Compositions and methods for FOXG1 syndrome

Abstract

Provided herein are compositions and methods for delivering FOXG1 to the central nervous system. Also provided are compositions and methods for preventing or treating FOXG1 deficiency in a subject in need thereof.

Description

Docket No.062686-510001WO COMPOSITIONS AND METHODS FOR FOXG1 SYNDROME CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Nos.63 / 681,018, filed Aug.8, 2024, and 63 / 754,216, filed Feb.5, 2025, which applications are incorporated herein by reference. BACKGROUND

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 062686-510001WO_seqs.xml, created July 28, 2025, which is 33,492 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND

[0003] FOXG1 syndrome is a rare neurodevelopmental disorder associated with heterozygous variants in the forkhead box G1 (FOXG1) gene and is characterized by impaired neurological development and / or altered brain physiology. Observed phenotypes of FOXG1 syndrome primarily include a particular pattern of structural alterations in the brain resulting from inherited de novo mutations in the FOXG1 gene. Such structural alterations include a thin or underdeveloped corpus callosum that connects between the right and left hemispheres of the brain, reduced sulci and gyri formation on the surface of the brain, and / or a reduced amount of white matter. FOXG1 syndrome affects most aspects of development in children and the main clinical features observed in association with FOXG1 variants comprise impairment of postnatal growth, primary (congenital) or secondary (postnatal) microcephaly, severe intellectual disability with absent speech development, epilepsy, stereotypies and dyskinesia, abnormal sleep patterns, unexplained episodes of crying, gastroesophageal reflux, and recurrent aspiration. Currently, there are no approved treatments for FOXG1 syndrome. New methods for the treatment of FOXG1 syndrome are needed. SUMMARY

[0004] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect.

[0005] In one aspect, a method of delivering a forkhead box G1 (FOXG1) protein to the central nervous system (CNS) in a human subject in need thereof is provided, the method comprising administering to the subject an effective amount of a recombinant adeno-associated virus (rAAV) encoding the FOXG1 protein, wherein the rAAV is administered by injection into the cerebrospinal fluid (CSF) via an intracerebroventricular (ICV) route.

[0006] In one aspect, a method of delivering a FOXG1 protein to telencephalon cells in a human subject in need thereof is provided, the method comprising administering to the subject an effective amount of an rAAV encoding the FOXG1 protein, wherein the rAAV is administered by injection into the CSF via an ICV route.Docket No.062686-510001WO

[0007] In one aspect, a method of treating FOXG1 deficiency in a human subject in need thereof is provided, the method comprising administering an effective amount of a rAAV encoding a FOXG1 protein to the subject by ICV injection.

[0008] In one aspect, a method of treating FOXG1 haploinsufficiency in a human subject in need thereof is provided, the method comprising administering an rAAV encoding a FOXG1 protein to the subject by ICV injection.

[0009] In one aspect, a method of preventing, improving, or treating FOXG1 syndrome in a human subject is provided, the method comprising administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection.

[0010] In one aspect, a method of reducing anxiety in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection.

[0011] In one aspect, a method of improving behavior deficits related to anxiety in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection. In some embodiments, the anxiety in the subject is related to a novel or unfamiliar environment and adapting to this novel environment.

[0012] In one aspect, a method of improving sleep patterns in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection. In some embodiments, the improvement in sleep pattern is increased time spent awake. In some embodiments, the improvement in sleep pattern is less fragmented NREM sleep.

[0013] In some embodiments, the subject has one or more mutations in a FOXG1 gene. In some embodiments, the subject has FOXG1 syndrome. In some embodiments, the human subject is an infant. In some embodiments, the human subject is a child. In some embodiments, the human subject is an adult.

[0014] In some embodiments, the ICV injection is a bilateral ICV injection.

[0015] In some embodiments, the rAAV payload is self-complementary (sc). In some embodiments, the rAAV payload is single-stranded (ss). In some embodiments, the rAAV comprises an isolated nucleic acid encoding a FOXG1 protein and at least one AAV capsid protein.

[0016] In some embodiments, the at least one capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.PHP-eB, or a variant of any of the foregoing. In some embodiments, the at least one capsid protein is an AAV9 capsid protein or an AAV.PHP-eB capsid protein. In some embodiments, the at least one AAV capsid protein has a tropism for central nervous system (CNS) cells. In some embodiments, the at least one AAV capsid protein has a tropism for telencephalon cells.Docket No.062686-510001WO

[0017] In some embodiments, the rAAV comprises an expression cassette having a transgene that encodes a FOXG1 protein. In some embodiments, the FOXG1 protein amino acid sequence is set forth in SEQ ID NO: 1. In some embodiments, the nucleic acid payload encoding the FOXG1 protein comprises the nucleic acid sequence set forth in any one of SEQ ID NOs: 2, 3, and 8-13.

[0018] In some embodiments, the rAAV further comprises adeno- associated virus (AAV) inverted terminal repeats (ITRs) operably linked to the transgene encoding FOXG1 protein. In some embodiments, the AAV ITRs are AAV2 ITR. In some embodiments, the AAV2 ITRs comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, at least one of the ITRs is a truncated ITR (AITR).

[0019] In some embodiments, the rAAV further comprises a promoter that is operably linked to the transgene encoding forkhead box G1 protein. In some embodiments, the promoter is a constitutive promoter, inducible promoter, or tissue-specific promoter. In some embodiments, the promoter comprises a chicken beta-actin (CB) promoter, a Ula promoter, or a neuron-specific promoter. In some embodiments, the neuron-specific promoter comprises a human synapsin 1 (hSynl) promoter or a human Ca2+ / calmodulin-dependent protein kinase II (hCAMKII) promoter. In some embodiments, the promoter comprises the sequence set forth in any one of SEQ ID NOs: 4 to 7.

[0020] In some embodiments, the transgene encoding forkhead box G1 protein comprises a codon- optimized nucleic acid sequence. In some embodiments, the codon-optimized nucleic acid sequence comprises the sequence set forth in SEQ ID NO: 3.

[0021] In some embodiments, the rAAV is formulated for ICV delivery.

[0022] In some embodiments, the administration restores at least 5%, 10%, 20%, 30%, or 40% FOXG1 protein in cells relative to untreated cells, or relative to a baseline measurement. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0024] FIG.1 shows a graph of the biweekly body weights from FOXG1 wild-type (WT) and heterozygous (Het) mice dosed with vehicle, or FOXG1 AAV (ALR-047) at 4e10 vector genomes (vg) / mouse, 1e11 vg / mouse, or 2.6e11 vg / mouse. There were no significant differences in body weight among the treatment groups as assessed by two‐way mixed effects analysis of variance (ANOVA) (F (4, 69) = 0.3412, p = 0.8492). Animals increased in body weight as the study progressed (F (24, 1245) = 4748, p < 0.0001). Means + / ‐ standard error of the mean (SEM) are displayed.

[0025] FIG.2 shows a schematic Kaplan‐Meier survival curve for the animals dosed with vehicle, or FOXG1 AAV (ALR-047) at 4e10 vg / mouse, 1e11 vg / mouse, or 2.6e11 vg / mouse. All study deaths occurred prior to weaning at post-natal day 21 (P21).Docket No.062686-510001WO

[0026] FIGs.3A-3C show graphs for the open field total distance traveled by all animals dosed with vehicle, or FOXG1 AAV (ALR-047) at 4e10 vg / mouse, 1e11 vg / mouse, or 2.6e11 vg / mouse. over time. FIG.3A shows the total distance traveled over time at 6 weeks of age. FIG.3B shows the total distance traveled over time at 8 weeks of age. FIG.3C shows the total distance traveled over time at 11 weeks of age. Means + / - SEM are displayed.

[0027] FIGs.4A-4C show graphs for the open field total distance traveled by all animals across the 30-minute trial. FIG.4A and FIG.4B show the total distance traveled at 6 and 8 weeks of age, respectively. Overall, there were no significant differences in total distance traveled among the treatment groups at either age (p = 0.2833 at 6 weeks of age, p = 0.0726 at 8 weeks of age). However, at 11 weeks of age, there were significant differences in total distance traveled among the treatment groups (FIG.4C); specifically, there was a significant increase in total distance traveled in the FOXG1 Het mice dosed with FOXG1 AAV (ALR-047) at 2.6e11 vg / mouse versus those dosed with vehicle (p < 0.0001). At all timepoints assessed, there was no significant difference between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with FOXG1 AAV (ALR-047) at 2.6e11 vg. At each age, one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant. **** p < 0.0001.

[0028] FIGs.5A-5C show graphs for the open field center distance traveled by all animals over time at 6, 8, and 11 weeks of age (FIG.5A, FIG.5B and FIG.5C, respectively). Means + / - SEM are displayed.

[0029] FIGs.6A-6C show graphs for the open field center distance traveled by all animals across the 30-minute trial at 6, 8 and 11 weeks of age (FIG.6A, FIG.6B and FIG.6C, respectively). There was a significant difference in center distance traveled between the vehicle-treated FOXG1 WT mice and vehicle-treated FOXG1 Het mice at 6 (p < 0.05), 8 (p < 0.05) and 11 (p < 0.01) weeks of age. With FOXG1 AAV (ALR-047) treatment of FOXG1 Het mice at 2.6e11 vg / mouse, there was a significant increase in center distance traveled at 11 weeks of age versus FOXG1 Het mice treated with vehicle (p < 0.001). At all timepoints assessed, there was no significant difference between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with FOXG1 AAV (ALR- 047) at 2.6e11 vg (p = 0.0001). At each age, one-way ANOVA with Tukey’s multiple comparisons test was used to assess statistical significance. Means + / - SEM are displayed. *p < 0.05; **p < 0.01, ***p < 0.001.

[0030] FIGs.7A-7C show graphs for the open field total rearing frequency by all animals over time at 6, 8 and 11 weeks of age (FIG.7A, FIG.7B and FIG.7C, respectively). Means + / - SEM are displayed.

[0031] FIGs.8A-8C show graphs for the open field total rearing frequency by all animals across the 30-minute trial at 6, 8 and 11 weeks of age (FIG.8A, FIG.8B and FIG.8C, respectively). Overall, there were no significant differences in total rearing frequency among the FOXG1 AAV (ALR-047) treatment groups at 6 (FIG.8A, p = 0.0981), 8 (FIG.8B, p = 0.0539), or 11 (FIG.8C, pDocket No.062686-510001WO = 0.0008) weeks of age. At 8 and 11 weeks of age, FOXG1 Het animals treated with FOXG1 AAV (ALR-047) at 2.6e11 vg / mouse reared significantly more frequently than FOXG1 Het animals treated with vehicle (p < 0.05 and p < 0.001, respectively). At each age, one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant *p < 0.05, ***p < 0.001.

[0032] FIGs.9A-9C show graphs for the open field center rearing frequency by all animals over time at 6, 8 and 11 weeks of age (FIG.9A, FIG.9B and FIG.9C, respectively). Means + / - SEM are displayed.

[0033] FIGs.10A-10C show graphs for the open field center rearing frequency by all animals across the 30-minute trial at 6, 8 and 11 weeks of age (FIG.10A, FIG.10B and FIG.10C, respectively). Overall, there were no significant differences in center rearing frequency among the treatment groups at 6 (p = 0.0847) or 8 weeks of age (p = 0.2258). However, at 11 weeks of age, there were significant differences in center rearing frequency among the treatment groups (p = 0.0247); specifically, there was a significant increase in open field center rearing frequency in the FOXG1 Het mice dosed with FOXG1 AAV (ALR-047) at 2.6e11 vg / mouse versus those dosed with vehicle (p < 0.05). At all timepoints assessed, there was no significant difference between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with FOXG1 AAV (ALR-047) at 2.6e11 vg. At each age, one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant, *p < 0.05.

[0034] FIGs.11A-11C show graphs for the open field velocity by all animals over time at 6, 8 and 11 weeks of age (FIG.11A, FIG.11B and FIG.11C, respectively). Means + / - SEM are displayed.

[0035] FIGs.12A-12C show graphs for the open field velocity by all animals across the 30-minute trial at 6, 8 and 11 weeks of age (FIG.12A, FIG.12B and FIG.12C, respectively). Overall, there were significant differences in velocity among the treatment groups at 6 (p = 0.0004) and 8 (p = 0.0011) weeks of age. However, there were no significant differences in velocity among the treatment groups at 11 weeks of age (p = 0.0633). At each age, one-way ANOVA with Tukey’s multiple comparison was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant, *p < 0.05, **p < 0.01.

[0036] FIGs.13A-13E show graphs for the grip strength performance of all animals by trial at 4, 6, 8, 10 and 12 weeks of age (FIG.13A, FIG.13B, FIG.13C, FIG.13D and FIG.13E, respectively). At each age, two-way ANOVA was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant

[0037] FIGs.14A-14E show graphs for the average grip strength performance of all animals at 4, 6, 8, 10 and 12 weeks of age (FIG.14A, FIG.14B, FIG.14C, FIG.14D and FIG.14E, respectively). Overall, there were no significant differences among the treatment groups in the grip strength at 4 (p = 0.02809), 6 (p = 0.4114), 8 (p = 0.3343), 10 (p = 0.4288) or 12 (p = 0.7754) weeksDocket No.062686-510001WO of age. At each age, one-way ANOVA with Tukey’s multiple comparison test was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant

[0038] FIGs.15A-15C show graphs for Fear Conditioning. FIG.15A shows the percent freezing over time in contextual fear conditioning. FIG.15B shows the average percent freezing in contextual fear conditioning. Overall, there were significant differences in contextual percent freezing among the treatment groups (p = 0.0002). FIG.15C shows the average percent freezing in cued fear conditioning by phase. There were significant differences in cued percent freezing among the treatment groups (F (4, 55) = 4.742, p = 0.0023). In FIG.15B, one-way ANOVA was used to assess statistical significance. In FIG.15C, two-way ANOVA was used to assess statistical significance. Means + / - SEM are displayed. ns: not significant. ** p < 0.01.

[0039] FIG.16 shows graphs for weekly body weights from FOXG1 WT and Het mice. Overall, the extent to which body weight changed over time varied by treatment group (F (24, 540) = 4.175, p < 0.0001), as assessed by two-way ANOVA with Tukey’s multiple comparison test. There were significant differences between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with either vehicle or FOXG1 AAV (ALR-047) at 4, 5, 6 and 7 weeks of age, as assessed by two-way ANOVA with Tukey’s multiple comparison test (* p < 0.05, ** p < 0.01, *** p < 0.001). Means + / - SEM are displayed. Note: n values in the legend reflect the total number of animals in each group at the time of enrollment.

[0040] FIGs.17A-17B show graphs for wire hang latency to fall at 10 weeks of age by all animals. FIG.17A shows latency to fall by trial. FIG.17B shows average latency to fall. There were no significant differences in latency to fall among the treatment groups, as assessed by one-way ANOVA (F (2, 44) = 2.782, p = 0.0728). Means + / - SEM are displayed. ns: not significant.

[0041] FIGs.18A-18B show graphs for open field data over time at 11 weeks of age for all animals. FIG.18A shows the total distance traveled over the 30-minute assessment, binned by 5- minute intervals. FIG.18B shows the total rearing frequency over the 30-minute assessment, as number of bouts binned by 5-minute intervals. Means + / - SEM are displayed.

[0042] FIGs.19A-19B show graphs for distance traveled in the open field test at 11 weeks of age for all animals. FIG.19A shows total distance traveled during the 30-minute assessment. Overall, there were differences in total distance traveled among the treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 4.845, p = 0.0125). FIG.19B shows center distance traveled during the 30-minute assessment. Overall, there were differences in center distance traveled among the treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 7.607, p = 0.0015). Means + / - SEM are displayed. ** p < 0.01, ns: not significant.

[0043] FIGs.20A-20B show graphs of rearing frequency in the open field test at 11 weeks of age. FIG.20A shows total rearing frequency during the 30-minute assessment. Overall, there were differences in total rearing frequency among the treatment groups, as assessed by one-way ANOVADocket No.062686-510001WO with Tukey’s multiple comparison test (F (2, 44) = 12.98, p < 0.0001). FIG.20B shows center rearing frequency during the 30-minute assessment. There were no differences in center rearing frequency among the treatment groups as assessed by with Tukey’s multiple comparison test (F (2, 44) = 0.9096, p = 0.4101). Means + / - SEM are displayed. ** p < 0.01, ns: not significant.

[0044] FIGs.21A-21B shows graphs of latency to turn on the tapered beam at 11 weeks of age. FIG.21A shows the latency to turn by trial. FIG.21B shows average latency to turn. There were no significant differences in average latency to turn among the treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 2.272, p = 0.1151). Means + / - SEM are displayed. ns: not significant.

[0045] FIGs.22A-22B show graphs of latency to traverse the tapered beam at 11 weeks of age. FIG.22A shows the latency to traverse the tapered beam by trial. FIG.22B shows the average latency to traverse. There were no significant differences in average latency to traverse the tapered beam among the treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 2.969, p = 0.0617). Means + / - SEM are displayed. ns: not significant.

[0046] FIGs.23A-23E show graphs of footslips on the tapered beam at 11 weeks of age. FIG. 23A shows left forelimb slip-step ratio. There were no significant differences in left forelimb slip-step ratio among treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 1.712, p = 0.1924). FIG.23B shows right forelimb slip-step ratio. There were no significant differences in right forelimb slip-step ratio among treatment groups, as assessed by one- way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 0.8371, p = 0.4398). FIG.23C shows left hindlimb slip-step ratio. There were no significant differences in left hindlimb slip-step ratio among treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 0.5693, p = 0.5700). FIG.23D shows right hindlimb slip-step ratio. There were no significant differences in right hindlimb slip-step ratio among treatment groups, as assessed by one- way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 1.639, p = 0.2058). FIG.23E shows total slip-step ratio. There were no significant differences in total slip- step ratio among treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 1.363, p = 0.2665). Means + / - SEM are displayed. ns: not significant.

[0047] FIG.24 shows graphs for running wheel time course data at 12 weeks of age. The distance traveled was plotted in one-hour bins. Means + / - SEM are displayed.

[0048] FIGs.25A-25B show graphs for total distance traveled during the light and dark cycles at 12 weeks of age. FIG.25A shows the total distance traveled during the animals’ light cycle (from 7AM to 7PM) on each day of the assessment. Overall, there were significant differences in the total distance traveled between the treatment groups, as assessed by two-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 14.75, p < 0.0001). Specifically, there were significant differences between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with vehicle on Day 1 and Day 2, as assessed by two-way ANOVA with Tukey’s multiple comparison test.Docket No.062686-510001WO There was also a significant difference between FOXG1 Het animals treated with FOXG1 AAV (ALR-047) versus those treated with vehicle on Day 1. FIG.25B shows the total distance traveled during the animals’ dark cycle (from 7PM to 7AM) on each day of the assessment. Overall, there were significant differences in the total distance traveled between the treatment groups, as assessed by two-way ANOVA with Tukey’s multiple comparison test (F (2, 44) = 13.03, p < 0.0001). Specifically, there were significant differences between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with vehicle on Night 2 and Night 3, as assessed by two-way ANOVA with Tukey’s multiple comparison test. Means + / - SEM are displayed. * p < 0.05, **** p< 0.0001, ns: not significant.

[0049] FIGs.26A and 26B shows graphs for DRFA (de-correlated ranked feature analysis) cloud analysis of SmartCube® results at 13 weeks of age. FIG.26A shows the significant differences between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with vehicle (p< 0.0001). There was significant recovery in FOXG1 Het animals treated with FOXG1 AAV (ALR- 047) versus those treated with vehicle (p = 0.0160). FIG.26B shows the top features that were significantly different between FOXG1 WT animals treated with vehicle and FOXG1 Het animals treated with vehicle. Treatment of FOXG1 Het animals with FOXG1 AAV (ALR-047) led to robust changes in 6 of these top features.

[0050] FIG.27 shows graphs for weekly body weights in vehicle treated WT (WT_Excipient), vehicle treated FOXG1 Het (HET_Excipient) and AAV treated FOXG1 Het (HET_ALR-047) mice. There were no significant differences in body weight among the treatment groups as assessed by two- way mixed effects ANOVA (F (2, 31) = 1.166, p = 0.3249). Body weights increased as the study progressed (F (2.792, 81.20) = 1988, p < 0.0001). Means + / - SEM are displayed.

[0051] FIGs.28A and 28B shows graphs for latency to the onset of nonrapid eye movement sleep (NREM, top panel) and rapid eye movement sleep (REM, bottom panel) from lights on following treatment of male FOXG1 Het mice with ALR-047 (HET_ALR-047) or vehicle (HET_Excipient) or male WT mice with vehicle (WT_Excipient). No significant differences were observed in latency to NREM or REM among the treatment groups as assessed by one-way ANOVA. Means + / - SEM are displayed. NREM: p=0.779; REM: p=0.518.

[0052] FIG.29 shows graphs for the average percentage time in wake, NREM and REM in light phase and dark phase. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. Top panel: vehicle treated FOXG1 Het mice (HET_Excipient) spent significantly less time awake during the dark phase compared to vehicle treated WT mice (WT_Excipient) (Dark phase p=0.0153). Middle panel: Vehicle treated FOXG1 Het mice (HET_Excipient) spent significantly more time in NREM during both the light and dark phases compared to vehicle treated WT mice (WT_Excipient) (Light phase p= 0.0342; Dark phase p= 0.0159). Bottom panel: REM was decreased in the AAV treated FOXG1 Het mice (HET_ALR-047) compared to the vehicle treated FOXG1 Het mice (HET_Excipient) (Light phase p=0.0158).Docket No.062686-510001WO Statistical significance within the light or dark phase was assessed by one-way ANOVA followed by a post hoc t test. * p < 0.05. Means + / - SEM are displayed.

[0053] FIG.30 shows graphs for the average hourly percentage time in wake, NREM and REM across the 24h light / dark period. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. No significant differences were observed among treatment groups as assessed by two-way (Group X Time bin) ANOVA. Means + / - SEM are displayed.

[0054] FIG.31 shows graphs for the average number of bouts for wake, NREM and REM in the 12h light and 12h dark phases. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. One way ANOVA showed significant changes for NREM in the dark phase and REM in the light phase. Post hoc t test analyses revealed the number of NREM bouts in the dark phase was significantly greater in the vehicle treated FOXG1 Het mice (HET_Excipient) compared to the vehicle treated WT mice (WT_Excipient) (p=0.0291 while the number of REM bouts in the light phase was significantly greater in the vehicle treated FOXG1 Het mice (HET_Excipient) compared to the AAV treated FOXG1 Het mice (HET_ALR-047) (p=0.0043). * p < 0.05. Means + / - SEM are displayed.

[0055] FIG.32 shows graphs for the average hourly number of bouts for wake, NREM and REM across the light and dark phases. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. Two-way (Group X Time) ANOVA analyses showed significant differences between vehicle treated FOXG1Het mice (HET_Excipient) and vehicle treated WT mice (WT_Excipient) (time bin 15; p=0.0042, time bin 18 ; p=0.0073), and between AAV treated FOXG1 Het mice (HET_ALR-047) and vehicle treated WT mice (WT_Excipient) (time bin 17; p=0.0336, time bin 18; p=0.0263) for REM in the dark phase * p < 0.05. Means + / - SEM are displayed.

[0056] FIG.33 shows graphs for the average bout durations for wake, NREM and REM in the 12h light and 12h dark phases. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. No significant differences were observed among treatment groups as assessed by one-way ANOVA (Wake; p=0.551, NREM; p=0.2185, REM; p=0.3381). Means + / - SEM are displayed.

