown to be associated with AD risk. PLD3 is a transcription factor EB (TFEB)-responsive gene and appears to affect amyloid precursor protein (APP) processing via lysosome-mediated mechanisms. There is data showing enrichment of predicted functional heterozygous variants in several additional lysosomal genes in late-onset sporadic AD. In attempts to validate these associations, a complementary role for the synaptic chaperone, cysteine string protein (CSPα), was discovered as a functional lysosome-related protein. In addition, data has been collected showing that CSPα transcript levels are decreased in the brains of AD patients and mouse models. Furthermore, mutations in CSPα affect autophagosome / lysosome fusion in vitro and Aβ production in vivo. Genes encoding the intracellular cholesterol transporter 1 (NPC1) and N-acetyl-α-glucosaminidase (NAGLU) have also been associated with AD risk in these analyses. In addition, significant age-related increases in NPC1 and NAGLU transcript levels were found in normal human brain samples. Interestingly, NPC1 and NAGLU transcript levels are also significantly higher in AD cases compared to age-matched controls. These results support the feasibility of the studies described herein and suggest that haploinsufficiency caused by functional heterozygous variants in the ALP gene likely affects the risk of developing AD by influencing APP metabolism, Aβ production, and Aβ degradation in vitro and in vivo.

[0276] (I) Identifying ALP genes enriched for rare functional variants in AD Single variant and gene-based analyses of all ALP genes (n = 430) are performed in 4,000 AD cases and 5,000 in-house controls. These results are replicated in independent samples (5,000 AD cases and 4,500 controls) from the Alzheimer's Disease Sequencing Project (ADSP). All ALP genes are analyzed using whole exome sequencing (WES) data from non-Finnish European individuals (n = 33,350) from the Exome Aggregation Consortium (ExAC) database to determine baseline gene variation in each ALP gene from European-derived individuals.

[0277] (II) Determine the functional effects of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro Using biochemical assays and cell-based assays, verify the effects of the selected variants on protein function, protein stability, and their effects on ALP function. Examine the effects of the verified functional variants on APP trafficking, APP half-life, APP processing machinery, and Aβ production in primary neurons. Aβ uptake and degradation are tested in glial cells from knockout or heterozygous mice (NAGLU, NPC1, and DNAJC5 genes).

[0278] (III) Determine the functional effects of haploinsufficiency in selected candidate ALP genes on the development of AD lesions in aged mice Determine whether mild lysosomal dysfunction accelerates Aβ production, plaque deposition, synapse loss, and gliosis in 5XFAD mice at the early (4 months) and late (8 months of age) stages of AD onset. Perform a quantitative pathological investigation of the effects of hereditary chronic lysosomal disorders on AD-related phenotypes in mice at 24 months of age in the absence of FAD mutations.

[0279] These studies form the proof-of-principle basis for the genetic contribution to ALP dysfunction associated with AD. This knowledge is currently lacking and it is possible to obtain new therapeutic targets.

[0280] Research strategy Significance The autophagy-lysosome pathway (ALP) is the major pathway for the degradation of organelles and aggregation-prone proteins. Autophagy (literally meaning "self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients via lysosomes. A true functional autophagic response induces the degradation of cytoplasmic materials of endogenous or exogenous origin within lysosomes. Lysosomes play an important role in the nutrient-sensing and signaling pathways involving the mammalian target of rapamycin complex 1 (mTORC1) kinase complex and transcription factor EB (TFEB) by a lysosome-nucleus signaling mechanism that controls cell clearance and energy metabolism. ALP is an intracellular quality control system that plays a protective role against neurodegeneration even in the absence of the expression of any disease-related mutant proteins. Neuron-specific deletion of the "core" autophagy genes (ATG5 and ATG7) causes abnormal protein accumulation, progressive neurodegeneration, and premature death.

[0281] ALP dysfunction in AD The familial form of AD is pathogenically caused by increased amyloid-β (Aβ) production, but recent studies in late-onset sporadic AD patients have shown impairment of Aβ clearance. Thus, the balance between production and clearance determines Aβ levels and the propensity for amyloid plaque development. The ALP "core" gene is transcriptionally downregulated during normal aging of the human brain. Surprisingly, in contrast to normal aging, transcriptional upregulation of ALP exists in the brains of AD patients, which may represent a compensatory attempt of the system to cope with the accumulation of abnormal proteins. There is a decrease in the level of beclin 1 (a multifunctional protein essential for autophagosome formation in ALP), an increase in the levels of rab5 and rab7 (small ras-related GTPase (rab) proteins that regulate vesicle transport along the endosome-lysosome pathway), and abnormal activation of macroautophagy (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase) in sporadic AD brains, as well as massive neuronal accumulation of autophagic vacuoles (AVs) and lysosomal dense bodies in degenerating neurites. Neuropathological studies have also found that autophagy-lysosome lesions in the AD brain contribute to AD pathogenesis, but the underlying mechanisms are not fully understood.

[0282] Hereditary ALP dysfunction exacerbates AD lesions Changes in ALP have also been found in multiple transgenic mouse AD models. Haploinsufficiency of beclin 1 (J20 and T41, hAPP751V171I, KM670 / 671NL) in two AD mouse models caused further disruption of their lysosomes, promoted intracellular and extracellular Aβ accumulation, and exacerbated neurodegeneration. The absence of lysosomal neuraminidase 1 (NEU1) worsened Aβ lesions in an AD model (5XFAD, APP KM670 / 671, I716V, V717I / PSEN1M146L / L286V). In contrast, overexpression of NEU1 reduced AD lesions. These results fully suggest that changes in the ALP "core" gene or lysosomal proteins exacerbate AD lesions.

[0283] Improvement of the ALP function reduces amyloid AD-related phenotypes APP / PS1 (APPK670M / N671L / PS1M146L) mice show abnormal macroautophagy activation in vulnerable neuronal populations even before extracellular deposition of Aβ. However, targeted expression of TFEB in both neurons and astrocytes reduced Aβ plaques in the APP / PS1 mouse model. TFEB expression causes upregulation of transcription of multiple lysosomal and trafficking genes, increasing lysosomal acidification and function. Activation of lysosomal cysteine proteases (by deleting cystatin B) in an AD mouse model (TgCRND8, hAPPK670N / M671L / V717F) rescued autophagy-lysosomal lesions, reduced the accumulation of abnormal Aβ and ubiquitinated proteins, decreased extracellular amyloid deposition and total brain Aβ40 / 42 levels, and prevented the onset of deficits in learning and memory tests. Pharmacological activation of lysosomal proteases decreased Aβ42 levels and improved performance in cognitive tests and synaptic deficits in two AD mouse models (J20 and APP / PS1). Pharmacological activation of ALP (mTOR inhibition) alleviated cognitive deficits and reduced the accumulation of β-amyloid in multiple AD models (J20, hAPP695,751,770V171F,KM670 / 671NL), (3xTg-AD, APPSwe / TauP301L), (APP-PS1, APPswe / PSEN1dE9). These results indicate that both overall activation of ALP or selective enhancement of lysosomal proteolysis promote amyloid clearance in multiple AD mouse models.

[0284] Aβ is generated in the endosome-autophagy-lysosome compartment Cell research suggests that the endosome-lysosome system is the major site of Aβ production. However, there is no consensus on where Aβ is actually produced. Aβ is generated after inducing macroautophagy both in vitro and in vivo. The accumulation of Aβ increases mTOR signaling, while decreasing mTOR signaling results in a reduction in Aβ levels, suggesting a negative feedback between ALP activation and Aβ levels. When autophagy is activated, autophagosomes become the cellular sites with the highest γ-secretase activity. PSEN2 and nicastrin (essential γ-secretase components for catalysis) are located in lysosomes. Indeed, PSEN1 regulates lysosomal pH. Pharmacological impairment of lysosomal function in vitro causes changes in Aβ production. Changes in lysosomal pH decrease Aβ secretion. Lysosomal protease inhibitors reduce the production of amyloidogenic APP fragments. All these studies suggest that overall lysosomal function plays an important role in normal and abnormal APP processing and subsequent amyloidogenesis. On the other hand, studies in mice lacking specific lysosomal genes have revealed the unique roles of each gene in APP processing and Aβ production. All evidence from human lesions, mouse, and cell models strongly suggests that defects in autophagy induction occur early in the disease, while defects in lysosomal clearance occur at a more advanced stage of the disease. However, it is not clear whether changes in ALP are the cause, result, or modifier of AD lesions.

