Human ependymal specific promoters and methods of use thereof

Abstract

Provided herein are compositions and methods for delivering molecular therapeutics to the ependyma of a subject.The method comprises administering an adeno-associated virus (AAV) to the subject.The AAV encodes a therapeutic transgene under the control of an ependyma-specific promoter. TIFF2025514794000003.tif69162

Description

[Technical field]

[0001] Priority claim This application claims the benefit of priority to U.S. Provisional Patent Applications 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 incorporated herein by reference.

[0002] 1. Field The present disclosure relates generally to the fields of medicine and virology. More specifically, the present disclosure relates to compositions and methods for the delivery of molecular therapeutics to a patient, particularly to the brain or central nervous system. [Background technology]

[0003] 2. Description of Related Art Adeno-associated viruses (AAV) are promising therapeutic candidates 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 into the host genome, but persists as an episome in the cell nucleus. Expression of the AAV cargo is controlled at the level of the packaging capsid and spatially by the transgene promoter. Also, the use of AAV for the treatment of diseases may necessitate intervention in diseased tissues, which may pose problems with target tissues containing gene expression profiles different from healthy tissues. Finding the right promoter sequence to drive the expression of a therapeutic transgene is an important goal. Summary of the Invention

[0004] overview Provided herein is a method of 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 the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or a promoter having at least about 80% sequence identity thereto. The promoter may comprise SEQ ID NO: 1, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or may 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.

[0005] The modified AAV may comprise a modified capsid protein, for example, the modified capsid protein comprises a targeting peptide, where the targeting peptide is 3-10 amino acids long, for example, 7 amino acids long. 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, where the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

[0006] The targeting peptide may be flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length, for example, these sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0007] The modified AAV capsid protein may be derived from the AAV2 capsid protein, where the targeting peptide is inserted into the AAV2 capsid protein after residue 587. The targeting peptide may be flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length, e.g., 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.

[0008] The modified AAV capsid protein may be derived from the AAV9 capsid protein, where the targeting peptide is inserted into the AAV9 capsid protein after residue 588. The targeting peptide is flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids in length, e.g., the linker sequences are AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0009] The therapeutic transgene may be an siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system. The therapeutic transgene may be ApoE2, and the subject is afflicted with Alzheimer's disease or at high risk of developing Alzheimer's disease compared to the population average. The administration may be direct intracerebroventricular or intraparenchymal injection. The modified AAV may be administered more than once, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. The modified AAV may be administered monthly, bimonthly, every 3 months, every 4 months, every 6 months, or annually. The method may further include providing a non-AAV therapy to the subject.

[0010] The method may include administration of a plurality of viral particles, for example, the virus is about 1×10 per kilogram. 6 ~Approx. 1×10 18 The virus is administered at a dose of approximately 1 × 10 vector genomes (vg / kg) per kg of subject. 7 ~1×10 17 , about 1×10 8 ~1×10 16 , about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , about 1×10 12 ~1×10 13 , or about 1×10 13 ~1×10 14 The therapeutic transgene may be administered in a dose of 1 vg. The subject may be a human or a non-human mammal. The human subject may be 50 years of age or older. The therapeutic transgene may be linked to a polyadenylation signal.

[0011] Also provided is a modified adeno-associated virus (AAV) encoding a therapeutic transgene operably linked to a promoter selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or a promoter having at least about 80% sequence identity thereto. The promoter may comprise SEQ ID NO:1, or a promoter having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or may 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.

[0012] The modified AAV may comprise a modified capsid protein, e.g., the modified capsid protein comprises a targeting peptide, where the targeting peptide is 3-10 amino acids in length, e.g., 7 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.

[0013] The modified AAV capsid protein may be derived from the AAV1 capsid protein, where the targeting peptide is inserted after residue 590 of the AAV1 capsid protein, for example, the targeting peptide is flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0014] The modified AAV capsid protein may be derived from the AAV2 capsid protein, where the targeting peptide is inserted after residue 587 of the AAV2 capsid protein, e.g., the targeting peptide is flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. The linker sequences may be AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide.

[0015] The modified AAV capsid protein may be derived from the AAV9 capsid protein, where the targeting peptide is inserted after residue 588 of the AAV9 capsid protein, for example, the targeting peptide is flanked by linker sequences, where the linker sequences on either side of the targeting peptide are 2 or 3 amino acids long. The linker sequences may be AAA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide.

[0016] The therapeutic transgene may be siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system.The therapeutic transgene may be linked to a polyadenylation signal.The therapeutic transgene may be transcriptionally linked to a detectable reporter, for example, a sequence that codes for a fluorescent protein, a peptide tag, or luciferase.

[0017] In another aspect, a pharmaceutical composition is provided comprising a modified AAV described herein and a pharma- ceutically acceptable carrier.

[0018] In yet another embodiment, an isolated and purified nucleic acid is provided comprising a sequence selected from the group consisting of 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 SEQ ID NO:1, or a sequence having at least about 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or may 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.

[0019] The sequence may be operably linked to a heterologous coding region. The nucleic acid may further comprise one or more of: (a) a multipurpose cloning site, (b) a transcription termination signal, (c) a polyadenylation sequence, and / or (d) an origin of replication. The nucleic acid may further comprise 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 repeat sequences. The nucleic acid may be contained in a replicable vector. The therapeutic transgene may be transcriptionally linked to the reporter by a sequence encoding a 2A "self-cleaving" peptide.

[0020] In yet another embodiment, a method of reducing or attenuating microglial inflammation is provided, comprising delivering ApoE2 to microglia in a subject in need thereof. Delivering ApoE2 to microglia comprises administering to the subject a modified AAV as defined herein or a pharmaceutical formulation comprising the same, wherein the therapeutic transgene is ApoE2. Microglial inflammation may be caused or 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 or associated with Alzheimer's disease.

[0021] Administration may be by direct intraventricular or intraparenchymal injection of ApoE2 or modified AAV and may involve two or more administrations, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, and / or monthly, bimonthly, quarterly, quarterly, six monthly, or yearly. The method may further include providing a non-AAV ApoE2 therapy to the subject.

[0022] In the case of AAV therapy, approximately 1 × 10 per kg of patient 7 ~1×10 17 , about 1×108 ~1×10 16 , about 1×10 9 ~1×10 15 , about 1×10 10 ~1×10 14 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 13 , about 1×10 10 ~1×10 11 , about 1×10 11 ~1×10 12 , about 1×10 12 ~1×10 13 , or approximately 1 × 10 13 ~1×10 14 Approximately 1 x 10 per kilogram, including the dose in vg. 6 ~Approx. 1×10 18 Multiple viral particles may be administered, such as a dose of 1 vector genome (vg / kg). The subject may be a human, a non-human mammal, or a human subject aged 50 or older. The therapeutic transgene may be linked to a polyadenylation signal.

[0023] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0024] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0025] The following drawings form part of the present specification and are provided to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0026] [Figure 1] Identifying ependymal-specific promoters. Due to the thin nature of the ependymal lining of the ventricles, we employed a subtractive approach to identify tissue-specific genes. Samples were taken from the white and grey matter adjacent to the ventricles, and from the region of the ventricular rim that contains white and grey matter along with the ependyma. Genes unique to white matter + ependyma and grey matter + ependyma samples were classified as enriched. To ensure active utilization of promoters in multiple relevant disease states, samples were sourced from healthy controls as well as patients with Alzheimer's disease (AD), Huntington's disease (HD), frontotemporal dementia, dementia with Lewy bodies, semantic dementia and dementia. [Diagram 2] Validate expression in the ependyma. Top gene hits were validated against published in situ hybridization data from the Allen Brain Institute. - is specific / not enriched in the ependyma; + is specific / enriched in the ependyma; NA is data not available. [Diagram 3] Validating expression in the ependyma. Cartoon representation of ependymal-specific promoter transgenes. Approximately 1100–2500 bp of potential promoter sequence of ependymal-enriched genes were cloned upstream of an eGFP reporter. Individual transgenes were identified using 3 bp barcodes in the 3' UTR of the RNA transcripts. [Figure 4] Use of the human ependymal (hEpendym) promoter library in mouse ependyma. Contribution of each transgene-associated barcode in mouse ependymal RNA and AAV4 viral library input following low-dose (5E10 vg), medium-dose (1E11 vg) and high-dose (5E11 vg) vector injection. [Diagram 5]Use of a human ependymal promoter library in mouse ependyma. Quantification of read enrichment in RNA output relative to viral library input. Significant enrichment was shown with the positive control ubiquitous iCAG promoter and the hVWA3a promoter. [Figure 6] Percentage contribution of the human ependymal promoter - eGFP in rhesus ependyma-containing samples. The same library used in Figure 4 was prepared as AAV2 and injected a total of 2E13 vg into the lateral ventricles of two adult rhesus macaques. Three weeks post-injection, the region of the ventricular rim containing the spinal cord was microdissected. RNA was isolated, converted to cDNA, and PCR products containing three-letter barcodes were subjected to amplicon sequencing. Colored bars indicate the relative contribution of each transgene. [Figure 7] Human ependymal library round 2 - Introducing a promoter intron to increase expression. The original transgene was modified to express human apolipoprotein 2 (ApoE2) cDNA and included a short (133 bp) and a long (951 bp) intron within the promoter region to increase expression through intron-mediated enhancement. Short flanking sequences known to promote efficient splicing were also included (blue and green bars). [Figure 8] The mRNA from the human ependymal promoter library is correctly spliced ​​and encodes the ApoE protein. To test the splicing efficiency of the intron-containing variants, the plasmids were transfected into HEK293 cells. Correct splicing was verified by amplifying before and after the intron-containing region in cDNA (C) versus plasmid DNA (D). [Figure 9] mRNA from a human ependymal promoter library is correctly spliced ​​and encodes ApoE protein. Western blot measuring ApoE protein output from intron-less (N) and intron-containing (short S, long L) variants in HEK293 cell lysates and media. [Figure 10]Intron-containing promoters used in the Rhesus monkey ependyma. A library of six different promoters containing intron-free, short, or long introns was prepared in a single AAV2 and injected into the lateral ventricles of two adult rhesus monkeys at a total dose of 2.8e13 vg. Four weeks later, the ventricular rim was microdissected. As before, amplicon sequencing of products containing unique three-letter barcodes in the 3' UTR was used to assess relative promoter usage in vivo. All hVWA3a variants showed relative enrichment in vivo over the input library. [Figure 11] The human ependymal promoter drives expression in mice. Adult APOE- / - (null) mice were injected into their right lateral ventricle with serotype AAV4 delivering APOE2 under the hVWA3a promoter. Ependymal tissue was microdissected, proteins extracted, and APOE2 quantified by automated Western blot technique (WES) compared to uninjected brain tissue. [Figure 12] Human ependymal promoter drives higher expression than ubiquitous CAG promoter in mice. APOE- / - (null) mice were injected with equal doses of serotype AAV4 delivering APOE2 under either ubiquitous CAG promoter or hVWA3a promoter into their right lateral ventricle. Proteins were extracted from microdissected ependymal tissue from all animals and subjected to automated Western blot technique (WES). From the band intensity, APOE2 driven by hVWA3a expressed higher amount of APOE2 protein than CAG promoter. [Figure 13] Peptide-modified AAV1 capsid with a human ependymal-specific promoter: ERDRpAAV1.hVWA3a.eGFP. Positive eGFP fluorescent signal is restricted to the ependymal cells lining the ventricles. [Figure 14] hVWA3a promoter segment (SEQ ID NO: 1). [Figure 15] hVWA3a promoter segment (SEQ ID NO: 2) with a short intron (underlined) (133 bp). [Figure 16] hVWA3a promoter segment (SEQ ID NO: 3) with a long intron (underlined) (951 bp). [Figure 17] Schematic diagram of an AAV transgene using upstream regulatory sequences of 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) was inserted downstream of the transcription start site to enhance transcription by intron-mediated enhancement, and a strong Kozak sequence was included to initiate APOE2 translation. The entire transgene is flanked by AAV2 inverted terminal repeats (ITRs). [Figure 18-1] hVWA3a promoter short intron hApoE2 expression construct for AAV (SEQ ID NO: 4). [Figure 18-2] hVWA3a promoter short intron hApoE2 expression construct for AAV (SEQ ID NO: 4). [Figure 19] 7E10 vg of pmAAV1.ERDR.hVWA3a.APOE2 significantly reduces ThioS positive signal in the cortex compared to vehicle treated controls. pmAAV1.ERDR.hVWA3a.APOE2 was delivered to a mouse model of Alzheimer's disease homozygous for human APOE4. There is a significant reduction in ThioS positive cell % in mice treated with high dose of 7E10 vg compared to vehicle treated control mice (p < 0.05). Low dose: 7E9 vg; Medium dose: 2E10 vg; High dose: 7E10 vg. ThioS stains B-pleated sheets found within amyloid plaques in mice (used as a marker for dense core plaques rather than diffuse plaques). The difference in ThioS staining but not oligomeric antibody staining (IBL) indicates that AAV is preventing the formation of dense core plaques rather than general plaque formation. [Figure 20]7E10 vg pmAAV1.ERDR.hVWA3a.APOE2 significantly reduces Aβ positive signal in the cortex compared to vehicle treated controls. pmAAV1.ERDR.hVWA3a.APOE2 was delivered to a mouse model of Alzheimer's disease homozygous for human APOE4. There is a significant reduction in Aβ stained cells in mice treated with 7E10 vg compared to vehicle treated mice (p < 0.05). Low dose: 7E9 vg; Mid dose: 2E10 vg; High dose: 7E10 vg. [Figure 21] Viral genome copies assayed by QPCR on 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 of mouse brains treated with all three doses of vector are positive when assayed on the nontranscribed region of the human VWA3a promoter sequence. Control (vehicle) treated AD mice and control treated WT mice showed only background levels of hVWA3a below the range detectable by the standard curve. Low dose: 7E9 vg; Mid dose: 2E10 vg; High dose: 7E10 vg. [Figure 22] Viral genome copies assayed by QPCR on DNA against the hVWA3a promoter sequence. Viral genome copies of pmAAV1.ERDR.hVWA3a.APOE2 are detectable in ependymal, cortical, and hippocampal tissues of the brain of non-human primates treated with three different doses of pmAAV1.ERDR.hVWA3a.APOE2. Brain tissue DNA lysates were assayed for total genome copies assayed at the non-transcribed region of the human VWA3a promoter sequence. Naïve samples are from NHPs that did not receive pmAAV1.ERDR.hVWA3a.APOE2. Top to bottom in the legend is the same as left to right in the graph. [Diagram 23]Expression of APOE2 in a triple transgenic mouse model of Alzheimer's disease. APOE4XAPP / PS1 is a triple transgenic mouse expressing chimeric mouse / human amyloid precursor protein, mutant human presenilin 1, and human APOE4, resulting in a model with many aspects of the human pathology. Images show cortical amyloid-β (Aβ; red) with neuronal staining (DAPI; blue) at 3, 4, 5, and 6 months of age when left untreated. In the study paradigm, mice were injected at 4 months of age, when plaque accumulation began, and necropsied at 6 months of age. Mice were injected intracerebroventricularly with increasing doses of pmAAV1.ERDR.hVWA3a.ApoE2 at 7E9, 2E10, and 7E10 vg. Readout of genome copy expression is shown in Figure 21. [Figure 24] APOE2 expression is beneficial for plaque deposition in APOE4XAPP / PS1 mice. ThioS staining on the left shows plaques 2 months after delivery of pmAAV1.ERDR.hVWA3a.ApoE2. Quantitative graphs show significant reduction in ThioS and soluble AB42 levels in mice dosed with 7E10 vg. [Diagram 25] APOE2 expression reduces plaque density and plaque size in AD mouse models. pmAAV1.ERDR.hVWA3a.ApoE2 in 7E10 vg significantly reduced plaque parameters compared to age-matched untreated AD mice. [Figure 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. [Figure 27] Virally expressed APOE2 prevents microgliosis near plaques. Using the images in Figure 26, two blinded scientists trained on the study scored microglia (Iba1; blue) near plaques (Aβ; red) in the brains of mice dosed with pmAAV1.ERDR.hVWA3a.ApoE2. [Figure 28] Staining of AD mouse brains at 3, 4, 5 and 6 months of age. The top panel shows cortical images stained with GFAP (green) and Aβ (red). The bottom panel (planel) shows cortical images stained with Iba1 (blue) and Aβ (red). [Figure 29] Preliminary evaluation of AD mice shows high variability in microgliosis. AD mice treated with pmAAV1.ERDR.hVWA3a.ApoE2 show even higher variability in microgliosis. [Diagram 30] Graphical representation of GFAP scoring near plaques. Delivery of APOE2 to AD mice does not significantly affect astrocytic reactivity near plaques as assessed by blinded pathological scoring. [Diagram 31] Virally expressed APOE2 prevents synapse loss near plaques. Brain images of AD mice injected with 7E10 vg pmAAV1.ERDR.hVWA3a.ApoE2 or vehicle control. PSD95 (postsynaptic density-95) staining in synaptic terminals is more evident in treated animals. [Diagram 32] Quantification of synaptic integrity in APOE2-dosed AD mice. Synapses proximal (near; red) and distal (far; blue) to the plaque were quantified from histological images. Only mice treated with 7E10 vg of therapeutic agent showed similar synaptic density near and far from the plaque. All other AD treatment groups had significantly lower synaptic density near the plaque compared to far from it (left graph). The right graph compares "near" and "far" synaptic density between groups, revealing that the high dose (7E10 vg) group had significantly higher synaptic density "near" the plaque compared to all other groups. [Diagram 33]Figures 33A-E. Ependymal cell expression of APOE2 driven by a novel AAV capsid and promoter. (Figure 33A) In situ hybridization showing human APOE expression in ependymal cells of the ventricles of APOE KO mice. (Figure 33B) Western blot of APOE showing that AAV-derived APOE2 produced from the ependyma in the cerebral cortex of APOE KO mice was approximately 10% of endogenous levels (Figure 33C). (Figure 33D) Viral genome copies in tissues extracted from each mouse [(F (4, 35) = 5.546 p = 0.0014) post hoc Tukey's multiple comparison test]. (Figure 33E) qRTPCR of human APOE normalized to vehicle-treated animals (F (4, 26) = 2.890 p = 0.0419 post hoc Dunnett's test vs vehicle). (E) Line graph showing significant correlation between APOE mRNA and viral genome copies (p=0.0445). Each mouse is an individual point, so N is shown. *p<0.05, **p<0.01, ***p<0.001. [Diagram 34] Figures 34A-E. POE2 reduces plaque deposition, number and size in a dose-dependent manner. (Figure 34A) IHC of ThioS in the cortex of dosed APP / PS1 / APOE4 animals. (Figure 34B) Cortical coverage by ThioS staining is significantly lower in high dose animals (F(3,27)=4.310 p=0.0329). (Figure 34C) Cortical coverage by ThioS correlates significantly with viral genome copy number in each mouse brain sample (p=0.0112). (Figure 34D) Plaque number (F(3,27)=3.597 p=0.0263) and (Figure 34E) plaque size (F(3,27)=4.113 p=0.0159) both show significant effects in the high dose group. Each mouse is an individual point, n is represented as open circles for females and closed circles for males. Post-hoc tests are shown as Dunnett's multiple comparison test compared to vehicle. p * p<0.05, ** p<0.01. [Diagram 35]Figures 35A-D. APOE2 reduces microgliosis near plaques. (Figure 35A) IHC for IBA1 and o Aβ in the cortex of dosed APP / PS1 / APOE4 animals. (Figure 35B) Mean microglial response scores (F(3,26)=4.529 p=0.0110) show reduced microglial activation in the high and mid dose groups (Figure 35C) significantly correlate with viral genome copy number in the mice (p=0.0081). (Figure 35D) This reduction is due to a decrease in the number of plaques scored as 4 and an increase in the number of plaques scored as 1. Each mouse is an individual point, with open circles representing females and closed circles representing males, n. Post-hoc tests are shown as Dunnett's multiple comparison test compared to vehicle. p *p<0.05. [Diagram 36] Figure 36A-D. APOE2 reduces nearby synapse loss. (Figure 36A) IHC of PSD95 and oAβ in the cortex of medicated APP / PS1 / APOE4 animals. (Figure 36B) Synaptic density was unchanged far from the plaques, (Figure 36C) but significantly increased near the plaques (F(3,27) = 5.153 p = 0.0060). (Figure 36D) This translates into a significant decrease in % synapse loss in high dose animals compared to vehicle (F(3,27) = 3.693 p = 0.0239). Each mouse is an individual point, n indicated as open circles for females and closed circles for males. Post-hoc tests are shown as Dunnett's multiple comparisons test compared to vehicle. p * p < 0.05. [Figure 37]Figure 37A-D. Effect of APOE2 on oligomeric Aβ. (Figure 37A) Cortical coverage by Aβ staining is significantly lower in high dose animals (F(3,27) = 6.336, p=0.0022). (Figure 37B) Cortical coverage by ThioS significantly correlates with viral genome copy number in that mouse (p=0.0173). (Figure 37C) ELISA shows that both SDS-soluble (F(3,28) = 3.497 p=0.0285) and (Figure 37D) formic acid-soluble (F(3,30) = 3.741 p=0.0214) Aβ show significant effects in the high dose group. Each mouse is an individual point, n as open circles represent females and closed circles represent males. Post-hoc tests are shown as Dunnett's multiple comparison test compared to vehicle. p *p<0.05, **p<0.01. [Figure 38] Figures 38A-E. APOE2 does not affect astrocyte reactivity near plaques. (Figure 38A) Representative images of the 4-point scale used to assess microglial and astrocytic reactivity to plaques. (Figure 38B) IHC for GFAP and oAβ in the cortex of dosed APP / PS1 / APOE4 animals. (Figure 38C) Mean astrocyte response scores (F(3,26)=1.634 p=0.2057) did not change between groups and (Figure 38D) did not significantly correlate with viral genome number (p=0.1904). (Figure 38E) There was no difference between groups in the number of plaques scored in each category. Each mouse is an individual point, n indicated as open circles for females and closed circles for males. Post-hoc tests are shown as Dunnett's multiple comparison test compared to vehicle. p *p<0.05. [Figure 39] Figures 39A-C. APOE2 does not affect neuritic dystrophy. (Figure 39A) IHC for Smi-312 and oAβ in the cortex of medicated APP / PS1 / APOE4 animals. (Figure 39B) Analysis shows no difference in the number of dystrophy counted per plaque (F(3,25) = 0.4710; p = 0.7052), even when taking into account plaque size (F(3,25) = 1.127 p = 0.3569) (Figure 39C). [Diagram 40] The human ependymal promoter drives ependymal-localized APOE transcription in NHPs after ICV delivery. A total of 1E13 vg of pmAAV1.ERDR.hVWA3a.ApoE2 vector was unilaterally injected into the lateral ventricle of adult African green monkeys. Tissues were harvested for sectioning 60 days after injection, and transgene expression was monitored by RNA fluorescent in situ hybridization (RNA-FISH). Probes designed to target human APOE show strong overlap with endogenous African green APOE due to high sequence homology. To identify the origin of the transcript, we relied on location. Because endogenous APOE transcription is exclusively in astrocytes and microglia, we can attribute the ependymal localization signal, defined by overlap with the ependymal-specific gene FoxJ1 (outlined by white dashed line), to transgenic-derived APOE2. Hoechst H33258 identifies tissue DNA. [Diagram 41] The human ependymal promoter increases CSF APOE in NHPs after ICV delivery. A total of 1E13 vg of pmAAV1.ERDR.hVWA3a.ApoE2 vector was unilaterally injected into the lateral ventricle of adult African green monkeys. CSF was collected at baseline, 30, 45, and 60 days post-injection, and APOE protein was measured by automated Western blot. All values ​​were normalized to baseline. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Detailed Description Here, we sought to identify promoter sequences that can drive ependymal specific expression in mouse and human brain and can be used in gene therapy to drive expression of secreted proteins to treat neurological diseases. We first sought to identify genes that are not affected by neurological disease status or age, and then to identify surrogate relevant promoters. Such promoters can be used to drive transgene expression in the ependyma, a layer of epithelial cells lining the ventricles of the brain. After infection of these cells with AAV, secreted proteins can enter the ventricles and be distributed throughout the brain via the cerebrospinal fluid. Moreover, stronger transgene expression can be achieved by eliminating promoters that may be adversely affected by altered gene expression patterns in diseased tissues.