[0057] FIG.34 shows graphs for the average hourly bout durations for Wake, NREM and REM in the 12h light and 12h dark phases. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. No significant differences were observed among treatment groups as assessed by two way ANOVA (Means + / - SEM are displayed).

[0058] FIG.35 shows graphs for the average normalized (to WT) electroencephalogram (EEG) spectral power during wake in the frontal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. No significant differences were observed among treatment groups as assessed by one-way ANOVA. (Means + / - SEM are displayed).Docket No.062686-510001WO

[0059] FIG.36 shows graphs for the average hourly normalized EEG spectral power during wake in the frontal cortex binned into the standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. Two way ANOVA showed a significant effect for beta in the dark phase. Post hoc analysis with t test revealed one data point with a significant reduction in beta during the 21st hour (9th hour of the dark) in the AAV treated FOXG1 Het mice (HET_ALR-047) compared to the vehicle treated FOXG1 Het mice (HET_Excipient) (p = 0.0295). * p < 0.05. Means + / - SEM are displayed.

[0060] FIG.37 shows graphs for the average normalized (to WT) EEG spectral power during NREM in the frontal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. One-way ANOVA showed significant effects for alpha in the light phase and high gamma in both the light and dark phases. Alpha in the light phase was reduced in the AAV treated FOXG1 Het mice (HET_ALR-047) compared to the vehicle treated WT mice (WT_Excipient) (t test post-hoc analysis, p =0.0200). High gamma in the light and dark phases was reduced for both the AAV (HET_ALR-047) and vehicle treated FOXG1 Het (HET_Excipient) groups compared to the vehicle treated WT mice (WT_Excipient) (t test post-hoc analysis, p = 0.0294, 0.0279, 0.0027 and <0.0001, respectively). * p <0.05. Means + / - SEM are displayed.

[0061] FIG.38 shows graphs for the average hourly normalized EEG spectral power during NREM in the frontal cortex binned into standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. Two-way ANOVA showed a significant effect for beta power in the dark phase. Post hoc analysis with t test revealed that the AAV treated FOXG1 Het mice (HET_ALR-047) had reduced beta compared to vehicle treated WT mice (WT_Excipient) for hours 13, 16, and 20-22 (p = 0.0197, 0.0250, 0.0255, 0.0353 and 0.0427, respectively). AAV treated FOXG1 Het mice (HET_ALR-047) also had reduced beta power compared to the vehicle treated FOXG1 Het mice (HET Excipient) for hour 13 (p = 0.0180). * p < 0.05. Means + / - SEM are displayed.

[0062] FIG.39 shows graphs for the average normalized (to WT) EEG spectral power during REM in the frontal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. One-way ANOVA showed significant effects for low gamma and high gamma in both the light and dark phases. For both low gamma and high gamma, power was greater in the vehicle treated WT mice (WT_Excipient) in both the light and dark phases compared to the AAV treated FOXG1 Het mice (HET_ALR-047) (Low gamma: p = 0.0279 and 0.0074 respectively, High gamma: p=0.0041 and <0.0001 respectively), and in the dark phase compared to the vehicle treated FOXG1 Het mice (HET_Excipient) for both low and high gamma (p =0.0077 and 0.0027 respectively). * p <0.05. Mean + / - SEM are displayed.Docket No.062686-510001WO

[0063] FIG.40 shows graphs for the average hourly normalized EEG spectral power during REM in the frontal cortex binned into the standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. Time points without data were due to the lack of REM to perform spectral analyses on. Two-way ANOVA showed significant effects for beta power in the light and dark phases. Post hoc analysis with t test revealed that beta was reduced during the first hour in the vehicle treated FOXG1 Het mice (HET_Excipient) compared to the vehicle treated WT mice (WT_Excipient) (p = 0.0030) and during hour 20 in the AAV treated FOXG1 Het mice (HET_ALR-047) compared to the vehicle treated WT mice (WT_Excipient) (p = 0.0144). * p < 0.05. Means + / - SEM are displayed.

[0064] FIG.41 shows graphs of the average normalized (to WT) EEG spectral power during wake in the parietal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. No significant differences were observed among treatment groups as assessed by one-way ANOVA. Means + / - SEM are displayed.

[0065] FIG.42 shows graphs for the average hourly normalized EEG spectral power during wake in the parietal cortex binned into the standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. No significant differences were observed among treatment groups as assessed by two-way ANOVA. Means + / - SEM are displayed.

[0066] FIG.43 shows graphs for the average normalized (to WT) EEG spectral power during NREM in the parietal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. No significant differences were observed among treatment groups as assessed by one-way ANOVA. Means + / - SEM are displayed.

[0067] FIG.44 shows graphs for the average hourly normalized EEG spectral power during NREM in the parietal cortex binned into the standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. No significant differences were observed among treatment groups as assessed by two-way ANOVA. Means + / - SEM are displayed.

[0068] FIG.45 shows graphs for the average normalized (to WT) EEG spectral power during REM in the parietal cortex in the 12h light and 12h dark phases binned into standard Greek power bands. The left set of 3 bars represents the light phase and the right set of 3 bars within the grey panel represents the dark phase. No significant differences were observed among treatment groups as assessed by one-way ANOVA. Means + / - SEM are displayed.

[0069] FIG.46 shows graphs for the average hourly normalized EEG spectral power during REM in the parietal cortex binned into the standard Greek power bands. The unshaded part represents the light phase and the part in the gray panel represents the dark phase. No significant differences wereDocket No.062686-510001WO observed among treatment groups as assessed by two-way ANOVA. Means + / - SEM are displayed. Time points without data were due to the lack of REM to perform spectral analyses on. DETAILED DESCRIPTION

[0070] The disclosure is based, in part, on the discovery, that adeno-associated virus (AAV) compositions encoding functional forkhead box G1 (FOXG1) can be delivered to a subject with FOXG1 deficiency by the intracerebroventricular (ICV) route. The AAV system employs a nucleic acid encoding FOXG1 for targeted gene expression in the subject’s cells in the brain. Various embodiments include administration of a recombinant adeno-associated virus (rAAV) encoding a FOXG1 protein, for example an rAAV comprising a nucleic acid payload encoding the FOXG1 protein.

[0071] Before the embodiments of the disclosure are described, it is to be understood that such embodiments are provided by way of example only, and that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the invention. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention.

[0072] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Various scientific dictionaries that include the terms included herein are well known and available to those in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice or testing of the disclosure, some preferred methods and materials are described. Accordingly, the terms defined immediately below are more fully described by reference to the specification as a whole. Definitions

[0073] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0074] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated cases, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0075] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.Docket No.062686-510001WO

[0076] In this application, the use of “or” means “and / or” unless stated otherwise. The terms “and / or” and “any combination thereof” and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C”. The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0077] Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting.

[0078] Reference in the specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0079] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the present disclosure can be used to achieve methods of the present disclosure.

[0080] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the amount “about 10” includes 10 and any amounts from 9 to 11. In yet another example, the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. Alternatively, particularly with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0081] The term “at least” followed by a number is used herein to denote the start of a range beginning with that number (which may be a range having an upper limit or no upper limit, depending on the variable being defined). For example, “at least 1” means 1 or more than 1.Docket No.062686-510001WO

[0082] The term “at most” followed by a number is used herein to denote the end of a range ending with that number (which may be a range having 1 or 0 as its lower limit, or a range having no lower limit, depending upon the variable being defined). For example, “at most 4” means 4 or less than 4, and “at most 40%” means 40% or less than 40%. When, in this specification, a range is given as “(a first number) to (a second number)” or “(a first number)-(a second number)” this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm, and whose upper limit is 100 mm.

[0083] As may be used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment” and “polynucleotide” are used interchangeably and are intended to include, but are not limited to, a polymeric form of nucleotides covalently linked together that may have various lengths, either deoxyribonucleotides or ribonucleotides, or analogs, derivatives or modifications thereof. Different polynucleotides may have different three-dimensional structures, and may perform various functions, known or unknown. Non- limiting examples of polynucleotides include a gene, a gene fragment, an exon, an intron, intergenic DNA (including, without limitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, a branched polynucleotide, a plasmid, a vector, isolated DNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, and a primer. Polynucleotides useful in the methods of the disclosure may comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or a combination of such sequences.

[0084] As used herein with respect to AAVs, the term “isolated” refers to an AAV that has been artificially produced or obtained. Isolated AAVs may be produced using recombinant methods. Such AAVs are referred to herein as “recombinant AAVs”.

[0085] The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence. Methods of alignment of sequences for comparison are well known in the art. Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches in the alignment by the length of the reference sequence, followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554 * 100=75.0). As the terms are used herein, gaps in the alignment do not decrease the percent sequence identity. Unless otherwise specified, optimal alignment of sequences for comparison is conducted by the global alignment algorithm of Needleman and Wunsch, Mol. Biol. 48:443 (1970) as implemented by EMBOSS Needle (on the World Wide Web at ebi.ac.uk / Tools / psa / emboss_needle / ) (Madeira et al. Nucleic Acids Res. 50(W1):W276-W279 (2022)). In embodiments, other alignment methods may be used, includingDocket No.062686-510001WO without limitation those described in Devereux, et al., Nucleic Acids Res. 12:387-95 (1984) ; Altschul et al., J. Mol. Biol. 215:403-10 (1990) (BLAST); Carrillo and Lipman Siam J. Appl. Math. 48(5) (1988); Computational Molecular Biology (Lesk, AM, ed., 1989); Biocomputing Informatics and Genome Projects, (Smith, DW, ed., 1993); Computer Analysis of Sequence Data, Part I, (Griffin and Griffin, eds., 1994); Sequence Analysis in Molecular Biology (von Heijne, 2012); Sequence Analysis Primer (Gribskov and Devereux, J., eds. 1993). Sequence identity is calculated using the implementation of the Needleman-Wunsch algorithm provided by the National Library of Medicine (on the World Wide Web at blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=GlobalAln)

[0086] For example, sequence identity can be determined by standard methods that are commonly used to compare the similarity of two polypeptide or two polynucleotide sequences. Using a computer program such as EMBOSS Needle or BLAST, two polypeptide or two polynucleotide sequences are aligned for optimal matching of their respective residues (either along the full length of one or both sequences, or along a pre-determined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 (a standard scoring matrix; see Dayhoff et al., in Atlas of Protein Sequence and Structure, vol. 5, supp. 3 (1978)) that can be used in conjunction with the computer program.

[0087] The term "binding," as used herein, refers to the process of attaching by a covalent bond or a non-covalent bond. Non-covalent bonds include those formed by van der Waals forces, hydrogen bonds, ionic bonds, entrapment or physical encapsulation, absorption, adsorption, and / or other intermolecular forces. Binding can be effectuated by any useful means, such as by enzymatic binding (e.g., enzymatic ligation) or by chemical binding (e.g., chemical ligation).

[0088] The term “promoter,” as used herein, generally refers to the regulatory DNA region which controls transcription or expression of a gene, and which may be located adjacent to or overlapping a nucleotide or region of nucleotides at which RNA transcription is initiated. A promoter may contain specific DNA sequences which bind protein factors, often referred to as transcription factors, which facilitate binding of RNA polymerase to the DNA leading to gene transcription. A ‘basal promoter’, also referred to as a ‘core promoter’, may generally refer to a promoter that contains all the basic elements to promote transcriptional expression of an operably linked polynucleotide. Eukaryotic basal promoters can contain a TATA-box or a CAAT box.

[0089] The term “expression,” as used herein, generally refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.Docket No.062686-510001WO

[0090] As used herein, “operably linked,” “operable linkage,” “operatively linked”, or grammatical equivalents thereof generally refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which may comprise promoter or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.

[0091] A “vector” as used herein, generally refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which may be used to mediate delivery of the polynucleotide to a cell. Examples of vectors include plasmids, viral vectors, liposomes, and other gene delivery vehicles. The vector generally comprises genetic elements, e.g., regulatory elements, operatively linked to a gene to facilitate expression of the gene in a target.

[0092] As used herein, “an expression cassette” and “a nucleic acid cassette” are used interchangeably generally to refer to a combination of nucleic acid sequences or elements that are expressed together or are operably linked for expression. In some cases, an expression cassette refers to the combination of regulatory elements and a gene or genes to which they are operably linked for expression.

[0093] As used herein, an “engineered” object generally indicates that the object has been modified by human intervention. According to non-limiting examples: a nucleic acid may be modified by changing its sequence to a sequence that does not occur in nature; a nucleic acid may be modified by ligating it to a nucleic acid that it does not associate with in nature such that the ligated product possesses a function not present in the original nucleic acid; an engineered nucleic acid may synthesized in vitro with a sequence that does not exist in nature; a protein may be modified by changing its amino acid sequence to a sequence that does not exist in nature; an engineered protein may acquire a new function or property. An “engineered” system comprises at least one engineered component.

[0094] Included in the current disclosure are variants of any of the FOXG1 polypeptides described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally, or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least aboutDocket No.062686-510001WO 98%, at least about 99% identity to any one of the FOXG1 sequences described herein. In some embodiments, such conservatively substituted variants are functional variants. Some embodiments include a FOXG1 variant with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to any one of the FOXG1 sequences described herein. Such functional variants can encompass sequences with substitutions such that the activity of the FOXG1 polypeptide is not disrupted.

[0095] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993)). The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)

[0096] The term “viral vector”, as used herein, refers to tools commonly used to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Examples of viral vectors include AAV vectors, lentiviral vectors, and adenoviral vectors.

[0097] As used herein “treatment” or “treating,” includes any beneficial or desirable effect, and may include even minimal improvement in symptoms. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0098] As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of a symptom of disease. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and / or burden of disease prior to onset or recurrence.

[0099] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a composition to achieve a beneficial or desired therapeutic result, including clinical results.

[0100] A “therapeutically effective amount” of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit aDocket No.062686-510001WO desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or cell are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient).

[0101] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. Methods

[0102] The disclosure provides, inter alia, methods for treating or preventing forkhead box G1 (FOXG1) deficiency in a subject in need thereof. Some embodiments include administering a recombinant adeno-associated virus (rAAV) encoding a FOXG1 protein to a subject. Some embodiments include administering an rAAV comprising a nucleic acid payload encoding the FOXG1 protein. The nucleic acid may include DNA (e.g. single stranded DNA).

[0103] FOXG1 protein is a member of the forkhead transcription factor family that is characterized by a distinct forkhead domain and is expressed specifically in neural progenitor cells of the forebrain. FOXG1 plays a role in the development of the brain and telencephalon. The telencephalon ultimately develops into several critical structures, including the largest part of the brain (i.e., cerebrum), which controls most voluntary activity, language, sensory perception, learning, and memory. In humans, forkhead box G1 is encoded by a FOXG1 gene, for example as set forth in NCBI Reference Sequence No. NM_005249.5: (SEQ ID NO: 2). AATTGTGGCTATAGCCGCCTCGATCGCTGTCTCCCCAGCCTCGCCGCGGCCGCTCCGGGA CGCGCCCGCCCGCCGCCCGGCTCTCCCCCCCTTTGGGCTGCTGCTGCTGCTGCTGTGACTG CTGCTGCGAGAGGAGGAGGAGGAGGAGGAAGCAGCGGGGGGGGGAGCGGGGGGTGGG GGGGGAGACCAAGAAGTACAGTTGGGAGCGAGGGAGCTTCACCCCCGGGGCGGTGGTT GTTTCTTTTTTCTTTCTTTCTTTTTTCTTTTCCTTTTTTTTTTTTTTTCTAATTCCTGAGGGGT GGTTGCTGCTTTTGCTACATGACTTGCCAGCGCCCGAGCCTGCGGTCCAACTGCGCTGCT GCCGGAGCGCTCAGTGCCGCCGCTGCCGCCCGCGCCCCCCGCGCCCCGTTCGGCACCCAC CGGTCGCCGCCGCCCGCCGCGCCGCTGTCCCGCTCCCGCGCCGCCGCCGCCGTTTCCCCC CGACGACTGGGTGATGCTGGACATGGGAGATAGGAAAGAGGTGAAAATGATCCCCAAGT CCTCGTTCAGCATCAACAGCCTGGTGCCCGAGGCGGTCCAGAACGACAACCACCACGCG AGCCACGGCCACCACAACAGCCACCACCCCCAGCACCACCACCACCACCACCACCATCA CCACCACCCGCCGCCGCCCGCCCCGCAACCGCCGCCGCCGCCGCAGCAGCAGCAGCCGC CGCCGCCGCCGCCCCCGGCACCGCAGCCCCCCCAGACGCGGGGCGCCCCGGCCGCCGAC GACGACAAGGGCCCCCAGCAGCTGCTGCTCCCGCCGCCGCCACCGCCACCACCGGCCGC CGCCCTGGACGGGGCTAAAGCGGACGGGCTGGGCGGCAAGGGCGAGCCGGGCGGCGGGDocket No.062686-510001WO CCGGGGGAGCTGGCGCCCGTCGGGCCGGACGAGAAGGAGAAGGGCGCCGGCGCCGGGG GGGAGGAGAAGAAGGGGGCGGGCGAGGGCGGCAAGGACGGGGAGGGGGGCAAGGAGG GCGAGAAGAAGAACGGCAAGTACGAGAAGCCGCCGTTCAGCTACAACGCGCTCATCATG ATGGCCATCCGGCAGAGCCCCGAGAAGCGGCTCACGCTCAACGGCATCTACGAGTTCAT CATGAAGAACTTCCCTTACTACCGCGAGAACAAGCAGGGCTGGCAGAACTCCATCCGCC ACAATCTGTCCCTCAACAAGTGCTTCGTGAAGGTGCCGCGCCACTACGACGACCCGGGC AAGGGCAACTACTGGATGCTGGACCCGTCGAGCGACGACGTGTTCATCGGCGGCACCAC GGGCAAGCTGCGGCGCCGCTCCACCACCTCGCGGGCCAAGCTGGCCTTCAAGCGCGGTG CGCGCCTCACCTCCACCGGCCTCACCTTCATGGACCGCGCCGGCTCCCTCTACTGGCCCA TGTCGCCCTTCCTGTCCCTGCACCACCCCCGCGCCAGCAGCACTTTGAGTTACAACGGCA CCACGTCGGCCTACCCCAGCCACCCCATGCCCTACAGCTCCGTGTTGACTCAGAACTCGC TGGGCAACAACCACTCCTTCTCCACCGCCAACGGCCTGAGCGTGGACCGGCTGGTCAAC GGGGAGATCCCGTACGCCACGCACCACCTCACGGCCGCCGCGCTAGCCGCCTCGGTGCC CTGCGGCCTGTCGGTGCCCTGCTCTGGGACCTACTCCCTCAACCCCTGCTCCGTCAACCTG CTCGCGGGCCAGACCAGTTACTTTTTCCCCCACGTCCCGCACCCGTCAATGACTTCGCAG AGCAGCACGTCCATGAGCGCCAGGGCCGCGTCCTCCTCCACGTCGCCGCAGGCCCCCTCG ACCCTGCCCTGTGAGTCTTTAAGACCCTCTTTGCCAAGTTTTACGACGGGACTGTCTGGG GGACTGTCTGATTATTTCACACATCAAAATCAGGGGTCTTCTTCCAACCCTTTAATACATT AACATCCCTGGGACCAGACTGTAAGTGAACGTTTTACACACATTTGCATTGTAAATGATA ATTAAAAAA ATAAGTCCAGGTATTTTTTATTAAGCCCCCCCCTCCCATTTCTGTACGTTTGTTCAGTCTCT AGGGTTGTTTATTATTCTAACAAGGTGTGGAGTGTCAGCGAGGTGCAATGTGGGGAGAA TACATTGTAGAATATAAGGTTTGGAAGTCAAATTATAGTAGAATGTGTATCTAAATAGTG ACTGCTTTGCCATTTCATTCAAACCTGACAAGTCTATCTCTAAGAGCCGCCAGATTTCCAT GTGTGCAGTATTATAAGTTATCATGGAACTATATGGTGGACGCAGACCTTGAGAACAACC TAAATTATGGGGAGAATTTTAAAATGTTAAACTGTAATTTGTATTTAAAAAGCATTCGTA GTAAAGGTGCCCAAGAAATTATTTTGGCCATTTATTGTTTTGTCCTTTTCTTTAAAGAACT GTTTTTTTTTCTTTTGTTTACTTTTAGACCAAAGATTGGGTTCTAGAAAATGCACTTGGTA TACTAAGTATTAAAACAAACAAAAAGGAAAGTTGTTTCAGTTGGCAACACTGCCCATTC AATTGAATCAGAAGGGGACAAAATTAACGATTGCCTTCAGTTTGTGTTGTGTATATTTTG ATGTATGTGGTCACTAACAGGTCACTT TTATTTTTTCTAAATGTAGTGAAATGTTAATACCTATTGTACTTATAGGTAAACCTTGCAA ATATGTAACCTGTGTTGCGCAAATGCCGCATAAATTTGAGTGATTGTTAATGTTGTCTTA AAATTTCTTGATTGTGATACTGTGGTCATATGCCCGTGTTTGTCACTTACAAAAATGTTTA CTATGAACACACAGAAATAAAAAATAGGCTAAATTCATATATATCTTGATACTTTTGTCT CTTTTATTAAGTAGAGCTAATTTTTTAAAGACCAATCAACTTATAGGGAATTCAAAGGCT TTTTCAGCCAAACTAAAATTTAAACTGCTCCTTTAATTTGAACTGACTCTAAAAATGAAA ATAGTATTTTTCCCTTTGTGAACAAATTTTACAAGGAGCAGCCTATTTAATAAACACTAG CTTTAAACAAAGTATAGGCTTTTCAGCTGATACCTGTAAGTTTCTGTGGATATACAGCAA AAAGAGATATAATTTAATTTTCTGTGCATAGCTCTTTACCCTGTGTTTATTTCCAAATCCA TTAATAGAATGCCATTTATATATTTTGTTTCAGGTATATTGTTAATAGAGCTTGGCAAATT ATAAATAAATATATGTATATGGTTAGATAGAAGTGACTATAATGCACACATATGTAATAT ATATAGACACACAGAGCCCTTCAGTTCAGGTACAATTTGCGCTATGAATGCTGCAAACAT TTTTGTTTAAATATTTGTATTTATACTTTCTAAGTCAGCATTTATTTTTGTGGCTGTTTACC CACAATGAAAGAGTTCTAATAAAGATGTGCTGAAGTTGCAATATA (SEQ ID NO: 2)

[0104] In some embodiments, a FOXG1 protein comprises the amino acid sequence set forth in NCBI Reference Sequence No. NP_005240.3: MLDMGDRKEVKMIPKSSFSINSLVPEAVQNDNHHASHGHHNSHHPQHHHHHHHHHHHPPPP APQPPPPPQQQQPPPPPPPAPQPPQTRGAPAADDDKGPQQLLLPPPPPPPPAAALDGAKADGL GGKGEPGGGPGELAPVGPDEKEKGAGAGGEEKKGAGEGGKDGEGGKEGEKKNGKYEKPPF SYNALIMMAIRQSPEKRLTLNGIYEFIMKNFPYYRENKQGWQNSIRHNLSLNKCFVKVPRHYDocket No.062686-510001WO DDPGKGNYWMLDPSSDDVFIGGTTGKLRRRSTTSRAKLAFKRGARLTSTGLTFMDRAGSLY WPMSPFLSLHHPRASSTLSYNGTTSAYPSHPMPYSSVLTQNSL GNNHSFSTANGLSVDRLVNGEIPYATHHLTAAALAASVPCGLSVPCSGTYSLNPCSVNLLAG QTSYFFPHVPHPSMTSQSSTSMSARAASSSTSPQAPSTLPCESLRPSLPSFTTGLSGGLSDYFTH QNQGSSSNPLIH (SEQ ID NO: 1).

