Methods and compositions for treatment of hydrocephalus

Abstract

Disclosed are compositions and methods for the treatment of hydrocephalus, for example pediatric hydrocephalus. The disclosed methods employ a non-surgical tool that may be used to control CSF production rate by inducing choroid plexus-specific apoptosis. In aspects, the non-surgical tools employ an AAV vector, a promoter, a suicide gene, and a suicide gene activator to achieve targeted choroid-plexus ablation and subsequent reduction of CSF volume.

Description

METHODS AND COMPOSITIONS FOR TREATMENT OF HYDROCEPHALUSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Pat. App. No. 63 / 564,178, entitled “Methods and Compositions for Treatment of Hydrocephalus," filed March 12, 2024, the disclosure of which is incorporated by reference herein.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under NS127177 and NS138647 awarded by the National Institutes of Health. The government has certain rights in the inventionBACKGROUND

[0003] Pediatric hydrocephalus is a medical condition characterized by the accumulation of cerebrospinal fluid (CSF) within the cavities (ventricles) of the brain in children. Hydrocephalus is one of the most common brain disorders, with nearly 400,000 new cases in children worldwide every year, and 88 cases per 100,000 population under 18 years of age. Hydrocephalus occurs when there is an imbalance between the production and absorption of CSF, leading to an excessive buildup of fluid.

[0004] There is no cure for hydrocephalus, only treatments, all of which require brain surgery. Currently, no medicine or biological tools exist to ablate or control the functions of the ChP in vivo. Neonatal patients with hydrocephalus of various etiologies undergo about 40,000 cerebrospinal fluid (CSF) diversion surgeries annually in the US. Surgical treatment via ventriculoperitoneal shunt or endoscopic third ventriculostomy is currently the most commonly used strategy to treat pediatric hydrocephalus. However, shunt- treated hydrocephalic patients continue to experience significant lifelong neurological problems and high malfunctioning shunt replacement surgery rates. The endoscopic choroid plexus (ChP) coagulation helps manage CSF volume by surgically removing the primary CSF production organ in the brain ventricles. However, this surgical procedure requires neurosurgeons with extensive training and still achieves only partial ChP removal. Further, although surgical CSF diversion, either by shunting or via endoscopic third ventriculostomy, may effectively control the ventricular size and intracranial pressure, over 80% of patients still suffer from one or more neurological sequelae, due to the shuntfailure and multiple shunt revisions (>80%) in surgically treated patients, which significantly diminishes patient quality of life. Therefore, the majority of patients often suffer from motor and cognitive impairment, epilepsy, neuropsychiatric problems, chronic headaches, and depression.

[0005] Improved methods of treating pediatric hydrocephalus are needed. The instant disclosure seeks to address one or more of the aforementioned needs in the art.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0007] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0008] FIG. 1. Choroid plexus (ChP) epithelial cells undergo apoptosis in response to Dtx treatment in iDTR mice (A) Schematic of experimental paradigm. (B) Expression levels of DTR ORF mRNA in the ChP and brain and peripheral tissues of 5-7 month-old iDTR+ / WT(heterozygous) and iDTR+ / +(homozygous) mice (mean ± SEM, n= 3-4 per tissue per genotype, Student’s t-test or nonparametric equivalent between genotypes within each tissue). CTX - cortex, HC - hippocampus, CB - cerebellum. No DTR expression was detected in WT mice (data not shown). (C, D) -presence of DTR protein (red) in the ChP of iDTR (D) but not WT mice (C). (E- G) TUNEL staining (green) showing apoptosis specifically in the ChP of Dtx-treated iDTR mice (G), but not Dtx-treated WT mice (E) or saline-treated iDTR mice (F), scale bar - 100 pm. (H-I) Magnified confocal image of a lateral ventricle ChP of a Dtx-treated iDTR animal stained with TUNEL (green, H), and ChP epithelium cell marker (al subunit of Na+ / K+ATPase, red, 1) and merged image (J). (K) - quantification of TUNEL+ cells that co-express different cell type markers (al - apical ChP epithelial cells, CD68 - macrophages, IB4 - endothelial cells, PDGFRp - pericytes) in the ChP of Dtx-treated iDTR animals (mean ± SD, n = 3. Scale bar - 20 pm). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0009] FIG. 2. Dose-dependent loss of ChP area in response to Dtx treatment in iDTR mice (A-I) Representative images of coronal sections showing ventricle and the ChP stained for TTR (red)and DAPI (blue) 2-5 weeks after Dtx treatment, 3-5 month-old animals. LV - lateral ventricle. Scale bar - 100 pm. (C, F, I) Magnified portions of B, E, H to show loss of TTR+ cells and the ventricle space in Dtx-treated iDTR mice. (J) Quantification of TTR+ area in coronal sections, normalized to WT mean (n = 3-6 per group, mean ± SEM, One-way ANOVA). (K-M) Whole mount ChPs at one month post-Dtx stained for TTR (ChP epithelial cells, green) and CD31 (blood vessel endothelium, white), 2-month-old animals. Right side - magnified portion of the whole mount to show blood vessel density. (N) Quantification of blood vessel length density (normalized to WT mean, n= 3-6 per group, mean ± SEM, Two-Way ANOVA). * - p < 0.05, ** - p < 0.01, *** - p < 0.001.

[0010] FIG. 3. Time course of CSF volume loss after Dtx treatment. (A) Schematic of experimental design to track daily CSF volume change. (B) T2- weighted MRI showing ventricular spaces in WT (left side) and iDTR (right side) brain. Scale bar - 1 cm. (C) - planimetric quantification of ventricle volume from 9 T2 MRI slices in WT and iDTR mice one day before Dtx, and at 1, 2, and 3 days post-Dtx, n = 4 per group, mean ± SEM, Two-Way ANOVA with genotype and timepoint as factors. Comparisons between genotypes: ns - p > 0.05, * - p < 0.05, *** - p < 0.001. Comparisons within genotype to pre-Dtx volume: ## - p < 0.01. (H, I) - example images of the cistema magna (CM) of WT (D) and iDTR (E) mice at 6 months post-Dtx during CSF collection. (F) - quantification of the volume of CSF collected from the CM (mean ± SEM, n = 7-8 per group, Mann- Whitney U test). *** - p < 0.001.

[0011] FIG. 4. Dose-dependent CSF volume loss after Dtx treatment. Top panel - representative 3D reconstructions of ventricular spaces from T2 fluid-sensitive MRI data acquired one day before Dtx treatment and 5-18 days after the start of Dtx treatment in 2-10 month-old animals. LV - lateral ventricles (blue), 3V - third ventricle (green), PR - pineal recess (orange), 4V - fourth ventricle (red). Scale bar - 100 pm. Bottom panel - quantification of ventricular volume from 3D reconstructions of T2 fluid-sensitive MRI data (normalized to pre-Dtx mean ventricular volume, n = 10-12 per group, mean ± SEM, One-Way ANOVA) * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001.

[0012] FIG. 5. Behavioral characterization of ChP-ablated mice at 1 month post Dtx treatment. (A) - change in body weight in a cohort of animals treated with Dtx at P30, followed up to day 30 post-Dtx, and a separate cohort weighed at 5 months post-Dtx. (B, D, F) - behavioral data from acohort treated with 4 ng / g / day x 3, n = 6-8 per group. (C, E, G, H) - data from a cohort treated with 20 ng / g / day x 3, n = 10-15 per group. (B, C) - total distance travelled in a 23 hour period in an automated open field paradigm. (F) - percentage of time spent, and distance travelled in the target quadrant in Barnes maze. (G, H) - total arm entries (G) and alternation rate (H) in a spontaneous alternation Y-maze. (E, F) - hourly motor activity over a 72 hour period in the automated open field test. Experiments conducted in 2-4 month-old animals. Data presented as mean ± SEM. (A) - Tw-Way RM ANOVA with timepoint and genotype as factors. B, F - Oneway ANOVA. C, G, H - Student’s t-test or Mann-Whitney U test. Data are presented as mean ± SEM. n.s - p > 0.05.

[0013] FIG. 6. Proliferating cells and newly-born neuroblasts in the SVZ at 3 and 6 months post- Dtx. (A-F) - data from coronal tissue sections at 3 months post Dtx. (A, B) - representative scans of coronal sections containing the SVZ area and the dorsal corpus callosum. Scale bar - 100 pm. LV - lateral ventricle, (a’, a”, b, b”) - magnified portions of A and B, containing the dorsal corpus callosum (a’, b’) and the SVZ (a”, b”) stained for DAPI (blue), Ki67 (green), DCX (red). (C, E) - quantification of ki67+ cells (normalized to length) in the dorsal corpus callosum (C) and the ventricular' wall. (D, F) - quantification of DCX+ area (normalized to SVZ length) in the dorsal corpus callosum (D) and the ventricular SVZ (F). Data are mean ± SEM, n = 8 per group, Two- way ANOVA with sex and genotype as factors. No statistically significant sex differences were detected. (G-L) - data from SVZ whole mount preparations at 3 months (G-I) and 6 months (J-L) post Dtx. Scale bar - 1 mm. (G, J) - representative SVZ whole mounts for the 3 month cohort (G) and the 6 month cohort (J) stained for DCX (red) and ki67 (green). (H, K) - DCX+ area fraction at 3 and 6 months post ablation. (I, L) - Ki67+ cell density at 3 and 6 months post ablation. Animals were 6-7 months old at the time of tissue collection. Data are mean ± SEM, n = 3-4 per group, Student's t-test or Mann-Whiney U test), n.s - p > 0.05, ** - p < 0.01, *** - p < 0.001.

[0014] FIG. 7. Disrupted ependymal cilia structure in ChP-ablated iDTR mice. (A) - schematic of the SVZ wall with the path of ependymal flow (blue arrow) and the attachment point (grey ceircle with asterisk). RMS - rostral migratory stream. B, D, E - representative areas in the ependymal flow path (A) with distinct directional cilia bundles at 3 months post-Dtx, 6 month-old animals. Stained for acetylated tubulin (green). Scale bar - 10 qm (C) - representative 3Dreconstructions of the ventricular surface stained for acetylated tubulin (green), DCX (red) and ki67 (gray). Scale bar - 50 pm.

[0015] FIG. 8. Effects of ChP ablation on migration of newly -born neuroblasts into the OB. A - experimental design schematic. BrdU labeling was performed at 5 months post-Dtx and tissue was collected at 1 week and 1 month post-BrdU. Representative OB sagittal sections from Dtx-treated WT (B, D) and iDTR (C, E) animals at 1 week (B, C) and 1 month (D, E) post-labeling, stained for BrdU (green) and NeuN (red), with magnified portions showing representative areas of the RMS and GCL. (F, H, J, L) - quantification of RMS BrdU-i- area fraction (F, J) and RMS area (H, L) at 1 week post-BrdU (F, H) or 1 month post-BrdU (J, L). (G, I, K, M) - quantification of GCL BrdU-i- cell density (G, K) and GCL area (I, M) at 1 week post-BrdU (G, I) or 1 month post-BrdU (K, M). Scale bar - 100 pm. Data are mean ± SEM, n = 4 per group or 1 week (F - I), n = 6-7 per group for 1 month (J - M), 3-4 sections per animal. Student’s t-test or Mann-Whitney U test, ns - p > 0.05, ** - p < 0.01.

[0016] FIG. 9. Effect of ChP ablation on SVZ neurogenesis at 1 month after ischemic stroke. A - schematic of the experimental design. (B, C) - representative T2- weighed MR images showing induction of focal cerebral ischemia in the striatum of WT (B) and iDTR (C) mice. Scale bar - 1 mm. (D, E, F) - planimetric quantification of edema volume (D), percentage of edema expansion (E), and percentage of infarct volume (F). Data are mean ± SEM, n = 4 - 6 per group, Student’s t- test, or Mann-Whitney U test, ns - p > 0.05. (G-K) - representative images of coronal sections of the ipsilateral SVZ of WT (G, H) and iDTR (I, K), immunostained for DCX (G, I) and DCX / GFAP / DAPI (H, J). Dashed grey line shows lesion border. Dashed yellow line shows distance travelled by the furthest migrating neuroblast (yellow boxes and magnified inserts in panels G and I). LV - lateral ventricle, SVZ - subventricular zone, CC - corpus callosum. Scale bar - 100 pm. (K - M) - quantification of DCX immunopositive area in the ipsilateral SVZ (K), ipsilateral corpus callosum (L), normalized to SVZ or CC length, respectively, and ipsilateral striatum, as a measure of post-stroke neuroblast migration into the lesion site (M). (N) - quantification of the average maximum distance from the SVZ travelled by a neuroblast per each section. Data are mean ± SEM, n = 5-7 per group, Student’s t-test or Mann-Whitney U test, ns - p > 0.05, * - p < 0.05, ** - p < 0.01.

[0017] FIG. 10. Rapid loss of ventricular spaces in Dtx-treated iDTR mice following ChP ablation. (A) Schematic of experimental timeline. (B) T2-wcighcd MRI one day before start of Dtx treatment and 3 days after the start of Dtx treatment, collapsed lateral and 3rd ventricles shown in red and green arrows, respectively, scale bar - 1 cm. (C) Quantification of the volume of the lateral and third ventricles calculated from 9 T2 slices per animal (mean ± SEM, n = 11-13 per group, Two-way repeated measures ANOVA with genotype and timepoint as factors). (D, E) - images of coronal brain sections showing the ventricle (red arrows). (F, G) - magnified image of coronal brain sections stained with DAPI showing the ventricle lumen arrows). Scale bar - 100 pm.

[0018] FIG. 11. Validation of ventricular volume loss in multiple iDTR allele-containing mouse lines from different labs. (A). Histological tissue sections collected in 2015-2017 (provided by Crone lab) showing ventricle loss in Chxl0-Cre+ iDTR-i- and ROSA26iDTR only animals. (B, D) Phenotype of ventricular volume loss seen in T2- weighted MRI in the Cx3crl-creER;iDTR line (expressed in macrophages and microglia) and the mGFAP-Cre;iDTR line (neural stem cells and astrocytes). (C) Histological brain sections stained with DAPI showing ventricle collapse in the GLAST-CreER;iDTR line (astrocytes), scale bar - 100 pm. In all panels the ventricle is indicated by red arrows.

[0019] FIG. 12. Stable and sustained CSF volume loss in Dtx-treated iDTR mice. (A, B) representative 3D reconstructions of fluid-filled ventricular spaces of WT (A) and iDTR (B) mice from T2 fluid-sensitive MRI data (lateral ventricles - blue, third ventricle - green, pineal recess - orange, fourth ventricle - red). (C-F) - quantification of ventricle volumes at day (-1) before Dtx treatment, day 3 after the start of Dtx treatment and at 8 and 20 months after the start of Dtx treatment at the age of 2 months. LV - lateral ventricles, 3V - third ventricle, PR - pineal recess, 4V - fourth ventricle (mean ± SEM, n = 3-4 per group, Student’s t-test, Welch’s t-test or Mann- Whitney U test within timepoints), n.s. - p > 0.05, * - p < 0.05, ** - p < 0.01, ** - p < 0.001, *** - p < 0.0001. Scale bar - 2 mm.

[0020] FIG. 13. ChP ablation induces CSF volume loss in different ages across adulthood in iDTR mice treated with Dtx. (A, B) - representative 3D reconstructions and quantification of ventricular spaces of WT and iDTR mice treated with Dtx at one month of age (A) and 20-24 months of age (B), scanned at 1 month after Dtx treatment. LV - lateral ventricles (blue), 3V - third ventricle(green), PR - pineal recess (orange), 4V - fourth ventricle (red). Scale bar - 2 mm. Data are presented as mean ± SEM, n = 3-5 per group, Student’s t-test or Mann-Whitney U test. * - p < 0.05, ** - p < 0.01, *** - p < 0.001, «« - p < 0.0001.

[0021] FIG. 14. Dose-dependent effects of ChP ablation on adult SVZ newly-born neuroblast pool at 1 month post-Dtx. (A, B, C) - Whole-mount immunostaining for DCX at 1 month post-Dtx in WT (A) mice that received 20 ng X3 of Dtx, iDTR mice that received 4 ng X3 of Dtx (B), and iDTR mice that received 20 ng X3 of Dtx (C) Scale bar - 1 mm. (a’, b’, c’) - magnified inserts showing representative areas of the SVZ whole mounts with migrating DCX+ newly-born neuroblasts. Scale bar - 100 pm. (D) - quantification of DCX+ area fraction of the SVZ whole mounts, Student’s t-test. * - p < 0.05.

[0022] FIG. 15. Validation of neuroblast migration phenotype using a different Dtx dosage, BrdU labeling regimen and sectioning plane (coronal). (A) - schematic of experimental design. (B, C) - representative coronal OB sections from Dtx-treated WT (B) and iDTR (C) mice stained for BrdU (green) and NeuN (red). Scale bar - 100 pm. D, E - quantification of BrdU-i- cell density (D) in the GCL and GCL area (E). n = 5 - 6 per group, 4 sections per animal. Data are mean ± SEM, Student’s t-test (D) or Mann- Whitney U test (E). ns - p > 0.05, * - p < 0.05.

[0023] FIG. 16. BrdU+ cell population in SVZ whole-mounts at 1 week post labeling. A - experimental design schematic. Representative images of SVZ whole mounts stained for BrdU (B) and DCX (C) Scale bar - 1000 pm. D - overlay. E - representative magnified area, scale bar - 100 pm. (F, G) - Representative images of SVZ whole mounts at 6 months post-Dtx showing similar Ki67+ cells in Dtx-treated WT (F) and iDTR (G) mice (quantification of Ki67 cells is presented in Fig. 61 and 6L). H - unbiased stereological quantification of BrdU-i- cell population in SVZ whole mounts. I - quantification of SVZ whole mount area. Data are mean ± SEM, n = 4 per group. Student’s t-test. ns, p > 0.05, * p < 0.05.