[0028] To achieve this, we obtained ependyma and adjacent ependyma-free samples from normal and diseased brains (e.g., Alzheimer's disease, Huntington's disease, frontotemporal dementia). Using RNA sequencing, we identified genes whose expression was enriched in samples containing the ependyma and whose expression was maintained regardless of disease state. Further evidence of its specificity was validated using publicly available data sets, including Allen Brain Institute in situ hybridization libraries. Because promoters are loosely defined structures, genomic sequences (approximately 1100-2500 bp) upstream of the transcription start sites of the top gene candidates (11 promoters) were isolated and placed upstream of a GFP reporter and a unique three-letter RNA barcode within an AAV-compatible transgene (flanked by ITRs). Plasmids containing the different promoters were pooled, prepared as AAV4 or AAV2, and injected directly into the ventricles of mice or rhesus monkeys, respectively. Tissues containing the ependyma were microdissected, and amplicon sequencing was performed in the region surrounding the three-letter barcodes. The output was compared to the library input to determine enrichment. Inclusion of an upstream intron was shown to increase tissue expression. The top hits (six promoters) identified in the initial screen were further modified to include short (133 bp) or long (951 bp) introns. Individual versions were again identified by unique three-letter barcodes in the 3' UTR. A transgene containing a variant of human von Willebrand factor A domain-containing 3a (hVWA3a) was most highly enriched in the final screen and was selected for further study.

[0029] These and other aspects of the disclosure are discussed in further detail below.

[0030] I. Adeno-associated virus (AAV) vectors Adeno-associated viruses (AAVs) are small, non-pathogenic viruses in the Parvoviridae family. To date, numerous serologically distinct AAVs have been identified, with more than a dozen identified from humans or primates. AAVs differ from other members of the family in that they depend on a helper virus for replication.

[0031] The AAV genome exists in an extrachromosomal state without integration into the host cell genome, has a broad host range, can transduce both dividing and non-dividing cells in vitro and in vivo, and can maintain high levels of expression of transduced genes. AAV viral particles are thermostable, resistant to solvents, detergents, pH, and temperature changes, and can be column purified and / or concentrated by CsCl gradients or other means. The AAV genome contains positive or negative single-stranded deoxyribonucleic acid (ssDNA). The approximately 4.7 kb genome of AAV consists of a segment of single-stranded DNA with positive or negative polarity. Both ends of the genome are short inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as origins of viral DNA replication.

[0032] AAV "genome" refers to the recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. AAV particles often contain an AAV genome packaged with AAV capsid proteins. When a recombinant plasmid is used to construct or produce a recombinant vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid is referred to as the "plasmid backbone," which is important for plasmid cloning and amplification, a process necessary for plasmid growth and production, but is not itself packaged or encapsulated into the viral particle. Thus, AAV vector "genome" refers to the nucleic acid that is packaged or encapsulated by the AAV capsid proteins.

[0033] AAV virions (particles) are non-enveloped icosahedral particles with a diameter of approximately 25 nm that contain the AAV capsid. AAV particles contain icosahedral symmetry composed of three related capsid proteins, VP1, VP2, and VP3, which interact with each other to form the capsid. The genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as the left-hand and right-hand ORFs. The right-hand ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1:1:10, respectively, although the ratios can vary, and all originate from the right-hand ORF. The VP1, VP2, and VP3 capsid proteins differ from each other by alternative splicing and the use of unusual start codons. Deletion analysis has shown that removal or remodeling of VP1, which is translated from messages that undergo alternative splicing, results in reduced yields of infectious particles. Mutations in the VP3 coding region result in failure to produce single-stranded progeny DNA or infectious particles. In certain embodiments, the genome of an AAV particle encodes one, two or all three of the VP1, VP2, and VP3 polypeptides.

[0034] The left ORF often encodes non-structural Rep proteins Rep40, Rep52, Rep68, and Rep78, which are involved in regulating replication and transcription as well as producing single-stranded progeny genomes. Two of the Rep proteins are associated with preferential inclusion of AAV genomes in a region of the q arm of human chromosome 19. Rep68 / 78 has been shown to have NTP binding activity as well as DNA and RNA helicase activity. Some Rep proteins have nuclear localization signals, along with several potential phosphorylation sites. In certain embodiments, the genome of an AAV (e.g., rAAV) encodes some or all of the Rep proteins. In certain embodiments, the genome of an AAV (e.g., rAAV) does not encode a Rep protein. In certain embodiments, one or more of the Rep proteins are not included in the AAV particle that contains the nucleic acid encoding a polypeptide, since they can be delivered in trans.

[0035] The ends of the AAV genome contain short inverted terminal repeats (ITRs) that have the potential to fold into T-shaped hairpin structures that serve as origins of viral DNA replication. Thus, the genome of AAV contains one or more (e.g., a pair) ITR sequences that flank the single-stranded viral DNA genome. The ITR sequences are often approximately 145 bases long each. Within the ITR region, two elements that are thought to be central to ITR function have been described: the GAGC repeat motif and the terminal resolution site (trs). The repeat motif has been shown to bind Rep when the ITR is in either a linear or hairpin conformation. This binding is thought to position Rep68 / 78 for cleavage at the trs in a site- and strand-specific manner. In addition to their role in replication, these two elements are also thought to be central to viral inclusion. The chromosome 19 integration locus contains a Rep binding site flanked by trs. These elements have been shown to be functional and necessary for locus-specific inclusion.

[0036] The term "recombinant" as a modifier of a vector, such as a recombinant viral vector, e.g., a recombinant lentivirus or recombinant parvovirus (e.g., AAV) vector, and as a modifier of a sequence, such as a recombinant nucleic acid sequence or a recombinant polypeptide, means that the composition has been manipulated (i.e., engineered) in a manner that does not generally occur in nature. Particular examples of recombinant vectors, such as AAV, retrovirus, or lentivirus vectors, are those in which a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted into the viral genome. One example of a recombinant nucleic acid sequence is one in which a nucleic acid (e.g., a gene) encodes an inhibitory RNA cloned into the vector with or without the 5', 3', and / or intron regions that are normally associated with the gene in the viral genome. The term "recombinant" is not always used herein with respect to vectors, such as viral vectors, and sequences, such as polynucleotides, but "recombinant" forms, including nucleic acid sequences, polynucleotides, transgenes, and the like, are clearly encompassed, even with such omissions.

[0037] A recombinant viral "vector" is derived from a wild-type viral genome by using molecular methods to remove a portion of the wild-type genome from the virus and replace it with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, in the case of AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the recombinant AAV vector. A "recombinant" viral vector (e.g., rAAV) is distinguished from a viral (e.g., AAV) genome in that a portion of the viral genome is replaced with a non-native sequence, such as a nucleic acid encoding a transactivator or a nucleic acid encoding an inhibitory RNA or a nucleic acid encoding a therapeutic protein, compared to the viral genome nucleic acid. Thus, the incorporation of such a non-native nucleic acid sequence defines the viral vector as a "recombinant" vector, and in the case of AAV, it can be referred to as a "rAAV vector."

[0038] In certain embodiments, the AAV (e.g., rAAV) comprises two ITRs. In certain embodiments, the AAV (e.g., rAAV) comprises a pair of ITRs. In certain embodiments, the AAV (e.g., rAAV) comprises a pair of ITRs adjacent to a nucleic acid sequence encoding a polypeptide having at least a function or activity (i.e., at the 5' and 3' ends of the nucleic acid sequence, respectively).