[0105] Mutations in FOXG1 are associated with FOXG1 syndrome, a severe neurological disease characterized by microcephaly and brain malformations, and severe cognitive and developmental deficiencies. The FOXG1 gene is composed of one coding exon and notably, the location or type of FOXG1 mutation can be associated with or indicative of clinical severity. Deletions or mutations in a single allele of the forkhead box G1 (FOXG1) gene, can reduce the amount of functional FOXG1 (i.e., wild type, unmutated FOXG1) and cause FOXG1 syndrome in a subject. FOXG1 syndrome is a rare disease characterized by developmental delay, severe intellectual disability, epilepsy, absent language, and dyskinesis. Hallmarks of altered brain physiologies associated with FOXG1 syndrome include cortical atrophy and agenesis of the corpus callosum.

[0106] FOXG1 syndrome is a rare neurodevelopmental disorder associated with severe cognitive dysfunction, autistic behavior, and early-onset hyperkinetic movement disorders. Patients have also been reported to experience sleep disturbances (see, e.g., Autism Res. Wong et al., Autism Res. (2023) 16(5):953-966). In Wong et al., subjects with FOXG1 syndrome were reported to show short sleep durations, impaired sleep efficiency, longer wake after sleep onset, and frequent night-waking. Sleep disturbances, especially in initiating and maintaining sleep, are reported to be common in subjects with FOXG1 syndrome.

[0107] The stages of sleep can be classified following the guidelines of the American Academy of Sleep Medicine (AASM). Sleep is roughly classified into Non Rapid Eye Movement (NREM) and Rapid Eye Movement (REM) sleep. The NREM sleep refers to the stage in which there is no rapid eye movement. The REM sleep refers to the stage in which there is rapid eye movement and during which most dreams occur. The sleep phases can be measured in a subject for example with electroencephalogram (EEG) measurements. The AASM manual for the scoring of sleep and associated events: rules, terminology and technical specifications (2015) (www.aasm.org / resources / pdf / scoring-manual-preface.pdf) can be used to score sleep and any associated events in subjects, for example as collected in data from EEG measurements.

[0108] In one aspect, the disclosure provides for compositions of functional FOXG1 and methods of modulation of the status, activity, or expression of functional FOXG1 in a cell, tissue, or in a subject. Also provided are compositions and methods for treating pathological conditions and diseases in a subject caused by a deficiency of FOXG1 (i.e., functional FOXG1). Accordingly, disclosed herein are compositions and methods useful for increasing an amount of FOXG1 (e.g., functional FOXG1 protein or functional FOXG1 messenger ribonucleic acid (mRNA)) in a cell having a shortage of functional FOXG1 (e.g., having a mutation, deletion, or insertion in a FOXG1 allele).Docket No.062686-510001WO Such compositions and methods are useful in their application for preventing a FOXGl-related disease or disorder or treating a subject having a FOXGl-related disease or disorder wherein the lack or shortage of functional FOXG1 protein can be remedied.

[0109] In some embodiments, the disclosure provides for a method of delivering a forkhead box G1 (FOXG1) protein to the central nervous system (CNS) in a human subject in need thereof, the method comprising administering to the subject an effective amount of a recombinant adeno- associated virus (rAAV) encoding the FOXG1 protein. The rAAV is administered by injection into the cerebrospinal fluid (CSF) via an intracerebroventricular (ICV) route.

[0110] In some embodiments, the disclosure provides for a method of delivering a FOXG1 protein to telencephalon cells in a human subject in need thereof, the method comprising administering to the subject an effective amount of an rAAV encoding the FOXG1 protein, wherein the rAAV is administered by injection into the CSF via an ICV route.

[0111] In some embodiments, the disclosure provides for a method of treating FOXG1 deficiency in a human subject in need thereof, the method comprising administering an effective amount of a rAAV encoding a FOXG1 protein to the subject by ICV injection.

[0112] In some embodiments, the disclosure provides for a method of treating FOXG1 haploinsufficiency human subject in need thereof, the method comprising administering an rAAV encoding a FOXG1 protein to the subject by ICV injection.

[0113] In some embodiments, a method of preventing, improving, or treating FOXG1 syndrome in a human subject is provided, the method comprising administering an effective amount of an rAAV encoding the FOXG1 protein.

[0114] In some embodiments, a method of reducing anxiety in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection.

[0115] In some embodiments, a method of improving behavior deficits related to anxiety in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection. In some embodiments, the anxiety in the subject is related to a novel or unfamiliar environment and adapting to this novel environment.

[0116] In some embodiments, a method of improving sleep patterns in a human subject suffering from FOXG1 syndrome is provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein to the subject by ICV injection. In some embodiments, the improvement in sleep pattern is increased time spent awake. In some embodiments, the improvement in sleep pattern is less fragmented NREM sleep.

[0117] In some embodiments, a method of improving behavioral dysfunction such as increased anxiety and reduced social interaction in a human subject suffering from FOXG1 syndrome isDocket No.062686-510001WO provided, the method comprising, administering an effective amount of an rAAV encoding the FOXG1 protein.

[0118] In some embodiments, the method comprises administering an effective amount of a rAAV encoding a FOXG1 protein to the subject by ICV injection.

[0119] In some embodiments, the ICV injection is a bilateral ICV injection. In some embodiments, the ICV injection is performed once.

[0120] In some embodiments, the subject has one or more mutations in a FOXG1 gene. Methods of determining mutations in the FOXG1 gene are known in the art, for example PCR (polymerase chain reaction) and RT-PCR (reverse transcription – polymerase chain reaction), or Western Blot. In some embodiments, the subject has FOXG1 syndrome.

[0121] In some embodiments, the subject is between 0 months and 12 months old. In some embodiments, the subject is between 12 months and 24 months old. In some embodiments, the subject is between 12 months and 36 months old. In some embodiments, the subject is between 12 months and 48 months old. In some embodiments, the subject is between 12 months and 60 months old. In some embodiments, the subject is between 12 months and 72 months old. In some embodiments, the subject is between 12 months and 84 months old. In some embodiments, the subject is between 12 months and 96 months old. In some embodiments, the subject is between 2 years and 3 years old. In some embodiments, the subject is between 2 years and 4 years old. In some embodiments, the subject is between 2 years and 5 years old. In some embodiments, the subject is between 2 years and 6 years old. In some embodiments, the subject is between 2 years and 7 years old. In some embodiments, the subject is between 2 years and 8 years old. In some embodiments, the subject is between 2 years and 9 years old. In some embodiments, the subject is between 2 years and 10 years old. In some embodiments, the subject is between 5 years and 10 years old. In some embodiments, the subject is between 6 years and 10 years old. In some embodiments, the subject is between 7 years and 10 years old. In some embodiments, the subject is between 8 years and 10 years old. In some embodiments, the subject is between 9 years and 10 years old. In some embodiments, the subject is between 10 years and 18 years old. In some embodiments, the subject is an infant. In some embodiments, the subject is a child. In some embodiments, the child is a human subject between 1 and 17 years old. In some embodiments, the subject is an adult.

[0122] Treating FOXG1 deficiency can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population of untreated subjects. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with the compositions of the disclosure.

[0123] Alleviating a disease associated with aberrant FOXG1 activity includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, "delaying" the development of a diseaseDocket No.062686-510001WO (such as a disease associated with aberrant FOXG1 activity) means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that "delays" or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0124] In some embodiments, administering the compositions described herein to a cell or subject increases FOXG1 expression in the cell or subject by between 2-fold and 100-fold (e.g., 2-fold, 5- fold, 10-fold, 20-fold, 50-fold, 75-fold, 100-fold, etc.) compared to a control subject.

[0125] In some embodiments, the administration restores at least 5%, 10%, 20%, 30%, or 40% FOXG1 protein in cells compared to, or relative to, untreated cells. In some embodiments, the administration restores at least 5%, 10%, 20%, 30%, or 40% FOXG1 protein in cells compared to, or relative to, a baseline measurement. Compositions

[0126] The disclosure provides compositions for treating or preventing forkhead box G1 (FOXG1) deficiency in a subject in need thereof.

[0127] In some embodiments, the FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a FOXG1 protein comprises an amino acid sequence that is at least 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, a FOXG1 protein comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1.

[0128] In some embodiments, the FOXG1 protein is encoded by a nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, a FOXG1 protein is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2.In some embodiments, the nucleic acid sequence is a codon-optimized nucleic acid sequence. In certain aspects, such codon-optimized nucleic acid variants provide for increased transcription and / or translation of the encoded transgene. Such codon-optimized nucleic acid variants can exhibit increased expression, e.g., 0.5-10 fold for certain codon optimized nucleic acid variants compared non-codon optimized nucleic acid encoding a transgene.

[0129] In some embodiments, a FOXG1 protein is encoded by a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any of SEQ ID NOs: 3 or 8-13. InDocket No.062686-510001WO some embodiments, the codon-optimized nucleic acid sequence comprises nucleic acid sequence set forth in SEQ ID NO: 3. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises (or consists of) the amino acid sequence set forth in SEQ ID NO: 13.

[0130] In some embodiments, the disclosure relates to isolated nucleic acids comprising an expression cassette having a transgene that encodes a FOXG1 protein. In some embodiments, an isolated nucleic acid encoding a FOXG1 protein comprises the nucleic acid sequence set forth in NCBI Reference Sequence No. NM_005249.5 (SEQ ID NO: 2). In some embodiments, an isolated nucleic acid encoding a FOXG1 protein comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence set forth in NCBI Reference Sequence No. NM_005249.5 (SEQ ID NO: 2). In some embodiments, an isolated nucleic acid encoding a FOXG1 protein comprises a nucleic acid sequence that is at least 70%, 75%, 80%, 90%, 95%, or 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 2. In some embodiments, an isolated nucleic acid encoding a FOXG1 protein comprises at least one (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, 100, 150, 200, 250, or more) nucleotide substitutions, insertions, deletions, or any combination thereof, relative to the nucleic acid sequence set forth in NCBI Reference Sequence No. NM_005249.5 (SEQ ID NO: 2). In some embodiments, an isolated nucleic acid encoding a FOXG1 protein comprises a codon-optimized nucleic acid sequence. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 3. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 9. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 11. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments an isolated nucleic acid encoding a FOXG1 protein comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0131] In some embodiments, the FOXG1 encoding sequence is operably connected to one or more regulatory sequences. In some embodiments, the nucleic acid further comprises one or moreDocket No.062686-510001WO promoters. In some embodiments, the nucleic acid further comprises one promoter driving the expression of the FOXG1 transgene.

[0132] In some embodiments, the promoter is a constitutive promoter, inducible promoter, or tissue-specific promoter.

[0133] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) FTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the b-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 a promoter. In some embodiments, a promoter is an RNA pol II promoter. In some embodiments, a promoter is an RNA pol III promoter, such as U6 or HI. In some embodiments, a promoter is an RNA pol II promoter. In some embodiments, a promoter is a chicken b-actin (CBA) promoter. In some embodiments, a promoter comprises a U1 a promoter.

[0134] Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et ah, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline -repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), the tetracycline-inducible system (Gossen et al., Science, 268:1766-1769 (1995), see also Harvey et al., Curr. Opin. Chem. Biol., 2:512-518 (1998)), the RU486-inducible system (Wang et al., Nat. Biotech., 15:239-243 (1997) and Wang et al., Gene Ther., 4:432-441 (1997)) and the rapamycin-inducible system (Magari et al., J. Clin. Invest., 100:2865-2872 (1997)). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0135] In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue- specific regulatory sequences bind tissue- specific transcription factors that induce transcription in a tissue specific manner. Such tissue- specific regulatory sequences (e.g., promoters, enhancers, etc..) are well known in the art. Exemplary tissue-specific regulatory sequences include, but are not limited to the following tissue specific promoters: retinoschisin proximal promoter, interphotoreceptor retinoid-binding protein enhancer (RS / IRBPa), rhodopsin kinase (RK), liver- specific thyroxin binding globulin (TBG) promoter, an insulin promoter, a glucagon promoter, a somatostatin promoter, a pancreatic polypeptide (PPY) promoter, a synapsin-1Docket No.062686-510001WO (Syn) promoter, a creatine kinase (MCK) promoter, a mammalian desmin (DES) promoter, a a- myosin heavy chain (a-MHC) promoter, or a cardiac Troponin T (cTnT) promoter. Other exemplary promoters include Beta-actin promoter, hepatitis B virus core promoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin promoter (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein promoter (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)), CD2 promoter (Hansal et al., J. Immunol., 161:1063-8 (1998); immunoglobulin heavy chain promoter; T cell receptor a-chain promoter, neuronal such as neuron- specific enolase (NSE) promoter (Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene promoter (Piccioli et al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and the neuron- specific vgf gene promoter (Piccioli et al., Neuron, 15:373-84 (1995)), among others which will be apparent to the skilled artisan. In some embodiments, the promoter preferentially drives transgene expression in certain tissues. In some embodiments, the disclosure provides a nucleic acid comprising a tissue-specific promoter operably linked to a transgene. As used herein, “tissue-specific promoter” refers to a promoter that preferentially regulates (e.g., drives or up-regulates) gene expression in a particular cell type relative to other cell types. A cell-type-specific promoter can be specific for any cell type, such as central nervous system (CNS) cells (e.g., neurons, astrocytes or oligodendrocytes), liver cells (e.g., hepatocytes), heart cells, muscle cells, etc. In some embodiments, a tissue-specific promoter is a muscle tissue or cell-specific promoter. Examples of CNS-specific promoters include but are not limited to synapsin (Syn), GFAP, Ca2+ / calmodulin-dependent protein kinase II (hCAMKII), etc.

[0136] In some embodiments, the promoter comprises a chicken beta-actin (CB) promoter, a Ula promoter, or a neuron-specific promoter.

[0137] In some embodiments, the promoter comprises a human synapsin 1 (hSynl) promoter or a human Ca 2+ / calmodulin-dependent protein kinase II (hCAMKII) promoter. In some embodiments, the promoter comprises a CB6-PI promoter nucleic acid sequence (SEQ ID NO: 4). In some embodiments, the promoter comprises a U1 promoter nucleic acid sequence (SEQ ID NO: 5). In some embodiments, the promoter comprises a hSynl promoter nucleic acid sequence (SEQ ID NO: 6). In some embodiments, the promoter comprises a hCAMKII promoter nucleic acid sequence (SEQ ID NO: 7). In some embodiments, the promoter comprises the sequence set forth in any one of SEQ ID NOs: 4 to 7. In some embodiments, a promoter comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 4-7.

[0138] In some embodiments, the polynucleotide further comprises non-coding sequences at 3' to the coding sequence. Non-limiting examples of non-coding sequences at 3' to the coding sequence include a poly(A) signal and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). An exemplary poly(A) signal is the SV40 late polyadenylation signal.Docket No.062686-510001WO Recombinant AAV

[0139] In some aspects, the disclosure provides isolated recombinant adeno-associated viruses (AAVs) comprising nucleic acid sequences encoding FOXG1 protein.

[0140] The isolated nucleic acids of the disclosure may be recombinant adeno-associated virus (AAV) vectors (rAAV vectors). The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into an AAV capsid and administered to a subject and / or delivered to a selected target cell. Recombinant AAV (rAAV) vectors are typically composed of, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). The transgene may comprise a region encoding, for example, a protein and / or an expression control sequence (e.g., a poly-A tail), as described elsewhere in the disclosure.

[0141] In some embodiments, rAAVs of the disclosure comprise a nucleotide sequence that is 99% identical, 95% identical, 90% identical, 85% identical, 80% identical, 75% identical, 70% identical, 65% identical, 60% identical, 55% identical, or 50% identical to a nucleotide sequence as set forth in SEQ ID NO: 2, 3, 8, 9, 10, 11, 12, or 13. In some embodiments, an rAAV comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 2-3 or 8-13.

[0142] In some embodiments, the rAAV payload is self-complementary (sc). In some embodiments, the rAAV payload is single-stranded (ss).

[0143] Recombinant AAVs (rAAVs) of the disclosure have tissue- or cell type specific targeting capabilities, such that a transgene encoded in the rAAV can be delivered specifically to one or morepredetermined tissue(s) (e.g., brain cortex etc.) or cell types (e.g., neurons etc.).· The AAV capsid isan important element in determining these tissue- or cell type specific targeting capabilities (e.g., tissue or cell tropism). Thus, an rAAV having a capsid appropriate for the tissue or cell type being targeted can be selected. In some embodiments, the at least one capsid protein is an AAV9 capsid protein or an AAV.PHP-eB capsid protein. In some embodiments, the at least one AAV capsid protein has a tropism for central nervous system (CNS) cells. In some embodiments, the at least one AAV capsid protein has a tropism for telencephalon cells.

[0144] Methods for obtaining recombinant AAVs having a desired capsid protein are well known in the art. (see, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, capsid proteins are structural proteins encoded by the cap gene of an AAV. AAVs comprise three capsid proteins, virion proteins 1 to 3 (named VP1, VP2 and VP3), all of which areDocket No.062686-510001WO transcribed from a single cap gene via alternative splicing. In some embodiments, the molecular weights of VP1, VP2 and VP3 are respectively about 87 kDa, about 72 kDa and about 62 kDa. In some embodiments, upon translation, capsid proteins form a spherical 60-mer protein shell around the viral genome. In some embodiments, the functions of the capsid proteins are to protect the viral genome, deliver the genome and interact with the host. In some aspects, capsid proteins deliver the viral genome to a host in a tissue specific manner.

[0145] In some embodiments, an AAV capsid protein has a tropism for central nervous system (CNS) tissues. In some embodiments, an AAV capsid protein targets telencephalon cells, neuronal cell types, astrocytes, oligodendrocytes, glial cells, etc. In some embodiments, an AAV capsid protein targets cochlear cells. In some embodiments, an AAV capsid protein is of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV.PHP-eB, AAVrh39, AAVrh43, and variants of any of the foregoing.

[0146] In some embodiments, rAAV vectors include ITRs and / or capsids based upon or having sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1, and / or AAV-2i8 ITRs and / or capsids. In certain embodiments, rAAV vectors include variants having less than 100% sequence identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1 and / or AAV-2i8 ITRs and / or capsids. Variants include amino acid insertions, additions, substitutions, and deletions. In particular aspects, variants are set forth in WO 2013 / 158879 (International Application PCT / US2013 / 037170), WO 2015 / 013313 (International Application PCT / US2014 / 047670) and US 2013 / 0059732 (U.S. application Ser. No.13 / 594,773, discloses LK01, LK02, LK03, etc.).

[0147] In certain embodiments, rAAV vector comprises one or more ITRs and / or capsids proteins (e.g., VP1, VP2, and / or VP3) having at least 70-80%, 80-90% or 90-99% identity to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1 and / or AAV-2i8. In certain embodiments, a rAAV vector comprises ITR(s) and / or capsid(s) (e.g., VP1, VP2 and / or VP3) having 75% or more sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1 and / or AAV-2i8 ITRs and / or capsids.

[0148] In some embodiments, a rAAV vector comprises an AAV serotype or an AAV pseudotype comprising an AAV capsid serotype different from an ITR serotype. Pseudotype rAAV in which an AAV capsid serotype is different from an ITR serotype can be composed of ITR(s) and / or capsid(s) (VP1, VP2 and / or VP3) having 75% or more sequence identity (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, etc.) to any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01,Docket No.062686-510001WO LK02, LK03, AAV 4-1 and / or AAV-2i8 ITRs and / or capsids, provided the serotypes of the ITRs and capsids are different.

[0149] In some embodiments, the rAAV vector comprises one or more ITRs and / or capsids (e.g., VP1, VP2, and / or VP3) with 100% identity to AAV2 capsid. In some embodiments, the AAV ITRs are AAV2 ITRs. In some embodiments, at least one of the AAV2 ITRs comprises the sequence set forth in SEQ ID NO: 14. In some embodiments, at least one AAV ITR is a truncated AAV ITR, for example a AITR as described, for example by McCarty (2008) Molecular Therapy 16(10): 1648- 1656.

[0150] rAAV vectors can include additional components or elements that are operably linked to the transgene and act in cis or in trans. In particular embodiments, a vector such as rAAV vector further includes an intron, an expression control element, one or more ITRs (e.g., any of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1 and / or AAV-2i8 serotypes, or a combination thereof), a filler polynucleotide sequence and / or poly A signal. In particular aspects, an intron is within or flanks a nucleic acid encoding a transgene, and / or an expression control element is operably linked to the nucleic acid encoding a transgene, and / or an AAV ITR(s) flanks the 5′ or 3′ terminus of the nucleic acid encoding a transgene, and / or a filler polynucleotide sequence flanks the 5′ or 3′terminus of the nucleic acid encoding a transgene.

[0151] Modified AAV capsid proteins provide an improved way to treat diseases with greater transduction efficiency and enhanced tropism for specific tissues and cell types of interest. The modified AAV capsid proteins can increase delivery of a construct encoding a therapeutic gene (e.g., FOXG1) with improved CNS tropism and / or reduced liver tropism. The modified AAV capsids proteins can increase delivery of a construct encoding a therapeutic gene (e.g., FOXG1) to CNS neurons, glial cells or both CNS neurons and glial cells (on-target delivery), and / or reduce delivery to liver cells (off-target delivery). Additionally, the construct could drive higher and more specific FOXG1 expression at the target by virtue of appropriate expression regulatory elements (ERE) (e.g., promoter sequences) and / or codon optimized coding sequences. Accordingly, one aspect of the present disclosure provides a modified AAV capsid protein, comprising: (i) a reference AAV capsid protein, and (ii) a 7-mer peptide having the sequence RGDLLLS (SEQ ID NO: 15) inserted into a site within VR VIII of the reference AAV capsid protein. In some embodiments, the AAV capsid protein is selected from one or more of VP1, VP2 and VP3. In some embodiments, the modified AAV capsid protein comprises a targeting peptide (i) PLQGAVHLY (SEQ ID NO: 16); (ii) PLQGAVRLY (SEQ ID NO: 17); (iii) PLQGAVKLY (SEQ ID NO: 18); (iv) PINGAVHLY (SEQ ID NO: 19); (v) PVNGAVHLY (SEQ ID NO: 20); (vi) PANGAVHLY (SEQ ID NO: 21); or (vii) PLNGAVHLY (SEQ ID NO: 22).

[0152] In some embodiments, the reference AAV capsid protein is a capsid protein of an AAV variant selected from the group consisting of: AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7;Docket No.062686-510001WO AAV8; AAV hu.37; AAV rh.10; AAV9; AAV hu.68; AAV10; AAV5, AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6-A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45- B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51-B; rh.19-B; rh.49-B; rh.52-B; rh.13-B; AAV2-B; rh.20- B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22-B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu.1-C; hu.18-C; hu.3-C; hu.25-C; hu.15-C; hu.16-C; hu.11-C; hu.10-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; rh74; hu.42-E; rh.57-E; rh.40-E; hu.67- E; hu.17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; Anc113; Anc126; Anc127; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80L1; Anc110; and Anc80DI.

