Human ventricular canal membrane specific promoter and application thereof

Abstract

Provided herein are compositions and methods for delivering a molecular therapeutic agent to the ventricular canal membrane of a subject. The methods comprise administering an adeno-associated virus (AAV) to the subject. The AAV encodes a therapeutic transgene under the control of a ventricular canal membrane specific promoter.

Description

[0001] Priority declaration

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial Nos. 63 / 482 / 155, 63 / 381,689, and 63 / 333,979, filed on January 30, 2023, October 31, 2022, April 22, 2022, and January 30, 2023, respectively, the entire contents of each of which are hereby incorporated by reference. Background Art 1. Technical Field

[0003] The present disclosure relates generally to the fields of medicine and virology. More specifically, it relates to compositions and methods for delivering molecular therapeutic agents to a patient, particularly to the brain or central nervous system.

[0004] 2. Description of Related Technology

[0005] Adeno-associated virus (AAV) represents a powerful therapeutic candidate for the treatment of neurological diseases. AAV is a non-enveloped single-stranded DNA virus that can infect both dividing and non-dividing cells. After infection, the virus does not show robust integration in the host genome, but persists in the nucleus as an episome. The expression of AAV cargo is spatially controlled by the level of the packaging capsid and the transgenic promoter. And because the use of AAV to treat diseases may require intervention in diseased tissues, problems may arise in target tissues containing gene expression profiles different from their healthy counterparts. Finding the right promoter sequence to drive therapeutic transgenic expression is an important goal. Summary of the invention

[0006] Provided herein are methods of expressing a therapeutic transgene in ependymal tissue of a subject, comprising administering to the subject a modified adeno-associated virus (AAV) encoding a therapeutic transgene under the control of a promoter selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a promoter having at least about 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. The promoter may comprise or consist of SEQ ID NO: 1, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The promoter may comprise or consist of SEQ ID NO: 2, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The promoter may comprise or consist of SEQ ID NO: 3, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0007] The modified AAV may comprise a modified capsid protein, for example wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide is three to ten amino acids in length, for example seven amino acids in length. The modified AAV capsid protein may be a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein. The modified AAV capsid protein may be derived from an AAV1 capsid protein, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

[0008] The targeting peptide may be flanked by linker sequences, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long, for example wherein the sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0009] The modified AAV capsid protein may be derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long, for example wherein the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

[0010] The modified AAV capsid protein can be derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. The flanking portion of the targeting peptide is a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long, for example, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0011] The therapeutic transgene can be siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein or CRISPR system. The therapeutic transgene can be ApoE2, and the subject suffers from Alzheimer's disease or is at an increased risk of developing Alzheimer's disease compared to the population average. Administration can be direct intraventricular or intraparenchymal injection. The modified AAV can be administered more than once, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. The modified AAV can be administered monthly, every other month, every three months, every four months, every six months or every year. The method may also include providing non-AAV therapy to the subject.

[0012] The method may include administering a plurality of viral particles, for example wherein the virus is present at about 1×10 6 About 1×10 18 The virus is administered at a dose of about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to 1×10 13 or about 1×10 13 Up to 1×10 14 vg / kg subject. The subject can be a human or a non-human mammal. The human subject can be 50 years of age or older. The therapeutic transgene can be linked to a polyadenylation signal.

[0013] Also provided is a modified adeno-associated virus (AAV) encoding a therapeutic transgene operably linked to a promoter selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a promoter having at least about 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. The promoter may comprise or consist of SEQ ID NO: 1, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The promoter may comprise or consist of SEQ ID NO: 2, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. The promoter may comprise or consist of SEQ ID NO: 3, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0014] The modified AAV may comprise a modified capsid protein, for example wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide is three to ten amino acids in length, for example seven amino acids in length. The modified AAV capsid protein may be a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

[0015] The modified AAV capsid protein can be derived from an AAV1 capsid protein, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein, for example, wherein the flanking portion of the targeting peptide is a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long. The linker sequence can be SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0016] The modified AAV capsid protein can be derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein, for example, wherein the flanking portion of the targeting peptide is a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long. The linker sequence can be AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

[0017] The modified AAV capsid protein can be derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein, for example, wherein the flanking portion of the targeting peptide is a linker sequence, wherein the linker sequence on each side of the targeting peptide is two or three amino acids long. The linker sequence can be AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0018] The therapeutic transgene can be siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein or CRISPR system. The therapeutic transgene can be linked to a polyadenylation signal. The therapeutic transgene can be transcriptionally linked to a detectable reporter, for example, a sequence encoding a fluorescent protein, a peptide tag or a luciferase.

[0019] In another embodiment, a pharmaceutical composition is provided, comprising a modified AAV as described herein and a pharmaceutically acceptable carrier.

[0020] In yet another embodiment, an isolated and purified nucleic acid is provided, comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a sequence having at least about 80% sequence identity thereto. The sequence may comprise or consist of SEQ ID NO: 1, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The sequence may comprise or consist of SEQ ID NO: 2, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The sequence may comprise or consist of SEQ ID NO: 3, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

[0021] The sequence may be operably linked to a heterologous coding region. The nucleic acid may also contain one or more of: (a) a general-purpose cloning site, (b) a transcription termination signal, (c) a polyadenylation sequence and / or (d) an origin of replication. The nucleic acid may also contain one or more of: (a) a sequence encoding a detectable marker, (b) a sequence encoding an affinity tag, and / or (c) one or two adeno-associated virus inverted terminal repeats. The nucleic acid may be contained in a replicable vector. The therapeutic transgene may be transcriptionally linked to the reporter via a sequence encoding a 2A "self-cleaving" peptide.

[0022] In yet another embodiment, a method for reducing or weakening microglial inflammation is provided, which includes delivering ApoE2 to microglia in an object in need thereof. ApoE2 is delivered to microglia, including administering a modified AAV as defined herein or a pharmaceutical preparation comprising it to the object, wherein the therapeutic transgene is ApoE2. Microglial inflammation may be caused by or is associated with neurodegenerative diseases, such as Huntington's disease, Parkinson's disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, progressive supranuclear palsy, amyotrophic lateral sclerosis, multiple sclerosis, Batten disease, and Creutzfeldt-Jakob disease. Microglial inflammation may be caused by or is associated with Alzheimer's disease.

[0023] Administration can be by direct intraventricular or intraparenchymal injection of ApoE2 or modified AAV, and can involve more than one administration, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, and / or monthly, every other month, every three months, every four months, every six months or yearly. The method can also include providing the subject with a non-AAV ApoE2 therapy.

[0024] For AAV therapy, multiple viral particles can be administered, for example, at about 1×10 per kg. 6 About 1×10 18 The dosage of vector genomes (vg / kg) includes the following dosages: about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to 1×10 13 or about 1×10 13 Up to 1×10 14vg / kg patient. The subject can be a human, a non-human mammal, or a human subject 50 years of age or older. The therapeutic transgene can be linked to a polyadenylation signal.

[0025] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, although indicating some preferred embodiments of the present disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from the detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0027] The following drawings constitute part of this specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments shown herein.

[0028] Figure 1 . Identification of ependymal-specific promoters. Due to the thin nature of the ependymal lining of the ventricles, the inventors adopted a subtractive approach to identify tissue-specific genes. Samples were obtained from white matter and gray matter adjacent to the ventricles, as well as areas at the ventricular margins that include white matter and gray matter as well as ependyma. Genes unique to white matter + ependyma and gray matter + ependyma samples were classified as enriched genes. To ensure the use of active promoters in a variety of related disease states, samples were derived from healthy controls as well as from patients with Alzheimer's disease (AD), Huntington's Disease (HD), frontotemporal dementia, Lewy body dementia, semantic dementia, and dementia.

[0029] Figure 2 . Validation of expression in ependyma. The best gene hits were validated based on in situ hybridization data published by the Allen Brain Institute. - Not ependyma-specific / enriched; +od ependyma-specific / enriched; NA No data available.

[0030] Figure 3. Validation of expression in ependyma. Cartoon of ependyma-specific promoter transgenes. Approximately 1100 to 2500 bp of potential promoter sequence from ependyma-enriched genes were cloned upstream of the eGFP reporter. A 3-bp barcode in the 3'UTR of the RNA transcript was used to identify individual transgenes.

[0031] Figure 4 hEpendymal promoter library usage in mouse ependyma. Fractional contribution of each transgene-associated barcode in mouse ependymal RNA and AAV4 viral library input following injection at low (5E10vg), medium (1E11vg), and high (5E11vg) vector doses.

[0032] Figure 5 .hEpendymal promoter library usage in mouse ependyma. Quantification of readout enrichment in RNA output relative to viral library input. Significant enrichment is shown by the positive control (ubiquitous iCAG promoter) and by the hVWA3a promoter.

[0033] Figure 6 . The fractional contribution of the h-ependymal promoter in samples containing rhesus monkey ependyma-eGFP. Figure 4 The same library used in was prepared as AAV2 and injected into the lateral ventricles of two adult rhesus monkeys at a total of 2E13 vg. At 3 weeks after injection, the area at the edge of the ventricle (including down through the spinal cord) was microdissected. RNA was isolated and converted to cDNA, and amplicon sequencing was performed on PCR products containing 3-letter barcodes. The colored bars indicate the relative contribution of each transgene.

[0034] Figure 7 .h Ependymal library round 2 - introduction of promoter introns to improve expression. The original transgene was modified to express human apolipoprotein 2 (ApoE2) cDNA and included short (133 bp) and long (951 bp) introns in the promoter region to improve expression through intron-mediated enhancement. Short flanking sequences (blue and green bars) known to promote efficient splicing were also included.

[0035] Figure 8 .h mRNA from the ependymal promoter library is correctly spliced ​​and encodes ApoE protein. To test the splicing efficiency in the intron-containing variants, the plasmids were transfected into HEK293 cells. Correct splicing was verified by amplifying the intron-containing region in the cDNA (C) and plasmid DNA (D).

[0036] Fig. 9.h Ependymal promoter library-derived mRNA is correctly spliced ​​and encodes ApoE protein. Western blots measured ApoE protein output from intron-less (N) and intron-containing (short S, long L) variants in HEK293 cell lysates and culture medium.

[0037] Fig.10 .Intron-containing promoters for Rh ependyma. Libraries of six different promoters containing no introns, short introns, or long introns were prepared in a single AAV2 and injected into the lateral ventricles of two adult rhesus monkeys with a total dose of 2.8e13 vg. Four weeks later, the ventricular margins were microdissected. Amplicon sequencing of products containing a unique 3-letter barcode in the 3'UTR was performed as previously described to assess relative promoter usage in vivo. All hVWA3a variants showed relative enrichment in vivo relative to the input library.

[0038] Fig.11 .h ependymal promoter drives expression in mice. Adult APOE- / - (null) mice were injected with serotype AAV4 to deliver APOE2 under the hVWA3a promoter to the right lateral ventricle. Ependymal tissue was microdissected and protein was extracted for quantification of APOE2 by automated Western blot technology (WES) compared to uninjected brain tissue.

[0039] Fig.12 .h ependymal promoter drives higher expression in mice than the ubiquitous CAG promoter. APOE- / - (null) mice were injected with serotype AAV4, and APOE2 under the ubiquitous CAG promoter or hVWA3a promoter was delivered to their right lateral ventricle at the same dose. Protein was extracted from ependymal tissue microdissected from all animals and subjected to automated Western blotting (WES). From the intensity of the band, APOE2 driven by hVWA3a expressed higher amounts of APOE2 protein than APOE2 driven by the CAG promoter.

[0040] Fig.13 . Peptide-modified AAV1 capsid with a human ependyma-specific promoter: ERDRpAAV1.hVWA3a.eGFP. Positive eGFP fluorescence signal is restricted to ependymal cells lining the ventricles.

[0041] Fig.14 .hVWA3a promoter segment (SEQ ID NO: 1).

[0042] Fig.15. hVWA3a promoter segment (SEQ ID NO: 2) with a short intron (underlined) (133 bp).

[0043] Fig.16 . hVWA3a promoter segment (SEQ ID NO: 3) with long intron (underlined) (951 bp).

[0044] Fig.17 Schematic diagram of an AAV transgene that uses upstream regulatory sequences from the human Von Willebrand Factor A Domain 3A (VWA3a) gene to drive ependymal-specific expression of human APOE2. A short β-globin / IgG chimeric intron (133 bp) is inserted downstream of the transcription start site to enhance transcription by intron-mediated enhancement and contains a strong Kozak sequence to initiate APOE2 translation. The entire transgene is flanked by AAV2 inverted terminal repeats (ITRs).

[0045] Fig.18 . hVWA3a promoter short intron hApoE2 expression construct for AAV (SEQ ID NO: 4).

[0046] Fig.19 .pmAAV1.ERDR.hVWA3a.APOE2@7E10vg significantly reduced ThioS positive signals in the cortex compared to vehicle-treated controls. pmAAV1.ERDR.hVWA3a.APOE2 was delivered to an Alzheimer's disease mouse model homozygous for human APOE4. The % ThioS positive cells in mice treated with a high dose of 7E10vg were significantly reduced relative to vehicle-treated control mice (p<0.05). Low dose: 7E9vg; medium dose: 2E10vg; high dose: 7E10vg. ThioS stains B-folds (used as a marker for dense core plaques as opposed to diffuse plaques) within amyloid plaques present in mice. The difference in ThioS but not oligo antibody staining (IBL) suggests that AAV prevents the formation of dense core plaques, rather than general plaque formation.

[0047] Fig. 20.pmAAV1.ERDR.hVWA3a.APOE2@7E10vg significantly reduced amyloid beta positive signals in the cortex compared to vehicle treated controls.pmAAV1.ERDR.hVWA3a.APOE2 was delivered to an Alzheimer's disease mouse model homozygous for human APOE4. Amyloid beta stained cells were significantly reduced in mice treated with 7E10vg relative to vehicle treated mice (p<0.05). Low dose: 7E9vg; Medium dose: 2E10vg; High dose: 7E10vg.

[0048] Fig.21 . Viral genome copies determined by QPCR of DNA against the hVWA3a promoter sequence. pmAAV1.ERDR.hVWA3a.APOE2 was delivered to an Alzheimer's disease mouse model homozygous for human APOE4. DNA lysates from mouse brains treated with all three doses of vector were positive when assayed at the non-transcribed region of the human VWA3a promoter sequence. Control (vehicle) treated AD mice as well as control treated WT mice showed only background levels of hVWA3a that were below the detectable range by the standard curve. Low dose: 7E9vg; Medium dose: 2E10vg; High dose: 7E10vg.

[0049] Fig. 22 . Viral genome copies determined by QPCR of DNA against the hVWA3a promoter sequence. Viral genome copies of pmAAV1.ERDR.hVWA3a.ApoE2 are detectable in ependymal, cortical, and hippocampal tissues in the brains of non-human primates treated with three different doses of pmAAV1.ERDR.hVWA3a.APOE2. Brain tissue DNA lysates were assayed for total genome copies determined at the non-transcribed region of the human VWA3a promoter sequence. The original sample was from NHPs that did not receive pmAAV1.ERDR.hVWA3a.APOE2. The keys from top to bottom in the figure are the same as the keys from left to right.

[0050] Fig.23.APOE2 expression in a triple transgenic mouse model of Alzheimer's disease. APOE4XAPP / PS1 is a triple transgenic mouse that expresses chimeric mouse / human amyloid precursor protein, mutant human presenilin 1, and human APOE4, establishing a model with many aspects of the human condition. Images show cortical amyloid beta (Aβ; red) and neuronal staining (DAPI; blue) at 3, 4, 5, and 6 months of age if untreated. The study paradigm was that mice were injected at 4 months of age when plaques began to accumulate and autopsied at 6 months of age. Mice were injected intraventricularly with pmAAV1.ERDR.hVWA3a.ApoE2 in increasing doses of 7E9, 2E10, and 7E10 vg. The readout of genomic copy expression is at Fig.21 Shown in.

[0051] Fig.24 .APOE2 expression is beneficial for plaque deposition in APOE4XAPP / PS1 mice. The ThioS staining on the left shows plaques 2 months after delivery of pmAAV1.ERDR.hVWA3a.ApoE2. The quantitative graph shows a significant reduction in ThioS levels and soluble AB42 levels in mice receiving the 7E10 vg dose.

[0052] Fig.25 APOE2 expression reduces plaque density and plaque size in AD mouse models. 7E10 vg of pmAAV1.ERDR.hVWA3a.ApoE2 significantly reduced plaque parameters relative to untreated age-matched AD mice.

[0053] Fig.26 Training images for glial grading parameters. Brain sections were stained for Iba1 (a marker for microglia; blue), GFAP (a marker for astrocytes; green), and amyloid-β (a marker for plaques; red), and the relative level of glial staining near plaques was scored.