[0024] FIG. 17. Effect of ChP ablation on the stimulation of SVZ neurogenesis in the contralateral SVZ at 1 month post-stroke. (A, B) - representative images of the contralateral SVZ of WT (A) and iDTR (B) mice, stained for DCX (red) and DAPI (blue). LV - lateral ventricle, CC - corpus callosum, SVZ - subventricular zone. Scale bar - 100 pm. (C, D) - quantification of DCX immunopositive area in the contralateral SVZ (C) and contralateral corpus callosum (D),normalized to SVZ or CC length, respectively. Data are mean ± SEM, n = 5-7 per group, Student’ s t-tcst. ns - p > 0.05, * - p < 0.05.

[0025] FIG. 18. Proof-of-concept study demonstrating the feasibility of choroid plexus (ChP) ablation in the neonate. (A) - experimental design. (B) - T2 MRI data collected at P21 and P30 (ventricular spaces visible in white). (C) - 3D reconstruction of fluid-sensitive MRI collected at P30, LVs -lateral ventricles, 3V - third ventricle, PR - pineal recess, 4 V - fourth ventricle. (D) - quantification of ventricle volume from (C), mean ± SEM, n = 4 per group, Student’s t-test, *** - p < 0.001.

[0026] FIG. 19 depicts the timeline of bilateral intracerebroventricular (ICV) injections in vivo of AAV-CMVp-cGFP and AAV-CMVp-rapaCas9 and data collection time. P, postnatal days.

[0027] FIG. 20 depicts the P0 injections of AAV-CMV-eGFP and AAV-CMV-rapaCasp9 visualized in the ChP.

[0028] FIG. 21 demonstrates that administration of AAV-CMV-rapaCasp9 and AAV-CAG- rapaCasp9 results in signs of ChP cell death after rapamycin treatment. Cleaved caspase-3 (c- Casp3) and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) show ongoing ChP cell death via the rapaCasp9 and rapamycin dependent apoptosis pathway (Panel A- D); c-Casp3 and TUNEL levels are higher in AAV-ChP-rapaCasp9 and rapamycin treated mouse (A and C)

[0029] FIG. 22 shows the results of AAV administration in cultured mouse ChP cells using the CMV (A) and the CAG (CMV-early enhancer / chicken B-actin) promoter (B). AAV-CAG-eGFP showed higher gene expression of eGFP in mouse ChP cells.

[0030] FIG. 23 depicts a graph showing dPCR results in P5 and P14 AAV-eGFP treated mice that show preferred biodistribution. dPCR data from P5 and P14 mice show preferred high AAV accumulation in the ChP (200-600 viral genome copy / cell), after P0 ICV injections. 30 ng of tissue DNA were used from each sample and demonstrates data regarding low AAV distribution in the brain and peripheral organs (less than 25 viral genome copy / cell) after ICV injections.

[0031] FIG. 24 demonstrates that ChP organoids infected with AAV-rapaCasp9 show ongoing cell death after administration of rapamycin. Organoids were cultured from human embryonic stemcells (hESCs) with choroid plexus protocol and infected with AAV-rapaCasp9 at approximately 30 days. AAV incubated on organoids for 6 days and were harvested. On day 7, organoids were treated with 200 nM rapamycin and harvested 24 hours later. Panel A. Transthyretin (TTR) is a ChP-specific transport protein. TTR IHC confirms ChP identity of epithelial protrusions in the organoids. Panel B. Cleaved-Caspase3 (c-Casp3)+ cells are seen in ChP organoids after rapamycin administration. The pink dots indicate ongoing cell death in the epithelia- like areas of the organoid. Panel C. Caspase9+ cells in an untreated AAV-infected organoid show successful infection after 6 days of incubation.

[0032] FIG. 25. Progressive hydrocephalus (pr ) mouse model. MRI-based ventricular volume analysis (left) and histological analysis (right) show significantly enlarged lateral ventricles (LV) in the prh mutant mice.

[0033] FIG. 26. Depicts data showing smaller ventricles after ChP ablation in the prh mice. MRI- based brain ventricular size analysis showed that significant reduction of ventricular volume in prh mutant mice after the ChP ablation.DETAILED DESCRIPTION

[0034] Disclosed are compositions and methods for the treatment of hydrocephalus in an individual in need thereof. In particular, a recombinant adeno-associated virus, (rAAV) such as AAV2 / 5 (a combination of replication deficient AAV2 genome and AAV5 capsid) that can be used to target the ChP epithelia and which can be used to ablate the ChP using a suicide gene system is disclosed. The disclosed methods employ AAV vectors to target the ChP for alleviating one or more symptom of hydrocephalus, including, but not limited to, CSF accumulation. The disclosed methods may be used alone, or in combination with surgical procedures for the reduction of accumulated CSF in the brain of an individual. In aspects, the methods may be used to achieve ablation of ChP using an AAV as a gene delivery tool to selectively target the ChP to achieve significant reduction in cerebrospinal fluid (CSF) volume in the individual. The AAV-mediated gene delivery methods may be used for ChP ablation as a therapeutic intervention to reverse excessive CSF accumulation. The methods may be used to treat, for example, neonatal hydrocephalus, which is deleterious to normal brain development and which causes debilitating neurological symptoms. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional oroptional element described herein or otherwise useful in the treatment of hydrocephalus using AAV vectors in combination with a suicide gene.

[0035] Gene therapy systems utilizing AAV vectors and suicide genes typically comprise a promoter (which can be tissue-specific or inducible) to drive expression of the suicide gene. After delivery, the vector enters target cells and is transported to the nucleus, where the host transcription machinery recognizes the promoter and transcribes the suicide gene into mRNA, which is then exported to the cytoplasm and translated into protein. The suicide gene typically functions by, for example, either encoding a cytotoxic protein that directly disrupts essential cellular processes or by expressing an enzyme that converts a subsequently administered non-toxic prodrug into its active, cytotoxic form, ultimately leading to apoptosis.

[0036] It has been found that non- surgical ChP removal can be achieved using an AAV-mediated targeting of ChP cells, and that this targeted ablation permits rapid and permanent CSF volume loss. Further, following the targeted ChP ablation, it was found that the ventricular size of the brain of treated mice reduced following treatment. In contrast, surgical methods which ablate the ChP, primarily accomplished by endoscopic ChP cauterization, has a 50% or greater failure rate, with no observation of ventricular resizing. Yet further, it was found that the targeted ChP ablation using in iDTR mice treated with Dtx did not cause any major neurological problems, in contrast to traditional methods used to ablate ChP. The disclosed methods were further found to achieve rapid and permanent reduction of CSF volume, in contrast to surgical methods which ablate ChP.Gene Therapy Vector

[0037] Disclosed are gene therapy vectors for the ablation of ChP in an individual in need thereof, particularly useful for the treatment of hydrocephalus. In embodiments, the gene therapy vector includes an AAV vector, a promoter, and a suicide gene. The gene therapy vectors may further comprise one or more additional components for functioning of the gene therapy vectors, for example, AAV-specific inverted Terminal Repeats (ITRs), regulatory elements, capsid proteins, and selection markers / reporter genes, as would be understood by one of ordinary skill in the art.AAV Vectors

[0038] The disclosed gene therapy vectors and related methods utilize an adeno-associated vims vector (AAV). AAV vectors are known in the art, and described in, for example, WO2020132466A1. Recombinant AAV described herein may be generated using known techniques. The term “AAV” as used herein refers to naturally occurring adeno-associated viruses, adeno-associated viruses available to one of skill in the art, as well as recombinant AAVs including chimeric and pseudotyped AAV vectors. In general, an AAV viral vector is an AAV DNase- resistant particle having an AAV protein capsid into which is packaged expression cassette flanked by AAV inverted terminal repeat sequences (ITRs) for delivery to target cells.

[0039] The disclosed gene therapy vectors may utilize a variety of different AAV vectors. In embodiments, the AAV vector is a ChP-preferred subtype which preferentially directs, or restricts, the infection of AAV to the ChP. In embodiments, the AAV vector is an recombinant AAV with replication-deficient AAV2 genome and AAV5 capsid, often referred to as AAV2 / 5, AAV5, or rAAV5. In embodiments, the AAV vector is an AAV5 vector. The AAV5 vector is also known in the art and is described in, for example, Jang, A., Lehtinen, M.K. Experimental approaches for manipulating choroid plexus epithelial cells. Fluids Barriers CNS 19, 36 (2022), doi.org / 10.1186 / sl2987-022-00330-2 and Ozelo, Margareth C., et al. "Valoctocogene roxaparvovec gene therapy for hemophilia A." New England Journal of Medicine 386.11 (2022): 1013-1025. The full sequence of AAV2 gene groups are disclosed in accession no. NC_001401.2 (www.ncbi.nlm.nih.gov / nuccore / NC_001401.2); an exemplary AAV5 capsid sequence is disclosed in accession no. NC_006152.1 (www.ncbi.nlm.nih.gov / nuccore / NC_006152.1).

[0040] In embodiments, the gene therapy vector is a chimeric AAV containing genetic elements from two or more serotypes. For example, pseudotyped vectors, wherein the capsid of one AAV is replaced with another natural or recombinant capsid protein, may be used. Such vectors are known in the art and described in, for example, Chen X, He Y, Tian Y, et al. Different Serotypes of Adeno-Associated Virus Vector- and Eentivirus-Mediated Tropism in Choroid Plexus by Intracerebroventricular Delivery. Hum Gene Ther. 2020;31(7-8):440-447. doi:10.1089 / hum.2019.300. In embodiments, the AAV vector is a chimeric vector and is AAV2 / 5. AAV2 / 5 is known in the art and is described in, for example, Chen X, et al (Hum. Gene. Ther. 2020), above. The AAV2 / 5 pseudotype has tropism to ChP epithelial cells, but not other neuralcell types, such as neurons, astrocytes, oligodendrocytes, microglia, or vascular endothelial cells. The AAV2 / 5 vector rarely targets other neural cell types, such as neurons, astrocytes, oligodendrocytes, and microglia when administered intracerebroventricularly. Therefore, the AAV2 / 5 pseudotype, which has the ChP-preferring AAV5 capsid in the CNS stable AAV2 genome, archived selective and prolonged (12 months) gene expression in mouse ChP epithelial cells in vivo, when it is applied to embryonic, newborn, or adult rodent brains. The AAV2 / 5 vector has been used to deliver ChP-specific exogenous NKCC1 ion transporter (see, e.g., Mazucanti, C.H. et al. AAV5-mediated manipulation of insulin expression in choroid plexus has long-term metabolic and behavioral consequences. Cell Rep 42, 112903 (2023)), superoxide dismutase, or insulin in ChP in vivo via intracerebroventricular (ICV) injection. AAV2 / 5 preferentially targets human ChP epithelial cells among many other human neural cell types in cultured human brain organoids which appear with cuboidal epithelium with transthyretin (TTR) and epithelial tissue organization, and which have a similar set of gene expression profiles to the human and mouse ChP.Promoter

[0041] The disclosed compositions and methods further utilize a promoter. The term “promoter,” as used herein, refers to any nucleic acid sequence that regulates the expression of genes by driving transcription of the nucleic acid sequence, which can be a heterologous target gene encoding a protein or an RNA. Promoters can be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter is a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. A promoter can also contain genetic elements at which regulatory proteins and molecules can bind, such as RNA polymerase and other transcription factors. In some embodiments of the aspects described herein, a promoter can drive the expression of a transcription factor that regulates the expression of the promoter itself. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive the expression of transgenes in vectors disclosed herein. A promoter sequence may be bounded at its 3' terminus by thetranscription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.

[0042] In embodiments, the promoter is an active promoter. As used herein, an “active” promoter refers to a promoter that drives high levels of gene transcription, in that the promoter efficiently recruits RNA polymerase and associated transcription factors, leading to robust mRNA production. In embodiments, the active promoter has one or more of the following characteristics: high transcriptional activity (produces abundant mRNA and high protein expression); efficient binding of RNA polymerase (contains sequences that strongly attract RNA polymerase and transcription factors); optimized core promoter elements (e.g., including well-defined TATA boxes, GC-rich regions, and initiator (Inr) sequences that enhance transcription initiation); the presence of enhancers (enhancer sequences that further boost transcription levels); and / or minimal repression (less likely to be silenced by chromatin modifications or repressor proteins). In embodiments, a promoter that permits expression to the ChP is used in the recombinant AAV vector, which activates transcription of suicide genes in ChP and restricts suicide gene expression to the ChP.

[0043] In embodiments, the promoter is a CAG (CMV-early enhancer / chicken beta-actin) promoter. In embodiments, the promoter is a CBh (CMV early enhancer fused to modified chicken P-actin promoter), which can be used to ensure strong expression of components of the Tet-On system.

[0044] In embodiments, the promoter is a CMV (Cytomegalovirus) Promoter.

[0045] In embodiments, the promoter is a tetracycline -responsive promoter element (TRE) Promoter. This promoter allows for the control of suicide gene expression by the components of the Tet-On system.

[0046] In embodiments, the promoter is a EF-la (Elongation Factor- 1 Alpha) Promoter.

[0047] Suicide Gene / Pro-Drug

[0048] In embodiments, the gene therapy vector comprises a suicide gene, operative connected to a promoter. As used herein, the term “suicide gene” refers to a gene that causes the cell expressing the suicide gene to die. The suicide gene can be a gene that confers sensitivity to an agent, e.g., adrug, upon the cell in which the gene is expressed, and causes the cell to die when the cell is contacted with or exposed to the agent. Suicide genes arc known in the art and include, for example, rapaCasp9, diphtheria toxin subunit a (Dta), Herpes Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine deaminase, purine nucleoside phosphorylase, and nitroreductase.

[0049] In embodiments, the suicide gene is a rapamycin-induced caspase 9 suicide gene (RapaCasp9). RapaCasp9 is a drug-inducible suicide gene system that utilizes a modified form of human caspase-9, a key initiator of the intrinsic apoptotic pathway, which has been engineered to include a drug-binding domain derived from FKBP12, a protein that interacts with rapamycin and its non-immunosuppressive analogs, such as AP1903. When a rapamycin analog (e.g., AP1903 or rimiducid, a small-molecule drug) is introduced into the system, the engineered rapaCasp9 protein is activated, resulting in dimerization and activation of exogenous caspase 9. Upon administration of the small-molecule, the modified caspase-9 molecules undergo rapid forced homodimerization, leading to autocatalytic activation of the apoptotic cascade. This results in the selective induction of caspase-dependent apoptosis in the engineered cells, thereby eliminating potentially harmful or overactive therapeutic cells which could result in an unfavorable outcome. RapaCasp9 is described in, for example, Stavrou M, et al., A Rapamycin-Activated Caspase 9-Based Suicide Gene. Mol Ther. 2018 May 2;26(5):1266-1276. doi: 10.1016 / j.ymthe.2018.03.001. Epub 2018 Mar 9. PMID: 29661681; PMCID: PMC5993966.

[0050] In one embodiment, the suicide gene is cytosine deaminase. Cytosine deaminase (CD) converts cytosine to uracil, in FDA-approved cancer gene therapy. CD is delivered to tumor cells, where it converts a non-toxic prodrug, such as 5-fluorocytosine into an active cytotoxic drug 5-(5- FU). 5-FU is an antimetabolite that inhibits thymidylate synthase, disrupting DNA and RNA synthesis, leading to cell death. Although ChP cells are non-dividing cells, 5-FU’s RNA synthesis inhibition is expected to cause cell death, therefore it can be the alternative ChP-ablation methods via AAV5 vector.

[0051] In embodiments, the suicide gene is the Diphtheria Toxin subunit A (DTA) suicide gene system. DTA combined with the tet-ON system in promoter is a drug-inducible suicide gene system that induces cell death in cells by expressing DTA, which is the active component of C. cliphtheriae toxin. To allow for more precise and dosc-dcpcndcnt induction of cell ablation, DTA is expressed under the control of the Tet-On system, composed of the tetracycline transcriptionsilencer (tTS), and the reverse tetracycline-responsive transcriptional activator (rtTA), as well as the TRE promoter. In the absence of the small molecule activator, tTS binds to the TRE, preventing suicide gene expression. In the presence of the FDA-approved drug tetracycline (or analogues such as doxycycline), rtTA binds to TRE, activating the expression of the suicide gene (DTA), thus resulting in apoptosis through arrest of protein synthesis. The use of this system would allow for both temporally controllable, and dose-dependent ablation of the ChP. The Tet-On system is described, for example, in Das AT, Tenenbaum L, Berkhout B. Tet-On Systems For Doxycycline- inducible Gene Expression. Cun' Gene Then 2016;16(3): 156-67. doi: 10.2174 / 1566523216666160524144041. PMID: 27216914; PMCID: PMC5070417.

