Methods to increase transduction of ependyma cells in brain

Abstract

The present disclosure is directed to the delivery of AAV vectors into the brain of a subject by introducing said AAV into the posterior region and / or temporal horn of the lateral ventricle of a subject are described.

Description

DESCRIPTIONMETHODS TO INCREASE TRANSDUCTION OF EPENDYMA CELLS IN BRAINREFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 641,689, filed May 2, 2024, the entire contents of which are hereby incorporated by reference.REFERENCE TO A SEQUENCE LISTING

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on May 2, 2025, is named CHOPPOOSOWO.xml and is 53,702 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates generally to the fields of gene therapy, medicine and brain biology. More particularly, the disclosure relates to the development AAV vectors targeting specific regions of the brain for deep brain gene therapy, and methods of delivering such vectors to specific regions of the brain by focused administration.2. Background

[0004] Lysosomal storage disorders (LSDs) are genetic diseases that result from deficiencies in individual enzymes that break down carbohydrates or lipids, leading to accumulation of undigested substrates within cells (Platt et al., 2018). In many cases, the peripheral manifestations of these disorders can be addressed by enzyme replacement therapy (ERT) where the enzyme is periodically infused, generally bi-weekly or monthly, into the bloodstream for uptake by affected organs (Fernandez-Pereira et al., 2021). Tissues that are difficult to treat in this manner include the brain, because ERT generally does not cross the blood-brain barrier. This can be overcome, in part, by direct infusion into the cerebrospinal fluid (CSF) through a chronically implanted device; however, periodic infusions require patients to live close to infusion centers, and the implanted device is prone to serious infections (Ziegler et al., 2011). The cross-correction properties of many lysosomal enzymes make them attractive candidates for gene therapy (Barton et al., 1971 ; Sands et al., 2006; Chang et al.,2008; Passini et al., 2006), because genetically corrected cells can synthesize and secrete product for uptake by other cells (Sands et al., 2006). Ependymal cells, the epithelial cells that line the ventricular spaces of the brain, provide an ideal site for secretion of recombinant proteins into the CSF and distribution throughout the brain, aided by perivascular spaces (Ichimura et al., 1991 ; Wolak et al., 2013; Liu et al., 2005). One LSD for which cross correction can be beneficial is late infantile neuronal ceroid lipofuscinosis (CLN2) disease, which is caused by recessively inherited mutations in TPP1, which encodes tripeptidylpeptidase 1. TPP1 is a soluble lysosomal hydrolase that when absent causes a debilitative and fatal childhood neurological disease (Sleat et al., 1997). TPP1 is synthesized as a proenzyme that is activated to the holoenzyme at low pH in the lysosome. When over-expressed, the pro-enzyme is secreted and can enter non-expressing cells by receptor mediated endocytosis, resulting in cross-correction (Sohar et al., 1999; Sondhi et al., 2005). Indeed, prior work in a canine model of CLN2 disease showed that TPP1 secretion from transduced ependyma could markedly extend lifespan and reduce symptoms through brain-wide biodistribution of the secreted product (Katz et al., 2015).

[0005] Cerebrospinal fluid is produced by the choroid plexus located in the lateral ventricles of the brain. Flow in the lateral ventricle moves caudal to rostral then down through the third and fourth ventricles toward the spinal column. The most common surgical access point for the brain’s ventricular system occurs at the anterior horn, the most rostral portion of the lateral ventricle. This is the location where shunts are placed to treat hydrocephalus and where the FDA-approved drug Brineura, a tripeptidyl peptidase 1 (TPP1) protein therapeutic, is delivered to treat CLN2 disease. Experimental AAV therapies are also commonly targeted to the anterior lateral ventricle, although no information is available on whether more optimal localized administration in this general region can be achieved.SUMMARY

[0006] Thus, in accordance with the present disclosure, there are provided methods of administering an adeno-associated virus (AAV) to the brain comprising introducing said AAV into the posterior region and / or temporal horn of the lateral ventricle of a subject. Also provided is a method of expressing a heterologous gene in the brain, comprising introducing an AAV comprising a genome that carries a coding sequence for the heterologous gene into the posterior region and / or temporal horn of the lateral ventricle of a subject thereby expressing the heterologous gene in the brain.

[0007] The modified AAV may comprise a modified capsid protein. The modified capsid protein may comprise a targeting peptide, which may be three to ten amino acids in length. The targeting peptide may be seven amino acids in length. The AAV may comprise a modified capsid protein comprising the targeting sequence of any one of SEQ ID NOs: 7-10. The modified AAV may be derived from AAV1, AAV2, or AAV9. The targeting sequence may be inserted near or after position 590 of SEQ ID NO: 1 or after position 587 of SEQ ID NO: 2 or after position 588 of SEQ ID NO: 3. The targeting sequence may be flanked by linker sequences, wherein the linker sequences on each side of the targeting sequence may be two or three amino acids long. The linker sequences may be SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence, or the linker sequences may be AAA on the N-terminal side of the targeting sequence and AA on the C- terminal side of the targeting sequence, or the linker sequences may be AAA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence. The modified capsid protein may comprise the sequence of any one of SEQ ID NOs: 4-6. The capsid protein may consist of the sequence of any one of SEQ ID NOs: 4-6.

[0008] The modified AAV capsid protein may be derived from an AAV 1 capsid protein (e.g., SEQ ID NO: 1), where the targeting peptide is inserted after residue 590 of the AAV1 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. For example, 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. The modified AAV1 capsid proteins may have a sequence at least 95% identical to SEQ ID NO: 4. The targeting peptide may be one of the peptides of SEQ ID NOs: 7-10. The targeting peptide may be SEQ ID NO: 7.

[0009] The modified AAV capsid protein may be derived from an AAV2 capsid protein (e.g., SEQ ID NO: 2), where the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. For example, 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. The modified AAV2 capsid proteins may have a sequence at least 95% identical to SEQ ID NO: 5. The targeting peptide may be one of the peptides of SEQ ID NOs: 7-10. The targeting peptide may be SEQ ID NO: 8 or 9.

[0010] The modified AAV capsid protein may be derived from an AAV9 capsid protein (e.g., SEQ ID NO: 3), where the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. The targeting peptide may be flanked by linker sequences, wherein the linker sequences on each side of the targeting peptides are two or three amino acids long. For example, 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. The modified AAV9 capsid proteins may have a sequence at least 95% identical to SEQ ID NO: 6. The targeting peptide may be one of the peptides of SEQ ID NOs: 7-10. The targeting peptide may be SEQ ID NO: 10.

[0011] The AAV maybe introduced more than once, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations, and / or administering occurring monthly, every other month, every two months, every three months, every four months, every six months, annually every other year, every three years, every four year or every five years. A plurality of AAV particles may be administered, such as about IxlO6to about IxlO14AAV vector genomes per kilogram (vg / kg), or a dosing from about 1X107-1X1014, about IxlO xlO14, about 1X109-1X1014, about IxlO10- IxlO14, about 1X 101O-1X1013, about 1X1O1O-1X1O13, about 1X1010-1X 1011, about lxlOn-lxlO12, or about 1X1012-1X1013AAV vector genomes per kilogram (vg / kg) of the patient. The subject may suffer from Epileptic encephalopathy and the AAV carries a coding sequence for Syntaxin-binding protein 1 (STXBP1). The subject may suffer from Batten’s Disease and the AAV carries a coding sequence for tripeptidyl peptidase- 1 (TPP1), and optionally the administering results in a reduction or improvement in a symptom in said subject caused by Batten’s Disease. The subject may be a human, a non-human primate, a non-primate mammal. The methods may be performed by introducing the AAV by way of bilateral administration.

[0012] Also provided are (a) a use of an adeno-associated virus (AAV) for delivery into the posterior region and / or temporal horn of the lateral ventricle of a subject; (b) a use of an adeno-associated virus (AAV) for delivery into the posterior region and / or temporal horn of the lateral ventricle of a subject for the treatment of a brain disease, such as Batten Disease or Epileptic encephalopathy.

[0013] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0014] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. 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 the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The 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.

[0016] FIGS. 1A-1C. AAV capsid library enrichment and candidate selection. (A) Schematic depicting the AAV capsid selection pipeline. AAV1, AAV2, and AAV9 libraries were generated that contained a semi-random heptapeptide insert within the variable region of loop 8, yielding an initial ~6.8 million capsid variants (1.3M for AAV1-, 2.3M for AAV2- and 3.25M for AAV9-based libraries). Variant enrichment was achieved by two sequential passages through a rhesus macaque brain. The first primate (black arrows) received a single ICV infusion of the Round 1 input library containing AAV capsid libraries from three parental serotypes. DNA extracted from collected tissues from the first animal and recovered AAV variant sequences were used to generate a Round 2 AAV input library that was delivered by ICV infusion into a second rhesus macaque from which both viral DNA and cellular RNA were collected. Successfully enriched capsids were identified using Illumina sequencing of viral amplicons. Capsid performance was assessed using a combination of total detection in target tissues and enrichment, which was defined as the ratio of a capsid’s relative abundance in a target region vs the input library. (B) Exemplar heatmap showing AAV1 capsid variant abundance rankings for DNA data; rank 1 = highest UMI count (out of 7900 AAV1 variants above threshold in these regions). Cx cerebral cortex, Cb cerebellum, HC hippocampus, Th thalamus, Ep ependyma. (C) Four highly enriched capsid variants were selected for fluorescence validation. Selected capsids expressing fluorescent payloads were individually cloned and produced, then pooled for subsequent validation. NHP images from A and C are from BioRender.com

[0017] FIGS. 2A-2C. AAV-Ep reporter evaluation. (A) Fluorescent reporter expression detection in the lateral (top) and fourth (bottom) ventricles of an adult rhesus macaque that received a unilateral ICV infusion of the four top candidate capsids 4 weeks earlier. DIC (differential interference contrast microscopy), AAV-EP1 (AAV1 variant; CAG.mTFPl), AAV-Ep2 (AAV2 variant: CAG.mRuby3, AAV-Ep3 (AAV2 variant; mNG), and AAV-Ep4 (AAV9 variant; CAG:BFP). (B) Fluorescent reporter expression for AAV-Epl in extra- ependymal regions of the same animal examined in (A). Scale bars are 50 pm. Coronal images are adapted from (38). (C) Depiction depicting the site of infusion (magenta arrow) into the right anterior lateral ventricle.

[0018] FIGS. 3A-3B. Low peripheral off-target expression of transgenes delivered by AAV-Ep+ in a rhesus macaque. (A) RNA-FISH detection of mTFPl transcript in liver and heart. (B) RNA-FISH detection of mTFPl transcripts in cervical, thoracic and lumbar DRGs from the same animal examined in (A). UBC detection included as a positive assay control. Nuclei are stained blue with DAPI. Scale bars are 50 pm.

[0019] FIGS. 4A-4E. Posterior lateral ventricle infusion of AAV-Ep+ improves ependymal transduction over anterior infusion in African green monkeys. (A) Depiction of the NHP ventricular system. Arrows indicate anterior (magenta) and posterior (cyan) injection sites. (B) Representative high magnification images of Hoechst (left) and mRuby3 (right) fluorescence of AGM ependyma in two locations in the lateral ventricles and the third ventricle 4 weeks after delivery of AAV-Ep+.mRuby3 at anterior (top) or posterior (bottom) coordinates. (C) Quantification of ependymal cell transduction at indicated locations. Dots indicate individual animals. (D) Representative images of mRuby3 fluorescence in parenchymal areas 4 weeks after posterior ICV delivery. (E) Representative images of mRuby3 fluorescence in cynomolgus monkey ependyma 4 weeks after infusion of AAV-Ep+.mRuby3 in the posterior lateral ventricle. Scale bar is 50 pm in A-D, 500 pm in E.

[0020] FIGS. 5A-5D. AAV-Ep+ transduces mouse brain ependyma and parenchyma and human iPSC-derived cortical neurons. (A) Representative tile scan and high magnification images demonstrating Hoechst (blue) and mRuby3 (red) fluorescence in the brain of an adult FVB mouse infused 4 weeks earlier with AAV-Ep+.mRuby3 at 1E+10 vg. Similar transduction profiles were observed in three animals. Scale bars: 1 mm or 100 pm (zoom). (B) Representative images of the hippocampus (left) and subiculum (right) demonstrating mRuby3 fluorescence in neurons. Scale bars: 100 pm. (C) Representative Hoechst (blue), mRuby3 (red)or MAP2 (green) fluorescence in human iPSCderived cortical neurons 10 days after transduction with AAV-Ep+.mRuby3 or AAVl.mRuby3 at indicated MOIs. Scale bars are 50 pm. (D) Transgene expression quantified by RT-qPCR. All samples were normalized to GAPDH and are presented relative to AAVl.eGFP at a dose of 1E+3. Each dot represents a biological replicate, p < 0.0001 for AAV-Ep+ vs. AAV1 at 1+E4 and 1+E5, respectively; two- way Anova with Bonferroni’s correction.