[0153] In some embodiments, the capsid is formed with VP1, VP2 and VP3 capsid proteins of Anc80L65 (described in WO2015 / 054653, which is incorporated by reference in its entirety herein). In some embodiments, the targeting peptide is inserted (i) between S586 and T589 of an Anc80L65 capsid protein, thereby replacing A587 and N588 of the Anc80L65 capsid protein; (ii) between Q585 and N588 of an Anc80L65 capsid protein, thereby replacing S586 and A587 of the Anc80L65 capsid protein; (iii) between L584 and A587 of an Anc80L65 capsid protein, thereby replacing Q585 and S586 of the Anc80L65 capsid protein; (iv) between A587 and A590 of an Anc80L65 capsid protein, thereby replacing N588 and T589 of the Anc80L65 capsid protein; or (v) between S586 and A587 of an Anc80L65 capsid protein.

[0154] In some embodiments, adjunctively administered neutralizing antibodies can restrict biodistribution of rAAVs to target tissues and organs by neutralizing AAVs in non-target organs and tissues. This adjunctive, systemic administration of neutralizing antibodies reduces non-target transduction of rAAVs administered non-systemically that escape the target organs and tissues, systemic toxicity, and systemic immune responses that travel to the target tissue and organs transduced by rAAVs. In some embodiments, the systemic administration of neutralizing antibodies reduces non-target transduction of rAAVs administered by ICV injection or infusion that escape the target organs and tissues, systemic toxicity, and systemic immune responses that travel to the target tissue and organs transduced by rAAVs. In some embodiments, the systemic administration of neutralizing antibodies reduces non-target transduction of rAAVs administered by ICV injection or infusion that escape the target CNS, systemic toxicity, and systemic immune responses that travel to the CNS transduced by rAAVs. In some embodiments, the at least one antibody is an IgG antibody. In some embodiments, the composition is intravenous immunoglobulin (IVIG). In some embodiments, the IVIG is a pool of antibodies obtained from: Gammagard™ Liquid® (Baxter Healthcare Corp), Gammagard™ S / D, Gammaplex™, Bivigam™, Carimune™ NF, Gamunex-C, Gammaked™, Flebogamma™ DIF, Octagam™, and Privigen™.

[0155] In some embodiments, the composition is capable of neutralizing an rAAV selected from AAV2; AAV1; AAV6; AAV3; AAV LK03; AAV7; AAV8; AAV hu.37; AAV rh.10; AAV9; AAVDocket No.062686-510001WO hu.68; AAV10; AAV5; AAV3-3; AAV4-4; AAV1-A; hu.46-A; hu.48-A; hu.44-A; hu.43-A; AAV6- A; hu.34-B; hu.47-B; hu.29-B; rh.63-B; hu.56-B; hu.45-B; rh.57-B; rh.35-B; rh.58-B; rh.28-B; rh.51- B; rh,19-B; rh.49-B; rh.52-B; rh,13-B; AAV2-B; rh.20-B; rh.24-B; rh.64-B; hu.27-B; hu.21-B; hu.22- B; hu.23-B; hu.7-C; hu.61-C; rh.56-C; hu.9-C; hu.54-C; hu.53-C; hu.60-C; hu.55-C; hu.2-C; hu. l-C; hu, 18-C; hu.3-C; hu.25-C; hu, 15-C; hu, 16-C; hu.l l-C; hu.lO-C; hu.4-C; rh.54-D; rh.48-D; rh.55-D; rh.62-D; AAV7-D; rh.52-E; rh.51-E; hu.39-E; rh.53-E; hu.37-E; rh.43-E; rh.50-E; rh.49-E; rh.61-E; hu.41-E; rh.64-E; hu.42-E; rh.57-E; rh.40-E; hu.67-E; hu,17-E; hu.6-E; hu.66-E; rh.38-E; hu.32-F; AAV9 / hu; hu.31-F; Anc80; Anc81; Anc82; Anc83; Anc84; Anc94; And 13; An 26; And 27; Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; Anc80L44; Anc80Ll; And 10; and Anc80DI.

[0156] Additional exemplary modified rAAV vectors, capsids, and targeting peptides are described in International Parent Application Publication No. WO / 2024 / 086747, International Parent Application Publication No. WO / 2024 / 040193, International Parent Application Publication No. WO / 2023 / 113805, International Parent Application Publication No. WO / 2023 / 113806, International Parent Application Publication No. WO / 2022 / 221193, International Parent Application Publication No. WO / 2022 / 099179, and International Parent Application Publication No. WO / 2022 / 173847.

[0157] In some embodiments, an expression control element comprises a constitutive or regulatable control element, or a tissue-specific expression control element or promoter. In particular aspects, an expression control element comprises an enhancer. In certain aspects, an expression control element (e.g., promoter or enhancer) confers expression in a neuronal cell (e.g., a human synapsin 1 promoter).

[0158] In addition to the major elements identified above for the recombinant AAV vector, the vector also includes conventional control elements which are operably linked with elements of the transgene in a manner that permits its transcription, translation and / or expression in a cell transfected with the vector or infected with the virus produced by the disclosure. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A number of expression control sequences, including promoters which are native, constitutive, inducible and / or tissue-specific, are known in the art and may be utilized.

[0159] In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No.6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with an AAV vector (comprising a transgene flanked by ITRDocket No.062686-510001WO elements) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (e.g., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes). Non- limiting examples of vectors suitable for use with the disclosure include pHLP19, described in U.S. Pat. No.6,001,650 and pRep6cap6 vector, described in U.S. Pat. No.6,156,303, the entirety of both incorporated by reference herein. The accessory function vector encodes nucleotide sequences for non- AAV derived viral and / or cellular functions upon which AAV is dependent for replication (e.g., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpes virus (other than herpes simplex virus type-1), and vaccinia virus.

[0160] The isolated nucleic acids, rAAVs, and compositions of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject via the ICV route. Pharmaceutical Compositions

[0161] The disclosure provides pharmaceutical compositions comprising immune cells comprising the engineered receptors of the disclosure and a pharmaceutically acceptable diluent, carrier or excipient.

[0162] In some embodiments, the pharmaceutical composition is formulated for local administration to the CNS or for systemic administration. In some embodiments, the pharmaceutical composition comprises a CSF, e.g., ultrafiltrate of plasma or synthetic cerebrospinal fluid. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0163] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteriaDocket No.062686-510001WO and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0164] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 mL of isotonic NaCl solution and either added to 1000 mL of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0165] Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0166] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0167] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the compositions of the disclosureDocket No.062686-510001WO into suitable host cells. In particular, the rAAV vector delivered transgenes may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like. Cellular Delivery

[0168] The isolated nucleic acids, rAAVs, and compositions of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a pharmaceutical composition), may be administered to a human subject.

[0169] The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects. Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the selected organ (e.g., intraportal delivery to the liver), intraocular injection, subretinal injection, oral, inhalation (including intranasal and intratracheal delivery), intravenous, intramuscular, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired. In some embodiments, the delivery is an injection into the cerebrospinal fluid (CSF) via an intracerebroventricular (ICV) route. In some embodiments, the ICV injection is a bilateral ICV injection.

[0170] The dose of rAAV virions required to achieve a particular "therapeutic effect," e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can determine a rAAV virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. In some embodiments, an effective amount of an rAAV is administered to the subject during a pre- symptomatic stage of degenerative disease. In some embodiments, a subject is administered an rAAV or composition after exhibiting one or more signs or symptoms of degenerative disease. Kits and Articles of Manufacture

[0171] The disclosure provides kits and articles of manufacture comprising the polynucleotides, vectors, and viral vectors comprising a sequence encoding the FOXG1 protein described herein. In some embodiments, the kit comprises articles such as vials, syringes and instructions for use.

[0172] In some embodiments, the kit comprises a polynucleotide, vector, or viral vector comprising a sequence encoding one or more FOXG1 protein of the disclosure.Docket No.062686-510001WO EXAMPLES

[0173] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein. EXAMPLE 1. Efficacy of Intracerebroventricular (ICV) Injection of FOXG1 AAV in FOXG1 HET mice in vivo

[0174] This example describes the determination of the efficacy of intracerebroventricular (ICV) injection of FOXG1 AAV (ALR-047) in FOXG1 HET (Het) mice in vivo using behavioral and molecular endpoints. Animals

[0175] Briefly, FOXG1 Q84 HET (Het) mice, on a C57BL / 6 background, were generated using CRISPR editing to insert a G nucleotide into the noncoding (antisense) strand of the mouse FOXG1 locus, yielding a complementary inserted C nucleotide immediately 3’ of position 250 in the FOXG1 coding sequence sense sequence. This insertion mutated the 84th codon, changing the encoded amino acid from Q (glutamine) to P (proline), shifted the frame of the coding sequence, and introduced a stop codon after an additional 44 codons. Beyond the 84th codon, the novel frameshifted sequence bears very little resemblance to the wildtype FOXG1 protein; the mutated protein totals 113 amino acids in length, compared to the wildtype 481 amino acids.

[0176] For the duration of the study, animals’ body weights were recorded twice per week and animals were checked for survival twice per day.

[0177] All animals were examined, manipulated, and weighed prior to initiation of the study to assure adequate health and suitability and to minimize non-specific stress associated with manipulation. FOXG1 AAV

[0178] The gene encoding FOXG1 (SEQ ID NO: 1) was cloned into an AAV9 vector.

[0179] FOXG1 AAV (scAAV9.hSyn1-opthFOXG1; ALR-047) (test article) was produced and aliquoted at a concentration of 2.69E13 genome copies (GC) / mL in formulation buffer (vehicle: PBS with 5% sorbitol and 0.001% F-68). Test articles and formulation buffer were stored at -80°C. On the first day of dose administration, test articles were thawed on wet ice. Once thawed, test articles were stored at 4°C and did not undergo any additional freeze‐thaw cycles. Study design

[0180] To conduct the in vivo study, one (1) group of n=15 male FOXG1 WT animals and four (4) groups of N=15 male FOXG1 Q84 HET (Het) animals were enrolled at P6 (postnatal day 6). Details for the study are listed in Table 1 and Table 2.Docket No.062686-510001WO Table 1. Study group composition.Genotyping

[0181] Genomic DNA was isolated from neonate tail clippings. From the genomic DNA prep, 2.5µL was used for PCR. PCR reactions were set up using QuantaBio’s sparQ HiFi PCR Master Mix (cat# 95192‐050) in 25µL reaction volume.

[0182] Briefly, 12.5µL 2X Master Mix was mixed with 1µL of 10µM Foxg1_Q86_253F (5’ CCA AGT CCT CGT TCA GCA TC3’) and 1µL of 10µM Foxg1_Q86_856R (5’TGC TTG TTC TCG CGG TAG TA3’), 8µL of water and 2.5µL of gDNA. The following PCR parameters were used: 94°C 3 min; [94 °C 30s, 60 °C 35s 72 °C 35s] x35 cycles; 72°C 5 min; 12 °C hold. After PCR cycles were completed, 15µL of PCR reaction was used for electrophoresis to ensure a dominant approximately 600bp DNA amplicon was present in each loaded lane.

[0183] Remaining PCR reactions were cleaned up and 10µL of PCR reaction was mixed with 4µL of Exosap‐IT reagent and incubated at 37°C for 15 minutes to degrade the remaining primers and nucleotides, followed by an inactivation step at 80°C for 15 minutes.

[0184] After PCR cleanup, 3µL of reaction was mixed with 7µL of water, and 5µL of 5µM sequencing primer (Foxg1_Q86_856R (5’TGC TTG TTC TCG CGG TAG TA3’)) for total of 15µL. Samples were DNA sequenced.

[0185] Upon completion of sequencing, DNA sequence files were reviewed individually to assign genotype. Table 2. Study timeline.Docket No.062686-510001WOICV Injections

[0186] Prior to injections, animals were anesthetized via cryoanesthesia. Animals were dosed via bilateral intracerebroventricular injection (ICV) at P6 with a dose volume of 5.0 µl per hemisphere (10.0 µL per animal). Treatments were administered using 10 µl Hamilton Syringes and custom Hamilton 301 / 2 g needles (point style 4, bevel 12°). A fresh syringe was used for each individual treatment. Animals were then placed on a warm (~36°C) heating pad immediately following the ICV injection. Once all animals from the litter were dosed and warmed, they were returned to the dam. Animals were closely monitored after the ICV injection for any observable adverse drug effects. Open Field

[0187] The open field test (OF) was used to assess both anxiety‐like behavior and motor activity at 6, 8, and 11 weeks of age. The open field chambers are plexiglass square chambers (27.3 x 27.3 x 20.3 cm) surrounded by infrared photobeam sources (16 x 16 x 16). The enclosure was configured to split the open field into a center and periphery zone and the photocell beams were set to measure activity in the center and in the periphery of the OF chambers. Animals having higher levels of anxiety or lower levels of activity tend to stay in the corners of the OF enclosures. On the other hand, mice that have high levels of activity and low levels of anxiety tend to spend more time in the center of the enclosure. Horizontal activity (distance traveled) and vertical activity (rearing) were measured from consecutive beam breaks. Animals were placed in the OF chambers for 30 minutes. Total ambulatory distance, ambulatory distance in center, total rearing, and rears in the center were measured. After testing, animals were placed back into their home cage.Docket No.062686-510001WO Grip Strength

[0188] Grip strength was used to assess the muscular strength in the limb muscles of the animals at 4, 6, 8, 10, and 12 weeks of age. Mice were held by the tail and lowered toward the mesh grip piece on the push-pull gauge (San Diego Instruments, San Diego, CA) until the animal grabbed with all four (4) paws. The animal was lowered toward the platform and gently pulled backwards with consistent force by the experimenter until the animal released its grip. The combined grip force of all four (4) limbs was recorded on the strain gauge. Animals were assessed across five (5) consecutive trials. After testing, animals were placed back into their home cage. Fear Conditioning

[0189] Testing was conducted when the animals were 12 weeks of age in the fear conditioning system manufactured by Coulbourn Instruments (PA, USA).

[0190] On day one 1, mice were placed into the conditioning chambers to habituate to the context for 120 sec where they were exposed to a three 20 second 80 dB tone (conditioned stimulus, CS) spaced 100 seconds apart. Fifteen seconds after each tone; mice received a foot shock (0.5mA for 1 sec), the unconditioned stimulus (US). The mouse remained in the conditioning chamber for another 60 seconds and then was returned to its home cage. Twenty-four hours after training the mice were tested for contextual memory where they were placed into the same chamber they were trained in for a period of 5 minutes without shock or any other interference.

[0191] Twenty-four hours after contextual fear conditioning, animals were tested for cued memory. Mice were placed in a novel context for 2 min (Pre-Cue). Then the CS (80dB tone) was presented for 3 minutes. Freezing behavior, defined as the complete lack of movement, was captured automatically with a video system and FreezeView software (Coulbourn Instruments, PA, USA). After testing, animals were placed back into their home cage. Biological Sample Collection

[0192] Upon completion of the study, when the animals were 13 weeks of age, all animals were euthanized for tissue collection. Animals were anesthetized with isoflurane and placed in stereotaxic frame. Following a skin incision inferior to the occiput and resection of subcutaneous tissue and muscle, CSF was collected by cisternae magna puncture with pulled glass microcapillaries.

[0193] After CSF collection, whole blood was harvested via cardiac puncture and processed for both plasma and serum. For plasma, whole blood was transferred to plasma K-EDTA tubes which were then left on wet ice for no more than 15 minutes prior to centrifugation at 5000G for 10 minutes at 4°C. The supernatant containing the plasma was then pipetted out and placed in separate 1.5mL Eppendorf tubes to generate one (1) 100 µL aliquot from each animal. Plasma was then frozen on dry ice and stored at -80°C. For serum, whole blood was transferred to serum collector tubes which were then left at room temperature for a minimum of 30 minutes prior to centrifugation at 5000G for 10Docket No.062686-510001WO minutes at 4°C. The supernatant containing the serum was then pipetted out and placed in separate 1.5mL Eppendorf tubes to generate one (1) 100 µL aliquot from each animal. Serum was then frozen on dry ice and stored at -80°C.

[0001] Before further tissue harvest, animals were flush perfused with ambient temperature PBS to remove blood cells. The brain was then removed and hemisected. The left hemisphere was drop fixed in freshly prepared 4% phosphate buffered PFA overnight at 4°C, then transferred to PBS and stored at 4°C. The right hemisphere was microdissected into the cortex, striatum, hippocampus, cerebellum, brain stem, amygdala, and pituitary gland. All right brain segments were snap frozen in liquid nitrogen and then kept on dry ice until long term storage at -80°C.

[0194] The gastrocnemius and soleus were collected from each leg. Samples from each side were snap frozen separately in liquid nitrogen and then kept on dry ice until long term storage at -80°C.

[0195] The following peripheral organs were collected: liver [one right lobe, one left lobe], heart [split into left and right atria and ventricles], diaphragm [split into 2 samples], thymus [split into 2 samples], and testis [split into left and right]. Each sample was snap frozen separately in liquid nitrogen and then kept on dry ice until long term storage at -80°C. Tissue weights were recorded for all samples. DNA and RNA Isolation

[0196] DNA and RNA samples were prepared with the Qiagen AllPrep DNA / RNA Kit for 96-well format. Frozen tissue from mouse cortex (approximately 75 mg) or hippocampus (approximately 15 mg) was added to 700 µL room temperature RLT Buffer (containing 10 µL / mL β-mercaptoethanol) in 2 mL homogenization tubes containing one 5mm stainless steel bead. Samples were homogenized using the FastPrep-24 at 6 m / s for 20 seconds for each of 2 cycles (40 seconds total), and then centrifuged at 6,000 rpm for 5 minutes at room temperature. Supernatants from cortex homogenates were diluted 1:5 (70µL homogenate, 280µL buffer RLT for a final volume of 350µL), whereas supernatants from hippocampus homogenates were used as is (350 µL, undiluted). These supernatants were then processed according to the Qiagen AllPrep DNA / RNA isolation kit’s protocol (Simultaneous Purification of DNA and RNA from Tissues using Spin Technology).

[0197] Isolated DNA was quantified using a Nanodrop 8000 (Thermo Scientific), diluted to 40 ng / 4 µL, and stored at -20°C until use in droplet digital PCR for vector genome quantitation.

[0198] Isolated RNA remained on ice and was quantified using Nanodrop 8000 to ensure relatively equal RNA isolation among samples. RNA was then immediately used for cDNA synthesis using High-Capacity RNA-to-cDNA™ Kit (Applied Biosystems) according to the kit’s protocol, or frozen at -80°C for later cDNA synthesis. Ten (10) µL of RNA (30-40 ng / µL) from all samples was added to each reaction vessel (20 µL total volume). Newly synthesized cDNA was stored at -20°C until use in RT-qPCR for mRNA measurements.Docket No.062686-510001WO Vector Genome Quantitation

[0199] Vector DNA levels were quantified using duplex TaqMan-based droplet digital PCR (ddPCR) assays. The TaqMan reagents used included mTfrc:TaqMan® Copy Number Reference Assay, mouse Tfrc (Thermo Scientific), and codon-optimized human FOXG1 (opthFOXG1): forward primer (AGCACAGGCCTGACCTTTAT), reverse primer (CATAGGGTGGCTGGGATAGG), and probe (CCCTGCACCACCCAAGGGCC) (Thermo Scientific). ddPCR was performed using the QX200 Droplet Digital PCR System (Bio-Rad). Human FOXG1 mRNA Quantitation

[0200] cDNA was thawed on ice and then 1 µL of cDNA was added to 9 µL Taqman Fast Advance Master mix (Applied Biosystems) as specified in the kit’s protocol with 1:1 ratio of housekeeper (mouse glucuronidase beta, GUSB) to gene of interest (human FOXG1). Thermocycler settings were selected according to the Taqman Fast Advance protocol using QuantStudio 6 (ThermoFisher): Step 1: 50°C, 2 min; Step 2: 95°C, 20 sec.; Step 3: 40 cycles with 95°C, 1 sec. and 60°C, 20 sec.; Step 4: 60°C, 30 sec. The primers and probe for human FOXG1 (Custom TaqMan Gene Expression Assay, VIC 750 RxNs (Life Technologies Corporation) were: forward primer: 5’- AGCACAGGCCTGACCTTTAT, reverse primer: 5’-CATAGGGTGGCTGGGATAGG, probe: 5’- CCCTGCACCACCCAAGGGCC. The primers and probe for mouse GUSB were obtained commercially [Mouse GUSB Assay ID: Mm01197698_m1, cat. # 4331182 (ThermoFisher / Life Technologies Corporation)].

[0201] ΔCq values were calculated by subtracting the housekeeper Cq (mouse GUSB) from the gene of interest Cq (human FOXG1). ΔΔCq values were calculated by subtracting average ΔCq of the appropriate Het vehicle group from each individual sample treated with FOXG1 AAV (ALR-047). Fold change values (2-ΔΔCq) were calculated for samples treated with FOXG1 AAV relative to the corresponding Het vehicle group (either cortex or hippocampus). Statistical Analysis

[0202] An alpha level of .05 was selected for all inferential statistics. Data from each cohort of animals was combined together prior to analysis. Ordinary one-way ANOVA and two-way mixed effects ANOVAs were used to assess differences in behavioral performance throughout the study. Tukey’s multiple comparisons test was employed to make appropriate post-hoc comparisons after the identification of main effects. RESULTS Body Weights

[0203] FOXG1 WT and HET (Het) male mice were enrolled at P6. Animals were body weighed twice weekly until tissue collection at 13 weeks of age. There were no significant differences in body weight between FOXG1 WT animals injected with vehicle and FOXG1 HET (Het) animals injected with vehicle (FIG.1).Docket No.062686-510001WO Survival

[0204] All FOXG1 WT and HET (Het) animals survived until the scheduled tissue collection time point at 13 weeks of age with the exception of one (1) FOXG1 WT animal and thirteen (13) FOXG1 HET (Het) animals which were found dead throughout the course of the study. See FIG.2 and Table 3 below for additional details. Animals were checked twice daily for survival. Throughout the course of the study, one animal from Group 1 (FOXG1 WT, vehicle), three animals from Group 2 (FOXG1 HET (Het), vehicle), two animals from Group 3 (FOXG1 HET (Het), FOXG1 AAV [ALR-047] low dose), four animals from Group 4 (FOXG1 HET (Het), FOXG1 AAV [ALR-047] mid dose), and four animals from Group 5 (FOXG1 HET (Het), FOXG1 AAV [ALR-047] high dose) were found dead (FIG.2). All deaths occurred prior to P21. There was no significant difference in the probability of survival among the treatment groups. Table 3. Survival Details.Open FieldDocket No.062686-510001WO

[0205] The open field test (OF) was conducted at 6, 8, and 11 weeks of age. Total ambulatory distance, ambulatory distance in center, total rearing, and rears in the center were measured and analyzed.

[0206] When open field parameters were analyzed, FOXG1 HET (Het) animals treated with vehicle exhibited significantly decreased center distance traveled and velocity as compared to FOXG1 WT treated with vehicle at 6, 8 and 11 weeks of age, and at 6 and 8 weeks of age, respectively, demonstrating a measurable FOXG1 phenotype in the open field. There were no significant differences in total distance traveled, total rearing frequency, and center rearing frequency between FOXG1 HET (Het) animals treated with vehicle and FOXG1 WT treated with vehicle, although there were decreases in average total distance traveled and center rearing frequency in FOXG1 HET (Het) animals treated with vehicle compared with FOXG1 WT treated with vehicle, at 11 weeks of age.