[0054] Fig. 27 . Virally expressed APOE2 prevents microglial proliferation near plaques. Fig.26 Two blinded scientists trained on images of the MRI machine scored microglia (Iba1; blue) near plaques (Aβ; red) in brains from mice dosed with pmAAV.ERDR.hVWA3a.ApoE2.

[0055] Fig.28 .Staining of AD mouse brains at 3, 4, 5 and 6 months of age. The upper panel shows images of cortex stained with GFAP (green) and Aβ (red). The lower panel shows images of cortex stained with Iba1 (blue) Aβ (red).

[0056] Fig.29 Preliminary assessment that AD mice have high variability in microgliosis AD mice dosed with pmAAV.ERDR.hVWA3a.ApoE2 showed even higher variability in microgliosis.

[0057] Fig.30 . Graphical representation of GFAP scores near plaques. Delivery of APOE2 to AD mice had no significant effect on astrocyte reactivity near plaques, as assessed by blinded pathology scoring.

[0058] Fig.31 Virally expressed APOE2 prevents synaptic loss near plaques. Brain images of AD mice injected with 7E10vg of pmAAV.ERDR.hVWA3a.ApoE2 or vehicle control. PSD95 (post-synaptic density-95) staining at synaptic terminals was more pronounced in animals that received the drug.

[0059] Fig.32 .Quantification of synaptic integrity in AD mice dosed with APOE2. Synapses proximal (near; red) and distal (far; blue) to plaques were quantified from histological images. Mice dosed with only the 7E10vg treatment showed similar amounts of synaptic density both near and far from plaques. In all other AD treatment groups, synaptic density near plaques was significantly lower relative to synaptic density far from plaques (left figure). The figure on the right compares the "near" and "far" synaptic density between groups, revealing that the synaptic density of "near" plaques was significantly higher in the high dose (7E10vg) group relative to all other groups.

[0060] Figures 33A to E. Ependymal cell expression of APOE2 driven by a novel AAV capsid and promoter. (Figure 33A) In situ hybridization shows expression of human APOE in ependymal cells of the midbrain ventricle of APOE KO mice. (Figure 33B) Western blot of APOE shows that AAV-derived APOE2 produced in the ependyma of the APOE KO mouse cortex is about 10% of the endogenous level (Figure 33C). (Figure 33D) Viral genome copies in tissues extracted from each mouse [(F(4,35)=5.546p=0.0014) post hoc Tukey multiple comparison test]. (Figure 33E) qRTPCR of human APOE normalized to vehicle-treated animals (F(4,26)=2.890p=0.0419 post hoc Dunnett test relative to vehicle]). (E) Line graph shows a significant correlation between mRNA and viral genome copies of APOE (p=0.0445). N represents a single point per mouse. *p<0.05, **p<0.01, ***p<0.001.

[0061] Figure 34A to E.POE2 reduces plaque deposition, number and size in a dose-dependent manner. (Figure 34A) IHC of thioS in the cortex of animals administered APP / PS1 / APOE4. (Figure 34B) In high-dose animals, the percentage of cortical coverage stained by ThioS was significantly lower (F(3,27)=4.310p=0.0329). (Figure 34C) The percentage of cortical coverage by ThioS was significantly correlated with the number of viral genome copies in brain samples from each mouse (p=0.0112). (Figure 34D) Both the number of plaques (F(3,27)=3.597p=0.0263) and (Figure 34E) plaque size (F(3,27)=4.113p=0.0159) showed significant effects in the high-dose group. n is represented by a single point per mouse, where the open circles are females and the solid ones are males. Post hoc tests show Dunnett's multiple comparison test compared to vehicle. p*p<0.05, **p<0.01.

[0062] Figure 35A to D. APOE2 reduces microglial proliferation near plaques. (Figure 35A) IHC of IBA1 and o Aβ in the cortex of animals administered APP / PS1 / APOE4. (Figure 35B) The average microglial response score (F(3,26)=4.529p=0.0110) shows that microglial activation in the high-dose group and the medium-dose group is reduced, and (Figure 35C) is significantly correlated with the number of viral genome copies in the mouse (p=0.0081). (Figure 35D) This reduction is due to a decrease in the number of plaques with a score of 4 and an increase in the number of plaques with a score of 1. n is represented by a single point per mouse, with open circles for females and solid for males. Post hoc tests are shown as Dunnett's multiple comparison tests compared with vehicle. p*p<0.05.

[0063] Figure 36A to D. APOE2 reduces synaptic loss nearby. (Figure 36A) IHC of PSD95 and oAβ in the cortex of animals administered APP / PS1 / APOE4. (Figure 36B) Synaptic density did not change away from plaques, but (Figure 36C) significantly increased near plaques (F(3,27)=5.153p=0.0060). (Figure 36D) This resulted in a significant reduction in the percentage of synaptic loss in high-dose animals compared to the vehicle (F(3,27)=3.693p=0.0239). n is represented as a single point per mouse, with open circles for females and solid circles for males. Post hoc tests are shown as Dunnett's multiple comparison tests compared to the vehicle. p*p<0.05.

[0064] Figure 37A to D. Effect of APOE2 on oligomeric Aβ. (Figure 37A) In high-dose animals, the percentage of cortical coverage by Aβ staining was significantly lower (F(3,27)=6.336, p=0.0022). (Figure 37B) The percentage of cortical coverage by ThioS was significantly correlated with the number of viral genome copies in the mouse (p=0.0173). (Figure 37C) ELISA showed that both SDS-soluble (F(3,28)=3.497p=0.0285) and (Figure 37D) formic acid-soluble (F(3,30)=3.741p=0.0214) Aβ showed significant effects in the high-dose group. n represents a single point per mouse, with open circles for females and solid circles for males. Post hoc tests are shown as Dunnett's multiple comparison tests compared to vehicle. p*p<0.05, **p<0.01.

[0065] Figure 38A to E. APOE2 does not affect the reactivity of astrocytes near plaques. (Figure 38A) Representative images of a four-point scale for assessing the reactivity of microglia and astrocytes to plaques. (Figure 38B) IHC of GFAP and oAβ in the cortex of animals administered APP / PS1 / APOE4. (Figure 38C) The average astrocyte response score (F(3,26)=1.634p=0.2057) showed no change between groups, and (Figure 38D) there was no significant correlation with the number of viral genomes (p=0.1904). (Figure 38E) There was no difference in the number of plaques counted in each category between groups. n is represented as a single point per mouse, with open circles for females and solid for males. Post hoc tests are shown as Dunnett's multiple comparison test compared to vehicle. P*p<0.05.

[0066] Figures 39A to C. APOE2 does not affect neuritic dystrophy. (Figure 39A) IHC of Smi-312 and oAβ in the cortex of animals dosed with APP / PS1 / APOE4. (Figure 39B) Analysis showed no difference in the number of dystrophy counted per plaque (F(3,25)=0.4710; p=0.7052) even (Figure 39C) when plaque size was considered (F(3,25)=1.127p=0.3569).

[0067] Fig.40 .h ependymal promoter drives ependymal localized APOE transcription in NHP after ICV delivery. A total of 1E13vg of pmAAV.ERDR.hVWA3a.ApoE2 vector was injected unilaterally into the lateral ventricle of adult African green monkeys. Tissues were harvested 60 days after injection for sectioning and transgenic expression was monitored by RNA fluorescent in situ hybridization (RNA-FISH). Probes designed for targeting human APOE showed strong overlap with endogenous African green APOE due to high sequence homology. In order to specify the source of transcription, the inventors rely on positioning. Endogenous APOE transcription only occurs in astrocytes and microglia, so the inventors can attribute the signal of ependymal localization to APOE2 of transgenic origin, which is defined by the overlap with the ependymal-specific gene FoxJ1 (outlined with white dotted lines). Hoechst H33258 identifies tissue DNA.

[0068] Fig.41.h ependymal promoter increases CSF APOE in NHP after ICV delivery. A total of 1E13 vg of pmAAV1.ERDR.hVWA3a.APOE2 vector was injected unilaterally into the lateral ventricle of adult African green monkeys. CSF was collected at baseline, 30, 45 and 60 days after injection, and APOE protein was measured by automated western blotting. All values ​​were normalized to baseline. DETAILED DESCRIPTION

[0069] Here, the inventors attempt to identify promoter sequences that can drive ependyma-specific expression in mouse and human brains, which can then be used in gene therapy models to drive the expression of secreted proteins to treat neurological diseases. The inventors choose to first identify genes that are insensitive to neurological disease states and age, and then identify relevant promoters through proxies. Such promoters can be used to drive transgene expression in the ependyma (the epithelial cell layer lining the ventricles of the brain). After infecting these cells with AAV, secreted proteins can enter the ventricles and be distributed throughout the brain by means of cerebrospinal fluid. And by eliminating promoters that may be negatively affected by altered gene expression patterns in diseased tissues, stronger transgene expression can be achieved.

[0070] To achieve this, the inventors obtained ependyma and adjacent ependyma-free samples from normal and diseased brains (Alzheimer's disease, Huntington's disease, frontotemporal dementia, etc.). Using RNA sequencing, the inventors identified genes whose expression was enriched in samples containing ependyma and maintained expression regardless of the disease state. Other evidence of its specificity was verified using public data sets (including the Allen Brain Institute in situ hybridization library). Since the promoter is a loosely defined structure, the inventors isolated the genomic sequence (about 1100 to 2500bp) upstream of the transcription start site from the best gene candidates (11 promoters) and placed it upstream of the GFP reporter and unique 3-letter RNA barcode in the AAV compatible transgene (flanked by ITR). The plasmids containing different promoters were merged, prepared as AAV4 or AAV2, and injected directly into the ventricles of mice or rhesus monkeys (rhesusmacaque), respectively. The tissue containing the ependyma was microdissected and the region around the 3 letter barcode was subjected to amplicon sequencing. The output was compared with the library input to determine enrichment. It has been shown that the inclusion of upstream introns improves tissue expression. The best hits (6 promoters) identified in the first screening were further modified to include short (133bp) or long (951bp) introns. The individual forms were identified again by the unique 3 letter barcodes in the 3'UTR. The transgenic comprising a variant of the human von Willebrand factor A domain (hVWA3a) containing 3a was the highest enriched in the final screening and was selected for further study.

[0071] These and other aspects of the disclosure are discussed in greater detail below.

[0072] I. Adeno-associated virus (AAV) vector

[0073] Adeno-associated virus (AAV) is a small, non-pathogenic virus of the Parvoviridae family. To date, many serologically different AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV differs from other members of the family in that it relies on a helper virus for replication.

[0074] The AAV genome can exist in an extrachromosomal state without integrating into the host cell genome; has a wide host range; transduces both dividing and non-dividing cells in vitro and in vivo, and maintains high levels of expression of transduced genes. AAV virus particles are thermostable; are resistant to solvents, detergents, pH changes and temperature; and can be column purified and / or concentrated on a CsCl gradient or by other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7kb genome of AAV consists of a segment of single-stranded DNA at the positive or negative pole. The end of the genome is a short reverse terminal repeat (ITR), which can be folded into a hairpin structure and serves as the starting point for viral DNA replication.

[0075] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form AAV particles. AAV particles typically contain an AAV genome packaged with AAV capsid proteins. In the case of using a recombinant plasmid to construct or manufacture a recombinant vector, the AAV vector genome does not include a "plasmid" portion that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is called a "plasmid backbone," which is important for plasmid cloning and amplification (the process required for plasmid proliferation and production), but it itself is not packaged or encapsulated in viral particles. Therefore, the AAV vector "genome" refers to a nucleic acid packaged or encapsulated by an AAV capsid protein.

[0076] AAV virions (particles) are non-enveloped icosahedral particles with a diameter of about 25 nm containing AAV capsids. AAV particles contain icosahedral symmetry, which is composed of three related capsid proteins VP1, VP2 and VP3, which interact to form capsids. The genome of most natural AAVs usually contains two open reading frames (ORF), sometimes also referred to as left ORF and right ORF. The right ORF usually encodes capsid proteins VP1, VP2 and VP3. These proteins are usually present in a ratio of 1:1:10, respectively, but can exist in different ratios and are all derived from the right ORF. VP1, VP2 and VP3 capsid proteins are different from each other by using alternative splicing and unusual start codons. Deletion analysis shows that removing or changing the VP1 translated from the alternative splicing information leads to a reduced yield of infectious particles. Mutations in the VP3 coding region result in the inability to produce any single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of the AAV particle encodes one, two or all three VP1, VP2 and VP3 polypeptides.

[0077] The left ORF usually encodes nonstructural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which, in addition to the production of single-stranded progeny genomes, are also involved in the regulation of replication and transcription. Two of the Rep proteins are related to the preferential integration of the AAV genome into the region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity and DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals and several potential phosphorylation sites. In certain embodiments, the genome of AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of AAV (e.g., rAAV) does not encode Rep proteins. In certain embodiments, one or more of the Rep proteins can be delivered in trans, and are therefore not included in the AAV particles comprising nucleic acids encoding polypeptides.

[0078] The end of the AAV genome comprises short reverse terminal repeats (ITRs), which have the potential to fold into a T-shaped hairpin structure, which serves as the starting point for viral DNA replication. Therefore, the genome of AAV comprises one or more ITR sequences (e.g., ITR sequence pairs), which are located on the flank of the single-stranded viral DNA genome. The length of the ITR sequence is generally about 145 bases per sequence. In the ITR region, two elements considered to be the core of ITR function, the GAGC repeat motif and the terminal resolution site (trs), have been described. When ITR is in a linear or hairpin conformation, the repeat motif demonstrates binding to Rep. This combination is considered to locate Rep68 / 78 at trs for cutting, which occurs in a site-specific and strand-specific manner. In addition to its role in replication, these two elements demonstrate the core of viral integration. Contained in chromosome 19 integration loci is a Rep binding site with adjacent trs. These elements demonstrate functionality, and are essential to locus-specific integration.

[0079] The term "recombinant" as a modifier of a vector, such as a recombinant virus, such as a lentiviral or parvoviral (e.g., AAV) vector, and a modifier of a sequence, such as a recombinant nucleic acid sequence and a polypeptide, means that the composition has been manipulated (i.e., modified) in a manner that does not normally occur in nature. A specific example of a recombinant vector (e.g., an AAV, retroviral, or lentiviral vector) is the insertion of a nucleic acid sequence that is not normally present in the wild-type viral genome into the viral genome. An example of a recombinant nucleic acid sequence is an inhibitory RNA that is encoded by a nucleic acid (e.g., a gene) cloned into a vector with or without the 5', 3', and / or intron regions that are normally associated with the gene in the viral genome. Although the term "recombinant" is not always used herein to refer to vectors, such as viral vectors, and sequences such as polynucleotides, "recombinant" forms including nucleic acid sequences, polynucleotides, transgenes, etc. are explicitly included, despite any such omissions.

[0080] Recombinant virus "vector" is derived from the wild-type genome of the virus, by using molecular methods to remove a portion of the wild-type genome from the virus, and replaced with non-natural nucleic acids (e.g., nucleic acid sequences). Typically, for example, for AAV, one or two reverse terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. "Recombinant" viral vectors (e.g., rAAV) are different from viral (e.g., AAV) genomes because a portion of the viral genome has been replaced by non-natural sequences for viral genomic nucleic acids (e.g., nucleic acids encoding transactivators or nucleic acids encoding inhibitory RNAs or nucleic acids encoding therapeutic proteins). Therefore, the incorporation of such non-natural nucleic acid sequences defines viral vectors as "recombinant" vectors, which, in the case of AAV, can be referred to as "rAAV vectors."

[0081] In certain embodiments, AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair. In certain embodiments, AAV (e.g., rAAV) comprises an ITR pair flanking (i.e., at each 5' and 3' end) at least a nucleic acid sequence encoding a polypeptide having a function or activity.

[0082] AAV vectors (e.g., rAAV vectors) can be packaged and referred to herein as "AAV particles" for subsequent ex vivo, in vitro, or in vivo infection (transduction) of cells. When a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle may also be referred to as a "rAAV particle". In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle typically comprises an rAAV vector or a portion thereof. The rAAV particle may be one or more rAAV particles (e.g., a plurality of AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encapsulates or packages the rAAV vector genome. It should be noted that reference to rAAV vectors may also be used to refer to rAAV particles.

[0083] Any suitable AAV particles (e.g., rAAV particles) can be used for the methods or purposes herein. The rAAV particles and / or the genomes contained therein can be derived from any suitable AAV serotype or strain. The rAAV particles and / or the genomes contained therein can be derived from two or more AAV serotypes or strains. Therefore, rAAV can include proteins and / or nucleic acids or parts thereof of any serotype or strain of AAV, wherein the AAV particles are suitable for infection and / or transduction of mammalian cells. Some non-limiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, and AAV-2i8.