[0021] FIGS. 6A-6E. Functional correction of Cln2- / - mice and hTPPl expression in NHPs with AAVEp+. hTPPl treatment. (A) TPP1 enzymatic activity in tissue lysates from mice infused with 5E+10 vg AAV-Ep+ at 7 weeks of age and harvested 5 weeks later (Cnl2- / - + AAV-Ep+.hTPPl n = 10, Cln2- / - = 6, Cln2+ / - = 8). No enzymatic activity was observed in vehicle-treated Cln2- / - mice (N.D., not detected). (****p<0.0005, by two-tailed Mann- Whitney U test. (B) Resting tremor amplitude at the indicated frequencies during a five-minute recording interval (Cln2- / - + AAV-Ep+.hTPPl n - 10, Cln2- / - = 7, Cln2+ / - - 9 at 12-weeks, Cln2- / - + AAV-Ep+.hTPPl n = 8, Cln2- / - = 7 , Cln2+ / - = 9 at 13 weeks, and Cln2- / - + AAV- Ep+.hTPPl n = 6, Cln2- / - = 5 , Cln2+ / - = 9 at 16 weeks). Panel B, lower right graph represents area under the curve of whole resting tremor amplitude spectrum (0-63 Hz) over time for each treatment group that was analyzed (* p<0.05, f p<0.01 by two-way ANOVA [mixed-effect model] followed by Bonferroni multiple comparisons post-hoc test). (C) Survival curves for mice treated with 5E+10 vg AAV-Ep+.hTPPl at 7-weeks of age (Cln2- / - + AAVEp+. hTPPl n = 10, Cln2- / - = 10, Cln2+ / - = 9, * p<0.05 by log rank Mantel-Cox test). (D) Pro-TPPl protein abundance in CSF normalized to each animal’s baseline values from 1, 2, and 4 weeks after ICV injection of AAV-Ep+.hTPPl into cynomolgus monkeys (5E+11 vg AAVEp+. hTPP 1 / hemisphere, 1 E+ 12 vg total). (E) Human TPP concentrations (ng / mL) in CSF expressed from AAV-Ep+.hTPPl-treated NHP compared to endogenous TPP1 in 9 human CSF samples. All data are expressed as mean + SEM.

[0022] FIGS. 7A-7F. Analysis pipeline flowchart. (A and B) Following Illumina sequencing of PM- AAV amplicon libraries, reads were parsed to identify properly formed amplicon reads by testing for perfect string matches to serotype specific constant regions flanking the nucleic acid sequences encoding the heptamer peptide insert. (C) Variant abundance was quantified as the UMI-collapsed read count of unique heptamer-encoding nucleic acid sequences. (D) In addition to abundance (UMIs), an enrichment metric was also calculated that quantifies the fold change in a variant's relative (percent) abundance in tissuesamples vs the input vector. (E and F) Variant performance was assessed using single-region (targeted) and cross-region analyses of DNA and RNA data. No single metric / analysis was used to identify top performing capsids for validation. The totality of available data was considered when selecting AAV-Ep candidates.

[0023] FIG. 8. Comparison of AAV-Ep+ and parental AAV1 performance. Box and whisker plots comparing transduction (UMI counts) of ependymal tissue by AAV-EP+ vs wildtype parental capsid, AAV 1 in a NHP (Table 1). Data come from sequencing of 10 ependymal tissue samples taken from the lateral ventricle (n=4 DNA, n=4 RNA), third ventricle (n=l RNA), and fourth ventricle (n=l RNA). Transduction efficiency for AAV-EP+ vs WT-AAV1 was measured within each dataset. The average of these fold change values for the 10 ependymal tissue datasets was 22.05.

[0024] FIG. 9. Comparative ependymal cell transduction efficiency in African green monkeys after ICV injection of AAV-Ep+ or AAV9. Two African green monkeys were ICV injected with AAV-Ep+.mRuby3 and two with AAV9.mRuby3, each at a dose of 1.0E+13 vg, into the posterior lateral ventricle. mRuby3 positive ependymal cells were quantified from 40pm thick brain slices taken every 4 mm.

[0025] FIGS. 10A-10B. AAV-Ep+ transgene expression in AGM DRGs. (A) RNA- FISH detection of AAV-Ep+-derived mRuby3 transcripts in cervical (top), thoracic (middle), and lumbar (bottom) DRGs in two AGMs infused with 1E+13 vg. (B) mRuby3 direct fluorescence in brain ventricle ependyma cells transduced with AAV-Ep+.mRuby3. Scale bar: 50 pm. Left and right grouped panels (A, B) are two separate animals.

[0026] FIGS. 11A-11C. Transduction pattern of AAV-Ep+ in cynomolgus macaque. Representative images of mRuby3 fluorescence following bilateral infusion of AAV- Ep+.mRuby3 at 5E+12 vg (1E+13 vg total) to the posterior lateral ventricle. (A) Positive cells in the ependyma of the lateral ventricles (Lvs). (B) Positive cells in the ependyma of the lateral ventricle and cerebral aqueduct (Aq) (C) Positive cells in the fourth ventricle (4v). Scale bars, 500 pm. Microscopy images in B are the same as in 4E.

[0027] FIGS. 12A-12C. AAV-Ep+ tissue transduction patterns are conserved across mouse strains. (A) Tile scan and high magnification images showing AAV-Ep+ transduction in the brain of an adult C57B16 mouse infused with 1E+10 vg. Fluorescence distribution was similar in three animals. Asterisks highlight regions of strong ependymal transduction. (B)AAV-Ep+-associated expression in extra-ependymal areas of adult C57B16 / J mice following ICV infusion (C) Direct fluorescence of AAV-Ep+ expression in cortical areas of adult FVB mice. Scale bars: 100 pm in panels. Left panel in A is 1 mm.

[0028] FIG. 13. Relative TPP1 activity in the brain parenchyma of TPP1 -deficient mice treated with AAV-Ep+.hTPPl, compared to data from TPP1 -deficient canines treated with AAV2.caTPPl. TPP1 activity in the striatum, thalamus, cerebellum and medulla oblongata in Cln2-I- mice after AAV-Ep+.hTPPl infusion (same data as in FIG. 6A) and in TPP1 -deficient dogs treated with AAV2.caTPP. Recombinant TPP1 activities were normalized to endogenous TPP1 activity in heterozygous mice or wild type dogs, respectively.

[0029] FIGS. 14A-14C. Bilateral infusion of AAV-EP+.mRuby3 into the posterior lateral ventricles of each hemisphere yields uniform distribution of ependymal transduction. FIG. 14A is a representative native fluorescence image showing a cross section of the anterior lateral ventricles from both hemispheres of a primate brain infused bilaterally with AAV- EP+.mRuby3 into the posterior lateral ventricles. The images in columns FIGS. 14B and 14C show enlarged merged and individual channels illustrating broad and robust transduction of the ependymal cells lining the lateral ventricles in both hemispheres.DETAILED DESCRIPTION

[0030] As discussed above, the most common surgical access point for the brain’s ventricular system occurs at the anterior horn, the most rostral portion of the lateral ventricle. This is the location where shunts are placed to treat hydrocephalus and it has also served as the point for introduction of experimental AAV treatments.

[0031] The inventors previously developed a modified AAV capsid, Ep+, that strongly targets the ependyma, the single layer of epithelial cells lining the walls of the ventricle. Using this capsid, they sought to transduce these cells for translation and release of secreted proteins into the CSF for broad distribution throughout the brain. The inventors hypothesized that delivery in the posterior lateral ventricle would increase dwell time and exposure to more of the ventricular space, and improve overall ependyma transduction as compared to anterior delivery, although no information on this sort of approach was known.

[0032] Using a primate model, anterior or posterior intracerebroventricular infusion of AAV-Ep+ was tested. Transduction quantification was performed using automated cellcounting across images from four animals. The inventors found that the site of ventricular infusion strongly impacts ependymal transduction by AAV-Ep+. The occipital infusion meaningfully improved ependymal cell transduction particularly in the posterior region and temporal horn of the lateral ventricle.

[0033] Lysosomal storage disorders (LSDs) are genetic diseases that result from deficiencies in individual enzymes that break down carbohydrates or lipids, leading to accumulation of undigested substrates within cells (Platt et al., 2018). In many cases, the peripheral manifestations of these disorders can be addressed by enzyme replacement therapy (ERT) where the enzyme is periodically infused, generally bi-weekly or monthly, into the bloodstream for uptake by affected organs (Fernandez-Pereira et al., 2021). Tissues that are difficult to treat in this manner include the brain, because ERT generally does not cross the blood-brain barrier. This can be overcome, in part, by direct infusion into the cerebrospinal fluid (CSF) through a chronically implanted device; however, periodic infusions require patients to live close to infusion centers, and the implanted device is prone to serious infections (Ziegler et al., 2011). The cross-correction properties of many lysosomal enzymes make them attractive candidates for gene therapy (Barton et al., 1971 ; Sands et al., 2006; Chang et al., 2008; Passini et al., 2006), because genetically corrected cells can synthesize and secrete product for uptake by other cells (Sands et al., 2006). Ependymal cells, the epithelial cells that line the ventricular spaces of the brain, provide an ideal site for secretion of recombinant proteins into the CSF and distribution throughout the brain, aided by perivascular spaces (Ichimura et al., 1991 ; Wolak et al., 2013; Liu et al., 2005). One LSD for which cross correction can be beneficial is late infantile neuronal ceroid lipofuscinosis (CLN2) disease, which is caused by recessively inherited mutations in TPP1, which encodes tripeptidylpeptidase 1. TPP1 is a soluble lysosomal hydrolase that when absent causes a debilitative and fatal childhood neurological disease (Sleat et al., 1997). TPP1 is synthesized as a proenzyme that is activated to the holoenzyme at low pH in the lysosome. When over-expressed, the pro-enzyme is secreted and can enter non-expressing cells by receptor mediated endocytosis, resulting in cross-correction (Sohar et al., 1999; Sondhi et al., 2005). Indeed, prior work in a canine model of CLN2 disease showed that TPP1 secretion from transduced ependyma could markedly extend lifespan and reduce symptoms through brain-wide biodistribution of the secreted product (Katz et al., 2015).

[0034] To translate this work clinically, the inventors undertook a screen to identify adeno-associated virus (AAV) capsid variants that effectively targeted ependymal cells for CSF distribution as well as transduced parenchymal neurons for enhanced effects. The inventors developed capsid libraries based on a number of AAV serotypes, infused the library mix into wildtype, adult old world nonhuman primates (NHPs), and, after 2 rounds of enrichment, identified a lead candidate, AAV-Ep+, that robustly transduced ependymal cells throughout the entire ventricular system. Moreover, AAV-Ep+ transduced neurons throughout brain cortices and other brain regions, with similar transduction patterns in three different adult NHP species, in two mouse strains and in human neurons derived from induced pluripotent stem cells (iPSCs), demonstrating, cross-species tropism. In NHPs, there was limited gene expression in sampled dorsal root ganglia, a common site of toxicity seen with AAV9-based vectors administered to the CSF (Hordeaux et al., 2020)

[0035] When applied to a murine model of CLN2 disease, AAV-Ep+.hTPPl improved the disease phenotype and prolonged lifespan at doses lower than used in previous publications using wild type capsids. When evaluated in healthy NHPs, AAV-Ep+.hTPPl generated hTPPl protein concentrations in CSF and parenchyma that were 9-fold greater than in CSF from children without CLN2 disease, supporting the translatability of this approach for CLN2 disease gene therapy as well as other disorders that would benefit from steady state protein delivery to the CSF.

[0036] ICV delivery of ependyma- specific AAV capsids enables broad distribution of lysosomal enzymes for therapeutic benefit in mouse and dog models of LSDs (Liu et al., 2005; Katz et al., 2015). Herein, the inventors identified AAV capsid variants enriched for transduction of NHP ependyma and brain parenchyma and showed that the lead candidate, V-Ep+, could improve survival and tremor in a mouse model of CLN2 disease. Further, ICV delivery of AAV-Ep+.TPPl to NHPs resulted in similar or greater concentrations of proTPPl in the CSF as observed after intracisterna magna delivery of AAV.rhlO (Sondhi et al., 2005) with 50x less virus, or AAV9 (Buss et al., 2022) with 30x less virus.

[0037] The inventors screened capsids in NHPs because selection strategies in murine model systems do not translate to larger mammals (Matsuzaki et al., 2018; Liguore et al., 2019). The initial screening library was comprised of variants derived from three different parental serotypes (AAV1, AAV2 and AAV9), all known to transduce the brain. The top variant was derived from AAV1 rather than the more commonly used AAV9, highlighting the value ofincluding multiple serotypes in capsid-discovery efforts. Similar to our other capsid enrichment campaign (Leib et al., 2024), the NHP-derived top candidate (AAV-Ep+) achieved high on- target and low off-target transduction. AAV-Ep-i- transduced large portions of cells lining the ventricles with greater than 20-fold higher potency than its parental serotype, AAV 1 , and with even higher efficiency relative to AAV9. The high transduction efficiency in mice, multiple NHP species, and human iPSC neurons supports the clinical translation of AAV-Ep+.