[0052] Further suicide genes are known in the art and may be employed. For example, the suicide gene may encode an inducible HSV thymidine kinase. In this embodiment, ganciclovir is administered to cause cellular apoptosis in the choroid plexus. In one embodiment, the suicide gene may encode an inducible cytosine deaminase. In this embodiment, 5-fluorocytosine is administered to generate 5-fluorouracil to cause cellular apoptosis in the choroid plexus. In one embodiment, the suicide gene may encode an inducible nitroreductase. In this embodiment, CB1954 is administered to produce a DNA-crosslinking cytotoxin to cause cellular apoptosis in the choroid plexus. In one embodiment, the suicide gene may encode an inducible diphtheria toxin subunit A (DTA). In this embodiment, the expression of DTA alone in the choroid plexus is sufficient to induce apoptosis through the arrest of protein synthesis. An appropriate activator is administered to inhibit protein synthesis and induce apoptosis in the choroid plexus.Methods of Treating

[0053] In one aspect, a method of treating hydrocephalus in an individual in need thereof is disclosed, the method comprising administering to the choroid plexus (ChP) of the individual a ChP targeting adeno-associated virus vector (AAV) that targets the choroid plexus (AAV-ChP) comprising a suicide gene, further comprising administering a suicide gene activator to the individual.

[0054] In embodiments, the method includes administering to an individual in need thereof an AAV-based gene therapy vector as described herein.

[0055] In embodiments, the individual is one who is diagnosed with hydrocephalus. In embodiments, the individual is a pediatric individual diagnosed with hydrocephalus. In embodiments, the individual is a neonate diagnosed with hydrocephalus. In embodiments, the individual is one who is determined to be likely to benefit from targeted ablation of the ChP. In embodiments the hydrocephalus is neonatal hydrocephalus.

[0056] In embodiments, the method includes administering to an individual in need thereof a recombinant AAV-CHP gene therapy vector comprising a suicide gene and a suicide gene activator. In aspects, the method may be canned out in conjunction with the placement of a shunt in the individual. In embodiments, the method includes administering to an individual in need thereof, a recombinant AAV-CHP gene therapy vector comprising a suicide gene and a suicide gene activator, wherein the suicide gene is a rapamycin-induced caspase 9 suicide gene (rapaCasp9). In this aspect, the method further includes administering a suicide gene activator, wherein the suicide gene activator is rapamycin. In further aspects, the suicide gene activator is rapamycin and the suicide gene is rapaCasp9, and the rapamycin is administered after administering the AAV-ChP. The administration of the suicide gene activator may be immediately following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one hour following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one day following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least two days following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be more than two days following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, the rapamycin may be administered prior to the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, the rapamycin may be administered at the same time as the recombinant AAV-CHP gene therapy vector comprising a suicide gene.

[0057] In embodiments, the method includes administering to an individual in need thereof, a recombinant AAV-CHP gene therapy vector comprising a suicide gene and a suicide geneactivator, wherein the suicide gene is diphtheria toxin a (Dta). In this aspect, the method further includes administering to the individual a suicide gene activator, wherein the suicide gene activator is doxycycline. In further aspects, the suicide gene activator is doxycycline and the suicide gene is Dta, and the doxycycline is administered after administering the AAV-ChP. The administration of the suicide gene activator may be immediately following the administration of the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one hour following the administration of the recombinant AAV-CHP comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one day following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least two days following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be more than two days following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, the doxycycline may be administered prior to the recombinant AAV-CHP gene therapy vector comprising a suicide gene. In further aspects, the doxycycline may be administered at the same time as the recombinant AAV- ChP gene therapy vector comprising a suicide gene.

[0058] In further embodiments, the method includes administering to an individual in need thereof a recombinant AAV-CHP gene therapy vector comprising a suicide gene and a suicide gene activator, wherein the suicide gene is cytosine deaminase. In this aspect, the method further includes administering to the individual a suicide gene activator, wherein the suicide gene activator is 5-fluorocytosine. In further aspects, the suicide gene activator is 5-fluorocytosine and the suicide gene is cytosine deaminase, and the 5-fluorocytosine is administered after administering the AAV- CHP gene therapy vector. The administration of the suicide gene activator may be immediately following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one hour following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least one day following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, administration of the suicide gene activator may be at least two days following the administration of the recombinant AAV-ChP gene therapy vector comprisinga suicide gene. Tn further aspects, administration of the suicide gene activator may be more than two days following the administration of the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, the 5-fluorocytosine may be administered prior to the recombinant AAV-ChP gene therapy vector comprising a suicide gene. In further aspects, the 5-fluorocytosine may be administered at the same time as the recombinant AAV-ChP gene therapy vector comprising a suicide gene.

[0059] The method may employ any mechanism by which the AAV-ChP gene therapy vector can be administered to a ventricle of the brain of the individual in need of treatment. In particular, the administration generally comprises any mechanism by which the AAV-ChP gene therapy vector can be contacted with the choroid plexus epithelial cells of the individual. For example, in aspects, the AAV-ChP gene therapy vector can be administered via intracerebro ventricular (ICV) injections, for example bilateral intracerebroventricular (ICV) injections. Both the AAV-ChP gene therapy vector and the suicide gene activator may be administered via the same technique.

[0060] The administration of the AAV-ChP gene therapy vector and the suicide gene activator may be administered for an amount and for a duration of time effective to ablate the choroid plexus of the individual. For example, the administration may be carried out sufficient to achieve either partial ablation or full ablation of the choroid plexus. Ablation may be determined by choroid plexus size after treatment. In aspects, the ablation is a reduction in size of at least about 50%, or at least about 60%, or at least about 70% or more as compared to the size of the choroid plexus prior to treatment. Efficacy of choroid plexus ablation applying this standard is shown in FIG. 2, J.

[0061] The administration of the AAV-ChP gene therapy vector and the suicide gene activator may be administered for an amount and for a duration of time effective to reduce production of cerebral spinal fluid (CSF) in the individual, which may be approximated by observation of a reduction of size of the ventricle in the treated individual. Reduction in production of CSF is determined by total CSF present post-treatment as compared to CSF present pre-treatment. In embodiments, the reduction is at least about 50%, or about 60%, or about 70%, or about 80% or more. Efficacy of the AAV-ChP vectors in reducing CSF production is shown in FIG 3, C.

[0062] The administration of the AAV-ChP gene therapy vector and the suicide gene activator may be administered for an amount and for a duration of time effective to reduce cerebral spinal fluid (CSF) volume in the individual, which may be approximated by observation of a reduction of size of the ventricle in the treated individual. The reduction in CSF volume may be approximated by an observed reduction in choroid plexus size. In aspects, the reduction in CSF volume (as determined by ventricle size) is a reduction of at least about 50%, or about 60%, or about 70%, or about 80% or more. See FIG. 3, C.

[0063] In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof monthly. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof monthly. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof every two months. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof every three months. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof every four months. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof every five months. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof every six months. In embodiments the method may include administration of one or both of the AAV-ChP and suicide gene activator to the individual in need thereof performed yearly.Route of Administration / Dosing

[0064] The formulated compositions may be delivered to the cell using routes of administration known in the art and described herein. For example, the disclosed compositions may be administered by any route that results in a therapeutically effective outcome. In one aspect, the administration is intracerebroventricular (into the cerebral ventricles). In embodiments, the disclosed compositions may be delivered to an individual in need thereof via injection. In embodiments, the disclosed compositions may be delivered to an individual in need thereof via intracerebroventricular injection (1CV). In embodiments, the disclosed composition is delivered via a small subgaleal ventricular access port, similar to Ommaya reservoir, that is clinically usedto manage neonatal hydrocephalus patients. Access to the cerebral ventricles can be performed with or without stereotactic guidance and is a relatively common neurosurgical procedure.

[0065] In embodiments, a single dose of the AAV is administered, followed by at least one, or at least two, or more than two doses of the suicide gene activator (small molecule drug, e.g. rapamycin, doxycycline / tetracycline, or GCV, etc). In embodiments, at least two doses are administered. In embodiments, at least three doses are administered. In embodiments, an initial dose of the suicide gene activator is administered, followed by a subsequent dose of the suicide gene activator, the subsequent dose being administered at least one week after the initial dose, or at least two week safter the initial dose.Virus Titer

[0066] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The dose of AAV viral vectors, e.g., the units of dose in vector genomes / per kilogram of body weight (vg / kg), required to achieve a desired effect or “therapeutic effect” (e.g., a certain serum concentration of a replacement enzyme) will vary based on several factors including, but not limited to: the route of AAV administration, the level of expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the expression multidomain therapeutic protein. One of skill in the art can readily determine a AAV virion dose range to treat a subject having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art, see, e.g., ODER “Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers,” July 2005, incorporated herein in its entirety by reference. An effective amount of the AAV is generally in the range of from about 10 pl to about 100 ml of solution containing from about 109to 1016genome copies per subject. Other volumes of solution may be used. The volume used will typically depend, among other things, on the size of the subject, the dose of the AAV, and the route of administration. In some embodiments, a dosage between about 1010to 1012AAV viral genome per subject is appropriate. In some embodiments the AAV is administered at a dose of 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject. In some embodiments the AAV is administered at a dose of 1010, 1011,1012, 1013, or 1014viral genomes per kg. In some embodiments, at least 2 x 1012viral genomes per kilogram (vg / kg) is administered.

[0067] Virus titer for treatment may be determined by one of ordinary skill in the art. For example, in embodiments, a virus titer higher than 1 x 1013GC / mL ICV is administered, at a volume of from about 2 to about 5 pL. In embodiments, the dose is 1-2 x IO10of the gene therapy vector. In embodiments, the dose is 4-10 xlO10of the gene therapy vectorSuicide Gene Activator Administration

[0068] Following administration of the AAV-rapaCasp9 administration, the appropriate suicide gene activator is administered. For example, in embodiments, the suicide gene activator is rapamycin. In this embodiment, the suicide gene is activated via rapamycin, and rapamycin is administered following administration of the AAV-rapaCasp9. Rapamycin (R-5000: CAS No. 6823-69-4, Purity >99%), available from the LC Laboratories. In embodiments, the rapamycin is administered at least one day following AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered from about 1 day to about 30 days following AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered within one week of the AAV- rapaCasp9 administration. In embodiments, the rapamycin is administered within two weeks of the AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered about 1 week following AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered about two weeks following AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered about three weeks following AAV-rapaCasp9 administration. In embodiments, the rapamycin is administered about four weeks following AAV-rapaCasp9 administration. Administration of rapamycin may be determined by one of ordinary skill in the art. In embodiments, the dosage may be about 0.1 to about 10 mg / kg, based on patient weight.

[0069] In embodiments, the suicide gene activator is tetracycline. In this embodiment, the suicide gene is activated via tetracycline, and tetracycline is administered following administration of the AAV-Tet-On-DTA. Tetracycline (T3258: CAS No. 60-54-8, Purity >99%), available from Millipore-Sigma. The dosing regimen of tetracycline is that sufficient to produce plasma concentrations of 0.5 ug / mL, or 10 mg / kg as used in preclinical studies on rats. In embodiments, the tetracycline is administered at least one day following AAV-Tet-On-DTA administration. Inembodiments, the tetracycline is administered from about 1 day to about 30 days following AAV- Tct-On-DTA administration. In embodiments, the tetracycline is administered within one week of the AAV-Tet-On-DTA administration. In embodiments, the tetracycline is administered within two weeks of the AAV-Tet-On-DTA administration. In embodiments, the tetracycline is administered about 1 week following AAV-Tet-On-DTA administration. In embodiments, the tetracycline is administered about two weeks following AAV-Tet-On-DTA administration. In embodiments, the tetracycline is administered about three weeks following AAV-Tet-On-DTA administration. In embodiments, the tetracycline is administered about four weeks following AAV-Tet-On-DTA administration. Administration of tetracycline may be determined by one of ordinary skill in the art. In embodiments, the dosage may be about 0.1 to about 10 mg / kg, based on patient weight.

[0070] In embodiments, the tetracycline is administered about 1 to 2 weeks following AAV-Tet- On-DTA administration to allow the suicide gene system to fully express. In embodiments, the tetracycline is administered daily for at least two days, or at least three days, or at least four days, or more than four days. In embodiments the administration is on consecutive days.

[0071] In embodiments, the suicide gene activator is doxycycline. In this embodiment, the suicide gene is activated via doxycycline, and doxycycline is administered following administration of the AAV-Tet-On-DTA. Doxycycline (D3447: CAS No. 10592-13-9, Purity >99%), available from Millipore-Sigma. The dosing regimen of doxycycline is that amount sufficient to produce plasma concentrations of 0.5 ug / mL, or 10 mg / kg as used in preclinical studies on rats. In embodiments, the doxycycline is administered at least one day following AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered from about 1 day to about 30 days following AAV- Tet-On-DTA administration. In embodiments, the doxycycline is administered within one week of the AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered within two weeks of the AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered about 1 week following AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered about two weeks following AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered about three weeks following AAV-Tet-On-DTA administration. In embodiments, the doxycycline is administered about four weeks following AAV-Tet-On-DTA administration. Administration of doxycycline may be determined by one ofordinary skill in the art. In embodiments, the dosage may be about 0.1 to about 10 mg / kg, based on patient weight.

[0072] In embodiments, the doxycycline is administered about 1 to 2 weeks following AAV-Tet- On-DTA administration to allow the suicide gene system to fully express. In embodiments, the doxycycline is administered daily for at least two days, or at least three days, or at least four days, or more than four days. In embodiments the administration is on consecutive days. Formulations

[0073] In embodiments, the disclosed compositions can be delivered via a formulation that contain one or more additional components suitable for administration to an individual. For example, the polynucleotides may be formulated to contain cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a biocrodiblc or biocompatiblc polymer, a solvent, and / or a sustained-release delivery depot.

[0074] Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosing may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0075] In embodiments, the disclosed compositions may include a surfactant, preservative, excipients, and / or buffer dissolved in the aqueous suspending liquid. In embodiments, the buffer is PBS. In embodiments, the buffer is an artificial cerebrospinal fluid (aCSF), e.g., Eliott’s formulation buffer; or Harvard apparatus perfusion fluid (an artificial CSF with final Ion Concentrations (in mM): Na 150; K 3.0; Ca 1.4; Mg 0.8; P 1.0; Cl 155). Various suitable solutions are known including those which include one or more of: buffering saline, a surfactant, and a physiologically compatible salt or mixture of salts adjusted to an ionic strength equivalent to about100 mM sodium chloride (NaCl) to about 250 mM sodium chloride, or a physiologically compatible salt adjusted to an equivalent ionic concentration.

[0076] In embodiments, the formulation is adjusted to a physiologically acceptable pH, e.g., in the range of pH 6 to 8, or pH 6.5 to 7.5, pH 7.0 to 7.7, or pH 7.2 to 7.8. As the pH of the cerebrospinal fluid is about 7.28 to about 7.32, for intrathecal delivery, a pH within this range may be desired; whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desired. However, other pHs within the broadest ranges and these subranges may be selected for other routes of delivery.

[0077] In one example, the formulation may contain, e.g., buffered saline solution comprising one or more of sodium chloride, sodium bicarbonate, dextrose, magnesium sulfate (e.g., magnesium sulfatc-7H20), potassium chloride, calcium chloride (e.g., calcium chloride -2H20), dibasic sodium phosphate, and mixtures thereof, in water. Suitably, for intrathecal delivery, the osmolarity is within a range compatible with cerebrospinal fluid (e.g., about 275 to about 290); see, e.g., emedicine.medscape.com / article / 2093316-overview. Optionally, for intrathecal delivery, a commercially available diluent may be used as a suspending agent, or in combination with another suspending agent and other optional excipients. See, e.g., Elliotts B® solution [Lukare Medical], In other embodiments, the formulation may contain one or more permeation enhancers. Examples of suitable permeation enhancers may include, e.g., mannitol, sodium glycocholate, sodium taurocholate, sodium deoxycholate, sodium salicylate, sodium caprylate, sodium caprate, sodium lauryl sulfate, polyoxyethylene-9-laurel ether, or EDTAExample 1

[0078] The following example supports the concept that CSF volume can be reduced by removing ChP without causing major neurological outcomes. CSF-producing choroid plexus epithelial cells express diphtheria toxin receptor (DTR) independent of Cre expression, and undergo apoptosis in response to Dtx in ROSA26iDTR mice.

[0079] The ROSA26iDTR line (referred to as iDTR mice in this study, Jax #007900) was originally developed to enable cell-type-specific ablation in mice. Specifically, it carries a floxed transcription stop-cassette preceding the gene for the simian Heparin-binding EGF-like growth factor, HB-EGF, which acts as a receptor (DTR) for C. diphlheriae toxin (Dtx). Cre-driven excision of the stop cassette induces cell-type specific DTR expression sensitizing cells to thetoxin. Upon binding of Dtx to DTR and its subsequent internalization, apoptosis is induced through the arrest of protein synthesis, resulting in cell type-specific ablation. An unexpected and pronounced loss of ventricular volume in the ROSA26iDTR mouse line upon administration of Dtx is reported by Applicant showing that Dtx induced ChP cell death in iDTR mice (Taranov et al 2024 PNAS).

