Gene therapy platform to restore gabaergic inhibition and improve cognition

Abstract

Described herein is a gene therapy platform to restore GABAergic inhibition and improve cognition in neurodevelopmental disorders, epilepsies, sensory disorders, aging, and Alzheimer's disease, by increasing expression of the transcription factor Meis2 in parvalbumin-expressing inhibitory neurons (PV INs).

Description

[0001]Attorney Docket No.29539-0804WO1 / MGH 2023-602 Gene Therapy Platform to Restore GABAergic Inhibition and Improve Cognition CLAIM OF PRIORITY This application claims the benefit of U.S. Provisional Application Serial Nos. 63 / 697,722, filed on September 23, 2024, and 63 / 734,053, filed on December 14, 2024. The entire contents of the foregoing are incorporated herein by reference. TECHNICAL FIELD Described herein is a gene therapy platform to restore GABAergic inhibition and improve cognition in neurodevelopmental disorders, epilepsies, sensory disorders, aging, and Alzheimer's disease, by increasing expression of the transcription factor Meis2 in parvalbumin-expressing inhibitory neurons (PV INs). BACKGROUND The hippocampus plays a crucial role in formation, storage and retrieval of memories, allowing for calibration of motivated and defensive behaviors2. During the early postnatal period, experience refines hippocampal circuitry to influence cognitive trajectory22, disruption of which results in maladaptive neural circuit functions such as cognitive impairments and epilepsy that characterize different neurodevelopmental disorders (NDDs), including autism spectrum disorders (ASDs). The identification of ultra-high confidence genetic risk factors for NDDs underscores the need to understand how experience and genetic risk conspire to compromise experience- dependent mechanisms instrumental for critical hippocampal functions. SUMMARY Provided herein are methods of treating a subject to reduce or reverse cognitive impairment and reduce seizures in disorders associated with excitation / inhibition imbalance. The methods comprise administering a therapeutically effective amount of an expression vector comprising a promoter that directs expression of a transgene sequence encoding an experience-dependent parvalbumin-expressing inhibitory neuron (PV IN) plasticity (XP) protein selected from human Meis2, Tbr1, Bcl11a, and Herc1 in parvalbumin-expressing inhibitory neurons (PV INs), and optionally an SE52 enhancer. Alternatively, the methods can Attorney Docket No.29539-0804WO1 / MGH 2023-602 include administering a dCas9 activator fusion protein (e.g., dCas9-VP64) with a gRNA targeting the activator to TBR1, BCL11A, MEIS2, or HERC1. Also provided herein are the expression vectors described herein for use in a method of treating a subject to reduce or reverse cognitive impairment and reduce seizures in disorders associated with excitation / inhibition imbalance. In some embodiments, the disorder is a neurodevelopmental disorder (NDD). In some embodiments, the NDD is autism spectrum disorder (ASD), epilepsy (e.g., Dravet syndrome, Scn2a-associated epilepsy, or seizures associated with NDDs), or schizophrenia (SCZ). In some embodiments, the disorder is Alzheimer's disease (AD), cognitive impairment in aging, or hearing loss. In some embodiments, the promoter comprises a parvalbumin promoter, a GAD65 promoter, or a beta actin promoter. In some embodiments, the vector is a viral vector, e.g., a recombinant adeno- associated virus (AAV), retroviruses, adenovirus, lentivirus, and herpes simplex virus-1. In some embodiments, the viral vector is an AAV, optionally selected from AAV2, AAV9, AAV-F, AAV.CPP.16, and AAVPhP.eB, and capsid and serotype variants thereof. In some embodiments, the expression vector comprises from 5’ -3’: inverted terminal repeat (ITR) – an optional S5E2 enhancer - promoter– XP protein coding transgene – optional woodchuck hepatitis virus post-transcriptional regulatoryelement (WPRE) - pA – ITR.In some embodiments, the XP protein is human