[0038] Another feature of the screen was the choice of the ICV route of administration to better expose vector to the largely postmitotic ependymal cells (Spassky et al., 2005; Rodrigo et al., 2023) and brain parenchyma for secretion of therapeutic molecules into the CSF. The location of infusion impacts transduction efficiency, with increased transduction being achieved when the direction of CSF flow was considered. It is presumably due to the direction of CSF flow that AAVs delivered via the cisterna magna do not efficiently transduce brain (Hinderer et al., 2014).

[0039] Potential applications for AAV-Ep-i- include treatment of genetic diseases caused by loss of enzyme function, including LSDs wherein the deficient enzyme is a soluble lysosomal hydrolase, such as TPP1 as shown here. AAV-based therapies applied thus far for CLN2 disease utilize direct parenchymal injection of AAV2 or AAVrh.10, with more recent efforts delivering AAVrh.10 via intracisterna magna injection (Sondhi et al., 2020; De et al., 2023). AAV-Ep+-based gene therapies would be delivered via ICV injection, a well- established method for delivering therapeutics to the central nervous system that results in broad brain distribution (Cohen- Pfeffer et al., 2017). In previous work, the inventors treated TPP1 -deficient dogs with 1.7E+13 vg of AAV2.caTPPl, which resulted in normalized enzyme activity in the brain, markedly extended lifespans and improved motor skills and social interactions (Katz et al., 2015). Unlike AAV2, AAV-Ep-i- transduced brain parenchyma as well as ependyma, which may provide greater therapeutic benefit with a similar approach and at lower doses.

[0040] The lower vector genome doses permitted by AAV-Ep-i- may lead to an improved safety profile and larger therapeutic index than conventional AAV gene therapies. Beyond the treatment of LSDs, AAV-Ep+ may also be suitable for expression of nanobodies or other small, secreted antibody fragments to target extracellular proteins or intracellular disease targets, or to increase their effect via cross-correction from transduced cells in the brain (Marino et al., 2022; Goodwin et al., 2021; Marino et al., 2022). It may also be well suited forbroad distribution of secreted growth factors as a treatment for Parkinson’s disease or amyotrophic lateral sclerosis (Parambi et al., 2022). AAV-Ep+ is a potent vector for gene therapy applications where CSF protein expression is required.

[0041] These and other aspects of the disclosure are described in detail below.I. AAV Vectors

[0042] Adeno-associated virus (AAV) is a small nonpathogenic virus of the parvoviridae family. To date, numerous serologically distinct AAVs have been identified, and more than a dozen have been isolated from humans or primates. AAV is distinct from other members of this family by its dependence upon a helper virus for replication.

[0043] AAV genomes can exist in an extrachromosomal state without integrating into host cellular genomes; possess a broad host range; transduce both dividing and non-dividing cells in vitro and in vivo and maintain high levels of expression of the transduced genes. AAV viral particles are heat stable; resistant to solvents, detergents, changes in pH, and temperature; and can be column purified and / or concentrated on CsCl gradients or by other means. The AAV genome comprises a single-stranded deoxyribonucleic acid (ssDNA), either positive- or negative-sensed. The approximately 4.7 kb genome of AAV consists of one segment of single stranded DNA of either plus or minus polarity. The ends of the genome are short-inverted terminal repeats (ITRs) that can fold into hairpin structures and serve as the origin of viral DNA replication.

[0044] An AAV “genome” refers to a recombinant nucleic acid sequence that is ultimately packaged or encapsulated to form an AAV particle. An AAV particle often comprises an AAV genome packaged with AAV capsid proteins. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, 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 cloning and amplification of the plasmid, a process that is needed for plasmid propagation and production but is not itself packaged or encapsulated into viral particles. Thus, an AAV vector “genome” refers to nucleic acid that is packaged or encapsulated by AAV capsid proteins.

[0045] The AAV virion (particle) is a non-enveloped, icosahedral particle approximately 25 nm in diameter that comprises an AAV capsid. The AAV particle comprises an icosahedral symmetry comprised of three related capsid proteins, VP1 , VP2 and VP3, which interact together to form the capsid. The genomes of most native AAVs often contain two open reading frames (ORFs), sometimes referred to as a left ORF and a right ORF. The right ORF often encodes the capsid proteins VP1, VP2, and VP3. These proteins are often found in a ratio of 1 :1:10 respectively, but may be in varied ratios, and are all derived from the right-hand ORF. The VP1 , VP2 and VP3 capsid proteins differ from each other by the use of alternative splicing and an unusual start codon. Deletion analysis has shown that removal or alteration of VP1 which is translated from an alternatively spliced message results in a reduced yield of infectious particles. Mutations within the VP3 coding region result in the failure to produce any singlestranded progeny DNA or infectious particles. The genome of an AAV particle may encode one, two or all three VP1, VP2 and VP3 polypeptides.

[0046] The left ORF often encodes the non- structural Rep proteins, Rep 40, Rep 52, Rep 68 and Rep 78, which are involved in regulation of replication and transcription in addition to the production of single-stranded progeny genomes. Two of the Rep proteins have been associated with the preferential integration of AAV genomes into a region of the q arm of human chromosome 19. Rep68 / 78 have been shown to possess NTP binding activity as well as DNA and RNA helicase activities. Some Rep proteins possess a nuclear localization signal as well as several potential phosphorylation sites. In one example, the genome of an AAV (e.g., an rAAV) encodes some or all of the Rep proteins. In another example the genome of an AAV (e.g., an rAAV) does not encode the Rep proteins. In some aspects of the present disclosure one or more of the Rep proteins can be delivered in trans and are therefore not included in an AAV particle comprising a nucleic acid encoding a polypeptide.

[0047] The ends of the AAV genome comprise short-inverted terminal repeats (ITR) which have the potential to fold into T-shaped hairpin structures that serve as the origin of viral DNA replication. Accordingly, the genome of an AAV comprises one or more (e.g., a pair of) ITR sequences that flank a single stranded viral DNA genome. The ITR sequences often have a length of about 145 bases each. Within the ITR region, two elements have been described which are believed to be central to the function of the ITR, a 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 forcleavage at the trs which occurs in a site- and strand-specific manner. In addition to their role in replication, these two elements appear to be central to viral integration. Contained within the chromosome 19 integration locus is a Rep binding site with an adjacent trs. These elements have been shown to be functional and necessary for locus specific integration.

[0048] The term “recombinant,” as a modifier of vector, such as recombinant viral, e.g., lentivirus or parvovirus (e.g., AAV) vectors, as well as a modifier of sequences such as recombinant nucleic acid sequences and polypeptides, means that the compositions have been manipulated (i.e., engineered) in a fashion that generally does not occur in nature. A particular example of a recombinant vector, such as an AAV, retroviral, or lentiviral vector would be where a nucleic acid sequence that is not normally present in the wild-type viral genome is inserted within the viral genome. An example of a recombinant nucleic acid sequence would be where a nucleic acid (e.g., gene) encodes an inhibitory RNA cloned into a vector, with or without 5', 3' and / or intron regions that the gene is normally associated within the viral genome. Although the term “recombinant” is not always used herein in reference to vectors, such as viral vectors, as well as sequences such as polynucleotides, “recombinant” forms including nucleic acid sequences, polynucleotides, transgenes, etc. are expressly included in spite of any such omission.

[0049] A recombinant viral “vector” is derived from the wild-type genome of a virus by using molecular methods to remove part of the wild-type genome from the virus, and replacing it with a non-native nucleic acid, such as a nucleic acid sequence. Typically, for example, for 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, since part of the viral genome has been replaced with a non-native sequence with respect to the viral genomic nucleic acid such a nucleic acid encoding a transactivator or nucleic acid encoding an inhibitory RNA or nucleic acid encoding a therapeutic protein. Incorporation of such non-native nucleic acid sequences therefore defines the viral vector as a “recombinant” vector, which in the case of AAV can be referred to as a “rAAV vector.”

[0050] An AAV (e.g., a rAAV) may comprise two ITRs. An AAV (e.g., a rAAV) may comprise a pair of ITRs. An AAV (e.g., a rAAV) may comprise a pair of ITRs that flank (i.e., are at each 5' and 3' end) of a nucleic acid sequence that at least encodes a polypeptide having function or activity.

[0051] An AAV vector (e.g., rAAV vector) can be packaged and is referred to herein as an “AAV particle” for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector is encapsulated or packaged into an AAV particle, the particle can also be referred to as a “rAAV particle.” An AAV particle may be a rAAV particle. A rAAV particle often comprises a rAAV vector, or a portion thereof. A rAAV particle can be one or more rAAV particles (e.g., a plurality of AAV particles). rAAV particles typically comprise proteins that encapsulate or package the rAAV vector genome (e.g., capsid proteins). It is noted that reference to a rAAV vector can also be used to reference a rAAV particle.

[0052] Any suitable AAV particle (e.g., rAAV particle) can be used for a method or use herein. A rAAV particle, and / or genome comprised therein, can be derived from any suitable serotype or strain of AAV. A rAAV particle, and / or genome comprised therein, can be derived from two or more serotypes or strains of AAV. Accordingly, a rAAV can comprise proteins and / or nucleic acids, or portions thereof, of any serotype or strain of AAV, wherein the AAV particle is suitable for infection and / or transduction of a mammalian cell. Nonlimiting examples of AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-rh74, AAV-rhlO and AAV-2i8.

[0053] In some aspects of the present disclosure, a plurality of rAAV particles comprises particles of, or derived from, the same strain or serotype (or subgroup or variant). In yet another aspect of the present disclosure, a plurality of rAAV particles comprise a mixture of two or more different rAAV particles (e.g., of different serotypes and / or strains).

[0054] As used herein, the term “serotype” is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants may not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0055] A rAAV vector may be based upon a first serotype genome corresponds to the serotype of one or more of the capsid proteins that package the vector. For example, theserotype of one or more AAV nucleic acids (e.g., ITRs) that comprises the AAV vector genome corresponds to the serotype of a capsid that comprises the rAAV particle.

[0056] A rAAV vector genome may be based upon an AAV (e.g., AAV2) serotype genome distinct from the serotype of one or more of the AAV capsid proteins that package the vector. For example, a rAAV vector genome can comprise AAV1 derived nucleic acids (e.g., ITRs), whereas at least one or more of the three capsid proteins are derived from a different serotype, e.g., an AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 or AAV-2i8 serotype or variant thereof.

[0057] In some aspects of the present disclosure, a rAAV particle or a vector genome thereof related to a reference serotype has a polynucleotide, polypeptide or subsequence thereof that comprises or consists of a sequence 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, RhlO, Rh74 or AAV-2i8 particle. In particular embodiments, a rAAV particle or a vector genome thereof related to a reference serotype has a capsid or ITR sequence that comprises or consists of a sequence at least 60% or more (e.g., 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to a capsid or ITR sequence of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 or AAV-2i8 serotype.

[0058] A method herein may comprise use, administration or delivery of an rAAVl, rAAV2, rAAV3, rAAV4, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, rAAVIO, rAAVl 1, rAAV12, rRhlO, rRh74 or rAAV-2i8 particle.

[0059] A method herein may comprise use, administration or delivery of a rAAVl particle. In some aspects of the present disclosure, a rAAVl particle comprises an AAV1 capsid. In another aspects of the present disclosure, a rAAV 1 particle comprises one or more capsid proteins (e.g., VP1, VP2 and / or VP3) that are at least 60%, 65%, 70%, 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 a corresponding capsid protein of a native or wild-type AAV1 particle. In certain aspects of the present disclosure a rAAV 1 particle comprises VP1 , VP2 and VP3 capsid proteins that are at least 75%or more identical, e.g., 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 to a corresponding capsid protein of a native or wild-type AAV1 particle. In another aspect, a rAAV2 particle is a variant of a native or wild-type AAV1 particle. In some aspects, one or more capsid proteins of an AAV2 variant have 1, 2, 3, 4, 5, 5-10, 10-15, 15-20 or more amino acid substitutions compared to capsid protein(s) of a native or wild-type AAV1 particle.

[0060] A rAAV particle may comprise 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 corresponding ITRs of a native or wild-type AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV-rh74, AAV-rhl O or AAV- 2i8, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired).

[0061] A rAAVl particle may comprise 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 corresponding ITRs of a native or wild-type AAV 1 particle, as long as they retain one or more desired ITR functions (e.g., ability to form a hairpin, which allows DNA replication; integration of the AAV DNA into a host cell genome; and / or packaging, if desired).

[0062] A rAAV particle can comprise an ITR having any suitable number of “GAGC” repeats. In certain aspects, an ITR of an AAV2 particle comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more “GAGC” repeats. In another aspect, a rAAV2 particle comprises an ITR comprising three “GAGC” repeats. In yet another aspect, a rAAV2 particle comprises an ITR which has less than four “GAGC” repeats. In certain embodiments a rAAV2 particle comprises an ITR which has more than four “GAGC” repeats. In certain embodiments an ITR of a rAAV2 particle comprises a Rep binding site wherein the fourth nucleotide in the first two “GAGC” repeats is a C rather than a T.