[0285] Human Lysosomal Genes and Complete Loss of Function in AD The few studies examining AD lesions in lysosomal storage disorders (LSDs) have shaped the current understanding of the relationship between lysosomal genes and AD. These studies have not found Aβ plaques in the brains of mucopolysaccharidosis (MPS), Niemann-Pick disease type C (NPC), or neuronal ceroid lipofuscinosis (NCL). However, MPS, NPC, and NCL patients show intense diffuse Aβ signals in the cytoplasm of cells throughout the brain (see, e.g., FIGS. 13A and 13B). MPS patients show a significant increase in the levels of soluble Aβ compared to normal control brains. Increases in the CSF levels of Aβ38, Aβ40, and Aβ42 suggest increased γ-secretase-dependent Aβ release in the brains of NPC patients. Human patients with NCL showed a significant decrease in Aβ40 and Aβ42 levels compared to controls (see, e.g., FIGS. 13A and 13B). In addition, mice deficient in the NPC1 (Niemann-Pick disease), CLN3 (Batten disease), and HEXB (Sandhoff disease) genes, in the absence of overexpression of FAD mutations, show increases in both intracellular APP fragments (α-CTF / β-CTF) and Aβ40 / 42 levels. Increases in intracellular APP / Aβ levels without Aβ plaques have been reported in mice deficient in the IDUA (MPS-I), SGSH (Sanfilippo A), GBA (Gaucher disease), and TPP1 (late infantile Batten disease) genes. A three-fold increase in Aβ40 levels was found in mice deficient in the IDUA (MPS-I) and SGSH (Sanfilippo disease type A) genes compared to controls without detectable Aβ42. In contrast, a significant decrease in intracellular APP / Aβ levels was found in mice deficient in the ASAH1 (Farber disease) or PPT1 (infantile Batten disease) genes. These studies suggest that APP trafficking or processing is affected in both human patients and mouse models with complete loss of function of lysosomal genes, even in the absence of Aβ plaques. Interestingly, cognitive decline in both humans and AD transgenic mice does not correlate with Aβ plaque load but correlates with soluble Aβ species. Data from transgenic AD mice indicate that intracellular Aβ is more neurotoxic than extracellular Aβ.Intracellular accumulation of Aβ has been shown to precede extracellular deposition in both human and mouse AD models. Therefore, the short lifespan of both humans and mice lacking lysosomal genes may prevent researchers from finding Aβ plaques in addition to abnormal production of Aβ40 or Aβ42.

[0286] Gene variations in the human ALP gene The human genome contains at least 430 genes related to ALP (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Mutations in 38% of all ALP genes (157 genes) cause human Mendelian genetic diseases (OMIM). A recent analysis of the frequency and types of mutations present in 60,000 individuals found that most of the ALP genes have mutations that are "intolerant to loss of function" and have fewer potentially harmful variants than predicted by the neutral model of evolution. This is consistent with the lethality of most "core" ALP genes during embryogenesis or neonatal period in knockout mice, as well as their importance for cell maintenance and survival. The most studied and well-known ALP genes are lysosomal genes that, when mutated, cause LSDs. Interestingly, most of these genes (about 50) that cause LSDs are not intolerant to LoF mutations and show considerable genetic coding variation in humans. Thus, epidemiological studies have shown a 10-fold range of differences in the levels of lysosomal enzyme activity in healthy humans. Individuals with genetic variants that cause haploinsufficiency in multiple lysosomal genes, including the GBA, NPC1, GALC, GAA, GLA, and IDUA genes, show significantly lower levels of enzyme activity compared to controls. In addition, haploinsufficiency in the NPC1 gene causes significant additional metabolic abnormalities in human carriers. Having one single normal copy of an ALP gene has long been assumed not to have health consequences. However, substantial genetic evidence supports the role of functional variants in the GBA gene as a major genetic risk factor for developing Parkinson's disease and Lewy body disease. In the homozygous situation, the same variants that cause LSD (Gaucher disease) in children, when present in a heterozygous fashion, affect the risk of adult-onset neurodegenerative diseases. These studies suggest that haploinsufficiency in lysosomal genes predisposes to common neurodegenerative disorders in adult humans.There is a lack of systematic evaluation of the contribution of functional heterozygous variants in the ALP gene that affect the risk of AD.

[0287] Innovation The studies described herein systematically and comprehensively evaluate genetic variations associated with the well-known dysfunction in ALP related to AD (see, e.g., Chapter (I)), and are conceptually innovative in understanding the impact of rare functional heterozygous variants in the ALP gene related to AD in vitro (see, e.g., Chapter (II)) and in vivo (see, e.g., Chapter (III)). Furthermore, these studies carefully examine neuronal lesions and determine their impact on amyloidosis with respect to the results of genetically altering ALP and clinically relevant endpoints. The current understanding of genetic variations in the ALP gene in AD is dominated by limited studies reporting spurious associations in isolated populations with very low replication rates. The studies described herein can overcome this limitation because their design comprehensively evaluates the contribution of genetic variations in each ALP gene to the risk of developing AD in a very large sample representing the general population. Thus, for a total of about 4000 AD cases (n) and about 5000 controls (n), in addition to in-house WES and exome chip data, data from two large publicly available databases of WES data, the Exome Aggregation Consortium (ExAC) (n of about 61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n of about 10,000) are used. Using these multiple datasets, a series of analyses are designed to resolve the genetic architecture of the well-known dysfunction in ALP related to AD. The studies described herein incorporate an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. Cell-based assays are complemented with biochemical data, live cell assays, RNAseq data from samples specific to brain cell types in mice, genome-wide gene expression data in human AD cases and controls, genome-wide gene expression data at different stages in human AD cases, genome-wide gene expression data from humans of different ages, and genome-wide gene expression data from an AD mouse model correlated with Aβ plaques.Therefore, as a result of these data mining efforts, the experiments described in Chapter (II) are performed not only in primary neurons but also in microglial cells. RNAseq data specific to cell types show that most of the ALP genes exhibit higher levels of expression in microglial cells than in neurons. The approach described herein overcomes previous uncertain studies on the role of the ALP gene in AD. The studies described in Chapter (III) can address the question of how age and haploinsufficiency of the ALP gene in vulnerable brain regions affect APP processing and transport, Aβ plaque load, and Aβ40 / 42 levels. These studies can overcome the limitations of previous studies using knockout mice of the ALP gene, which show rapid neurodegeneration and short lifespan, complicating the interpretation of the effects of the deleted gene on AD. These studies are made possible by an innovation through a collaborative study involving researchers with expertise in neurogenetics, lysosome biology, LSD animal models, and AD lesions across cell models and mouse models.

[0288] Approach Here, state-of-the-art genomic tools and large datasets are used to reveal the genetic architecture underlying the AD-related decline in ALP function. In addition, the effects of selected variants and genes on APP metabolism, Aβ production, and Aβ degradation can be verified in vitro and in vivo.

[0289] Data and Results Heterozygous variants in lysosomal genes affect the risk of developing AD. The list of autophagy lysosome gene sets (about 430 genes) derived by mining existing annotations in public databases and literature has been manually curated previously. All ALP genes within the AD cohort can be analyzed. However, lysosomal genes in which loss-of-function (LoF) mutations cause LSD are the best-studied models of ALP dysfunction causing neurodegeneration in human and mouse models. In addition, small sample size studies on single lysosomal genes in isolated (geographically and genetically) populations have supported the role of genetic variants in lysosomal genes for the risk of AD. However, these studies have not been replicated. Therefore, the analysis of the genetic contribution of ALP genes to AD has been initiated by focusing on 46 lysosomal genes using a large dataset that is a good representative of European populations. The drawback of the current available set of bioinformatics tools is that there is no perfect algorithm to predict whether coding variants with unknown functions have functional (biological) consequences. Therefore, the inclusion and exclusion criteria for potential functional variants were defined based on common features between coding variants with unknown functions and known complete or nearly complete LoF variants that cause LSD when they are homozygous or compound heterozygous.