[0084] In certain embodiments, a plurality of rAAV particles comprise particles of the same strain or serotype (or subgroup or variant), or are derived from the same strain or subtype (or subgroup or variant). In certain embodiments, a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., different serotypes and / or strains).

[0085] As used herein, the term "serotype" is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serological distinctness is determined based on the lack of cross-reactivity between antibodies to one AAV and antibodies to another AAV. Such cross-reactivity differences are typically due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes). Although there is the possibility that AAV variants (including capsid variants) may not be serologically distinguishable from a reference AAV or other AAV serotypes, they differ in at least one nucleotide or amino acid residue compared to the reference or other AAV-serotypes.

[0086] In certain embodiments, the rAAV vector based on the first serotype genome corresponds to the serotype of one or more capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) comprising the AAV vector genome corresponds to the serotype of the capsid that comprises the rAAV particle.

[0087] In certain embodiments, the rAAV vector genome may be based on an AAV (e.g., AAV2) serotype genome that is different from the serotype of one or more AAV capsid proteins that package the vector. For example, the rAAV vector genome may comprise an AAV2-derived nucleic acid (e.g., ITR), while at least one or more of the three capsid proteins are derived from a different serotype, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotypes or variants thereof.

[0088] In certain embodiments, the rAAV particles or their vector genomes associated with a reference serotype have polynucleotides, polypeptides, or subsequences thereof comprising or consisting of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a polynucleotide, polypeptide, or subsequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 particle. In some specific embodiments, the rAAV particles or their vector genomes associated with a reference serotype have a capsid or ITR sequence comprising or consisting of a sequence that is at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, or AAV-2i8 serotype.

[0089] In certain embodiments, the methods herein comprise the use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0090] In certain embodiments, the method herein includes using, applying or delivering rAAV2 particles. In certain embodiments, rAAV2 particles include AAV2 capsids. In certain embodiments, rAAV2 particles include one or more capsid proteins (e.g., VP1, VP2 and / or VP3), which have at least 60%, 65%, 70%, 75% or higher homogeneity with the corresponding capsid proteins of natural or wild-type AAV2 particles, such as 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% homogeneity. In certain embodiments, the rAAV2 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% identical to the corresponding capsid proteins of native or wild-type AAV2 particles, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity. In certain embodiments, the rAAV2 particles are variants of native or wild-type AAV2 particles. In some aspects, one or more capsid proteins of the AAV2 variants have 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20 or more amino acid substitutions compared to the capsid proteins of native or wild-type AAV2 particles.

[0091] In certain embodiments, the rAAV9 particles comprise an AAV9 capsid. In certain embodiments, the rAAV9 particles comprise one or more capsid proteins (e.g., VP1, VP2, and / or VP3) that are at least 60%, 65%, 70%, 75% or more identical to the corresponding capsid proteins of native or wild-type AAV9 particles, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical. In certain embodiments, the rAAV9 particles comprise VP1, VP2 and VP3 capsid proteins, which have at least 75% or higher identity with the corresponding capsid proteins of natural or wild-type AAV9 particles, such as 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity. In certain embodiments, the rAAV9 particles are variants of natural or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variants have 1, 2, 3, 4, 5, 5 to 10, 10 to 15, 15 to 20 or more amino acid substitutions compared to the capsid proteins of natural or wild-type AAV9 particles.

[0092] In certain embodiments, the rAAV particle comprises one or two ITRs (e.g., an ITR pair) that have at least 7 sequences identical to the corresponding ITRs of native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rh10, or AAV-2i8. 5% or more identity, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0093] In certain embodiments, the rAAV2 particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% or more identical to the corresponding ITRs of a native or wild-type AAV2 particle, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identical, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0094] In certain embodiments, the rAAV9 particle comprises one or two ITRs (e.g., an ITR pair) that are at least 75% or more identical to the corresponding ITRs of a native or wild-type AAV2 particle, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc., up to 100% identity, so long as it retains one or more desired ITR functions (e.g., the ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into the host cell genome; and / or packaging, if desired).

[0095] rAAV particles may comprise ITRs having any suitable number of "GAGC" repeats. In certain embodiments, the ITRs of AAV2 particles comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs containing three "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs having less than four "GAGC" repeats. In certain embodiments, rAAV2 particles comprise ITRs having more than four "GAGC" repeats. In certain embodiments, the ITRs of rAAV2 particles comprise a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0096] An exemplary suitable length of DNA that can be incorporated into a rAAV vector for packaging / encapsulation into rAAV particles can be about 5 kilobases (kb) or less. In some specific embodiments, the length of the DNA is less than about 5 kb, less than about 4.5 kb, less than about 4 kb, less than about 3.5 kb, less than about 3 kb, or less than about 2.5 kb.

[0097] The rAAV vector comprising the nucleic acid sequence for guiding the expression of RNAi or polypeptide can be produced using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are usually packaged into AAV particles with transduction ability and propagated using the AAV virus packaging system. AAV particles with transduction ability can bind to and enter mammalian cells, and then deliver nucleic acid carriers (e.g., heterologous genes) to the nucleus of cells. Therefore, complete rAAV particles with transduction ability are configured to transduce mammalian cells. The rAAV particles configured to transduce mammalian cells do not generally have replication ability, and require additional protein mechanisms to replicate themselves. Therefore, the rAAV particles configured to transduce mammalian cells are modified to bind to and enter mammalian cells, and nucleic acid is delivered to cells, wherein the nucleic acid for delivery is generally located between the AAV ITR pairs in the rAAV genome.

[0098] Suitable host cells for producing transduction-capable AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can or have been used as recipients of heterologous rAAV vectors. Cells from a stable human cell line HEK293 (easily obtained, for example, by the American Type Culture Collection with accession number ATCCCRL1573) can be used. In certain embodiments, a modified human embryonic kidney cell line (e.g., HEK293) transformed with type 5 adenovirus DNA fragments and expressing adenovirus E1a and E1b genes is used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected and provides a particularly convenient platform for producing rAAV particles therein. Methods for producing high-titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be prepared as described in Wright, 2008 and Wright, 2009.

[0099] In certain embodiments, before or simultaneously with transfection of the AAV expression vector, the AAV helper function is introduced into the host cell by transfecting the host cell with an AAV helper construct. Therefore, the AAV helper construct is sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the AAV function of the deletion necessary for productive AAV transduction. The AAV helper construct generally lacks the AAV ITR and can neither replicate nor package itself. These constructs can be in the form of plasmids, phages, transposons, clays, viruses or virions. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both Rep and Cap expression products. Many other vectors encoding Rep and / or Cap expression products are known.

[0100] An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0101] II. Therapeutic Agents

[0102] In some embodiments, viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding inhibitory RNA, noncoding RNA and / or therapeutic proteins to cells in culture or in a host organism.

[0103] A. Inhibitory RNA

[0104] "RNA interference (RNAi)" is a process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces degradation of target mRNA and subsequently leads to sequence-specific inhibition of gene expression.

[0105] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecules, "short hairpin RNA" or "shRNA" molecules or "miRNA" are RNA duplexes that target nucleotides of a target nucleic acid sequence. The term "siRNA" as used herein is a general term covering subsets of shRNA and miRNA. "RNA duplex" refers to a structure formed by complementary pairing between two regions of an RNA molecule. siRNA "targets" a gene because the nucleotide sequence of the siRNA duplex portion is complementary to the nucleotide sequence of the target gene. In certain embodiments, siRNA targets a sequence encoding huntingtin. In some embodiments, the length of the siRNA duplex is less than 30 base pairs. In some embodiments, the length of the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs. In some embodiments, the length of the duplex is 19 to 25 base pairs in length. In certain embodiments, the length of the duplex is 19 or 21 base pairs in length. The RNA duplex portion of the siRNA can be a part of a hairpin structure. In addition to the duplex portion, the hairpin structure can include a loop portion between the two sequences that form the duplex. The length of the loop can vary. In some embodiments, the length of the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In certain embodiments, the length of the loop is 18 nucleotides. The hairpin structure can also include 3' and / or 5' overhangs. In some embodiments, the overhang is a 3' and / or 5' overhang of 0, 1, 2, 3, 4 or 5 nucleotides in length.

[0106] shRNA is composed of a stem-loop structure, which is designed to include a 5' flanking region, a siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies utilize short-hairpin RNA (shRNA) driven by a strong polIII-based promoter. Many shRNAs show effective knockdown of target sequences in vitro and in vivo, however, some shRNAs that show effective knockdown of target genes are also found to be toxic in vivo.

[0107] miRNA is a small cell RNA (about 22nt) processed from a precursor stem-loop transcript. Known miRNA stem-loops can be modified to contain RNAi sequences that are specific to the target gene. MiRNA molecules can be superior to shRNA molecules because miRNA is endogenously expressed. Therefore, miRNA molecules are less likely to induce the interferon pathway of dsRNA response, they are more effectively processed than shRNA, and have been shown to be 80% more effective in silencing.

[0108] A recently discovered alternative is to use artificial miRNA (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more naturally similar to endogenous RNAi substrates and are more susceptible to Pol-II transcription (e.g., allowing tissue-specific expression of RNAi) and polycistronic strategies (e.g., allowing delivery of multiple siRNA sequences). See U.S. Patent No. 10,093,927, which is incorporated by reference.

[0109] The transcription unit of "shRNA" consists of sense and antisense sequences connected by a loop of unpaired nucleotides. shRNA is exported from the nucleus by exportin-5, and once in the cytoplasm, it is processed by Dicer to produce functional siRNA. "miRNA" stem-loop consists of sense and antisense sequences connected by a loop of unpaired nucleotides, which are usually expressed as part of a larger primary transcript (pri-miRNA), excised by the Drosha-DGCR8 complex, producing an intermediate product called a miRNA precursor, which is then exported from the nucleus by exportin-5, and once in the cytoplasm, it is processed by Dicer to produce functional siRNA. "Artificial miRNA" or "artificial miRNA shuttle vector" used herein refers interchangeably to a primary miRNA transcript having a region of a duplex stem loop (at least about 9 to 20 nucleotides), which is excised by Drosha and Dicer processing and replaced by the siRNA sequence of the target gene, while retaining the structural elements within the stem loop necessary for effective Drosha processing. The term "artificial" originates from the fact that the flanking sequences (about 35 nucleotides upstream and about 40 nucleotides downstream) are derived from restriction enzyme sites within the multiple cloning site of the siRNA. The term "miRNA" as used herein encompasses both naturally occurring miRNA sequences as well as artificially generated miRNA shuttle vectors.

[0110] The siRNA may be encoded by a nucleic acid sequence, and the nucleic acid sequence may further comprise a promoter. The nucleic acid sequence may further comprise a polyadenylation signal. In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six Ts.

[0111] When designing RNAi, several factors need to be considered, such as the properties of siRNA, the persistence of the silencing effect and the selection of the delivery system. In order to produce the RNAi effect, the siRNA introduced into the organism will usually contain exon sequences. In addition, the RNAi process is homology-dependent, so the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous sequences rather than gene-specific sequences. Preferably, siRNA shows greater than 80%, 85%, 90%, 95%, 98% or even 100% identity between the sequence of siRNA and the gene sequence to be inhibited. Sequences with less than about 80% identity to the target gene are not so effective in fact. Therefore, the greater the homology between siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0112] In addition, the size of siRNA is an important consideration. In some embodiments, the disclosure relates to siRNA molecules comprising at least about 19 to 25 nucleotides and capable of regulating gene expression. In the context of the disclosure, the length of siRNA is preferably less than 500, 200, 100, 50 or 25 nucleotides. More preferably, the length of siRNA is about 19 nucleotides to about 25 nucleotides.

[0113] siRNA target generally means a polynucleotide comprising a region encoding a polypeptide or a polynucleotide region regulating replication, transcription, or translation or other processes important to polypeptide expression; or a polynucleotide comprising a region encoding a polypeptide and a region operably linked thereto regulating expression. Any gene expressed in a cell can be targeted. Preferably, the target gene is a gene related to a cell activity progression that is important for a disease or is particularly interesting as a research subject or is associated with the cell activity progression.

[0114] B. Non-coding RNA

[0115] As demonstrated by cDNA cloning projects and genomic tile arrays, more than 90% of the human genome undergoes transcription but does not encode proteins. These transcription products are called non-protein coding RNA (ncRNA). A variety of ncRNA transcripts, such as ribosomal RNA, transfer RNA, competing endogenous RNA (ceRNA), small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA), are essential for cellular function. Similarly, a large number of short ncRNAs, such as microRNA (miRNA), endogenous short interfering RNA (siRNA), PIWI-interacting RNA (piRNA) and small nucleolar RNA (snoRNA), are also known to play important regulatory roles in eukaryotic cells. Recent studies have shown that groups of long ncRNA (lncRNA) transcripts exhibit cell type-specific expression and are located in specific subcellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the idea that they were selected during the evolutionary process.

[0116] LncRNAs have been shown to have many different functions. In many cases, they have been shown to play a role in regulating the activity or localization of proteins, or to act as an organizing framework for subcellular structures. In other cases, lncRNAs are processed to produce a variety of small RNAs, or they can regulate how other RNAs are processed. The latest version of data (version #27) produced by the public research consortium GenCode catalogued just under 16,000 lncRNAs in the human genome, yielding nearly 28,000 transcripts; when other databases are included, more than 40,000 lncRNAs are known.

[0117] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, modulate the activity of protein-binding partners, direct chromatin-modifying complexes to their sites of action, and be post-transcriptionally processed to generate a large number of 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0118] There is also increasing evidence that abnormally expressed lncRNAs play an important role in normal physiological processes as well as in a variety of disease states. lncRNAs are dysregulated in a variety of diseases, including ischemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This dysregulation is also manifested in a variety of cancer types, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Several lncRNAs, such as gadd74 and lncRNA-RoR5, regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, and thus provide an additional layer of flexibility and robustness for cell cycle progression. In addition, some lncRNAs are associated with mitotic processes, such as centromeric satellite RNAs, which are essential for kinetochore formation and are therefore essential for chromosome segregation during mitosis in humans and Drosophila. Another nuclear lncRNA, MA-lincl, functions in cis to inhibit the expression of its neighboring gene Pura (a regulator of cell proliferation) to regulate M phase exit.

[0119] lncRNA is a group of transcripts generally defined as more than 200 nucleotides (e.g., about 200 to about 1200 nt, about 2500 nt or more) that lack an extended open reading frame (ORF). The term "non-coding RNA" (ncRNA) includes lncRNA as well as shorter transcripts (e.g., less than about 200 nt, e.g., about 30 to 200 nt).

[0120] Therefore, in some embodiments, the delivery of ncRNA, such as delivery to a specific brain structure of interest, corrects abnormal RNA expression levels or regulates the level of pathogenic lncRNA. Therefore, in some embodiments, the disclosure provides rAAV, wherein the viral genome is modified to encode therapeutic non-coding RNA (ncRNA). In some embodiments, ncRNA is a long non-coding RNA (lncRNA) of about 200 nucleotides (nucleotide, nt) or longer. In some embodiments, the therapeutic agent is an ncRNA of about 25nt or about 30nt to about 200nt in length. In some embodiments, the length of lncRNA is about 200nt to about 1,200nt. In some embodiments, the length of lncRNA is about 200nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400 or about 300nt.

[0121] C. CRISPR system

[0122] Gene editing is a technology that allows the modification of targeted genes within living cells. Recently, the use of the bacterial immune system of CRISPR to perform gene editing on demand has revolutionized the way scientists perform genome editing. The Cas9 protein of the CRISPR system is an RNA-guided DNA endonuclease that can be relatively easily engineered to target new sites by changing the sequence of its guide RNA. This discovery made sequence-specific gene editing functionally effective.

[0123] In general, a "CRISPR system" collectively refers to transcripts and other elements involved in the expression or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr partner sequences (including "direct repeats", and partial direct repeats processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), and / or other sequences and transcripts from the CRISPR locus.

[0124] CRISPR / Cas nuclease or CRISPR / Cas nuclease system can comprise non-coding RNA molecule (guide) RNA, which binds specifically to DNA sequence; and Cas protein (e.g., Cas9), which has nuclease functionality (e.g., two nuclease domains). One or more elements of CRISPR system can be derived from type I, type II or type III CRISPR system, for example, from a specific organism comprising endogenous CRISPR system, such as Streptococcus pyogenes.