[0207] FOXG1 HET (Het) animals treated with FOXG1 AAV (ALR-047) only exhibited significant differences in the open field as compared to FOXG1 HET (Het) animals treated with vehicle when treated with the highest dose (2.6e11 vg / mouse). When assessed at 11 weeks of age, FOXG1 HET (Het) animals treated with FOXG1 AAV (ALR-047) at 2.6e11 vg / mouse demonstrated significant increases in total distance traveled, center distance traveled, total rearing frequency, and center rearing frequency as compared to FOXG1 HET (Het) animals treated with vehicle, suggesting a possible beneficial effect of FOXG1 AAV (ALR-047) on motor performance in the open field when administered at the highest dose. In addition, at 8 weeks of age, the total rearing frequency showed a significant increase with 2.6e11 vg / mouse FOXG1 AAV (ALR-047) compared with FOXG1 HET (Het) animals treated with vehicle. FOXG1 HET (Het) animals dosed with either 1e11 vg / mouse or 4e10 vg / mouse FOXG1 AAV (ALR-047) were not observed to be significantly different from FOXG1 HET (Het) animals dosed with vehicle for any of the open field parameters assessed at any of the timepoints (FIGs.3A-3C, FIGs.4A-4C, FIGs.5A-5C, FIGs.6A-6C, FIGs.7A-7C, FIGs.8A- 8C, FIGs.9A-9C, FIGs.10A-10C, FIGs.11A-11C, and FIGs.12A-12C).

[0208] Taken together, the data suggest that FOXG1 AAV (ALR-047) ameliorates the motor deficits observed in the open field at 11 weeks of age in the FOXG1 mouse model when administered at P6 via ICV at a dose of 2.6e11 vg / mouse. Grip Strength

[0209] Grip strength was assessed across five (5) trials at 4, 6, 8, 10, and 12 weeks of age. Performance was analyzed by trial and by averaging the trials.

[0210] When grip strength data were analyzed, there were no significant differences in grip strength between FOXG1 HET (Het) animals treated with vehicle and FOXG1 WT animals treated with vehicle at any time point assessed, suggesting that there is no grip strength deficit in FOXG1 HET (Het) animals. There was no significant effect of FOXG1 AAV (ALR-047) on FOXG1 HETDocket No.062686-510001WO (Het) mice compared with FOXG1 HET (Het) animals treated with vehicle at any dose or age evaluated FIGs.13A-13E and FIGs.14A-14E). Fear Conditioning

[0211] The fear conditioning assessment was conducted across three days when the animals were 12 weeks of age. On day 1, mice were exposed to the cues and foot shocks. On day 2, the mice were placed into the same chamber they were trained in for 5-minutes without exposure to shocks or cues in order to assess contextual memory. On day 3, the animals were introduced to a novel environment and the percent freezing was assessed prior to, during, and after exposure to the same cues they were exposed to on the first day.

[0212] Analysis of the average percent freezing in contextual fear conditioning found that FOXG1 HET (Het) animals treated with vehicle freeze significantly less than FOXG1 WT animals treated with vehicle, suggesting a deficit in learning and memory in the FOXG1 HET (Het) animals. FOXG1 HET (Het) animals treated with FOXG1 AAV (ALR-047) at any of the doses assessed did not exhibit significant improvement in average percent freezing in contextual fear conditioning, signifying no effect of FOXG1 AAV (ALR-047) in FOXG1 HET (Het) animals in fear conditioning. When the data from the cued fear conditioning were analyzed, no phenotypic differences in percent freezing were detected between FOXG1 HET (Het) animals treated with vehicle and FOXG1 WT animals treated with vehicle. There was no significant effect of FOXG1 AAV (ALR-047) on FOXG1 HET (Het) animals compared with FOXG1 HET (Het) animals treated with vehicle on cued fear conditioning at 12 weeks of age (FIGs.15A-15C). Vector Genome and Human FOXG1 mRNA Levels

[0213] In vehicle treated FOXG1 WT and FOXG1 Het mice, vector genome levels in cortex and hippocampus (Table 4) were less than 0.05 vector genome per diploid cell (VG / DC), as expected since these animals were not treated with FOXG1 AAV (ALR-047). In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), vector genome levels in cortex were 1.1 to 19, 1.0 to 3.5 and 6.8 to 41 VG / DC and in hippocampus were 0.4 to 5.8, 2.4 to 7.2 and 0.8 to 45 after ICV administration of 4e10, 1e11 and 2.6e11 vg / mouse FOXG1 AAV (ALR-047), respectively (Table 4). Table 4 Vector genome and human FOXG1 mRNA levels.Docket No.062686-510001WOa: fold change [2^-(ΔΔCq)] relative to Het / vehicle group average; b: 4e10 vg / mouse FOXG1 AAV (ALR-047); c: 1e11 vg / mouse FOXG1 AAV (ALR-047); d: 2.6e11 vg / mouse FOXG1 AAV (ALR-047)

[0214] nd: no data because Cq >40 for human FOXG1 (huFOXG1), VG / DC: vector genome per diploid cell. In vehicle treated FOXG1 WT and FOXG1 Het mice, human FOXG1 mRNA levels in cortex and hippocampus, normalized to mouse GUSB mRNA levels (Table 4), were very low as expected since these animals were not treated with FOXG1 AAV (ALR-047) and thus, not expected to express human FOXG1. Some samples in these vehicle groups had human FOXG1 Cq’s that were undetermined due to exceeding the Cq cut-off of 40, resulting in incalculable ∆Cq’s for these samples, which would skew the average ∆Cq per vehicle group higher. In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), normalized human FOXG1 mRNA levels in cortex and hippocampus (Table 4) were very high relative to the vehicle treated FOXG1 Het group; the fold changes relative to the average of the vehicle treated FOXG1 Het group in cortex were 2,986 to 39,928, 2,342 to 12,790 and 27,097 to 97,121 and in hippocampus were 8,968 to 91,918, 58,072 to 109,562 and 14,307 to 821,069 after ICV administration of 4e10, 1e11 and 2.6e11 vg / mouse FOXG1 AAV (ALR-047), respectively.Docket No.062686-510001WO EXAMPLE 2. Behavioral effects in FOXG1 HET mice in vivo after Intracerebroventricular (ICV) Injection of FOXG1 AAV

[0215] The goal of this study was to evaluate the efficacy of AAV gene replacement therapy (scAAV9.hSyn1-opthFOXG1, lot ALR-047, test article) in male FOXG1 Het mice using behavioral and molecular endpoints. To conduct the study, FOXG1 Q84 Het male mice were bred with WT (C57BL / 6) females to generate FOXG1 WT and Het animals for study enrollment as described in Example 1. Only male mice were enrolled in the study.

[0216] During the course of the study, 12 / 12 light / dark cycles were maintained. All testing was performed during the animal’s light cycle phase. The room temperature was maintained between 20 and 23˚C with a relative humidity maintained around 50%. All mice were housed on OptiMICE® racks in an enriched environment containing nesting material. Litters were provided an igloo. Chow and water were provided ad libitum for the duration of the study. Study design

[0217] To conduct the study, one (1) group of n=18 male FOXG1 WT animals and two (2) groups of n=18 male FOXG1 Q84 Het animals were enrolled at P6 (postnatal day 6). Animals were bred and enrolled in two rounds of breeding, with representation from each treatment group in each breeding cohort. The cohorts were separated by one (1) week. Genotyping of the mice was performed as described in Example 1.

[0218] Animals were dosed via bilateral ICV injection at P6 with a dose volume of 5.0 µl per hemisphere (10.0 µL per animal) as described in Example 1. Details for the study are listed in Table 5 and Table 6. Table 5. Study group composition.Docket No.062686-510001WO Table 6. Study timeline.Wire Hang

[0219] The wire hang test of motor function was conducted when the animals were 10 weeks of age by following a modified protocol as described in Santa-Maria et al., (2012). Mice were placed on top of a standard wire cage lid. The lid was lightly shaken to cause the animal to tighten its grip and the lid was then turned upside down. The latency of mice to fall off the wire grid was measured, and average values were computed from three trials (30 seconds apart). Trials were stopped if the mouse remained on the lid after 5 minutes. Open Field

[0220] The open field test (OF) was used to assess both anxiety-like behavior and motor activity at 11 weeks of age. The open field chambers were plexiglass square chambers (27.3 x 27.3 x 20.3 cm; surrounded by infrared photobeam sources (16 x 16 x 16). The enclosure was configured to split the open field into a center and periphery zone and the photocell beams were set to measure activity in the center and in the periphery of the OF chambers. Animals having higher levels of anxiety or lower levels of activity tend to stay in the corners of the OF enclosures. On the other hand, mice that have high levels of activity and low levels of anxiety tend to spend more time in the center of the enclosure. Horizontal activity (distance traveled) and vertical activity (rearing) were measured from consecutive beam breaks. Animals were placed in the OF chambers for 30 minutes. Total ambulatory distance, ambulatory distance in center, total rearing, and rears in the center were measured. After testing, animals were placed back into their home cage. Tapered Balance Beam

[0221] The tapered balance beam assessment was performed at 11 weeks of age. This assessment proves to be a sensitive measure of motor impairment and is capable of detecting motor deficits early and even when other measures of motor function, such as the rotarod, fail to show any deficits (Heng et al., 2007; Brooks et al., 2011).Docket No.062686-510001WO

[0222] The balance beam consists of a strip of smooth black acrylic 100 cm in length, with a square cross section that tapers from a width of 1.5 cm to 0.5 cm. The beam also consists of a 0.5 cm safety ledge located 2 cm below the beam. The ledge maintains a constant width of 0.5 cm as the beam tapers. The angle of the beam is 17° from horizontal running from low to high. The highest point of the beam is approximately 58 cm from the floor. At the opposite side of the balance beam (‘end’ portion) there is a goal box which rests on the aforementioned support stand. The goal box is constructed from black acrylic, measuring 10.5 cm3 and containing a 3 cm² entrance hole.

[0223] On the training day, each animal completed 4 traversals in order to be considered trained for the beam. On testing day (24 hours later), mice received 3 trials of testing with an inter trial interval (ITI) of 30 seconds. Mice were placed on the bottom of the beam, facing away from the goal box. The time from placement on the beam to turning to face the goal box was recorded as the latency to turn. The maximum amount of time an animal had to complete the turn was 120 seconds. If the animal was unable or refused to turn after 120 seconds, it was positioned on the beam facing the goal box for the next phase of the experiment. Once the animal was facing the goal box, the latency to traverse the beam was recorded. The maximum amount of time an animal had to complete the traversal was 120 seconds. During the beam traversal, the number of foot slips was recorded. The slip-step ratio was calculated by dividing the total number of slips by the total number of steps taken per limb. Running Wheel

[0224] Running wheel activity was assessed at 12 weeks of age. The wheels used were from Lafayette Instrument®, Mouse Activity Wheel with Dual Licometer. The polycarbonate chambers measured 13.9”L x 9.25”W x 7.875”H (35.323.5 x 20 cm) and the aluminum running wheels measured 5.0” ID (12.7 cm) by 2.25” (5.72 cm) width (inside) for a Run Distance of 0.40 meters / revolution. The run surface consisted of 38 rods 0.188” diameter on 0.4298” centers with a 0.2418” gap. The metric equivalent is approximately 4.8 mm diameter on 10.9 mm centers with a 6.14 mm gap. The activity wheels were connected to an interface which electronically recorded the animal’s activity (running interval, average speed, total distance).

[0225] Animals were loaded into the chambers containing the running wheels at approximately 11AM on Day 1 and subsequently removed from the chambers at the same time on Day 4, 72 hours later. Mice remained in the activity wheel chambers 24 hours / day the three consecutive testing days, after which they were returned to their home cages. SmartCube®

[0226] The SmartCube® assessment was performed at 13 weeks of age. SmartCube® is a platform that employs computer vision to detect changes in body geometry, posture, and behavior (both spontaneous and in response to specific challenges). Mice were taken in their home cage to the SmartCube® suite of experimental rooms where they remained until they were placed in the apparatus. The standard SmartCube® protocol was run for aDocket No.062686-510001WO single session lasting 45 minutes. After the session, mice were placed back into to their home cage and were returned to the colony room. Any abnormal behavior was noted. Biological Sample Collection

[0227] Fecal samples were collected from all animals at 3 timepoints: baseline (prior to dose administration), at week 9, and at week 13. Animals were placed in standard cages and given one hour to produce feces. One to three pellets were collected per animal and placed in a 2.0 mL Eppendorf. Samples were snap frozen in liquid nitrogen and stored at -80°C. Mice were selected based on behavioral performance in open field, wire hang, and tapered balance beam.

[0228] Upon completion of the study, when the animals were 13 weeks of age, all remaining animals were euthanized for tissue collection. Animals were anesthetized with isoflurane and placed in stereotaxic frame. Following a skin incision inferior to the occiput and resection of subcutaneous tissue and muscle, CSF was collected by cisternae magna puncture with pulled glass microcapillaries.

[0229] After CSF collection, whole blood was harvested via cardiac puncture and processed for both plasma and serum (25 µL each). For plasma, whole blood was transferred to plasma K-EDTA tubes which were then left on wet ice for no more than 15 minutes prior to centrifugation at 5000G for 10 minutes at 4°C. The supernatant containing the plasma was then pipetted out and placed in separate 1.5mL Eppendorf tubes to generate one (1) 25 µL aliquot from each animal. Plasma was then frozen on dry ice and stored at -80°C. For serum, whole blood was transferred to serum collector tubes which were then left at room temperature for a minimum of 30 minutes prior to centrifugation at 5000G for 10 minutes at 4°C. The supernatant containing the serum was then pipetted out and placed in separate 1.5mL Eppendorf tubes to generate one (1) 25 µL aliquot from each animal. The serum was then frozen on dry ice and stored at -80°C

[0230] Additionally, approximately 250 µl of whole blood was placed in K3-EDTA microtainer tubes and diluted with DPBS in a 1:1 ratio. Tubes were gently mixed by hand and placed on wet ice until PBMC isolation. To isolate PBMC, 3mL of Histopaque-1119 (Sigma 11191- 100ML) was added to the bottom of a 15mL polypropylene conical centrifuge tube (Corning Falcon). Slowly, 3mL of Histopaque-1077 (Sigma 10771-100ML) was layered over the Histopaque-1119. The DPBS diluted blood was layered over the Histopaque-1077 and the gradient was spun at 870xg in centrifuge for 20 minutes at 24 C, acceleration 1, deceleration 1. The cloudy layer was collected in 1.5 mL tubes (~1.5mL) and centrifuged in the Eppendorf centrifuge for 5 mins at 1000xg at RT. The supernatant was aspirated without disturbing the pellet.1mL of ACK Lysis Buffer (ThermoFisher, A10492-01) was then added. PBMCs were allowed to stand in the ACK Lysis Buffer for ~3 mins at RT and then centrifuged in the Eppendorf centrifuge for 5 mins at 1000G at RT.1 mL of PBS+2%FBS (StemCell Technologies, #07905) was added, and the pellet was resuspended and centrifuged in the Eppendorf centrifuge for 5 mins at 1000G at RT. The supernatant was aspirated without disturbing the pellet (PBMC are in the pellet). Samples were then frozen on dry ice and stored at -80°C until shipment.Docket No.062686-510001WO

[0231] Before further tissue harvest, animals were flush perfused with ambient temperature PBS to remove blood cells. The brain was then removed and hemisected. Each hemisphere was microdissected into the cortex, striatum, hippocampus, midbrain cerebellum, inferior colliculus, and brain stem. All brain segments were snap frozen in liquid nitrogen and then kept on dry ice until long term storage at -80°C.

[0232] From the liver, one (1) sample was taken from the right lobe and one (1) sample was taken from the left lobe and placed in separate tubes. The large intestine was removed, flushed, and snap frozen in liquid nitrogen. Each sample was snap frozen separately in liquid nitrogen and then kept on dry ice until long term storage at -80°C.

[0233] Tissue weights were recorded for all samples. DNA and RNA Isolation

[0234] DNA and RNA samples were prepared with the Qiagen AllPrep DNA / RNA Kit for 96-well format. Frozen tissue from mouse cortex (approximately 75 mg) or hippocampus (approximately 15 mg) was added to 700 µL room temperature RLT Buffer (containing 10 µL / mL β-mercaptoethanol) in 2 mL homogenization tubes containing one 5mm stainless steel bead. Samples were homogenized using the FastPrep-24 at 6 m / s for 20 seconds, then frozen for storage. Immediately before processing, tissue homogenate samples were thawed at room temperature. Cortex homogenates were diluted 1:5 (70µL homogenate, 280µL buffer RLT for a final volume of 350µL). Hippocampus homogenates were diluted 2.5:3.5 (250µL homogenate, 100µL buffer RLT for a final volume of 350µL). Samples were centrifuged at 6,000 rpm for 5 minutes at room temperature. The supernatants were removed and then processed according to the Qiagen AllPrep DNA / RNA isolation kit’s protocol (Simultaneous Purification of DNA and RNA from Tissues using Spin Technology).

[0235] Isolated DNA was stored at -20°C until use in droplet digital PCR for vector genome quantitation.

[0236] Isolated RNA remained on ice and was immediately used for cDNA synthesis using High- Capacity RNA-to-cDNA™ Kit (Applied Biosystems) according to the kit’s protocol.10 µL of RNA (30-40 ng / µL) from all samples was added to each reaction vessel (20 µL total volume). Newly synthesized cDNA was stored at -20°C until use in RT-qPCR for mRNA measurements. Vector Genome Quantitation

[0237] Vector DNA levels were quantified as described in Example 1. Human FOXG1 mRNA Quantitation Human FOXG1 mRNA levels were quantified as described in Example 1. Statistical Analysis

[0238] An alpha level of .05 was selected for all inferential statistics. Data from each cohort of animals was combined together prior to analysis. Ordinary one-way ANOVA and two-way mixed effects ANOVAs were used to assess differences in behavioral performance throughout the study.Docket No.062686-510001WO Tukey’s multiple comparisons test was employed to make appropriate post-hoc comparisons after the identification of main effects. Similarity Analysis Using “Clouds Framework”

[0239] The outcome from SmartCube is a large set of features (behavioral parameters) that can be used for various analyses. Many of these features are correlated. Therefore, statistically independent combinations were formed of the original features (further referred to as de-correlated features). Each de-correlated feature extracts information from the whole cluster of the original features, so the new feature space has lower dimensionality (DRFA: de-correlated ranked feature analysis). A feature ranking algorithm was applied to score each feature for its discrimination power (ability to separate the two groups, e.g., reference vehicle and AAV gene therapy). Ranking is an important part of the analyses because it weighs each feature change by its relevance. A feature ranking algorithm was applied, derived from support vector in the support vector machine learning method, to rank each feature. RESULTS Body Weights

[0240] FOXG1 WT and Het male mice were enrolled at P6. Animals were body weighed once weekly until tissue collection at 13 weeks of age.

[0241] FOXG1 WT animals treated with vehicle were found to weigh significantly more than FOXG1 Het animals treated with either vehicle or FOXG1 AAV (ALR-047) at 4 (p = 0.0084), 5 (p = 0.0009), 6 (p = 0.0069), and 7 (p = 0.0494) weeks of age, as assessed by two-way ANOVA with Tukey’s multiple comparison test. No significant differences were detected between the body weights of FOXG1 Het mice treated with vehicle and those treated with FOXG1 AAV (ALR-047) at any point over the course of the study (FIG.16). Survival

[0242] All FOXG1 WT and Het animals survived until the scheduled tissue collection time point at 13 weeks of age with the exception of two (2) FOXG1 WT animals and five (5) FOXG1 Het animals which were found dead throughout the course of the study. See Table 7 below for additional details. Table 7 Survival details.Docket No.062686-510001WO Wire Hang

[0243] Animals were assessed for motor function using the wire hang test at 10 weeks of age. The latency to fall off the wire grid was measured, and average values were computed from three trials with a 30 second inter-trial interval. Trials were stopped if the mouse remained on the lid after 5 min.

[0244] There were no significant differences in latency to fall off the wire among the treatment groups, as assessed by one-way ANOVA. Due to the lack of a behavioral phenotype between the vehicle treated FOXG1 WT animals and vehicle treated FOXG1 Het animals, it was not possible to evaluate the effects of FOXG1 AAV (ALR-047) treatment (FIGs.17A-17B). Open Field

[0245] The open field test (OF) was conducted at 11 weeks of age. Total ambulatory distance, ambulatory distance in center, total rearing, and rears in the center were measured and analyzed across the 30-minute assessment.

[0246] At 11 weeks of age, FOXG1 Het mice treated with vehicle traveled significantly less in the center of the apparatus (p = 0.0014) and reared significantly more overall (p = 0.0057) as compared to FOXG1 WT animals treated with vehicle, as assessed by one-way ANOVA with Tukey’s multiple comparison test. No significant treatment effects were detected between FOXG1 Het animals that received vehicle and those that received FOXG1 AAV (ALR-047) in either center distance or total rearing. Due to the lack of a behavioral phenotype in total distance and center rearing, it was not possible to evaluate the effects of FOXG1 AAV (ALR-047) treatment on these measures. (FIGs 18A- 18B, FIGs 19A-19B, and FIGs 20A-20B). Tapered Balance Beam

[0247] Animals were assessed in the tapered balance beam at 11 weeks of age. Each animal was tested in 3 trials with a 30 second intertrial interval. The latency to turn on the beam and the latency to traverse the beam were recorded for all trials. Additionally, video scoring was employed to count the total number of footslips made while traversing the beam in all 3 trials, which was expressed as a ratio to the total number of steps.

[0248] There were no significant differences in latency to turn, latency to traverse, or slip-step ratio among treatment groups, as assessed by one-way ANOVA with Tukey’s multiple comparison test. Due to the lack of a behavioral phenotype between vehicle treated FOXG1 WT animals and vehicle treated FOXG1 Het animals, it was not possible to evaluate the effects of FOXG1 AAV (ALR-047) treatment (FIGs 21A-21B, FIGs 22A-22B, and FIGs 23A-23E) Running Wheel

[0249] Animals were assessed in the running wheels for three (3) consecutive days during the last week of the study when the animals were 12 weeks of age. The distance traveled (m) was recorded throughout the three days and graphed in one-hour bins. The total distance traveled (m) during the animals’ light cycle (from 7AM-7PM) and dark cycle (from 7PM-7AM) were calculated separatelyDocket No.062686-510001WO (FIG.24, FIGs 25A-25B). Statistical significance was assessed by two-way ANOVA with Tukey’s multiple comparison test.

[0250] When distance traveled during the day cycle was analyzed, FOXG1 WT animals treated with vehicle traveled significantly more compared to FOXG1 Het animals treated with vehicle over the course of the first (p < 0.0001) and second (p = 0.0294) day. A significant treatment effect was detected on day 1 with FOXG1 Het mice treated with FOXG1 AAV (ALR-047) traveling significantly more than FOXG1 Het mice treated with vehicle (p = 0.0288). No treatment effect was detected on day 2.

[0251] A significant treatment effect of FOXG1 AAV (ALR-047) on FOXG1 Het animals compared with vehicle treated FOXG1 Het animals was only detected in the running wheel during Day 1. Notably, this is a time period that is associated with acclimating to and exploring a novel environment, suggesting that FOXG1 AAV treatment reduces anxiety-like behavior and improves habituation to a new environment or acclimatization.

[0252] When distance traveled during the night cycle was analyzed, FOXG1 WT animals treated with vehicle traveled significantly more than FOXG1 Het animals treated with vehicle during the second (p < 0.0001) and third (p < 0.0001) night of testing. No treatment effects were detected between FOXG1 Het mice treated with vehicle and FOXG1 Het mice treated with FOXG1 AAV (ALR-047) during the second and third night of testing.

[0253] Due to the lack of phenotypic differences between FOXG1 WT and FOXG1 Het animals treated with vehicle on Night 1, Day 3, and Day 4, it was not possible to assess treatment effects of FOXG1 AAV (ALR-047) at these time points. SmartCube®

[0254] Animals were assessed in the SmartCube® apparatus at 13 weeks of age. The testing session lasted 45 minutes during which animals were exposed to various challenges, while their spontaneous behaviors and responses were analyzed.