[0290] The discovery sample was composed of WES data from 523 unrelated AD cases and 386 controls. Table 5 shows the top lysosomal genes associated with AD. As expected, the gene-specific cumulative minor allele frequency (cMAF) from the ExAC dataset (European, non-Finnish) was highly concordant with the cMAF from the in-house AD database (European) (r 2= 0.97). In the AD cohort, coding variants in each lysosomal gene that met the inclusion criteria were collated gene by gene. Eighty-two percent were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The amount of variant (cMAF) that changes rare proteins was compared to the amounts observed in controls and ExAC. For most genes, there is excessive variation in cases compared to controls, but only slight associations were found with the SGSH gene (p = 4.2 × 10 -3 −6, OR = 3.7, 95% CI 1.4–9.6) and the CLN8 gene (p = 1.0 × 10 -2 −4, OR = 8.9, 95% CI 1.1–68.1) (see, for example, Table 5).

[0291] When compared to the cMAF of the ExAC samples, 14 genes passed a very stringent multiple-testing correction threshold of p < 1.0 × 10 -4 (0.05 / 450), and 13 genes causing LSDs passed a gene-level significance threshold of p < 2.4 × 10 -6 (0.05 / 20,000) (see, for example, Table 5). Next, the ADSP cohort was used to replicate the findings listed in Table 5, which included 5045 AD cases and 4500 controls (see, for example, Table 7).

Table 7

[0292] Nine genes replicated the association with AD in this independent sample (see, for example, Table 7). Of note is the fact that the associations found in the replication sample were in the same direction and had the same effect size.

[0293] NPC1 transcript levels associated with age and AD status Based on the criteria defined above, three lysosomal genes replicated in two samples, such as the gene encoding intracellular cholesterol transporter 1 (NPC1), the gene encoding N-acetyl-α-glucosaminidase (NAGLU), and the DNAJC5 gene encoding cysteine string protein α (CSPα), were selected for functional analysis. NPC1 transports low-density lipoproteins to the late endosome / lysosome compartment, where they are hydrolyzed and released as free cholesterol. LoF in this gene causes Niemann-Pick type C disease. NPC1 transcripts show higher expression levels (about 4.5-fold) in microglia and astrocytes than in neurons. In neuropathologically normal human brain samples, there was a highly significant increase in NPC1 transcript levels with age (p < 0.0001) (see, for example, Figure 10A). NPC1 transcript levels were significantly higher in AD cases compared to age-matched controls (p = 0.01, see, for example, Figure 10B).

[0294] NAGLU transcript levels in relation to age, AD status, and in an AD mouse model NAGLU degrades heparan sulfate, and complete LoF of this gene causes mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome B. RNAseq data from brain cell types in mice indicate that NAGLU transcripts are expressed at higher levels (by about 20-fold) in microglia than in neurons. In neuropathologically normal human brain samples, there was a significant age-dependent increase in NAGLU transcript levels (p = 0.02) (see, e.g., FIG. 1A). NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (p = 0.007) (see, e.g., FIG. 1B). NAGLU transcript levels also showed an age-dependent proportional increase with the onset of AD lesions in the cortex of an AD mouse model (APP, p.K670N / p.M671L / PSEN1, p.M146V, heterozygous [HET] or homozygous [HO], see, e.g., FIG. 1C) compared to levels in wild-type mice (black line, see, e.g., FIG. 1C) (right panel, see, e.g., FIG. 1C).

[0295] DNAJC5 transcript levels as a function of age, AD status, and in an AD mouse model CSPα is a synaptic chaperone involved in endocytosis and maintenance of protein homeostasis at synapses. Heterozygous mutations in CSPα cause autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL). The DNAJC5 transcript is highly expressed in neurons and in brain regions most affected by AD lesions. A decrease in DNAJC5 transcript levels with age was found in neuropathologically normal brain samples from the prefrontal cortex region (see, e.g., FIG. 8A). DNAJC5 transcript levels were significantly lower in AD cases compared to age-matched controls in laser capture microdissected, tangle-free neurons from AD and controls (p < 0.0001, see, e.g., the left graph in FIG. 8B) (GEO database, Series GSE5281). This finding was replicated in a different study (GEO database, Series GSE15222) (see, e.g., the right graph in FIG. 8B). Also, DNAJC5 transcript levels showed an age-dependent decrease and were found to be inversely proportional to the development of AD lesions in the cortex of an AD mouse model (APP, p.K670N / p.M671L, FIG. 8) compared to levels in wild-type mice (see, e.g., FIG. 8). All of these results suggest that NPC1, NAGLU, and CSPα are involved in the development of AD.

[0296] Endogenous CSPα localizes to lysosomes, and mutant CSPα affects autophagy protein levels (LC3-II and p62). Endogenous CSPα co-localized with lysosomal markers in both cell bodies, neurites, and synaptic boutons in primary cortical neurons and a neuron-like cell type (N2A) (see, e.g., FIG. 12A). Subcellular fractionation showed that a significant proportion of CSPα co-precipitated with another lysosomal marker (LAMP1) (see, e.g., FIG. 12B). These results suggest that endogenous CSPα is a lysosome-associated protein. Expression of the ANCL-causing mutation (p.L115R) resulted in high molecular weight CSPα aggregates and caused an increase in the levels of the lysosomal proteins LAMP1 and SNAP23. There was a decrease in p62 and a persistent conversion from LC3-I to LC3-II, suggesting activation of autophagy and a block in autophagosome and lysosome fusion (see, e.g., FIG. 12C). This ANCL-causing mutation significantly increased the level of the LysoTracker signal compared to the empty vector. In contrast, overexpression of CSPα-WT significantly decreased the LysoTracker signal (see, e.g., FIG. 12D).

[0297] CSPα affects APP processing in vivo Brains of ANCL patients did not show Aβ plaques or neurofibrillary tangles. However, there was significant intracellular accumulation of APP / Aβ in cortical neurons of ANCL patients (ANCL, see, e.g., FIG. 13A). Quantification of Aβ in the detergent-soluble and insoluble (guanidine) fractions of brain samples from ANCL, AD, and healthy control samples revealed a significant decrease in Aβ40 and Aβ42 levels in ANCL patients compared to both control and AD samples (see, e.g., FIG. 13B). These results suggest that CSPα plays a role in Aβ production and AD pathogenesis.

[0298] Study design and methods (I) Identify the ALP gene enriched for rare functional variants in AD The hypothesis that haploinsufficiency caused by functional heterozygous variants in the ALP gene affects the risk of developing AD can be tested. As described herein, an innovative approach that combines the analysis of predicted rare functional variants in the ALP gene in large datasets (both in-house databases and publicly available ones) is used to prioritize candidate genes in ALP with evidence of involvement in AD risk.

[0299] (I) Methodology and Analysis in Chapter Define the allele frequency threshold for rare variants Initial analysis focuses on lysosomal genes. The same methodology is applied to all ALP genes. However, in the absence of human diseases caused by LoF mutations in the remaining ALP genes, the inclusion criteria are adjusted based on experience with lysosomal genes. In the NCBI ClinVar database, variants causing approximately 2,740 LSDs have been reported. Most of the AD samples are of European origin. Therefore, non-Finnish samples (about 33,000 individuals) were selected from ExAc to establish a baseline for genetic variation in lysosomal genes in populations with similar genetic backgrounds. 288 LoF variants were found in the tested lysosomal genes annotated as heterozygous in the ExAc samples, 76% being missense variants, 10% affecting alternative splicing, and 12% being nonsense mutations. Most LoF variants are predicted to be deleterious (87%) by SIFT and predicted to be damaged (84%) by polyphen2. Most (73%) LoF variants are located within highly conserved nucleotides (GREP score > 4). Some LoF variants reported in ExAc are not classified as variants causing LSDs in the NCBI ClinVar database. Heterozygous LoF variants were found in each of the tested lysosomal genes, but the number of heterozygous LoF variants varies from 1 found in the HYAL1 gene to 20 found in the ARSA gene. The cMAF of these heterozygous LoF variants per gene ranges from 1.5 - 05 in the CSTD gene to 0.003 in the NPC2 gene. Therefore, as a conservative upper limit, 1×10 -3The cMAF threshold of was applied. Using the information obtained from the analysis of LoF variants, including the maximum MAF, SIFT, Polyphen2, or GERP score of LoF variants, the following inclusion criteria were defined: 1) call rate > 98% in AD cases, 2) Maf < 0.01% per variant, 3) only variants likely to change the protein in the designated canonical transcript annotated as missense by ExAc or Ensemble, 4) frameshift, 5) nonsense, 6) variants affecting splice donor and acceptor regions, and 7) whether there is evidence of pathogenicity in the NCBI ClinVar database and it is located in the 3' or 5' UTR region. Variants not found in ExAC, or not present in the NCBI ClinVar database, or with a synonymous intronic 3' or 5' UTR variant with Maf > 0.01%, and miscalls in ExAC and ClinVar were excluded. Individuals were selected based on the results of the major components to ensure that population-specific variants had no confounding effect on this analysis.