[0125] The CRISPR system can induce double strand breaks (DSB) at the target site, followed by destruction as discussed herein. In other embodiments, a Cas9 variant considered to be a "nickase" is used to nick a single strand at the target site. Paired nickases can be used, for example, to improve specificity, each enzyme being guided by a different gRNA targeting sequence pair so that when the nick is introduced at the same time, a 5' overhang is introduced. In other embodiments, a catalytically inactive Cas9 is fused to a heterologous effector domain (e.g., a transcriptional repressor (e.g., KRAB) or activator) to affect gene expression. Alternatively, a CRISPR system with a catalytically inactive Cas9 further comprises a transcriptional repressor or activator fused to a ribosome binding protein.

[0126] In some aspects, Cas nuclease and gRNA (including a fusion of crRNA and fixed tracrRNA specific to the target sequence) are introduced into the cell. In general, the target site at the 5' end of gRNA targets the Cas nuclease to the target site, such as a gene, using complementary base pairing. The target site can be selected based on the 5' position of its adjacent protospacer adjacent motif (protospacer adjacent motif, PAM) sequence (e.g., usually NGG or NAG). In this aspect, the gRNA is targeted to the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11 or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, the CRISPR system is characterized by promoting the elements of the formation of CRISPR complexes at the target sequence site. Generally speaking, "target sequence" generally refers to a sequence designed to have complementarity in the guide sequence, wherein the hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. Perfect complementarity is not necessarily required if there is sufficient complementarity to cause hybridization and promote formation of the CRISPR complex.

[0127] The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as within an organelle of a cell. In general, a sequence or template that can be used to reorganize a target locus comprising a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence". In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, recombination is homologous recombination.

[0128] Generally speaking, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). A tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), can also form part of a CRISPR complex, for example by hybridizing along at least a portion of the tracr sequence to all or a portion of a tracr partner sequence operably linked to a guide sequence. The tracr sequence has sufficient complementarity to the tracr partner sequence to hybridize and participate in the formation of the CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr partner sequence when optimally aligned.

[0129] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into the cell so that the expression of the elements of the CRISPR system guides the formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, the Cas enzyme, the guide sequence connected to the tracr partner sequence, and the tracr sequence can each be operably connected to a separate regulatory element on a separate vector. The Cas enzyme can be a target gene under the control of a regulated alternative splicing event, as disclosed herein, as a chimeric target gene minigene or as a target gene of a chimeric minigene transactivator. The gRNA can be under the control of a constitutive promoter.

[0130] Alternatively, two or more elements expressed by the same or different regulatory elements can be combined in a single vector, while one or more additional vectors provide any components of the CRISPR system not contained in the first vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in the cell.

[0131] The vector may comprise a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme (e.g., a Cas protein). Some non-limiting examples of Cas proteins include

[0132] Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the S. pyogenes Cas9 protein can be found in the SwissProt database under accession number Q99ZW2.

[0133] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or Streptococcus pneumoniae (S.pneumonia)). The CRISPR enzyme can guide the cutting of one or both chains at the location of the target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence. The vector can encode a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme so that the mutated CRISPR enzyme lacks the ability to cut one or both chains of the target polynucleotide comprising the target sequence. For example, the substitution of aspartate to alanine (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cuts two chains to a nickase (cutting a single strand). In some embodiments, the Cas9 nickase can be used in combination with a guide sequence (e.g., two guide sequences) that target the sense and antisense strands of a DNA target, respectively. This combination allows the two chains to be nicked and used to induce NHEJ or HDR.

[0134] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon optimized for expression in a specific cell, such as a eukaryotic cell. Eukaryotic cells can be those of a specific organism or derived from a specific organism, such as a mammal, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates. In general, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in a target host cell by replacing at least one codon of a native sequence with a more frequent or most frequently used codon in the genes of the target host cell while maintaining the native amino acid sequence. Various species show specific preferences for certain codons of specific amino acids. Codon bias (differences in codon usage between organisms) is generally related to the translation efficiency of messenger RNA (mRNA), which in turn is believed to depend on the properties of the codons being translated and the availability of specific transfer RNA (tRNA) molecules, etc. The advantage of the selected tRNA in the cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized based on codon optimization for optimal gene expression in a given organism.

[0135] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm.

[0136] Optimal alignment can be determined using any suitable algorithm for aligning sequences, some non-limiting examples of which include the Smith-Waterman algorithm; the Needleman-Wunsch algorithm; algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner); Clustal W; Clustal X; BLAT; Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.)); SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).

[0137] The CRISPR enzyme can be part of a fusion protein comprising one or more heterologous protein domains. The CRISPR enzyme fusion protein may include any additional protein sequence, and optionally a linker sequence between any two domains. Some examples of protein domains that can be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Some non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (influenza hemagglutinin, HA) tags, Myc tags, VSV-G tags, and thioredoxin (thioredoxin, Trx) tags. Some examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including but not limited to maltose binding protein (MBP), S tag, Lex A DNA binding domain (DBD) fusion, GAL4A DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0138] D. Therapeutic proteins

[0139] Some embodiments relate to the expression of recombinant proteins and polypeptides, such as those listed below.

[0140] Apolipoprotein E2. Apolipoprotein E (Apolipoprotein E, APOE) is a protein involved in fat metabolism in mammals. Subtypes are associated with Alzheimer's disease and cardiovascular disease. APOE belongs to a family of fat-binding proteins known as apolipoproteins. In the circulation, it exists as part of several types of lipoprotein particles, including chylomicron remnants, VLDL, IDL, and some HDL. APOE significantly interacts with the low-density lipoprotein receptor (LDLR), which is essential for the normal processing (catabolism) of triglyceride-rich lipoproteins. In peripheral tissues, APOE is mainly produced by the liver and macrophages and regulates cholesterol metabolism. In the central nervous system, APOE is mainly produced by astrocytes and cholesterol is transported to neurons through the APOE receptor, which is a member of the low-density lipoprotein receptor gene family. APOE is the main cholesterol carrier in the brain. APOE is required for the transport of cholesterol from astrocytes to neurons. APOE qualifies as a checkpoint inhibitor of the classical complement pathway by forming a complex with activated C1q. APOE is a protein involved in fat metabolism in mammals. Isoforms are associated with Alzheimer's disease and cardiovascular disease.

[0141] APOE is 299 amino acids long and contains multiple amphipathic α-helices. According to crystallographic studies, the hinge region connects the N-terminal and C-terminal regions of the protein. The N-terminal region (residues 1 to 167) forms an antiparallel four-helix bundle, so that the non-polar side faces the interior of the protein. At the same time, the C-terminal domain (residues 206 to 299) contains three α-helices, which form a large exposed hydrophobic surface and interact with the α-helices in the N-terminal helical bundle domain through hydrogen bonds and salt bridges. The C-terminal region also contains a low-density lipoprotein receptor (LDLR) binding site.

[0142] APOE is polymorphic, with three major alleles (ε2, ε3, and ε4): APOE-ε2 (cys112, cys158), APOE-ε3 (cys112, arg158), and APOE-ε4 (arg112, arg158). Although these allelic forms differ from each other by only one or two amino acids at positions 112 and 158, these differences also alter APOE structure and function.

[0143] As of 2012, the E4 variant is the largest known genetic risk factor for late-onset sporadic Alzheimer's disease (AD) in multiple ethnic groups. However, the E4 variant is not associated with risk in every group. Nigerians have the highest APOE4 allele frequency observed in the world population, but AD is rare among them. This may be due to their low cholesterol levels. Caucasians and Japanese who carry two E4 alleles have a 10 to 30 times greater risk of developing AD before the age of 75 compared to those who do not carry any E4 alleles. This may be caused by interaction with amyloid. Alzheimer's disease is characterized by the accumulation of peptide beta-amyloid aggregates. Apolipoprotein E enhances the proteolytic breakdown of this peptide both within and between cells. The isoform APOE-ε4 is not as effective as other isoforms in promoting these reactions, resulting in increased vulnerability to AD in individuals with this genetic variant.

[0144] Although 40% to 65% of AD patients have at least one copy of the ε4 allele, APOE4 is not a determinant of the disease. At least one-third of patients with AD are APOE4 negative, and some APOE4 homozygotes never develop the disease. However, those with two ε4 alleles have a risk of developing AD up to 20 times. There is also evidence that the APOE2 allele can play a protective role in AD. Therefore, the genotype with the highest risk of Alzheimer's disease and occurring in early life is APOE4,4. Using the genotype APOE3,3 as a benchmark (people with this genotype are considered to have a risk level of 1.0) and only for the Caucasian population, individuals with the genotype APOE4,4 have an odds ratio of 14.9 for developing Alzheimer's disease. Individuals with the APOE3,4 genotype face an odds ratio of 3.2, and those with copies of the 2 allele and the 4 allele (APOE2,4) have an odds ratio of 2.6. The odds ratio for people with one copy of the 2 allele and one copy of the 3 allele (APOE2,3) is 0.6. The odds ratio for people with two copies of the 2 allele (APOE2,2) is also 0.6.

[0145] Additional exemplary therapeutic proteins include secreted antibodies, nanobodies, tripeptidyl peptidase 1 (TPP1), sulfamidase (SGSH), palmitoyl-protein thioesterase 1 (PPT1), β-glucuronidase (GUSB), α-L-iduronidase (IDUA), galactocerebrosidase (GALC), CLN6 transmembrane ER protein (CLN6), β-galactosidase (GLB1), β-hexosaminidase A α subunit (HEXA), sulfatase modifying factor 1 (SUMF1), α-d-mannosidase (MAN2B1), N-acetylglucosamine-6-sulfatase (GNS), heparan-α-glucosaminide N-acetyltransferase (HGSNAT), and α-N-acetylglucosaminidase (NAGLU).

[0146] When the present application refers to the function or activity of a "modified protein" or "modified polypeptide", one of ordinary skill in the art will understand that this includes, for example, a protein or polypeptide that has additional advantages relative to an unmodified protein or polypeptide. It is specifically contemplated that embodiments regarding a "modified protein" may be implemented relative to a "modified polypeptide", and vice versa.

[0147] Recombinant proteins may have deletions and / or replacements of amino acids; therefore, proteins with deletions, proteins with replacements, and proteins with deletions and replacements are modified proteins. In some embodiments, these proteins may also include inserted or added amino acids, such as proteins with fusion proteins or with joints. "Modified deletion proteins" lack one or more residues of the native protein, but may have the specificity and / or activity of the native protein. "Modified deletion proteins" may also have reduced immunogenicity or antigenicity. An example of a modified deletion protein is a protein that lacks amino acid residues from at least one antigenic region (i.e., a region of a protein that is determined to be antigenic in a specific organism (e.g., an organism to which the modified protein is administered)).

[0148] Replacement or substitution variants generally comprise an exchange of one amino acid with another amino acid at one or more sites within a protein, and can be designed to modulate one or more properties of a polypeptide, particularly its effector function and / or bioavailability. Replacements may or may not be conservative, i.e., one amino acid is replaced by one of similar shape and charge. Conservative replacements are well known in the art and include, for example, the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartic acid to glutamic acid; cysteine ​​to serine; glutamine to asparagine; glutamic acid to aspartic acid; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine, or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.

[0149] In addition to deletions or substitutions, modified proteins may have insertions of residues, which typically involve adding at least one residue in a polypeptide. This may include inserting a targeting peptide or polypeptide or just a single residue. The terminal additions referred to as fusion proteins are discussed below.

[0150] Gonzalez et al., BMC Neurosci 10.1186 / 1471-2202-12-4 (2011) A peptide library of M13 phage was screened for ligands that can be internalized into CP epithelial cells by incubating phage with choroid plexus explants and recovering particles with targeting ability. The specific binding and dose-dependent binding and internalization of three peptides identified after four rounds of screening were analyzed. The binding was considered specific because internalization was prevented by co-incubation with homologous synthetic peptides. In addition, after icv injection into the rat brain, it was found that each peptide targeted phage to epithelial cells in the CP and the ependyma lining the ventricles. Such peptides can be used in the AAV vectors described herein.

[0151] The term "biological functional equivalent" is well known in the art and is further defined in detail herein. Thus, sequences in which about 70% to about 80%, or about 81% to about 90%, or even about 91% to about 99% of the amino acids are identical or functionally equivalent to the amino acids of the reference polypeptide are included, provided that the biological activity of the protein is maintained. In certain aspects, the recombinant protein and its natural counterpart may be biologically functionally equivalent.

[0152] It will also be understood that amino acid and nucleic acid sequences may contain additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, and still be substantially as shown in one of the sequences disclosed herein, as long as the sequence meets the criteria set forth above, including, where protein expression is concerned, maintaining biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences, which may, for example, contain various non-coding sequences flanking the 5' or 3' portion of the coding region, or may contain various internal sequences known to exist within genes, i.e., introns.

[0153] Protein or peptide used herein generally refers to, but is not limited to, proteins greater than about 200 amino acids to the full-length sequence translated from a gene; polypeptides greater than about 100 amino acids; and / or peptides of about 3 to about 100 amino acids. For convenience, the terms "protein", "polypeptide" and "peptide" are used interchangeably herein.

[0154] As used herein, "amino acid residue" refers to any naturally occurring amino acid, any amino acid derivative, or any amino acid mimetic known in the art. In certain embodiments, the residues of a protein or peptide are continuous, without any non-amino acid interrupting the sequence of amino acid residues. In other embodiments, the sequence may contain one or more non-amino acid moieties. In some specific embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0155] Thus, the term "protein or peptide" encompasses an amino acid sequence comprising at least one of the 20 common amino acids found in naturally occurring proteins or at least one modified or unusual amino acid.

[0156] Certain embodiments of the present disclosure relate to fusion proteins. These molecules may have therapeutic proteins connected to heterologous domains at the N- or C-termini. For example, fusions may also use leader sequences from other species to allow recombinant expression of proteins in heterologous hosts. Another useful fusion includes adding protein affinity tags, for example, serum albumin affinity tags or six histidine residues, or immunologically active domains, for example, antibody epitopes, preferably cleavable, to facilitate purification of the fusion protein. Non-limiting affinity tags include polyhistidine, chitin binding protein (CBP), maltose binding protein (MBP), and glutathione-S-transferase (GST).

[0157] Methods for producing fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of the complete fusion protein, or by attaching a DNA sequence encoding a heterologous domain and then expressing the complete fusion protein.

[0158] By connecting the gene with a bridging DNA fragment encoding a peptide linker (splicing between the tandemly linked polypeptides), the production of a fusion protein that restores the functional activity of the parent protein can be facilitated. The linker will be of sufficient length to allow the correct folding of the resulting fusion protein.

[0159] III. Methods of Treatment and Administration

[0160] In some aspects, viral vectors can be directly administered to a patient (in vivo), or they can be used to treat cells in vitro or ex vivo and then administered to a patient. In particular, methods for inducing transgene expression in the ependyma are provided herein. In some of these embodiments, the subject has a brain or neurological disorder, and the transgene is delivered in a therapeutically effective amount. In some embodiments, the AAV vector transduces cells of at least about 70% of the target tissue; AAV targets inner and outer hair cells with an efficiency of at least about 70%, 80%, 90%, 95% or more, even up to 100%. In some embodiments, the cells are ventricular cells of the brain, e.g., ependymal cells.

[0161] Ependyma is the thin neuroepithelium (single layer of columnar ciliated epithelium) lining of the ventricular system of the brain and the central canal of the spinal cord.Ependyma is one of four types of neuroglia in the central nervous system (CNS). It participates in the production of cerebrospinal fluid (CSF) and shows as a reservoir for nerve regeneration.Ependyma is composed of ependymal cells (ependymal cell) referred to as ependymocytes (ependymocyte) (a class of glial cells). These cells line the ventricles in the brain and the central canal of the spinal cord, so that the ventricles in the brain and the central canal of the spinal cord are filled with cerebrospinal fluid. These are neural tissue cells with a simple columnar shape, much like the shape of some mucosal epithelial cells. Early monociliated ependymal cells differentiate into multiciliated ependymal cells because of their function in circulating cerebrospinal fluid. The basement membrane of these cells is characterized by the tentacle-like extension attached to astrocytes. The top side is covered with cilia and microvilli.

[0162] The term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by insertion or incorporation of nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells so that the nucleic acid sequence therein is transcribed and, if encoding a protein, is subsequently translated by the cell.

[0163] These compositions are useful for treating disorders of the brain or central nervous system. Thus, in some embodiments, the methods described herein are used to treat disorders listed in Table A using the corresponding sequence listing in Table A in a subject in need thereof.

[0164] Any suitable cell or mammal can be applied or treated by the methods or uses described herein. Generally, the mammals that need the methods described herein are suspected of having or expressing abnormal or abnormal proteins associated with disease states. Alternatively, the mammal recipient may have a condition suitable for gene replacement therapy. "Gene replacement therapy" used herein refers to the administration of exogenous genetic material encoding therapeutic agents to the recipient and the subsequent in situ expression of the administered genetic material. Therefore, the phrase "conditions suitable for gene replacement therapy" covers such conditions, such as hereditary diseases (i.e., disease conditions attributed to one or more gene defects) and acquired pathologies (i.e., pathological conditions not attributed to congenital defects). Therefore, the term "therapeutic agent" used herein refers to any agent or substance that has a beneficial effect on a mammal recipient. Therefore, "therapeutic agent" covers both therapeutic and preventive molecules with nucleic acid or protein components.