[0039] AAV vectors (e.g., rAAV vectors) can be packaged and are referred to herein as "AAV particles" for infecting (transducing) cells ex vivo, in vitro or in vivo. When a recombinant AAV vector is enclosed or packaged in an AAV particle, the particle can also be referred to as a "rAAV particle". In certain embodiments, the AAV particle is a rAAV particle. The rAAV particle often comprises a rAAV vector or a portion thereof. The rAAV particle can be one or more rAAV particles (e.g., multiple AAV particles). The rAAV particle typically comprises a protein (e.g., a capsid protein) that encloses or packages the rAAV vector genome. It should be noted that references to rAAV vectors can also be used to refer to rAAV particles.

[0040] Any suitable AAV particle (e.g., rAAV particle) can be used in the method or use herein. The rAAV particle and / or genome contained therein can be derived from any suitable serotype or strain of AAV. The rAAV particle and / or genome contained therein can be derived from two or more serotypes or strains of AAV. Thus, the rAAV can comprise the protein and / or nucleic acid or parts thereof of any serotype or strain of AAV, and the AAV particle is suitable for infection and / or transduction of mammalian cells. 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.

[0041] In certain embodiments, the plurality of rAAV particles comprises particles of the same strain or serotype (or subgroup or variant), or particles derived from the same strain or serotype (or subgroup or variant). In certain embodiments, the plurality of rAAV particles comprises a mixture of two or more different (e.g., different serotypes and / or different strains) rAAV particles.

[0042] As used herein, the term "serotype" is a characteristic used to refer to an AAV having a capsid that is serologically different from other AAV serotypes. Serological characteristics are determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. Such differences in cross-reactivity are usually 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 AAV variants, including capsid variants, may not be serologically different from a reference AAV serotype or other AAV serotypes, they differ from the reference AAV serotype or other AAV serotypes by at least one nucleotide or amino acid residue.

[0043] In certain embodiments, an rAAV vector based on a first serotype genome corresponds to one or more serotypes of the capsid proteins that package the vector. For example, the serotype of one or more AAV nucleic acids (e.g., ITRs) that make up the AAV vector genome corresponds to the serotype of the capsid that makes up the rAAV particle.

[0044] In certain embodiments, rAAV vector genome can be based on AAV (e.g., AAV2) serotype genome, which is derived from one or more serotypes of AAV capsid proteins that package the vector.For example, rAAV vector genome can include nucleic acid (e.g., ITR) from AAV2, while at least one or more of the three capsid proteins are derived from different serotypes, such as AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotypes or variants thereof.

[0045] In certain embodiments, an rAAV particle or vector genome thereof associated with a reference serotype has a polynucleotide, polypeptide, or subsequence thereof that comprises or consists 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 certain embodiments, an rAAV particle or its vector genome related to a reference serotype has a capsid or ITR sequence that comprises or consists 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 the capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74 or AAV-2i8 serotype.

[0046] In certain embodiments, the methods herein include use, administration, or delivery of rAAV1, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAV10, rAAV11, rAAV12, rRh10, rRh74, or rAAV-2i8 particles.

[0047] In certain embodiments, the method herein comprises the use, administration or delivery of rAAV2 particles.In certain embodiments, the rAAV2 particles comprise AAV2 capsid.In certain embodiments, the rAAV2 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 protein of native AAV2 particles 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%, and up to 100% identical. In certain embodiments, the rAAV2 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical to the corresponding capsid proteins of native or wild-type AAV2 particles, for example, 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% identical. 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 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of a native or wild-type AAV2 particle.

[0048] 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, for example, 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 to the corresponding capsid protein of a native or wild-type AAV9 particle. In certain embodiments, the rAAV9 particles comprise VP1, VP2 and VP3 capsid proteins that are at least 75% or more identical to the corresponding capsid proteins of a native or wild-type AAV9 particle, for example, 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% identical. In certain embodiments, the rAAV9 particles are variants of native or wild-type AAV9 particles. In some aspects, one or more capsid proteins of the AAV9 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions compared to the capsid proteins of a native or wild-type AAV9 particle.

[0049] In certain embodiments, the rAAV particles are designed to incorporate one or more desired ITR functions (e.g., the ability to form a hairpin to allow DNA replication, the ability to integrate the AAV into the host cell genome, etc.). In some embodiments, the AAV-2i8 vector comprises one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, 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, 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, so long as the vector retains DNA inclusion, and / or packaging, if desired.

[0050] In certain embodiments, the rAAV2 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, 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, to the corresponding ITRs of a native or wild-type AAV2 particle, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin to enable DNA replication, inclusion of the AAV DNA into the host cell genome, and / or packaging if desired).

[0051] In certain embodiments, the rAAV9 particles contain one or two ITRs (e.g., a pair of ITRs) that are at least 75% or more identical, 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, to the corresponding ITRs of native or wild-type AAV2 particles, so long as they retain one or more desired ITR functions (e.g., the ability to form a hairpin to enable DNA replication, inclusion of the AAV DNA into the host cell genome, and / or packaging if desired).

[0052] rAAV particles can include an ITR with any suitable number of "GAGC" repeats. In certain embodiments, the ITR of an AAV2 particle includes 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with three "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with fewer than four "GAGC" repeats. In certain embodiments, the rAAV2 particle includes an ITR with more than four "GAGC" repeats. In certain embodiments, the ITR of an rAAV2 particle includes a Rep binding site in which the fourth nucleotide in the first two "GAGC" repeats is C instead of T.

[0053] Examples of suitable lengths of DNA that can be incorporated into rAAV vectors for packaging / encapsidation into rAAV particles can be about 5 kilobases (kb) or less. In certain 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.

[0054] rAAV vectors containing nucleic acid sequences directing the expression of RNAi or polypeptides can be produced using suitable recombinant techniques known in the art (see, for example, Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transducible AAV particles and propagated using AAV viral packaging systems. Transducible AAV particles have the ability to bind and enter mammalian cells and subsequently deliver their nucleic acid cargo (e.g., heterologous genes) to the nucleus of the cells. Thus, intact transducible rAAV particles are configured to transduce mammalian cells. rAAV particles configured to transduce mammalian cells are often not replicable and require additional protein machinery to replicate themselves. Thus, rAAV particles configured to transduce mammalian cells are engineered to bind and enter mammalian cells and deliver nucleic acid to the cells, with the delivered nucleic acid often being located between a pair of AAV ITRs in the rAAV genome.

[0055] Suitable host cells for producing transducible AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be used as or have been used as recipients of heterologous rAAV vectors. The stable human cell line HEK293 (e.g., readily available from the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, modified human embryonic kidney cell lines (e.g., HEK293) that are transformed with adenovirus type 5 DNA fragments and express adenovirus E1a and E1b genes are used to produce recombinant AAV particles. The modified HEK293 cell line is easily transfected, making it a particularly convenient platform for producing rAAV particles. Methods for producing high titer AAV particles capable of transducing mammalian cells are known in the art. For example, AAV particles can be made as described in Wright, 2008 and Wright, 2009.

[0056] In certain embodiments, AAV helper functions are introduced into host cells by transfecting AAV helper constructs into host cells before or simultaneously with transfection of AAV expression vector. Thus, in some cases, AAV helper constructs are used to at least transiently express AAV rep and / or cap genes to complement missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and cannot replicate or package themselves. These constructs can take the form of plasmids, phages, transposons, cosmids, viruses, or virions. Several AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45, which code for both Rep and Cap expression products. Several other vectors are also known that code for Rep and / or Cap expression products.

[0057] An "expression vector" is a special vector that contains a gene or nucleic acid sequence together 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 heterologous nucleic acid sequences, expression control elements (e.g., promoters, enhancers), introns, ITRs, and polyadenylation signals.

[0058] II. Therapeutic substances 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 RNAs, non-coding RNAs, and / or therapeutic proteins to cells in culture or in a host organism.

[0059] A. Inhibitory RNA "RNA interference (RNAi)" is the process of sequence-specific post-transcriptional gene silencing initiated by siRNA. During RNAi, siRNA induces the degradation of target mRNA, resulting in sequence-specific inhibition of gene expression.

[0060] "Inhibitory RNA", "RNAi", "small interfering RNA" or "short interfering RNA" or "siRNA" molecule, "short hairpin RNA" or "shRNA" molecule, or "miRNA" is an RNA duplex of nucleotides that targets a nucleic acid sequence of interest. As used herein, the term "siRNA" is a general term that encompasses a subset of shRNA and miRNA. "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. The nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene, so that the siRNA "targets" that gene. In certain embodiments, the siRNA targets the sequence that codes for Huntington. In some embodiments, the length of the duplex of the siRNA is less than 30 base pairs. In some embodiments, 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 length. In some embodiments, the length of the duplex is 19-25 base pairs long. In certain embodiments, the length of the duplex is 19 base pairs long or 21 base pairs long. The RNA duplex portion of the siRNA can be part of a hairpin structure. The hairpin structure contains, in addition to the duplex portion, a loop portion located between the two sequences forming the duplex. The length of the loop can vary. In some embodiments, 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 long. In certain embodiments, the loop is 18 nucleotides long. The hairpin structure can also contain a 3' overhang and / or a 5' overhang. In some embodiments, the overhang is a 3' overhang and / or a 5' overhang that is 0, 1, 2, 3, 4, or 5 nucleotides long.

[0061] shRNAs consist of stem-loop structures designed to contain a 5' flanking region, an siRNA region segment, a loop region, a 3' siRNA region, and a 3' flanking region. Most RNAi expression strategies have used short hairpin RNAs (shRNAs) driven by strong polIII promoters. Many shRNAs have shown effective knockdown of target sequences both in vitro and in vivo, but some shRNAs that show effective knockdown of target genes have also been found to be toxic in vivo.

[0062] miRNA is a small cellular RNA (approximately 22 nt) that is processed from precursor stem-loop transcript.Known miRNA stem-loop can be modified to contain RNAi sequence specific to gene of interest.miRNA molecule may be preferred to shRNA molecule because miRNA is endogenously expressed.Therefore, miRNA molecule is less likely to induce dsRNA-responsive interferon pathway, is more efficiently processed than shRNA, and has been shown to silence 80% more effectively than shRNA.

[0063] An alternative approach that has been discovered recently is the use of artificial miRNAs (pri-miRNA scaffolds that shuttle siRNA sequences) as RNAi vectors. Artificial miRNAs are more similar to endogenous RNAi substrates in nature and are more suitable for 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 herein by reference.

[0064] The transcription unit of an "shRNA" consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides. shRNAs are exported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. The stem-loop of an "miRNA" consists of a sense sequence and an antisense sequence connected by a loop of unpaired nucleotides, which are typically expressed as part of a larger primary transcript (pri-miRNA), which is excised by the Drosha-DGCR8 complex to generate an intermediate known as a pre-miRNA, which is then exported from the nucleus by exportin-5 and processed by Dicer in the cytoplasm to generate functional siRNAs. As used interchangeably herein, "artificial miRNA" or "artificial miRNA shuttle vector" refers to a primary miRNA transcript in which the region of the double-stranded stem-loop (at least about 9-20 nucleotides) excised by Drosha and Dicer processing is replaced with an siRNA sequence for a target gene while retaining the structural elements in the stem-loop required for effective Drosha processing. The term "artificial" comes from the fact that the flanking sequences (approximately 35 nucleotides upstream and approximately 40 nucleotides downstream) are derived from restriction enzyme sites within the multiple cloning site of the siRNA. As used herein, the term "miRNA" encompasses both naturally occurring miRNA sequences and artificially created miRNA shuttle vectors.

[0065] siRNA can be encoded by a nucleic acid sequence, and the nucleic acid sequence can also include a promoter.The nucleic acid sequence can also include a polyadenylation signal.In some embodiments, the polyadenylation signal is a synthetic minimal polyadenylation signal or a sequence of six T's.

[0066] In the design of RNAi, there are several factors that need to be considered, such as the nature of the siRNA, the permanence of the silencing effect and the choice of delivery system. To produce the RNAi effect, the siRNA that is introduced into the organism typically contains exonic sequences. Furthermore, since the RNAi process depends on homology, the sequence must be carefully selected to maximize gene specificity while minimizing the possibility of cross-interference between homologous but non-gene-specific sequences. Preferably, the siRNA exhibits greater than 80%, 85%, 90%, 95%, or 98% identity, or even 100% identity, between the sequence of the siRNA and the gene to be inhibited. Sequences with less than about 80% identity to the target gene are much less effective. Thus, the higher the homology between the siRNA and the gene to be inhibited, the less likely the expression of unrelated genes will be affected.

[0067] In addition, the size of the siRNA is also an important consideration. In some embodiments, the present disclosure relates to siRNA molecules that contain at least about 19-25 nucleotides and can regulate gene expression. In the context of the present disclosure, the siRNA is preferably less than 500, 200, 100, 50, or 25 nucleotides in length. More preferably, the siRNA is about 19 nucleotides to about 25 nucleotides in length.

[0068] By siRNA target is generally meant a polynucleotide that comprises a region that codes for a polypeptide, or a polynucleotide that comprises a region that regulates the replication, transcription or translation or other processes important for the expression of a polypeptide, or a polynucleotide that comprises both a region that codes for a polypeptide and a region that is functionally linked thereto and regulates its expression.Any gene that is expressed in cells can be targeted.Preferably, the target gene is involved or associated with the progression of a cellular activity that is important for disease or that is of particular interest for research.

[0069] B. Non-coding RNA As evidenced by cDNA cloning projects and genome tiling arrays, over 90% of the human genome is transcribed but does not code for proteins. These transcripts are referred to as non-protein-coding RNAs (ncRNAs). Various ncRNA transcripts, such as ribosomal RNAs, transfer RNAs, competitive endogenous RNAs (ceRNAs), small nuclear RNAs (snRNAs), and small nucleolar RNAs (snoRNAs), are essential for cellular functions. Similarly, a number of short ncRNAs, such as microRNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), and small nucleolar RNAs (snoRNAs), are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that show cell-type-specific expression and localize to specific subcellular compartments. lncRNAs are also known to play important roles during cell development and differentiation, supporting the view that they have been selected during evolution.

[0070] LncRNAs appear to have many different functions. In many cases, they appear to play a role in regulating protein activity or localization or function as an organizational framework for subcellular structures. In other cases, lncRNAs may be processed to produce multiple small RNAs or modulate how other RNAs are processed. The latest version of the data generated by the public research consortium GenCode (version number 27) catalogs just under 16,000 lncRNAs in the human genome, generating nearly 28,000 transcripts; when other databases are included, more than 40,000 lncRNAs are known.

[0071] Interestingly, lncRNAs can affect the expression of specific target proteins at specific genomic loci, modulate the activity of protein-binding partners, recruit chromatin-modifying complexes to their sites of action, and are post-transcriptionally processed to generate a large number of 5'-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs.

[0072] Increasing evidence also suggests that aberrantly expressed lncRNAs play important roles in normal physiological processes and multiple disease states. lncRNAs are misregulated in a variety of diseases, including ischemia, cardiac disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation has also been shown in various types of cancer, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma, and leukemia. Some lncRNAs, such as gadd74 and lncRNA-RoR5, regulate cell cycle regulators such as cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors, and p53, thus providing additional flexibility and robustness to cell cycle progression. In addition, some lncRNAs are associated with mitotic processes, such as centromeric satellite RNAs, which are essential for kinetochore formation and thus important for chromosome segregation during mitosis in humans and flies. Another nuclear lncRNA, MA-lincl, regulates M-phase exit by functioning in cis to repress the expression of the neighboring gene Pura, a regulator of cell proliferation.

[0073] lncRNAs are a group generally defined as transcripts of 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 lncRNAs and short transcripts of less than about 200 nt, e.g., about 30 to 200 nt.