Meis 2. In some embodiments, the human Meis 2 protein sequence is at least 90% or 95% identical to SEQ ID NO:23. In some embodiments, the expression vector is administered by intravenous, intrathecal, intracerebroventricular, intracisternal, intranerve, subcutaneous, intradermal, epidural, transforaminal, selective nerve root, or stereotactic intraparenchymal administration. Also provided herein are expression vectors comprising a promoter, an optional SE52 enhancer, and a transgene sequence encoding an experience-dependent parvalbumin-expressing inhibitory neuron (PV IN) plasticity (XP) protein selected from human Meis2, Tbr1, Bcl11a, and Herc1. In some embodiments, the promoter comprises a parvalbumin promoter, a GAD65 promoter, or a beta actin promoter. Attorney Docket No.29539-0804WO1 / MGH 2023-602 In some embodiments, the vector is a viral vector, e.g., a recombinant adeno- associated virus (AAV), retroviruses, adenovirus, lentivirus, and herpes simplex virus-1. In some embodiments, the viral vector is an AAV, optionally selected from AAV2, AAV9, AAV-F, AAV.CPP.16, and AAVPhP.eB, and capsid and serotype variants thereof. In some embodiments, the expression vector comprises from 5’ -3’: inverted terminal repeat (ITR) – optional S5E2 enhancer - promoter– XP transgene - woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) - pA – ITR. In some embodiments, the XP protein is human Meis 2. In some embodiments, the human Meis 2 protein sequence is at least 95% identical to SEQ ID NO:23. Unless otherwise defined, 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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, willcontrol.Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims. DESCRIPTION OF DRAWINGS FIGs.1A-D. Screen for experience-dependent PV IN plasticity genes in adult CA2 / CA3. a, Schematic of experimental workflow to biochemically isolate and sequence the translatome from naïve- and mossy fiber-triggered activated PV INs in adult PVCre:Rpl22HAf / fmice. Lentiviruses expressing Ablim3 shRNA-GFP or non- targeting shRNA (shNT-GFP) were injected into the DG and two weeks later CA2 / CA3 regions were microdissected. n=6 mice (3M and 3F) per sample, 5 samples / 30 mice for shNT; 4 samples / 24 mice for shRNA group. b, Left,Heatmap of expression values for differentially expressed genes, DEGs, shown as normalized Z- scores relative to the average expression of a given gene across all samples. Right, top, Principal component analysis (PCA) plot of PV IN translatomes. The first two Attorney Docket No.29539-0804WO1 / MGH 2023-602 principal components are shown with the corresponding fractions of variance. Bottom, volcano plot showing statistical significance (-log10 P-value) vs. magnitude of change (log2 of fold change) of gene expression. DEGs are marked in red. c, Pie chart showing numbers of upregulated and downregulated XPGs. Upregulated XPGs linked to NDDs / epilepsies are highlighted. XPGs in blue font are implicated in epilepsies. d, Left, Experimental design. S5E2 enhancer AAV expressing control (dTomato) or XPGs (Tbr1 or Bcl11a or Meis2) were injected into CA2 / CA3 of 2- month-old wildtype mice 2 weeks prior to perfusion. Right, representative images and quantification of PV+puncta density in CA3 (N = 6 mice for vector control; 5 mice for Tbr1; 4 mice for Bcl11a; 6 mice for Meis2) and CA2 (N=4 mice for each group). Scale bar, 10 μm. *p < 0.05; **p < 0.01; ***p < 0.001 using one-way ANOVA with Bonferroni post hoc test. All experiments are performed in male and female mice, and all data are displayed as mean ± SEM. FIGs.2A-F. Reduced social experience-dependent induction of PV IN synapses and Meis2 upregulation in CA3 / CA2 PV INs of Cntnap2 KO mice. a, Experimental design. Following habituation to a context, mice were exposed to a habituated context or a novel mouse for 10 min. b, Representative images and quantification of PV IN+puncta (green, N = 3 mice for Cntnap2+ / +, context and social groups; N = 3 mice for Cntnap2- / -, context