[0080] Considering that the phenotype described above could present a unique opportunity to examine the role of the ChP and CSF in vivo in the adult brain, the underlying mechanism behind CSF loss in this model was investigated. Since the ChP has long been considered to be the main CSF-producing organ, it was hypothesized that the loss of CSF could be due to the Dtx-induced apoptosis in the CSF-producing ChP epithelium which might have “leaky” expression of the DTR gene in a Cre-independent manner. To test whether DTR could be expressed in the ChP or other brain regions and peripheral tissues in iDTR mice, RNA was harvested from ChPs of three cerebral ventricles and a variety of brain regions as well as some peripheral epithelial organs (skin from the ear, lung, and small intestine) from WT, iDTR heterozygous, and homozygous mice (none of which express Cre recombinase). Indeed, using quantitative reverse transcription-PCR (qRT- PCR), it was observed that a substantial amount of DTR mRNA is transcribed in iDTR heterozygous and homozygous mice (FIG. 18, B), particularly enriched in the ChP from the lateral ventricle (LV), 3rdventricle (3V) and 4thventricle (4V). The quantity of DTR RNA in the ChP generally shows higher levels in homozygous iDTR mice than heterozygous mice with a similar expression pattern among tested regions / organs. Interestingly, the levels of DTR expression in ChP arc much higher than in various brain parenchymal regions (Fig IB) and peripheral epithelial tissues (FIG. 18, B), with the gut having the highest level amongst the peripheral tissues examined, but still substantially lower than ChP levels. Importantly, all RNA samples were treated with DNase, and RT(-) control shows no amplification in the qPCR reaction, validating that the RNA levels of DTR detected in our study are not due to DNA contamination. As expected, DTR expression was not detectable in any of the WT tissues examined. To further validate the presence of DTR in the ChP of iDTR mice, immunohistochemistry was performed to detect DTR protein with an antibody specific for simian and not mouse HB-EGF and only detected DTR immunoreactive signal in the ChP but not from other brain regions in the iDTR mice (FIG. 1, C, D). Next, whether apoptosis occurs in the ChP of iDTR mice after Dtx administration was examined. TUNEL staining on day 3 was performed after the first Dtx injection (20ng / g / day),which showed extensive apoptosis within the ChP in iDTR + Dtx cohort but not in WT + Dtx or iDTR + saline cohort (FIG. 1, E-G). In WT + Dtx or iDTR + saline controls, sparse TUNEL positive cells were only detected in the regions where ongoing adult neurogenesis is present (the subventricular zone - SVZ and the subgranular zone - SGZ) consistent with previous reports39,40. However, in the iDTR + Dtx mice, in addition to the sparse TUNEL+ cells in the SVZ and SGZ regions, substantial TUNEL positive cells were also specifically detected in the ChPs. To examine which cell types in the ChP undergo apoptosis after Dtx treatment (FIG. 1, H - J), co- immuno staining was performed for the al subunit of Na+ / K+ ATPase (marker for the apical surface of the ChP epithelial cells), CD68 (macrophage marker), IB4 (blood vessel endothelium) and PDGFR (blood vessel pericytes). It was determined that compared to other cell types in the ChP, apoptotic cell death occurred predominantly (93.5+5.7%) in al-positive ChP epithelial cells (FIG. 1,K), indicating that it is the loss of CSF-producing ChP epithelial cells that leads to the loss of CSF volume and ventricular spaces in iDTR mice after Dtx treatment.[00811 Next, the degree of ChP epithelial cell ablation following different dosages of Dtx administered to iDTR mice was evaluated. Adult iDTR heterozygous mice were subjected to three different dosages of Dtx (20ng / g / day for 3 days, 4ng / g / day for 3 days, lOng / g / day for 2 days) and a control group of WT mice were treated with 20ng / g / day for 3 days. Brain tissue was collected at one month post-Dtx injection (a time point at which it was observed that the loss of ventricular volume is stable after the initial Dtx injection) to evaluate extent of ChP epithelial cell loss. First, serial coronal sections were collected and quantified ChP area by transthyretin (TTR) immuno staining, which is an additional marker for the entire ChP epithelium (FIG. 2, A- 1). A marked decrease in TTR+ ChP epithelium area was observed with all dosages of Dtx to a similar extent in the 3rdventricle ChP, while in the lateral ventricle ChP the 20 ngX3 dosage produced the greatest degree of ablation (FIG. 2, J). However, considering that the plane of coronal sections could obscure changes occurring along the entire length of the ChP, entire lateral ventricle ChPs were extracted from a cohort of WT and iDTR mice one month post- ablation and performed ChP whole mount immunohistochemistry to stain for TTR (ChP epithelial cells) and CD31 (blood vessel endothelium marker). Consistent with the coronal section evaluation, ChP whole mount immuno staining also shows (FIG. 2, K-M) that the total surface area of ChP is decreased following the high (20ng / g / day X3) more so than the low (4ng / g / dayX3) dose of Dtx and, interestingly, loss of ChP epithelial cells and shrinkage of total ChP surface area resulted in to increased blood vessellinear length normalized to ROT area (defined as blood vessel length density in our study) in ChP- ablatcd mice (FIG. 2, N). This suggests that loss of ChP epithelial cells might lead to secondary changes in other cell types in the ChP, which warrants further investigation in future studies. At 3 or 6 months post-Dtx treatment, almost complete absence of ChP in iDTR mice was observed, therefore ChP whole mounts could not be collected at these time points to evaluate the remaining ChP size. Interestingly, this finding demonstrates the absence of regeneration of the adult ChP epithelium after ablation, which has not been specifically tested previously.

[0082] Thus, it was demonstrated that administration of Dtx to iDTR mice is a non-invasive way to selectively ablate the CSF-producing epithelial cells. Taking advantage of this model, the role of ChP / CSF in general brain health and some key neurological processes that have been long speculated to be dependent on normal ChP / CSF functions was investigated.

[0083] Rapid loss of ventricular spaces in Dtx-treated iDTR mice following ChP ablation.

[0084] To examine the timeline of CSF loss following ChP ablation, 20 ng / g / day of Dtx was administered over three days in young adult mice (3 - 4 months old, female and male animals, FIG. 10, A). The loss of ventricle volume was visible on T2-weighted MRI scans as early as three days after the first Dtx injection (FIG. 1, B). Both female and male iDTR mice showed a similar pattern and extent of CSF loss following ChP ablation. The reconstruction of ventricular volume from T2- weighted slices showed a pronounced decrease in the volume of lateral and third ventricles on day 3 after start of Dtx treatment in iDTR but not in WT mice. (FIG. 1, C). The ventricular size phenotype was also observed in PFA-fixed histologically processed coronal brain sections (FIG. 1, D - G). These results confirm that the loss of ventricular space occurred in a Cre-independent manner, demonstrating that only the ROSA26iDTR allele is required to induce ventricle loss. Next, to further investigate how fast CSF is depleted in Dtx-treated iDTR mice, Dtx was administered at 20 ng / g / day X 3 to 3-4-month-old mice and acquired T2-weighted MRI scans one day before the start of Dtx treatment and at one, two, and three days after the first Dtx injection (FIG. 3, A). In Dtx-treated iDTR mice, the loss of ventricular volume has already initiated by day 1 after the start of Dtx treatment, exacerbating by day 2 and proceeding to almost complete loss of ventricular volume on day 3 post-Dtx (FIG. . 3, B, C). That suggests either the requirement of at least three consecutive Dtx doses or 3 days for the apoptosis in the ChP epithelium to reach its peak or it could also reflect the delayed relationship between the loss of ChP epithelium and the loss of CSF,albeit the latter appears less likely due to rapid rates of CSF circulation in the brain. Furthermore, to validate the loss of CSF volume seen on MRI, CSF was collected from the cistcma magna (CM) of WT and iDTR mice post-Dtx (FIG. 3, D-F). On average, WT mice yielded 7.3 pl of CSF, while the iDTR mice only yielded 1.66 pl. Additionally, the CSF volume loss was visually apparent due to the flat, collapsed appearance of the CM in iDTR mice, as opposed to WT mice (FIG. 3, D, E). Given the potentially broad impact of this phenotype on numerous prior studies (see, e.g., Mouse Genome Informatics. References associated with Gt(ROSA)26Sortml(HBEGF)Awai Allele 2021; Available from: http: / / www.informatics.jax.org / reference / allele / MGI:3772576?typeFilter=Literature), to validate this as an authentic phenotype, an independent lab (Goto lab) replicated these findings from iDTR breeders recently purchased from Jackson Laboratories. Additionally, these findings were validated in tissue sections from utilizing the ROSA26iDTR line with or without Cx3crlCreER(expressed primarily in peripheral macrophages and microglia), GlastCreER(expressed in astrocytes) or mGFAP-Cre lines (expressed primarily in astrocytes and neural stem cells) maintained and treated with Dtx in the Luo lab (FIG. 11). Moreover, retrospectively examining tissue preserved in 2015-2017 from a third independent lab (Crone lab), the same phenotype of loss of ventricular spaces in the Dtx-treated ROSA26iDTR mice with or without ChxlOCrewas observed (in this line, see e.g., Romer, S. H. et al. Accessory respiratory muscles enhance ventilation in ALS model mice and are activated by excitatory V2a neurons. Exp Neurol 287, 192- 204 (2017), Cre expression is restricted to the spinal cord, brainstem, and the eye but absent in the forebrain) (FIG. 11). Thus, multiple independently maintained iDTR mouse lines dating back to at least 2015 demonstrate loss of ventricular volume following Dtx treatment, independent of Cre expression.

[0085] Lateral and 3rdventricles experience more profound volume loss compared to 4thventricle and pineal recess after Dtx treatment

[0086] To quantify the CSF volume loss in each ventricle in vivo, a T2 fluid-sensitive MRI sequence that allows for the detection of CSF in live animals and reconstruction of 3D CSF-filled ventricular spaces in mouse brain was developed. Utilizing this sequence, the distribution of CSF volume in cohorts of WT and iDTR mice treated with either 4 or 20 ng / g / day of Dtx for three consecutive days at 5-18 days after Dtx treatment was examined. As seen in 3D reconstructions ofthe T2 fluid-sensitive MRI data (FIG. 4, A), the lateral and third ventricles undergo profound volume loss upon Dtx administration in iDTR mice, with 4 ng / g / day exerting a partial effect compared to 20 ng / g / day (FIG. 4, B). The 4thventricle and the pineal recess seem to be less susceptible to CSF volume loss, with 4 and 20 ng / g / day affecting their volume to a similar extent (FIG. 4 B).

[0087] Stable and sustained loss of CSF volume after ChP ablation

[0088] Next, to investigate whether the loss of CSF volume was sustained in this model, 20 ng / g / day of Dtx was administered for 3 days to cohorts of 2-month-old WT and iDTR mice and aged them for up to 20 months post Dtx. Using the T2 fluid-sensitive MRI sequence, the animals were scanned before Dtx administration, at day 3 after the start of Dtx treatment and at 8 and 20 months post-Dtx (FIG. 12, A and B). The rapid and profound loss of ventricular volume observed at day 3 post-Dtx was present for up to 20 months (the longest timepoint tested). The lateral and third ventricles exhibited a nearly total loss of CSF (FIG. 12, C, D), while the 4thventricle and the pineal recess displayed a smaller decrease in CSF volume (FIG. 12, E). These results indicate that alternative sources of CSF production or regeneration of ChP after ablation do not occur or are not sufficient to restore ventricular volume. Over the span of 20 months in this small cohort (n=3-4 per group), no unusually high mortality or gross motor abnormalities in iDTR mice that received Dtx was observed.

[0089] CSF volume reduction after ChP ablation is observed in both young (1 month) and old (20- 24 month) iDTR mice treated with Dtx.

[0090] Evidence from human and rodent studies shows profound changes in the production rate and composition of the CSF with aging, which has been linked to numerous pathological processes. Considering the potential relevance of this model to the role of CSF in aging, whether the capacity of Dtx to induce CSF volume loss in iDTR mice changes with age was evaluated. To this end, Dtx (20 ng / g / day x 3) was administered to cohorts of young (1 month old) and old (20- 24 month old) WT and iDTR mice. On T2 fluid-sensitive MRI scans performed on day 3 after the first Dtx injection, a similarly pronounced loss of CSF volume was observed in both cohorts, as well as a similar pattern of CSF volume loss by ventricle in young mice (FIG. 13, A) and aged mice (FIG. 13, B).

[0091] ChP / CSF loss does not cause an acute neurobehavioral phenotype.

[0092] Unexpectedly, ChP ablation and subsequent loss of CSF after Dtx treatment did not lead to gross and acute neurological and motor deficits in our model. In fact, ChP-ablated animals seem to survive for up to 20 months post-Dtx (FIG. 12) without any visually apparent differences from age-matched WT controls, including in body weight at day 4, 7, and 30 post-Dtx (short-term), as well as at 5 months post-Dtx (long-term) in a separate cohort of animals (FIG. 5, A). To investigate whether ChP ablation could induce a more subtle neurological phenotype, the general behavioral functioning of the animals at 1 month post ablation was examined (FIG. 5, B-H). A comprehensive battery of tests were conducted to examine general motor function, circadian locomotor activity rhythms, and cognitive function in two different cohorts of ChP-ablated mice given either 4 ng / g / day x 3 (FIG. 5, B, D, F) or 20 ng / g / day x 3 (FIG. 5, C, E, G, H) of Dtx. At one-month postablation, ChP-ablated mice exhibited similar locomotor activity in a 23-hour time period (FIG. 5, B, C), as well as a normal light-dark circadian locomotor activity rhythm (FIG. 5, D, E). Cognitive function measured either by Barnes Maze (FIG. 5, F) or spontaneous alternation Y-maze (FIG. 5, G, H) showed no differences at 1 month post ChP ablation. These findings in themselves are surprising given the numerous essential homeostatic functions ascribed to the CSF and the ChP, such as maintenance of electrolyte levels that are essential for sustaining and generating membrane potentials, as well as carrying essential hormones and neuromodulators and possibly removing waste products, as well as due to the fact that some of the key brain regions responsible for motor control (striatum and cerebellum), or cognitive function (hippocampus), lie in close proximity to the ventricular system.

[0093] The choroid plexus / CSF are essential for newly-born neuroblast retention in the SVZ.

[0094] Choroid plexus ablation does not affect the Ki67+ proliferating cell number but leads to reduced newly born neuroblast (doublecortin, DCX+) number in the SVZ in the adult mouse brain. Given the close proximity of the subventricular zone (SVZ) neural stem cells to the CSF and the lateral ventricle ependymal cell layers, this model was used to evaluate the long-proposed role of CSF in regulating adult neurogenesis. WT and iDTR heterozygous mice were treated with 4 or 20 ng / g / dayX3 dosage regimen of Dtx. Doublecortin (DCX) was used as a marker for newly born ncuroblasts and Ki67 as a marker for proliferating cells. For DCX and Ki67 quantification, the animals were treated with Dtx and harvested at 4 weeks (FIG. 14) or either 3 months or 6 monthspost Dtx injection (FIG. 6). Previous studies have shown that the SVZ newly bom neuroblasts can be added into the olfactory bulb between 2-4 weeks after their birth as new granule neurons, an earlier timepoint of 4 weeks post-ablation was first evaluated (FIG. 14) by immunostaining for the newly bom neuroblast cell marker (DCX) in coronal sections containing the SVZ. The high (20 ng / g / day, FIG. 14, B, b’) dose of Dtx resulted in a significant decrease in DCX+ area fraction in iDTR mice compared to WT mice (FIG. 14, A, a’), albeit characterized by considerable variability (FIG. 14, C). To test whether this initial trend of decrease in the SVZ neuroblast pool will be sustained over time, a cohort of animals was evaluated at 3 months post Dtx by immunostaining for neuroblasts (DCX) and proliferating cells (Ki67) (FIG. 6, A-F). Previous studies have suggested that ChP provides factors in the CSF that are critical for maintaining the proliferation of SVZ neural stem cells and rapidly dividing progenitor cells16'51, however, the results show that at 3 months post ChP / CSF ablation, the total Ki67+ cell number was not affected by ChP ablation in iDTR mice (FIG. 6, C, E). DCX immunoreactive area in the dorsal corpus callosum, which is not in direct contact with the CSF was not affected in the 3-month post-Dtx group (FIG. 6, D). However, in the ventral SVZ which directly contacts the CSF, DCX immunoreactive area was significantly decreased in ChP-ablated mice, indicating a reduced number of DCX+ newly born neuroblasts (FIG. 6, F). Since ChP ablation leads to shrinkage of lateral ventricles which might affect the overall shape or the anterior-posterior position of the coronal section, we further examined the SVZ in whole mount preparations in both 3-month and 6-month ChP-ablated mice compared to Dtx-treated WT mice (FIG. 6, G-L). Strikingly, the results from SVZ whole mount immuno staining further validate the absence of an effect of ChP ablation on Ki67+ cell density in the lateral wall of the SVZ (FIG. ,6 I, L). However, consistent with the results from coronal section quantification, SVZ whole mounts show significantly decreased DCX immunoreactive area in iDTR mice both 3 and 6 months after Dtx treatment (FIG. 6, H, K). This decrease in DCX+ cells in the SVZ whole mounts was not observed in age-matched iDTR mice that did not receive Dtx treatment (data not shown). Additionally, a dense migratory network of DCX+ cells were observed in WT SVZ whole mounts, which is mostly absent in the SVZ of ChP-ablated mice. The results indicate that ChP / CSF-derived factors might not be critical for maintaining the proliferative potential of SVZ neural stem cells and / or transient amplifying progenitors but instead are critical for the maintenance of neuroblast number in the SVZ and / or the migration within the network formed by DCX+ neuroblasts.

[0095] Previous reports suggest that the directional flow of CSF and the polarity of motile cilia of the ependymal cells lining the lateral ventricular wall might be critical for ncuroblast migration in the SVZ (see, e.g., Sawamoto, K. et al. New Neurons Follow the Flow of Cerebrospinal Fluid in the Adult Brain. Science 311, 629-632 (2006)) (FIG. 7 A). It was further hypothesized that that the loss of CSF-borne factors and overall CSF circulation could affect the motile cilia of SVZ ependymal cells as well, which could lead to disruption of the migratory network and reduced neuroblast number in the SVZ after ChP ablation. Applicant stained SVZ whole mount preparations at 3 months post ablation for acetylated tubulin (FIG. 7, B-E) which visualizes motile cilia and their polarity and direction. Interestingly, the results show that in ChP-ablated mice, Applicant observed a disorganized pattern and loss of the directional orientation of acetylated tubulin-positive bundles of motile cilia, which could underlie the differences in DCX+ neuroblast number in the SVZ, as well as the altered migratory network that Applicant observed in ChP- ablated mice (FIG. 6, G, J).