[0300] Table 8 shows an overview of the available samples for performing genetic analysis. [Table 8]

[0301] There is access to phenotypic data, DNA, and / or genetic data for over 17,000 individuals from the Knight Alzheimer’s Disease Research Center (Knight ADRC), Alzheimer’s Disease Neuroimaging Initiative (ADNI), NIA-LOAD Study, Spanish dataset, and Alzheimer’s Disease Sequencing Project (ADSP). In addition, 10,000 samples from the ADSP are currently being sequenced (WGS). All ALP genes within the in-house database can be analyzed. Descriptions of these datasets have been published previously. Each case received a diagnosis of Alzheimer's type dementia using criteria equivalent to those of the National Institute of Neurological and Communication Disorders and Stroke-Alzheimer’s Disease and Related Disorders Association for probable AD. Controls received the same evaluations as cases but were cognitively normal. All individuals were of European ancestry, and written consent was obtained from all participants.

[0302] Alzheimer's Disease Sequencing Project (ADSP) Using WES data from AD cases and controls, all genetic analyses described herein are performed on the ALP gene. The data were downloaded from the ADSP in December 2015. The discovery stage dataset includes WGS data for 584 subjects from 113 families, pedigree data for over 4,000 subjects, WES data for 5,096 cases and 4,965 controls, and whole exome sequence data from an additional 853 subjects (682 cases [510 non-Hispanic, 172 Hispanic]) and 171 Hispanic control subjects from families with multiple AD-affected members. The ADSP replication stage is already underway and includes WGS data for an additional 10,000 individuals.

[0303] Publicly available WES data The allele frequencies from non-Finnish European samples included in ExAc are used as the allele frequency reference for the analysis of all ALP genes. The data were downloaded from ExAC (version 0.3.1, March 2015). Only data from ALP genes containing a high proportion of coding regions extending beyond a median sequence depth of 30-fold, and only high-quality (PASS filter) variants were included in this analysis.

[0304] Technical annotations regarding whole exome sequencing data collection There is WES from 2,000 individuals. Exome enrichment is performed using the SureSelect 52Mb Target Enrichment System (Agilent). DNA was sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling are performed using Novoalign and SAM tools. Once the data are processed and the sequence variants detected by exome sequencing are confirmed by genotyping, efficient and effective methods for data management, quality control, cleaning, annotation, and analysis developed and used for other exome sequencing projects are applied.

[0305] Statistical tests To account for the effects of rare variants with moderate effect sizes, validated statistical methods developed to analyze the association with rare variants are used. Briefly, gene-based methods collapse rare variants within a region into a single value and then test the association between rare variants within a region and the trait of interest. The Sequence Kernel Association Test (SKAT) is used to test the association between the context within a gene region and rare variants. SKAT can account for variants that have effects in different directions within the same gene and adjust for confounding covariates, including population markers. Independent case-control samples are then used to replicate findings from the discovery dataset. Analyses of each different dataset are performed separately. Joint analysis is done to combine p-values and ORs. This method has been successfully used in previous studies to identify novel genes for AD.

[0306] Population structure This analysis defines the European ancestry of the in-house samples included in the analysis. Eigenstrat is used as an anchor on the samples together with HapMap samples to confirm self-reported race / ethnicity.

[0307] Bioinformatics analysis The following publicly available databases are used for complementary analysis: Online Mendelian Inheritance in Man (OMIM), ExAC, GWAS Catalog, ClinVar database, Human Autophagy Database.

[0308] Output analysis To determine the output for detecting genetic variants associated with AD, the analysis was performed using Proc Power in SAS. Analyses were performed using minor allele frequencies in the range of 0.01 - 0.50, ORs in the range of 1.2 - 3.6, and sample sizes in the range of 4,000 - 7,000. α was set at 5×10 -8 for single variant analysis and 5×10 -6It was adjusted to. There is approximately 80% output to detect the influence of OR > 1.19 (or < 0.84).

[0309] Expected result A new association between the AD risk and the ALP gene is expected to be revealed by the study described herein. This study can identify novel genes in ALP associated with the AD risk (see, e.g., Table 5), and shows that these can be replicated in independent samples with appropriate coverage depth (see, e.g., Table 7). Consistent with the minimal genetic variation found in most of the "core" ALP genes in the large dataset (ExAC) and, in addition, the absence of associated human diseases, an enrichment of functional variants in the "core" ALP genes in AD patients is not expected.

[0310] Alternative approach This result and output calculation suggest that there is sufficient output to detect an average odds ratio greater than 2.7 in gene-based analysis. If the association of the ALP gene fails to replicate by a case-control design, an endophenotype design can be used. Thus, the effect of variants in the ALP gene on CSF biomarker levels for AD can be determined by performing single variant and gene-based analyses for each of the ALP gene and CSF biomarkers such as t-tau, p-tau and Aβ42. To evaluate whether the regulatory genomic regions of the ALP gene may be involved in the risk of developing AD, the association of the ALP gene can be analyzed in data from a previously published GWAS by the International Genomics of Alzheimer’s Project (I-GAP) consisting of a total of 25,580 AD cases and 48,466 controls. The complementary approach will examine whether the ALP gene affects the age at onset (AAO).

[0311] Analysis of the genetic variations of the remaining approximately 384 ALP genes and their potential association with AD risk has been completed. The sample size can be increased using the ADSP replication phase that includes WGS data for an additional 10,000 individuals (both AD cases and controls). Appropriate collaboration with the organizers of the Genome Aggregation Database (gnomAD) is established. gnomAD is an expansion of ExAc that contains exome sequence data from 123,136 individuals and whole-genome sequencing from 15,496 individuals, and replicates findings in a larger dataset. ADSP data is included in gnomAD, and the use of the public version of gnomAD is excluded from the current analysis.

[0312] (II)(a) Determine the functional effects of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro (I) Evaluating all variants of each ALP gene associated with AD identified in section (I) is beyond the scope of the study described herein. Therefore, the top 3 - 5 variants identified in section (I) of the NAGLU, NPC1, and DNAJC5 genes are prioritized. The top variants are defined based on their frequency in AD patients, predicted effects on proteins by SIFT and Polyphen2, and GERP conservation scores. These genes are selected based on the strength of the data from both discovery and replication samples. A hypothesis has been proposed that novel ALP genes associated with AD risk affect APP metabolism, Aβ production, and Aβ degradation in vitro.

[0313] Evaluate the effects on protein products Variants that share many of the in-silico features of the Lof variant have been selected. The experiments outlined in this specification can generate experimental data to verify the functional effects on each protein. The selected variants are engineered using site-directed mutagenesis of the cDNA of the NAGLU, NPC1, and DNAJC5 genes. Wild-type and mutant cDNAs are subcloned into lentiviral vectors as described above. Lentiviral vectors are produced, handled, and disposed of in a BSL2 facility according to Section III-E-1 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Primary neuron cultures are performed as described above. The NAGLU, NPC1, and DNAJC5 genes are expressed in primary neurons from knockout mice, and the effect on protein stability is quantified by Western blot. The intracellular localization of the mutated proteins is evaluated by confocal images of immunofluorescence, and the selected proteins are co-localized within lysosome (LAMP-1 or -2), early endosome (EEA1), late endosome (Rab7), ER (KDel), and Golgi (Giantin) markers. The effect on the enzyme activity of the NAGLU variant is tested using a fluorescence assay as described above. The effect of the selected variants on NPC1 is performed as previously published. The functional effects of the selected variants on DNAJC5 are tested by their ability to prevent the degradation of SNAP-25.