[0063] Exemplary suitable length of DNA can be incorporated in rAAV vectors for packaging / encapsidation into a rAAV particle can about 5 kilobases (kb) or less. In particular,embodiments, length of DNA is less than about 5kb, 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.

[0064] rAAV vectors that include a nucleic acid sequence that directs the expression of an RNAi or polypeptide can be generated using suitable recombinant techniques known in the art (e.g., see Sambrook et al., 1989). Recombinant AAV vectors are typically packaged into transduction competent AAV particles and propagated using an AAV viral packaging system. A transduction competent AAV particle is capable of binding to and entering a mammalian cell and subsequently delivering a nucleic acid cargo (e.g., a heterologous gene) to the nucleus of the cell. Thus, an intact rAAV particle that is transduction-competent is configured to transduce a mammalian cell. A rAAV particle configured to transduce a mammalian cell is often not replication competent and requires additional protein machinery to self-replicate. Thus, a rAAV particle that is configured to transduce a mammalian cell is engineered to bind and enter a mammalian cell and deliver a nucleic acid to the cell, wherein the nucleic acid for delivery is often positioned between a pair of AAV fTRs in the rAAV genome.

[0065] Suitable host cells for producing transduction competent AAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells that can be, or have been, used as recipients of a heterologous rAAV vectors. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. In certain aspects a modified human embryonic kidney cell line (e.g., HEK293), which is transformed with adenovirus type-5 DNA fragments and expresses the adenoviral Ela and Elb genes is used to generate recombinant AAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform in which to produce rAAV particles. Methods of generating high titer AAV particles capable of transducing mammalian cells are known in the art.

[0066] In certain aspects of the present disclosure, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. A number of other vectors are known which encode Rep and / or Cap expression products.

[0067] An “expression vector” is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions needed for expression in a host cell. An expression vector may contain at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous nucleic acid sequence, expression control element (e.g., a promoter, enhancer), intron, ITR(s), and polyadenylation signal.

[0068] The viral vectors may comprise a modified capsid, wherein the modified capsid comprises an amino acid sequence that targets the viral vector. The following table correlates the insertion sites for various A A Vs mentioned herein:

[0069] The AAV may be derived from AAV1. An exemplary wild-type reference AAV1 capsid protein sequence is provided in SEQ ID NO: 1. A targeting peptide may be inserted at position 590 of the AAV 1 capsid. An exemplary modified AAV 1 capsid protein sequence is provided in SEQ ID NO: 4, which shows the targeting peptide insertion after position 590 as SSAXXXXXXXXAS (SEQ ID NO: 11), where the leading SSA and the trailing AS are linker sequences and X7 represents the targeting peptide.

[0070] The AAV may be derived from AAV2. An exemplary wild-type reference AAV2 capsid protein sequence is provided in SEQ ID NO: 2. A targeting peptide may be inserted at position 587 of the AAV2 capsid. An exemplary modified AAV2 capsid protein sequence is provided in SEQ ID NO: 5, which shows the targeting peptide insertion after position 587 as AAAXXXXXXXAA (SEQ ID NO: 12), where the leading AAA and the trailing AA are linker sequences and X7 represents the targeting peptide.

[0071] The AAV may be derived from AAV9. An exemplary wild-type reference AAV9 capsid protein sequence is provided in SEQ ID NO: 3. A targeting peptide may beinserted at position 588 of the AAV9 capsid. An exemplary modified AAV9 capsid protein sequence is provided in SEQ ID NO: 6, which shows the targeting peptide insertion after position 588 as AAAXXXXXXXAS (SEQ ID NO: 23), where the leading AAA and the trailing AS are linker sequences and X7 represents the targeting peptide.

[0072] Reference capsid sequences correlating to the serotypes above are set out below:Wild type AAV1 capsid amino acid sequenceMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLG PFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTS FGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQP AKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGV GNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYS TPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIA NNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRT QNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWT GASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMIT DEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQG PIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYST GQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYL TRPL (SEQ ID NO: 1)Wild type AAV2 capsid amino acid sequenceMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFG GNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPA RKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIAN NLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTG ATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPI WAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTR NL (SEQ ID NO: 2)Wild type AAV9 capsid amino acid sequenceMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL YKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKR LNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPA DVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSY AHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPS YRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLS GSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTG WVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYK SNNVEFAVNTEGVYSEPRP1GTRYLTRNL (SEQ ID NO: 3)AAV 1 capsid library amino acid sequenceMAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLG PFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTS FGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQP AKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGV GNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYS TPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIA NNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWT GASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMIT DEEEIKATNP VATERFGTV AVNFQSS STDS S AXXXXXXX ASPATGD VH AMGALPGM VWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFS ATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNG LYTEPRPIGTRYLTRPL (SEQ ID NO: 4)AAV2 capsid library amino acid sequenceMAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPF NGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFG GNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPA RKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVG NSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTP WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIAN NLTST VQ VFTDS EYQLP Y VLGS AHQGCLPPFPAD VFM VPQ YG YLTLNNGS Q AVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLTDQYLYYLSRT NTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITD EEEIRTTNPVATEQYGSVSTNLQRGNAAAXXXXXXXAARQAATADVNTQGVLPGM VWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFS AAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGV YSEPRPIGTRYLTRNL (SEQ ID NO: 5)AAV9 capsid library amino acid sequenceMAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLG PGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDT SFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQ PAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGV GSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTI ANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVG RS SFYCLEYFPS QMLRTGNNFQFS YEFENVPFHS S Y AHS QSLDRLMNPLIDQYL Y YLS KTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMI TNEEEIKTTNPVATESYGQVATNHQSAQAAAXXXXXXXASAQAQTGWVQNQGILP GMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPT AFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNT EGVYSEPRPIGTRYLTRNL (SEQ ID NO: 6)

[0073] Note: “XXXXXXX” represents 7 random amino acids and indicates the site where the 7-mer targeting peptide is located.

[0074] In some aspects, the modified AAV capsid proteins are derived from an AAV1 capsid protein (see SEQ ID NO: 1), and the targeting peptide is inserted after residue 590 of the AAV 1 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, the linker sequences are SSA on the N-terminal side of the targeting peptide and AS on the C-terminal side of the targeting peptide. In some aspects, the modified AAV1 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 4. In some aspects, the targeting peptide has a sequence as provided herein. In some aspects, the targeting peptide has a sequence of any one of SEQ ID NOs: 7-10.

[0075] In some aspects, the modified AAV capsid proteins are derived from an AAV2 capsid protein (see SEQ ID NO: 2), and the targeting peptide is inserted after residue 587 of the AAV2 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, the linker sequences are AAA on the N-terminal side of the targeting peptide and AA on the C-terminal side of the targeting peptide. In some aspects, the modified AAV2 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 5. In some aspects, the targeting peptide has a sequence as provided herein. In some aspects, the targeting peptide has a sequence of any one of SEQ ID NOs: 7-10.

[0076] In some aspects, the modified AAV capsid proteins are derived from an AAV9 capsid protein (see SEQ ID NO: 3), and the targeting peptide is inserted after residue 588 of the AAV9 capsid protein. In some aspects, the targeting peptide is flanked by linker sequences, and the linker sequences on each side of the targeting peptides are two or three amino acids long. In some aspects, 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. In some aspects, the modified AAV9 capsid proteins have a sequence at least 95% identical to SEQ ID NO: 6. In some aspects, the targeting peptide has a sequence as provided herein. In some aspects, the targeting peptide has a sequence of any one of SEQ ID NOs: 7-10.II. Methods for Treatment Administration

[0077] Viral vectors in some aspects may be administered directly to patients (in vivo) or they can be used to treat cells in vitro or ex vivo, and then administered to patients. The term “vector” refers to small carrier nucleic acid molecule, a plasmid, virus (e.g., AAV vector,retroviral vector, lentiviral vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Vectors, such as viral vectors, can be used to introduce / transfer nucleic acid sequences into cells, such that the nucleic acid sequence therein is transcribed and, if encoding a protein, subsequently translated by the cells.

[0078] Any suitable cell or mammal can be administered or treated by a method or use described herein. Typically, a mammal in need of a method described herein is suspected of having or expressing an abnormal or aberrant protein that is associated with a disease state. Alternative, the mammalian recipient may have a condition that is amenable to gene replacement therapy. As used herein, “gene replacement therapy” refers to administration to the recipient of exogenous genetic material encoding a therapeutic agent and subsequent expression of the administered genetic material in situ. Thus, the phrase “condition amenable to gene replacement therapy” embraces conditions such as genetic diseases (i.e., a disease condition that is attributable to one or more gene defects), acquired pathologies (i.e., a pathological condition which is not attributable to an inborn defect), cancers and prophylactic processes (i.e., prevention of a disease or of an undesired medical condition). Accordingly, as used herein, the term “therapeutic agent” refers to any agent or material, which has a beneficial effect on the mammalian recipient. Thus, “therapeutic agent” embraces both therapeutic and prophylactic molecules having nucleic acid or protein components.

[0079] Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In certain examples a mammal is a human. In certain aspects a mammal is a non-rodent mammal (e.g., human, pig, goat, sheep, horse, dog, or the like). In other aspect a non-rodent mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. A mammal may be an animal disease model, for example, animal models having or expressing an abnormal or aberrant protein that is associated with a disease state or animal models with insufficient expression of a protein, which causes a disease state.

[0080] Mammals (subjects) treated by a method or composition described herein include adults (18 years or older) and children (less than 18 years of age). Adults include the elderly. Representative adults are 50 years or older. Children range in age from 1-2 years old,or from 2-4, 4-6, 6-18, 8-10, 10-12, 12-15 and 15-18 years old. Children also include infants.Infants typically range from 1-12 months of age.

[0081] A method may include administering a plurality of viral particles to a mammal as set forth herein, where severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro-degenerative disease, decreased, reduced, prevented, inhibited or delayed. A method may include administering a plurality of viral particles to a mammal to treat an adverse symptom of a disease state, such as a neuro- degenerative disease. A method may include administering a plurality of viral particles to a mammal to stabilize, delay or prevent worsening, or progression, or reverse and adverse symptom of a disease state, such as a neuro-degenerative disease.

[0082] A method may include administering a plurality of viral particles to the central nervous system, or portion thereof as set forth herein, of a mammal and severity, frequency, progression or time of onset of one or more symptoms of a disease state, such as a neuro- degenerative disease, are decreased, reduced, prevented, inhibited or delayed by at least about 5 to about 10, about 10 to about 25, about 25 to about 50, or about 50 to about 100 days.

[0083] A symptom or adverse effect may comprise an early stage, middle or late stage symptom; a behavior, personality or language symptom; swallowing, movement, seizure, tremor or fidgeting symptom; ataxia; and / or a cognitive symptom such as memory, ability to organize.III. Pharmaceutical Compositions

[0084] As used herein the term “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable composition, formulation, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or 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 composition, “pharmaceutically acceptable” and “physiologically acceptable” formulations and compositions can be sterile. Such pharmaceutical formulations and compositions may be used, for example in administering a viral particle to a subject.

[0085] Such formulations and compositions include solvents (aqueous or non aqueous), solutions (aqueous or non aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the formulations and compositions.

[0086] Pharmaceutical compositions typically contain a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, Tween80, and liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the 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 be present in such vehicles.

[0087] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration or delivery by various routes.

[0088] Pharmaceutical forms suitable for injection or infusion of viral particles can include sterile aqueous solutions or dispersions which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate form should be a sterile fluid and stable under the conditions of manufacture, use and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The 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. Isotonic agents, for example, sugars, buffers orsalts (e.g., sodium chloride) can be included. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0089] Solutions or suspensions of viral particles can optionally include one or more of the following components: a sterile diluent such as water for injection, saline solution, such as phosphate buffered saline (PBS), artificial CSF, a surfactants, fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), 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 the adjustment of tonicity such as sodium chloride or dextrose.

[0090] Pharmaceutical formulations, compositions and delivery systems appropriate for the compositions, methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, PA; The Merck Index (1996) 12th 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) 11th ed., Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[0091] Viral particles and their compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The dosage unit forms are dependent upon the number of viral particles believed necessary to produce the desired effect(s). The amount necessary can be formulated in a single dose, or can be formulated in multiple dosage units. The dose may be adjusted to a suitable viral particle concentration, optionally combined with an anti-inflammatory agent, and packaged for use.

[0092] In one embodiment, pharmaceutical compositions will include sufficient genetic material to provide a therapeutically effective amount, i.e., an amount sufficient toreduce or ameliorate symptoms or an adverse effect of a disease state in question or an amount sufficient to confer the desired benefit.

[0093] A “unit dosage form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dosage forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. Thus, for example, viral particles, and pharmaceutical compositions thereof, can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.

[0094] Formulations containing viral particles typically contain an effective amount, the effective amount being readily determined by one skilled in the art. The viral particles may typically range from about 1% to about 95% (w / w) of the composition, or even higher if suitable. The quantity to be administered depends upon factors such as the age, weight and physical condition of the mammal or the human subject considered for treatment. Effective dosages can be established by one of ordinary skill in the art through routine trials establishing dose response curves.IV. Disease States

[0095] STXBP1 and Epileptic Encephalopathy. Syntaxin-binding protein 1 (also known as Muncl8-1) is a protein that in humans is encoded by the STXBP1 gene (Ref Seq NG_016623.2). This gene encodes a syntaxin-binding protein. The encoded protein appears to play a role in release of neurotransmitters via regulation of syntaxin, a transmembrane attachment protein receptor. Mutations in this gene have been associated with neurological disorders including epilepsy, intellectual disability, and movement disorders.