[0255] Phenotypic profiling in SmartCube® revealed significant differences between FOXG1 WT and FOXG1 Het animals treated with vehicle (Discrimination = 97.1%, p < 0.0001). Small but significant recovery was detected in FOXG1 Het mice treated with FOXG1 AAV (ALR-047) (Recovery = 23.2%, p = 0.0160) (FIG.26A) compared with FOXG1 Het mice treated with vehicle.

[0256] SmartCube® provides approximately 3,000 features per animal per timepoint, resulting in a grand total of more than 100,000 features throughout the assessment. Of the top features of the SmartCube® that were statistically different between FOXG1 WT and FOXG1 Het animals treated with vehicle (FIG.26B), six features were robustly improved by treatment with FOXG1 AAV (ALR- 047): Sniffing to Mobility transition during the Shock phase, Immobility to Supported Rearing transition during the first rest, Supported Rearing to Immobility transition during the first rest, Horizontal to Vertical Mobility transition during the Shock phase, Exploring to Horizontal MobilityDocket No.062686-510001WO transition during the Coordination task, and Mobility Counts in the Habituation phase. In general, these features reflect an improvement in habituation to a novel environment.

[0257] Taken together, these data suggest that scAAV9.hSyn1-opthFOXG1 (lot ALR-047), administered at P6 via ICV at a dose of 2.6e11 vg / mouse, improves acclimatization to and exploration of a novel environment, and reduces anxiety-like behavior assessed at 12 to 13 weeks of age. Vector Genome and Human FOXG1 mRNA Levels

[0258] In vehicle treated FOXG1 WT and FOXG1 Het mice, vector genome levels in cortex and hippocampus (Table 8) were less than 0.01 vector genome per diploid cell (VG / DC), as expected since these animals were not treated with FOXG1 AAV (ALR-047). In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), vector genome levels in cortex and hippocampus (Table 8) ranged from 0.3 to 33 VG / DC and 1.2 to 68 VG / DC, respectively. Table 8 Vector genome and human FOXG1 mRNA levels.Docket No.062686-510001WOa: fold change [2^-(ΔΔCq)] relative to Het / vehicle group average; nd: no data because Cq >40 for human FOXG1 (huFOXG1), VG / DC: vector genome per diploid cell

[0259] In vehicle treated FOXG1 WT and FOXG1 Het mice, human FOXG1 mRNA levels in cortex and hippocampus, normalized to mouse GUSB mRNA levels (Table 8), were very low as expected since these animals were not treated with FOXG1 AAV (ALR-047) and thus, not expected to express human FOXG1. Some samples in these vehicle groups had human FOXG1 Cq’s that were undetermined due to exceeding the Cq cut-off of 40, resulting in incalculable ∆Cq’s for these samples, which would skew the average ∆Cq per vehicle group higher. In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), normalized human FOXG1 mRNA levels in cortex and hippocampus (Table 8) were very high relative to the vehicle treated FOXG1 Het group; the fold changes relative to the average of the vehicle treated FOXG1 Het group ranged from 814 to 74,916 and 1,095 to 49,799 in cortex and hippocampus, respectively.

[0260] In the FOXG1 Het group treated with FOXG1 AAV (ALR-047), 3 of the 11 mice had more than 10-fold higher levels of vector genome in cortex and hippocampus tissues (animals FOXG1_0142.01, FOXG1_0146.01 and FOXG1_0149.03) than the other 8 mice in this group. The higher levels of vector genome in cortex and hippocampus of animals FOXG1_0142.01, FOXG1_0146.01 and FOXG1_0149.03 are similar to these levels in another study (see Example 3) which used the same FOXG1 AAV and dose, suggesting that the remaining 8 mice in this group (Table 8) had received lower than intended doses. EXAMPLE 3. Evaluation of Electroencephalogram in FOXG1 HET Mice in vivo after Intracerebroventricular (ICV) Injection of FOXG1 AAV

[0261] This example describes the determination of the efficacy of intracerebroventricular (ICV) injection of FOXG1 AAV (ALR-047) in FOXG1 Q84 heterozygous (FOXG1 Het) mice in vivo using behavioral and molecular endpoints. The goals of the present study were to evaluate FOXG1 Het male mice for (1) electroencephalogram (EEG) phenotype and (2) efficacy of AAV gene replacement therapy (scAAV9.hSyn1-opthFOXG1) administered by bilateral ICV injection on the EEG phenotype.Docket No.062686-510001WO

[0262] To conduct the study, FOXG1 Het male mice were bred with WT (C57BL / 6) females to generate FOXG1 WT and Het animals for study enrollment as described in Example 1. During the course of the study, 12 / 12 light / dark cycles were maintained. Study design

[0263] To conduct the study, one group of n=10 male FOXG1 WT animals and two groups of n=12 male FOXG1 Het animals were enrolled at P6 (postnatal day 6), as shown in Table 9, after assessment of genotype. Genotyping of the mice was performed as described in Example 1.

[0264] Animals were dosed via bilateral ICV injection at P6 with a dose volume of 5.0 µl per hemisphere (10.0 µL per animal) as described in Example 1. Details for the study are listed in Table 9 and Table 10. Table 9 Study group composition.PBS with 5% sorbitol and 0.001% F-68 was used as the vehicle in this study. ICV: intracerebroventricular, N: number of animals, N / A: not applicable, WT: wild-type. Table 10. Study timeline.EEG SurgeriesDocket No.062686-510001WO

[0265] Adult mice were implanted with electrodes around 7 weeks of age and allowed to recover for approximately 10 days. EEG recording was performed at approximately 8 weeks of age and again at approximately 12 weeks of age.

[0266] Mice were implanted for 2 EEG channels and 1 electromyography (EMG) recording using a headmount for tethered data collection. EEG Recordings

[0267] EEG data acquisition was performed with the Pinnacle recording system.

[0268] After acclimating to the recording room, mice were habituated to the EEG recording chamber and tether for at least 20 hours prior to the start of the recording. Following all acclimation, EEG / EMG activity was recorded for 2 days. Although only one 24-hour period was scored and analyzed, 48 hours were recorded for protection. EEG Data Analysis

[0269] EEG data were evaluated for sleep / wake states, spectral power within sleep / wake states, and for any unusual activity such as seizures, aberrant spiking, and other hypersynchronous activity like spike-and-wave or spindle spike events.

[0270] EEG data were read into Neuroscore (Data Sciences International) software or similar program for visualization and processing and subsequent analyses. Offline, artifacts were removed from the data and sleep stages were assigned manually for every 10-second epoch using EEG and EMG by conventional methods as previously described (Morairty et al., 2011, 2012, 2013, 2014; Fisher et al., 2013, 2016) as: wake (W; less regular, low amplitude EEG with high and sometimes phasic EMG activity); nonrapid eye movement sleep (NREM; high-amplitude EEG waves with predominant delta (1-4 Hz), low EMG activity); rapid eye movement sleep (REM; stable, low- amplitude EEG waves dominated by theta (4–8 Hz) with near absent EMG activity). Unusual EEG events were marked and analyzed.

[0271] Data for the study were analyzed for the following: • Hourly percent time in W, NREM and REM. • Latency to the onset of NREM and REM (from light onset) • Cumulative time in W, NREM and REM • Measures of sleep / wake consolidation (hourly average bout duration and the number of bouts for W, NREM and REM) • Quantitative EEG analysis (spectral analysis) with full statistical analysis of standard power bands (such as delta 1-4 Hz, theta 4-8 Hz, alpha 8-12 Hz, sigma 12-16 Hz, beta 16-24 Hz, low gamma 24-60 Hz, high gamma 60-100 Hz) in W, NREM and REM • Quantification of unusual EEG patterns Spectral analysis

[0272] High pass filter with 1Hz was used to remove the low frequency noise in the data. Welch fast Fourier transform with fast Fourier transform size of 2048 was used to calculate power spectra forDocket No.062686-510001WO each 10-s epoch. Band power was calculated by integrating power spectra over the frequency range based on Simpson’s rule. Then the hourly average power spectra and band power were extracted separately for W, NREM, and REM. Epochs with artifacts were excluded from calculating the average. Statistical Analysis

[0273] An alpha level of .05 was selected for all inferential statistics. Body weight differences were assessed with two-way mixed effects ANOVA. EEG data were evaluated with two-way mixed effects ANOVA that were performed separately for light and dark phases to test the effects of group and time. One-way ANOVA was used to test the group effect in latency and cumulative time. Post- hoc t-test was performed on overall values between each group pair for significant group effect, and post-hoc t-test was performed on hourly values between each group for significant group-time interaction. Biological Sample Collection

[0274] Upon completion of the study, when the animals were 13 weeks of age, all animals were euthanized for tissue collection. Samples were collected and processed as described in Example 2. DNA and RNA Isolation

[0275] DNA and RNA samples were prepared as described in Example 2. Vector Genome Quantitation

[0276] Vector DNA levels were quantified as described in Example 1. Human FOXG1 mRNA Quantitation

[0277] Human FOXG1 mRNA levels were quantified as described in Example 1. Human FOXG1 Protein Quantitation

[0278] Human FOXG1 protein concentrations were quantified by ultra-high-performance liquid chromatography - tandem mass spectrometry (UPLC-MS / MS). Frozen tissues from mouse cortex or hippocampus were thawed, homogenized, denatured for 10 minutes at 100°C, alkylated with iodoacetamide, and digested with trypsin at 37°C. After digestion, the samples were analyzed by UPLC-MS / MS. The human FOXG1 signature peptide (GEPGGGPGELAPVGPDEK) in the mouse cortex and hippocampus tissues were measured. The analysis was accomplished with a SCIEX Triple Quad™ 7500 LC-MS / MS System, which features an IonDrive™ Turbo V source operating in positive electrospray ionization (ESI) mode, along with an ultra-high-performance liquid chromatograph (UPLC) system. RESULTS Body Weight

[0279] FOXG1 WT and Het male mice were enrolled at P6, and body weights measured weekly until euthanasia at 13 weeks of age. There were no significant differences in body weight among the treatment groups (FIG.27).Docket No.062686-510001WO Survival

[0280] All WT and FOXG1 Het animals survived until the scheduled euthanasia at 13 weeks of age with the exception of three (3) FOXG1 Het animals. One animal (FOXG1_0187.03) treated with vehicle (Group 2) was found dead at 3 weeks of age. One animal (FOXG1_0198.02) treated with FOXG1 AAV (ALR-047 (Group 3) was also found dead at 3 weeks of age. Additionally, one animal (FOXG1_0187.01) treated with FOXG1 AAV (ALR-047) (Group 3) was euthanized at 12 weeks of age due to head mount dislodge which is a criterion for humane endpoint. Sleep

[0281] Data were analyzed to compare the three groups of mice: 1) WT administered vehicle (the excipient), 2) FOXG1 Het administered vehicle, and 3) FOXG1 Het administered FOXG1 AAV (ALR-047).

[0282] Latency to the onset of NREM and REM showed no statistically significant difference between the three groups (FIG.28).

[0283] The average percent time during the 12 h light and 12 h dark phases spent in each of the three sleep / wake states revealed significant differences between the groups (FIG.29). Average hourly percent time in wake, NREM and REM showed no significant differences between the groups (FIG. 30). Based on the average percent time during the 12 h light and 12 h dark phases (FIG.29), vehicle treated FOXG1 Het mice spent significantly less time awake during the dark phase compared to vehicle treated WT mice. Vehicle treated FOXG1 Het mice also spent significantly more time in NREM during both the light and dark phases compared to vehicle treated WT mice. However, there were no significant differences between the vehicle treated WT mice and the AAV treated FOXG1 Het mice in time spent awake during the dark phase, time spent in NREM during the light phase or time spent in NREM during the dark phase, indicating that the treatment with scAAV9.hSyn1- opthFOXG1 had normalized these changes in the FOXG1 Het mice. In addition, REM was decreased significantly in the AAV treated FOXG1 Het mice compared to the vehicle treated FOXG1 Het mice in the light phase. However, neither the AAV nor vehicle treated FOXG1 Het mice were significantly different from the vehicle treated WT mice.

[0284] The average number of bouts and bout duration for wake, NREM and REM in the 12 h light and dark phases and the average hourly number of bouts and bout durations are shown in FIGs.31- 34. The decrease in wake time in vehicle treated FOXG1 Het mice during the 12 h dark phase occurred primarily via an approximately 35% reduction (though not significant) in wake bout duration with no change in bout number (FIG.31 and FIG.33).

[0285] NREM time increased in vehicle treated FOXG1 Het mice due to a higher number of NREM bouts in both the light and dark phases (significant in the dark phase, FIG.31), although there were small reductions in NREM bout duration (not significant, FIG.33). In other words, although there was more NREM sleep in the vehicle treated FOXG1 Het mice, it was slightly more fragmented compared to the vehicle treated WT mice.Docket No.062686-510001WO

[0286] REM time in the light phase decreased in AAV treated FOXG1 Het mice due to a significantly reduced number of REM bouts compared to the vehicle treated FOXG1 Het mice (FIG. 31). However, neither the AAV nor vehicle treated Het mice were significantly different from the vehicle treated WT mice in REM bout number. No significant differences in REM bout durations were found (FIG.33). Spectral Analysis

[0287] Only a few significant spectral power changes were observed in the vehicle treated FOXG1 Het mice compared to vehicle treated WT mice. Frontal Cortex

[0288] During waking, no significant differences in spectral power were found between the vehicle treated FOXG1 Het and the vehicle treated WT groups (FIGs.35-36).

[0289] During NREM, general decreases in EEG power were observed with some data points reaching statistical significance (FIGs.37-38). NREM alpha was decreased in the light phase in the AAV treated FOXG1 Het group compared to the vehicle treated WT group. NREM high gamma was reduced in both the vehicle and AAV treated FOXG1 Het groups compared to the vehicle treated WT group in both the light and dark phases. For the hourly data, NREM beta was decreased during the first hour of the light phase in the AAV treated FOXG1 Het group compared to the vehicle treated WT group. NREM beta was decreased in the vehicle treated FOXG1 Het group during the 1st, 4th, and 8-10th hours of the dark phase. NREM beta was also reduced in vehicle treated FOXG1 Het mice compared to the AAV treated FOXG1 Het mice during the 1st hour of the dark phase.

[0290] A few changes in spectral power during REM were found (FIGs.39-40). REM is expressed much less than either waking or NREM and provides the least interesting spectral data. REM low and high gamma were reduced in the AAV treated FOXG1 Het group in both the light and dark phases compared to vehicle treated WT mice, while the vehicle treated FOXG1 Het group was significantly reduced only during the dark phase. Parietal Cortex

[0291] There were no significant differences between any experimental group for EEG power in the parietal cortex likely due to high variance in the WT signal amplitude (FIGs.41-46). However, clear trends can be seen with generalized decreases in power for both AAV and vehicle treated FOXG1 Het groups during waking and during sleep (both NREM and REM) for the AAV treated FOXG1 Het mice compared to the vehicle treated WT mice.

[0292] In summary, a moderate but significant sleep phenotype was found in the vehicle treated FOXG1 Het mice compared to the vehicle treated WT mice. The FOXG1 Het mice were awake less and slept more during both the light and dark phases. However, the NREM sleep occurred in a more fragmented pattern. No significant differences in sleep patterns were found in the AAV treated FOXG1 Het mice compared to the vehicle treated WT mice, suggesting that the treatment with scAAV9.hSyn1-opthFOXG1 had normalized the sleep patterns in FOXG1 Het mice.Docket No.062686-510001WO

[0293] Although relatively few data points reached significance, EEG power was generally lower in both AAV and vehicle treated FOXG1 Het groups during waking compared to the vehicle treated WT mice in both the frontal and parietal channels. Power was also reduced during NREM sleep for both AAV and vehicle treated FOXG1 Het groups in the frontal channel, particularly in the gamma ranges. In the parietal cortex during NREM, only the AAV treated FOXG1 Het mice had reduced power. During REM, only the AAV treated FOXG1 Het mice had reduced power in both the frontal and parietal channels. The lack of significance in the EEG power data is largely due to an unusually high variation in the raw power within the vehicle treated WT group. Vector Genome and Human FOXG1 mRNA Levels

[0294] In vehicle treated WT and FOXG1 Het mice, vector genome levels in cortex and hippocampus (Table 10) were less than 0.01 vector genome per diploid cell (VG / DC), as expected since these animals were not treated with FOXG1 AAV (ALR-047). In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), vector genome levels in cortex and hippocampus (Table 10) ranged from 1.8 to 41 VG / DC and 0.5 to 68 VG / DC, respectively. Table 10 Vector genome and human FOXG1 mRNA levels.Docket No.062686-510001WOa: fold change [2^-(ΔΔCq)] relative to Het / vehicle group average; nd: no data because Cq >40 for human FOXG1 (huFOXG1), VG / DC: vector genome per diploid cell

[0295] In vehicle treated WT and FOXG1 Het mice, human FOXG1 mRNA levels in cortex and hippocampus, normalized to mouse GUSB mRNA levels (Table 10), were very low as expected since these animals were not treated with FOXG1 AAV (ALR-047) and thus, not expected to express human FOXG1. Some samples in these vehicle groups had human FOXG1 Cq’s that were undetermined due to exceeding the Cq cut-off of 40, resulting in incalculable ∆Cq’s for these samples, which would skew the average ∆Cq per vehicle group higher. In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), normalized human FOXG1 mRNA levels in cortex and hippocampus (Table 10) were very high relative to the vehicle treated FOXG1 Het group; the fold changes relative to the average of the vehicle treated FOXG1 Het group ranged from 1,371 to 21,599 and 554 to 45,497 in cortex and hippocampus, respectively.

[0296] In the FOXG1 Het group treated with FOXG1 AAV (ALR-047), 1 of the 10 mice (animal FOXG1_0202.02) had approximately 3-fold and 36-fold lower levels of vector genome in cortex and hippocampus tissues, respectively, than the other 9 mice in this group (Table 10). The levels of vector genome in cortex and hippocampus of animal FOXG1_0202.02 suggested that this animal had received a lower than intended dose. Human FOXG1 Protein Levels

[0297] Human FOXG1 protein levels were evaluated by UPLC-MS / MS in cortex and hippocampus tissues from approximately half of the mice in each group in the study. In vehicle treated FOXG1 WT and FOXG1 Het mice, human FOXG1 protein levels in cortex and hippocampus (Table 11) were below the limit of quantitation (BLQ) in all samples that were tested, as expected since these animals were not treated with FOXG1 AAV (ALR-047). In FOXG1 Het mice treated with FOXG1 AAV (ALR-047), human FOXG1 protein levels in cortex and hippocampus (Table 11) wereDocket No.062686-510001WO measurable in all samples that were tested and ranged from 0.009 to 0.021 ng / mL and 0.001 to 0.024 ng / mL, respectively. Table 11 Human FOXG1 protein levels.Docket No.062686-510001WO na: not available (samples not evaluated), BLQ: below the limit of quantitation EXAMPLE 4. Behavioral effects in FOXG1 Het Mice in vivo after Intracerebroventricular (ICV) Injection of FOXG1 AAV

[0298] The goal of this study is to evaluate the efficacy of AAV gene replacement therapy (scAAV9.hSyn1-opthFOXG1, lot ANL-034-035, test article) in male and female FOXG1 Het mice using behavioral and molecular endpoints. To conduct the study, FOXG1 Q84 Het male mice are bred with WT (C57BL / 6) females to generate WT and FOXG1 Het animals for study enrollment as described in Example 1. Study design

[0299] Sixty (60) FOXG1 Het and thirty (30) WT mice are distributed into study groups. Pups are balanced by litter and body weight and distributed according to Table 12 below. Animals (both males and females) are dosed at P2 (postnatal day 2). Details for the study are listed in Table 12 and Table 13. Animals

[0300] Mice are assigned unique identification numbers (ear notched) and housed in polycarbonate OptiMICE cages. All animals are examined, manipulated and weighed prior to initiation of the study to assure adequate health and suitability and to minimize non-specific stress associated with manipulation.

[0301] During the course of the study, 12 / 12 light / dark cycles are maintained. The room temperature is maintained between 20 and 23°C with a relative humidity maintained around 50%. Chow and water are provided ad libitum for the duration of the study.