[0074] Thus, in some embodiments, delivery of ncRNA, for example to a particular brain structure of interest, corrects aberrant RNA expression levels or modulates the levels of disease-causing lncRNA. Thus, in some embodiments, the present disclosure provides rAAVs in which the viral genome is engineered to encode a therapeutic non-coding RNA (ncRNA). In some embodiments, the ncRNA is a long non-coding RNA (lncRNA) about 200 nucleotides (nt) or more in length. In some embodiments, the therapeutic is an ncRNA about 25 nt or about 30 nt to about 200 nt in length. In some embodiments, the lncRNA is about 200 nt to about 1,200 nt in length. In some embodiments, the lncRNA is about 200 nt to about 1,100, about 1,000, about 900, about 800, about 700, about 600, about 500, about 400, or about 300 nt in length.

[0075] C. CRISPR Systems Gene editing is a technique that allows targeted genetic modification in living cells. In recent years, the implementation of on-demand gene editing using the bacterial CRISPR immune system has revolutionized the way scientists approach genome editing. The Cas9 protein of the CRISPR system, an RNA-guided DNA endonuclease, can be relatively easily engineered to target new sites by altering its guide RNA sequence. With this discovery, sequence-specific gene editing has become functionally enabled.

[0076] In general, "CRISPR system" refers collectively to transcripts and other elements involved in directing the expression or activity of CRISPR-associated ("Cas") genes, such as sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and, in the case of endogenous CRISPR systems, partial direct repeats that have been processed by tracrRNA), guide sequences (also referred to as "spacers" in the case of endogenous CRISPR systems), and / or other sequences and transcripts from a CRISPR locus.

[0077] A CRISPR / Cas nuclease or CRISPR / Cas nuclease system can include a non-coding RNA molecule (guide) RNA that binds to DNA in a sequence-specific manner and a Cas protein (e.g., Cas9) with nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system can be derived from a Type I, Type II, or Type III CRISPR system, for example, from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes.

[0078] The CRISPR system can induce a double stranded break (DSB) at the target site and subsequent scission, as discussed herein. In another embodiment, a Cas9 variant considered a "nickase" is used to nick one strand at the target site. A pair of nickases can be used, each directed by a pair of different gRNAs that target sequences in such a way that a 5' overhang is introduced when the nicks are introduced simultaneously, for example to improve specificity. In another embodiment, catalytically inactive Cas9 is fused to a heterologous effector domain, such as a transcriptional repressor (e.g., KRAB) or transcriptional activator, to affect gene expression. Alternatively, the CRISPR system with catalytically inactive Cas9 further comprises a transcriptional repressor or transcriptional activator fused to a ribosome binding protein.

[0079] In some aspects, Cas nuclease and gRNA (comprising a fusion of a target sequence-specific crRNA and an invariant tracrRNA) are introduced into cells. In general, a target site at the 5' end of the gRNA targets the Cas nuclease to the target site, e.g., a gene, using complementary base pairing. The target site can be selected based on its location, immediately 5' to a protospacer adjacent motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, the gRNA targets 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, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the site of the target sequence. Generally, "target sequence" generally refers to the sequence that guide sequence is designed to have complementarity with, and hybridization between target sequence and guide sequence promotes the formation of CRISPR complex.Full complementarity is not necessarily required, as long as there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complex.

[0080] The target sequence may comprise any polynucleotide, such as a DNA polynucleotide or an RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in a cell organelle. In general, the sequence or template that can be used for recombination into the targeted locus that comprises the target sequence is referred to as an "editing template" or an "editing polynucleotide" or an "editing sequence". In some aspects, the exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is a homologous recombination.

[0081] Typically, in the case of endogenous CRISPR systems, the formation of a CRISPR complex (including a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in the cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 base pairs or more of the target sequence). The tracr sequence, which may include or consist of all or a portion of the wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence, or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence), may also form part of a CRISPR complex, for example, by hybridization to all or a portion of the tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity over the entire length of the tracr mate sequence when optimally aligned.

[0082] 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 those elements of the CRISPR system directs the formation of CRISPR complexes at one or more target sites. Components can also be delivered to the cell as proteins and / or RNA. For example, Cas enzyme, guide sequence linked to tracr-mate sequence, and tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Cas enzyme can be a target gene that is controlled by a regulated alternative splicing event as disclosed herein as a chimeric target gene minigene or as a target gene for a chimeric minigene transactivator. gRNA can be controlled by a constitutive promoter.

[0083] Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in one vector, providing any components of the CRISPR system that are not included in the first vector with one or more additional vectors. The vector may include one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. Using multiple different guide sequences, a single expression construct may be used to target CRISPR activity to multiple corresponding target sequences in a cell.

[0084] The vector may include a regulatory element operably linked to an enzyme coding sequence encoding a CRISPR enzyme, such as a Cas protein, non-limiting examples of which include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, etc. (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Csel, 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, Csfl, 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.

[0085] The CRISPR enzyme can be Cas9 (e.g., from Streptococcus pyogenes or S. pneumonia). The CRISPR enzyme can direct cleavage of one or both strands at the location of the target sequence, e.g., within the target sequence and / or within the complementary strand of the target sequence. The vector can encode a CRISPR enzyme that is mutated such that the mutant CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence, as compared to the corresponding wild-type enzyme. For example, an aspartic acid to alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from Streptococcus pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (one strand cleavage). 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 both strands to be nicked and used to induce NHEJ or HDR.

[0086] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is codon-optimized for expression in a particular cell, such as a eukaryotic cell. The eukaryotic cell may be of or derived from a particular organism, such as a mammal, including but not limited to a human, mouse, rat, rabbit, dog, or non-human primate. In general, codon optimization refers to the process of modifying a nucleic acid sequence by replacing at least one codon of the native sequence with a codon that is more or most frequently used in the host cell's genes while maintaining the native amino acid sequence, in order to enhance expression in the host cell of interest. Different species exhibit a particular bias for certain codons of a particular amino acid. Codon bias (the difference in codon usage between organisms) is often correlated with the efficiency of messenger RNA (mRNA) translation, and is believed to depend, among other things, on the properties of the codon being translated and the availability of a particular transfer RNA (tRNA) molecule. The predominance of a selected tRNA in a cell is generally a reflection of the codon that is most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0087] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more, when optimally aligned using a suitable alignment algorithm.

[0088] Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of such algorithms include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transformation (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).

[0089] CRISPR enzymes may be part of a fusion protein that includes one or more heterologous protein domains. CRISPR enzyme fusion proteins may include any additional protein sequences and, optionally, a linker sequence between any two domains. Examples of protein domains that may be fused to CRISPR enzymes include, but are not limited to, epitope tags, reporter gene sequences, and protein domains that have one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. 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), autofluorescent proteins including HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP). CRISPR enzymes can be fused to gene sequences encoding proteins or fragments of such proteins that bind to DNA molecules or other cellular molecules, such as, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Further domains that may form part of fusion proteins comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference.

[0090] D. Therapeutic Proteins Some embodiments relate to the expression of recombinant proteins and polypeptides, such as those listed below.

[0091] Apolipoprotein E2. Apolipoprotein E (APOE) is a protein involved in fat metabolism in mammals. Subtypes are associated with Alzheimer's and cardiovascular disease. APOE belongs to a family of fat-binding proteins called apolipoproteins. In the circulation, it is present as part of several classes of lipoprotein particles, including chylomicron remnants, VLDL, IDL, and some HDL. APOE interacts significantly with the low-density lipoprotein receptor (LDLR), which is essential for normal processing (catabolism) of triglyceride-rich lipoproteins. In peripheral tissues, APOE is produced primarily by the liver and macrophages, where it mediates cholesterol metabolism. In the central nervous system, APOE is produced primarily by astrocytes, which transport cholesterol to neurons via the APOE receptor, a member of the low-density lipoprotein receptor gene family. APOE is the major cholesterol transporter in the brain. APOE is required for cholesterol transport from astrocytes to neurons. APOE is eligible as a checkpoint inhibitor of the classical complement pathway due to its complex formation with activated C1q. APOE is a protein involved in fat metabolism in mammals. Subtypes are associated with Alzheimer's disease and cardiovascular disease.

[0092] APOE is 299 amino acids long and contains multiple amphipathic α-helices. Crystallographic studies have shown that a hinge region connects the N-terminal and C-terminal regions of the protein. The N-terminal region (residues 1–167) forms an antiparallel four-helix bundle with the nonpolar side facing the inside of the protein. Meanwhile, the C-terminal domain (residues 206–299) contains three α-helices that form a large exposed hydrophobic surface and interact with the α-helices of the N-terminal helix bundle domain through hydrogen bonds and salt bridges. The C-terminal region also contains the low-density lipoprotein receptor (LDLR) binding site.

[0093] APOE is polymorphic, with three major alleles (ε2, ε3 and ε4): APOE-ε2 (cys112, cys158), APOE-ε3 (cys112, arg158) and APOE-ε4 (arg112, arg158). These allelic forms differ by only one or two amino acids at positions 112 and 158, but these differences alter the structure and function of APOE.

[0094] As of 2012, the E4 variant was the largest known genetic risk factor for late-onset sporadic Alzheimer's disease (AD) in various ethnic groups. However, the E4 variant does not correlate with risk in all populations. Although Nigerians have the highest observed frequency of APOE4 alleles among global populations, AD is rare among them. This may be due to their lower cholesterol levels. Caucasian and Japanese carriers of the two E4 alleles have 10-30 times the risk of developing AD by age 75 compared to those who do not carry either E4 allele. This may be caused by interactions with amyloid. Alzheimer's disease is characterized by the accumulation of aggregates of the peptide beta-amyloid. Apolipoprotein E enhances the proteolysis of this peptide both intracellularly and intercellularly. The isoform APOE-ε4 is not as effective as the others at promoting these responses, resulting in higher vulnerability to AD in individuals who carry that genetic variant.

[0095] Although 40-65% of patients with AD 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 do not develop the disease. However, individuals with two ε4 alleles have up to 20 times the risk of developing AD. There is also evidence that the APOE2 allele may play a protective role in AD. Thus, the genotype with the highest risk of Alzheimer's disease and onset at an earlier age is APOE4,4. Using the genotype APOE3,3 as the reference (individuals with this genotype are considered to have a risk level of 1.0), in the Caucasian population only, individuals with the genotype APOE4,4 have an odds ratio of 14.9 for developing Alzheimer's disease. The odds ratio for individuals with the APOE3,4 genotype is 3.2, and for those with two and four copies of the APOE genotype (APOE2,4), the odds ratio is 2.6. For those with one copy each of the APOE genotype (APOE2,3), the odds ratio is 0.6. For those with two copies of the APOE genotype (APOE2,2), the odds ratio is also 0.6.

[0096] Further 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).

[0097] It will be understood by those of skill in the art that when the application refers to a function or activity of a "modified protein" or "modified polypeptide," it encompasses, for example, a protein or polypeptide that has additional advantages over the unmodified protein or polypeptide. It is specifically contemplated that embodiments relating to a "modified protein" can also be implemented with respect to a "modified polypeptide," and vice versa.

[0098] Recombinant proteins may carry amino acid deletions and / or substitutions. Thus, proteins with deletions, proteins with substitutions, and proteins with deletions and substitutions are modified proteins. In some embodiments, these proteins may further include inserted or added amino acids, such as fusion proteins or proteins with linkers. A "modified deleted protein" may lack one or more residues of the native protein but may have the specificity and / or activity of the native protein. A "modified deleted protein" may also have reduced immunogenicity or antigenicity. An example of a modified deleted protein is one in which amino acid residues are deleted from at least one antigenic region, i.e., from a region of the protein that has been determined to be antigenic in a particular organism, e.g., the organism to which the modified protein is administered.

[0099] Substitution or replacement variants typically contain the exchange of one amino acid for another at one or more sites within the protein and can be designed to modulate one or more properties of the polypeptide, particularly its effector functions and / or bioavailability. Substitutions may or may not be conservative substitutions, i.e., one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, an alanine to serine change, an arginine to lysine change, an asparagine to glutamine or histidine change, an aspartic acid to glutamic acid change, a cysteine ​​to serine change, a glutamine to asparagine change, a glutamic acid to aspartic acid change, a glycine to proline change, a histidine to asparagine or glutamine change, an isoleucine to leucine or valine change, a leucine to valine or isoleucine change, a lysine to arginine change, a methionine to leucine or isoleucine change, a phenylalanine to tyrosine, leucine or methionine change, a serine to threonine change, a threonine to serine change, a tryptophan to tyrosine change, a tyrosine to tryptophan or phenylalanine change, and a valine to isoleucine or leucine change.

[0100] In addition to deletion or substitution, modified proteins may have insertion of residues. This typically involves the addition of at least one residue in the polypeptide. This may include the insertion of a targeting peptide or targeting polypeptide or simply the insertion of a single residue. Terminal additions, called fusion proteins, are described below.

[0101] Gonzalez et al., BMC Neurosci 10.1186 / 1471-2202-12-4 (2011) screened a peptide library of M13 bacteriophage for ligands that could be internalized into CP epithelial cells by incubating the phage with choroid plexus tissue slices and recovering targeting-competent particles. Three peptides identified after four rounds of screening were analyzed for specific and dose-dependent binding and internalization. Binding was considered specific because internalization was prevented by co-incubation with the cognate synthetic peptide. Furthermore, after icv injection into rat brain, each peptide was found to target the phage to epithelial cells of the CP and the ependyma, which lines the ventricles. Such peptides can be employed in the AAV vectors described herein.

[0102] The term "biologically functional equivalent" is well understood in the art and is further described 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 those of a reference polypeptide are encompassed, so long as the biological activity of the protein is maintained. A recombinant protein may, in certain aspects, be a biologically functional equivalent to the corresponding native protein.

[0103] It will also be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, and still be essentially as described in one of the sequences disclosed herein, so long as the sequence meets the above criteria, including, where protein expression is concerned, maintenance of biological protein activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences which may include, for example, various non-coding sequences adjacent to either the 5' or 3' portion of the coding region, or may include various internal sequences, i.e., introns, that are known to be present within genes.

[0104] As used herein, a protein or peptide generally refers to, but is not limited to, a protein of more than about 200 amino acids, up to the full-length sequence translated from a gene; a polypeptide of more than about 100 amino acids; and / or a peptide of about 3 to about 100 amino acids. For convenience, the terms "protein," "polypeptide," and "peptide" are used interchangeably herein.

[0105] As used herein, "amino acid residue" refers to any natural amino acid, any amino acid derivative, or any amino acid mimic known in the art. In certain embodiments, the residues of a protein or peptide are contiguous, without any non-amino acid sequence interrupting the sequence of amino acid residues. In other embodiments, the sequence may include one or more non-amino acid moieties. In certain embodiments, the sequence of residues of a protein or peptide may be interrupted by one or more non-amino acid moieties.

[0106] Thus, the term "protein or peptide" encompasses amino acid sequences that include at least one of the 20 common amino acids found in naturally occurring proteins, or at least one modified or unusual amino acid.

[0107] Certain aspects of the present disclosure relate to fusion proteins. These molecules may have a therapeutic protein linked to a heterologous domain at the N-terminus or C-terminus. For example, fusions may also use leader sequences from other species to allow recombinant expression of the protein in a heterologous host. Other useful fusions include the addition of a protein affinity tag, preferably cleavable, such as a serum albumin affinity tag or six histidine residues, or an immunologically active domain, such as an antibody epitope, to facilitate purification of the protein. Non-limiting examples of affinity tags include polyhistidine, chitin-binding protein (CBP), maltose-binding protein (MBP), and glutathione-S-transferase (GST).

[0108] Methods for making fusion proteins are well known to those skilled in the art. Such proteins can be produced, for example, by de novo synthesis of an entire fusion protein, or by attachment of a DNA sequence encoding a heterologous domain followed by expression of the intact fusion protein.

[0109] The production of fusion proteins that restore the functional activity of the parent protein can be facilitated by ligating the genes with a bridging DNA segment that encodes a peptide linker joined between the tandemly linked polypeptides, the linker being of sufficient length to allow proper folding of the resulting fusion protein.