[0096] The STXBP1 gene is located on the q arm of chromosome 9 in position 34.11 and has 19 exons spanning 80,510 base pairs. The encoded protein is a peripheral membrane protein located in the cytosol. In the retina and cerebellum, an alternatively spliced transcript variant is expressed, containing an additional exon and totaling 594 amino acids. Alternativesplicing can produce an isoform with exon 20 and an isoform without. Exemplary STXBP1 protein sequences are provided as Ref Seqs NP_003156.1 and NP_001032221.6, which are incorporated herein by reference.

[0097] The encoded protein may participate in the regulation of synaptic vesicle docking and fusion, possibly through interaction with GTP-binding proteins. It is essential for neurotransmission and binds syntaxin, a component of the synaptic vesicle fusion machinery, probably in a 1:1 ratio. It can interact with syntaxins 1, 2, and 3 but not syntaxin 4 and may play a role in determining the specificity of intracellular fusion reactions. This protein functions in a late stage of the intracellular membrane fusion process of exocytosis. Dissociation of this protein from syntaxin determines the kinetics of postfusion events. This protein is essential for presynpatic vesicle release and is rapidly phosphorylated hy protein kinase C upon neuronal depolarization. The protein participates in the secretory pathway between the Golgi apparatus and cell membrane.

[0098] Mutations in the STXBP1 cause early infantile epileptic encephalopathy type 4 (EIEE4), a severe form of epilepsy characterized by frequent tonic seizures or spasms beginning in infancy with a specific EEG finding of suppression-burst patterns, characterized by high-voltage bursts alternating with almost flat suppression phases. Affected individuals have neonatal or infantile onset of seizures, profound intellectual disability, and MRI evidence of brain hypomyelination. Inheritance of EIEE4 is autosomal dominant, but due to the severity of the condition most cases are de novo.

[0099] This gene was initially discovered in 2008 as a cause for the severe form of epilepsy also called Ohtahara syndrome. Since then, it has become one of the most prominent genes for epileptic encephalopathies, and is increasingly being associated with other forms of epilepsy. STXBP1 variants are increasingly being identified in people with wider neurological problems, including intellectual disability or movement disorders without epilepsy.

[0100] In melanocytic cells STXBP1 gene expression may be regulated by MITF. The STXBP1 gene is expressed in the brain and spinal cord and highly enriched in axons. Expression of this protein is highest in the retina and cerebellum. The encoded protein binds SYTL4. STXBP1 has been shown to interact with STX2, STX4 and STX1A.

[0101] TPP1 and CLN2 / Batten Disease / TPPI Deficiency. In some aspects, the present disclosure may be directed to the use of treating a neurological disorder, such as late infantileneuronal ceroid lipofuscinosis (CLN2) disease / Batten disease. Batten disease is a fatal disease of the nervous system that typically begins in childhood, typically between 5 and 10 years of age. Often, it is autosomal recessive, a common name for the neuronal ceroid lipofuscinoses (NCLs). Although Batten disease is usually regarded as the juvenile form of NCL (or “type 3”), some physicians use the term Batten disease to describe all forms of NCL. Historically, the NCLs were classified by age of disease onset as infantile NCL (INCL), late infantile NCL (LINCL), juvenile NCL (JNCL) or adult NCL (ANCL). At least 20 genes have been identified in association with Batten disease, but juvenile NCL, the most prevalent form of Batten disease, has been linked to mutations in the CLN3 gene.

[0102] Batten disease is a terminal illness. Brineura is the first FDA-approved treatment to slow loss of walking ability (ambulation) in symptomatic pediatric patients 3 years of age and older with late infantile neuronal ceroid lipofuscinosis (CLN2), also known as tripeptidyl peptidase- 1 (TPP1) deficiency. TPP1, also known as Lysosomal pepstatin- insensitive protease, is an enzyme that in humans is encoded by the TPP1 gene (Ref Seq NG_008653.1). Mutations in the TPP1 gene lead to late infantile neuronal ceroid lipofuscinosis. The human gene TPP1 encodes a member of the sedolisin family of serine proteases. The human gene has 13 exons and locates at the chromosome band l lpl5. Exemplary TPP1 protein sequences are provided as Ref Seq NP_000382.3, which is incorporated by reference herein, and SEQ ID NO: 21. An exemplary coding sequence for TPP1 is provided as SEQ ID NO: 22.

[0103] Human TPP1 comprises tripeptidyl-peptidase I activity (TPP1 enzyme activity). TPP1 activity comprises a non-specific lysosomal peptidase activity which generates tripeptides from the breakdown products produced by lysosomal proteinases. Substratespecificity studies indicate that TPP1 primarily cleaves tripeptides from unsubstituted amino termini in peptides and proteins. Endogenously expressed TPP1 is synthesized as a catalytically inactive enzyme. After targeting lysosomes, because of the acidic environment, TPP1 is autocatalytically processed into a mature active enzyme. The activity of TPP1 can be measured and / or quantitated in vitro using known methods.

[0104] The human TPP1 is 61kDa in size and composed of 563 amino acids. An isoform of 34.5kDa and 320 amino acids is generated by alternative splicing and a peptide fragment of 1-243 amino acid is missing. TPP1 contains a globular structure with a subtilisin- like fold, a Ser 475-Glu 272-Asp 360 catalytic triad. It also contains an octahedrallycoordinated Ca2+-binding site that are characteristic features of the S53 sedolisin family of peptidases. Unlike other S53 peptidases, it has steric constraints on the P4 substrate pocket, which might contribute to its preferential cleavage of tripeptides from the unsubstituted N- terminus of proteins. Two alternative conformations of the catalytic Asp276 are associated with the activation status of TPP1.

[0105] High expression of TPP1 is found in bone marrow, placenta, lung, pineal and lymphocytes. The protease functions in the lysosome to cleave N-terminal tripeptides from substrates and has weaker endopeptidase activity. It is synthesized as a catalytically inactive enzyme which is activated and autoproteolyzed upon acidification.

[0106] The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders with pathological phenotypes that auto fluorescent lipopigments present in neurons and other cell types. Over the past two decades, accumulating evidences indicates that NCLs are caused by mutations in eight different genes, including genes encoding several soluble proteins (cathepsin D, PPT1 and TPP1). Mutations of gene TPP1 result in late- infantile neuronal ceroid lipofuscinosis which is associated with the failure to degrade specific neuropeptides and a subunit of ATP synthase in the lysosome. Mutations in the TPP1 gene lead to late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood. It has been demonstrated that a single injection of intravitreal implantation of autologous bone marrow derived stem cells transduced with a TPP1 expression construct at an early stage in the disease progression could substantially inhibit the development of disease-related retinal function deficits and structural changes. This result implies that ex vivo gene therapy using autologous stem cells may be an effective means of achieving sustained delivery of therapeutic compounds to tissues such as the retina for which systemic administration would be ineffective.

[0107] In some embodiments, TPP1 may be under the control of a promoter, such as a CM V early enhancer / chicken P actin (CAG) promoter. The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors. The CAG promoter was constructed from the following sequences:(C) the cytomegalovirus (CMV) early enhancer element,(A) the promoter, the first exon and the first intron of chicken beta-actin gene,(G) the splice acceptor of the rabbit beta-globin gene.

[0108] The resulting synthetic element was used in the pCAGGS expression vector. The initiation codon located at the proximal region of the second exon was disrupted by digesting with Ncol restriction enzyme and replacing the site with a Hindlll linker. Although the whole construct is commonly referred to as the “CAG promoter”, it includes a part of the transcribed sequence (the first exon and the first intron of chicken beta-actin gene) and enhancer elements. In addition to the CMV immediate early enhancer, the intron of the chicken beta actin gene contains an enhancer element, which is highly conserved among vertebrates. The 3' portion of the promoter has high GC content and is thus refractory to PCR amplification.V. Definitions

[0109] The terms “polynucleotide,” “nucleic acid” and “transgene” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and polymers thereof. Polynucleotides include genomic DNA, cDNA and antisense DNA, and 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 -splicing RNA, or antisense RNA). Polynucleotides can include naturally occurring, synthetic, and intentionally modified or altered polynucleotides (e.g., variant nucleic acid). Polynucleotides can be single stranded, double stranded, or triplex, linear or circular, and can be of any suitable length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0110] A nucleic acid encoding a polypeptide often comprises an open reading frame that encodes the polypeptide. Unless otherwise indicated, a particular nucleic acid sequence also includes degenerate codon substitutions.[0 II I] Nucleic acids can include one or more expression control or regulatory elements operably linked to the open reading frame, where the 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, a TATA box, and the like), translation initiation sequences, mRNA stability sequences, poly A sequences, secretory sequences, and the like. Expression control / regulatory elements can be obtained from the genome of any suitable organism.

[0112] A “promoter” refers to a nucleotide sequence, usually upstream (5') of a coding sequence, which directs and / or controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. A pol II promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA- box and optionally other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A type 1 pol III promoter includes three cis-acting sequence elements downstream of the transcriptional start site: a) 5'sequence element (A block); b) an intermediate sequence element (I block); c) 3' sequence element (C block). A type 2 pol III promoter includes two essential cis-acting sequence elements downstream of the transcription start site: a) an A box (5' sequence element); and b) a B box (3’ sequence element). A type 3 pol III promoter includes several cis-acting promoter elements upstream of the transcription start site, such as a traditional TATA box, proximal sequence element (PSE), and a distal sequence element (DSE).

[0113] An “enhancer” is a DNA sequence that can stimulate transcription activity and may be an innate element of the promoter or a heterologous element that enhances the level or tissue specificity of expression. It is capable of operating in either orientation (5 ’->3’ or 3’- >5’), and may be capable of functioning even when positioned either upstream or downstream of the promoter.

[0114] Promoters and / or enhancers may be derived in their entirety from a native gene, or be composed of different elements derived from different elements found in nature, or even be comprised of synthetic DNA segments. A promoter or enhancer may comprise DNA sequences that are involved in the binding of protein factors that modulate / control effectiveness of transcription initiation in response to stimuli, physiological or developmental conditions.

[0115] Non-limiting examples of promoters include SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, pol II promoters, pol III promoters, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from non-viral genes, such as the murine metallothionein gene, will also find use herein. Exemplary constitutive promoters include the promoters for the following genes which encode certain constitutive or “housekeeping” functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolate reductase (DHFR), adenosine deaminase,phosphoglycerol kinase (PGK), pyruvate kinase, phosphoglycerol mutase, actin promoter, U6, and other constitutive promoters known to those of skill in the art. In addition, many viral promoters function constitutively in eukaryotic cells. These include: the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney Leukemia Virus and other retroviruses; and the thymidine kinase promoter of Herpes Simplex Virus, among many others. Accordingly, any of the above-referenced constitutive promoters can be used to control transcription of a heterologous gene insert.

[0116] A “transgene” is used herein to conveniently refer to a nucleic acid sequence / polynucleotide that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene that encodes an inhibitory RNA or polypeptide or protein, and are generally heterologous with respect to naturally occurring AAV genomic sequences.

[0117] The term “transduce” refers to introduction of a nucleic acid sequence into a cell or host organism by way of a vector (e.g., a viral particle). Introduction of a transgene into a cell by a viral particle is can therefore be referred to as “transduction” of the cell. The transgene may be integrated into genomic nucleic acid of a transduced cell. If an introduced transgene becomes integrated into the nucleic acid (genomic DNA) of the recipient cell or organism it can be stably maintained in that cell or organism and further passed on to or inherited by progeny cells or organisms of the recipient cell or organism. Finally, the introduced transgene may exist in the recipient cell or host organism extra chromosomally, or only transiently. A “transduced cell” is therefore a cell into which the transgene has been introduced by way of transduction. Thus, a “transduced” cell is a cell into which, or a progeny thereof in which a transgene has been introduced. A transduced cell can be propagated, transgene transcribed and the encoded inhibitory RNA or protein expressed. For gene therapy uses and methods, a transduced cell can be in a mammal.

[0118] Transgenes under control of inducible promoters are expressed only or to a greater degree, in the presence of an inducing agent, (e.g., transcription under control of the metallothionein promoter is greatly increased in presence of certain metal ions). Inducible promoters include responsive elements (REs) which stimulate transcription when their inducing factors are bound. For example, there are REs for serum factors, steroid hormones, retinoic acid and cyclic AMP. Promoters containing a particular RE can be chosen in order to obtain an inducible response and in some cases, the RE itself may be attached to a differentpromoter, thereby conferring inducibility to the recombinant gene. Thus, by selecting a suitable promoter (constitutive versus inducible; strong versus weak), it is possible to control both the existence and level of expression of a polypeptide in the genetically modified cell. If the gene encoding the polypeptide is under the control of an inducible promoter, delivery of the polypeptide in situ is triggered by exposing the genetically modified cell in situ to conditions for permitting transcription of the polypeptide, e.g., by intraperitoneal injection of specific inducers of the inducible promoters which control transcription of the agent. For example, in situ expression by genetically modified cells of a polypeptide encoded by a gene under the control of the metallothionein promoter, is enhanced by contacting the genetically modified cells with a solution containing the appropriate (i.e., inducing) metal ions in situ.