[0165] Some non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cattle, goats, sheep, pigs), and experimental animals (e.g., mice, rats, rabbits, guinea pigs). In certain embodiments, the mammal is a human. In certain embodiments, the mammal is a non-rodent mammal (e.g., a human, pig, goat, sheep, horse, dog, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in utero). The mammal can be male or female. In certain embodiments, the mammal can be an animal disease model, for example, an animal model having or expressing an abnormal or abnormal protein associated with a disease state or an animal model that does not adequately express a protein and causes a disease state.

[0166] Mammals (subjects) treated by the methods or compositions described herein include adults (18 years or older) and children (less than 18 years old). Adults include elderly people. Representative adults are 50 years or older. Children range in age from 1 to 2 years old, or 2 to 4, 4 to 6, 6 to 18, 8 to 10, 10 to 12, 12 to 15, and 15 to 18 years old. Children also include infants. Infants are generally 1 to 12 months old.

[0167] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, wherein the severity, frequency, progression or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, reduced, blocked, inhibited or delayed. In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat adverse symptoms of a disease state (e.g., a neurodegenerative disease). In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to stabilize, delay or prevent the deterioration or progression of adverse symptoms of a disease state (e.g., a neurodegenerative disease), or to reverse adverse symptoms of a disease state (e.g., a neurodegenerative disease).

[0168] In certain embodiments, the methods comprise administering a plurality of viral particles to the central nervous system, or a portion thereof, of a mammal as described herein, and the severity, frequency, progression, or onset of one or more symptoms of a disease state (e.g., a neurodegenerative disease) is reduced, diminished, arrested, inhibited, or delayed by at least about 5 to about 10 days, about 10 to about 25 days, about 25 to about 50 days, or about 50 to about 100 days.

[0169] In some embodiments, a composition comprising a therapeutically effective number of viral particles containing a transgene, or comprising one or more different groups of viral particles, wherein each particle in a group may contain the same type of transgene, but wherein each group of particles contains a different type of transgene than the other groups, as described herein, can be delivered.

[0170] Formulations according to the present disclosure may be used for CNS delivery by a variety of techniques and routes including, but not limited to, intraparenchymal, intracerebral, intracerebroventricular (ICV), intrathecal (e.g., IT-lumbar, IT-thoracic, IT-cisternaria), and any other techniques and routes for direct or indirect injection into the CNS and / or CSF.

[0171] In some embodiments, the formulation is delivered to the CNS by administering the formulation into the cerebrospinal fluid (CSF) of a subject in need of treatment. In some embodiments, intrathecal administration is used to deliver viral particles into the CSF. Intrathecal administration (also referred to as intrathecal injection) as used herein refers to injection into the spinal canal (the intrathecal space surrounding the spinal cord). A variety of techniques can be used, including but not limited to intraventricular injection through a burrhole or a cisternal or lumbar puncture. Exemplary methods are described in Lazorthes et al. Advances in Drug Delivery Systems and Applications in Neurosurgery, 18: 143-192 (1991) and Ommaya et al., Cancer Drug Delivery, 1: 169-179 (1984), the contents of which are incorporated herein by reference.

[0172] According to the present disclosure, viral particles can be injected in any area around the spinal canal. In some embodiments, viral particles are injected into the lumbar region or the cisterna magna or intraventricularly into the ventricular space. The term "lumbar region" or "lumbar area" used herein refers to the area between the third and fourth lumbar (lower back) vertebrae and further includes the L2-S1 region of the spine. Typically, intrathecal injections performed through the lumbar region are also referred to as "lumbar IT delivery" or "lumbar IT administration". The term "cistern" refers to the space around and below the cerebellum via an opening between the skull and the top of the spine. Typically, intrathecal injections via the cisterna magna are also referred to as "cistern delivery". The term "ventricle" refers to a cavity in the brain that is continuous with the central canal of the spinal cord. Therefore, intrathecal administration includes any infusion into the central canal. Typically, injections via the ventricular cavity are referred to as intraventricular (ICV) delivery.

[0173] A variety of devices can be used for intrathecal delivery according to the present disclosure. In some embodiments, the device for intrathecal administration includes a fluid access port (e.g., an injectable port); a hollow body (e.g., a catheter) having a first flow orifice in communication with the fluid access port flow and a second flow orifice configured to be inserted into the spinal cord; and a fixing mechanism for fixing the insertion of the hollow body in the spinal cord. In multiple embodiments, the fluid access port includes a reservoir. In some embodiments, the fluid access port includes a mechanical pump (e.g., an infusion pump). In some embodiments, the implanted catheter is connected to a reservoir (e.g., for push injection delivery) or an infusion pump. The fluid access port can be implanted or external.

[0174] In some embodiments, intrathecal administration can be performed by lumbar puncture (i.e., slow push) or through a port-catheter delivery system (i.e., infusion or push). In some embodiments, the catheter is inserted between the lamina of the lumbar spine and the tip is passed up through the thecatheter space to the desired level (usually L3 to L4).

[0175] The single dose volume suitable for intrathecal administration is usually smaller.Usually, the intrathecal delivery according to the disclosure maintains the balance of CSF composition and the intracranial pressure of the object.In some embodiments, the intrathecal delivery is carried out without removing CSF accordingly from the object.In some embodiments, the suitable single dose volume can be, for example, less than about 10ml, 8ml, 6ml, 5ml, 4ml, 3ml, 2ml, 1.5ml, 1ml or 0.5ml.In some embodiments, the suitable single dose volume can be about 0.5 to 5ml, 0.5 to 4ml, 0.5 to 3ml, 0.5 to 2ml, 0.5 to 1ml, 1 to 3ml, 1 to 5ml, 1.5 to 3ml, 1 to 4ml or 0.5 to 1.5ml.In some embodiments, the intrathecal delivery according to the disclosure relates to the step of first removing the CSF of the desired amount. In some embodiments, prior to IT administration, less than about 10 ml (e.g., less than about 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1 ml) of CSF is first removed. In those cases, a suitable single dose volume may be, for example, more than about 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml.

[0176] A variety of additional devices can be used to achieve intrathecal administration of therapeutic compositions. For example, a preparation containing the desired enzyme can be administered using an Ommaya reservoir, which is commonly used for intrathecal administration of drugs for meningeal cancer (Ommaya, Lancet 2: 983-84, 1963). More specifically, in this method, a ventricular tube is inserted through a hole formed in the anterior horn and connected to an Ommaya reservoir installed under the scalp, and the reservoir is punctured subcutaneously to deliver the replaced specific enzyme intrathecally, which is injected into the reservoir. Other devices for administering therapeutic compositions or preparations intrathecally to individuals are described in U.S. Patent No. 6,217,552, which is incorporated herein by reference. Alternatively, viral particles can be administered intrathecally, for example, by a single injection or continuous infusion. It should be understood that dosage therapy can be administered in a single dose or in multiple doses.

[0177] In one embodiment of the present disclosure, the viral particles are administered by intracerebroventricular injection into the brain of the subject. The injection can be accomplished, for example, through a burr hole made in the skull of the subject. In another embodiment, the viral particles and / or additional pharmaceutical preparations are administered into the ventricles of the subject through a surgically inserted shunt. For example, injection can be performed into the larger lateral ventricle. In some embodiments, injection can also be performed into the third and fourth smaller ventricles. In yet another embodiment, the pharmaceutical composition used in the present disclosure is administered by injection into the cisterna magna or lumbar region of the subject.

[0178] IV. Pharmaceutical Compositions

[0179] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, formulation, liquid or solid, or a mixture thereof, which is suitable for one or more routes of administration, routes of in vivo delivery or routes of contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is not a biologically or otherwise undesirable substance, for example, the substance can be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions can be sterile. Such pharmaceutical preparations and compositions can be used, for example, to administer viral particles to a subject.

[0180] Such formulations and compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic agents, and absorption enhancers or absorption delaying agents that are compatible with drug administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions, and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial agents, antiviral agents, and antifungal agents) may also be incorporated into the formulations and compositions.

[0181] Pharmaceutical compositions generally include pharmaceutically acceptable excipients. Such excipients include any agent that does not itself induce the production of antibodies that are harmful to the individual receiving the composition and can be administered without excessive toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. Pharmaceutically acceptable salts such as inorganic acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, etc., and salts of organic acids such as acetates, propionates, malonates, benzoates, etc., may be included therein. In addition, auxiliary substances such as surfactants, wetting agents or emulsifiers, pH buffer substances, etc. may be present in such carriers.

[0182] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery indicated herein or known to those skilled in the art. Thus, the pharmaceutical composition comprises a carrier, diluent, or excipient suitable for administration or delivery by a variety of routes.

[0183] The pharmaceutical form suitable for injection or infusion of viral particles may include sterile aqueous solution or dispersion, which is suitable for the temporary preparation of sterile injectable or infusible solution or dispersion optionally encapsulated in liposome. In all cases, the final form should be sterile fluid and stable under preparation, use and storage conditions. Liquid carrier or carrier can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, non-toxic glyceride and their suitable mixture. Suitable fluidity can be, for example, by forming liposome, by keeping the required particle size in the case of dispersion or by using a surfactant to keep. Isotonic agents may be included, for example, sugar, buffer or salt (for example, sodium chloride). The extended absorption of injectable compositions can be realized by using delayed absorption agents such as aluminum monostearate and gelatin in the compositions.

[0184] The solution or suspension of viral particles may optionally include one or more of the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), artificial CSF, a surfactant, a fixed oil, a polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), glycerin, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, etc.; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose.

[0185] Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the present disclosure are known in the art (see, e.g.,

[0186] Remington: The Science and Practice of Pharmacy(2003)20 th ed., MackPublishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996)12th ed., Merck Publishing Group, Whitchouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11 th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), RL Juliano, ed., Oxford, NY, pp. 253-315).

[0187] The viral particles and compositions thereof can be formulated in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as single doses for individuals to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit form depends on the number of viral particles believed to be necessary to produce the desired effect. The necessary amount can be formulated in a single dose or can be formulated in multiple dosage units. The dosage can be adjusted to a suitable viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0188] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, ie, an amount sufficient to reduce or alleviate the symptoms or adverse effects of the disease state in question or an amount sufficient to confer a desired benefit.

[0189] As used herein, "unit dosage form" refers to physically discrete units suitable as unit doses for subjects to be treated; each unit containing a predetermined amount is optionally associated with a pharmaceutical carrier (excipient, diluent, carrier or filler) that is calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. The unit dosage form may be in ampoules and vials that may contain, for example, liquid compositions or compositions in a freeze-dried or lyophilized state; for example, a sterile liquid carrier may be added prior to in vivo administration or delivery. A single unit dosage form may be contained in a multi-dose kit or container. Thus, for ease of administration and uniformity of dosage, for example, viral particles and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage forms.

[0190] The formulation comprising the viral particles generally comprises an effective amount, which is readily determined by one skilled in the art. The viral particles may generally be from about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount to be administered depends on factors such as the age, weight and physical condition of the mammalian or human subject being treated. The effective dose may be determined by one of ordinary skill in the art by routine experiments establishing a dose response curve.

[0191] V. Definition

[0192] The terms "polynucleotide", "nucleic acid" and "transgenic" are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced ​​or unspliced ​​mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, micro RNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA or antisense RNA). Polynucleotides may include naturally occurring, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides may be single-stranded, double-stranded or triple-stranded, linear or circular, and may have any suitable length. When discussing polynucleotides, the sequence or structure of a specific polynucleotide may be described herein according to the convention of providing a sequence in the 5' to 3' direction.

[0193] A nucleic acid encoding a polypeptide generally comprises an open reading frame encoding the polypeptide. Unless otherwise indicated, a specific nucleic acid sequence also includes degenerate codon substitutions.

[0194] The nucleic acid may comprise one or more expression control or regulatory elements operably linked to the open reading frame, wherein the one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in mammalian cells. Some non-limiting examples of expression control / regulatory elements include transcription initiation sequences (e.g., promoters, enhancers, TATA boxes, etc.), translation initiation sequences, mRNA stability sequences, poly A sequences, secretion sequences, etc. Expression control / regulatory elements may be obtained from the genome of any suitable organism.

[0195] "Promoter" refers to a nucleotide sequence that is usually located upstream (5') of a coding sequence and directs and / or controls the expression of a coding sequence by providing recognition for RNA polymerase and other factors required for proper transcription. Pol II promoters contain a minimal promoter, which is a short DNA sequence containing a TATA box and optionally other sequences for specifying a transcription start site, to which regulatory elements are added to control expression. Type 1 pol III promoters contain three cis-acting sequence elements downstream of the transcription start site: a) 5' sequence element (A block); b) middle sequence element (I block); c) 3' sequence element (C block). Type 2 pol III promoters contain two essential cis-acting sequence elements downstream of the transcription start site: a) A box (5' sequence element); and b) B box (3' sequence element). Type 3 pol III promoters contain several cis-acting promoter elements upstream of the transcription start site, such as the traditional TATA box, proximal sequence element (PSE), and distal sequence element (DSE).

[0196] An "enhancer" is a DNA sequence that stimulates transcriptional activity and can be an intrinsic element of a promoter or a heterologous element that enhances expression levels or tissue specificity. It can operate in either direction (5'->3' or 3'->5') and can function even if located upstream or downstream of a promoter.

[0197] Enhancers may be derived entirely from a native gene, or be composed of different elements derived from different elements found in nature, or even comprise synthetic DNA segments. Enhancers may comprise DNA sequences that participate in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0198] As used herein, "transgene" conveniently refers to a nucleic acid sequence / polynucleotide that is intended to be or has been introduced into a cell or organism. A transgene includes any nucleic acid, such as a gene encoding an inhibitory RNA or a polypeptide or protein, and is generally heterologous to the naturally occurring AAV genomic sequence.

[0199] The term "transduction" refers to the introduction of a nucleic acid sequence into a cell or host organism by means of a vector (e.g., a viral particle). Therefore, the introduction of a transgene into a cell by a viral particle can be referred to as the "transduction" of a cell. The transgene may or may not be integrated into the genomic nucleic acid of the transduced cell. If the introduced transgene is integrated into the nucleic acid (genomic DNA) of the recipient cell or organism, it can be stably maintained in the cell or organism, and further transmitted to the daughter cells or organism of the recipient cell or organism or inherited by it. Finally, the introduced transgene may be present outside the chromosome of the recipient cell or host organism, or only exist temporarily. Therefore, "transduced cells" are cells into which transgenes are introduced by means of transduction. Therefore, "transduced" cells are cells or their offspring into which transgenes have been introduced. Transduced cells can proliferate, transcribe transgenes and express encoded inhibitory RNA or protein. For gene therapy purposes and methods, transduced cells can be in mammals.

[0200] A nucleic acid / transgene is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, wherein the promoter is capable of controlling the transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element may also be referred to as an expression cassette.

[0201] In certain embodiments, the expression control element comprises a CMV enhancer.

[0202] As used herein, the terms "modification" or "variant" and grammatical variations thereof refer to deviations of a nucleic acid, polypeptide, or subsequence thereof from a reference sequence. Thus, modified and variant sequences may have substantially the same, higher or lower expression, activity, or function as a reference sequence, but retain at least some of the activity or function of the reference sequence. A specific type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, such as a missense or nonsense mutation.

[0203] A "nucleic acid" or "polynucleotide" variant refers to a modified sequence that has been genetically altered compared to the wild type. A sequence can be genetically modified without changing the encoded protein sequence. Alternatively, a sequence can be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combined sequence that has been codon-modified to encode a protein that still retains at least partial sequence identity with a reference sequence (e.g., a wild-type protein sequence) and has also been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without changing the amino acids of the protein encoded thereby, and some codons of the nucleic acid variant will be changed, which in turn changes the amino acids of the protein encoded thereby.

[0204] The terms "protein" and "polypeptide" are used interchangeably herein. "Polypeptides" encoded by "nucleic acids" or "polynucleotides" or "transgenes" disclosed herein include partial or full-length native sequences, such as naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments), and sequence variants thereof, as long as the polypeptide retains a certain degree of function or activity. Therefore, in the methods and uses of the present disclosure, such polypeptides encoded by nucleic acid sequences do not need to be identical to endogenous proteins that are defective or whose activity, function or expression is insufficient, missing or absent in the treated mammal.