[0110] III. Methods of Treatment and Administration In some aspects, viral vectors can be administered directly (in vivo) to patients, or can be used to treat cells in vitro or ex vivo, and then administered to patients. In particular, provided herein is a method for inducing expression of a transgene in the ependyma. 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 at least about 70% of the cells of the target tissue; the AAV vector targets inner and outer hair cells with at least about 70%, 80%, 90%, 95% or more efficiency, and even as high as 100% efficiency. In some embodiments, the cells are cells of the brain ventricles, such as ependymal cells.

[0111] The ependyma is a thin neuroepithelial (simple columnar ciliated epithelium) lining of the ventricular system of the brain and the central canal of the spinal cord. The ependyma is one of the four types of neuroglia in the central nervous system (CNS). It has been shown to be involved in the production of cerebrospinal fluid (CSF) and to function as a reservoir for neural regeneration. The ependyma is composed of ependymal cells, a type of glial cell called ventricular ependymal cells. These cells line the ventricles of the brain and the central canal of the spinal cord, which fill with CSF. They are simple columnar neural tissue cells, much like some mucosal epithelial cells. Early single-ciliated ependymal cells differentiate into multiciliated ependymal cells for the function of circulating CSF. The basement membrane of these cells is characterized by tentacle-like extensions that attach to astrocytes. The apical side is covered with cilia and microvilli.

[0112] The term "vector" refers to a small carrier nucleic acid molecule, a plasmid, a virus (e.g., AAV vector, retroviral vector, lentiviral vector), or other vehicle that can be manipulated by inserting or incorporating a nucleic acid. A vector, such as a viral vector, can be used to introduce / transfer a nucleic acid into a cell such that the nucleic acid sequence within the nucleic acid is transcribed by the cell and, if it encodes a protein, is subsequently translated.

[0113] These compositions can be used to treat brain or central nervous system conditions. Thus, in some embodiments, the methods described herein are used to treat a condition listed in Table A, using the corresponding sequence listing in Table A, in a subject in need thereof.

[0114] Any suitable cell or mammal can be administered or treated by the methods or uses described herein. Typically, a mammal in need of the methods described herein is suspected of having or expressing an abnormal or aberrant protein associated with a disease state. Alternatively, the mammalian recipient may have a condition suitable for gene replacement therapy. As used herein, "gene replacement therapy" refers to the administration of exogenous genetic material encoding a therapeutic agent to a recipient and the subsequent expression of the administered genetic material in situ. Thus, the phrase "condition suitable for gene replacement therapy" encompasses conditions such as genetic diseases (i.e., disease states resulting from one or more gene defects) and acquired pathologies (i.e., pathological conditions not resulting from congenital defects). Thus, as used herein, the term "therapeutic agent" refers to any agent or material that has a beneficial effect on a mammalian recipient. Thus, "therapeutic agent" encompasses both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0115] Non-limiting examples of mammals include humans, non-human primates (apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g. dogs and cats), farm animals (e.g. horses, cows, goats, sheep, pigs), and laboratory 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. humans, pigs, goats, sheep, horses, dogs, etc.). In certain embodiments, the non-rodent mammal is a human. The mammal can be of any age or stage of development (e.g. 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, such as an animal model having or expressing an abnormal or aberrant protein associated with a disease state, or an animal model in which the expression of a protein is insufficient and causes the disease.

[0116] Mammals (subjects) treated with the methods or compositions described herein include adults (18 years or older) and children (under 18 years). Adults include the elderly. A typical adult is 50 years or older. Children range in age from 1-2 years or 2-4 years, 4-6 years, 6-18 years, 8-10 years, 10-12 years, 12-15 years, and 15-18 years. Children also include infants. Infants are typically in the 1-12 month age range.

[0117] In certain embodiments, the method comprises administering a plurality of viral particles to a mammal as described herein, and reducing, reducing, preventing, inhibiting, or delaying the severity, frequency, progression, or onset time of one or more symptoms of a disease state, such as a neurodegenerative disease.In certain embodiments, the method comprises administering a plurality of viral particles to a mammal to treat a deleterious symptom of a disease state, such as 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 or reversal of a disease state, such as a neurodegenerative disease, and the deleterious symptoms.

[0118] In certain embodiments, the methods include administering a plurality of viral particles to the central nervous system or a portion thereof as described herein, and reducing, reducing, preventing, inhibiting, or delaying 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 the severity, frequency, progression, or time to onset of one or more symptoms of a disease state, such as a neurodegenerative disease.

[0119] In some embodiments, a composition can be delivered that includes a therapeutically effective number of transgene-containing viral particles, or a composition that includes one or more sets of different viral particles, where each particle in the set can include the same type of transgene, but each set of particles includes a different type of transgene than in the other sets, as described herein.

[0120] Formulations according to the present disclosure can be used for CNS delivery via a variety of techniques and routes, including, but not limited to, intraparenchymal, intracerebral, intracerebroventricular (ICV), intrathecal (e.g., IT-lumbar, IT-thoracic, IT-cisternomagna) administration, as well as any other technique and route for direct or indirect injection into the CNS and / or CSF.

[0121] In some embodiments, the formulation is delivered to the CNS by administration into the cerebrospinal fluid (CSF) of the subject in need of treatment. In some embodiments, intrathecal administration is used to deliver viral particles into the CSF. As used herein, intrathecal administration (also referred to as intrathecal injection) refers to injection into the spinal canal (the intrathecal space surrounding the spinal cord). Various techniques can be used, including but not limited to, lateral ventricular injection, such as by cranial puncture or cisternal puncture 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.

[0122] According to the present disclosure, the viral particles may be injected into any area surrounding the spinal canal. In some embodiments, the viral particles are injected into the lumbar region or cisterna magna, or intraventricularly into the ventricular cavity. As used herein, the term "lumbar region" or "lumbar area" refers to the area between the third and fourth lumbar vertebrae (lower back), more generally the L2-S1 region of the spine. Intrathecal injection via the lumbar region or lumbar area is usually also referred to as "lumbar IT delivery" or "lumbar IT administration". The term "cisterna magna" refers to the space around and below the cerebellum via the opening between the skull and the upper spine. Intrathecal injection via the cisterna magna is usually also referred to as "cisterna magna delivery". The term "ventricle" refers to the cavity in the brain that is continuous with the central canal of the spinal cord. Thus, intrathecal administration includes any injection into the central canal. Injection via the ventricular cavity is commonly referred to as intracerebroventricular (ICV) delivery.

[0123] Various 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 injection port); a hollow body (e.g., a catheter) having a first flow orifice in fluid communication with the fluid access port and a second flow orifice configured for insertion into the spinal cord; and a fixation mechanism for fixing the insertion of the hollow body in the spinal cord. In various 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, an implanted catheter is connected to either a reservoir (e.g., for bolus delivery) or an infusion pump. The fluid access port can be implanted or external.

[0124] In some embodiments, intrathecal administration can be performed either by lumbar puncture (i.e., slow bolus) or via a port-catheter delivery system (i.e., infusion or bolus). In some embodiments, the catheter is inserted into the lumbar interdiscal space and the tip is threaded up the intrathecal cavity to the desired level (generally L3-L4).

[0125] A single dosage volume suitable for intrathecal administration is typically small. Typically, intrathecal delivery according to the present disclosure maintains a balance of the composition of the CSF and the intracranial pressure of the subject. In some embodiments, intrathecal delivery is performed without a corresponding removal of CSF from the subject. In some embodiments, a suitable single dosage volume can be, for example, less than about 10 ml, 8 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, 1.5 ml, 1 ml, or 0.5 ml. In some embodiments, a suitable single dosage volume can be about 0.5-5 ml, 0.5-4 ml, 0.5-3 ml, 0.5-2 ml, 0.5-1 ml, 1-3 ml, 1-5 ml, 1.5-3 ml, 1-4 ml, or 0.5-1.5 ml. In some embodiments, intrathecal delivery according to the present disclosure involves first removing a desired amount of CSF. In some embodiments, 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 initially removed prior to IT administration. In such cases, an appropriate single dosage 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.

[0126] A variety of other devices can be used to achieve intrathecal administration of therapeutic compositions. For example, a formulation containing the desired enzyme can be administered using an Ommaya reservoir, which is commonly used to intrathecally administer drugs for meningeal carcinomatosis (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 placed under the scalp, and the reservoir is subcutaneously punctured to deliver the specific enzyme to be replaced that is injected into the reservoir into the intrathecal cavity. Other devices for intrathecal administration of therapeutic compositions or formulations 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 the administration treatment can be in the form of a single dose administration or multiple doses.

[0127] In one embodiment of the present disclosure, viral particles are administered by injection into the lateral ventricle of the subject's brain. Injection can be, for example, through a burr hole drilled in the subject's skull. In another embodiment, viral particles and / or other pharmaceutical preparations are administered into the subject's ventricle through a surgically inserted shunt. For example, injection can be into the larger lateral ventricle. In some embodiments, injection can also be into the third and fourth 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.

[0128] IV. Pharmaceutical Compositions As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" refer to a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery, or in vivo contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Such compositions, "pharmaceutically acceptable" and "physiologically acceptable" formulations and compositions may be sterile. Such pharmaceutical formulations and pharmaceutical compositions may be used, for example, in administering viral particles to a subject.

[0129] 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 suspending media, coatings, isotonicity and absorption enhancing or absorption delaying agents that are compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions can include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the formulations and compositions.

[0130] Pharmaceutical compositions typically contain pharma- ceutically acceptable excipients. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition and that may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween 80, and liquids such as water, saline, glycerol, and ethanol. They may include pharma-ceutically acceptable salts, such as mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulfates, and salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as surfactants, wetting or emulsifying agents, pH buffering substances, and the like, may also be present in such vehicles.

[0131] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as described herein or known to those of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0132] Suitable pharmaceutical forms for injection of viral particles can include sterile aqueous solutions or dispersions, adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In any case, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture, use, and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media, including, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. Isotonicity agents, such as sugars, buffers, or salts (such as sodium chloride), can be included. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, such as aluminum monostearate and gelatin, in the composition.

[0133] The solution or suspension of viral particles may optionally contain one or more of the following components: sterile diluents, such as water for injection, saline solutions, such as phosphate-buffered saline (PBS), artificial CSF, surfactants, fixed oils, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), glycerin, or other synthetic solvents, antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and the like; 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.

[0134] Pharmaceutical formulations, compositions and delivery systems suitable for the compositions, methods and uses of the present disclosure are known in the art (e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington's Pharmaceutical Sciences (1990) 18 th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12 th ed., Merck Publishing Group, Whitehouse, 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), R.L. Juliano, ed., Oxford, NY, pp. 253-315).

[0135] Viral particles and their compositions can be formulated into dosage unit form to facilitate administration and ensure uniformity of dosage. As used herein, dosage unit form refers to a physically discrete unit suitable as a unitary dosage to an individual to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect together with a necessary pharmaceutical carrier. The dosage unit form depends on the number of viral particles that are considered to be required to produce a desired effect. The required amount can be formulated in a single dose or in multiple dosage units. The dose can be adjusted to the appropriate viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0136] In one embodiment, the pharmaceutical composition will contain sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient to reduce or ameliorate the symptoms or deleterious effects of the disease state in question, or an amount sufficient to provide the desired benefit.

[0137] As used herein, "unit dosage form" refers to a physically discrete unit suitable for a unitary dosage to a subject to be treated, each unit containing a predetermined amount calculated to produce a desired effect (e.g., a prophylactic or therapeutic effect) when administered once or multiple times, optionally together with a pharmaceutical carrier (excipient, diluent, vehicle or filler). Unit dosage forms may be, for example, in ampoules and vials that may contain liquid compositions, or compositions in a freeze-dried or lyophilized state, and may, for example, be added with a sterile liquid carrier before in vivo administration or in vivo delivery. Individual unit dosage forms may be included in a multi-dose kit or container. Thus, for example, viral particles and their pharmaceutical compositions may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0138] A formulation containing viral particles typically contains an effective amount, which is easily determined by one skilled in the art. The viral particles can typically be in the range of about 1% to about 95% (w / w) of the composition, or even higher if appropriate. The amount administered depends on factors such as the age, weight and health of the mammalian or human subject for which treatment is being considered. Those skilled in the art can establish effective dosages by routine testing to establish dose-response curves.

[0139] V. Definition The terms "polynucleotide", "nucleic acid" and "transgene" are used interchangeably herein to refer to any form of nucleic acid, oligonucleotide, 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, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-spliced ​​RNA or antisense RNA). Polynucleotides can include natural, synthetic and intentionally modified or altered polynucleotides (e.g., variant nucleic acids). Polynucleotides can be single-stranded, double-stranded or triplexed, linear or circular, and of any suitable length. In discussing polynucleotides, the sequence or structure of a particular polynucleotide may be described herein according to the convention of describing the sequence in the 5' to 3' direction.

[0140] A nucleic acid that encodes a polypeptide often includes an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.

[0141] The nucleic acid can include one or more expression control or expression regulatory elements operably linked to the open reading frame, which one or more regulatory elements are configured to direct the transcription and translation of the polypeptide encoded by the open reading frame in a mammalian cell. 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, polyA sequences, secretion sequences, etc. Expression control / regulatory sequences can be obtained from the genome of any suitable organism.

[0142] A "promoter" refers to a nucleotide sequence, usually upstream (5') of a coding sequence, that directs and / or controls the expression of the coding sequence by providing recognition sites for RNA polymerase and other factors required for proper transcription. Pol II promoters contain a minimal promoter, a short DNA sequence consisting of a TATA box and, optionally, other sequences that serve to specify the 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) the 5' sequence element (A block); b) the intermediate sequence element (I block); c) the 3' sequence element (C block). Type 2 pol III promoters contain two essential cis-acting sequence elements downstream of the transcription start site: a) the A box (5' sequence element); and b) the 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 conventional TATA box, the proximal sequence element (PSE), and the distal sequence element (DSE).

[0143] An "enhancer" is a DNA sequence capable of stimulating transcriptional activity and can be a promoter-specific or heterologous element that enhances the level or tissue specificity of expression. It can operate in either orientation (5'→3' or 3'→5') and has the ability to function whether placed upstream or downstream of the promoter.

[0144] Enhancers may be derived in their entirety from a native gene, or may be composed of different elements derived from different elements found in nature, or even composed of synthetic DNA segments. Enhancers may contain DNA sequences involved in the binding of protein factors that regulate / control the effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0145] "Transgene" is used herein for convenience to refer to a nucleic acid sequence / polynucleotide that is or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as an inhibitory RNA or a gene encoding a polypeptide or protein, which is generally heterologous to the native AAV genome sequence.

[0146] The term "transducing" refers to the introduction of a nucleic acid sequence into a cell or host organism by a vector (e.g., a viral particle). Thus, the introduction of a transgene into a cell by a viral particle can be referred to as "transduction" of the 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 recipient organism, it can be stably maintained in that cell or organism and can be further transmitted or inherited by the descendant cells or descendant organisms of the recipient cell or recipient organism. Finally, the introduced transgene may be present extrachromosomally or only transiently in the recipient cell or recipient host organism. Thus, a "transduced cell" is a cell into which a transgene has been introduced by transduction. Thus, a "transduced" cell is a cell into which a transgene has been introduced or its descendants. The transduced cell can be propagated, transcribed, and the encoded inhibitory RNA or protein expressed. For gene therapy uses and methods, the transduced cell can be present in a mammal.

[0147] A nucleic acid / transgene is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence, where the promoter is capable of controlling transcription of the encoded polypeptide. A nucleic acid operably linked to an expression control element can also be referred to as an expression cassette.

[0148] In certain embodiments, the expression control element comprises a CMV enhancer.