[0302] Animals are dosed via bilateral ICV injection at P2 with a dose volume of 3 µl per hemisphere (6 µL per animal). Table 12. Study group composition.Docket No.062686-510001WO Table 13. Study timeline.ICV Injections

[0303] Prior to injection, animals are anesthetized via cryoanesthesia. Animals are dosed via bilateral ICV injection at P2, with a dose volume of 3 µl / side (6 µl / mouse). Animals are then placed on a warm (~36°C) heating pad immediately following the ICV injection. Animals are closely monitored after the ICV injection for any observable adverse effects. Body weight and survival Animals are weighed once per week starting at the time of injection (immediately prior to dosing) and are checked for survival twice per day. Ultra Sonic Vocalizations

[0304] Mice produce Ultra Sonic Vocalizations (USVs) to convey information related to positive or negative emotional states and to mediate social interactions. USV deficits in mice reflect communication and social interaction deficits. A normalization of USV deficits with AAV treatment would translate to improved communication and social interaction in humans. See e.g., Premoli et al. Neural Regen. Res. (2020) 16,(6).Docket No.062686-510001WO Neonatal:

[0305] At post-natal days 9 and 14 (P9, P14), all enrolled animals are assessed in the ultrasound sound vocalization assay using the Metris Sonotrack and SmartChamber system (Hoofddorp, Netherlands). The cage with the mother and the litter is taken to the testing room at a room temperature of about 24° C. The mothers are taken out of the cages at least 30 minutes (min) prior to test, placed in new cages with water and food available and taken to the colony room until the pups have all been tested. Pups remain in the home cage with familiar bedding and nest material. During the test, one pup at a time is taken to a clean Plexiglas chamber within the USV chamber for a 5 min trial. The isolation response is measured by means of a built-in ultrasound microphone and recorded using Metris software. After testing is complete, dams and pups are reunited in the home cage. Adult:

[0306] At 8 weeks of age, all enrolled animals are again assessed. Adult Males

[0307] Male mice are housed in a male-only room for a minimum of 1 week prior to USV test. Two days prior to USV testing, male mice are brought to the USV testing room where they acclimate for a minimum of 1 hour. After acclimation, a sexually mature female mouse is placed into each male cage. Males are paired with females for a minimum of 1 hour after which the females are removed from the cage. At the conclusion of the male-female pairing, males are brought back to their male- only colony room. On the day of USV testing, males are brought to the USV testing room and allowed to acclimate for a minimum of 1 hour. A single cage is placed into each soundproof USV detector. A gauze pad moistened with at least 30 μL of fresh sexually mature female urine is placed into each cage. The USV recording session begins as soon as the gauze pad has been placed into the cage. The recording session ends after 300 seconds. Adult Females

[0308] Female mice are single housed 3 days prior to USV testing. On the day of USV testing, the female mice are brought to the USV testing room and allowed to acclimate for a minimum of 1 hour. Females are tested for USV in pairs. One female is placed into the cage of another female that she was originally cage mates with (prior to the 3 day single housing). The USV recording begins as soon as the former female cage mate is placed into the other cage mate’s cage. The USV recording ends after 300 seconds. During the recording sessions, all USVs are recorded from the female pair. Wire Hang

[0309] The wire hang test of motor function is conducted by following a modified protocol as described in Santa-Maria et al., (2012). Mice are placed on top of a standard wire cage lid. The lid is lightly shaken to cause the animal to tighten its grip and the lid is then turned upside down. The latency of mice to fall off the wire grid is measured, and average values are computed from three trials (30 seconds apart). Trials are stopped if the mouse remains on the wire grid after 5 minutes.Docket No.062686-510001WO Open Field Test

[0310] The open field test (OF) is used to assess both anxiety-like behavior and motor activity. The open field chambers are plexiglass square chambers The enclosure is configured to split the open field into a center and periphery zone and the photocell beams are set to measure activity in the center and in the periphery of the OF chambers. Animals having higher levels of anxiety or lower levels of activity tend to stay in the corners of the OF enclosures. On the other hand, mice that have high levels of activity and low levels of anxiety tend to spend more time in the center of the enclosure. Horizontal activity (distance traveled) and vertical activity (rearing) are measured from consecutive beam breaks. Animals are placed in the OF chambers for 30 minutes. Total distance traveled and rearing frequency is measured in the open field arena. Tapered Balance Beam

[0311] The tapered balance beam (TBB) consists of a beam angled and elevated from the floor. At the opposite side of the balance beam (‘end’ portion) there is a goal box, which rests on the support stand. Following habituation to the testing room, mice are placed on the ‘starting’ end of the balance beam. Mice receive 3 trials per day, with an intertest interval (ITI) of 30 sec, and are returned to the home cage between trials. Latency to turn on the beam (sec), latency to traverse the beam (sec), and number of foot slips (left / right; fore / hind) are recorded. All tests are also recorded using a video camera to aid in scoring. Running Wheel

[0312] The activity wheels are connected to an interface which electronically records the animal’s activity (running interval, average speed, total distance). Animals spend 72 consecutive hours in the running wheels. Data are continuously recorded. SmartCube®

[0313] SmartCube® is a platform that employs computer vision to detect changes in body geometry, posture, and behavior (both spontaneous and in response to specific challenges). Mice are taken in their home cage to the SmartCube® suite of experimental rooms where they remain until they are placed in the apparatus. The standard SmartCube® protocol is run for a single session lasting 45 minutes. After the session, mice are placed back into to their home cage and returned to the colony room. Any abnormal behavior is noted. Similarity Analysis Using “Clouds Framework”

[0314] The outcome from SmartCube is a large set of features (behavioral parameters) that can be used for various analyses. Many of these features are correlated. Therefore, statistically independent combinations are formed of the original features (further referred to as de-correlated features). Each de-correlated feature extracts information from the whole cluster of the original features, so the new feature space has lower dimensionality (DRFA: de-correlated ranked feature analysis). A feature ranking algorithm is applied to score each feature for its discrimination power (ability to separate the two groups, e.g., reference vehicle and AAV gene therapy). Ranking is an important part of theDocket No.062686-510001WO analyses because it weighs each feature change by its relevance. A feature ranking algorithm is applied, derived from support vector in the support vector machine learning method, to rank each feature. Biological Sample Collection

[0315] At 13 weeks of age, all surviving animals per group have tissues collected. Animals are anesthetized with isoflurane and placed in a stereotaxic frame. Following a skin incision inferior to the occiput and resection of subcutaneous tissue and muscle, approximately 2ul of clean CSF are collected by cisternae magna puncture with pulled glass microcapillaries. After CSF collection, whole blood is harvested via cardiac puncture and processed for plasma and serum (25 μL aliquots of each) and PBMCs are isolated. These samples are frozen and stored at -80°C.

[0316] Before further tissue harvest, animals are flush perfused with ambient temperature PBS to remove blood cells. The brain is removed and hemisected. From 8 mice per group, the left hemisphere is drop fixed in freshly prepared 4% phosphate buffered PFA overnight at 4°C, then transferred to PBS and stored at 4°C until shipment or immunohistochemical processing. Samples are sectioned and stained for markers that may include FOXG1, NeuN, GFAP and IBA1, as well as H&E. All remaining hemispheres are microdissected into the cortex, striatum, hippocampus, midbrain, cerebellum, inferior colliculus, and brain stem.

[0317] Organs: Liver [one right lobe, one left lobe] – each sample is frozen separately.

[0318] The large intestines are removed, flushed, and flash frozen.

[0319] Tissue weight is recorded for each sample.

[0320] Tissue samples are snap-frozen and stored at -80°C, and may be evaluated for vector genome levels, FOXG1 mRNA levels and FOXG1 protein levels. Statistical Analysis

[0321] Data are analyzed by analysis of variance (ANOVA) followed by post-hoc comparisons where appropriate. An effect is considered significant if p < .05. Data are represented as the mean and standard error of the mean (s.e.m).

[0322] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein. INCORPORATION BY REFERENCE

[0323] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes. EQUIVALENTS

[0324] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. The scope of the inventionDocket No.062686-510001WO is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.Docket No.062686-510001WO SEQUENCES Forkhead box G1 (FOXG1) protein [Homo sapiens] amino acid sequence NCBI NP_005240.3 (SEQ ID NO:1) MLDMGDRKEVKMIPKSSFSINSLVPEAVQNDNHHASHGHHNSHHPQHHHHHHHHHHHPPPPAPQPPPP PQQQQPPPPPPPAPQPPQTRGAPAADDDKGPQQLLLPPPPPPPPAAALDGAKADGLGGKGEPGGGPGEL APVGPDEKEKGAGAGGEEKKGAGEGGKDGEGGKEGEKKNGKYEKPPFSYNALIMMAIRQSPEKRLTL NGIYEFIMKNFPYYRENKQGWQNSIRHNLSLNKCFVKVPRHYDDPGKGNYWMLDPSSDDVFIGGTTG KLRRRSTTSRAKLAFKRGARLTSTGLTFMDRAGSLYWPMSPFLSLHHPRASSTLSYNGTTSAYPSHPMP YSSVLTQNSLGNNHSFSTANGLSVDRLVNGEIPYATHHLTAAALAASVPCGLSVPCSGTYSLNPCSVNLL AGQTSYFFPHVPHPSMTSQSSTSMSARAASSSTSPQAPSTLPCESLRPSLPSFTTGLSGGLSDYFTHQNQG SSSNPLIH FOXGl nucleic acid sequence; NCBI NM_005249.5 (SEQ ID NO: 2) AATTGTGGCTATAGCCGCCTCGATCGCTGTCTCCCCAGCCTCGCCGCGGCCGCTCCGGGACGCGCC CGCCCGCCGCCCGGCTCTCCCCCCCTTTGGGCTGCTGCTGCTGCTGCTGTGACTGCTGCTGCGAGA GGAGGAGGAGGAGGAGGAAGCAGCGGGGGGGGGAGCGGGGGGTGGGGGGGGAGACCAAGAAGT ACAGTTGGGAGCGAGGGAGCTTCACCCCCGGGGCGGTGGTTGTTTCTTTTTTCTTTCTTTCTTTTTT CTTTTCCTTTTTTTTTTTTTTTCTAATTCCTGAGGGGTGGTTGCTGCTTTTGCTACATGACTTGCCAG CGCCCGAGCCTGCGGTCCAACTGCGCTGCTGCCGGAGCGCTCAGTGCCGCCGCTGCCGCCCGCGCC CCCCGCGCCCCGTTCGGCACCCACCGGTCGCCGCCGCCCGCCGCGCCGCTGTCCCGCTCCCGCGCC GCCGCCGCCGTTTCCCCCCGACGACTGGGTGATGCTGGACATGGGAGATAGGAAAGAGGTGAAAA TGATCCCCAAGTCCTCGTTCAGCATCAACAGCCTGGTGCCCGAGGCGGTCCAGAACGACAACCAC CACGCGAGCCACGGCCACCACAACAGCCACCACCCCCAGCACCACCACCACCACCACCACCATCA CCACCACCCGCCGCCGCCCGCCCCGCAACCGCCGCCGCCGCCGCAGCAGCAGCAGCCGCCGCCGC CGCCGCCCCCGGCACCGCAGCCCCCCCAGACGCGGGGCGCCCCGGCCGCCGACGACGACAAGGGC CCCCAGCAGCTGCTGCTCCCGCCGCCGCCACCGCCACCACCGGCCGCCGCCCTGGACGGGGCTAA AGCGGACGGGCTGGGCGGCAAGGGCGAGCCGGGCGGCGGGCCGGGGGAGCTGGCGCCCGTCGGG CCGGACGAGAAGGAGAAGGGCGCCGGCGCCGGGGGGGAGGAGAAGAAGGGGGCGGGCGAGGGC GGCAAGGACGGGGAGGGGGGCAAGGAGGGCGAGAAGAAGAACGGCAAGTACGAGAAGCCGCCG TTCAGCTACAACGCGCTCATCATGATGGCCATCCGGCAGAGCCCCGAGAAGCGGCTCACGCTCAA CGGCATCTACGAGTTCATCATGAAGAACTTCCCTTACTACCGCGAGAACAAGCAGGGCTGGCAGA ACTCCATCCGCCACAATCTGTCCCTCAACAAGTGCTTCGTGAAGGTGCCGCGCCACTACGACGACC CGGGCAAGGGCAACTACTGGATGCTGGACCCGTCGAGCGACGACGTGTTCATCGGCGGCACCACG GGCAAGCTGCGGCGCCGCTCCACCACCTCGCGGGCCAAGCTGGCCTTCAAGCGCGGTGCGCGCCT CACCTCCACCGGCCTCACCTTCATGGACCGCGCCGGCTCCCTCTACTGGCCCATGTCGCCCTTCCTG TCCCTGCACCACCCCCGCGCCAGCAGCACTTTGAGTTACAACGGCACCACGTCGGCCTACCCCAGC CACCCCATGCCCTACAGCTCCGTGTTGACTCAGAACTCGCTGGGCAACAACCACTCCTTCTCCACC GCCAACGGCCTGAGCGTGGACCGGCTGGTCAACGGGGAGATCCCGTACGCCACGCACCACCTCAC GGCCGCCGCGCTAGCCGCCTCGGTGCCCTGCGGCCTGTCGGTGCCCTGCTCTGGGACCTACTCCCT CAACCCCTGCTCCGTCAACCTGCTCGCGGGCCAGACCAGTTACTTTTTCCCCCACGTCCCGCACCC GTCAATGACTTCGCAGAGCAGCACGTCCATGAGCGCCAGGGCCGCGTCCTCCTCCACGTCGCCGCA GGCCCCCTCGACCCTGCCCTGTGAGTCTTTAAGACCCTCTTTGCCAAGTTTTACGACGGGACTGTCT GGGGGACTGTCTGATTATTTCACACATCAAAATCAGGGGTCTTCTTCCAACCCTTTAATACATTAA CATCCCTGGGACCAGACTGTAAGTGAACGTTTTACACACATTTGCATTGTAAATGATAATTAAAAA AATAAGTCCAGGTATTTTTTATTAAGCCCCCCCCTCCCATTTCTGTACGTTTGTTCAGTCTCTAGGG TTGTTTATTATTCTAACAAGGTGTGGAGTGTCAGCGAGGTGCAATGTGGGGAGAATACATTGTAGA ATATAAGGTTTGGAAGTCAAATTATAGTAGAATGTGTATCTAAATAGTGACTGCTTTGCCATTTCA TTCAAACCTGACAAGTCTATCTCTAAGAGCCGCCAGATTTCCATGTGTGCAGTATTATAAGTTATC ATGGAACTATATGGTGGACGCAGACCTTGAGAACAACCTAAATTATGGGGAGAATTTTAAAATGT TAAACTGTAATTTGTATTTAAAAAGCATTCGTAGTAAAGGTGCCCAAGAAATTATTTTGGCCATTT ATTGTTTTGTCCTTTTCTTTAAAGAACTGTTTTTTTTTCTTTTGTTTACTTTTAGACCAAAGATTGGG TTCTAGAAAATGCACTTGGTATACTAAGTATTAAAACAAACAAAAAGGAAAGTTGTTTCAGTTGGC AACACTGCCCATTCAATTGAATCAGAAGGGGACAAAATTAACGATTGCCTTCAGTTTGTGTTGTGT ATATTTTGATGTATGTGGTCACTAACAGGTCACTTTTATTTTTTCTAAATGTAGTGAAATGTTAATA CCTATTGTACTTATAGGTAAACCTTGCAAATATGTAACCTGTGTTGCGCAAATGCCGCATAAATTT GAGTGATTGTTAATGTTGTCTTAAAATTTCTTGATTGTGATACTGTGGTCATATGCCCGTGTTTGTC ACTTACAAAAATGTTTACTATGAACACACAGAAATAAAAAATAGGCTAAATTCATATATATCTTGA TACTTTTGTCTCTTTTATTAAGTAGAGCTAATTTTTTAAAGACCAATCAACTTATAGGGAATTCAAADocket No.062686-510001WO GGCTTTTTCAGCCAAACTAAAATTTAAACTGCTCCTTTAATTTGAACTGACTCTAAAAATGAAAAT AGTATTTTTCCCTTTGTGAACAAATTTTACAAGGAGCAGCCTATTTAATAAACACTAGCTTTAAAC AAAGTATAGGCTTTTCAGCTGATACCTGTAAGTTTCTGTGGATATACAGCAAAAAGAGATATAATT TAATTTTCTGTGCATAGCTCTTTACCCTGTGTTTATTTCCAAATCCATTAATAGAATGCCATTTATAT ATTTTGTTTCAGGTATATTGTTAATAGAGCTTGGCAAATTATAAATAAATATATGTATATGGTTAGA TAGAAGTGACTATAATGCACACATATGTAATATATATAGACACACAGAGCCCTTCAGTTCAGGTAC AATTTGCGCTATGAATGCTGCAAACATTTTTGTTTAAATATTTGTATTTATACTTTCTAAGTCAGCA TTTATTTTTGTGGCTGTTTACCCACAATGAAAGAGTTCTAATAAAGATGTGCTGAAGTTGCAATAT A Codon optimized FOXG1 nucleic acid sequence (SEQ ID NO: 3) ATGCTGGACATGGGCGATAGGAAGGAAGTCAAGATGATCCCCAAGAGTAGTTTCTCAATCAATAGCC TGGTGCCCGAAGCCGTGCAGAACGATAATCACCACGCCAGCCACGGCCACCACAACTCCCACCACC CTCAGCACCATCATCACCATCATCACCACCACCACCACCCACCTCCACCAGCACCACAGCCTCCACC CCCTCCACAGCAGCAGCAGCCTCCTCCTCCACCTCCACCAGCACCCCAGCCTCCACAGACCCGCGG CGCCCCTGCCGCCGACGATGACAAGGGACCACAGCAGCTGCTGCTGCCTCCTCCACCCCCTCCACC CCCTGCCGCCGCCCTGGATGGCGCCAAGGCCGACGGCCTGGGAGGCAAGGGAGAGCCTGGAGGAG GACCAGGCGAGCTGGCCCCAGTGGGCCCCGATGAGAAGGAGAAGGGAGCAGGAGCAGGAGGAGA GGAGAAGAAGGGCGCCGGCGAGGGCGGCAAGGATGGAGAGGGCGGCAAGGAGGGCGAGAAGAA GAACGGCAAGTACGAGAAGCCACCCTTCTCTTATAATGCCCTGATCATGATGGCCATCAGACAGAGC CCCGAGAAGAGGCTGACCCTGAACGGCATCTATGAGTTCATCATGAAGAATTTTCCTTACTATCGCG AGAACAAGCAGGGCTGGCAGAATTCTATCCGGCACAACCTGAGCCTGAATAAGTGCTTCGTGAAGG TGCCCAGACACTATGATGACCCTGGCAAGGGCAATTACTGGATGCTGGATCCCAGCTCCGATGACGT GTTTATCGGCGGCACCACAGGCAAGCTGCGGAGAAGGAGCACCACATCCAGGGCAAAGCTGGCCT TCAAGAGGGGAGCAAGGCTGACCAGCACAGGCCTGACCTTTATGGACAGAGCCGGCTCCCTGTATT GGCCTATGAGCCCATTCCTGTCCCTGCACCACCCAAGGGCCTCTAGCACACTGAGCTACAACGGCA CCACATCTGCCTATCCCAGCCACCCTATGCCATACTCCTCTGTGCTGACCCAGAATAGCCTGGGCAAC AATCACTCTTTTAGCACAGCAAACGGCCTGTCCGTGGACAGGCTGGTGAATGGCGAGATCCCATAC GCTACCCACCACCTGACAGCAGCCGCCCTGGCAGCATCCGTGCCATGCGGCCTGTCCGTGCCCTGT TCTGGCACCTATAGCCTGAACCCCTGCTCCGTGAATCTGCTGGCCGGCCAGACATCTTACTTCTTTCC TCACGTGCCCCACCCTTCTATGACCAGCCAGAGCTCCACATCCATGTCTGCCAGGGCAGCATCTAGC TCCACCTCCCCACAGGCCCCTTCTACACTGCCTTGTGAGTCCCTGCGGCCATCCCTGCCCTCTTTTAC CACAGGCCTGTCTGGCGGCCTGTCCGATTACTTCACCCACCAGAACCAGGGCTCCTCCTCAAACCC ACTGATTCACTAA CB6-PI promoter nucleic acid sequence (SEQ ID NO: 4) CTAGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCAC TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTTATTGC GGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGC TGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGA CCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTT ACTGACATCCACTTTGCCTTTCTCTCCACAG U1 promoter nucleic acid sequence (SEQ ID NO: 5) ATGGAGGCGGTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAATAT TTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCTAAGTGTTTTAAAATGDocket No.062686-510001WO TGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGAGGCTTAAATATTTACCGTAACTATGAAA TGCTACGCATATCATGCTGTTCAGGCTCCGTGGCCACGCAACTCATACT hSynl promoter nucleic acid sequence (SEQ ID NO: 6) GAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACC TGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATC AGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGG ACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGAC GTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGG CCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCG GCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGA GCGCAGTCGAGA hCAMKII promoter nucleic acid sequence (SEQ ID NO: 7) ACTTGTGGACTAAGTTTGTTCGCATCCCCTTCTCCAACCCCCTCAGTACATCACCCTGGGGGAACAG GGTCCACTTGCTCCTGGGCCCACACAGTCCTGCAGTATTGTGTATATAAGGCCAGGGCAAAGAGGA GCAGGTTTTAAAGTGAAAGGCAGGCAGGTGTTGGGGAGGCAGTTACCGGGGCAACGGGAACAGG GCGTTTCGGAGGTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGC CACAGTGCCCTGCTCAGAAGCCCCAAGCTCGTCAGTCAAGCCGGTTCTCCGTTTGCACTCAGGAGC ACGGGCAGGCGAGTGGCCCCTAGTTCTGGGGGCAG pAAV.CB6-PI-hFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 8) CTAGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCC ATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCC CCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAG TACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGT TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTAT TTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGG CGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTC CTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGC AAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGT CTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGAC AGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAG GCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATT ACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGGCCACCATGCTGG ACATGGGAGATAGGAAAGAGGTGAAAATGATCCCCAAGTCCTCGTTCAGCATCAACAGCCTGGTG CCCGAGGCGGTCCAGAACGACAACCACCACGCGAGCCACGGCCACCACAACAGCCACCACCCCCA GCACCACCACCACCACCACCACCATCACCACCACCCGCCGCCGCCCGCCCCGCAACCGCCGCCGC CGCCGCAGCAGCAGCAGCCGCCGCCGCCGCCGCCCCCGGCACCGCAGCCCCCCCAGACGCGGGGC GCCCCGGCCGCCGACGACGACAAGGGCCCCCAGCAGCTGCTGCTCCCGCCGCCGCCACCGCCACC ACCGGCCGCCGCCCTGGACGGGGCTAAAGCGGACGGGCTGGGCGGCAAGGGCGAGCCGGGCGGC GGGCCGGGGGAGCTGGCGCCCGTCGGGCCGGACGAGAAGGAGAAGGGCGCCGGCGCCGGGGGGG AGGAGAAGAAGGGGGCGGGCGAGGGCGGCAAGGACGGGGAGGGGGGCAAGGAGGGCGAGAAGA AGAACGGCAAGTACGAGAAGCCGCCGTTCAGCTACAACGCGCTCATCATGATGGCCATCCGGCAG AGCCCCGAGAAGCGGCTCACGCTCAACGGCATCTACGAGTTCATCATGAAGAACTTCCCTTACTAC CGCGAGAACAAGCAGGGCTGGCAGAACTCCATCCGCCACAATCTGTCCCTCAACAAGTGCTTCGT GAAGGTGCCGCGCCACTACGACGACCCGGGCAAGGGCAACTACTGGATGCTGGACCCGTCGAGCG ACGACGTGTTCATCGGCGGCACCACGGGCAAGCTGCGGCGCCGCTCCACCACCTCGCGGGCCAAG CTGGCCTTCAAGCGCGGTGCGCGCCTCACCTCCACCGGCCTCACCTTCATGGACCGCGCCGGCTCC CTCTACTGGCCCATGTCGCCCTTCCTGTCCCTGCACCACCCCCGCGCCAGCAGCACTTTGAGTTACA ACGGCACCACGTCGGCCTACCCCAGCCACCCCATGCCCTACAGCTCCGTGTTGACTCAGAACTCGC TGGGCAACAACCACTCCTTCTCCACCGCCAACGGCCTGAGCGTGGACCGGCTGGTCAACGGGGAGDocket No.062686-510001WO ATCCCGTACGCCACGCACCACCTCACGGCCGCCGCGCTAGCCGCCTCGGTGCCCTGCGGCCTGTCG GTGCCCTGCTCTGGGACCTACTCCCTCAACCCCTGCTCCGTCAACCTGCTCGCGGGCCAGACCAGT TACTTTTTCCCCCACGTCCCGCACCCGTCAATGACTTCGCAGAGCAGCACGTCCATGAGCGCCAGG GCCGCGTCCTCCTCCACGTCGCCGCAGGCCCCCTCGACCCTGCCCTGTGAGTCTTTAAGACCCTCTT TGCCAAGTTTTACGACGGGACTGTCTGGGGGACTGTCTGATTATTTCACACATCAAAATCAGGGGT CTTCTTCCAACCCTTTAATACATTAAGGTACCTCTAGAGTCGAGGACGGGGTGAACTACGCCTGAG GATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTC TGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG pAAV.CB6-PI-opthFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 9) CTAGTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCAT ATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCG CCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGTCGAGGCCACGTTCTGCTTCAC TCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG ATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGG CGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCG AGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCAAGCTTTATTGC GGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGC TGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGA CCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTT ACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTA GAGTACTTAATACGACTCACTATAGGCTAGCCTCGAGGCCACCATGCTGGACATGGGCGATAGGAAG GAAGTCAAGATGATCCCCAAGAGTAGTTTCTCAATCAATAGCCTGGTGCCCGAAGCCGTGCAGAAC GATAATCACCACGCCAGCCACGGCCACCACAACTCCCACCACCCTCAGCACCATCATCACCATCATC ACCACCACCACCACCCACCTCCACCAGCACCACAGCCTCCACCCCCTCCACAGCAGCAGCAGCCTC CTCCTCCACCTCCACCAGCACCCCAGCCTCCACAGACCCGCGGCGCCCCTGCCGCCGACGATGACA AGGGACCACAGCAGCTGCTGCTGCCTCCTCCACCCCCTCCACCCCCTGCCGCCGCCCTGGATGGCG CCAAGGCCGACGGCCTGGGAGGCAAGGGAGAGCCTGGAGGAGGACCAGGCGAGCTGGCCCCAGT GGGCCCCGATGAGAAGGAGAAGGGAGCAGGAGCAGGAGGAGAGGAGAAGAAGGGCGCCGGCGA GGGCGGCAAGGATGGAGAGGGCGGCAAGGAGGGCGAGAAGAAGAACGGCAAGTACGAGAAGCC ACCCTTCTCTTATAATGCCCTGATCATGATGGCCATCAGACAGAGCCCCGAGAAGAGGCTGACCCTG AACGGCATCTATGAGTTCATCATGAAGAATTTTCCTTACTATCGCGAGAACAAGCAGGGCTGGCAGA ATTCTATCCGGCACAACCTGAGCCTGAATAAGTGCTTCGTGAAGGTGCCCAGACACTATGATGACCC TGGCAAGGGCAATTACTGGATGCTGGATCCCAGCTCCGATGACGTGTTTATCGGCGGCACCACAGGC AAGCTGCGGAGAAGGAGCACCACATCCAGGGCAAAGCTGGCCTTCAAGAGGGGAGCAAGGCTGA CCAGCACAGGCCTGACCTTTATGGACAGAGCCGGCTCCCTGTATTGGCCTATGAGCCCATTCCTGTC CCTGCACCACCCAAGGGCCTCTAGCACACTGAGCTACAACGGCACCACATCTGCCTATCCCAGCCA CCCTATGCCATACTCCTCTGTGCTGACCCAGAATAGCCTGGGCAACAATCACTCTTTTAGCACAGCA AACGGCCTGTCCGTGGACAGGCTGGTGAATGGCGAGATCCCATACGCTACCCACCACCTGACAGCA GCCGCCCTGGCAGCATCCGTGCCATGCGGCCTGTCCGTGCCCTGTTCTGGCACCTATAGCCTGAACC CCTGCTCCGTGAATCTGCTGGCCGGCCAGACATCTTACTTCTTTCCTCACGTGCCCCACCCTTCTATG ACCAGCCAGAGCTCCACATCCATGTCTGCCAGGGCAGCATCTAGCTCCACCTCCCCACAGGCCCCTT CTACACTGCCTTGTGAGTCCCTGCGGCCATCCCTGCCCTCTTTTACCACAGGCCTGTCTGGCGGCCT GTCCGATTACTTCACCCACCAGAACCAGGGCTCCTCCTCAAACCCACTGATTCACTAAGGTACCTCT AGAGTCGAGGACGGGGTGAACTACGCCTGAGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGG GACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGT GTGTTGGAATTTTTTGTGTCTCTCACTCG pAAVsc.Ula-hFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 10) ATGGAGGCGGTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAATAT TTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCTAAGTGTTTTAAAATG TGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGAGGCTTAAATATTTACCGTAACTATGAAA TGCTACGCATATCATGCTGTTCAGGCTCCGTGGCCACGCAACTCATACTACCGGTGCCACCATGCTGDocket No.062686-510001WO GACATGGGAGATAGGAAAGAGGTGAAAATGATCCCCAAGTCCTCGTTCAGCATCAACAGCCTGGTG CCCGAGGCGGTCCAGAACGACAACCACCACGCGAGCCACGGCCACCACAACAGCCACCACCCCCA GCACCACCACCACCACCACCACCATCACCACCACCCGCCGCCGCCCGCCCCGCAACCGCCGCCGCC GCCGCAGCAGCAGCAGCCGCCGCCGCCGCCGCCCCCGGCACCGCAGCCCCCCCAGACGCGGGGCG CCCCGGCCGCCGACGACGACAAGGGCCCCCAGCAGCTGCTGCTCCCGCCGCCGCCACCGCCACCA CCGGCCGCCGCCCTGGACGGGGCTAAAGCGGACGGGCTGGGCGGCAAGGGCGAGCCGGGCGGCG GGCCGGGGGAGCTGGCGCCCGTCGGGCCGGACGAGAAGGAGAAGGGCGCCGGCGCCGGGGGGGA GGAGAAGAAGGGGGCGGGCGAGGGCGGCAAGGACGGGGAGGGGGGCAAGGAGGGCGAGAAGAA GAACGGCAAGTACGAGAAGCCGCCGTTCAGCTACAACGCGCTCATCATGATGGCCATCCGGCAGAG CCCCGAGAAGCGGCTCACGCTCAACGGCATCTACGAGTTCATCATGAAGAACTTCCCTTACTACCGC GAGAACAAGCAGGGCTGGCAGAACTCCATCCGCCACAATCTGTCCCTCAACAAGTGCTTCGTGAAG GTGCCGCGCCACTACGACGACCCGGGCAAGGGCAACTACTGGATGCTGGACCCGTCGAGCGACGA CGTGTTCATCGGCGGCACCACGGGCAAGCTGCGGCGCCGCTCCACCACCTCGCGGGCCAAGCTGG CCTTCAAGCGCGGTGCGCGCCTCACCTCCACCGGCCTCACCTTCATGGACCGCGCCGGCTCCCTCTA CTGGCCCATGTCGCCCTTCCTGTCCCTGCACCACCCCCGCGCCAGCAGCACTTTGAGTTACAACGGC ACCACGTCGGCCTACCCCAGCCACCCCATGCCCTACAGCTCCGTGTTGACTCAGAACTCGCTGGGC AACAACCACTCCTTCTCCACCGCCAACGGCCTGAGCGTGGACCGGCTGGTCAACGGGGAGATCCC GTACGCCACGCACCACCTCACGGCCGCCGCGCTAGCCGCCTCGGTGCCCTGCGGCCTGTCGGTGCC CTGCTCTGGGACCTACTCCCTCAACCCCTGCTCCGTCAACCTGCTCGCGGGCCAGACCAGTTACTTT TTCCCCCACGTCCCGCACCCGTCAATGACTTCGCAGAGCAGCACGTCCATGAGCGCCAGGGCCGCG TCCTCCTCCACGTCGCCGCAGGCCCCCTCGACCCTGCCCTGTGAGTCTTTAAGACCCTCTTTGCCAA GTTTTACGACGGGACTGTCTGGGGGACTGTCTGATTATTTCACACATCAAAATCAGGGGTCTTCTTC CAACCCTTTAATACATTAAGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGC CCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTT TGTGTCTCTCACTCG pAAVsc.Ula-opthFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 11) ATGGAGGCGGTACTATGTAGATGAGAATTCAGGAGCAAACTGGGAAAAGCAACTGCTTCCAAATAT TTGTGATTTTTACAGTGTAGTTTTGGAAAAACTCTTAGCCTACCAATTCTTCTAAGTGTTTTAAAATG TGGGAGCCAGTACACATGAAGTTATAGAGTGTTTTAATGAGGCTTAAATATTTACCGTAACTATGAAA TGCTACGCATATCATGCTGTTCAGGCTCCGTGGCCACGCAACTCATACTACCGGTGCCACCATGCTG GACATGGGCGATAGGAAGGAAGTCAAGATGATCCCCAAGAGTAGTTTCTCAATCAATAGCCTGGTG CCCGAAGCCGTGCAGAACGATAATCACCACGCCAGCCACGGCCACCACAACTCCCACCACCCTCAG CACCATCATCACCATCATCACCACCACCACCACCCACCTCCACCAGCACCACAGCCTCCACCCCCTC CACAGCAGCAGCAGCCTCCTCCTCCACCTCCACCAGCACCCCAGCCTCCACAGACCCGCGGCGCCC CTGCCGCCGACGATGACAAGGGACCACAGCAGCTGCTGCTGCCTCCTCCACCCCCTCCACCCCCTG CCGCCGCCCTGGATGGCGCCAAGGCCGACGGCCTGGGAGGCAAGGGAGAGCCTGGAGGAGGACC AGGCGAGCTGGCCCCAGTGGGCCCCGATGAGAAGGAGAAGGGAGCAGGAGCAGGAGGAGAGGAG AAGAAGGGCGCCGGCGAGGGCGGCAAGGATGGAGAGGGCGGCAAGGAGGGCGAGAAGAAGAAC GGCAAGTACGAGAAGCCACCCTTCTCTTATAATGCCCTGATCATGATGGCCATCAGACAGAGCCCCG AGAAGAGGCTGACCCTGAACGGCATCTATGAGTTCATCATGAAGAATTTTCCTTACTATCGCGAGAA CAAGCAGGGCTGGCAGAATTCTATCCGGCACAACCTGAGCCTGAATAAGTGCTTCGTGAAGGTGCC CAGACACTATGATGACCCTGGCAAGGGCAATTACTGGATGCTGGATCCCAGCTCCGATGACGTGTTT ATCGGCGGCACCACAGGCAAGCTGCGGAGAAGGAGCACCACATCCAGGGCAAAGCTGGCCTTCAA GAGGGGAGCAAGGCTGACCAGCACAGGCCTGACCTTTATGGACAGAGCCGGCTCCCTGTATTGGCC TATGAGCCCATTCCTGTCCCTGCACCACCCAAGGGCCTCTAGCACACTGAGCTACAACGGCACCAC ATCTGCCTATCCCAGCCACCCTATGCCATACTCCTCTGTGCTGACCCAGAATAGCCTGGGCAACAATC ACTCTTTTAGCACAGCAAACGGCCTGTCCGTGGACAGGCTGGTGAATGGCGAGATCCCATACGCTA CCCACCACCTGACAGCAGCCGCCCTGGCAGCATCCGTGCCATGCGGCCTGTCCGTGCCCTGTTCTG GCACCTATAGCCTGAACCCCTGCTCCGTGAATCTGCTGGCCGGCCAGACATCTTACTTCTTTCCTCAC GTGCCCCACCCTTCTATGACCAGCCAGAGCTCCACATCCATGTCTGCCAGGGCAGCATCTAGCTCCA CCTCCCCACAGGCCCCTTCTACACTGCCTTGTGAGTCCCTGCGGCCATCCCTGCCCTCTTTTACCAC AGGCCTGTCTGGCGGCCTGTCCGATTACTTCACCCACCAGAACCAGGGCTCCTCCTCAAACCCACT GATTCACTAAGGATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGC ATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCT CACTCGDocket No.062686-510001WO pAAVsc.hSynl-opthFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 12) GAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACC TGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATC AGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGG ACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGAC GTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGG CCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCG GCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGA GCGCAGTCGAGAACCGGTGCCACCATGCTGGACATGGGCGATAGGAAGGAAGTCAAGATGATCCCC AAGAGTAGTTTCTCAATCAATAGCCTGGTGCCCGAAGCCGTGCAGAACGATAATCACCACGCCAGC CACGGCCACCACAACTCCCACCACCCTCAGCACCATCATCACCATCATCACCACCACCACCACCCA CCTCCACCAGCACCACAGCCTCCACCCCCTCCACAGCAGCAGCAGCCTCCTCCTCCACCTCCACCA GCACCCCAGCCTCCACAGACCCGCGGCGCCCCTGCCGCCGACGATGACAAGGGACCACAGCAGCT GCTGCTGCCTCCTCCACCCCCTCCACCCCCTGCCGCCGCCCTGGATGGCGCCAAGGCCGACGGCCT GGGAGGCAAGGGAGAGCCTGGAGGAGGACCAGGCGAGCTGGCCCCAGTGGGCCCCGATGAGAAG GAGAAGGGAGCAGGAGCAGGAGGAGAGGAGAAGAAGGGCGCCGGCGAGGGCGGCAAGGATGGA GAGGGCGGCAAGGAGGGCGAGAAGAAGAACGGCAAGTACGAGAAGCCACCCTTCTCTTATAATGC CCTGATCATGATGGCCATCAGACAGAGCCCCGAGAAGAGGCTGACCCTGAACGGCATCTATGAGTT CATCATGAAGAATTTTCCTTACTATCGCGAGAACAAGCAGGGCTGGCAGAATTCTATCCGGCACAAC CTGAGCCTGAATAAGTGCTTCGTGAAGGTGCCCAGACACTATGATGACCCTGGCAAGGGCAATTAC TGGATGCTGGATCCCAGCTCCGATGACGTGTTTATCGGCGGCACCACAGGCAAGCTGCGGAGAAGG AGCACCACATCCAGGGCAAAGCTGGCCTTCAAGAGGGGAGCAAGGCTGACCAGCACAGGCCTGAC CTTTATGGACAGAGCCGGCTCCCTGTATTGGCCTATGAGCCCATTCCTGTCCCTGCACCACCCAAGG GCCTCTAGCACACTGAGCTACAACGGCACCACATCTGCCTATCCCAGCCACCCTATGCCATACTCCT CTGTGCTGACCCAGAATAGCCTGGGCAACAATCACTCTTTTAGCACAGCAAACGGCCTGTCCGTGG ACAGGCTGGTGAATGGCGAGATCCCATACGCTACCCACCACCTGACAGCAGCCGCCCTGGCAGCAT CCGTGCCATGCGGCCTGTCCGTGCCCTGTTCTGGCACCTATAGCCTGAACCCCTGCTCCGTGAATCT GCTGGCCGGCCAGACATCTTACTTCTTTCCTCACGTGCCCCACCCTTCTATGACCAGCCAGAGCTCC ACATCCATGTCTGCCAGGGCAGCATCTAGCTCCACCTCCCCACAGGCCCCTTCTACACTGCCTTGTG AGTCCCTGCGGCCATCCCTGCCCTCTTTTACCACAGGCCTGTCTGGCGGCCTGTCCGATTACTTCAC CCACCAGAACCAGGGCTCCTCCTCAAACCCACTGATTCACTAAGGATCCGATCTTTTTCCCTCTGCC AAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTT CATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG pAAVsc.hCAMKII-opthFOXGl nucleic acid sequence; no ITRs (SEQ ID NO: 13) ACTTGTGGACTAAGTTTGTTCGCATCCCCTTCTCCAACCCCCTCAGTACATCACCCTGGGGGAACAG GGTCCACTTGCTCCTGGGCCCACACAGTCCTGCAGTATTGTGTATATAAGGCCAGGGCAAAGAGGA GCAGGTTTTAAAGTGAAAGGCAGGCAGGTGTTGGGGAGGCAGTTACCGGGGCAACGGGAACAGG GCGTTTCGGAGGTGGTTGCCATGGGGACCTGGATGCTGACGAAGGCTCGCGAGGCTGTGAGCAGC CACAGTGCCCTGCTCAGAAGCCCCAAGCTCGTCAGTCAAGCCGGTTCTCCGTTTGCACTCAGGAGC ACGGGCAGGCGAGTGGCCCCTAGTTCTGGGGGCAGACCGGTGCCACCATGCTGGACATGGGCGATA GGAAGGAAGTCAAGATGATCCCCAAGAGTAGTTTCTCAATCAATAGCCTGGTGCCCGAAGCCGTGC AGAACGATAATCACCACGCCAGCCACGGCCACCACAACTCCCACCACCCTCAGCACCATCATCACC ATCATCACCACCACCACCACCCACCTCCACCAGCACCACAGCCTCCACCCCCTCCACAGCAGCAGC AGCCTCCTCCTCCACCTCCACCAGCACCCCAGCCTCCACAGACCCGCGGCGCCCCTGCCGCCGACG ATGACAAGGGACCACAGCAGCTGCTGCTGCCTCCTCCACCCCCTCCACCCCCTGCCGCCGCCCTGG ATGGCGCCAAGGCCGACGGCCTGGGAGGCAAGGGAGAGCCTGGAGGAGGACCAGGCGAGCTGGC CCCAGTGGGCCCCGATGAGAAGGAGAAGGGAGCAGGAGCAGGAGGAGAGGAGAAGAAGGGCGC CGGCGAGGGCGGCAAGGATGGAGAGGGCGGCAAGGAGGGCGAGAAGAAGAACGGCAAGTACGA GAAGCCACCCTTCTCTTATAATGCCCTGATCATGATGGCCATCAGACAGAGCCCCGAGAAGAGGCTG ACCCTGAACGGCATCTATGAGTTCATCATGAAGAATTTTCCTTACTATCGCGAGAACAAGCAGGGCT GGCAGAATTCTATCCGGCACAACCTGAGCCTGAATAAGTGCTTCGTGAAGGTGCCCAGACACTATG ATGACCCTGGCAAGGGCAATTACTGGATGCTGGATCCCAGCTCCGATGACGTGTTTATCGGCGGCAC CACAGGCAAGCTGCGGAGAAGGAGCACCACATCCAGGGCAAAGCTGGCCTTCAAGAGGGGAGCA AGGCTGACCAGCACAGGCCTGACCTTTATGGACAGAGCCGGCTCCCTGTATTGGCCTATGAGCCCAT TCCTGTCCCTGCACCACCCAAGGGCCTCTAGCACACTGAGCTACAACGGCACCACATCTGCCTATCC CAGCCACCCTATGCCATACTCCTCTGTGCTGACCCAGAATAGCCTGGGCAACAATCACTCTTTTAGCDocket No.062686-510001WO ACAGCAAACGGCCTGTCCGTGGACAGGCTGGTGAATGGCGAGATCCCATACGCTACCCACCACCTG ACAGCAGCCGCCCTGGCAGCATCCGTGCCATGCGGCCTGTCCGTGCCCTGTTCTGGCACCTATAGCC TGAACCCCTGCTCCGTGAATCTGCTGGCCGGCCAGACATCTTACTTCTTTCCTCACGTGCCCCACCC TTCTATGACCAGCCAGAGCTCCACATCCATGTCTGCCAGGGCAGCATCTAGCTCCACCTCCCCACAG GCCCCTTCTACACTGCCTTGTGAGTCCCTGCGGCCATCCCTGCCCTCTTTTACCACAGGCCTGTCTG GCGGCCTGTCCGATTACTTCACCCACCAGAACCAGGGCTCCTCCTCAAACCCACTGATTCACTAAGG ATCCGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTG GCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCG AAV2 ITR nucleic acid sequence (SEQ ID NO: 14) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCG ACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCAC TAGGGGTTCCT