[0205] Some non-limiting examples of modifications include one or more nucleotide or amino acid replacements (e.g., about 1 to about 3, about 3 to about 5, about 5 to about 10, about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 500, about 500 to about 750, about 750 to about 1000, or more nucleotides or residues).

[0206] An example of an amino acid modification is a conservative amino acid substitution or deletion.In some embodiments, the modified sequence or variant sequence retains at least part of the function or activity of the unmodified sequence (eg, wild-type sequence).

[0207] Another example of amino acid modification is the introduction of targeting peptides into the capsid protein of the viral particle.Peptides have been identified that target recombinant viral vectors to the central nervous system (eg, different brain regions).

[0208] The recombinant virus modified in this way can preferentially bind to one type of tissue (e.g., CNS tissue) rather than another type of tissue (e.g., liver tissue). In certain embodiments, the recombinant virus carrying the modified capsid protein can "target" the cerebrovascular epithelial tissue by binding at a higher level than the comparable unmodified capsid protein. For example, the recombinant virus with the modified capsid protein can bind to the ependymal tissue at a level 50% to 100% higher than the unmodified recombinant virus.

[0209] "Nucleic acid fragment" is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in most organisms is the genetic material, while ribonucleic acid (RNA) is involved in transferring the information contained in the DNA to the protein. Fragments and variants of the disclosed nucleotide sequences and proteins or partial length proteins encoded therefrom are also encompassed in the present disclosure. "Fragment" or "portion" means the full length or less than the full length of the nucleotide sequence or amino acid sequence encoding a polypeptide or protein. In certain embodiments, a fragment or portion is biologically functional (i.e., retaining 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the activity or function of the wild type).

[0210] A "variant" of a molecule is a sequence that is substantially similar to the sequence of a natural molecule. For nucleotide sequences, variants include those sequences that encode the same amino acid sequence as the natural protein due to the degeneracy of the genetic code. For example, these naturally occurring allelic variants can be determined using molecular biology techniques (such as, for example, using polymerase chain reaction (PCR) and hybridization techniques). Variant nucleotide sequences also include nucleotide sequences of synthetic origin, such as those produced by using site-directed mutagenesis (which encodes natural proteins), and those encoding polypeptides with amino acid substitutions. In general, the nucleotide sequence variants of the present disclosure will have at least 40%, 50%, 60% to 70%, such as 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% to 79%, usually at least 80%, such as 81% to 84%, at least 85%, such as 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% to 98% sequence identity with the native (endogenous) nucleotide sequence. In certain embodiments, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the activity or function of the wild-type).

[0211] "Conservative variation" of a particular nucleic acid sequence refers to those nucleic acid sequences that encode the same or substantially the same amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For example, codons CGT, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Therefore, at each position where arginine is specified by a codon, the codon can be changed to any corresponding codon described without changing the encoded protein. Such nucleic acid variation is a "silent variation", which is a "conservatively modified variation". Unless otherwise indicated, each nucleic acid sequence encoding a polypeptide described herein also describes each possible silent variation. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is typically the only codon for methionine) can be modified to produce functionally identical molecules by standard techniques. Therefore, each "silent variation" of a nucleic acid encoding a polypeptide is implicit in each described sequence.

[0212] The term "significant identity" of a polynucleotide sequence means that the polynucleotide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% or 89%, or at least 90%, 91%, 92%, 93% or 94%, or even at least 95%, 96%, 97%, 98% or 99% sequence identity compared to a reference sequence using one of the alignment programs using standard parameters. One skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc. Significant identity of amino acid sequences for these purposes generally means at least 70%, at least 80%, 90% or even at least 95% sequence identity.

[0213] In the case of polypeptides, the term "substantially identical" means that the polypeptide comprises a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunoreactive with antibodies raised against a second polypeptide. Thus, for example, a polypeptide is identical to a second polypeptide where the two peptides differ only in conservative substitutions.

[0214] "Disease" means a condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0215] The term "treatment" and variations thereof also refer to both therapeutic treatment and preventive or precautionary measures, wherein the purpose is to prevent, inhibit, alleviate or reduce undesirable physiological changes or disorders, such as the occurrence, progression or deterioration of disorders. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, attenuation of disease extent, stabilization of symptoms or adverse effects of the disease (i.e., no deterioration or progression), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or total), whether detectable or undetectable. "Treatment" also means prolonged survival compared to the expected survival without treatment. Those in need of treatment include those already suffering from the disease or disorder and those susceptible to the disease (e.g., as determined by genetic assays).

[0216] As used herein, "substantially free" with respect to a specified component is used herein to mean that none of the specified component is purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the specified component resulting from any accidental contamination of the composition is much less than 0.05%, preferably less than 0.01%. Most preferred are compositions wherein the amount of the specified component cannot be detected by standard analytical methods.

[0217] As used herein in the specification, a noun without a quantifier may mean one or more. As used herein in the claims, when used in conjunction with the word "comprises", a noun without a quantifier may mean one or more than one.

[0218] Unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, the use of the term "or / or" in the claims is used to mean "and / or", but the present disclosure supports a definition referring to only alternatives and "and / or." As used herein, "another" may mean at least a second or more.

[0219] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation in error for the device being employed to determine the value, the inherent variation in the method being employed to determine the value, the variation that exists between study subjects, or a value within 10% of the stated value.

[0220] VI.Medicine Box

[0221] The present disclosure provides a kit having packaging material and having one or more components therein. The kit typically includes a label or package insert containing a description of the components or instructions for use of the components therein in vitro, in vivo, or ex vivo. The kit may include a collection of such components (e.g., nucleic acids, recombinant vectors, and / or viral particles).

[0222] A kit refers to a physical structure that houses one or more components of the kit. The packaging material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0223] The label or insert may include information on one or more of the components, dosage amounts, clinical pharmacology (including mechanism of action, pharmacokinetics and pharmacodynamics) of the active ingredient. The label or insert may include information indicating the manufacturer, batch number, place of production and date of production, and expiration date. The label or insert may include information indicating manufacturer information, batch number, manufacturer location and date. The label or insert may include information on the diseases for which the kit components can be used. The label or insert may include instructions for clinicians or subjects to use one or more of the kit components in methods, uses, treatment schemes or therapeutic regimens. The instructions may include dosage amounts, frequency or duration, and instructions for implementing any method, use, treatment scheme or prevention or therapeutic regimen described herein.

[0224] The label or insert may contain information about any benefits that the components may provide, such as preventive or therapeutic benefits. The label or insert may contain information about potential adverse side effects, complications, or reactions, such as warning the subject or clinician about situations in which a particular composition is not suitable for use. Adverse side effects or complications may also occur when the subject has, will, or is taking one or more other drugs, which may be incompatible with the composition or with another treatment regimen or therapeutic regimen that the subject has, will, or is experiencing that is incompatible with the composition, and therefore, the instructions may contain information about such incompatibilities.

[0225] The label or insert includes "printed matter", such as paper or cardboard, either alone or fixed to a component, a kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial containing a kit component. The label or insert may additionally include a computer-readable medium, such as a barcode printed label, a disk, an optical disk (e.g., CD- or DVD-ROM / RAM, DVD), an MP3, or an electrical storage medium (e.g., RAM and ROM) or a mixture of these such as magnetic / optical storage media, flash memory, hybrid, and storage type cards.

[0226] VII. Examples

[0227] The following examples are included to illustrate some preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for the practice of the present disclosure. However, it will be appreciated by those skilled in the art that, without departing from the spirit and scope of the present disclosure, many changes may be made to some of the disclosed specific embodiments, and still the same or similar results may be obtained.

[0228] Example 1

[0229] Identify ependyma-enriched genes or deletion methods for ependyma-specific genes. The ependyma constitutes a thin epithelial layer lining the ventricles of the brain and the central canal of the spine. These cells are adjacent to the cerebrospinal fluid (CSF) of the ventricles, which is a liquid that not only fills these cavities but also diffuses into the substance by transporting to the subarachnoid space and along the perivascular space, and is widely distributed throughout the brain. Therefore, proteins secreted from the ependyma into the CSF can be widely delivered to the entire brain. In therapy, infection of ependymal cells with adeno-associated viruses (AAVs) encoding secretory proteins has been shown to promote widespread expression of proteins in rodents, dogs, and non-human primates brains. To ensure the robust, long-term expression of ependyma-localized AAVs, the inventors attempted to identify endogenous gene features and their respective regulatory regions that can be incorporated into the AAV transgene. The importance of this study is to identify genes whose expression is insensitive to disease states, ensuring that even when the gene is introduced into pathological brains, there is strong expression. Therefore, the inventors obtained samples from healthy patients as well as samples from patients with frontotemporal dementia, semantic dementia, Lewy body dementia, Alzheimer's disease (AD) and Huntington's disease (HD). Clean separation of this specific tissue type is very difficult due to the limited depth of the ependymal lining. To circumvent this problem, the inventors used a subtraction method to identify ependymal enriched genes or ependymal specific genes. Tissue samples were obtained from white matter and gray matter adjacent to the ventricles and white matter and gray matter at the edges of the ventricles containing ependyma. Genes that were specific to white matter + ependyma and gray matter + ependyma samples were considered to be ependymal enriched or ependymal specific ( Figure 1 ). Top hits were compared against in situ hybridization data published by the Allen Institute for Brain Science ( Figure 2 ).

[0230] Isolation of predicted regulatory regions in ependymal-specific genes. In order to isolate functional promoter segments, the UCSC genome browser was used to retrieve promoter-like features in the upstream sequences of identified genes. Of particular interest are segments containing H3K4me3, H3K27Ac, ​​CpG islands, and transcription factor binding sites. Segments of approximately 1100 to 2500 base pairs (base pair, bp) were amplified by PCR from human (HT1080) genomic DNA (genomicDNA, gDNA) and placed upstream of the eGFP reporter. Promoters were identified using the 3-bp barcode in the 3'UTR for high-throughput sequencing. Individual plasmids were merged in an equimolar ratio and transgenics were packaged in AAV4 or AAV2 capsids as libraries for testing in mice and rhesus monkeys, respectively.

[0231] In vivo testing of isolated promoter segments driving RNA expression in the ependyma of mice and rhesus monkeys. A library of 13 transgenes containing ependyma-enriched promoters prepared as a single AAV4 was injected into the lateral ventricles of adult FVBn mice at high doses (1e11vg), medium doses (5e10vg) and low doses (1e10vg). After three weeks, the ventricular lining and restricted surrounding tissues were microdissected and RNA was isolated using Trizol. A two-step PCR protocol was used to amplify the region around the 3-bp barcode from cDNA to connect to the Illumina sequencing adapter. The final product was subjected to amplicon sequencing to quantify the relative contribution. Figure 4 The graphs in show the fractional contribution of each barcode to the total read counts in the tissue and input virus.

[0232] To quantify the function of the isolated segments as promoters in vivo, the relative contribution of RNA output was normalized to that of the input virus. As expected, the ubiquitous iCAG promoter showed strong expression in vivo ( Figure 5 ). Transgenes containing regulatory sequences from the human von Willebrand factor A domain containing 3a (hVWA3a) gene were also enriched relative to the viral input.

[0233] Will Figure 4 The same library used in was prepared into AAV2 and injected into the lateral ventricle of two adult rhesus monkeys at 2e13 total vg per animal. Three weeks later, the region at the ventricular margin (including the spinal cord) was microdissected ( Figure 6 ). RNA was isolated, converted to cDNA, and amplicon sequencing was performed on PCR products containing 3-bp barcodes. The colored bars indicate the relative contribution of each transgene in tissue RNA or viral DNA. The RNA enrichment pattern in matching positions was similar between two different macaques and in different regions of the same structure.

[0234] The original transgene was modified to contain a promoter-located intron and the ApoE2 coding sequence. Genetic variations within the apolipoprotein E gene (APOE) on chromosome 19 are associated with differential risk for late-onset Alzheimer's disease. APOE3, the most common allele, is considered neutral, neither increasing nor decreasing a person's risk of developing the disease. APOE4 increases risk and is associated with earlier disease onset, while APOE2, a relatively rare variant, provides protection against the disease. Intraventricular injection of AAV4-APOE2 into a mouse model of early-onset AD resulted in transduction of the ependyma and choroid plexus and detectable protein deposition in the cortical parenchyma, which led to reversal of Aβ deposition and improved clearance from the CNS (PMID: 24259049). In order to make the inventors' design suitable for therapeutic applications, the inventors replaced the eGFP reporter with human ApoE2 cDNA in a construct containing six different ependymal-enriched promoters ( Figure 7 and 8 These six libraries were further extended by introducing either a short (133 bp) β-globin / IgG chimeric intron or a long (951 bp) chicken β-actin / rabbit β-globin intron to test intron-mediated expression enhancement, for a total of 3 variants per gene.

[0235] To measure intron splicing, individual plasmids were transfected into HEK293 cells and harvested at 24 h. RNA was isolated using Trizol according to the manufacturer's instructions and reverse transcribed using MultiScribe reverse transcriptase. Correct splicing was verified by amplification of intron-containing regions from cDNA (C) and plasmid DNA (D). Complete intron removal was observed in most transgenes, except for the short intron from hANXA1 ( Figure 8 ). Protein export from intron-containing transgenes was also measured in HEK293 cell lysates and culture medium. Fig. 9 Western blots for APOE are included, a protein that is not readily detected endogenously in HEK293 cells. Consistent with intron-mediated enhancement, all variants showed increased protein export in the presence of introns, with the increase being greater for long introns relative to short introns.

[0236] In vivo measurement of intron-containing promoter activity. All 18 ApoE2 transgenic variants (intronless, short and long) were prepared as separate plasmids and combined in equimolar ratios to generate a single AAV2 library. Virus was injected into the lateral ventricle of two adult rhesus monkeys at a total dose of 2.8e13 vg / animal. Four weeks later, the ventricular margins were microdissected from 4 mm thick coronal slabs and RNA was isolated using Trizol. cDNA amplicons containing a unique 3 letter barcode in the 3'UTR were sequenced for in vivo assessment of relative promoter usage ( Fig.10 ). Numbers in brackets indicate separations of equivalent plates in the larger specified construct. All hVWA3a variants showed relative enrichment in vivo relative to the input library.

[0237] hVWA3a promoter drives transgene expression in mouse models. Adult APOE- / - (null) mice were injected with serotype AAV4 to deliver APOE2 under the hVWA3a promoter to the right lateral ventricle of the mice. Ependymal tissue was microdissected and protein was extracted for quantification of APOE2 by automated Western blotting (WES) compared to uninjected brain tissue.

[0238] hVWA3a promoter shows higher expression of APOE2 relative to previous constructs. APOE- / - (deficient) mice were injected with serotype AAV4, and APOE2 under the ubiquitous CAG promoter or hVWA3a promoter was delivered to the right lateral ventricle of the mice in equal doses. Protein was extracted from microdissected ependymal tissue from all animals and subjected to automated Western blotting (WES). Based on the intensity of the bands, APOE2 driven by hVWA3a expressed higher amounts of APOE2 protein than APOE2 driven by the CAG promoter.

[0239] Biodistribution of the novel capsid ERDRpAAV1 in combination with hVWA3a restricted expression to ependymal cells in a mouse model as shown by the eGFP transgene. Peptide-modified AAV1 capsid with a human ependyma-specific promoter: ERDRpAAV1.hVWA3a.eGFP. Positive eGFP fluorescence signal was restricted to ependymal cells lining the ventricles.

[0240] The introduction of ApoE2 via ependymal expression in the APP / PS1 / ApoE4 mouse model reduced the size and density of cortical amyloid plaques and reduced the concentration of oligomeric Aβ in the brains of these animals. In addition, the inventors observed that the introduction of ApoE2 in the APP / PS1 / ApoE4 mouse model significantly reduced the activation of microglia near plaques in the cortex. This suggests that ApoEε4 may drive microglia toward an inflammatory phenotype and that exogenous ApoE2 is able to interrupt or reduce this abnormal activation. The inventors also found that ApoE2 was able to prevent or reduce the synaptic loss that occurs in the halo around amyloid β plaques in these mice. In summary, this suggests that the effect of ApoE2 on microglia may be to protect against abnormal engulfment of synapses near plaques that may occur due to amyloid deposition. See Figure 23 to 3 4.

[0241] These results suggest that viral delivery of secreted ApoE2 into ependymal ApoE4 vectors may be an effective therapeutic strategy to affect both the classic pathologies of AD (eg, plaque deposition and neurodegeneration) and the enhanced neuroinflammatory spectrum observed in sporadic AD.