[0149] As used herein, the term "modify" or "variant" and its grammatical variants refer to nucleic acid, polypeptide or its subsequence deviating from reference sequence.Thus, modified and variant sequences may have substantially the same, greater or less expression, activity or function than reference sequence, but at least partially retain the activity or function of reference sequence.A particular type of variant is mutant protein, which refers to the protein encoded by gene with mutation, such as missense mutation or nonsense mutation.

[0150] A "nucleic acid" variant or a "polynucleotide" variant refers to a modified sequence in which genes are altered compared to the wild type. A sequence can be genetically altered without altering the encoded protein sequence. Alternatively, a sequence can be genetically altered to encode a variant protein. A nucleic acid variant or a polynucleotide variant can also refer to a combination sequence that is codon-altered to encode a protein that still retains at least partial sequence identity to a reference sequence, such as a wild type protein sequence, and is also codon-altered to encode a variant protein. For example, some codons of such a nucleic acid variant are altered so as not to alter the amino acid of the protein encoded thereby, and some codons of the nucleic acid variant are altered so as to alter the amino acid of the protein that it encodes.

[0151] The terms "protein" and "polypeptide" are used interchangeably herein. The "polypeptide" encoded by the "nucleic acid" or "polynucleotide" or "transgene" disclosed herein includes partial or full-length native sequences, as well as naturally occurring wild-type and functional polymorphic proteins, their functional subsequences (fragments), and sequence variants thereof, so long as the polypeptide retains some function or activity. Thus, in the methods and uses of the present disclosure, such polypeptides encoded by nucleic acid sequences need not be identical to endogenous proteins that are defective or whose activity, function or expression is insufficient, deficient, or absent in the mammal being treated.

[0152] Non-limiting examples of modifications include substitution of one or more nucleotides or amino acids (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).

[0153] Examples of amino acid modifications are conservative amino acid substitutions or deletions. In certain embodiments, the modified or variant sequence retains at least part of the function or activity of the unmodified sequence (e.g., the wild-type sequence).

[0154] Another example of an amino acid modification is a targeting peptide introduced into the capsid protein of the viral particle. Peptides have been identified that target recombinant viral vectors to the central nervous system, for example to different brain regions.

[0155] Such modified recombinant viruses may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). In certain embodiments, recombinant viruses bearing modified capsid proteins may "target" brain vascular epithelial tissue by binding at a higher level than comparable unmodified capsid proteins. For example, recombinant viruses with modified capsid proteins may bind to brain ependymal tissue at a 50%-100% higher level than unmodified recombinant viruses.

[0156] A "nucleic acid fragment" is a portion of a given nucleic acid molecule. In most organisms, deoxyribonucleic acid (DNA) is the genetic material, while ribonucleic acid (RNA) is responsible for the transfer of the information contained in DNA into proteins. Fragments and variants of the disclosed nucleotide sequences and the proteins or partial length proteins encoded thereby are also encompassed by the present disclosure. By "fragment" or "portion" is meant a full-length or less than full-length nucleotide sequence encoding a polypeptide or protein, or an amino acid sequence of a polypeptide or protein. In certain embodiments, the fragment or portion 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 wild-type activity or function).

[0157] A "variant" of a molecule is a sequence that is substantially similar to the sequence of the native molecule. In the case of nucleotide sequences, variants include sequences that code for the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Such natural allelic variants can be identified using molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those that code for native proteins, as well as those that code for polypeptides with amino acid substitutions, created, for example, using site-directed mutagenesis. Generally, nucleotide sequence variants of the present disclosure have at least 40%, 50%, 60% or 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79%, typically at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity to the native (endogenous) nucleotide sequence. In certain embodiments, the variants are biologically functional (i.e., retain 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).

[0158] A "conservative substitution" of a particular nucleic acid sequence refers to a nucleic acid sequence that codes for the same or essentially the same amino acid sequence. Because the genetic code is degenerate, any given polypeptide is encoded by a large number of functionally identical nucleic acids. For example, the codons CGT, CGC, CGA, CGG, AGA, and AGG all code for the amino acid arginine. Thus, wherever a codon specifies arginine, the codon can be changed to any of the corresponding codons described without changing the encoded protein. Such nucleic acid variations are "silent variations," which are a type of "conservatively modified variations." Any nucleic acid sequence described herein that codes for a polypeptide represents all possible silent variations, unless otherwise noted. Those skilled in the art will recognize that each codon in a nucleic acid (except ATG, which is usually the only methionine codon) can be modified by standard techniques to obtain a functionally identical molecule. Thus, each "silent variation" of a nucleic acid that codes for a polypeptide is implicit in each described sequence.

[0159] The term "substantial 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 when compared to a reference sequence using one of the alignment programs described with standard parameters. It will be understood by those skilled in the art that these values ​​can be appropriately adapted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame position, etc. For these purposes, substantial amino acid sequence identity typically means at least 70%, at least 80%, 90% or even at least 95% sequence identity.

[0160] The term "substantial identity" with respect to a polypeptide indicates that the polypeptide comprises a sequence with 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 in a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with an antibody raised against the other polypeptide. Thus, one polypeptide is identical to another polypeptide, for example, when the only difference between the two polypeptides is a conservative substitution.

[0161] "Disease" means a condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0162] The terms "treat" and "treatment" refer to both therapeutic and prophylactic or preventative measures, the purpose of which is to prevent, inhibit, reduce, or diminish undesirable physiological changes or disorders, such as the onset, progression, or worsening of disorders. For purposes of this disclosure, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, diminishment of the extent of disease, stabilization (i.e., no worsening or progression) of symptoms or adverse effects of disease, whether detectable or undetectable, delay or slowing of disease progression, improvement or mitigation of disease state, and remission (whether partial or complete). "Treatment" can also mean a prolongation of survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those with a predisposition (e.g., as determined by genetic assay).

[0163] As used herein, "essentially free" of a particular component means that the particular component is not intentionally formulated in the composition and / or is present only as a contaminant or in trace amounts.The total amount of the particular component resulting from unintentional incorporation of the composition is therefore significantly lower than 0.05%, preferably lower than 0.01%.Most preferred is a composition in which the amount of such particular component cannot be detected by standard analytical methods.

[0164] As used herein, "a" or "an" can mean one or more. As used herein in the claims, the words "a" or "an," when used with the word "comprising," can mean one or more than one.

[0165] The use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or mutually exclusive alternatives, but the present disclosure supports the definition to refer to alternatives only and "and / or." As used herein, "another" can mean at least a second or more.

[0166] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation of the device, the method being employed to determine the value, the variation that exists among study subjects, or a value that is within 10% of the stated value.

[0167] VI. Kits The present disclosure provides a kit with packaging material and one or more components therein.The kit typically includes a label or insert that includes a description of the components contained therein or an instruction for the in vitro, in vivo, or ex vivo use of the components contained therein.The kit can include a collection of such components, such as nucleic acids, recombinant vectors, and / or viral particles.

[0168] A kit refers to a physical structure that contains 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, cardboard, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0169] The label or package insert may include the identity of one or more components contained therein, dosage, clinical pharmacology of the active ingredient, such as mechanism of action, pharmacokinetics and pharmacodynamics. The label or package insert may include information identifying the manufacturer, lot number, place and date of manufacture, expiration date. The label or package insert may include information identifying the manufacturer information, lot number, place and date of manufacture. The label or package insert may include information regarding the disease for which the kit components may be used. The label or package insert may include instructions for the clinician or subject to use one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. The instructions may include dosage, frequency or duration of administration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0170] The label or package insert may include information about benefits that the components may provide, such as preventive or therapeutic benefits. The label or package insert may include information about potentially harmful side effects, complications, or reactions, such as warnings to the subject or clinician about situations in which it may not be appropriate to use a particular composition. Adverse side effects or complications may also occur if the subject has previously taken, will take, or is currently taking one or more other medications that may be incompatible with the composition, or if the subject has previously taken, will take, or is currently taking another treatment protocol or therapeutic regimen that may be incompatible with the composition, and the instructions may also include information about such incompatibilities.

[0171] A label or package insert includes "printed matter", such as paper or cardboard, either separate or attached to a component, kit or packaging material (e.g., a box), or attached to an ampoule, tube or vial that the kit component is in. The label or package insert can further include a computer readable medium, such as a printed label with a bar code, a disk, an optical disk, such as a CD- or DVD-ROM / RAM, a DVD, an MP3, or an electronic storage medium, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage media, flash memory, hybrids and memory type cards. EXAMPLES

[0172] VII. Working Examples The following examples are provided to demonstrate preferred embodiments of the present disclosure. As will be understood by those skilled in the art, the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for its implementation. However, those skilled in the art will understand in light of the present disclosure that many changes can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0173] Example 1 A subtractive approach to identify ependymal-enriched or ependymal-specific genes. The ependyma constitutes a thin epithelial layer lining the ventricles of the brain and the central canal of the spinal column. These cells are in close proximity to the cerebrospinal fluid (CSF) in the ventricles, a body fluid that not only fills these cavities but also distributes widely throughout the brain by migrating into the subarachnoid space and diffusing along the perivascular spaces into the parenchyma. Therefore, proteins secreted from the ependyma to the CSF can be delivered widely throughout the brain. Therapeutically, it has been demonstrated that infection of ependymal cells with adeno-associated viruses (AAV) encoding secreted proteins promotes widespread protein expression in rodent, canine and non-human primate brains. To ensure robust and long-term expression of AAV localized to the ependyma, we sought to identify endogenous gene signatures and their respective regulatory regions that could be incorporated into AAV transgenes. A key part of this search was to identify genes whose expression was insensitive to disease state, such that robust expression would be ensured even when introduced into diseased brains. Therefore, we obtained samples from healthy patients as well as patients with frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Alzheimer's disease (AD) and Huntington's disease (HD), among others. The limited depth of the ependymal lining makes it very difficult to cleanly separate this specific tissue type. To circumvent this problem, we employed a subtractive approach to identify ependymal-enriched or ependymal-specific genes. Tissue samples were obtained from white and gray matter, including the white and gray matter adjacent to the ventricles, as well as the ependyma at the periventricular margin. Genes specific to white matter + ependyma and gray matter + ependyma samples were considered enriched or specific to the ependyma (Figure 1). The top hits were compared to in situ hybridization data published by the Allen Brain Institute (Figure 2).

[0174] Isolation of predicted regulatory regions in ependymal-specific genes. To isolate functional promoter segments, the UCSC Genome Browser was used to search for promoter-like signatures in the upstream sequences of the identified genes. Of particular interest were segments containing H3K4me3, H3K27Ac, ​​CpG islands, and transcription factor binding sites. Segments of approximately 1100–2500 base pairs (bp) were PCR amplified from human (HT1080) genomic DNA (gDNA) and placed upstream of an eGFP reporter. Promoters were identified using 3 bp barcodes in the 3' UTR for high-throughput sequencing (Figure 3). Individual plasmids were pooled in equimolar ratios and transgenes were packaged as libraries into AAV4 or AAV2 capsids and tested in mice and rhesus monkeys, respectively.

[0175] In vivo testing of isolated promoter segments to drive RNA expression in mouse and rhesus monkey ependyma. A library of 13 transgenes containing ependymal-enriched promoters, prepared as a single AAV4, was injected into the lateral ventricles of adult FVBn mice at high (1e11 vg), medium (5e10 vg) and low doses (1e10 vg). After 3 weeks, the ventricular lining and limited surrounding tissue were microdissected and RNA was isolated using Trizol. Regions surrounding the 3 bp barcodes were amplified from cDNA using a two-step PCR protocol to ligate Illumina sequencing adapters. Final products were subjected to amplicon sequencing to quantify relative contributions. The graphs in Figure 4 show the percent contribution of each barcode to the total number of reads for tissues and input viruses.

[0176] To quantify the promoter function of isolated segments in vivo, the relative contribution of RNA output was normalized to the relative contribution of 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 over the viral input.

[0177] The same library used in Figure 4 was prepared as AAV2 and injected into the lateral ventricles of two adult rhesus macaques at 2e13 total vg per animal. Three weeks later, the region of the ventricular rim containing the spinal cord was microdissected (Figure 6). RNA was isolated, converted to cDNA, and PCR products containing 3 bp barcodes were subjected to amplicon sequencing. Colored bars indicate the relative contribution of each transgene in tissue RNA or viral DNA. The enrichment patterns of RNA at matched positions were similar between two different rhesus macaques and within different regions of the same structure.

[0178] Modification of the original transgene to include a promoter-localized intron and ApoE2 coding sequence. Genetic variation within the apolipoprotein E gene (APOE) on chromosome 19 is associated with differential risk of late-onset Alzheimer's disease. The most common allele, APOE3, is considered neutral and does not increase or decrease an individual's risk of developing the disease. APOE4 increases risk and is associated with early onset of the disease, whereas the relatively rare variant, APOE2, protects against the disease. Intracerebroventricular injection of AAV4-APOE2 into an early-onset AD mouse model resulted in transduction of the ependyma and choroid plexus and detectable protein deposition in the cortical parenchyma, reversing Aβ deposition and improving clearance from the CNS (PMID: 24259049). To adapt our design for therapeutic applications, we replaced the eGFP reporter with human ApoE2 cDNA in constructs containing six different ependymal-enriched promoters (Figures 7 and 8). The six libraries were further expanded by introducing a short (133 bp) β-globin / IgG chimeric intron or a long (951 bp) chicken β-actin / rabbit β-globin intron to test for intron-mediated enhanced expression, for a total of three variants for each gene.

[0179] To measure intron splicing, individual plasmids were transfected into HEK293 cells and harvested at 24 hours. RNA was isolated using Trizol per manufacturer's instructions and reverse transcribed using MultiScribe reverse transcriptase. Correct splicing was verified by amplifying across the intron-containing region in cDNA (C) vs. plasmid DNA (D). Complete intron removal was observed in most transgenes, with the exception of the short intron in hANXA1 (Figure 8). Protein output from intron-containing transgenes was also measured in HEK293 cell lysates and media. Figure 9 includes a western blot for APOE, a protein that is not readily detected endogenously in HEK293 cells. Consistent with intron-mediated enhancement, all variants showed increased protein output in the presence of the intron, to a greater extent with the long intron versus the short intron.

[0180] In vivo measurement of promoter activity including introns. All 18 ApoE2 transgene variants (intronless, short, and long) were prepared as separate plasmids and pooled in equimolar ratios to create a single AAV2 library. Two adult rhesus macaques were injected with virus into the lateral ventricles at a total dose of 2.8e13 vg per animal. After 4 weeks, ventricular rims were microdissected from 4 mm thick coronal slabs and RNA was isolated using Trizol. Relative promoter usage in vivo was assessed using cDNA amplicon sequencing of products containing unique 3-letter barcodes in the 3' UTR (Figure 10). Numbers in brackets indicate isolation from equivalent slabs in the larger constructs shown. All hVWA3a variants showed relative enrichment in vivo over the input library.

[0181] Confirmation that the hVWA3a promoter can drive transgene expression in a mouse model. Adult APOE- / - (null) mice were injected into their right lateral ventricle with serotype AAV4 delivering APOE2 under the hVWA3a promoter. Ependymal tissue was microdissected, proteins were extracted, and APOE2 was quantified by automated Western blot technique (WES) and compared to uninjected brain tissue.

[0182] Confirmation that hVWA3a promoter shows higher APOE2 expression compared to previous constructs. APOE- / - (null) mice were injected into their right lateral ventricle with equal doses of serotype AAV4 delivering APOE2 under either the ubiquitous CAG promoter or the hVWA3a promoter. Proteins were extracted from microdissected ependymal tissue from all animals and subjected to automated Western blot technique (WES). From the band intensity, APOE2 driven by hVWA3a expressed higher amount of APOE2 protein than CAG promoter.

[0183] Biodistribution of the novel capsid ERDRpAAV1 in combination with hVWA3a restricts expression to ependymal cells in a mouse model as visualized by the eGFP transgene. Peptide-modified AAV1 capsid with a human ependymal-specific promoter: ERDRpAAV1.hVWA3a.eGFP. Positive eGFP fluorescent signal is restricted to ependymal cells lining the brain ventricles.