[0119] A nucleic acid / transgene is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. A nucleic acid / transgene encoding and RNAi or a polypeptide, or a nucleic acid directing expression of a polypeptide may include an inducible promoter, or a tissue-specific promoter for 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.

[0120] A cell-type-specific or inducible promoters, enhancers and the like, may be employed in the methods and uses described herein. Non-limiting examples of inducible promoters include DNA responsive elements for ecdysone, tetracycline, hypoxia and IFN.

[0121] An expression control element may comprise a CMV enhancer. An expression control element may comprise a beta actin promoter. An expression control element may comprise a chicken beta actin promoter. An expression control element may comprise a CMV enhancer and a chicken beta actin promoter.

[0122] As used herein, the terms “modify” or “variant” and grammatical variations thereof, mean that a nucleic acid, polypeptide or subsequence thereof deviates from a reference sequence. Modified and variant sequences may therefore have substantially the same, greater or less expression, activity or function than a reference sequence, but at least retain partial activity or function of the reference sequence. A particular type of variant is a mutant protein, which refers to a protein encoded by a gene having a mutation, e.g., a missense or nonsense mutation.

[0123] A “nucleic acid” or “polynucleotide” variant refers to a modified sequence which has been genetically altered compared to wild-type. The sequence may be genetically modified without altering the encoded protein sequence. Alternatively, the sequence may be genetically modified to encode a variant protein. A nucleic acid or polynucleotide variant can also refer to a combination sequence which has been codon modified to encode a protein that still retains at least partial sequence identity to a reference sequence, such as wild-type protein sequence, and also has been codon-modified to encode a variant protein. For example, some codons of such a nucleic acid variant will be changed without altering the amino acids of a protein encoded thereby, and some codons of the nucleic acid variant will be changed which in turn changes the amino acids of a protein encoded thereby.

[0124] The terms “protein” and “polypeptide” are used interchangeably herein. The “polypeptides” encoded by a “nucleic acid” or “polynucleotide” or “transgene” disclosed herein include partial or full-length native sequences, as with naturally occurring wild-type and functional polymorphic proteins, functional subsequences (fragments) thereof, and sequence variants thereof, so long as the polypeptide retains some degree of function or activity. Accordingly, in methods and uses of the invention, such polypeptides encoded by nucleic acid sequences are not required to be identical to the endogenous protein that is defective, or whose activity, function, or expression is insufficient, deficient or absent in a treated mammal.

[0125] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (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).

[0126] An example of an amino acid modification is a conservative amino acid substitution or a deletion. In particular embodiments, a modified or variant sequence retains at least part of a function or activity of the unmodified sequence (e.g., wild-type sequence).

[0127] Another example of an amino acid modification is a targeting peptide introduced into a capsid protein of a viral particle. Peptides have been identified that target recombinant viral vectors, to the central nervous system, such as to distinct brain regions.

[0128] A recombinant virus so modified may preferentially bind to one type of tissue (e.g., CNS tissue) over another type of tissue (e.g., liver tissue). A recombinant virus bearing a modified capsid protein may “target” brain vascular epithelia tissue by binding at level higher than a comparable, unmodified capsid protein. For example, a recombinant virus having a modified capsid protein may bind to brain vascular epithelia tissue at a level 50% to 100% greater than an unmodified recombinant virus.

[0129] A “nucleic acid fragment” is a portion of a given nucleic acid molecule. Deoxyribonucleic acid (DNA) in the majority of organisms is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins. Fragments and variants of the disclosed nucleotide sequences and proteins or partiallength proteins encoded thereby are also encompassed by the present invention. By “fragment” or “portion” is meant a full length or less than full length of the nucleotide sequence encoding, or the amino acid sequence of, a polypeptide or protein. In certain examples, 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 activity or function of wild-type).

[0130] A “variant” of a molecule is a sequence that is substantially similar to the sequence of the native molecule. For nucleotide sequences, variants include those sequences that, because of the degeneracy of the genetic code, encode the identical amino acid sequence of the native protein. Naturally occurring allelic variants such as these can be identified with the use of molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis, which encode the native protein, as well as those that encode a polypeptide having amino acid substitutions. Generally, nucleotide sequence variants of the invention will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequence identity to the native (endogenous) nucleotide sequence. In certain examples, the variant is biologically functional (i.e., retains 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of activity or function of wild-type).

[0131] “Conservative variations” of a particular nucleic acid sequence refers to those nucleic acid sequences that encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGT, CGC, CGA, CGG, AGA and AGG all encode the amino acid arginine. Thus, at every position where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded protein. Such nucleic acid variations are “silent variations,” which are one species of “conservatively modified variations.” Every nucleic acid sequence described herein that encodes a polypeptide also describes every possible silent variation, except where otherwise noted. One of skill in the art will recognize that each codon in a nucleic acid (except ATG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule by standard techniques. Accordingly, each “silent variation” of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0132] The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, or at least 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, at least 80%, 90%, or even at least 95%.

[0133] The term “substantial identity” in the context of a polypeptide indicates that a 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 over a specified comparison window. An indication that two polypeptide sequences are identical is that one polypeptide is immunologically reactive with antibodies raised against the second polypeptide. Thus, a polypeptide is identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

[0134] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01 %. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0135] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0136] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.

[0137] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.VI. Kits

[0138] The invention provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a nucleic acid, recombinant vector, and / or viral particles.

[0139] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).|0140| Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can includeinformation identifying manufacturer, lot numbers, manufacture location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease for which a kit component may be used. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.

[0141] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another treatment protocol or therapeutic regimen which would be incompatible with the composition and, therefore, instructions could include information regarding such incompatibilities.

[0142] Labels or inserts include “printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD- ROM / RAM, DVD, MP3, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic / optical storage media, FLASH memory, hybrids and memory type cards.VII. Examples

[0143] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1 - Peptide-modified AAV library enrichment identifies capsid variants with high target tissue tropism

[0144] Capsids with improved tropism for ependymal cells and brain parenchyma were identified using a peptide-modified AAV capsid library screening approach (FIG. 1A). To increase capsid diversity, the screen included three different parental serotypes (AAV 1 , AAV2, and AAV9). Capsid sequences were modified by insertion of a random heptamer peptide into the variable region of loop 8 at amino acid positions 590, 587, and 588 for AAV1, AAV2, and AAV9, respectively. The three serotype libraries were pooled and injected into the lateral ventricle of an adult rhesus macaque. Brain regions of interest, including ependyma and cerebral cortices, were collected and a second-round AAV library was generated from the viral DNA recovered from the collected tissues. The newly prepared AAV library was injected into the lateral ventricle of a second adult rhesus macaque. Illumina amplicon sequencing was performed on tissues from the second round of screening and analyzed to assess capsid variant performance (FIG. 7). After two rounds of selection, variants were evaluated based on enrichment in ependyma and other brain regions. Based on this analysis, the 4 top performing capsids were selected for fluorescence validation (2 variants from serotype 2 and 1 variant each from serotypes 1 and 9; see Materials and Methods). These were independently produced and assessed for ependymal, cortical and subcortical tropism (FIGS. IB and 1C).Example 2 - AAV candidate validation identifies a lead variant with broad cortical, subcortical and ependymal transduction

[0145] The four capsid variants selected for broad enrichment across ependymal and parenchymal regions (AAV-Ep’s) were packaged as single stranded (ss) DNA genomes with four distinct fluorescent reporter transgenes driven by the ubiquitous CAG promoter. These reporters were selected based on spectral compatibility and brightness to minimize potential biases on the fluorescence readout. All four capsid variants were produced and purified at titers greater than 5E+12 vg / mL with total yields as expected for other similar research grade virus preparations. The AAV variants were pooled and injected at a total dose 4E+13 vg into the right anterior lateral ventricle of a single adult rhesus macaque (FIG. 2). After 3 weeks, the animal was euthanized, and tissues were processed for histology. Vector distribution wasassessed using direct epifluorescence of the four reporter transgenes. Whereas AAV-Ep4 and AAV-Epl transduced ependyma in the lateral ventricle near the site of injection, only AAV- Epl, (noted as AAV-Ep+ in all subsequent uses) was found in the more distant fourth ventricle (FIG. 2A). AAV-Ep+ also transduced numerous cells in cortex and deep brain regions including the thalamus, subiculum and parasubiculum (FIG. 2B). A comparison of capsid performance in sequenced tissues showed a 22-fold enrichment of AAV-Ep+ against its parental capsid AAVI in ependyma (FIG. 8).

[0146] A substantial portion of AAVs delivered by the intracerebroventricular (ICV) route can target peripheral tissues including the liver (Meseck el al., 2022; Ballon el al., 2020), with high-dose AAVs inducing toxicity (Hudry et al., 2023). Recently, a comprehensive analysis of NHPs infused with CSF-delivered AAVs has shown nonclinical dorsal root ganglion (DRG) pathology in 83% of samples (Hordeaux et al., 2020). DRG toxicity in NHPs has been linked to high transgene expression rather than AAV transduction (Hordeaux et al., 2020; Buss et al., 2022). To examine peripheral, off-target transduction, the inventors performed RNAscope against AAV-derived transcripts in a rhesus macaque injected with 8.8E+12 vg of AAV-Ep+.mTFPl (FIG. 3). The inventors observed little transgene expression in the liver and no detectable transcripts in the heart or DRGs at multiple levels of the spinal cord (FIGS. 3A and 3B). These data supported AAV-Ep+ for further analysis based on high on-target transgene expression in brain ependyma and neurons with low off target expression in the periphery.Example 3 - Delivery of AAV-Ep+ to the posterior lateral ventricles yields superior ependymal transduction

[0147] Based on the direction of CSF flow through the ventricular system (FIG. 4A), the inventors hypothesized that a posterior site of administration would lead to higher transduction efficiency of ependymal cells in posterior regions of the lateral ventricles with similar transduction efficiency in anterior regions. The inventors tested this hypothesis by delivering 1E+13 vg AAV-Ep+.mRuby3 bilaterally into either the anterior or posterior lateral ventricles of four African green monkeys (AGMs), two infused at each anatomical location (FIG. 4A). Qualitatively, posterior infusions yielded more uniform and robust expression throughout the ventricular system (FIG. 4B). As expected, the site of injection did not affect the percentage of transduced ependymal cells in the anterior horn of the lateral ventricles, where transduction percentages averaged 68% and 70% after the anterior and posteriorinfusion, respectively. The site of injection minimally impacted transduction of the 3rd ventricle (Ant. 58%, Post. 64%), and the 4th ventricle (Ant. 28%, Post. 44%). In contrast, posterior infusions reproducibly increased the percentage of transduced cells in the lateral ventricle body and in the temporal and posterior regions of the lateral ventricles, with over 47% of cells transduced and transduction performance over anterior infusion increased by 19- and 11 -fold respectively (FIG. 4C. Temporal Ant. 1.09%, Post. 21.35%; Posterior Ant. 4.05%, Post. 47.50%). The hippocampus and temporal cortex were also transduced following posterior infusion (FIG. 4D). The inventors infused AAV9.mRuby3 into the posterior lateral ventricle of another animal at the same dose for comparison. At this dose, less than 2% of the ependymal cells were transduced with AAV9 (FIG. 9). In a separate study with two additional AGMs that received ICV injections of 1E+13 vg AAV-Ep+.mRuby3, transcripts were present in only 0.3 or 3.3% of numerous sampled DRG neurons at all spinal cord regions (FIG. 10). Lastly, 1E+13 vg AAV-Ep+.mRuby3 was infused bilaterally into the posterior lateral ventricle of an adult cynomolgus monkey (FIG. 4E and FIG. 11). Transduction of the ependyma was observed throughout the ventricular system (FIGS. 11A-11C), consistent with results from rhesus macaques and AGMs (FIGS. 1 and 2). Taken together, these data showed robust on- target transduction of ependymal cells following posterior lateral ventricle delivery in NHPs and demonstrated conserved tropism of AAV-Ep+ for ependymal cells across three old world NHP species.Example 4 - AAV-Ep+ transduces mouse ependyma and human iPSC-derived neurons