[0314] Evaluate the effects on APP trafficking, endocytosis, and intracellular localization Multiple studies have shown that APP endocytosis is essential for the co-localization of APP with β- and γ-secretases in endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impairment of endosomal flux following lysosomal dysfunction increases the transit time within this organelle, increases the tendency for β-cleavage and γ-cleavage, and thus increases the tendency for Aβ production. To determine whether selected variants in the NAGLU, NPC1, and DNAJC5 genes affect the steady-state levels of APP, APP endocytosis, or the enhancement of APP flux to lysosomes for degradation, the dynamics of intracellular APP appearance and the levels of APP at the cell surface can be determined using a cell surface biotinylation assay as previously published. The effect on the full-length APP half-life is measured by Western blot at 0, 5, 10, 30 minutes under treatment with the protein synthesis inhibitor cycloheximide. Immunohistochemical co-localization microscopy techniques are performed to study the effects on APP and SorL1 intracellular localization. The transcript levels of proteins and the APP processing machinery, including α-secretases (ADAM10 and ADAM17), β-secretase 1 (BACE1), and the γ-secretase complex (PSEN1 and Nicastrin), are measured by Western blot and RT-qPCR, respectively.

[0315] Evaluate the effect on Aβ production A significant proportion of APPs target lysosomes, and intracellular APP levels rapidly accumulate in the presence of lysosomal acidification inhibitors, suggesting that lysosomal degradation causes APP proteolysis and eliminates the formation of Aβ peptides. In addition, human Sanfilippo patients (NAGLU - deficient) show a significant increase in the level of soluble Aβ compared to normal control brains. In the brains of NPC (NPC1 - deficient) patients, it has not been reported that the CSF levels of Aβ40 and Aβ42 increase, and that the levels of β - cleaved soluble APP do not change. Human patients with mutations in DNAJC5 show a significant decrease in Aβ40 and Aβ42 levels compared to controls. Therefore, it is possible to evaluate whether the selected variants affect Aβ production in cell culture. Aβ species in cell lysates and cell - derived media are detected by sandwich ELISA as previously published. Briefly, Aβx - 40 and Aβx - 42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti - Aβ35 - 40) and HJ7.4 (anti - Aβ37 - 42). HJ5.1 (anti - Aβ13 - 28), a biotinylated antibody targeting the central domain, or HJ3.5 (anti - Aβ1 - 13) targeting the N - terminal amino acid is used as the detection antibody, and then streptavidin - poly - HRP - 40 (Fitzgerald Industries) is used. APP - derived proteolytic fragments such as α - CTF and β - CTF, and sAPPα and sAPPβ are measured by Western blot. The level of full - length APP is also monitored by Western blot.

[0316] Evaluate the effect on Aβ degradation Microglia proliferate around amyloid plaques and phagocytose amyloid substances, but subsequent degradation is impaired, contributing to progressive amyloid accumulation in AD. Although microglial cells can take up fibrillar Aβ, the reason why they cannot degrade it is not clear. However, microglial cells derived from AD patients show a decrease in beclin-1 and subsequent impairment of ALP function. In addition, insoluble fibrillar Aβ affects the transport of the chloride channel CIC-7 to lysosomes in primary microglia, impairing lysosomal degradation. However, when lysosomal acidification is restored, Aβ degradation is enhanced. Collectively, this evidence suggests that ALP deficiency in microglial cells may contribute to the development of AD. The data mining efforts presented here have revealed that the NAGLU and NPC1 genes are expressed at higher levels in microglial cells than in neurons. Therefore, it is possible to evaluate whether primary microglial cells derived from NAGLU- and NPC1-deficient and heterozygous mice transduced with the selected variants can take up and degrade exogenous Aβ. Aβ uptake and Aβ degradation are performed as previously published.

[0317] Evaluate the effect on ALP function It is possible to test whether neurons from hemizygous mice exhibit ALP dysfunction and whether these changes are increased by selected variants. Western blots of LC3 and p62 are used as an indirect indicator of macroautophagy activation. Autophagic flux is evaluated by the amount of LC3-II present in cells in the absence or presence of autophagy activators (rapamycin and Torin1), autophagy inhibitors (bafilomycin A1), lysosome-directed agents (chloroquine, ammonium chloride), and E64 / pepstatin. This is complemented by live cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion is further evaluated by the co-localization of LC3 and LAMP1. Lysotracker is used to quantify the number of acidic compartments per cell.

[0318] Bioinformatics analysis For complementary analysis, the following publicly available databases are used: Gene Expression Omnibus, Brain RNA-seq2, gene expression data from Mouse Dementia Network (Mouse DemNet), PolyPhen2, SIFT, Mouse Genome Informatics.

[0319] Expected results (I) It is expected that the ALP gene risk variants identified in section (I) cause partial loss of function and alter ALP function. Residual activity of 5-20% is expected to be detected in the function of these proteins. It is also expected that overexpression or downregulation of the selected genes will affect APP transport, APP metabolism, Aβ production or Aβ degradation. In addition, it is expected that the experiments described herein will also enable the evaluation of the effect of selected variants in the ALP gene on the survival of neurons and microglial cells.

[0320] If no stable differences are observed between WT and risk variants in NAGLU, NPC1, and DNAJC5, this could be due to overexpression masking subtle changes in function. AD risk variants that can manifest as disease over a lifetime but cause small changes that can be a challenge to detect over the number of days of cell culture. Alternatively, 5XFAD transgenic mice or primary neurons from N2A695 cells could be used and transduced with selected variants in the NAGLU, NPC1, and DNAJC5 genes to test their effect on Aβ production.

[0321] The next step after identifying variants in the NAGLU, NPC1, and DNAJC5 genes that affect both the risk and onset of AD in vitro is to take advantage of the direct generation of iPSc from human-derived fibroblasts and advances in genome editing methods. Neurons or glial cells derived from iPSc from AD patients carrying variants in the NAGLU, NPC1, and DNAJC5 genes can be used to compare the effect of such variants on APP metabolism compared to CRISPr-corrected cells with the same genetic background. Once these tools are available, therapeutic strategies such as enzyme replacement (NAGLU), cyclodextrin (NPC1), or pharmacological modulation of ALP can be utilized to test their effect on Aβ production or Aβ degradation in vitro.

[0322] (II)(b) Determine the functional effect of haploinsufficiency in selected candidate ALP genes on the onset of AD lesions in aged mice The consequences of neurodegeneration due to complete loss of function of the NAGLU and NPC1 genes in mice and humans have been characterized, but little is known about the long-term consequences of single copies of these genes. Hemizygous mice and humans have always been assumed to be normal. However, recently, haploinsufficiency in the NPC1 gene has been shown to cause significant metabolic abnormalities in humans and mice. A hypothesis is proposed that AD lesions develop from a mild form of hereditary ALP dysfunction, and that further age-related ALP impairment may be required for their appearance. The main endpoints are soluble Aβ levels measured at 4 months of age (before plaque deposition) in the presence of mutations that cause FAD in mice, and plaque burden at 8 months of age. The effect on soluble Aβ levels is the main endpoint in NAGLU, NPC1, and DNAJC5 hemizygous mice at 24 months of age. Hemizygous mice are generated from commercially available knockout mice.