[0184] Introducing ApoE2 via epithelial expression in this 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. Furthermore, we observed that introducing ApoE2 in this APP / PS1 / ApoE4 mouse model dramatically reduced the activation of microglia near cortical plaques. This indicates that ApoEε4 induces microglia into a proinflammatory phenotype and that exogenous ApoE2 can interrupt or reduce this abnormal activation. We also found that ApoE2 can prevent or reduce the loss of synapses that occurs in the halo around amyloid β plaques in these mice. Taken together, these indicate that the effect of ApoE2 on microglia may protect against the abnormal phagocytosis of synapses near plaques that likely occurs as a result of amyloid deposition. See Figures 23-34.

[0185] These results suggest that viral delivery of secreted ApoE2 to ependymal ApoE4 carriers may be an effective therapeutic strategy to affect both the classical pathology of AD (e.g., plaque deposition and neurodegeneration) and the enhanced neuroinflammatory profile observed in sporadic AD.

[0186] Example 2 method. Study design We performed intracerebroventricular (ICV) injections of novel AAV vectors expressing APOE2 variants into the ventricular cavity of AD transgenic mouse models. Mice were injected with either AAV or vehicle control for 2 months. Using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), we assessed the effect of human APOE2 on amyloid deposition. Mice were randomly assigned to treatment groups. The nature of the injected vector was blinded until statistical analysis. We estimate that these studies require eight animals per condition (four of each sex). This will provide a power of >0.8 to confirm a 30% correction of the baseline phenotype when compared based on previous data (Hudry et al., 2013).

[0187] animal APP / SP1 (Radde et al., 2006) mice express human mutant APP KM670 / 671NL and PSEN1 L166P under the Thy1 promoter, causing a severe phenotype characterized by amyloid deposition at 3–4 months of age. APOE targeted replacement expresses human APOE4 (Huynh et al., 2019) in a mouse model under the control of the mouse APOE promoter. These animals were backcrossed until the APP / PS1 transgene was expressed together with two copies of human APOE4 (APPPS1 / APOE4) in place of the mouse apoE. We exposed 4-month-old APOE4-TR / APP / PS1 mice to either high (7E10vg, n=14), medium (2E10vg, n=11) or low (7E9vg, n=11) doses of AAVert-APOE2 or vehicle control (n=10) for 2 months. In addition, a cohort of APOE KO (Jackson labs) mice on a C57BL / 6 background was included as a comparative measure of Aβ levels in tissues and CSF. Experiments were performed in accordance with National Institutes of Health (NIH) and institutional guidelines, and both males and females were used. Due to the small size of mouse brains, not all animals were used for all analyses, and n is indicated by the number of points shown. Open circles indicate females and closed circles indicate males.

[0188] Viral vector construction and production . Research-grade production of AAV-based viral vectors is conducted by the Research Vector Core (RVC) at The Children's Hospital of Philadelphia. Certified Working Cell Bank (WCB) Adhesive Human Embryonic Renal epithelial cells (HEK293) Production was carried out by transient triple transfection of Cells were grown in tissue culture flasks and roller bottles prior to transfection. Testing of investigational products is performed in-house by RVC QC, and test methods, procedures, and results are reported on the Certificate of Analysis (CoA) for each lot.

[0189] Stereotaxic intraventricular injectionAAV intraventricular injections were performed as previously described (14, 30). Animals were anesthetized (O2 / isoflurane 0.2%) and placed in a stereotaxic frame (David Kopf Instruments). 5.25 μl of the virus preparation was injected into each lateral ventricle at 0.20 μl / min using a 33-gauge needle attached to a 10 μl Hamilton syringe (Hamilton Medical). Stereotaxic coordinates were calculated from the bregma (anteroposterior +0.3 mm, mediolateral ±1 mm, and dorsoventral -2 mm).

[0190] Western blot Mouse cortical tissue was homogenized in 10 parts by weight of ice-cold TBS containing 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 blot. Protein concentration was determined using a BCA assay. Total protein (5–10 μg) was loaded and separated on a 4–12% NuPAGE gel in MES buffer, and then proteins were separated by weight at 120 V for 2 h. Proteins were electrotransferred to nitrocellulose membranes in Tris-glycine transfer buffer using an XCell II™ Blot Module system at 30 V for 1.5 h. The membrane was incubated in blocking buffer (Li-Cor Biosciences) diluted 1:1 in TBS for 1 h to reduce background staining. The membranes were then incubated with primary antibodies; rb anti-APOE (Novus biologicals, NBP1-31123) and ms anti-GAPDH (Millipore MAB374) diluted in blocking buffer supplemented with 0.1% Tween-20 overnight at room temperature with shaking. The membranes were then washed and incubated with the appropriate 680 and 800 IR dye secondary antibodies (Li-Cor Biosciences). The membranes were imaged using an Odyssey infrared imaging system and analyzed using Odyssey software.

[0191] DNA and RNA extraction and analysisGenomic DNA was extracted from brain tissue using the QIAamp DNA Mini Kit (Qiagen) according to the manufacturer's protocol. Samples were run on a BioRad CFX384 Real Time System C1000 Touch using BioRad CFX Manager 3.1 software. Total genome copies were quantified against a six-point standard curve generated with a linearized plasmid containing the construct. Primers / probes (designed against non-coding regions in the construct) were used with TaqMan® Master Mix (Applied Biosystems).

[0192] Total RNA was extracted from brain tissue using TRIzol (Ambion by Life Technologies) according to the manufacturer's protocol. RNA (1 μg) was treated with DNase I, RNase-free (ThermoScientific) according to the manufacturer's protocol. Complementary DNA was generated using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Samples were run on a BioRad CFX384 Real Time System C1000 Touch using BioRad CFX Manager 3.1 software. APOE levels were quantified with primers / probes designed for use with TaqMan® Master Mix (Applied Biosystems). Exogenous mRNA levels of transgene-expressing human APOE (Hs00171168_m1) were determined using a commercially available TaqMan® primer / probe set (Applied Biosystems). Endogenous mouse β-actin (Mm02619580_g1) was used as a reference gene to normalize expression between samples.

[0193] ELISA quantitativeConcentrations 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 the TBS, SDS-soluble and SDS-insoluble fractions of each mouse. Mouse brain slices were homogenized in 10 volumes (w / v) of TBS buffer containing cOmplete protease inhibitor cocktail (Roche) and centrifuged at 1,000,000 × g for 30 min at 4 °C. The supernatant was collected and set aside as the TBS-soluble fraction. The pellet was then homogenized in 10 volumes (w / v) of TBS buffer containing 2% SDS and incubated at 37 °C for 30 min before centrifuging at 100,000 × g for 30 min 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, then centrifuged at 100,000 × g for 30 min at 4 °C. The formic acid-soluble supernatant was dried in a 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 (Source: Hashimoto et al., 2020) (Hashimoto et al., 2020).

[0194] RNAscopeDrop-fixed hemispheres from APOE KO mice were sectioned at 30 μm on a cryo-ultramicrotome. Three mice per experimental condition (sham vs. AAV-injected) were stained for APOE mRNA by RNAscope. RNAscope experiments were performed using the Manual Fluorescent Multiplex kit v2 (Advanced Cell Diagnostics) with minor adjustments according to the manufacturer's recommendations. Briefly, for each mouse, several sections were printed onto superfrost slides for use in APOE mRNA quantification. After target retrieval and protease digestion, probe hybridization was performed for 2 h at 40°C with hs-APOE (433091), 3-plex Positive Control Probe_Mm (320881) and Negative Control Probe- DapB (310043). After an amplification step to obtain the RNAscope signal, the signal was developed using TSA-cy3 (Perkin Elmer FP1170). Sections were counterstained at 1:1000 dapi, mounted with Immunomount, and scanned at 10x magnification using an Olympus VS120-S6-W virtual slide microscope.

[0195] Immunohistological analysisMice were euthanized by isoflurane inhalation. One cerebral hemisphere was fixed in 4% paraformaldehyde and 15% glycerol in PBS and switched to 30% glycerol in PBS after 48 hours. The remaining hemisphere was snap frozen for biochemical analysis. Drop-fixed hemispheres were processed by Neuroscience Associates. Forty 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 was performed overnight at 4°C in 0.05% Triton-X and 2.5% normal goat serum. Sections were then washed in TBS and the appropriate secondary antibody was diluted 1:500 in 0.05% Triton-X and 2.5% normal goat serum in TBS at room temperature. Sections were incubated with dapi 1:1000 in TBS for 10 min at room temperature, washed, and then mounted with Immunomount.

[0196] Plaque quantification Every 10th section was stained for amyloid-β using rabbit anti-Aβ (1:500, IBL, Cat. No. 18584) as described above. Amyloid-dense core plaques were labeled with 0.05% Thio-S (Sigma-Aldrich) in 50% ethanol before mounting. Sections were mounted and scanned at 40x using a nanozoom microscope. Sections were quantified using qupath (Bankhead et al., 2017). Cortical areas were selected for each section, plaques were identified using an object classifier, and plaque-covered area was assessed as a percentage of the measured cortical area. For plaque size and number, areas of equal size were selected in the cortex of each animal, and plaques were identified using an object classifier.

[0197] Glial assessmentSome sections were stained as above. Primary antibodies were biotinylated Ms anti-Aβ 82E1 (IBL 10326) at 1:1000, GFAP-488 (Millipore MAB 3402X) at 1:500, and rabbit anti-IBA1 (wako 019-19741) at 1:500. Secondary antibodies were streptavidin Alexa Fluor 568 (Invitrogen S11226) and donkey anti-rabbit 647 (A-31573). Areas containing five cortical plaques from the somatosensory cortex were randomly imaged at 40x using an Olympus FV3000 confocal laser scanning microscope. Z-stacks were generated and each plaque was graded by two blinded investigators on a four-point scale for the level of glial reactivity (Figure 38A). All plaques from individual mice were averaged to generate the graphs in Figures 35B-C and Figures 38C-D, while all plaques from a given experimental group were evaluated for Figures 35D and 38E.

[0198] Synapse quantificationSome sections were stained as above. Primary antibodies were rabbit anti-Aβ 1:500 (IBL, Cat. No. 18584) and goat anti-PSD95 1:500 (abcam ab12093). Secondary antibodies were donkey anti-goat 488 (Invitrogen A-11078) and donkey anti-rabbit 594 (A A-21207). Areas containing five cortical plaques from the somatosensory cortex were randomly imaged at 60x with an oil immersion objective on an Olympus FV3000 confocal laser scanning microscope. 5 μm Z-stacks were imaged with a slice size of 0.56 μm. Images were processed using custom Image J and Matlab Macros similar to Jackson et al. (Jackson et al., 2019). Briefly, 10 μm × 10 μm crops were taken from areas within 15 μm of the plaque halo or >40 μm from the plaque halo. Cellular debris and dapi were avoided. Crops were thresholded using a custom image J macro and synapses were quantified using a custom matlab macro. All crops were averaged to determine density proximal and distal to the plaque for each mouse.

[0199] Neurite quantification Some sections were stained as above. Primary antibodies were rabbit anti-Aβ 1:500 (IBL, Cat. No. 18584) and mouse anti-SMI312 1:500 (Biolegend 837904). Secondary antibodies were donkey anti-rabbit 488 (Invitrogen A-21206) and donkey anti-mouse 594 (A-21203). Areas containing five cortical plaques from the somatosensory cortex were randomly imaged at 60x with an oil immersion objective on an Olympus FV3000 confocal laser scanning microscope. Z-stacks of 20 μm were imaged with a slice size of 1 μm. Images were quantified using Image J by a blinded experimenter who counted the number of dystrophies per plaque and also quantified the plaque area. In images where more than one plaque was present, the largest plaque was quantified.

[0200] statistical analysis Statistical analysis was performed using Prism software. One-way ANOVA was used to perform all between-group analyses, followed by Dunnett's multiple comparison test between each group and vehicle control. Simple linear regression analysis was used to evaluate the correlation between factors and viral genome copy number, and p-values ​​represented whether the slope was significantly different from zero. Statistics were performed with each mouse as a single data point. Samples were blinded in each analysis.

[0201] result. Intracerebroventricular infusion of AAVert-APOE2 dose-dependently induces sustained production of APOE2 in the brain APOE is primarily produced by astrocytes and microglial cells in the CNS, where it can be secreted and then diffuse throughout the parenchyma. Previously, we have shown that APOE2 produced by cells in the ependyma, which lines the ventricles, can diffuse to the cerebral cortex, where it influences plaques and plaque-associated damage (Hudry et al., 2013).

[0202] We performed a single intracerebroventricular (ICV) injection of a novel ependymal-restricted AAV capsid expressing APOE2 (AAVert) into 4-month-old APOE KO mice, which were sacrificed 2 months later. 10 Injection of 1000 genomic units (vg) of virus into Apoe KO mice resulted in robust expression of APOE2 mRNA in the ependymal cell lining of the brain, as detected using RNAscope for human APOE (Figure 1A). Virus-driven APOE2 protein was also detected in TBS extracts of the cortex by Western blot, and was shown to be 10% of normal APOE (Figure 3B-C).

[0203] We then injected AAV carrying APOE2 into 4-month-old APPPS1 / APOE4 animals, which were sacrificed two months later. 9 vg, medium dose - 2E 10 vg, and high dose - 7E 10vg, as well as vehicle controls. DNA extraction from the hindbrain followed by qPCR demonstrated a dose-dependent effect of uptake, although three animals showed no uptake (Figure 33D). The viral genome copy number correlated with an increase in human APOE mRNA abundance, which in high-dose animals was approximately 50% higher 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.

[0204] APOE2 expression has a dose-dependent effect on AB plaque deposition levels The APP / PS1 / APOE4 model is a relatively aggressive model of amyloidosis. At 4 months of age, APPPS1 / APOE4 animals show moderate plaque deposition, and by 6 months of age, plaque deposition is established throughout the cortex. We therefore injected at 4 months and sacrificed at 6 months. Using ThioS as a marker of dense core amyloid plaques, we showed a dose-dependent effect of APOE2 on plaque deposition (Figure 34A-C). High-dose animals show an approximately 33% reduction in the percentage of cortex covered with ThioS positive staining compared to vehicle-treated animals (Figure 34B). The dose-dependent effect of APOE2 on plaques is evident when comparing plaque burden with viral genome copy numbers extracted from the hindbrain (Figure 34C). Staining with an anti-oligomeric Aβ (oAβ) antibody showed similar trends at both the group level (Supplementary Figure 1A) and individual level (Supplementary Figure 1B).

[0205] The observed reduction in cortical coverage was accompanied by a significant reduction in plaque density (Figure 34D), with high-dose animals showing 1.2 mm 2 The results showed a reduction in the number of plaques per 100 μg / mL and an even more significant reduction in the size of Aβ plaques (FIG. 34E).

[0206] Biochemical measurements of amyloid are consistent with the imaging measurements: concentrations of Aβ42 peptides measured from formic acid and SDS soluble extracts of mouse brain mimicked the changes observed histologically, such that high-dose animals showed an approximately 50% reduction in both SDS (Figure 3C) and formic acid (Supplementary Figure 1D) soluble Aβ42 amounts.

[0207] APOE4 has been shown to impair Aβ clearance and promote 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, resulting in reduced plaque.

[0208] APOE2 expression has a dose-dependent effect on plaque-associated neuroinflammation APOE4 mice have been shown to have a more aggressive neuroinflammatory response to plaques when compared to APOE2 or APOE3 mice. This mirrors 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, we performed IHC for Aβ, Iba1 (microglia) (Figure 35A) and GFAP (astrocytes in Figure 38B). The level of glial reactivity was assessed on a 4-point scale, with 1 being non-reactive and 4 being highly reactive (Figure 38A). Images were assessed by two blinded investigators, with a correlation R of 0.89. 2 We chose to evaluate the area immediately surrounding each individual plaque, rather than the entire cortex, because microglial activation in these mice is closely associated with plaques, and thus high-dose animals with fewer plaques have lower overall levels of activated microglia. Using this method, we can examine microglial reactivity partially dissociated from overall plaque density.