group; N = 4 mice for Cntnap2- / -, socialgroup) in CA3. *p < 0.05; **p < 0.01 using two-way ANOVA with Bonferroni posthoc test. Scale bar, 10 μm. c, Experimental design. Following habituation to a context, mice were exposed to a habituated context or a novel mouse for 10 min. Representative images of Meis2 expression (green) in PV INs in RGS14+labeling CA2 subfield. d, Top, quantification of Meis2 expression (N = 3 mice for each group; for total PV INs, n= 107 for Cntnap2+ / +, naïve group; n= 122 for Cntnap2+ / +, social group; n= 63 for Cntnap2- / -, naïve group; n= 69 for Cntnap2- / -, social group). Bottom, cumulative probability plots of Meis2 expression in PV INs. Kolmogorov-Smirnov test, *p < 0.05; **p < 0.01. e,f, 2-month old Cntnap2+ / +and- / -mice were injected with rAAV-S5E2-dTomato (Ctrl) or rAAV-S5E2-Meis2-nlsdTomato (Meis2) into CA2 / CA32 weeks prior to quantification of Syt2+puncta density in CA2 (N = 4 mice for each group). Scale bar, 10 μm. *p < 0.05; **p < 0.01 using two -way ANOVA with Bonferroni post hoc test. All experiments are performed in male and female mice, and all data are displayed as mean ± SEM. Attorney Docket No.29539-0804WO1 / MGH 2023-602 FIGs.3A-I. Meis2-dependent restoration of excitatory and inhibitory transmission upon CA2 PNs, PV IN excitability, and inhibitory-dependent synaptic plasticity in Cntnap2- / -mice. a, Schematic depicting mossy fiber pathway and whole-cell voltage-clamp recording of spontaneous excitatory postsynaptic current (sEPSC) from CA2 pyramidal neurons (PN). Cntnap2- / -or+ / +mice were injected with rAAV-S5E2-Meis2-P2A-nlsdTomato or rAAV-S5E2-dTomato (Ctrl) virus along the stratum lucidum mossy fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Cumulative probability plots of sEPSC frequency and amplitude from CA2 PNs. Kolmogorov-Smirnov test, *p < 0.05, n=8-9 cells, 1-3 cells per mouse, 5-9 mice per group. b, Cumulative probability plots of spontaneous inhibitory postsynaptic current (sIPSC) frequency and amplitude from CA2 PNs. Kolmogorov-Smirnov test, *p < 0.05, n=8-9 cells, 1-3 cells per mouse, 5-9 mice per group. c, Schematic depicting mossy fiber pathway and whole- cell voltage-clamp recording of miniature excitatory postsynaptic current (mEPSC) from CA2 PNs. Bath application of tetrodotoxin (TTX, 1 µM) was done to block voltage-gated sodium channels. Cumulative probability plots of mEPSC frequency and amplitude from CA2 PNs. Kolmogorov-Smirnov test, *p < 0.05, n=8-9 cells, 1-3 cells per mouse, 5-9 mice per group. d, Cumulative probability plots of miniature inhibitory postsynaptic current (sIPSC) frequency and amplitude from CA2 PNs.Kolmogorov-Smirnov test, *p < 0.05, n=8-9 cells, 1-3 cells per mouse, 5-9 mice pergroup. e, Schematic depicting whole-cell voltage-clamp recording of mEPSC from PV INs along the stratum lucidum of the dorsal hippocampus. Cumulative probability plots of mEPSC frequency and amplitude from PV INs. Kolmogorov-Smirnov test, *p < 0.05, n=9-15 cells, 2-4 cells per mouse, 3-6 mice per group. f, Representative traces depicting 10 burst optic stimulation train (473 nm blue light, 1 ms pulse duration, 100 ms inter-stim interval repeated 5 times with 20 sec between trains) delivered above the mossy fiber pathway proximal to the hilus of the DG. Optically evoked EPSCs were recorded by voltage-clamp at -70 mV and IPSCs by voltage-clamp at 0 mV. g, Schematic depicting mossy fiber driven optically evoked EPSC and IPSC recorded from CA2 PNs. Mice were injected with pAAV5-CamKIIa-hChR2-eYFP into the dorsal dentate gyrus. Line graphs depict EPSC, IPSC, and excitation / inhibition ratio across optic evoked stimulation train. Graphs were analyzed with Two-way RM ANOVA with Tukey posthoc, *p < 0.05, n=10-12 cells, 1-2 cells per mouse, 6-9 mice Attorney Docket No.29539-0804WO1 / MGH 2023-602 per group. h, Schematic depicting mossy fiber pathway and whole-cell voltage-clamp onto CA2 PNs. A bipolar stimulating electrode contained in a patch pipette filled with ACSF was placed