[0096] The choroid plexus / CSF are essential for newly-born neuroblast retention in the SVZ.

[0097] While it has previously been hypothesized that the CSF and CSF-bome factors are essential for adult neurogenesis and particularly for NSC proliferation in the SVZ, the results suggest that the presence of CSF flow or ChP-derived factors could instead be critical for maintaining the ventricular wall localization of DCX+ newly-born neuroblasts. In order to further examine the fate of newly-born neuroblasts after ChP ablation, 5-bromo-2’-deoxyuridine (BrdU) labeling was performed (2 injections 4 hours apart) at 5 months after Dtx treatment and harvested tissue at 1 week and 1 month post-labeling (FIG. 8, A). To evaluate whether the homeostatic continuous migration and addition of newly-born neuroblasts from the SVZ into the OB is affected, BrdU immuno staining was performed in sagittal brain sections (FIG. 8). At 1 week post-labeling, BrdU-i- cells were enriched predominantly in the rostral migratory stream (RMS) compared to the granular cell layer (GCL), while at 1 month they have largely transitioned into the GCL from the RMS. This temporal transition pattern was observed in both WT and ChP-ablated mice. However, in ChP-ablated animals, a pronounced increase in BrdU-labelled cells was observed in the RMS at 1 week post-labeling compared to WT mice (FIG. 8, B, C, F-I). At one-month post-labelling, this increase was sustained in ChP-ablated mice in the GCL, with the majority of BrdU-i- cells being NeuN+ granule neurons (FIGS. 8, D, E, J-M). BrdU labeling was performed using a differentdosage regimen to capture multiple cell division cycles (1 injection per day for 3 consecutive days) at 6 months after ChP ablation and collected OB sections in the coronal plane at 1 month postlabeling (FIG. 15). In this labeling paradigm in an independent cohort of mice, Applicant observed a similar phenotype of increased BrdU+NeuN+ cells in the GCL of ChP-ablated mice at 1 month post-BrdU pulse labeling.

[0098] These unexpected results suggest that the decreased number of DCX+ newly-born neuroblasts in the SVZ wall after ChP ablation may not be due to decreased differentiation or survival but may instead be due to enhanced migration into the OB / RMS at 1 week after pulse labeling (FIG. 8, B, C) and subsequent maturation into BrdU+ / NeuN+ adult-bom neurons at 1 month later (FIG. 8, D, E). Thus, the role of the ChP and the CSF in adult SVZ neurogenesis could be to retain the newly-born neuroblasts in the SVZ and delay their migration into the OB. Indeed, using whole mount SVZ immunostaining, at 1 week post-labeling, A corresponding pronounced decrease in BrdU-i- cell population was observed in the SVZ wall of ChP-ablated mice (FIG. 16, B, H), while total Ki67+ proliferating cell number was unaltered (FIG. 6, 1, L, FIG. 16, F, G), suggesting that the increase in labeled cells in the OB / RMS at 1 week post-labeling is likely not due to differences in BrdU labeling efficiency, but due to increased exit of BrdU labeled cells from the SVZ into the OB.

[0099] ChP ablation impairs the neurogenic response to ischemic stroke

[0100] It was next hypothesized that the decreased DCX+ neuroblast retention at the SVZ wall in ChP-ablated mice could potentially affect the dynamics of adult neurogenesis and the migration of neuroblasts into the injured area in response to CNS injury such as stroke. To determine the role of the ChP and CSF in post-stroke adult neurogenesis and neuroblast migration, transient middle cerebral artery occlusion (tMCAo) surgery was performed to induce focal cerebral ischemia in cohorts of WT and iDTR mice at 3 months post-Dtx (FIG. 9, A). Occlusion was maintained for 60 minutes and then the occluding microfilament was withdrawn to induce reperfusion. Successful infarct induction was validated via MRI at 24-48 hours post-MCAo (FIG. 9, B, C). To note, no statistically significant differences in edema volume, infarct volume and percentage of edema expansion were observed between WT and ChP-ablated mice (FIG. 9, D, E, F). Serial coronal brain sections were collected at 35 days post-stoke (a time point at which both SVZ neurogenesis and longitudinal neuroblast migration have been shown to be stimulated) andstained for DCX to evaluate the neurogenic response to ischemia and neuroblast migration into the lesion site (FIG. 9, G - J). On the side ipsilateral to the stroke, a marked decrease in DCX+ cells was observed in the striatum of ChP-ablated mice compared to WT stroke mice, indicating a reduction in neuroblast migration into the lesion site (FIG. 9, M). Furthermore, DCX immunopositive area was also profoundly reduced at the SVZ wall in iDTR mice compared to WT, in both the ipsilateral (FIG. 9, K) and contralateral SVZ (FIG. 17, C), indicating reduced stimulation of adult SVZ neurogenesis in response to stroke, an effect of ChP ablation on DCX+ cells in the ipsilateral (FIG. 9, L) and contralateral (FIG. 17, D) corpus callosum, a brain area that does not have direct contact with CSF, was not observed, consistent with the data from non-stroke animals (FIG. 6, C, D). These results suggest that, while corpus callosum neuroblast population might not rely on ChP / CSF, the decrease in SVZ wall neurogenesis following ChP ablation could be directly related to the depletion of signals coming from the adjacent CSF. Furthermore, the maximum neuroblast migration distance towards the lesion site was measured for each of the sections analyzed, and did not observe a difference between WT and ChP-ablated animals (FIG. 9, N), suggesting that the reduced neuroblast number in the striatum is not likely due to reduced migration of neuroblasts into the lesion site, but instead is likely caused by the decreased pool of available SVZ NBs at the SVZ wall.

[0101] ChP ablation improves the CSF volume in the neonatal hydrocephalus model. FIG. 25 depicts the hydrocephalus model as described in Abdelhamed Z, Vuong SM, Hill L, Shula C, Timms A, Beier D, Campbell K, Mangano FT, Stottmann RW, Goto J. Development. 2018 Jan 9;145(l):dcvl54500. doi: 10.1242 / dcv.l54500.PMID: 29317443 Progressive hydrocephalus (prh) mouse model. On left, is an image obtained via MRI of wild type (top) and Ccdc39 / prh (bottom) brain ventricular system. MRI-based ventricular volume analysis (left) and histological analysis (right) show significantly enlarged lateral ventricles (LV) in the prh mutant mice. On the right is shown is brain histology of the wild type (top) and neonatal hydrocephalus mouse model (bottom). This represents the enlarged lateral ventricles in the neonatal mouse hydrocephalus model used in testing of ChP ablation using the iDTR / Dtx treatment. The results of the treatment are shown in FIG. 26. FIG. 26 depicts data showing smaller ventricles after ChP ablation in the prh mice using iDTR / dtx. MRI-based brain ventricular size analysis showed significant reduction of ventricular volume in prh mutant mice after the ChP ablation. FIG. 26 shows CSF size reduction in the treatment of hydrocephalus model mice prh mice) with the Dtx-DTR transgenic system.

[0102] Discussion

[0103] ChP epithelial cells are the main contributor of CSF production. The ChP has in general been recognized as the main contributor to CSF production, but some studies attributed only 30% of CSF volume produced to the ChP, albeit the studies were performed by invasive surgical excision of the lateral ventricle ChP in mammalian species. Ependymal cells of the ventricular wall and the cerebral vascular endothelium have also been proposed as extrachoroidal CSF secretion sources. However, when ChP epithelial cells are ablated in adult brain, significant (>90%) loss of CSF volume was observed, directly supporting the proposed notion that ChP epithelium is the main contributor of CSF production in adult brain or, alternatively, is required for production of CSF by extrachoroidal cells in healthy mice.

[0104] Adult ChP epithelial cells have minimal regenerative capacity. The results show that once the ChP is ablated in adult brain, the loss of CSF is stable and irreversible for at least 20 months, suggesting that the ChP epithelial cells have minimal regenerative capacity in the adult mouse brain, which has not been specifically tested previously. This may carry implications for patients who underwent surgical ChP cauterization as a treatment for hydrocephalus, recognizing that there may be cross-species differences. Additionally, considering that damage to the ChP has been observed in ischemic and hemorrhagic stroke, as well as in traumatic brain injury, these findings raise a question on the role of the ChP in long-term sequelae of these conditions. Specifically, considering that newly-born neuroblasts migrate from the SVZ into the infarct site in ischemic stroke, and that their number is reduced in ChP-ablated animals (FIG. 6), it is possible that the degree of damage to the ChP in stroke could correlate with post-stroke recovery outcomes.

[0105] ChP ablation and CSF loss does not cause acute neuronal death or an adverse motor phenotype. At day 3 after the start of Dtx treatment, apoptotic cell death was observed only in the ChP epithelium and not in other brain tissues (except for sparse apoptotic cells in the SVZ and SGZ which is also observed in the WT control mice). Surprisingly, no overt neurobehavioral, survival, or growth differences were observed arising from ChP ablation at one month postablation. A comprehensive battery of behavioral tests for motor function, circadian locomotor activity patterns, and spatial learning and working memory were conducted, and no differences between WT and iDTR Dtx-trcatcd mice at one month post-ablation were observed (FIG. 5). This surprising finding raises questions about the extent to which the ChP and CSF are critical for CNShomeostasis in adult mice, as they have been previously considered essential for maintaining many aspects of normal CNS function. It is possible that other cell types may compensate for some aspects of ChP function, even if ventricular volume cannot be maintained. The lack of mortality or an overt adverse motor or cognitive phenotype further underscores the utility of this model in long-term studies on the role of the ChP and CSF in aging or aging-associated disease models, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), as well as brain injury models such as TBI, and stroke.

[0106] New inducible mouse model for choroid plexus ablation. The robust phenotype of ChP epithelial cell ablation and shrinkage of ventricular spaces with loss of CSF volume in the iDTR mice after Dtx administration makes it an attractive model for studying ChP and CSF function in the adult mouse brain. The ChP and CSF are thought to play a role in multiple developmental, homeostatic and disease processes. Despite the importance and therapeutic potential of these understudied CNS compartments, the tools to efficiently manipulate the ChP / CSF in the adult brain have been largely unavailable. To date, the commonly available tools to study the CSF system include invasive infusions or injections of artificially introduced solutes, which have been demonstrated to directly affect glymphatic-mediated CSF circulation. Another tool used in the field is astrocytic AQP4 knockout to study the dynamics of perivascular solute clearance. While this tool has produced interesting findings, it may have confounding phenotypes due to the broader role of astrocytic AQP4 in CNS physiology, including affecting astrocyte volume and migration, as well as modulating CNS immune responses. Thus, the scope of such studies is necessarily limited to the dynamics of CSF / intcrstitial fluid exchange within the perivascular space and clearance of artificially introduced solutes without examining the role of ChP beyond CSF production, such as the functions of its extensive secretome. While tools to manipulate the ChP, such as the Foxj 1 -CreERT2 line or rAAV 2 / 5, have also been utilized in the field, these approaches have limited specificity for ChP epithelial cells. While the Foxj l-CreERT2 line and adenoviral approaches allow for the manipulation of the ChP secretome or CSF absorption in hydrocephalus, these methods do not readily permit direct manipulation of normal CSF production and flow, which largely limits knowledge of the role of ChP / CSF in the adult brain. Hence, the newly identified specific expression of the DTR gene in the ChP epithelium could serve as a useful non-invasive tool that can break new ground in studies of the ChP-CSF system and glymphatic circulation in the mouse brain, especially considering the surprising lack of increasedmortality, differences in body weight or effects on overt motor and cognitive functioning in ChP- ablatcd mice at 1 month post-Dtx (FIG. 5). The key features of the iDTR mouse model include: A). Rapid and non-invasive ablation. Loss of CSF volume is apparent as early as day 3 after the start of Dtx treatment in heterozygous iDTR mice, allowing precise control of the timing of ChP / CSF ablation in a variety of genetic and non-genetic disease models. B) Stable ablation. Ablation of the ChP and CSF volume loss induced by a single 3-day Dtx regimen is stable for at least 20 months post Dtx injection, making it feasible to study long-term effects of CSF function in aging and aging-associated diseases. C) Dose-dependent ChP ablation and CSF volume loss. The 4 ng / gX3 or 20ng / gX3 Dtx dosage regimens used in the study produce different degrees of ChP ablation and CSF volume loss in adult mice at 1 month post ablation, allowing for more precision in modulating the severity of the phenotype. D) Wide age range in which the ChP can be successfully ablated. iDTR mice both at 1 and 24 months of age show substantial loss of CSF volume after Dtx administration (FIG. 13). This makes the model useful to study the role of the ChP and CSF throughout adulthood or only in aged mice, either in homeostasis or in combination with disease models. In summary, the iDTR mouse model has many key features to allow for precisely temporally controllable, dose-dependent and stable ChP ablation that can be easily combined with other animal models.

[0107] A potential caveat in previous cell-type specific ablation studies utilizing the ROSA26iDTR mouse line. Applicant further discovered a Cre-independent robust ChP / CSF phenotypes in ROSA26iDTR mice after Dtx administration. While the mechanism behind Cre- independent DTR expression in the ChP is unknown, it can be speculated that alternative splicing (skipping the poly-adenlyation sequence in the LoxP flanked “stop” cassette) specific to the ChP likely plays a role. Since the publication of this transgenic mouse line in 2005, its robust and timely Dtx-dependent ablation of the targeted cell types has been widely used in over 341 published studies. It is possible that ablation of ChP could account for some of the results previously attributed to ablation of other cell types in prior studies. Notably, our results indicate that ChP ablation does not impair survival or result in overt behavioral deficits in several commonly used tests. Importantly, this study emphasizes the need for including proper experimental controls (i.e. Cre-negative iDTR-i- mice + Dtx) in subsequent studies to ensure that any differences in phenotype are due to ablation of Cre expressing cells. Despite the wide application of this line, brain MRI was not utilized in prior studies, likely explaining the lack of any earlier report on this phenotype.

[0108] Given the growing understanding of the role of infiltrating peripheral immune cells in CNS pathology and homeostasis, and the proposed role of the ChP as the immune gateway to the CNS, this model will allow for direct testing of the proposed role of the ChP as a crucial regulator of immune function in the brain. The data demonstrate that a powerful mouse genetic tool was identified that will facilitate examining the role of CSF in aging, neurorepair, and brainperipheral immune interactions in CNS diseases, such as TBI, stroke, neurodegenerative and demyelinating diseases.

[0109] Materials and Methods

[0110] Table 1 . Resources and reagents used in the study.

[0111] Animals

[0112] All animal experiments were performed in accordance with procedures approved by the University of Cincinnati Institutional Animal Care and Use Committee in compliance with NIH guidelines. All mice were maintained on a C57BL / 6 background. Wildtype C57BL / 6 (Jax #000664) and ROSA26iDTR mice (Jax # 007900) were obtained from Jackson Laboratories and bred in-house. To further validate this phenotype, Applicant also retrospectively examined independent mouse lines that carried different Cre drivers, but all shared the iDTR allele in our own lab as well as in two independent labs (Goto lab and Crone lab). These mice lines include mGFAPcre-iDTR that contains the mGFAPcre allele (Jax #012886, Luo lab) the GLAST- CreER:iDTR (Jax #012586, Luo Lab), CX3CRl-CreER:iDTR (Jax # 021160, Luo lab), an independently maintained iDTR mouse colony in the Goto lab (Jax #007900, Goto lab), and (Chxl0-Cre;iDTR, Crone lab41).

[0113] WT or iDTR mice were subjected to Diphtheria toxin (Dtx) treatment according to details described herein at different ages. CSF was collected from cistema magna as describedpreviously in Kaur, A., Shuken, S., Yang, A. C. & Tram, T. A protocol for collection and infusion of cerebrospinal fluid in mice. STAR Protoc 4, 102015 (2023). Tissue from different brain regions and other peripheral organs are harvested and subjected to qRT-PCR. For mouse stroke model, transient middle cerebral artery occlusion (tMCAo) was induced in male and female WT and iDTR mice (7 months old, 6 months post- blation, 25— 35g) by intraluminal occlusion of the left MCA for 60 min with a silicone rubber-coated monofilament (Cat.602212PK10Re and 602312PK10Re, Doccol Corporation) as previously described in Longa, E. Z., Weinstein, P. R., Carlson, S. & Cummins, R. Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20, 84-91 (1989).

[0114] Tissue collection for Immunohistology

[0115] Animals were anesthetized and transcardially perfused with ice-cold 0.1 M phosphate buffer (pH 7.2) and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brain was dissected out and post-fixed in 4% paraformaldehyde overnight at 4 °C and sequentially dehydrated in 20% and 30% sucrose in 0.1 M phosphate buffer (pH 7.2) for cryoprotection. Preparation of choroid plexus (ChP) and SVZ whole mounts are carried out as previously described in Mirzadeh, Z., Han, Y.-G., Soriano-Navarro, M., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. Cilia organize ependymal planar polarity. J Neurosci 30, 2600-2610 (2010). . Whole mounts or coronal sections are subjected to various immuno staining protocol to detect DTR protein, DCX, Ki67, BrdU, CD31, TTR and other markers as described. The dosage and BrdU injection time are provided in each figure with an experimental timeline.