[0209] We found a clear, statistically significant reduction in microglial reactivity in the high- and medium-dose groups compared to vehicle-treated mice (Figure 35B), and that this attenuation correlated significantly with viral genome copy number in individual animals (Figure 35C). This reduction appears to be determined by the increased number of plaques that did not provoke a strong microglial reaction (score 1) in high- and medium-dose animals, and the reduction in plaques with a score of 4 compared to vehicle-treated animals (Figure 3D). In contrast, astrocytic reactivity around plaques is not affected by APOE2 levels or expression (Figures 38A-D), and is equally elevated around plaques in all groups.

[0210] APOE2 exposure modulates synaptic loss around amyloid deposits Synapse loss is known to correlate 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). We previously showed that APOE4 is associated with higher synapse loss near plaques in both carriers and mice compared to APOE3 mice or carriers. Because APOE4 has been shown not to prevent synapse loss, we tested the hypothesis that the addition of APOE2 could protect synaptic integrity in this model.

[0211] Postsynaptic densities (PSD95) were identified using immunohistochemistry and imaged by confocal microscopy (Figure 38A). Synaptic loss in this model has been shown to occur most significantly within 15 um from the plaque edge (Hudry et al., 2013; Koffie et al., 2012), therefore crops were taken from this region and analyzed similarly to crops taken >40 um from the plaque edge. Synaptic density distal to the plaque did not differ between groups (Figure 38B); however, animals injected with the highest dose showed increased levels of synapses proximal to the plaque compared to vehicle-treated animals (Figure 38C), restoring near-normal synaptic density. Percentage of synaptic loss was calculated by comparing synaptic density proximal to distal to the plaque in the same animals (Figure 38D). Vehicle-treated animals showed twice as much synaptic loss as high-dose animals, and 70% of the high-dose animals showed less than 10% loss near the plaque compared to the other groups, where all but one animal showed more than 10% loss.

[0212] We also assessed the number of dystrophic neurites associated with amyloid deposition by staining for the axon marker SMI312 in parallel with the oligomeric Aβ antibody (Figure 39A). No difference in the number of neuritic dystrophy was observed between groups (Figures 39B-C).

[0213] Discussion: APOE has long been of interest when considering risk modifiers or therapeutics for AD, as there are strong genetic and experimental associations between APOE genotype and AD (Serrano-Pozo, Das, et al., 2021). However, practical issues of distributing gene products throughout the brain have been a barrier to using gene therapy for 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. In this study, we tested an approach to overcome this barrier: expressing secreted proteins via transduction of the ependyma and associated structures, allowing the secreted proteins to diffuse throughout the cortical mantle. We apply this approach to the expression of APOE2 in the APOE4 / APP / PS1 model of Alzheimer's pathology and show that, at achievable doses, APOE2 expression can positively impact plaque deposition, neuroinflammation and neurodegeneration within 8 weeks of treatment.

[0214] Importantly, this improvement was observed in the setting of established disease (i.e., when plaque deposition has already begun) and models, from a neuropathological perspective, the changes thought to occur in patients with established early Alzheimer's disease. The results presented herein suggest that the influence of APOE in AD is not the result of APOE4-associated developmental or midlife changes, but is continuous during disease progression (Evans et al., 2020), and that the course of the disease can be altered by manipulating APOE even after AD is established.

[0215] We tested three doses of virus to determine the minimal effective dose of AAV and APOE2 required to see efficacy in this highly aggressive amyloidosis model (Figure 2). Based on the work of others (Castellano et al., 2011; Hashimoto et al., 2012; Serrano-Pozo, Das, et al., 2021), we hypothesize that the primary effect of APOE2 on the characteristic AD pathology of Aβ plaques is through increased clearance of Aβ through the BBB and reduced aggregation. Our study is consistent with this conclusion, showing that APOE2 production affects both dense-core fibrous plaques and biochemical measures of Aβ.

[0216] Neuroinflammation and AD have long been associated, in part, due to a number of microglial genes that are risk factors for AD, and also due to the marked increase in microgliosis and astrogliosis in the brains of both AD cases and mouse models (Leng & Edison, 2021). Recently, the impact of APOE genotype on neuroinflammation has been investigated (Hong et al., 2016). Microglia produce APOE under basal conditions, but production increases dramatically in these cells upon inflammation. This finding led to the hypothesis that microglial-produced APOE4 may have a feed-forward loop-induced or perpetuated gain of toxic functional effects (Krasemann et al., 2017), raising the possibility that the effects of APOE may be cell-autonomous. We show that exogenous APOE2 produced by ependymal cells suppresses plaque-proximal microglial activation in mice (Fig. 3), which is not consistent with this hypothesis and indicates that at least some of the effects of APOE on microglia are due to APOE produced in other cell types.

[0217] APOE4 is associated with more severe synapse loss near plaques in AD (Hudry et al., 2013; Koffie et al., 2012). In mice with the highest dose of AAV, we observed a reduction in the amount of synapse loss indicating that APOE2 can prevent this neurodegenerative phenotype. We have previously shown that APOE and oligomeric oAβ colocalize at synapses and that APOE4 is more efficient at delivering oAβ to synapses (Koffie et al., 2012). This synapse-protective effect of APOE2 may be due to several mechanisms that are not mutually exclusive. APOE2 may help reduce bioactive oAβ present in synapses, the action of APOE2 on microglia may cause a reduction in the level of synaptic pruning by reactive microglia, and increased clearance of oAβ may reduce the amount of oAβ within the plaque halo. The combination of these effects likely results in the absence of toxic oAβ at synapses, reducing both microglial pruning and the synaptic toxic effects of oAβ.

[0218] The data presented herein highlight the utility of ependymal-restricted AAVs for the expression of secreted proteins that can spread throughout the brain parenchyma once secreted into the neuropil and CSF. This approach demonstrates the practical potential of using this gene therapy for whole brain treatment, which could have considerable implications in lysosomal storage diseases and for diseases such as loss of function of progranulin in frontotemporal dementia associated with progranulin mutations. We ultimately envision the ability to introduce a variety of potential therapeutic agents into the brain, from single-chain antibody production to expression of other bioactive molecules. The current study establishes in principle two things: APOE2 protein as a therapeutic agent and an AAV platform for therapeutic agents that are currently hindered from use for CNS disorders by either blood-brain barrier issues or difficulties in peripheral expression (including clearance).

[0219] In conclusion, the data presented here suggest that gene-therapeutically introduced APOE2 has protective functions similar to well-established phenotypes in human patients inheriting the E2 or E4 allele. In this model, even modest levels of APOE2 expression impact Aβ deposition, attenuate neuroinflammation, and support the synaptic system. This speaks to the important potential of APOE modulation as a disease-modifying therapeutic agent in patients with Alzheimer's disease.

[0220] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present disclosure have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that various modifications may be made to the methods and steps or steps of the methods described herein without departing from the concept, spirit and scope of the present disclosure. More specifically, it will be apparent that certain specific agents that are chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present disclosure as defined by the appended claims.

[0221] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF2025514794000002.tif44146

Claims

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

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

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

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

5. The pharmaceutical product according to claim 1, wherein the modified AAV comprises a modified capsid protein.

6. The pharmaceutical product according to claim 5, wherein the modified capsid protein comprises a targeting peptide, the targeting peptide having a length of 3 to 10 amino acids, for example, 7 amino acids.

7. The pharmaceutical product according to 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 pharmaceutical product according to claim 6, wherein the modified AAV capsid protein is derived from the AAV1 capsid protein, and the targeting peptide is inserted after the 590th residue of the AAV1 capsid protein.

9. The pharmaceutical product according to claim 8, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

10. The pharmaceutical product according to claim 9, wherein the linker sequence is the N-terminal SSA of the targeting peptide and the C-terminal AS of the targeting peptide.

11. The pharmaceutical product according to claim 6, wherein the modified AAV capsid protein is derived from the AAV2 capsid protein, and the targeting peptide is inserted after the 587th residue of the AAV2 capsid protein.

12. The pharmaceutical product according to claim 11, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

13. The pharmaceutical product according to claim 12, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide.

14. The pharmaceutical product according to claim 6, wherein the modified AAV capsid protein is derived from the AAV9 capsid protein, and the targeting peptide is inserted after the 588th residue of the AAV9 capsid protein.

15. The pharmaceutical product according to claim 14, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

16. The pharmaceutical product according to claim 15, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide.

17. The pharmaceutical product according to claim 1, wherein the therapeutic transgene is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system.

18. The pharmaceutical product according to claim 1, wherein the therapeutic transgene is ApoE2, and the subject is suffering from Alzheimer's disease or has a higher risk of developing Alzheimer's disease compared to the population average.

19. The pharmaceutical product according to claim 1, wherein the administration is a direct intraventricular or intraparenchymal injection.

20. The pharmaceutical product according to claim 1, wherein the modified AAV is administered two or more times.

21. The pharmacopoeia according to claim 20, wherein the modified AAV is administered two, three, four, five, six, seven, eight, nine, ten times, or more times.

22. The pharmacopoeia according to 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 pharmaceutical product according to claim 1, further comprising the step of providing non-AAV therapy to the subject.

24. The pharmaceutical product according to claim 1, wherein multiple virus particles are administered.

25. The aforementioned multiple virus particles are approximately 1 × 10⁶ per kilogram. 6 ~Approx. 1×10 18 The pharmaceutical product according to claim 24, administered in a dose of one vector genome (vg / kg).

26. The plurality of virus particles are about 1×10 per kg of patient 7 to 1×10 17 ; about 1×10 8 to 1×10 16 ; about 1×10 9 to 1×10 15 ; about 1×10 10 to 1×10 14 ; about 1×10 10 to 1×10 13 ; about 1×10 10 to 1×10 13 ; about 1×10 10 to 1×10 11 ; about 1×10 11 to 1×10 12 ; about 1×10 12 to 1×10 13 ; or about 1×10 13 to 1×10 14 vg and are administered in a dose of, the medicament according to claim 24.

27. The pharmaceutical product according to claim 1, wherein the subject is a human.

28. The pharmaceutical product according to claim 1, wherein the subject is a non-human mammal.

29. The pharmaceutical product according to claim 27, wherein the human subject is 50 years of age or older.

30. The pharmaceutical product according to claim 1, wherein the therapeutic transgene is linked to a polyadenylation signal.

31. A modified adeno-associated virus (AAV) encoding a therapeutic transgene functionally linked to a promoter selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a promoter having at least approximately 80% sequence identity with them.

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

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

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

35. A modified AAV according to claim 31, comprising a modified capsid protein.

36. The modified AAV according to claim 35, wherein the modified capsid protein comprises a targeting peptide, the targeting peptide having a length of 3 to 10 amino acids, for example, 7 amino acids.

37. The modified AAV according to 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 according to claim 36, wherein the modified AAV capsid protein is derived from the AAV1 capsid protein, and the targeting peptide is inserted after residue 590 of the AAV1 capsid protein.

39. The modified AAV according to claim 38, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

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

41. The modified AAV according to claim 36, wherein the modified AAV capsid protein is derived from the AAV2 capsid protein, and the targeting peptide is inserted after the 587th residue of the AAV2 capsid protein.

42. The modified AAV according to claim 41, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

43. The modified AAV according to claim 42, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AA at the C-terminus of the targeting peptide.

44. The modified AAV according to claim 36, wherein the modified AAV capsid protein is derived from the AAV9 capsid protein, and the targeting peptide is inserted after the 588th residue of the AAV9 capsid protein.

45. The modified AAV according to claim 44, wherein the targeting peptide is adjacent to a linker sequence, and the linker sequences on both sides of the targeting peptide are 2 or 3 amino acid lengths long.

46. The modified AAV according to claim 45, wherein the linker sequence is AAA at the N-terminus of the targeting peptide and AS at the C-terminus of the targeting peptide.

47. The modified AAV according to claim 31, wherein the therapeutic transgene is siRNA, shRNA, miRNA, non-coding RNA, lncRNA, therapeutic protein, or CRISPR system.

48. The modified AAV according to claim 31, wherein the therapeutic transgene is linked to a polyadenylation signal.

49. The modified AAV according to claim 31, wherein the therapeutic transgene is transcriptionally linked to a detectable reporter, for example, a sequence encoding a fluorescent protein, a peptide tag, or luciferase.

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

51. Isolated and purified nucleic acids containing sequences selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or sequences having at least approximately 80% sequence identity with them.

52. The nucleic acid according to 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 according to 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 according to 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 according to claim 51, wherein the sequence is functionally linked to a heterogeneous coding region.

56. The nucleic acid according to claim 51, further comprising one or more of (a) a multipurpose cloning site, (b) a transcription termination signal, (c) a polyadenylated sequence, and / or (d) an origin of replication.

57. The nucleic acid according to claim 51, further comprising (a) a sequence encoding a detectable marker, (b) a sequence encoding an affinity tag, and / or (c) one or more of one or two adeno-associated virus reverse-terminal repeat sequences.

58. The nucleic acid according to claim 51, contained in a replicable vector.

59. The nucleic acid according to claim 51, wherein the nucleic acid is transcriptionally linked to a reporter by a sequence encoding a 2A "self-cleaving" peptide.

60. A pharmaceutical agent for use in a method of reducing or attenuating microglial inflammation, wherein the pharmaceutical agent comprises the ApoE2 protein or a modified AAV according to any one of claims 31 to 49, and the method comprises the step of delivering ApoE2 to the microglia of a target in need.

61. The pharmaceutical product according to claim 60, wherein the step of delivering ApoE2 to the microglia of the target comprises the step of administering ApoE2 protein or a modified AAV according to any one of claims 31 to 49 to the target, and the therapeutic transgene is ApoE2.

62. The pharmacopoeia according to claim 60, 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 disease, and Creutzfeldt-Jakob disease.

63. The pharmaceutical product according to claim 60, wherein the administration of the ApoE2 protein or modified AAV is by direct intraventricular injection or intraparenchymal injection.

64. The pharmaceutical product according to claim 60, wherein the ApoE2 protein or modified AAV is administered two or more times.

65. The pharmacopoeia according to claim 64, wherein the ApoE2 protein or modified AAV is administered two, three, four, five, six, seven, eight, nine, ten times, or more times.

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

67. The pharmaceutical agent according to claim 60, further comprising the step of providing a non-AAV ApoE2 therapy to the subject.

68. The pharmaceutical product according to claim 60, wherein multiple virus particles are administered.

69. The aforementioned multiple virus particles are approximately 1 × 10⁶ per kilogram. 6 ~Approx. 1×10 18 The pharmaceutical product according to claim 68, administered in a dose of one vector genome (vg / kg).

70. The aforementioned multiple virus particles amount to approximately 1 × 10⁶ per kg of patient weight. 7 ~1 x 10 17 , about 1×10 8 ~1 x 10 16 , about 1×10 9 ~1 x 10 15 , about 1×10 10 ~1 x 10 14 , about 1×10 10 ~1 x 10 13 , about 1×10 10 ~1 x 10 13 , about 1×10 10 ~1 x 10 11 , about 1×10 11 ~1 x 10 12 , about 1×10 12 ~1 x 10 13 , or approximately 1 x 10 13 ~1 x 10 14 The pharmaceutical product according to claim 68, administered in a dose of 1 VG.

71. The pharmaceutical product according to claim 60, wherein the subject is a human.

72. The pharmaceutical product according to claim 60, wherein the subject is a non-human mammal.

73. The pharmaceutical product according to claim 71, wherein the human subject is 50 years of age or older.

74. The pharmaceutical product according to claim 60, wherein the therapeutic transgene is linked to a polyadenylation signal.

75. The pharmacopoeia according to claim 60, wherein the microglial inflammation is caused by or associated with Alzheimer's disease.

Images