[0148] Cross-species incompatibility of AAV serotypes can pose challenges to translation of therapeutics from pre-clinical models to clinical application. In mice, AAV4 delivered to the CSF effectively targets the ependyma hut is not efficient in NHPs (Liu et al., 2005; Davidson et al., 2000; Hudry et al., 2023). In addition, AAV2 transduces ependyma in NHPs and dogs, but is not effective in mice (Katz et al., 2015; Ballon et al., 2020). To examine cross-species behavior of this NHP-evolved capsid, AAV-Ep+.mRuby3 was infused into the posterior lateral ventricle of adult C57BL / 6 and FVB mice (n=3 per strain). Fluorescent reporter expression was evaluated via epifluorescence and was seen in ependyma throughout the ventricular system (FIG. 5A and FIG. 12A) and in neurons of the hippocampus and subiculum (FIG. 5B and FIG. 12B). Transduced cells were also present in the cortex of both mouse strains (FIGS. 12B and 12C). The inventors also evaluated transduction efficiency of AAV-Ep+ in human neurons derived from iPSCs. At day 40 of differentiation, iPSC-derivedcortical neurons were transduced with AAV-Ep+.mRuby3 or its parental serotype AAV1- eGFP at several multiplicities of infection (MOIs; 1E+3, 1E+4 and 1E+5). 10 days after transduction, qualitative analysis by microscopy revealed higher transduction by AAV-Ep+ compared to AAV1 at equivalent doses (FIG. 5C). Quantification by RT-qPCR revealed a 34- fold increase over AAV1 at a MOI of 1E+3 [FIG. 5D; p < 0.0001 for AAV-Ep+ vs. AAV1 at 1+E4 and 1+E5, respectively; two-way analysis of variance (ANOVA) followed by Bonferroni’s correction for multiple comparisons]. These data showed that AAV-Ep+ can transduce cultured human neurons, 2 strains of mice, and 3 species of NHPs, and further supporting its potential use in pre-clinical testing for human disease applications.Example 5 - ICV infusion of AAV-Ep+.hTPPl delays phenotypes in a mouse model of CLN2 disease and results in therapeutic protein amounts in the CSF of NHPs

[0149] Given the observed cross-species compatibility, AAV-Ep+ vectors were tested in a mouse model of CLN2 disease (Sleat et al., 2004). AAV-Ep+.hTPPl, with TPP1 expressed from the CAG promoter as before (5E+10 vg) or vehicle was infused by ICV injection into seven- week-old mice. Five weeks after administration, TPP1 enzyme activity and TPP1 protein abundance was measured in different brain tissues and compared to that of vehicle- treated Cln2+ / - and Cln2- / - mice. Enzyme activity in Cln2- / - mice treated with AAV-Ep+.hTPPl was 7-27 times greater than in heterozygous carriers in superficial and deep regions of the cerebrum as well as the cerebellum (FIG. 6A). The supraphysiological enzyme activity achieved was greater than those achieved after ICV delivery of AAV2.caTPPl at a dose of 1.7E+13 vg to CLN2 disease dogs that resulted in marked phenotypic rescue (FIG. 13) (Katz et al., 2015). Cln2- / - mice have a survival deficit and a tremor phenotype that emerges at 12 weeks and is quantifiable at 24 and 52 Hz (Chang et al., 2008). To assess the effects of A AVEp+. hTPPl on these phenotypes, a second cohort of CLN2 mice was ICV-injected as above at 7 weeks of age for survival and tremor analysis. Comparison of tremor in control and treated mice revealed significant improvements with AAV-Ep+.hTPPl treatment at specific frequencies by 12 weeks of age that reached significance by area under the curve for the total spectrum by 16 weeks of age (FIG. 6B, p<0.05 by two-way ANOVA mixed-effect model followed by Bonferroni multiple comparisons post-hoc test). In addition, lifespan was modestly but significantly extended relative to vehicle-treated controls (FIG. 6C; p<0.05 by log rank Mantel-Cox test).

[0150] To further assess the translatability of this approach, 1E+12 vg of AAV- Ep+.hTPPl or vehicle was infused bilaterally into the posterior lateral ventricle of twocynomolgus monkeys. CSF was obtained before injection (baseline) and again at 7-, 14-, and 28-days post-infusion. CSF TPP1 concentrations were measured using a capillary western blot system with an antibody that cross-reacts with human and NHP TPP 1. Animals showed a timedependent increase in TPP1 proenzyme concentrations, reaching 3 and 6x baseline at 28 days (FIG. 6D). The concentration of human TPP1 produced minus the cynomolgus monkey baseline was 5- and 9-times higher than that found in the CSF from children without CLN2 disease (FIG. 6E). In contrast, earlier work reported that intracisterna magna infusion of 5E+13 vg AAVrhlO into NHPs achieved only 43-62% of normal human TPP1 concentrations in the CSF on average (Sondhi et al., 2020). Together, these mouse and NHP studies of AAV- Ep+.hTPPl support its further development for potential clinical application.Example 6 - Materials and Methods

[0151] Study Design. The overall objectives of the study were to identify AAV capsid variants with brain tropism following ICV injection in NHPs and to assess the translational potential of a top AAV capsid variant in NHPs, mice, and human iPSC-derived neurons. NHP experiments included 1-2 animals per group, and wildtype mouse experiments included 3 animals per group. Experimenters were not blinded to treatment group for NHP and wildtype mouse experiments. Human iPSC experiments were conducted with 3 biological replicates of 3-4 technical replicates. Cln2- / - mouse experiments included 7-10 animals per group. Experimenters were blinded to treatment group while conducting the tremor assay. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council), and mouse and NHP procedures were approved by the CHOP Research Institute Animal Care and Use Committee (protocol numbers 23-001358 and 25-001328, respectively).

[0152] Peptide-modified AAV library description and construction. Library plasmids were constructed by cloning the AAV2 Rep open reading frame and AAV Cap open reading frames of different serotypes into an AAV2 inverted terminal repeat-containing plasmid. Silent sequence modifications were made in the Cap coding sequence to introduce restriction enzyme (RE) sites around the peptide insertion site. Oligonucleotides encoding the random 7-mer (NNK)? were synthesized by Integrated DNA Technologies and cloned into the RE-digested recipient plasmids using Gibson Assembly. Pooled plasmids were transformed into Endura electrocompetent cells (LGC) and plasmid isolation performed using Qiagen Giga Prep Endofree kits (QIAGEN). For the second round of library generation, viral genomic DNA fromcollected tissues was isolated by QIAamp DNA mini kits (QIAGEN) and the variable region of the AAV library was amplified using serotype-specific primers. Amplicons were incorporated into recipient plasmids by Gibson Assembly and transformed into Endura electrocompetent cells(LGC). Pooled, purified variant plasmids were used to generate the Round 2 library.

[0153] AAV production. AAVs were generated by the CHOP Research Vector Core. For AAV library production, HEK 293 cells from ATCC (the American Type Culture Collection) were transfected with pAd helper and pAAV.Lib plasmids, the latter at an average of 1000 plasmid copies per cell to reduce crosspackaging. Recombinant virus was harvested and purified by two rounds of cesium chloride gradient centrifugation. Plasmids encoding the capsids selected for further evaluation were used in the final studies, each encoding a different fluorophore as noted in the figure legends. In all cases, the sequences were cloned into the AAV Cap gene in loop 8 of viral protein VP3 along with flanking Gly / Ser amino acids. Insertions were made at amino acid positions 590, 587, and 588 for AAV1, AAV2 and AAV9 respectively.

[0154] Gene blocks of hTPPl cDNA and multiple fluorescent reporters (mTFPl, mRuby3, mNeongreen and mTagBFP) were purchased from IDT (Integrated DNA Technologies) and cloned into an AAV backbone plasmid with CAG promoter and BGHpA. AAVs were titered by droplet digital PCR with ITR primer probe for AAV library vectors, CAG promoter primer probe for fluorescent vector, and hTPPl primer probe for hTPPl vectors.

[0155] NHP procedures. NHPs were housed at the CHOP Research Institute under a 12-hour light:dark cycle with ad libitum access to purified drinking water and twice daily feedings with Purina LabDiet Certified Primate Diet (5048) enriched with fruits and vegetables.

[0156] NHPs were injected with test articles or vehicle control as indicated in Table 1. AAV formulation buffer was used as vehicle control and to dilute viruses to the desired titer for injection. Before all ICV injections, animals were sedated with either ketamine and xylazine or Telazol followed by isofhirane anesthesia for the duration of the procedure. For stereotaxic ICV injections, baseline MRI scans were used to define trajectories. Cannula placement was confirmed by fluoroscopy using IsoVue M contrast before infusing test article. For ClearPoint ICV injections, the animal was sedated as above and positioned in the MRI scanner, and theClearPoint system was used to define target coordinates and calculate cannula tracks. The test article was mixed with ProHance gadolinium contrast (1.8 mM final concentration) to visualize the infusate and confirm cannula placement. After a ten-minute dwell period, the cannula was removed, the skin sutured, and the animal recovered. Buprenorphine SR was given as analgesia, and the animals were monitored postoperatively for pain and welfare.

[0157] NHPs were euthanized by exsanguination. Animals were sedated, transcardially perfused with ice-cold saline, and the brain removed and processed into 4-mm coronal slabs in a rhesus macaque or cynomolgus brain matrix, respectively. For AAV library experiments, tissue samples from ependyma, cerebral cortex, hippocampus, caudate, putamen, thalamus, pons and cerebellar cortex were micro-dissected, and flash frozen for recovery of AAV amplicons for the next round of evaluation.

[0158] NHP fluorescence validation. For fluorescence microscopy analyses, animals were euthanized by transcardially perfusion with ice-cold saline after sedation, and the brains removed and slabbed into 4-mm coronal slabs. Brain slabs, DRGs, and other tissues were postfixed in 4% paraformaldehyde, cryoprotected by sequential immersion in 15% and 30% sucrose in 0.1M PBS baths for 2 days each step at 4°C. 40 mm thick sections were prepared via with a Leica SM2010R microtome attached to a BSF-30MP freezing stage (Physitemp). Sections were rinsed in 0.1M PBS and incubated in 1 / 5000 Hoechst for one minute before being mounted in superfrost plus slides. Coverslips were mounted with Fluoromount-G (SouthemBiotech, #0100-01). Slides were analyzed with a Leica DM6000B epifluorescence microscope equipped with a Hammatsu ORCAFlash 4.0 monochrome camera and Leica LED 405, L5 ET, Y3 ET, TXT ET, Chroma 39001 AT-ECFP / C, and 49003 ET-EYFP filter cubes.

[0159] Amplicon sequencing of AAV libraries. Genomic DNA (gDNA) or RNA from harvested tissues (ependyma plus cortical, subcortical and cerebellar regions) was isolated using a QIAamp DNA mini kit or RNeasy Plus kit (QIAGEN). Variant amplicon libraries were generated using two rounds of PCR. In the first round, sequence encoding the modified capsid insertion was amplified using serotype-specific primers with PCR cycles less than 30 using the Q5 2x Master Mix (New England Biolabs). First-round PCR products were used as input for the second PCR that introduced Illumina i5 and i7 sequencingindices and adapters. Sizes of second round amplicon DNA were checked by agarose gel and purified using a MinElute Gel Extraction Kit (QIAGEN). For all libraries, sequencing was done on an Illumina NovaSeq 6000 using a 200-cycle reagent kit and paired end read chemistry yielding 100 bp paired end readoutputs. Initial quality control of Illumina FASTQs was performed by testing for perfect string matches to known constant regions of the amplicons. Capsid variant identity and abundance was quantified from passing reads by tabulating the occurrence of unique nucleic acid sequences encoding the heptamer peptide inserts. Variant abundance was quantified as the UMI collapsed read count for each unique sequence. A baseline variant performance threshold was applied by removing any variants with UMI counts < 100. In addition, potential sequencing errors were removed by filtering out variants that had < 1 % the UMI counts and were within 1 edit distance of another variant. Capsid variant performance within each tissue was assessed using two primary metrics: variant abundance (UMI counts) and an enrichment score. The enrichment score measures the fold change in a capsid variant’s abundance in a tissue compared to its abundance in the injected virus library (input virus).

[0160] Capsid variant performance was assessed using DNA and RNA data, UMI counts and enrichment metrics, and single-region (targeted) and cross-region analyses. Selection of capsids for validation was done after considering the totality of available data. No single data / metric / region combination served as a singular deciding factor.

[0161] RNAscope-FISH. 4 mm coronal post-fixed in 4% PFA brain slabs were sucrose cryoprotected and cut in 2X2 cm blocks and OCT embedded. 16 pm thick slices were collected on Superfrost Plus slides after microtomy with a Leica CM 1950 cryostat. Fluorescent in situ hybridization (FISH) was performed using RNAscope Multiplex Fluorescent Reagent Kit v2 Assay (Advanced Cell Diagnostics, Cat. 323100-USM) following manufacturer’s guidelines. A mTFPl and mRuby3 RNAscope probes (Advanced Cell Diagnostics Cat. 500271-C3 and 557101-C4) were used to detect fluorophore transcripts in the DRGs. Opal620 and Opal690 fluorophores (Akoya Biosciences, FP1495001KT and FP1497001KT) were associated to the mTFPl-C3 and mRuby3-C4 probes respectively. Slides were observed under a Leica SP8 confocal microscope.