[0116] T2-weighted MRI and brain ventricular measurement using T2-weighted MRI anatomical scans

[0117] WT and iDTR mice that were subjected to various dosages and different durations of Dtx treatment underwent an imaging session at 1 day before Dtx injection and different days after Dtx injection by staff that were blinded to the genotypes / treatment groups. MRI studies were carried out to detect ventricular volume as described.

[0118] Image acquisition and analyses

[0119] Fluorescence images were acquired using either a Leica DM5000B microscope equipped with a motorized stage and a Hamamatsu Cl 1440 fluorescent camera and configured tobe operated through the Neurolucida 2020 software suite, Nikon TiOE upright widefield microscope or a Leica Stcllaris 8 Widc-ficld confocal microscope with LAS-X software. For details on unbiased sampling and quantification for each markers. For all analyses, the sum or average of data from each mouse was treated as n = 1 for statistical analysis. For all analyses, the sum or average of data from each mouse was treated as n = 1 for statistical analysis.

[0120] Behavioral Tests

[0121] Open Field Test, Barnes Maze, Spontaneous alternation Y-maze tests were carried out in WT or iDTR mice subjected to Dtx treatment as described. Experimental timelines were provided in each figure.

[0122] Statistics

[0123] Results are expressed as mean + SEM. Statistical analyses were performed in Sigmaplot 12.0 and GraphPad Prism 10 using Student’s t test, and one- or two-way analysis of variance (ANOVA), as appropriate, with Tukey, Dunn’s, or Holm-Sidak post hoc tests. When the data did not meet the assumptions of normality or equal variance, the corresponding nonparametric tests were used. The significance level was set at a = 0.05. Number of animals used in each experiment plotted as single data point and are indicated in Figure legends. Graphs were made in GraphPad Prism and portions of figures were created with BioRender.com. The notation for p- valucs was as follows: n.s - p > 0.05, * - p < 0.05, ** - p < 0.01, *** - p < 0.001, **** - p < 0.0001.

[0124] Animals

[0125] All animal experiments were performed in accordance with procedures approved by the University of Cincinnati Institutional Animal Care and Use Committee in compliance with NIH guidelines. All mice were maintained on a C57BL / 6 background. Wildtype C57BL / 6 (Jax #000664) and ROSA26iDTR mice (Jax # 007900) were obtained from Jackson Laboratories and bred in-house. The mice were housed in the animal facility of University of Cincinnati on a 14-h light / 10-h dark diurnal cycle. Food and water were provided ad libitum. For PCR primer sequences used for iDTR genotyping, see Table 1. Both female and male adult mice were used in this study. The age of mice in each experiment is indicated in the individual figure legends. To further validate this phenotype, Applicant also retrospectively examined independent mouse lines that carrieddifferent Cre drivers, but all shared the iDTR allele in our own lab as well as in two independent labs (Goto lab and Crone lab). These mice lines include mGFAPcrc-iDTR that contains the mGFAPcre allele (Jax #012886, Luo lab) the GLAST-CreER:iDTR (Jax #012586, Luo Lab), CX3CRl-CreER:iDTR (Jax # 021160, Luo lab), an independently maintained iDTR mouse colony in the Goto lab (Jax #007900, Goto lab), and (Chxl0-Cre;iDTR, Crone lab, see Romer, S. H. et al. Accessory respiratory muscles enhance ventilation in ALS model mice and are activated by excitatory V2a neurons. Exp Neurol 287, 192-204 (2017).).

[0126] Administration of Diphtheria toxin (Dtx) to ablate the choroid plexus (ChP) in the iDTR mice

[0127] Recombinant Corynebacterium diphtheriae toxin was obtained from Fisher (List Biological Laboratories, Cat. #150) or Sigma (cat. # D0564-1MG) as a lyophilized powder and reconstituted in 0.9% saline at 2 mg / ml. The resulting stock solution was stored at -80 °C and diluted with ice-cold saline as needed to a final concentration of 2 ng / pl. The resulting working solution was kept on ice and used within one hour after preparation. As described previously in Bedolla, A. et al. Diphtheria toxin induced but not CSF1R inhibitor mediated microglia ablation model leads to the loss of CSF / ventricular spaces in vivo that is independent of cytokine upregulation. Journal of Neuroinflammation 19, 3 (2022)., Dtx was administered i.p. for three consecutive days with no more than 24 hours between injections. Dtx from Fisher or Sigma generated similar results in our studies.

[0128] Collection of CSF from the cisterna magna

[0129] Animals were anesthetized and the head was fixed in a stereotactic frame. As described in Kaur, A., Shuken, S., Yang, A. C. & Iram, T. A protocol for collection and infusion of cerebrospinal fluid in mice. STAR Protoc 4, 102015 (2023), the skin and the underlying muscle layers were separated to expose the atlanto-occipital membrane. A glass pipette was used to collect CSF over 12 minutes after which animals were euthanized. Next, the CSF was weighed, and the volume of the CSF was estimated by the weight of collected CSF (assumed to be approximately equal to that of water).

[0130] Examination of DTR expression in ChP and other tissues via qRT-PCR

[0131] To examine DTR expression in the ChP and other tissues in WT and iDTR mice, ChP, various brain regions and peripheral tissues were harvested from adult WT, homo- or heterozygous iDTR mice for quantitative RT-PCR. Mice were perfused with ice-cold phosphate buffer (pH 7.2) before being euthanized, and the brains were immediately removed and chilled on ice. The 3rd, 4th- and lateral ventricle ChP tissues, cortical, hippocampal, thalamus and cerebellar tissues were dissected, and total RNA was extracted following the instructions from the manufacturer (RNAqueous Micro or Mini RNA extraction kit, Thermo Fisher). Additionally, peripheral epithelial tissues (skin from the ear, small intestine, and lung) were also collected for total RNA extraction. Total RNA (80-300 ng) was treated with RQ-1 RNase-free Dnase I and reverse transcribed into cDNA using random hexamers by Superscript III reverse transcriptase (Life Sciences). cDNA levels for HPRT1 (hypoxanthine phosphoribosyl transferase 1), were determined using a specific universal probe Library primer probe set (Roche), and for DTR using a TaqMan gene expression assay (ThermoFisher, 4351372, assay Rh02840489_ml) by quantitative RT-PCR using a Roche Light Cycler II 480. Relative expression levels of DTR were calculated using the A Ct method compared to HPRT1 as a reference gene for each individual sample. Comparative DTR expression between tissues was calculated using the A A Ct method and the expression was normalized to the sample with the lowest DTR mRNA levels observed in heterozygous iDTR mice (thalamus). For primers, 6-carboxyfluorescein (FAM) labeled probes and the TaqMan assay used in the quantitative RT-PCR see Table 1. All RNA samples were treated with DNAse before reverse transcriptase reaction, and (-) RT reactions were included as negative control to ensure the absence of genomic DNA contamination.

[0132] Tissue collection for Immunohistology

[0133] Animals were anesthetized and transcardially perfused with ice-cold 0.1 M phosphate buffer (pH 7.2) and then with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The brain was dissected out and post-fixed in 4% paraformaldehyde overnight at 4 °C and sequentially dehydrated in 20% and 30% sucrose in 0.1 M phosphate buffer (pH 7.2) for cryoprotection.

[0134] Preparation of choroid plexus (ChP) and SVZ whole mounts

[0135] After transcardial PFA perfusion, the cerebellum, pons and medulla were separated, and a sagittal cut was made to separate the hemispheres. Using fine forceps and a stab knife, the cerebral cortex was peeled away, and the midbrain and thalamus dissected, exposing the ventricle wall. The lateral ventricle ChP was isolated and collected in 0.1 M PB for further staining. The whole lateral ventricular wall containing the thin layer of SVZ was dissected to prepare for SVZ whole mount IHC as described previously in Mirzadeh, Z., Han, Y.-G., Soriano-Navarro, M., Garcia- Verdugo, J. M. & Alvarez-Buylla, A. Cilia organize ependymal planar polarity. J Neurosci 30, 2600-2610 (2010) (also see SVZ whole-mount staining for characterization of neurogenesis and ependymal cell motile cilia for details below).

[0136] Immunostaining

[0137] Free-floating IF staining of coronal sections or whole choroid plexus tissues

[0138] For free-floating immunofluorescent staining, the brain tissue was frozen in OCT on dry ice and sectioned at 30 pm on a cryostat. Whole dissected ChP tissue (collected at 1 month post-ablation) or coronal brain sections were washed 3x and blocked in blocking buffer (4% BSA, 0.3% Triton X-100 (Acros Organics) in in 0.1 M phosphate buffer, pH 7.2) for 1 hr at RT with shaking and transferred into primary antibody solution (For antibodies used see Table 1) in blocking buffer for incubation overnight at 4 °C on a shaker.

[0139] Then, sections were washed in PB before incubated in secondary antibodies (conjugated with Alexa 488, Alexa 555, Alexa 647, Alexa 790 1:500-1000; Life Technologies, Carlsbad, CA, USA or Jackson Immuno Research, West Grove, PA, USA) dissolved in blocking buffer for 4 hours at RT with shaking. For the ChP whole mounts, the procedure was the same except for the secondary antibody incubation being done overnight at 4 °C with shaking. Free floating sections were mounted onto microscope slides (12-550-15, Fisherbrand) and coverslipped in Mowiol 4-88 (17951, Poly sciences Inc.) mounting medium.

[0140] Staining of directly mounted coronal cryosections

[0141] For ChP area quantification using coronal brain sections, brain tissue was sectioned coronally at 40 um into 4 sets on a cryostat and directly mounted onto glass slides to prevent potential loss of ChP tissue during free floating staining. Slides were washed 3x and blocked overnight at 4 °C in blocking buffer using CoverWell incubation chambers (645502, Grace Bio-labs), 800 l per chamber. Then, the slides were incubated for 72 hours (to ensure good penetration) at 4 °C in primary antibody diluted in blocking buffer, washed 3 x 30 min and then incubated in secondary antibody diluted in blocking buffer for 48 hours at 4°C. All incubations were done in CoverWell chambers. For antibodies used see Table 1.

[0142] TSA staining ofDTR protein

[0143] For diphtheria toxin receptor (DTR) staining, tyramide signal amplification (TSA) was performed. Briefly, 40 pm-thick coronal sections were treated with 3% H2O2 solution for 1 hour, blocked, and incubated with Goat anti-HB-EGF (human) antibody (1:500, R&D Systems, AF-259-NA) overnight. On the second day, after stringent washing, the tissue was incubated with anti-goat IgG biotinylated secondary antibody for 2 hours, then ABC mix solution from VECTASTAIN ABC kit™ (Vector) for 1 hour, and then tyramide dissolved in 0. IM borate buffer was applied for 10 min with stringent washing between the incubations. Streptavidin Alexa Fluor 594 (Thermo Fisher) was then applied for 1 hour, followed by DAPI (Sigma- Aldrich) staining and mounting with coverslip and DAPI Fluoromount-G mounting media (SouthemBiotech 0100-20).

[0144] Co-staining ofTUNEL and choroid plexus cell-type markers

[0145] To determine identity of the cell types undergoing apoptosis in response to Dtx, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed according to the manufacture’s protocol using ApopTag® Fluorescein in situ Apoptosis Detection Kit (Millipore). For the co-staining of TUNEL with alpha-1 subunit of Na+ / K+ ATPase (a-1), TUNEL assay was performed first except the final step of applying Anti-Digoxigenin Conjugate (FITC) to avoid the heat-induced inactivation of fluorescent signals, and antigen retrieval with citrate buffer (pH 6) at 95 °C for 45 min was performed. Blocking for 1 hour and anti-mouse a-1 antibody (1:50, Millipore, 05-369) incubation was performed overnight. Following day, after stringent washing, the tissue was incubated with anti-mouse secondary antibody along with Anti- Digoxigenin Conjugate (FITC). For the other cell type markers (CD68 Bio Rad, MCA1957T, 1:300; PDGFRP Cell Signaling 3169S, 1:100; Isolectin GS-IB4, AlexaFluor 568 conjugate, Invitrogen 121412, 1: 100) the procedure was identical except for not including the antigen retrieval step. The sections were then washed, counterstained with DAPI for 5 minutes and cover-slipped with DAPI Fluoromount-G mounting media (SouthemBiotech #0100-20).

[0146] 5 -bromo-2' -deoxyuridine (BrdU) immunostaining

[0147] Animals injected i.p. with BrdU labeling reagent (10 pl / g bodyweight, Invitrogen #000103) 1 week or 1 month prior were sacrificed by PB / PFA perfusion and sequentially dehydrated in sucrose solution. For FIG. 8, WT and iDTR animals were treated with 2x BrdU labeling reagent 4 hours apart at 5 months post-Dtx (20 ng X3). For FIG. 6, WT and iDTR animals were treated with 3x BrdU labeling reagent 24 hours apart at 6 months post-Dtx (4 ng X3). Coronal or sagittal cryosections were washed 3x at RT and incubated in pre-warmed freshly made DNA denaturation solution (50% formamide, 50% 2x SSC buffer) for 2 hours at 60 °C with shaking. After being washed 3x in 2x SSC at 60 °C with shaking, the tissue was incubated in 2N HC1 at 37 °C with shaking. Then, the sections were incubated lx in borate buffer (pH 8.5) at RT with shaking for 10 min and washed lx in PB at RT. The tissue was blocked in blocking buffer for 1 hr at RT and the standard immunofluorescent protocol was followed (see Free floating IF staining above).

[0148] For BrdU staining of SVZ whole mount preparations, the whole-mount immuno staining protocol was followed with an additional step of incubating the tissue in 2N HC1 at 37 °C for 30 minutes before the start of the IF protocol as described previously in Shen, Q. et al. Adult SVZ Stem Cells Lie in a Vascular Niche: A Quantitative Analysis of Niche Cell-Cell Interactions. Cell Stem Cell 3, 289-300 (2008).

[0149] Histological verification of brain ventricle loss in various iDTR allele-containing mouse lines.

[0150] The perfusion fixed brain cryosection with 30 pm in thickness were mounted (some counterstained with DAPI) and cover-slipped. The mouse brain tissue from Crone lab (ChxlO- Cre;iDTR) were collected in 2015-2017, thawed, mounted and cover- slipped. The brain sections were scanned using a PathScan microslide scanner to obtain images of brain sections from + 0.5 mm to - 2.0 mm in reference to Bregma, according to a coronal atlas of the mouse brain (Franklin and Paxinos, (Franklin KBJ 1997)). The image analyzer was blinded to group and treatment information.

[0151] T2-weighted MRI and brain ventricular measurement using T2-weighted MRI anatomical scans

[0152] WT and iDTR mice that were subjected to various dosages and different durations of Dtx treatment underwent an imaging session at 1 day before Dtx injection and different days after Dtx injection by staff that were blinded to the genotypes / treatment groups.

[0153] MRI studies were performed on a vertical wide bore 9.4T Bruker Avance III HD scanner with a 36 mm proton volume coil. Mice were anesthetized with 1.5 - 2% isoflurane and kept warm with circulating air. Temperature and respiration rate were monitored with equipment from Small Animal Instruments, Inc. (SAI, Inc., NY). T2-weighted anatomical coronal images of the brain were acquired with a fat suppressed two-dimensional (2D) rapid acquisition with relaxation enhancement (RARE) sequence (see Hennig, J., Nauerth, A. & Friedburg, H. RARE imaging: a fast imaging method for clinical MR. Magn Reson Med 3, 823-833 (1986) using the following parameters: TR 4 sec, TE 71.5 ms, echo spacing 6.5 ms, 9 or 15 slices, slice thickness / gap 0.75 / 0.3 mm, RARE factor 20, receiver bandwidth 67k, averages 4, matrix 192 x 192, FOV 28.4 x 28.4 mm, and total scan time 2:24 minutes.

[0154] Ventricular volumes were quantified by outlining and measuring ventricular space for each animal from the individual T2-weighted coronal images (sections ranging from Bregma AP: +1.5 mm to AP: - 4 mm) using ImageJ. CSF / ventricular volume was calculated by multiplying the area of ventricular space by the thickness of each slice (mm) of the acquisition.

[0155] Planimetric quantification of ischemic stroke infarct properties from T2-weighted MRI data

[0156] T2- weighted MR images were acquired with an interval of 1.0 mm (total of 15 slices per animal) at 24-48 hr post-MCAo. Eight consecutive slices per animal were quantified using ImageJ software. ROIs encompassing the contra- and ipsilateral sides, as well as the infarct area, were traced using ImageJ. Edema volume was calculated as a sum of all ROI areas of the edema lesion. Edema expansion was calculated as 100 * (edema volume) / ( areas of contralateral side - areas of ipsilateral side). Infarct volume percentage was calculated as 100 * ( areas of contralateral side - areas of ipsilateral side) I ( areas of contralateral side).

[0157] T2 Fluid- sensitive MRI and ventricle volume quantification to characterize doseresponse to Dtx and stability of CSF loss over time

[0158] To further confirm the 3D volume of each ventricle in WT or ChP-ablated mice, Applicant developed a 3D T2 Fluid-Sensitive MRI sequence to examine CSF volume in vivo before and after Dtx administration.

[0159] Fluid- sensitive images of the brain were acquired with a fat suppressed three- dimensional (3D) RARE sequence using the following parameters: TR 2 sec, TE 275 ms, echo spacing 11.5 ms, RARE factor 60, receiver bandwidth 104k, averages 4, matrix 320 x 108 x 80, FOV 48x16.2x12mm, and total scan time 7 minutes 44 seconds.