[0242] Example 2

[0243] method. Study Design . The inventors performed intracerebroventricular (ICV) infusions with a new AAV vector that expresses an APOE2 variant within the ventricular space of a transgenic mouse model of AD. Mice were injected with AAV or vehicle control for a period of 2 months. The inventors evaluated the effect of human APOE2 on amyloid deposition using immunohistochemistry (IHC) and enzyme-linked immunosorbant assay (ELISA). Mice were randomly assigned to treatment groups. The nature of the injected vector was blinded prior to statistical analysis. The inventors estimated that 8 animals (4 of each sex) would be required for these studies per condition. This would provide a power of >0.8 to see a 30% correction of baseline phenotype compared to previous data (Hudry et al., 2013).

[0244] animal. APP / SP1 (Radde et al., 2006) mice express the human mutants APPKM670 / 671NL and PSEN1L166P under the Thy1 promoter, which results in a severe phenotype characterized by amyloid deposition at 3 to 4 months of age. APOE-targeted alternative expression of human APOE4 under the control of the murine APOE promoter in a mouse model (Huynh et al., 2019). These animals were backcrossed until the APP / PS1 transgene was expressed together with two copies of human APOE4 to replace mouse apoe (APPPS1 / APOE4). The inventors exposed 4-month-old APOE4-TR / APP / PS1 mice to high (7E10vg, n=14), medium (2E10vg, n=11) or low (7E9vg, n=11) doses of AVert-APOE2 or vehicle control (n=10) for 2 months. In addition, APOE KO (Jackson labs) mice in C57BL / 6 background were included as a comparative measure of Aβ levels in tissues and CSF. Experiments were performed according to the National Institutes of Health (NIH) and institutional guidelines, and two genders were used. Due to the small size of the mouse brain, not all animals were used for each analysis, and n is represented by the number of points shown. Open circles represent females and solid circles represent males.

[0245] Viral vector construction and production . Research-grade production of AAV-based viral vectors is provided by Children's Hospital of Philadelphia Research Vectors The Research Vector Core (RVC) was obtained from a certified working cell bank by transient triple transfection The cells were prepared by using adherent human embryonic kidney epithelial cells (HEK293) from Working Cell Bank (WCB). Prior to transfection, cells were expanded in tissue culture flasks and roller bottles. Testing of study products was performed in-house by RVC QC, and the test methods, procedures, and results were documented on the Certificate of Analysis (CoA) for each batch.

[0246] Stereotactic intraventricular injection Intraventricular injections of AAV were performed as previously described (14, 30). Animals were anesthetized (O2 / isofluorane 0.2%) and placed on a stereotaxic frame (David Kopf Instruments). Injections were performed in each lateral ventricle at 0.20 μl / min using a 33G needle connected to a 10-μl Hamilton syringe (Hamilton Medical). Stereotactic coordinates were calculated from bregma (anteroposterior +0.3 mm, mediolateral ±1 mm, and dorsoventral -2 mm).

[0247] Western blottingMouse cortical tissue was homogenized in 10 volumes by weight of ice-cold TBS with protease and phosphatase inhibitors using a handheld electric homogenizer. The homogenate was then spun at 10 000 g for 10 min and the supernatant (TBS soluble fraction) was collected for western blotting. Protein concentration was determined using the BCA assay. Total protein (5 to 10 μg) was loaded and separated by 4% to 12% NuPAGE gel in MES buffer, and proteins were then separated by weight at 120 V for 2 h. XCell II in tris-glycine transfer buffer was used. TM The blotting module system electrotransferred the protein to a nitrocellulose membrane at 30V for 1.5 hours. The membrane was incubated in a blocking buffer (Li-Cor Biosciences) diluted with 1:1 TBS for 1 hour to reduce background staining. The membrane was then incubated with a primary antibody; rb anti-APOE (Novus biologicals, NBP1-31123) and ms anti-GAPDH (Millipore MAB374) were diluted in a blocking buffer supplemented with 0.1% Tween 20 and left overnight at room temperature with shaking. The membrane was then washed and incubated with appropriate 680 and 800IR dye secondary antibodies (Li-Cor Biosciences). The membrane was imaged using the Odyssey infrared imaging system and analyzed using Odyssey software.

[0248] DNA and RNA extraction and analysis Genomic DNA was extracted from brain tissue using the QIAamp DNA Mini Kit (Qiagen) following the manufacturer's protocol. Samples were run on a BioRad CFX384 Real-Time System C1000Touch using BioRad CFXManager 3.1 software. Total genomic copies were quantified against a 6-point standard curve generated using a linearized plasmid containing the construct. Primers / probes (designed for noncoding regions in the construct) were mixed with MasterMix (Applied Biosystems) was used together.

[0249] Total RNA was extracted from brain tissue using TRIzol (Ambion, Life Technologies) according to the manufacturer's protocol. RNA (1 μg) was treated with RNase-free DNase I (ThermoScientific) according to the manufacturer's protocol. Complementary DNA was generated using a high-capacity cDNA reverse transcription kit (LifeTechnologies). Samples were run on a BioRad CFX384 Real-Time System C1000Touch using BioRad CFX Manager 3.1 software. The designed primers / probes to be used were used to identify the target gene. APOE levels were quantified using Master Mix (Applied Biosystems). Transgenic expression of human APOE (Hs00171168_m1) commercial Exogenous mRNA levels of primer / probe sets (Applied Biosystems). Endogenous mouse β-actin (Mm02619580_g1) was used as a reference gene to normalize expression between samples.

[0250] ELISA quantification . The concentrations of Aβ40 and Aβ42 were determined by BNT-77 / BA-27 (for Aβ40) and BNT-77 / BC-05 (for Aβ42) sandwich ELISA (Wako) according to the manufacturer's instructions. Aβ40 and Aβ42 concentrations were measured in TBS, SDS-soluble, and SDS-insoluble fractions of each mouse. The sections of mouse brain were homogenized in 10 volumes (w / v) of TBS buffer containing a complete protease inhibitor cocktail (Roche) and centrifuged at 1000,000 × g for 30 minutes at 4°C. The supernatant was collected and retained as the TBS-soluble fraction. The precipitate was then homogenized in 10 volumes (w / v) of TBS buffer containing 2% SDS, incubated at 37°C for 30 minutes, and then centrifuged at 100,000 × g for 30 minutes at 20°C. The SDS-insoluble pellet was dissolved in 500 μl of 70% formic acid and sonicated on ice at 10% power for 1 min 30 s intervals until completely dissolved, and then centrifuged at 100,000 × g for 30 min at 4 °C. The formic acid-soluble supernatant was dried by Speed-Vac and then resuspended in 1 volume (w / v) of dimethyl sulfoxide (DMSO). The DMSO-soluble fraction was used as the SDS-insoluble fraction (adapted from Hashimoto et al 2020) (Hashimoto et al., 2020).

[0251] RNAscope. Drop fixed hemispheres of APOE KO mice were cut into 30 μm sections on a cryo-ultramicrotome. Three mice per experimental condition (sham surgery vs AAV injection) were stained for APOE mRNA by RNAscope. RNAscope experiments were performed using the Manual Fluorescence Multiplex Kit v2 (Advanced Cell Diagnostics) with slight adjustments following the manufacturer's recommendations. In brief, several sections from each mouse were baked on superfrost slides for APOE mRNA quantification. After target repair and protease digestion, probe hybridization was performed at 40°C for 2 hours with hs-APOE (433091), 3-plex positive control probe _Mm (320881) and negative control probe -DapB (310043). After the amplification step to obtain the RNAscope signal, the signal was visualized using TSA-cy3 (Perkin Elmer FP1170). Sections were counterstained with 1:1000 dapi and mounted using immunomount and scanned at ×10 magnification using an Olympus VS120-S6-W virtual slide microscope.

[0252] Immunohistological analysis . Mice were euthanized by isoflurane inhalation. One brain hemisphere was fixed in 4% paraformaldehyde and 15% glycerol in PBS and changed to 30% glycerol in PBS after 48 hours. The remaining hemisphere was snap frozen for biochemical analysis. Drop-fixed hemispheres were processed by the Society for Neuroscience. 40 hemispheres were embedded in gelatin blocks and sectioned at 30 μm. Sections were permeabilized in 0.5% Triton-X for 15 minutes and then blocked in 0.1% Triton-X and 5% normal goat serum for 1 hour at room temperature. Incubation with primary antibodies in 0.05% Triton-X and 2.5% normal goat serum overnight at 4°C. Sections were then washed in TBS and appropriate secondary antibodies were diluted 1:500 in 0.05% Triton-X and 2.5% normal goat serum in TBS at room temperature. Sections were incubated with 1:1000 DAPI in TBS for 10 minutes at room temperature, washed, and mounted using immunoslides.

[0253] Plaque quantification. Every 10th section was stained for amyloid beta using rabbit anti-Abeta (1:500, IBL, catalog number 18584) as described above. Amyloid dense core plaques were labeled with 0.05% Thio-S (Sigma-Aldrich) in 50% ethanol before mounting. The sections were mounted and scanned at 40× using a nanozoomer microscope. The sections were quantified using qupath (Bankhead et al., 2017). For each section, a cortical area was selected, and plaques were identified using an object classifier and the plaque coverage area was assessed as a percentage of the measured cortical area. For the size and number of plaques, areas of the same size were selected in the cortex of each animal and plaques were identified using an object classifier.

[0254] Glial Assessment . Several sections were stained as described above. The primary antibodies were 1:1000 biotinylated Ms anti-Abeta82E1 (IBL 10326), 1:500 GFAP-488 (Millipore MAB 3402X), and 1:500 rabbit anti-IBA1 (wako 019-19741). Secondary antibodies were streptavidin Alexa Fluor 568 (Invitrogen S11226) and donkey anti-rabbit 647 (A-31573). Five random areas containing cortical plaques from the somatosensory cortex were imaged at 40× using an Olympus FV3000 confocal laser scanning microscope. Z stacks were generated and the level of glial reactivity for each plaque was assessed by two blinded investigators using a 4-point scale (Figure 38A). All plaques from individual mice were averaged together to generate the graphs in Figures 35B to C and Figures 38C to D, while for Figures 35D and 38E, all plaques from a given experimental group were evaluated.

[0255] Synaptic quantification. Several sections were stained as described above. The primary antibodies used were 1:500 rabbit anti-Abeta1:500 (IBL, catalog number 18584) and 1:500 goat anti-PSD95 (abcam ab12093). The secondary antibodies were donkey anti-goat 488 (Invitrogen A-11078) and donkey anti-rabbit 594 (A-21207). Five areas containing cortical plaques were randomly imaged from the somatosensory cortex using an Olympus FV3000 confocal laser scanning microscope using an oil immersion objective at 60×. 5 μm Z stacks were imaged with a slice size of 0.56 μm. Images were processed using Matlab macros and custom Image J similar to Jackson et al. (Jackson et al., 2019). Briefly, 10 μm × 10 μm crops were obtained from areas within 15 μm of the plaque aperture or greater than 40 μm from the plaque aperture. Cell debris and DAPI were avoided. Harvests were thresholded using a custom Image J macro and synapses were quantified using a custom MATLAB macro. All harvests were averaged together to find the density near and far from plaques for each mouse.

[0256] Neurite quantification . Several sections were stained as described above. The primary antibodies used were 1:500 rabbit anti-Abeta 1:500 (IBL, catalog number 18584) and 1:500 mouse anti-SMI312 (Biolegend 837904). The secondary antibodies were donkey anti-rabbit 488 (Invitrogen A-21206) and donkey anti-mouse 594 (A-21203). Five areas containing cortical plaques were randomly imaged from the somatosensory cortex using an Olympus FV3000 confocal laser scanning microscope using an oil immersion objective at 60×. Z stacks of 20 μm were imaged with a slice size of 1 μm. Images were quantified using Image J in a blind experiment, which counted the number of dystrophies per plaque and also quantified the plaque area. The largest plaque was quantified in images where more than one plaque was present.

[0257] Statistical analysis . Statistical analysis was performed using Prism software. All intergroup analyses were analyzed using one-way ANOVA, followed by Dunnett's multiple comparison test between each group and vehicle control. Simple linear regression analysis was used to assess the correlation between factors and viral genome copy number, where the p value represents whether the slope is significantly non-zero. Statistics were performed with each mouse as a single data point. Samples were blinded for each analysis.

[0258] result. Intracerebroventricular injection of AAVert-APOE2 resulted in sustained production of APOE2 in the brain in a dose-dependent mannerAPOE is produced primarily by astrocytes and microglia within the CNS and, once produced, is secreted, after which it can diffuse throughout the parenchyma. Previously, the inventors showed that APOE2 produced by ependymal cells lining the ventricles can diffuse as far as the cortex, where it affects plaques and plaque-associated lesions (Hudry et al., 2013).

[0259] The present inventors injected a new ependymal restricted AAV capsid (AAVert) expressing APOE2 into four-month-old APOE KO mice by single intracerebroventricular (ICV) injection. Two months later, the mice were sacrificed. 10 Injection of virus containing 500 genomic units (vg) of APOE2 into Apoe KO mice resulted in robust expression of APOE2 mRNA in ependymal cells lining the ventricles, which was detected using RNAscope for human APOE ( Figure 1 A) Virus-driven APOE2 protein was also detected in TBS extracts of cortex by western blotting and was shown to be 10% of normal APOE (Fig. 33B to C).

[0260] The inventors then proceeded to inject AAV carrying APOE2 into four-month-old APPPS1 / APOE4 animals, which were sacrificed two months later. The inventors injected animals with three different doses: low-7E 9 vg, medium-2E 10 vg and high-7E 10 vg, and vehicle control group. DNA was extracted from the rhombencephalon, followed by qPCR, showing a dose-dependent uptake effect and three animals showing no uptake (Figure 33D). The number of viral genome copies correlated with the increase in the amount of human APOE mRNA, which was about 50% higher in high-dose animals than the endogenous levels observed in vehicle-treated animals (Figure 33E). Together, these data indicate that a single ICV injection induces APOE2 expression in ependymal cells in a dose-dependent manner.

[0261] APOE2 expression has a dose-dependent effect on the level of AB plaque deposition. The APP / PS1 / APOE4 model is a relatively aggressive model of amyloidosis. APPPS1 / APOE4 animals showed moderate plaque deposition at four months of age, and by six months of age, plaque deposition was well established throughout the cortex. Therefore, the inventors performed injections at 4 months and sacrificed at 6 months. Using ThioS as a marker for dense core amyloid plaques, they showed a dose-dependent effect of APOE2 on plaque deposition (Figures 34A to C). High-dose animals showed an approximately 33% decrease in the percentage of cortex covered by ThioS positive staining compared to vehicle-treated animals (Figure 34B). When plaque burden was compared to the number of viral genome copies extracted from the rhombomere, the dose-dependent effect of APOE2 on plaques was clear (Figure 34C). Staining using anti-oligomeric Aβ (oAβ) antibodies was not significantly different at the group level (Supplementary Figure 34A). Figure 1 A) and individual level (supplement Figure 1 B) Both showed similar trends.

[0262] The observed reduction in the percentage of cortex covered was accompanied by a significant reduction in plaque density ( FIG. 34D ), with high-dose animals showing 1.34% (95% CI, 1.2-1.4) per mm compared to vehicle-treated animals. 2 The number of plaques was reduced, and the size of Aβ plaques was even more significantly reduced ( FIG. 34E ).

[0263] Biochemical measurements of amyloid were consistent with imaging measurements. Concentrations of Aβ42 peptide measured from formic acid and SDS-soluble extracts of mouse brain mimicked the changes observed histologically, such that high-dose animals showed a decrease in SDS (Figure 36C) and formic acid (Supplementary Figure 36C). Figure 1 D) The amount of both soluble Aβ42 was reduced by about 50%.

[0264] APOE4 has been shown to impair Aβ clearance and promote Aβ aggregation, whereas APOE2 has been shown to have the opposite effect. The data presented herein are consistent with increased efflux of Aβ peptides across the BBB in the presence of APOE2 leading to reduced plaques.

[0265] APOE2 expression has a dose-dependent effect on plaque-associated neuroinflammation. APOE4 mice showed a more aggressive neuroinflammatory response to plaques compared to APOE2 or APOE3 mice. This reflects the human disease, as data show that APOE4 carriers have a more inflammatory phenotype (Serrano-Pozo, Li, et al., 2021). To see if the addition of APOE2 could attenuate the effects of plaques on local astrocytes and microglia, the inventors performed IHC for Aβ, Iba1 (microglia) (Figure 35A), and GFAP (astroglia in Figure 38B). The inventors assessed the level of glial reactivity on a 4-point scale, with 1 being unreactive and 4 being very reactive (Figure 38A). Images were evaluated by 2 blinded investigators, with the relevant R 2 The present inventors chose to evaluate the area immediately surrounding a single plaque rather than the entire cortex because microglial activation in these mice is closely associated with plaques, and therefore high-dose animals with fewer plaques would have lower overall levels of active microglia. By utilizing this approach, the present inventors were able to observe microglial reactivity that was partially independent of overall plaque density.