[0323] Effect on mouse models of AD lesions Complete loss of NAGLU protein function in human patients with Sanfilippo disease causes a significant three-fold increase in soluble Aβ levels compared to normal control brains. In patients with complete loss of NPC1 function, it has not been reported that CSF levels of Aβ40 and Aβ42 increase and that levels of β-cleaved soluble APP do not change. NAGLU transcript levels showed an age-dependent proportional increase with the onset of AD lesions in the cortex of AD mouse models (see, for example, Figure 1C). DNAJC5 transcript levels showed an age-dependent decrease and were inversely proportional to the onset of AD lesions in the cortex of AD mouse models (see, for example, Figure 8A). Human patients with heterozygous mutations in DNAJC5 showed a significant decrease in Aβ40 and Aβ42 levels compared to controls (see, for example, Figure 13). Similar to AD transgenic mice, cognitive decline in humans does not correlate with Aβ plaque load but correlates with soluble Aβ species. Considering data from human LSD patients and LoF mouse models that support the role of these genes in intracellular Aβ production, it is possible to determine whether mild lysosomal disorders (hemizygosity in NAGLU, NPC1, and DNAJC5) accelerate Aβ production in well-characterized mouse models of AD carrying FAD mutations that favor Aβ production. The 5XFAD model is a very aggressive amyloid deposition model in which intracellular Aβ42 develops at 1.5 months of age, plaques develop at 2 months, loss of synaptic markers and memory deficits develop at 4 months, and neuron loss develops at 9 months. The development of plaques is accompanied by reactive gliosis. To further confirm whether NAGLU, NPC1, and DNAJC5 haploinsufficiency exacerbates the existing amyloidogenic process, NAGLU, NPC1, and DNAJC5 mice are crossed with 5XFAD transgenic mice. For this experiment, four groups (30 mice / group) are generated. The effects of gene dosage on APP metabolism, Aβ plaque load, and Aβ40 / Aβ42 levels at 4 and 8 months are determined in 5XFAD / NAGLU(+ / -), 5XFAD / NPC-1(+ / -), and 5XFAD / DNAJC5(+ / -) mice.4 months is an early time point for amyloid plaque deposition in 5XFAD mice. 8 months represents the age when amyloid plaques are abundant.

[0324] Sample size Calculation of sample size indicates that at least n = 15 mice / group are required to detect a 20% increase (SD = 30%, α = 5%) in endpoints such as plaque burden, and detergent-soluble and insoluble Aβ40 and Aβ42, while testing an equal number of male and female mice at 80% power. Fifteen hemizygous mice from each gene crossed to 5XFAD [5XFAD / NAGLU(− / +), 15 5XFAD / NPC1(− / +) and 5XFAD / DNAJC5(− / +)] and 15 additional 5XFAD mice are anesthetized and euthanized at 4 and 8 months of age, and brains are collected for histological and biochemical studies.

[0325] The effect of haploinsufficiency in lysosomal genes on aged mice AD lesions (e.g., Aβ plaques) are typically age-dependent. However, published studies do not address the interaction between age and ALP dysfunction. Most studies evaluating the role of ALP in AD in vivo use pharmacological approaches or complete absence of the ALP gene and short-term endpoints. A genetic approach is used to reduce the endogenous levels of the NAGLU, NPC1, and DNAJC5 genes, and a quantitative pathological investigation is performed on the effects of hereditary chronic lysosomal disorders on AD-related phenotypes with Aβ. The results show that in normal human brain samples, with age, the NPC1 and NAGLU transcript levels increase very significantly (see, for example, FIGS. 10A and 1A). In addition, the NPC1 and NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (see, for example, FIGS. 10B and 1B). These results suggest that a compensatory response to aggregated proteins from the lysosomal genes NPC1 and NAGLU is part of the normal aging process. The abnormal increase found in the AD model suggests an attempt to control abnormal levels of Aβ. In contrast, in neuropathologically normal brain samples, there is a decrease in DNAJC5 transcript levels with age (see, for example, FIG. 8A), and the DNAJC5 transcript levels were significantly lower in AD cases compared to age-matched controls (see, for example, FIG. 8B). DNAJC5 encodes a neuroprotective synaptic chaperone whose mutation impairs ALP function (see, for example, FIG. 13B). Therefore, haploinsufficiency in these genes may exacerbate AD-related phenotypes in aged mice.

[0326] Sample size Fifteen hemizygous mice from each gene [NAGLU (− / +), NPC1 (− / +), and DNAJC5 (− / +)] and fifteen wild-type mice are anesthetized and euthanized at 24 months of age, and the brain is collected for histological and biochemical studies.

[0327] Quantification of amyloid plaques and Aβ production Fixed frozen brain sections (50 μm) are stained in a subcohort of mice with X-34, immunostained with the HJ3.4 (anti-Aβ) antibody, and plaque load is quantified (expressed as % area). Aβ levels in brain tissue homogenates from the contralateral hemisphere are fractionated into soluble (PBS) and insoluble (5M guanidine) fractions and quantified using ELISA. Evaluate whether haploinsufficiency of the selected gene affects the APP processing machinery.

[0328] Synaptic marker Synapse loss is a common finding in humans and AD mouse models. Evaluate whether a single copy of the selected gene accelerates synapse loss in 5XFAD mice by Western blot using antibodies against the following presynaptic markers. SNAP-25, vesicle-associated membrane protein 2, syntaxin 1, and synaptophysin, previously published.

[0329] ALP dysfunction Using the above brain sections, the sections are immunostained with anti-LAMP1, LC3, and p62 antibodies.

[0330] Effects on neurodegeneration and reactive gliosis Fixed frozen brain sections from the above cohort are immunostained with the reticulon-3 (RTN-3) antibody (RTN-3 selectively accumulates in degenerating neurites) and degenerating neurites are quantified as previously published. Previous studies have shown that NAGLU, NPC1, and DNAJC5-deficient mice exhibit increased astrogliosis. Therefore, in a parallel study, brain sections are stained with anti-CD11b and anti-GFAP antibodies to investigate the effect of a single copy of the selected gene on reactive gliosis.

[0331] Expected results Heterozygous mice for the NAGLU, NPC1, and DNAJC5 genes are expected to accelerate and exacerbate the burden of Aβ plaques in 5XFAD mice. Haploinsufficiency of the selected genes is expected to affect APP metabolism and Aβ production in aged mice, increasing synaptic loss and reactive gliosis without Aβ plaque formation.

[0332] If changes in APP metabolism and Aβ production are not found in the brains of heterozygous mice, an AAV2 / 9 vector with the most significant variant verified in the experiments described in Chapter (II) is generated and stereotactically injected into the hippocampus of neonatal heterozygous mice, and the presence of AD lesions is evaluated. Alternatively, transcripts of the NAGLU, NPC1, and DNAJC5 genes are knocked down in neonatal 5XFAD transgenic mice by injecting an AAV2 / 9 vector with shRNA / RNAi verified for them. Knock-in mice for variants in the selected genes with the most potent effects on in vitro assays can be generated using CRISPr technology.

[0333] Combining the results from the studies described herein with the availability of fluorescence assays for NAGLU activity and mass spectrometry assays for NPC1 biomarkers allows screening of CSF, plasma, serum, or dried blood spots from large cohorts of AD cases and controls to detect specific defects that can be used as biomarkers for AD.

[0334] Example 4: Determining the impact of genetic variations in NAGLU on the onset of Alzheimer's disease (AD) and Parkinson's disease (PD) This example describes the in vitro and in vivo effects of genetic variations in the NAGLU gene on the onset of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0335] Increasing evidence suggests clinical, pathological, and genetic overlaps between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) associated with Parkinson's disease (PD). Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms are not fully understood. There is no systematic evaluation of the role played by genetic variants in enzymes involved in the lysosomal degradation of HS in the pathogenesis of AD or PD. Here, genetic analysis was performed in large case-control AD and PD cohorts, and a significant enrichment of rare functional variants was found in lysosomal enzyme genes responsible for heparan sulfate (HS) degradation with a substantial effect size. Heparan sulfate proteoglycan (HSPG) regulates the oligomerization, clearance, endocytosis, and transport of various pathogenic proteins, including amyloid-β (Aβ) and α-synuclein (α-Syn). Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. So far, it is not clear whether the decrease in N-acetyl-α-glucosaminidase (NAGLU) activity and the resulting HSPG accumulation affect APP metabolism, Aβ plaque load, or the aggregation and spread of α-Syn. The studies described herein can determine the effects of hypomorphic missense variants in the NAGLU gene on Aβ precursor protein (APP) transport, Aβ production in neurons, and Aβ degradation by glial cells. An innovative approach for modeling genetic variants associated with human neurodegenerative diseases in mice is described. The use of a neurotropic adeno-associated virus (AAV) vector enables the non-invasive widespread distribution and long-lasting global neuronal expression of hypomorphic NAGLU variants initially injected in NAGLU heterozygous mice with long-term follow-up. Thus, the cellular effects of NAGLU variants, as well as the effects of aging and heterozygosity, can be studied, as seen in AD and PD patients.It can also be determined whether decreased or overexpressed NAGLU affects AD lesions present in a well-characterized mouse model of AD. It can be determined whether hypomorphic missense variants in the NAGLU gene affect the binding, internalization, and aggregation of α-Syn in primary neurons and whether those changes are rescued by substrate inhibition, recombinant enzyme supplementation, or gene therapy. Finally, using a well-established and reliable method for pathological α-Syn spreading, it can be determined whether hypomorphic NAGLU variants affect the formation of aggregates of phosphorylated α-Syn, connectivity-dependent spreading, and their effects on disease progression and lifespan. The results of the studies described herein have important implications for the identification of PD and AD patients with genetically determined lysosomal dysfunction, and restoration of such dysfunction can provide an effective therapy.