[0160] WT or iDTR mice that were treated with 4ng / gX3 or 20ng / gX3 dosage regimens were subjected to the 3D Fluid-sensitive MRI scan 1-3 days before the first Dtx injection and at 5- 18 days after end of Dtx treatment to examine the effects of different dosages of Dtx. To examine the stability and potential reversal of the phenotype, one cohort of mice (n=4 for control or iDTR+Dtx mice) were followed up by 3D-Fluid sensitive MRI scans for up to 20 months after Dtx injection. To examine the age range of iDTR mice in which ChP ablation can be achieved, Applicant examined a cohort of 1 -month-old iDTR mice or 24-month-old iDTR mice (Dtx dosage: 20ng / gX3) and subjected them to the same 3D-fluid sensitive MRI.

[0161] The resulting series of T2 Fluid-Sensitive images were imported into Imaris 9.8.0 and 3D ventricular surfaces were reconstructed from a series of DICOM files. The surfaces were generated using the Surfaces tool with surface detail of 0.1 and seed point diameter of 0.8. The resulting 3D ventricular surfaces were manually divided into separate ventricles where appropriate. The volumes were quantified using the Measurements tool in Imaris.

[0162] SVZ whole-mount staining for characterization of neurogenesis and ependymal cell motile cilia

[0163] Brain tissue was harvested after 4% PFA perfusion at 1, 3 and 6 months postablation. SVZ whole-mounts were dissected as described in Mirzadeh, Z., Han, Y.-G., Soriano- Navarro, M., Garcia-Verdugo, J. M. & Alvarez-Buylla, A. Cilia organize ependymal planar polarity. J Neurosci 30, 2600-2610 (2010) and washed 3x10 min in 0.1% Triton X-100 in 0.1 M PB. Then, the mounts were blocked in 4% BSA in 0.1 M PB (pH 7.2) with 2.3% Triton X-100 for 1 hour at RT. The whole mounts were transferred into the primary antibody solution and incubated for 72 hours at 4 °C with shaking. Then, the tissue was washed 3x30 min in 0.1% Triton X-100 in0.1 M PB and transferred into secondary antibody solution and incubated for 72 hours at 4 °C with shaking, washed 3x30 min in 0.1% Triton X-100 in 0.1 M PB, mounted on microscope slides and coverslipped.

[0164] Image acquisition and analyses

[0165] Fluorescence images were acquired using either a Leica DM5000B microscope equipped with a motorized stage and a Hamamatsu Cl 1440 fluorescent camera and configured to be operated through the Neurolucida 2020 software suite, Nikon TiOE upright widefield microscope or a Leica Stellaris 8 Wide-field confocal microscope with LAS-X software. For ChP and SVZ whole mount DCX and Ki67 quantification, tissue was scanned and automatically stitched using the Slide scanning workflow in Neurolucida. For doublccortin total immunopositivc area quantification in SVZ whole mounts and coronal sections, images were analyzed in Nikon Elements AR, and the DCX immunopositive area fraction was taken to estimate the density of DCX+ newly-born neuroblasts in the SVZ wall. For Ki67 counts, representative coronal section confocal images were acquired and Ki67+ cells were manually counted in ImageJ 1.53, or SVZ whole mounts were scanned using the MBF Setereolnvestigator at a z-stack thickness of 40 pm to fully capture the outer layer of the ventricular’ wall. The scanning sites were randomly selected using an unbiased sampling grid generated in Stereo Investigator to cover 10% of the total SVZ whole mount area and Ki67+ or BrdU-i- cells were counted, and the population density was estimated using the Optional Fractionator workflow in StereoInvestigator. For ChP ablation evaluation, an average of 15-20 coronal sections from 1 out of 4 sets per brain were scanned and TTR+ area for LV and 3V ChP was quantified using Nikon Elements AR. For CD31+ blood vessel length density, maximum projection images of scanned whole mount ChPs were quantified using ImageJ. Consistently located ROIs were selected based on large blood vessels as landmarks. Each blood vessel within the ROI was traced, and the total linear length of capillaries within each ROI was divided by the total ROI area to obtain the length density. For BrdU-i- cell number quantification in olfactory bulb sagittal sections, 3-4 sections per animal were chosen based on consistent landmarks. Using ImageJ, the images were thresholded and converted to binary masks. Then, ROIs containing the GCL and RMS were applied and the number of BrdU-i- cells was automatically calculated. For quantification of BrdU-i- NeuN-i- cells in coronal OB sections (FIG.15), a similar procedure was followed using NTS Elements. For all analyses, the sum or average of data from each mouse was treated as n = 1 for statistical analysis.

[0166] For quantification of post-stroke SVZ neurogenesis, scanned images of sections containing the ipsi- and contralateral SVZ and corpus callosum, as well as the ipsilateral striatum, including the lesion site, were acquired on the Leica DM5000B microscope. Three sections containing the most DCX+ migrating neuroblasts were chosen per animal. ROIs containing the ipsi- and contralateral SVZ, ipsi- and contralateral CC, as well as the ipsilateral striatum with the lesion site, were traced using ImageJ. The images were thresholded, and DCX+ immunopositive area was calculated. To calculate the maximum migration distance towards the lesion site, a perpendicular line was drawn from the SVZ wall to the furthest migrating neuroblast for each of the three sections per animal, and an average of the three distances was taken.

[0167] For all analyses, the sum or average of data from each mouse was treated as n = 1 for statistical analysis.

[0168] Behavioral Tests

[0169] Open Field Test. The open field apparatus (Omnitech electronics Inc, Columbus, OH) consisted of 2 sets of 8 photobeam arrays for animal horizontal activity detection and 1 set of 8 photobeam arrays for vertical activity detection. Each monitor was installed on a cube frame inside of a closed cabinet. A 42Lx42Wx31T cm Plexiglas box divided into 4 chambers to allow for simultaneous recording of two animals was placed inside of each frame. Mouse bedding was evenly placed on the bottom of the box and the feeder and water bottle were installed on the box lid. The cabinet was lit in a 12 h light / 12 h dark light cycle and equipped with ventilation fans. The chambers were washed using a steam mouse cage washer between tests. Animal activity was tracked using automated Fusion software (Omnitech electronics). The locomotor activity was analyzed over a 23-hour and 72-hour periods.

[0170] Barnes Maze. Barnes maze (Stoelting) consisted of a gray circular platform 91 cm in diameter, at an elevation of 90 cm from the floor. On the platform surface, 20 holes of 5 cm diameter were evenly distributed around the edge. Among them, 19 out of 20 holes had a small gray square tray 2 cm in depth underneath each hole, while the remaining “escape” hole led to a 5-cm deep, 5 cm-wide, 15 cm-long escape box underneath. During the probe trial, the escape holewas replaced with the same 2 cm-deep “sham” tray. The test room was lit by LED light strips at 40 lux intensity. The testing area was encircled by black curtains. Above the center of the platform, a 500-watt light bulb and a fan were set to provide additional aversive stimulation during the test. Four colored visual cues on a white background were hung surrounding the platform to provide spatial cues during the test. Between tests, the apparatus was rinsed with warm water and wiped with 20% ethanol to eliminate olfactory cues. The test included 3 stages: Habituation, Acquisition Learning and Acquisition Probe. Habituation consisted of a single 2-minute trial per animal without visual cues or additional overhead light or fan exposure. During the Acquisition learning stage, each animal was trained for 2 trials (3 minutes duration each and 15 min inter-trial interval) for 4 days with visual cues and with the overhead light and fan on. The acquisition probe stage involved a single 3-minute trial with visual cues and with the overhead light and fan on. Animal activity was tracked in real time and analyzed using the automated ANY-Maze 7.1 behavior analysis software (Stoelting). The animals were tested at 1 month after Dtx administration.[001711 Spontaneous alternation Y-maze. The Y-maze apparatus consisted of 3 identical arms angled at 120° (5 cm wide, 35 cm long) enclosed by dark plastic walls 10 cm tall. The testing area was enclosed by dark curtains and lit with LED light strips at 18 lux. The animal was placed into one of the arms and allowed to freely explore the maze for 8 minutes while its locomotor activity was recorded using an overhead- mounted camera and tracked in real time using ANY- maze 7.1. To measure spatial working memory, the alternation rate was calculated as follows: % Alternations = total number of three-arm sequences I (total entries - 2).Example 2

[0172] Example 2 demonstrates feasibility and efficiency of choroid plexus (ChP) ablation in the neonate using adeno-associated virus (AAV) as a gene delivery tool to selectively target the ChP and achieve significant reduction in cerebrospinal fluid (CSF) volume. AAV-mediated gene delivery for ChP ablation is proposed as a therapeutic intervention to reverse excessive CSF accumulation in neonatal hydrocephalus, which is deleterious to normal brain development and causes debilitating neurological symptoms.

[0173] Using the mouse as a mammalian model, loss of CSF volume in neonates was successfully demonstrated using the AAV2 / 5 ChP-targeting serotype, carrying either a controleGFP vector, or the DTR vector. Expression of DTR in the ChP allows for ablation of DTR- cxprcssing cells after diphtheria toxin (Dtx) administration. Neonates at postnatal day 1 (Pl) were injected with either control or DTR virus intracerebroventricularly to target the ChP, and Dtx was administered P4-P6, and brain MRI data was collected at P21 and P30 (FIG. 1 A). All study subjects survived to P30 (study endpoint), demonstrating the compatibility of this strategy with survival. In MRI data collected both at P21 and P30 (FIG. 1 B), the AAV-DTR-treated animals demonstrated complete loss of ventricle volume, which was not reversible. Furthermore, in 3D reconstructions of the MRI data collected at P30, complete loss of CSF volume is observed in the lateral and third ventricles, while only a small portion of CSF remains in the pineal recess and the fourth ventricle (FIG. 1, C, D), demonstrating the efficiency of this approach in inducing CSF loss throughout the ventricular system. These data also support the feasibility of using the inducible DTA suicide gene system with the AAV2 / 5 vector as a strategy to achieve regulatable ablation of the ChP due to the similarity of the mechanism of cell death induction between the DTA and DTR- Dtx systems.

[0174] Example 3 and Example 4

[0175] Currently, there are no existing medicines or clinically approved biological tools capable of ablating or controlling the functions of the choroid plexus (ChP). Partial ablation of the ChP via endoscopic procedures combined with endoscopic third ventriculostomy (ETV), however, has been shown to reverse hydrocephalus-related phenotypes. We lately discovered that diphtheria toxin can ablate 60% or more ChP in iDTR transgenic mice without causing any neurological problems, shown in example 1. A less invasive and versatile tool to ablate the ChP that offers an improved and prolonged treatment option for hydrocephalus and other cerebrospinal fluid (CSF) production disorders would be advantageous.

[0176] Examples 3 and 4 illustrate the successful and highly selective tropism and delivery of suicide genes such as rapaCasp9 using an AAV2 / 5 vector in the ChP, demonstrating the translational potential of this biological tool in controlling CSF production for the treatment of hydrocephalus. Novel ChP-targeting AAVs expressing rapaCasp9 were developed and tested. This approach represents a fundamental shift from traditional hydrocephalus treatments, which primarily focus on removing excess CSF from the ventricular system. While conceptually similar to the existing endoscopic ChP coagulation (CPC) surgery, this method is significantly lessinvasive, removes the risk of unwanted bleeding from the ChP vasculature, and requires a shorter operating time.

[0177] This gene therapy can be administered via a small subgaleal ventricular access port in a clinical setting, avoiding the risks associated with CPC, such as hemorrhage, choroidal artery coagulation, and ischemia. Furthermore, unlike other gene replacement therapies designed to supplement missing protein functions, this method does not require continuous gene expression and may provide long-term benefits for the patients.

[0178] By enabling ChP-specific delivery of a clinically validated suicide gene, this innovative ChP ablation tool offers a potent and minimally invasive option for treating hydrocephalus — particularly neonatal hydrocephalus — and other neurological disorders.

[0179] Example 3. AAV-rapaCasp9 Administration to Human ChP Cells in Brain Organoids

[0180] Human ESC-derived (hESC) brain organoids were used to demonstrate AAV tropism in human ChP. Human ChP epithelial cells in 3D brain organoids (Figure 24 D) were generated based on established protocols as described in Pellegrini L, et al., Human CNS barrierforming organoids with cerebrospinal fluid production. Science. 2020 Jul 10;369(6500):eaaz5626. doi: 10.1126 / science.aaz5626. Epub 2020 Jun 11. PMID: 32527923. The human ChP organoids, developed from human forebrain organoids, secreted CSF-likc fluid and featured cuboidal epithelium with transthyretin (TTR) expression and organized epithelial tissue. Gene expression profiles in the human ChP organoids were similar to those of the human and mouse ChP. Approximately 50% of the spheroids formed balloon-like CSF inclusions indicative of ChP epithelium, with immunohistochemistry (IHC) showing expression of FOXJ1 and TTR, confirming their ChP-like identity (FIG. 24, A, D-F). ChP epithelia were observed protruding from the organoids after cultivation, at which point the AAV-ChP-eGFP virus (AAV2 / 5 virus expressing eGFP) or AAV-ChP-rapaCasp9 (AAV2 / 5 expressing rapaCasp9) were introduced. AAV2 / 5 vectors targeted human ChP epithelial cells in the 3D ChP organoids but did not target other cell types that exist in the same organoids, such as human forebrain neurons, astrocytes, and neural progenitor cells (Figure 24 G).

[0181] The AAV2 / 5 pseudotype demonstrated strong tropism for ChP epithelial cells but did not target other neural cell types such as neurons, astrocytes, oligodendrocytes, microglia, or vascular- endothelial cells in the mouse brain. When applied to embryonic or newborn rodent brains, the AAV2 / 5 vector selectively and sustainably (up to 12 months) expressed genes in mouse ChP epithelial cells in vivo. Similarly, AAV2 / 5 preferentially targets human ChP epithelial cells in cultured human brain organoids. AAV2 / 5 vectors and the tropism thereof are described in the art, see, e.g., Jang, A. & Lehtinen, M.K. Experimental approaches for manipulating choroid plexus epithelial cells. Fluids Barriers CNS 19, 36 (2022); Jang, A. & Lehtinen, M.K. In utero intracerebroventricular delivery of adeno-associated viral vectors to target mouse choroid plexus and cerebrospinal fluid. STAR Protoc 4, 101975 (2022); Chen, X. et al. Different Serotypes of Adeno-Associated Virus Vector- and Lentivirus-Mediated Tropism in Choroid Plexus by Intracerebroventricular Delivery . Hum Gene Ther 31, 440-447 (2020); Chen 2020; Watson, D.J., Passini, M.A. & Wolfe, J.H. Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adenoassociated virus type 5 vectors. Hum Gene Ther 16, 49-56 (2005); Jang Fluids Barriers CNS 2022, Jang STAR Protoc 2022; Mazucanti, C.H. et al. AAV5-mediated manipulation of insulin expression in choroid plexus has longterm metabolic and behavioral consequences. Cell Rep 42, 112903 (2023).

[0182] To deliver the specific suicide gene to the ChP via AAV, the ChP-targeting AAV, AAV2 / 5, was used. RapaCasp9, a prodrug for rapamycin-induced apoptosis, was used to ablate ChP cells. RapaCasp9 is a new enzyme / prodrug system used to induce rapamycin-induced apoptotic cell death in targeted cells. This engineered suicide gene -based system uses exogenous activation of the Caspase 9 catalytic domain fused with rapamycin binding domains of FKBP12 (rapaCasp9) to induce apoptosis upon rapamycin treatment. It successfully eliminated antigenspecific transgenic T-cells through viral rapaCasp9 delivery and improved the adverse immunological effects of adoptive cell transfer therapy for treating cancer (M. Stavrou, B. Philip, C. Traynor- White, C. G. Davis, S. Onuoha, S. Cordoba, et al. Molecular Therapy 2018 Vol. 26 Issue 5 Pages 1266-1276). Rapamycin is a brain-penetrating agent clinically used to treat child epilepsy or astrocytoma. It is therefore suited as an activator of the prodrug to activate rapaCasp9 and induce apoptosis in ChP.

[0183] The AAV-ChP plasmid vector expressing rapaCasp9 in human codon sequence was designed and validated rapamycin-dcpcndcnt cell death in vitro using cultured HEK293 cells (Data not shown).— Rapamycin treatment (1 or 100 nM) induced pAAV-ChP— rapaCasp9 vector dependent cell death in HEK293 cells at similar rapamycin dosing and timing to those reported in previous studies in human T-cells and human leukemia cell line, Jurkat cells. (See, e.g., M. Stavrou, B. Philip, C. Traynor- White, C. G. Davis, S. Onuoha, S. Cordoba, et al. Molecular Therapy 2018 Vol. 26 Issue 5 Pages 1266-1276.) This result validated the rapamycin-inducible apoptosis function of the AAV2 genome DNA construct.

[0184] Referring to FIG. 24 A-C, organoids were cultured from human embryonic stem cells (hESCs) with choroid plexus protocol and infected with AAV-ChP-rapaCasp9 at approximately 30 days. AAV was incubated on organoids for 6 days. On day 7, organoids were treated with 200nM rapamycin and harvested 24 hours later. Transthyretin (TTR) is a ChP-specific transport protein. TTR IHC confirms ChP identity of epithelial protrusions in the organoids. Cleaved-Caspase3+ cells are seen in ChP organoids after rapamycin administration (Figure 24B). The pink dots indicate ongoing cell death in the epithelia-like areas of the organoid. Caspase9+ cells in an untreated AAV-infected organoid show successful infection after 6 days of incubation (FIG. 24, C). FIG. 24G] demonstrates successful uptake of the vector in ChP epithelial cells. Referring to FIG. 24G, visualization of organoids post-treatment show that hESC-derived ChP brain organoids were infected with AAV-eGFP and demonstrated ChP-specific gene delivery.