[0266] The inventors found a clear, statistically significant reduction in microglial reactivity in the high and medium dose groups compared to vehicle treated mice (Figure 35B), and this attenuation showed a significant correlation with the number of viral genome copies in individual animals (Figure 35C). This reduction was shown to be driven by an increase in the number of plaques that did not elicit a strong microglial response (score of 1) and a decrease in plaques with a score of 4 in high and medium dose animals compared to vehicle treated animals. Figure 3 D). In contrast, peri-plaque astrocyte reactivity was not affected by APOE2 levels or expression (Figure 38A to D) and was equally elevated around plaques in all groups.

[0267] APOE2 exposure modulates synaptic loss around amyloid deposits . Synaptic loss is known to be associated with cognitive impairment and has been shown to occur near plaques in human patients (Koffie et al., 2012) and in this mouse model (Hudry et al., 2013). The inventors have previously shown that APOE4 in both carriers and mice is associated with a higher amount of synaptic loss near plaques compared to APOE3 mice or carriers. Since APOE4 has been shown not to prevent synaptic loss, the inventors tested the hypothesis in this model that adding APOE2 would be able to protect synaptic integrity.

[0268] Postsynaptic density (PSD95) was identified using immunohistochemistry and imaged by confocal microscopy (Figure 38A). Synaptic loss in this model has been shown to occur most prominently within 15 μm of the plaque edge. Therefore, harvests collected from this area were analyzed the same as harvests collected greater than 40 μm from the edge of the plaque. There was no difference between the groups in synaptic density away from the plaque (Figure 38B); however, animals injected with the highest dose showed increased levels of synapses near the plaque (Figure 38C), returning to near-normal synaptic density compared to vehicle-treated animals. The percentage of synaptic loss was calculated by comparing the synaptic density near the plaque with the synaptic density away from the plaque in the same animal (Figure 38D). Vehicle-treated animals showed more than twice the synaptic loss of high-dose animals, with 70% of high-dose animals showing less than 10% loss near the plaque, compared to the other groups, where all but one animal showed greater than 10% loss.

[0269] The inventors also evaluated the number of dystrophic neurites associated with amyloid deposits by staining for the axonal marker SMI312 and oligomeric Aβ antibodies (Figure 39A). The inventors found no difference in the number of neuritic dystrophies between groups (Figures 39B to C).

[0270] Discussion. The strong genetic and experimental links between APOE genotype and AD have long made APOE an interesting subject when considering modulators or therapeutics for AD risk (Serrano-Pozo, Das, et al., 2021). However, the practical problem of distributing gene products throughout the brain is an obstacle to the use of gene therapy in diseases that affect large amounts of brain tissue, such as AD. This is especially true in large organisms such as non-human primates or humans. The current study has tested a method to overcome this obstacle: expression of secretory proteins by transduction of the ependyma and related structures, allowing the secretory proteins to diffuse throughout the cortical mantle. The inventors applied this approach to the expression of APOE2 in the APOE4 / APP / PS1 model of Alzheimer's pathology and showed that at achievable doses, expression of APOE2 can positively affect plaque deposition, neuroinflammation, and neurodegeneration within 8 weeks of treatment.

[0271] Importantly, this improvement was observed in an established disease setting (i.e., plaque deposition had already begun), neuropathologically mimicking the changes that occur in patients with established early-stage Alzheimer's disease. The results presented here suggest that the effects of APOE on AD are sustained throughout disease progression, rather than being a result of developmental or midlife changes associated with APOE4 (Evans et al., 2020), and that manipulation of APOE can alter the course of the disease even after AD is established.

[0272] The inventors tested three doses of virus to determine the minimum effective amount of AAV and APOE2 required to see an effect in this very aggressive model of amyloidosis ( Figure 2 ). Based on the work of others (Castellano et al., 2011; Hashimoto et al., 2012; Serrano-Pozo, Das, et al., 2021), the inventors hypothesized that the primary effect of APOE2 on the hallmark AD pathology of Aβ plaques is due to increased clearance of Aβ across the BBB and reduced aggregation. This study showed that APOE2 production has an effect on both dense core fibrous plaques and physiochemical measures of Aβ, consistent with this conclusion.

[0273] Neuroinflammation and AD have long been linked, in part due to the number of microglial genes that are risk factors for AD, and also due to the significant increase in microglial and astrocyte proliferation in the brain from both AD cases and mouse models (Leng & Edison, 2021). In recent years, the effect of APOE genotype on neuroinflammation has been studied (Hong et al., 2016). Microglia produce APOE under basal conditions, but when inflamed, the production in these cells increases dramatically. This observation led to the hypothesis that APOE4 produced by microglia has a gain of toxic functional effects in causing or maintaining a feedforward loop (Krasemann et al., 2017), which raises the possibility that the effect of APOE is cell autonomous. The inventors showed that exogenous APOE2 produced by ependymal cells inhibited microglial activation near mouse plaques ( Figure 3 ), which is inconsistent with this hypothesis and suggests that at least some of the effects of APOE on microglia are due to APOE produced in other cell types.

[0274] APOE4 is associated with greater synaptic loss near plaques in AD (Hudry et al., 2013; Koffie et al., 2012). In mice given the highest dose of AAV, the inventors saw a reduction in the amount of synaptic loss, suggesting that APOE2 can prevent this neurodegenerative phenotype. The inventors have previously shown that APOE and oligomeric oAβ co-localize at synapses and that APOE4 is more effective in delivering oAβ to synapses This synaptic protective effect of APOE2 may be due to a number of non-mutually exclusive mechanisms. APOE2 may help reduce the presence of bioactive oAβ at synapses, the effects of APOE2 on microglia may lead to reduced levels of synaptic pruning caused by reactive microglia, and enhanced oAβ clearance may reduce the amount of oAβ in the plaque aperture. The combination of these actions may result in a lack of toxic oAβ at synapses, reducing both microglial pruning and the synaptotoxic effects of oAβ.

[0275] The data provided herein emphasize the utility of ependymal restricted AAV for expressing secretory proteins, which, once secreted into neurites and CSF, can diffuse throughout the brain parenchyma. This method demonstrates the practical possibility of treating the entire brain using this gene therapy approach, which is of great significance for lysosomal storage diseases and diseases such as loss of progranulin function in frontotemporal dementia associated with progranulin mutations. The inventors envision that a variety of potential treatments will eventually be introduced into the brain, ranging from the production of single-chain antibodies to the expression of other bioactive molecules. The current research has in principle determined two things: APOE2 protein as a therapeutic agent, and an AAV platform for treatment, where blood-brain barrier issues or difficulties in peripheral expression (including clearance) currently hinder its use for CNS disorders.

[0276] In summary, the data presented here indicate that gene therapy-introduced APOE2 has protective functions that correspond to the well-recognized phenotype in human patients who have inherited the E2 or E4 alleles. In this model, even modest levels of APOE2 expression affect Aβ deposits, reduce neuroinflammation, and support synaptic systems. This suggests that APOE modulation is an important possibility for disease-modifying treatment of patients with Alzheimer's disease.

[0277] **************

[0278] According to the present disclosure, all methods disclosed and claimed herein can be carried out and implemented without excessive experimentation. Although the compositions and methods of the present disclosure have been described according to some preferred embodiments, it is obvious to those skilled in the art that, without departing from the concept, spirit and scope of the present disclosure, changes can be applied to the steps or step sequences of the methods described herein and the methods described herein. More specifically, it will be obvious that certain medicaments related in both chemistry and physiology can be used to replace the medicaments described herein, while achieving the same or similar results. All such similar substitutions and modifications obvious to those skilled in the art are considered to be within the spirit, scope and concept of the present disclosure defined by the appended claims.

[0279] References

[0280] The following references, to the extent they provide exemplary procedural or other details supplementary to those described herein, are specifically incorporated herein by reference.

[0281] US Patent Publication 20110059502

[0282] U.S. Patent 6,217,552

[0283] Gonzalez et al., BMC Neurosci 10.1186 / 1471-2202-12-4 (2011)

[0284] Lazorthes el al. Advances in Drug Delivery Systems and Applications inNeurosurgery, 18: 143-192 (1991)

[0285] Ommaya et al., Cancer Drug Delivery, 1: 169-179 (1984)

[0286] Ommaya, Lancet 2:983-84(1963)

Claims

1. A method for expressing a therapeutic transgene in ependymal tissue of a subject, the method comprising administering to the subject a modified adeno-associated virus (AAV) encoding a therapeutic transgene under the control of a promoter selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a promoter having at least about 80% sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:

3.

2. The method of claim 1, wherein the promoter is SEQ ID NO: 1 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

3. The method of claim 1, wherein the promoter is SEQ ID NO: 2 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

4. The method of claim 1, wherein the promoter is SEQ ID NO:3 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

5. The method of claim 1, wherein the modified AAV comprises a modified capsid protein.

6. The method of claim 5, wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide has a length of three to ten amino acids, such as a length of seven amino acids.

7. The method of claim 5, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

8. The method of claim 7, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein, and wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

9. The method of claim 8, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

10. The method of claim 9, wherein the linker sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

11. The method of claim 7, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

12. The method of claim 11, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

13. The method of claim 12, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

14. The method of claim 7, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

15. The method of claim 14, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

16. The method of claim 15, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

17. The method of any one of claims 1 to 16, wherein the therapeutic transgene is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system.

18. The method of any one of claims 1 to 17, wherein the therapeutic transgene is ApoE2 and the subject has Alzheimer's disease or is at an increased risk of developing Alzheimer's disease compared to a population average.

19. The method of any one of claims 1 to 18, wherein the administration is direct intracerebroventricular injection or intraparenchymal injection.

20. The method of any one of claims 1 to 19, wherein the modified AAV is administered more than once.

21. The method of claim 20, wherein the modified AAV is administered 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

22. The method of claim 20, wherein the modified AAV is administered monthly, every other month, every three months, every four months, every six months, or annually.

23. The method of any one of claims 1 to 22, further comprising providing the subject with a non-AAV therapy.

24. The method of any one of claims 1 to 23, wherein a plurality of viral particles are administered.

25. The method of claim 24, wherein the virus is present at about 1×10 6 About 1×10 18 The dose was administered at 1 vector genome (vg / kg).

26. The method of claim 24, wherein the virus is administered at a dose of about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to 1×10 13 or about 1×10 13 Up to 1×10 14 vg / kg patient.

27. The method of any one of claims 1 to 26, wherein the subject is a human.

28. The method of any one of claims 1 to 26, wherein the subject is a non-human mammal.

29. The method of any one of claims 27, wherein the human subject is 50 years of age or older.

30. The method of any one of claims 1 to 29, wherein the therapeutic transgene is linked to a polyadenylation signal.

31. A modified adeno-associated virus (AAV), wherein the modified adeno-associated virus (AAV) encodes a therapeutic transgene operably linked to a promoter selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or a promoter having at least about 80% sequence identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO:

3.

32. The modified AAV of claim 31, wherein the promoter is SEQ ID NO: 1 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

33. The modified AAV of claim 31, wherein the promoter is SEQ ID NO: 2 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

34. The modified AAV of claim 31, wherein the promoter is SEQ ID NO:3 or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

35. The modified AAV of claim 31, wherein the modified AAV comprises a modified capsid protein.

36. The modified AAV of claim 35, wherein the modified capsid protein comprises a targeting peptide, wherein the targeting peptide is three to ten amino acids in length, such as seven amino acids in length.

37. The modified AAV of claim 35, wherein the modified AAV capsid protein is a modified AAV1 capsid protein, a modified AAV2 capsid protein, or a modified AAV9 capsid protein.

38. The modified AAV of claim 37, wherein the modified AAV capsid protein is derived from an AAV1 capsid protein, wherein the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

39. The modified AAV of claim 38, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

40. The modified AAV of claim 39, wherein the linker sequence is SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

41. The modified AAV of claim 37, wherein the modified AAV capsid protein is derived from an AAV2 capsid protein, wherein the targeting peptide is inserted after residue 587 of the AAV2 capsid protein.

42. The modified AAV of claim 41, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

43. The modified AAV of claim 42, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

44. The modified AAV of claim 37, wherein the modified AAV capsid protein is derived from an AAV9 capsid protein, wherein the targeting peptide is inserted after residue 588 of the AAV9 capsid protein.

45. The modified AAV of claim 44, wherein the targeting peptide is flanked by linker sequences, wherein the linker sequences on each side of the targeting peptide are two or three amino acids long.

46. ​​The modified AAV of claim 45, wherein the linker sequence is AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

47. The modified AAV of any one of claims 31 to 46, wherein the therapeutic transgene is a siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or a CRISPR system.

48. The modified AAV of any one of claims 31 to 47, wherein the therapeutic transgene is linked to a polyadenylation signal.

49. The modified AAV of any one of claims 31 to 48, wherein the therapeutic transgene is transcriptionally linked to a detectable reporter, such as a sequence encoding a fluorescent protein, a peptide tag, or a luciferase.

50. A pharmaceutical composition comprising the modified AAV of any one of claims 31 to 49 and a pharmaceutically acceptable carrier.

51. An isolated and purified nucleic acid comprising a sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID ID: 3, or a sequence having at least about 80% sequence identity thereto.

52. The nucleic acid of claim 51, wherein the sequence is SEQ ID NO: 1 or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

53. The nucleic acid of claim 51, wherein the sequence is SEQ ID NO: 2 or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

54. The nucleic acid of claim 51, wherein the sequence is SEQ ID NO:3 or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

55. The nucleic acid of claim 51, wherein the sequence is operably linked to a heterologous coding region.

56. The nucleic acid of claims 51 to 55, further comprising one or more of: (a) a general-purpose cloning site, (b) a transcription termination signal, (c) a polyadenylation sequence, and / or (d) an origin of replication.

57. The nucleic acid of any one of claims 51 to 56, further comprising one or more of the following: (a) a sequence encoding a detectable marker, (b) a sequence encoding an affinity tag, and / or (c) one or two adeno-associated viral inverted terminal repeats.

58. The nucleic acid of any one of claims 1 to 57, wherein the nucleic acid is contained in a replicable vector.

59. The nucleic acid of any one of claims 1 to 58, wherein the therapeutic transgene is transcriptionally linked to the reporter via a sequence encoding a 2A "self-cleaving" peptide.

60. A method of reducing or attenuating microglial inflammation comprising delivering ApoE2 to microglia in a subject in need thereof.

61. The method of claim 60, wherein delivering ApoE2 to microglia comprises administering to the subject an ApoE2 protein or a modified AAV according to claims 31 to 49 or a pharmaceutical formulation according to claim 50, wherein the therapeutic transgene is ApoE2.

62. The method of claim 60 or 61, wherein the microglial inflammation is caused by or associated with a neurodegenerative disease, such as Huntington's disease, Parkinson's disease, motor neuron disease, spinocerebellar ataxia, spinal muscular atrophy, progressive supranuclear palsy, amyotrophic lateral sclerosis, multiple sclerosis, Batten's disease, and Creutzfeldt-Jakob disease.

63. The method of any one of claims 60 to 62, wherein administration of ApoE2 or modified AAV is by direct intracerebroventricular injection or intraparenchymal injection.

64. The method of any one of claims 60 to 63, wherein the ApoE2 protein or modified AAV is administered more than once.

65. The method of claim 64, wherein the ApoE2 protein or modified AAV is administered 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

66. The method of claim 64, wherein the ApoE2 protein or modified AAV is administered monthly, every other month, every three months, every four months, every six months, or annually.

67. The method of any one of claims 60 to 66, further comprising providing the subject with a non-AAV ApoE2 therapy.

68. The method of any one of claims 60 to 67, wherein a plurality of viral particles are administered.

69. The method of claim 68, wherein the AAV is present at about 1×10 6 About 1×10 18 The dose was administered at 1 vector genome (vg / kg).

70. The method of claim 68, wherein the virus is administered at a dose of about 1×10 7 Up to 1×10 17 , about 1×10 8 Up to 1×10 16 , about 1×10 9 Up to 1×10 15 , about 1×10 10 Up to 1×10 14 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 13 , about 1×10 10 Up to 1×10 11 , about 1×10 11 Up to 1×10 12 , about 1×10 12 Up to 1×10 13 or about 1×10 13 Up to 1×10 14 vg / kg patient.

71. The method of any one of claims 60 to 70, wherein the subject is a human.

72. The method of any one of claims 60 to 70, wherein the subject is a non-human mammal.

73. The method of claim 72, wherein the human subject is 50 years of age or older.

74. The method of any one of claims 60 to 73, wherein the therapeutic transgene is linked to a polyadenylation signal.

75. The method of any one of claims 60, 61, and 63 to 73, wherein the microglial inflammation is caused by or associated with Alzheimer's disease.

Images