[0336] The goal of the studies described herein is to determine the in vitro and in vivo effects of genetic variations in the NAGLU gene in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). These studies incorporate an innovative approach for modeling genetic variants associated with human neurodegenerative diseases in mice. The studies described herein can identify novel lysosomal genes associated with AD and PD and provide deeper insight into the mechanisms of lysosomal dysfunction in the pathogenesis of AD and PD.

[0337] Increasing evidence suggests clinical, pathological, and genetic overlap between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) with Parkinson's disease (PD). Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms are not fully understood. Heparan sulfate proteoglycan (HSPG) regulates the oligomerization, clearance, endocytosis, and transport of amyloid-β (Aβ) and α-synuclein (α-Syn) in cell cultures. HSPG binds to Aβ and accelerates its oligomerization and aggregation. HS independently stimulates the formation of α-Syn protofibrils in vitro and mediates cellular Aβ uptake. HSPG also mediates the uptake of α-Syn by macropinocytosis. Pharmacological inhibition of HSPG binding to pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. In addition, HSPG is present in Aβ plaques and Lewy bodies (LBs). These findings suggest that HS and HSPG play important roles in Aβ and α-Syn metabolism and subsequent AD and PD pathogenesis. However, there is no systematic evaluation of the role played by genetic variants in enzymes involved in the lysosomal degradation of HS in the development of AD or PD. Therefore, genetic analysis was performed in large case-control AD and PD cohorts, and significant enrichment of rare functional variants was identified in lysosomal enzyme genes responsible for heparan sulfate (HS) degradation with a considerable effect size. The most widely studied lysosomal enzyme involved in HS degradation is N-acetyl-α-glucosaminidase (NAGLU), deficiency of which causes mucopolysaccharidosis IIIB (MPS-IIIB) in humans. There is a well-characterized mouse model of NAGLU deficiency that recapitulates the features of human disease.The goal of the research described in this specification is to determine whether the hypomorphic missense variants in the NAGLU gene found in patients with AD (odds ratio = 3.7) and PD (odds ratio = 4.7) affect APP metabolism, Aβ production and degradation in vitro, and AD lesions in vivo, as well as α-Syn aggregation in vitro and α-Syn diffusion in vivo.

[0338] (I) Determine the effect of hypomorphic missense variants in the NAGLU gene on neuronal APP metabolism and Aβ production, Aβ glial degradation, and AD lesions in vivo Both human MPS-IIIB patients and NAGLU-deficient mice show an increase in the cortical level of intracellular full-length APP. MPS-IIIB patients also show a significant 3-fold increase in the level of soluble Aβ40 compared to control brains. The hypothesis is put forward that changes in NAGLU activity affect APP metabolism and Aβ production in neurons. The heterozygous state of AD patients with NAGLU variants is modeled in vitro. As described in this specification, the effects of hypomorphic NAGLU variants on APP trafficking, APP half-life, APP processing machinery and Aβ production can be determined in primary neurons.

[0339] Increasing evidence suggests that microglial cells contribute to the development of AD. Data mining efforts have revealed that NAGLU shows high levels of expression in microglial cells. However, little is known about the role of NAGLU in glial cells. Therefore, as described in this specification, it is possible to determine whether primary microglial cells stably expressing hypomorphic NAGLU variants can take up and degrade exogenous Aβ.

[0340] In the absence of familial AD mutations, NAGLU-deficient mice show intracellular accumulation of Aβ and HSPG in the inner olfactory cortex. The effects of NAGLU heterozygosity and aging on AD lesions are modeled in vivo. It can be determined whether the most deleterious NAGLU variants affect the AD-related phenotype in 24-month-old NAGLU heterozygous mice after injection into neonates using the AAV2 / 9-PHP.B vector.

[0341] It can be determined whether decreased or overexpressed NAGLU affects Aβ production, Aβ clearance, plaque deposition, synapse loss, and neuroinflammation in 5XFAD mice at 4 and 8 months.

[0342] (II) Determine the effect of a hypomorphic missense variant in the NAGLU gene on α-Syn aggregation in vitro and α-Syn spreading in vivo MPS-IIIB patients show severe neuronal loss in the substantia nigra (SN) and accumulation of pathological phosphorylated α-Syn (pSyn) in neurons in the temporal cortex, hippocampus, and SN. The hypothesis is put forward that reduced NAGLU activity affects α-Syn uptake, clearance, and aggregation in vitro and spreading in vivo.

[0343] As described herein, it can be determined whether the binding, internalization, and aggregation of preformed fibrils (PFFs) of α-Syn are affected in primary neurons stably expressing a hypomorphic NAGLU variant.

[0344] It can also be determined whether substrate inhibition (genistein), recombinant enzyme supplementation, or gene therapy rescues the effects on α-Syn PFF internalization and aggregation.

[0345] Intrastriatal injection of α-Syn PFFs recapitulates the accumulation of intracellular LB lesions, the selective loss of SN neurons, and the impairment of coordinated movement in transgenic mice expressing wild-type and mutant A53T human α-Syn. Intrastriatal inoculation of α-Syn PFFs is performed in hemizygous or NAGLU-deficient mice injected with the AAV2 / 9-PHP.B vector expressing the most deleterious NAGLU variant at birth. To quantify the formation of aggregates of phosphorylated α-Syn, connectivity-dependent spread, and their effects on disease progression and lifespan.

[0346] Significance Heparan sulfate in AD and PD Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms are not fully understood. Heparan sulfate proteoglycans (HSPGs), which consist of HS chains covalently attached to specific protein cores, are abundant molecules on the cell surface and extracellularly that interact with a spectrum of ligands. Membrane HSPGs function as endocytosis receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and their bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and transport of various pathogenic proteins, including Aβ and α-Syn. HSPGs are present in Aβ plaques, as well as in LBs and LNs. HSPGs have been shown to bind to Aβ and accelerate its oligomerization and aggregation. HS significantly stimulates the formation of α-Syn protofibrils in vitro. HS also mediates cellular Aβ uptake. HSPGs mediate the uptake of α-Syn by macropinocytosis. Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. These findings suggest that HS and HSPGs play important roles in Aβ and α-Syn metabolism and the pathogenesis of AD and PD.

[0347] Lysosomal Dysfunction in Alzheimer's Disease The familial forms of Alzheimer's disease (AD) are pathogenically caused by increased production of amyloid-β (Aβ) and subsequent aggregation of Aβ into soluble oligomers or insoluble Aβ plaques in the extracellular space (ISF, interstitial fluid). However, recent studies in late-onset sporadic AD patients have shown impaired clearance of Aβ. Thus, the balance between production and clearance determines Aβ levels and the propensity for Aβ plaque development. Lysosomes play a major role in the degradation of intracellular organelles and aggregation-prone proteins. Neuropathological studies have also found that lysosomal lesions in the AD brain contribute to AD pathogenesis, but the underlying mechanisms are not fully understood. Changes in lysosomes have been found in multiple transgenic mouse AD models. These results strongly suggest that changes in lysosomal proteins accelerate AD lesions. Cellular studies suggest that the endosome-lysosome system is a major site of Aβ production. Presenilin 2 (PSEN2) and nicastrin (an essential catalytic component of γ-secretase) are located in lysosomes. Indeed, PSEN1 regulates lysosomal pH. Pharmacological impairment of lysosomal function in vitro causes changes in Aβ production. Changes in lysosomal pH decrease Aβ secretion. Lysosomal protease inhibitors decrease the production of amyloidogenic APP fragments. All of these studies suggest that overall lysosomal function plays an important role in normal and abnormal amyloid precursor protein (APP) processing and subsequent amyloidogenesis.