[0185] In sum, based on the successful treatment of human ChP epithelial cells in organoids, the described ChP ablation methods should be capable of targeting human ChP epithelial cells with high specificity, reducing CSF volume in the brain immediately and permanently, improving ventricular volume of neonatal hydrocephalus, while posing minimal toxicity and risks to other neural cells and peripheral organs.

[0186] Example 4 Mouse brain ChP (in vivo)

[0187] Example 4 demonstrates the in vivo efficacy of the described AAV vectors delivering suicide genes in ablating the choroid plexus (ChP). Successful delivery of rapaCasp9 and subsequent ChP cell death were achieved in an in vivo system. The rapaCasp9 system, a widely used suicide gene technology described in, for example, Stavrou, Maria et al. “A Rapamycin-Activated Caspase 9-Based Suicide Gene.” Molecular therapy: the journal of the American Society of Gene Therapy vol. 26,5 (2018): 1266-1276,. The rapaCasp9 was integrated into the recombinant AAV2 / 5 (rAAV) to infect ChP epithelial cells and induce programmed cell death in the ChP cells upon rapamycin treatment. Rapamycin activates the catalytic domain of Caspase 9 by dimerization, which initiates apoptosis in the infected ChP cells. This study highlights the successful application of AAV-mediated suicide gene delivery (rapaCasp9) to selectively and efficiently ablate ChP epithelial cells, demonstrating its potential for managing cerebrospinal fluid (CSF) production in hydrocephalus.

[0188] Experiments were carried out according to the timeline as shown in FIG. 19. Experiments were conducted according to the timeline outlined in FIG. 19. C57BL / 6 wildtype (WT) mice were used to test the efficacy of AAV2 / 5 under the control of the CMV or CAG promoter. The viral constructs utilized included a control virus (AAV2 / 5-CMVp-eGFP, AAV2 / 5- CAG-eGFP expressing enhanced green fluorescent protein) and a test virus (AAV2 / 5-CMVp- rapaCasp9, AAV2 / 5-CAG-rapaCasp9 expressing rapaCasp9). eGFP is basic fluorescent protein that are encoded into the genome of the AAV as a reporter gene.

[0189] The AAV injection was carried out as follows: Postnatal day 0 (P0) mice were given bilateral intracerebroventricular (ICV) stereotaxic injections of 2 pL AAV into the brain ventricle of approximately 3.8xl010GC / mouse (AAV-ChP-eGFP and AAV-ChP-rapaCasp9). For rapamycin treatment: between P4-6, mice were given a total of 3 intraperitoneal (IP) rapamycin injections. Injections 1-3 were 10 mg / kg, alternating sides each day. ChP-specific targeting of the AAV2 / 5-ChP-EGFP vector was confirmed through P14 mouse brain sections, as illustrated in FIG. 20. Strong eGFP expression was observed in the ChP, with negligible expression in other cell types, ensuring specificity. Following rapamycin treatment, cleaved caspase-3 (c-Casp3) and TUNEL staining revealed ongoing ChP cell death, as depicted in FIG. 21. These results demonstrated the successful activation of rapaCasp9-mediated apoptosis in ChP epithelial cells via AAV-ChP delivering suicide genes. We confirmed that the CAG promoter is active and even stronger than the CMV promoter in cultured mouse ChP cells (FIG. 22).

[0190] Biodistribution analysis, shown in FIG. 23, indicated significant accumulation of AAV in the ChP (-600 GC / ccll) than the whole brain (-20 GC / ccll) 5 days after the ICV injection. Minimal presence (less than 5 GC / cell) was detected in peripheral tissues such as the kidney, heart,and lung, with moderate levels in the liver and spinal cord (-20 GC / cell). Digital PCR (dPCR) further confirmed low viral biodistribution in peripheral organs and was used to detect AAV and mouse genome DNAs. The AAV2 / 5 serotype's tropism for the liver was consistent with previously documented findings.

[0191] While both AAV2 / 5-CMVp-eGFP and AAV2 / 5-CMVp-rapaCasp9 viruses demonstrated successful infection of ChP epithelial cells, Caspase9+ cells were found in less ChP cells so far with longer AAV genome product (CMVp-mRFP-rapaCasp9) (FIG. 20), although full activation of the rapaCasp9 system was achieved with higher titer virus under the CAG promoter (FIG.21).

[0192] AAV2 / 5-CMV-cGFP (reporter virus) was used to evaluate efficacy, biodistribution and specificity. For immunohistochemistry (IHC) studies, slides were processed with primary antibody overnight, 2 hours of secondary antibody, and 5 mins of DAPI in between lx PBS washes before being cover slipped and imaged. Digital PCR (dPCR) was used to assay AAV genome copy number relative to mouse genome existing in the mouse tissue 5 or 14 days after the ICV delivery of AAV. In brief, fresh frozen samples of mouse brain, liver, spinal cord, lung, heart, and kidney were harvested, proceeded for DNA extraction, and analyzed at the CCHMC Transgenic Animal and Genome Editing (TAGE) core. Ventricular volumes were analyzed using Imaris with consistent pre-defined parameters. IHC images were processed and qualified in ImageJ / Fiji.

[0193] Targeted expression in ChP was observed in vivo. FIG. 20 depicts P14 mouse brain section after intraventricular injection of AAV5-CMV-EGFP at P0 (left) and AAV-CMV- rapaCasp9 (right) as visualized in the ChP, demonstrating that ChP-specific targeting of AAV2 / 5- CMVvector in the neonatal mouse brain was achieved in the ChP of the lateral ventricle. It is noted that eGFP signal was observed to be strong in the Chp and negligible in other cell types, demonstrating specificity of the vector for ChP. (Abbreviations: Olfactory bulb, Ob; Cerebral cortex, Cx; Lateral ventricle, Lv; Choroid plexus, ChP; Cerebellum (Cb); Ependymal cells, Ep; and Corpus callosum, CC.)

[0194] ChP cell death was observed following treatment of the suicide gene rapaCasp9- activating agent, rapamycin, in vivo. Referring to FIG. 21, administration of AAV-CMV-rapaCasp9 and AA V-CAG-rapaCasp9 results in signs of ChP cell death after rapamycin treatment. Cleaved caspasc-3 (c-Casp3) and terminal dcoxynuclcotidyl transferase dUTP nick end labeling (TUNEL) show ongoing ChP cell death via the rapaCasp9 and rapamycin dependent apoptosis pathway (FIG. 21, Panel A-D); c-Casp3 and TUNEL levels are higher in AAV-ChP-rapaCasp9 and rapamycin treated mouse (FIG. 21, Panels A and C).

[0195] All AAV2 / 5 viruses demonstrated successful infection. A positive infection by the AAV-ChP-eGFP vims in the ChP in C57B6 / J mice demonstrate the active function of the CMV and CAG promoter in the ChP. Mice that received AAV-ChP-rapaCasp9 vims show Caspase9+ cells in the ChP, meaning that the AAV-ChP can specifically deliver suicide gene in the ChP.

[0196] dPCR showed low peripheral biodistribution in mice. dPCR was used as an alternative to qtPCR due to its ability to detect smaller and rarer sequences as well as its reliance on the absolute copy number, allowing for more accuracy. Out of the 12 mice in this study, there was minimal virus detected in the lung, heart, and kidney, with moderate vims levels detected in the liver and spinal cord. The AAV2 / 5 serotype has been documented to have some accumulation detected in the liver. (Marsin, D. et al. “High- accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing” Molecular Therapy Methods and Clinical Development 15041, 2 (2015), doi: 10.1038 / mtm.2015.41), but it was confirmed that it was minimal in our hands as well.

[0197] It is noted that promoter activity was likewise demonstrated in cultured mouse cells. Referring to FIG. 22, AAV administration using AAV-CMV-eGFP and AAV-CAG-eGFP effectively achieved gene delivery in mouse ChP cells in vitro. AAV-CMV-eGFP (FIG. 22, A) and AAV-CAG-eGFP (FIG. 22, B), were both effective, AAV-CAG-eGFP showing higher gene expression as compared to AAV-CMV-eGFP.

[0198] Overall, this study underscores the specificity and efficacy of AAV2 / 5 vectors in targeting ChP epithelial cells and inducing programmed cell death through rapaCasp9. These findings demonstrate the potential of this approach for hydrocephalus management, although further study is needed to optimize vims production system and test ChP-ablation efficacy in large animals with human equivalent scale size.

[0199] Exemplary Combinations

[0200] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. The following examples arc not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0201] Example 1

[0202] A non-surgical method of treating hydrocephalus in an individual in need thereof, comprising administering to the choroid plexus (ChP) of the individual a ChP-targeting adeno- associated virus vector (AAV-ChP) comprising a suicide gene; and a suicide gene activator.

[0203] Example 2

[0204] The method of example 1, the AAV-ChP being a recombinant AAV2 / 5.

[0205] Example 3

[0206] The method of example 1 or 2, the suicide gene being selected from one or both of a rapamycin-induced caspase 9 suicide gene (rapaCasp9), and diphtheria toxin a (Dta).

[0207] Example 4

[0208] The method of any one of examples 1 to 3, the suicide gene activator being selected from rapamycin and doxycycline.

[0209] Example 5

[0210] The method of any one of examples 1 to 4, the AAV-ChP further comprising a promoter active in choroid plexus (ChP).

[0211] Example 6

[0212] The method of example 5, the AAV-ChP further comprising a promoter, the promoter being selected from a CAG promoter, a CMV promoter, a TRE promoter, and an EF-la promoter.

[0213] Example 7

[0214] The method of any one of examples 2 to 6, the recombinant AAV2 / 5 comprising an AAV5 capsid.

[0215] Example 8

[0216] The method of any one of examples 1 to 7, the suicide gene being cytosine deaminase.

[0217] Example 9

[0218] The method of example 8, the suicide gene activator being 5-fluorocytosine.

[0219] Example 10

[0220] The method of any one of examples 1 to 7, wherein the suicide gene activator is rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is administered after administering the AAV-ChP.

[0221] Example 11

[0222] The method of any of examples 1 to 7, further comprising administering rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is administered before administering the AAV-ChP.

[0223] Example 12

[0224] The method of any of examples 1 to 7, wherein the suicide gene activator is rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is being administered concurrently with the AAV-ChP and rapaCasp9 gene.

[0225] Example 13

[0226] The method of any preceding example, the administration comprising contact with choroid plexus epithelial cells of the individual.

[0227] Example 14

[0228] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to ablate the choroid plexus of the individual, wherein the ablation is either partial removal or complete removal, preferably wherein the ablation is determined by choroid plexus size after treatment, preferably wherein the ablation is a reduction in size of at least about 50%, or at least about 60%, or at least about 70% or more.

[0229] Example 15

[0230] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to reduce cerebral spinal fluid (CSF) production, preferably wherein the reduction is determined by the size of the ventricle, preferably wherein the reduction is determined by total CSF pre-treatment versus post-treatment, wherein the reduction is at least about 50%, or about 60%, or about 70%, or about 80% or more.

[0231] Example 16

[0232] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to reduce cerebral spinal fluid (CSF) volume, preferably wherein the reduction in volume is determined by choroid plexus size, preferably wherein the reduction is at least about 50%, or about 60%, or about 70%, or about 80% or more.

[0233] Example 17

[0234] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed monthly.

[0235] Example 18

[0236] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed every two months.

[0237] Example 19

[0238] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed every three months.

[0239] Example 20

[0240] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed every four months.

[0241] Example 21

[0242] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed every five months.

[0243] Example 22

[0244] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed every six months.

[0245] Example 23

[0246] The method of any preceding example, the administration of one or both of the AAV-ChP and suicide gene activator being performed yearly.

[0247] Example 24

[0248] The method of any preceding example, the hydrocephalus being neonatal hydrocephalus.

[0249] Example 25

[0250] The method of any preceding example, wherein the method is carried out in conjunction with a placement of a shunt in the individual.

[0251] Example 26

[0252] A choroid plexus (ChP) ablation tool for treating pediatric hydrocephalus comprising a ChP targeting adcno-associatcd virus vector (AAV-ChP) comprising a suicide gene.

[0253] Example 27

[0254] The choroid plexus ablation tool of example 26, the suicide gene being selected from one or both of a rapamycin-induced caspase 9 suicide gene (rapaCasp9), and diphtheria toxin a (Dta).

[0255] Example 28

[0256] The ChP ablation tool of example 26, the suicide gene being rapaCasp9.

[0257] Example 29

[0258] The ChP ablation tool of example 26, the suicide gene being diphtheria toxin a (Dta).

[0259] Example 30

[0260] The ChP ablation tool of any one of examples 26-29, further comprising a promoter.

[0261] Example 31

[0262] The ChP ablation tool of example 30, the promoter being selected from a CAG promoter, a CMV promoter, a TRE promoter, and an EF-la promoter.

[0263] Example 32

[0264] The ChP ablation tool of example 30 or 31 , the promoter being operatively linked to the suicide gene.

[0265] Example 33

[0266] The ChP ablation tool of any one of examples 26 to 32, the AAV-ChP being provided in a pharmaceutically acceptable carrier.

[0267] Example 34

[0268] A composition comprising the ChP ablation tool of any of examples 26 through 33, formulated for delivery to the ventricle of a human.

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Claims

CLAIMS1. A non- surgical method of treating hydrocephalus in an individual in need thereof, comprising administering to the choroid plexus (ChP) of the individual a. a ChP-targeting adeno-associated virus vector (AAV-ChP) comprising a suicide gene; and b. a suicide gene activator.

2. The method of claim 1, the AAV-ChP being a recombinant AAV2 / 5.

3. The method of claim 1 or 2, the suicide gene being selected from one or both of a rapamycin-induced caspase 9 suicide gene (rapaCasp9), and diphtheria toxin a (Dta).

4. The method of any one of claims 1 to 3, the suicide gene activator being selected from rapamycin and doxycycline.

5. The method of any one of claims 1 to 4, the AAV-ChP further comprising a promoter active in choroid plexus (ChP).

6. The method of claim 5, the AAV-ChP further comprising a promoter, the promoter being selected from a CAG promoter, a CMV promoter, a TRE promoter, and an EF-la promoter.

7. The method of any one of claims 2 to 6, the recombinant AAV2 / 5 comprising an AAV5 capsid.

8. The method of any one of claims 1 to 7, the suicide gene being cytosine deaminase.

9. The method of claim 8, the suicide gene activator being 5-fluorocytosine.

10. The method of any one of claims 1 to 7, wherein the suicide gene activator is rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is administered after administering the AAV-ChP.

11. The method of any of claims 1 to 7, further comprising administering rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is administered before administering the AAV-ChP.

12. The method of any of claims 1 to 7, wherein the suicide gene activator is rapamycin and the suicide gene is rapaCasp9, and wherein the rapamycin is being administered concurrently with the AAV-ChP and rapaCasp9 gene.

13. The method of any preceding claim, the administration comprising contact with choroid plexus epithelial cells of the individual.

14. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to ablate the choroid plexus of the individual, wherein the ablation is either partial removal or complete removal, preferably wherein the ablation is determined by choroid plexus size after treatment, preferably wherein the ablation is a reduction in size of at least about 50%, or at least about 60%, or at least about 70% or more.

15. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to reduce cerebral spinal fluid (CSF) production, preferably wherein the reduction is determined by the size of the ventricle, preferably wherein the reduction is determined by total CSF pre-treatment versus post-treatment, wherein the reduction is at least about 50%, or about 60%, or about 70%, or about 80% or more.

16. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being in an amount and for a duration of time effective to reduce cerebral spinal fluid (CSF) volume, preferably wherein the reduction in volume is determined by choroid plexus size, preferably wherein the reduction is at least about 50%, or about 60%, or about 70%, or about 80% or more.

17. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed monthly.

18. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed every two months.

19. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed every three months.

20. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed every four months.

21. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed every five months.

22. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed every six months.

23. The method of any preceding claim, the administration of one or both of the AAV-ChP and suicide gene activator being performed yearly.

24. The method of any preceding claim, the hydrocephalus being neonatal hydrocephalus.

25. The method of any preceding claim, wherein the method is carried out in conjunction with a placement of a shunt in the individual.

26. A choroid plexus (ChP) ablation tool for treating pediatric hydrocephalus comprising a ChP targeting adeno-associated virus vector (AAV -ChP) comprising a suicide gene.

27. The choroid plexus ablation tool of claim 26, the suicide gene being selected from one or both of a rapamycin-induced caspase 9 suicide gene (rapaCasp9), and diphtheria toxin a (Dta).

28. The ChP ablation tool of claim 26, the suicide gene being rapaCasp9.

29. The ChP ablation tool of claim 26, the suicide gene being diphtheria toxin a (Dta).

30. The ChP ablation tool of any one of claims 26-29, further comprising a promoter.

31. The ChP ablation tool of claim 30, the promoter being selected from a CAG promoter, a CMV promoter, a TRE promoter, and an EF- la promoter.

32. The ChP ablation tool of claim 30 or 31, the promoter being operatively linked to the suicide gene.

33. The ChP ablation tool of any one of claims 26 to 32, the AAV-ChP being provided in a pharmaceutically acceptable carrier.

34. A composition comprising the ChP ablation tool of any of claims 26 through 33, formulated for delivery to the ventricle of a human.

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