Claims

Docket No.062686-510001WO CLAIMS What is claimed is:

1. A method of delivering a forkhead box G1 (FOXG1) protein to the central nervous system (CNS) in a human subject in need thereof, comprising administering to the subject an effective amount of a recombinant adeno-associated virus (rAAV) comprising a nucleic acid payload encoding the FOXG1 protein, wherein the rAAV is administered by injection into the cerebrospinal fluid (CSF) via an intracerebroventricular (ICV) route.

2. A method of delivering a FOXG1 protein to telencephalon cells in a human subject in need thereof, comprising administering to the subject an effective amount of an rAAV comprising a nucleic acid payload encoding the FOXG1 protein, wherein the rAAV is administered by injection into the CSF via an ICV route.

3. A method of treating FOXG1 deficiency in a human subject in need thereof, the method comprising administering an effective amount of a rAAV comprising a nucleic acid payload encoding a FOXG1 protein to the subject by ICV injection.

4. A method of treating FOXG1 haploinsufficiency in a human subject in need thereof, the method comprising administering an rAAV comprising a nucleic acid payload encoding a FOXG1 protein to the subject by ICV injection.

5. A method of preventing, improving, or treating FOXG1 syndrome in a human subject, the method comprising administering an effective amount of an rAAV comprising a nucleic acid payload encoding the FOXG1 protein to the subject by ICV injection.

6. A method of reducing anxiety in a human subject suffering from FOXG1 syndrome, the method comprising, administering an effective amount of an rAAV comprising a nucleic acid payload encoding the FOXG1 protein to the subject by ICV injection.

7. The method of claim 6, wherein the anxiety in the subject is related to a novel or unfamiliar environment and adapting to this novel environment.

8. A method of improving behavior deficits related to anxiety in a human subject suffering from FOXG1 syndrome, the method comprising, administering an effective amount of an rAAV comprising a nucleic acid payload encoding the FOXG1 protein to the subject by ICV injection.

9. The method of claim 8, wherein the anxiety in the subject is related to a novel or unfamiliar environment and adapting to this novel environment.

10. A method of improving sleep patterns in a human subject suffering from FOXG1 syndrome, the method comprising, administering an effective amount of an rAAV comprising a nucleic acid payload encoding the FOXG1 protein to the subject by ICV injection.

11. The method of claim 10, wherein the improvement in sleep pattern is increased time spent awake.Docket No.062686-510001WO 12. The method of claim 10, wherein the improvement in sleep pattern is less fragmented NREM sleep.

13. The method of any one of claims 1-12, wherein the subject has one or more mutations in a FOXG1 gene.

14. The method of any one of claims 1-4, wherein the subject has FOXG1 syndrome.

15. The method of any one of claims 1-12, wherein the human subject is an infant.

16. The method of any one of claims 1-12, wherein the human subject is a child.

17. The method of any one of claims 1-12, wherein the human subject is an adult.

18. The method of any one of claims 1-12, wherein the ICV injection is a bilateral ICV injection.

19. The method of any one of claims 1-12, wherein the payload is self-complementary (sc).

20. The method of any one of claims 1-12, wherein the payload is single-stranded (ss).

21. The method of any one of claims 1-12, wherein the rAAV further comprises at least one AAV capsid protein.

22. The method of claim 21, wherein the at least one capsid protein is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.PHP-eB, or a variant of any of the foregoing.

23. The method of claim 21, wherein the at least one capsid protein is an AAV9 capsid protein or an AAV.PHP-eB capsid protein.

24. The method of claim 21, wherein the at least one AAV capsid protein has a tropism for central nervous system (CNS) cells.

25. The method of claim 21, wherein the at least one AAV capsid protein has a tropism for telencephalon cells.

26. The method of any one of claims 1-12, wherein the payload comprises an expression cassette having a transgene that encodes the FOXG1 protein.

27. The method of claim 26, wherein the expression cassette further comprises adeno- associated virus (AAV) inverted terminal repeats (ITRs) operably linked to the transgene encoding FOXG1 protein.

28. The method of claim 27, wherein the AAV ITRs are AAV2 ITR.

29. The method of claim 28, wherein at least one of the AAV2 ITRs comprises the sequence set forth in SEQ ID NO:

14.

30. The method of claim 27, wherein at least one of the ITRs is a truncated ITR (AITR).

31. The method of any one of claims 1-12, wherein the FOXG1 protein comprises the amino acid sequence as set forth in SEQ ID NO:

1.

32. The method of any one of claims 1-29, wherein the nucleic acid payload encoding the FOXG1 protein comprises the nucleic acid sequence as set forth in any one of SEQ ID NOs: 2, 3, and 8-13.Docket No.062686-510001WO 33. The method of claim 26, wherein the expression cassette further comprises a promoter that is operably linked to the transgene encoding the FOXG1 protein.

34. The method of claim 33, wherein the promoter is a constitutive promoter, inducible promoter, or tissue-specific promoter.

35. The method of claim 33, wherein the promoter comprises a chicken beta-actin (CB) promoter, a Ula promoter, or a neuron-specific promoter.

36. The method of claim 33, wherein the promoter comprises a neuron-specific promoter comprising a human synapsin 1 (hSynl) promoter or a human Ca2+ / calmodulin-dependent protein kinase II (hCAMKII) promoter.

37. The method of claim 33, wherein the promoter comprises the sequence set forth in any one of SEQ ID NOs: 4 to 7.

38. The method of claim 26, wherein the transgene encoding the FOXG1 protein comprises a codon-optimized nucleic acid sequence.

39. The method of claim 38, wherein the codon-optimized nucleic acid sequence comprises the sequence set forth in SEQ ID NO:

3.

40. The method of any one of claims 1-12, wherein the rAAV is formulated for intracerebroventricular (ICV) delivery.

41. The method of any one of claims 1-12, wherein the administration restores at least 5%, 10%, 20%, 30%, or 40% FOXG1 protein levels in cells relative to untreated cells, or relative to a baseline measurement.

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