[0348] APP Metabolism in Humans and Mice Deficient in HS Metabolic Genes The stepwise degradation of HS within lysosomes involves at least four enzymes. Loss-of-function (LoF) mutations in these genes cause the accumulation of partially degraded HS within lysosomes, leading to mucopolysaccharidosis type III (MPS III) subtypes A, B, C, and D (Sanfilippo syndrome). MPS patients show intense diffuse Aβ signals in the cytoplasm of cells throughout the brain. MPS patients exhibit a significant increase in the levels of soluble Aβ compared to normal control brains. In addition, increased intracellular APP / Aβ levels have been reported in MPS mouse models in the absence of overexpression of familial AD (FAD) mutations. A three-fold increase in Aβ40 levels has been found in MPS III mouse models compared to controls. Cognitive decline in both human and AD transgenic mice correlates with soluble Aβ species. Data from transgenic AD mice indicate that intracellular Aβ is more neurotoxic than extracellular Aβ. The accumulation of intracellular Aβ has been shown to precede extracellular deposition in both human and mouse AD models. These studies suggest that APP trafficking or processing is affected in both human patients and mouse models with complete loss of function of lysosomal genes involved in HS metabolism.

[0349] Lysosomal dysfunction in PD Post-mortem studies of humans and model systems suggest that defects in endocytic transport, lysosomal integrity, and lysosomal hydrolase activity play important roles in synucleinopathies. Lysosomal markers are components of Lewy bodies (LBs) in sporadic Parkinson's disease (PD) patients. Therefore, it has been suggested that LBs and Lewy neurites (LNs) can form seeds around damaged lysosomes as the disease progresses and increase in size by the continuous deposition of undegraded material derived from lysosomes. Multiple cell-based models converge on the importance of intercellular transfer of proteinopathic seeds in the progression of synucleinopathies, although mechanistic questions remain. It is still unclear whether specific α-Syn strains are internalized via distinct receptors or endocytic machinery. Macropinocytosis-mediated uptake of α-Syn by immortalized cells and primary neurons is mediated by heparan sulfate proteoglycan (HSPG). However, the role of HS in α-Syn spreading in vivo has not been evaluated. Lysosomal processing is the main fate of internalized α-Syn protofibrils in primary neurons. Severe pharmacological disruption of lysosomal function causes abnormal intracellular processing of α-Syn protofibrils, concomitant with increased inclusion formation rates due to mobilization of endogenous α-Syn. The processes governing this mobilization are still poorly understood, suggesting that pathogenic species must escape from intralysosomal transport. Thus, exogenous α-Syn species have been reported to cause rupture of vesicles and lysosomal membranes by endocytosis, thereby escaping endocytic transport and lysosomal degradation. Upon entry into the cytoplasmic matrix, these α-Syn protofibrils or oligomers can interact with soluble species and initiate mobilization of endogenous α-Syn. These results further support the idea that defects in lysosomal activity and integrity can accelerate pathological α-Syn aggregation and transmission. HSPG is present in LBs and LNs. HS significantly stimulates α-Syn protofibril formation in vitro. However, the role of lysosomal accumulation of HS in α-Syn aggregation and spreading has not yet been fully established.

[0350] Large-scale analysis of gene variations in human lysosomal genes Recent analyses of the frequency and types of mutations present in 60,000 exomes have found that lysosomal genes are "intolerant to loss-of-function" mutations and have fewer potentially harmful variants than predicted by the neutral model of evolution. This is consistent with the lethality during embryogenesis or the neonatal period of most "core" lysosomal genes in knockout mice. The most studied and well-known lysosomal genes are those that, when mutated, cause lysosomal storage diseases (LSDs). Interestingly, most of these genes that cause LSDs are not intolerant to LoF mutations and show substantial genetic coding variations in humans. Thus, there is a 10-fold range of difference in the levels of lysosomal enzyme activity in healthy humans. Having a single normal copy of a lysosomal gene has long been assumed not to have health consequences. However, recent studies in carriers of heterozygous missense pathogenic mutations in the NPC1 gene have revealed significant systemic metabolic abnormalities. In addition, substantial genetic evidence supports the role of heterozygous missense variants in the GBA gene as a major genetic risk factor for developing synucleinopathies. In the homozygous situation, the same pathogenic variants that cause LSD (Gaucher disease) in children, when present in a heterozygous fashion, affect the risk of adult-onset neurodegenerative diseases including PD and Lewy body dementia. These studies suggest that the combination of aging and haploinsufficiency in lysosomal genes predisposes to common neurodegenerative disorders in adult humans. There is a lack of systematic evaluation of the contribution of functional heterozygous variants in lysosomal genes that affect the risk of AD or PD.

[0351] Individuals with genetic variants causing haploinsufficiency in the NAGLU gene show significantly lower levels of enzyme activity than controls. Recent studies have reported the enzyme activity of 164 NAGLU missense "variants of uncertain significance (VUS)" in the ExAC dataset and 35 pathogenic missense mutations reported in HGMD, 17 of which were also found in ExAC. Approximately 90% of the pathogenic variants were shown to exhibit less than 15% enzyme activity compared to wild-type levels. These data suggest that individuals with hypomorphic heterozygous variants in the NAGLU gene exist in the general population. However, the long-term consequences of NAGLU haploinsufficiency have not been fully studied. Genetic analyses were performed in large case-control AD and PD cohorts, and significant enrichment of rare functional variants was identified in genes of lysosomal heparan sulfate (HS) metabolizing enzymes with substantial effect sizes. Hypomorphic missense variants in NAGLU were associated with AD (p = 3×10 -3 , OR = 2.3, 95% CI 1.2 - 5.2) and PD (p = 3.6×10 -7 , OR = 3.6, CI = 2.8 - 8.3). 【0352...

Claims

1. A pharmaceutical composition for preventing, treating, reversing, or delaying the onset of Alzheimer's disease (AD) dementia in a subject determined to have or be at risk of having AD dementia, detected to be heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of PPT1, DNAJC5, and NAGLU, the composition comprising an autophagy-lysosome pathway agent comprising a gene for gene therapy (GT), wherein the gene for gene therapy is a wild-type copy of the lysosomal gene.

2. The pharmaceutical composition according to claim 1, wherein the at least one lysosomal gene is NAGLU.

3. The pharmaceutical composition according to claim 1, wherein the lysosomal gene is DNAJC5.

4. The pharmaceutical composition according to claim 1, wherein the lysosomal gene is PPT1.

5. The pharmaceutical composition according to claim 1, wherein the subject is haploinsufficient for a heterozygous loss-of-function variant of PPT1 or has the heterozygous loss-of-function variant of PPT1.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the loss-of-function variant is one or more variants of insertion, substitution, or deletion of the lysosomal gene.

7. A pharmaceutical composition for preventing, treating, reversing, or delaying the onset of Alzheimer's disease (AD) in a subject determined to have or be at risk of having AD, detected to be heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of PPT1, DNAJC5, and NAGLU, the composition comprising an autophagy-lysosome pathway modulator comprising a gene for gene therapy (GT), wherein the gene for gene therapy is a wild-type copy of the lysosomal gene.

8. The pharmaceutical composition according to claim 7, wherein the lysosomal gene is DNAJC5.

9. The pharmaceutical composition according to claim 7, wherein the lysosomal gene is NAGLU.

10. The pharmaceutical composition according to claim 7, wherein the lysosomal gene is PPT1.

11. The pharmaceutical composition according to any one of claims 7 to 10, wherein the loss-of-function variant is one or more of insertion, substitution, or deletion of the lysosomal gene.

12. A method for determining the risk of Alzheimer's disease in a subject, the method comprising detecting the presence of at least one lysosomal gene that is heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of PPT1, DNAJC5, and NAGLU in a biological sample obtained from the subject, a method, wherein when at least one lysosomal gene heterozygote for the loss-of-function variant is detected, it indicates that the subject is at risk of Alzheimer's disease.

13. The method according to claim 12, wherein the at least one lysosomal gene is NAGLU.

14. The method according to claim 12, wherein the lysosomal gene is DNAJC5.

15. The method according to claim 12, wherein the lysosomal gene is PPT1.

16. The method according to any one of claims 12 to 15, wherein the loss-of-function variant is one or more of insertion, substitution, or deletion of the lysosomal gene.

Images