[0162] Mouse biodistribution studies. Mice were maintained on a 12 h light / dark cycle and ad libitum access to water and food. Adult C57BL / 6 and FVB mice (n=3, JAX strains #000664 and #01800) were injected with AAVEp+. mRuby3 at 1E+10 vg. Injections targeting lateral ventricle (from bregma in mm: AP, -2.18; ML, ±2.9; DV, 3.5) were done at a rate of 25 nL / min. Three weeks after injection, mice were transcardially perfused with 0.05 M PBS, pH 7.4, followed by 50 ml of 4% w / v paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Brains were cryoprotected and sectioned in the sagittal plane. For each brain, the sites of injection were verified, and sections analyzed using a Leica DM6000B epifluorescence microscope for low power and tiled images. Z-stacks were acquired using a Leica SP8 confocal microscope. Both microscopes were under the control of Leica LAS X v3.7 software.

[0163] Automated cell counting. Automated cell counting was used to evaluate transduction in confocal images from AAV-Ep+- (348 images) and AAV9- (230 images) treated NHPs (QuPath vO.4.4)(Bankhead et al., 2017). For quantitation of AAV-Ep+ transduction, 30,006 total ependymal cells were defined and counted as either AAVEp+. mRuby3 positive or negative using a centroid-based fluorescence intensity threshold. For quantitation of AAV9 transduction, 22,458 total ependymal cells were defined and counted as either AAV9.mRuby3 positive or negative. Nuclei within the region of interest were defined by Hoechst signal. Because no cytoplasmic marker was available, a small uniform cytoplasmic expansion was created around each defined nucleus. A threshold-based cell classifier was applied to define the positive / negative status of each cell. Aggregated cell counts from each image were exported and processed using a custom R pipeline for data analysis and visualization.

[0164] Human iPSC-derived neuron transduction. iPSC-derived cortical excitatory neurons were generated from iPSCs as previously described (Waxman et al., 2023). After 40 days of differentiation, cells were transduced with differing MOIs based on initial cell plating counts. Ten days later RNA was isolated for qRT-PCR using Quick-RNA miniprep kit according to manufacturer's instructions (Zymo Research). For microscopy, iPSCderived cortical neurons were seeded on coverslips and transduced as above. At 10 days posttransduction, cells were fixed, blocked, and fluorescence was assessed using epi- fluorescent microscopy (Leica, DM6000B).

[0165] Cln2- / - mice. Cln2- / - mice have been described previously (Sleat et al., 2004) and were obtained from Peter Lobel, Rutgers University, then backcrossed to C57BL / 6J miceat Children’s Hospital of Philadelphia Research Institute. This mouse model has a neo cassette insertion between exons 11 and 12 that in homozygosity results in no detectable TPP1 activity. Cln2- / - mice or heterozygous littermates were injected into the lateral ventricle with either 5.0E+10 vg of AAV-Ep+.hTPPl or vehicle control (AAV formulation buffer) at 7 weeks of age. Animals were anesthetized with isoflurane and the surgical depth of anesthesia was confirmed by toe / tail pinch and eyeblink reflex. The injection coordinates were 0.3 mm anterior, 1 mm lateral, 2mm depth, and a 10 pL volume was injected into the right ventricle at a 0.33 pL / min infusion rate. Following injection, the subject was allowed to recover in a warming chamber until a normal range of activity was regained. Mice were then returned to the home cage.

[0166] TPP1 activity assay. Five weeks after injection, mice were anesthetized with isoflurane and transcardially perfused with lOmL of ice-cold 0.1 M PBS, pH 7.4. Brains were harvested and microdissected. Samples were then stored at -80°C until processing. Thawed tissues were homogenized in ice-cold homogenization buffer (0.1% Triton X-100 in 0.1M PBS), with Complete Protease Inhibitor Cocktail (Roche). Insoluble material was removed from the homogenate by centrifugation at 21,000 x g at 4°C for 15 minutes, and protein content in the supernatant was quantified by DC Protein Assay (BioRad). 10 pL homogenate was added to wells of a 96 well black wall / clear bottom plate containing 80 pL sodium citrate buffer (pH 4.0). The enzyme reaction was initiated by addition of 10 pL substrate (250 pM Ala-Ala-Phe 7-amido-4 methylcoumarin in sodium citrate buffer, pH 4.0) and read every 3 minutes for 2 hours in a SpectraMax M5 microplate reader (Molecular Devices) at 37°C with a 460 nm emission filter. TPP1 activity was calculated as the change in fluorescence units (FU) per minute. Absolute quantification was calculated using a hTPPl standard curve (Novus Biologicals, 2237-SE-010) pre-activated in sodium citrate buffer for 12 hours at room temperature.

[0167] Tremor activity monitoring and survival. Tremor activity was quantified with San Diego Instruments Tremor Monitor (SDI). Animals were acclimated in the behavior room for 30 minutes before the experiment. Once inside the apparatus, the mouse was allowed to habituate in the animal enclosure for three min before recording tremor activity for five min. Tabulation of tremor activities was analyzed by SDI data software. Mice were randomly assigned to experimental groups using the RAND function in Excel (Microsoft) after genotyping. Cln2+ / -+ vehicle group consist of n=9 (five females and four males), Cln2- / -+vehicle with n=10 (five females and five males), Cln2- / -+AAV-Ep+.hTPPlwith n=10 (five females and five males).

[0168] hTPPl pro-enzyme quantification. TPP1 proenzyme in CSF were quantified using a Jess Simple Western blot system (Protein Simple, Bio-techne) and Compass software (Protein Simple) following the manufacturer’s indications. Absolute quantitation was calculated from a six-point standard curve (0.03 to 1 ng / mL) generated with human recombinant TPP1 (Novus Biologicals, 2237-SE-010) diluted with 0. IX sample buffer (Protein Simple, no. 042-195). CSF and standard curve samples were combined with 5x Fluorescent Master Mix at 4: 1 ratio, denatured at 95 °C for five minutes, spun down at 4°C, and then loaded into a 12-230 kDa separation capillary cartridge (Protein Simple, no. SM-W004). A ratio of 1 :50 mouse anti-TPPl monoclonal antibody (Abeam, ab54685) and anti-mouse detection module (Protein Simple, no. DM-002) was used for TPP1 detection. Note that this anti-TPPl antibody does not distinguish human and cynomolgus monkey TPP1. Therefore, human TPP1 protein concentration was estimated by subtracting each animal’s baseline TPP1 CSF concentration from the total CSF concentrations at the measurement timepoints.

[0169] Statistical analysis. GraphPad Prism 10 software (GraphPad Software) and G*Power 3.1.9.6 (Heinrich Heine Universitat, Dusseldorf, Germany) software packages were used for statistical analyses. None of the groups analyzed for TPP1 activity in the mouse experiment (FIG 6A) passed the Anderson-Darling normality test. Statistical significance was assessed by the two-tailed Mann-Whitney test. Resting tremor was analyzed by unbalanced mixed-eSects analysis followed by a Dunnett’s multiple comparation test. Resting tremor plots showing amplitude for the frequency range are mean± SD by two-way ANOVA (mixed-effect model) followed by Bonferroni multiple comparisons post-hoc test. Survival was analyzed by log-rank Mantel-Cox test.Table 1. NHP information

[0170] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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 disclosure as defined by the appended claims.REFERENCESThe 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.Ballon et al., Quantitative Whole-Body Imaging of I-124-Labeled Adeno-Associated Viral Vector Biodistribution in Nonhuman Primates. Hum Gene Ther 31, 1237-1259 (2020).Bankhead et al., QuPath: Open source software for digital pathology image analysis. Sci Rep 7, 16878 (2017).Barton & Neufeld, The Hurler corrective factor. Purification and some properties. J Biol Chem 246, 7773-7779 (1971).Buss et al., Characterization of AAV-mediated dorsal root ganglionopathy. Mol Ther Methods Clin Dev 24, 342-354 (2022).Calabrese et al., A diffusion tensor MRI atlas of the postmortem rhesus macaque brain. 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Brain Res 545, 103-113 (1991).Katz et al., AAV gene transfer delays disease onset in a TPP1 -deficient canine model of the late infantile form of Batten disease. Sci Transl Med 7, 313ral80 (2015).Leib et al., Optimized AAV capsids for diseases of the basal ganglia show robust potency and distribution in adult nonhuman primates, biorxiv, (2024).Liguore et al., AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice. Mol Ther 27, 2018-2037 (2019).Liu et al., Functional correction of CNS phenotypes in a lysosomal storage disease model using adeno-associated virus type 4 vectors. J Neurosci 25, 9321-9327 (2005).Marino et al., AAV-mediated delivery of an anti-BACEl VHH alleviates pathology in an Alzheimer's disease model. EMBO Mol Med 14, e09824 (2022).Marino & Holt, AAV Vector-Mediated Antibody Delivery (A-MAD) in the Central Nervous System. Front Neurol 13, 870799 (2022).Matsuzaki et al., Intravenous administration of the adeno-associated virus-PHP.B capsid fails to upregulate transduction efficiency in the marmoset brain. Neurosci Lett 665, 182- 188 (2018).Meseck et al., Intrathecal sc-AAV9-CB-GFP: Systemic Distribution Predominates Following Single-Dose Administration in Cynomolgus Macaques. Toxicol Pathol 50, 415-431 (2022).Parambi et al., Gene Therapy Approach with an Emphasis on Growth Factors: Theoretical and Clinical Outcomes in Neurodegenerative Diseases. Mol Neurobiol 59, 191-233 (2022).Passini et al., Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis. J Neurosci 26, 1334-1342 (2006).Platt et al., Lysosomal storage diseases. Nat Rev Dis Primers 4, 27 (2018).Rodrigo Albors et al., An ependymal cell census identifies heterogeneous and ongoing cell maturation in the adult mouse spinal cord that changes dynamically on injury. 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Claims

WHAT IS CLAIMED IS:

1. A method of administering an adeno-associated virus (AAV) to the brain comprising introducing said AAV into the posterior region and / or temporal hom of the lateral ventricle of a subject.

2. A method of expressing a heterologous gene in the brain, comprising introducing an AAV comprising a genome that carries a coding sequence for the heterologous gene into the posterior region and / or temporal horn of the lateral ventricle of a subject thereby expressing the heterologous gene in the brain.

3. The method of claim 1 or 2, wherein said AAV comprises a modified capsid protein comprising the targeting sequence of any one of SEQ ID NOs: 7-10.

4. The method of claim 3, wherein the modified AAV is derived from AAV1, AAV2, or AAV9.

5. The method of any one of claim 3 or claim 4, wherein the targeting sequence is inserted near or after position 590 of SEQ ID NO: 1, after position 587 of SEQ ID NO: 2, or after position 588 of SEQ ID NO: 3.

6. The method of any one of claims 2-5, wherein the targeting sequence is flanked by linker sequences, optionally wherein the linker sequences on each side of the targeting sequence are two or three amino acids long.

7. The method of claim 6, wherein the linker sequences are SSA on the N-terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence, or wherein the linker sequences are AAA on the N-terminal side of the targeting sequence and AA on the C-terminal side of the targeting sequence, or wherein the linker sequences are AAA on the N- terminal side of the targeting sequence and AS on the C-terminal side of the targeting sequence.

8. The method of claim 6 or 7, wherein the targeting sequence flanked by linker sequences comprises the sequence of SEQ ID NO: 11 or 12 or 23.

9. The method of any one of claims 2-8, wherein the modified capsid protein comprises the sequence of any one of SEQ ID NOs: 4-6.

10. The method of any one of claims 1-9, wherein the AAV is introduced more than once, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administrations, and / or administering occurring monthly, every other month, every two months, every three months, every four months, every six months, annually every other year, every three years, every four year or every five years.

11. The method of any one of claims 1-10 wherein a plurality of AAV particles is administered.

12. The method of claim 11, wherein dosing is at about IxlO6to about IxlO14AAV vector genomes per kilogram (vg / kg).

13. The method of claim 12, wherein dosing is from about 1X107-1X1014, about 1x10s- IxlO14, about 1X109-1X1014, about lxlOlo-lxlO14, about lxlOlo-lxlO13, about lxlOlo-lxlO13, about lxlOlo-lxlOn, about lxlOn-lxlO12, or about 1X1012-1X1013AAV vector genomes per kilogram (vg / kg) of the patient.

14. The method of any one of claims 1-13, wherein subject suffers from Epileptic encephalopathy and the AAV carries or the heterologous gene is a coding sequence for Syntaxin-binding protein 1 ( STXBP1).

15. The method of any one of claims 1-13, wherein said subject suffers from Batten’s Disease and the AAV carries or the heterologous gene is a coding sequence for tripeptidyl peptidase- i (TPP 1 ) .

16. The method of claim 15, wherein administering results in a reduction or improvement in a symptom in said subject caused by Batten’s Disease.

17. The method of any one of claims 1-16, wherein the subject is a human, a non-human primate, a non-primate mammal.

18. The method of any one of claims 1-17, wherein the AAV is introduced by way of bilateral administration.

19. Use of an adeno-associated virus (AAV) for delivery into the posterior region and / or temporal horn of the lateral ventricle of a subject.

20. Use of an adeno-associated virus (AAV) for delivery into the posterior region and / or temporal horn of the lateral ventricle of a subject for the treatment of a brain disease, such as Batten Disease or Epileptic encephalopathy.

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