along the hilus of the DG along the mossy fiber pathway. Plasticity was induced by electrically evoked theta-burst stimulation (TBS), which consisted of bursts of 4 pulses at 100 Hz, interburst interval was 200 ms, and repeated 4 times at 10 sec intervals. IPSCs or EPSCs were optically evoked. Line graph depicts long-term depression of optically evoked IPSC (iLTD) in a Cntnap2+ / +mouse. Significance (p < 0.05) was assessed by unpaired t test between baseline (1stbar at -5 to 0 min) and 35 minutes post theta-burst stimulation (2ndbar at 35 to 40 min). Two group iLTD graphs depict IPSC amplitude between baseline and post. Blue lines depict cells that depressed and red lines depict no statistical difference between baseline and post stimulation. Pie charts display the percent of total recorded neurons that depressed. Recordings were from n=9-10 cells, 1 cell per slice, 1-2 slices per mouse, 8-10 mice per group. i, Schematic depicting whole-cell current-clamp onto PV INs along the stratum lucidum adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Representative current traces from each group depict action potential response to current steps (200 pA, 500 ms). The line graph depicts the number of action potential (AP) spike responses to incremental current steps. Two-way RM ANOVA with Tukey posthoc, *p < 0.05, n=9-12 cells, 2-3 cells per mouse, 4-5 miceper group. The bar graph depicts the rheobase, the minimum current required toinduce an action potential. Two-way ANOVA with Uncorrected Fisher’s LSD posthoc, *p < 0.05, n=12-14 cells, 2-3 cells per mouse, 4-5 mice per group. FIGs.4A-I. Meis2-dependent rescue of cognition in adult Cntnap2 KO mice. a, 2-month old Cntnap2+ / +and- / -mice were injected with rAAV-S5E2-dTomato (Ctrl) or rAAV-S5E2-Meis2-nlsdTomato (Meis2, nls=nuclear localization signal) into CA2 / CA32 weeks prior to behavioral testing (N = 9 for + / + mice with Ctrl; 7 mice for + / + with Meis2; 7 mice for - / - with Ctrl; 10 mice for - / - with Meis2). Representative images of Ctrl and Meis2 expression in CA2 / CA3 subfield. Scale bar, 500 μm. b, Schematic of novel object location task (Fig.11 for behavioral testing schedule). c, Quantification of total distance travelled, and time spent investigating objects. d, Schematic of social cognition task depicting habituation trial T1, encoding / recognition trial, T2; and social memory discrimination trial, T3. e, Quantification of total distance travelled during T1 trial. f, Quantification of Attorney Docket No.29539-0804WO1 / MGH 2023-602 interaction time during habituation (T1), social recognition phase (T2) and social stimuli interaction time in the discrimination trial (T3) (N =11 mice per group). Emp, empty pencil cup; stim, stimulus mouse; F, familiar mouse; N, a new novel stimulus mouse. These experiments are performed in male and female mice. All data are displayed as mean ± SEM and analyzed using two-way ANOVA with Bonferroni post hoc test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. g, Experimental workflow for tagging neurons active during social interaction and assessing reactivation of tagged neuronal ensemble.2-month-old Cntnap2+ / +and- / -mice were injected with Cal-Light viruses (AAV-ST-KA2, AAV-M13-TEV-C-P2AtsTom, AAV- TetO-EGFP) into the CA2 / 3 region. One week later, mice were injected either AAV- S5E2-Meis2 (Meis2) or AAV-S5E2-dTomato (Ctrl) and were implanted with fiberoptic probes above CA2 / 314 days prior to behavioral testing. (N = 7 for + / + and - / - mice with Ctrl; 10 mice for + / + with Meis2; 8 mice for - / - with Meis2). h, Representative image of tagged neurons. Scale bar, 20 μm. i, Quantification of Cal- light tagged active neurons (GFP+), cFos+neurons, and reactivation ratio (cFOS+GFP+ / GFP+cells) in Cntnap2+ / +and- / -mice injected with either Ctrl or Meis2 expressing viruses. These experiments are performed in male and female mice. All data are displayed as mean ± SEM and analyzed using two-way ANOVA with Tukey post hoc test. ns, not significant; *p < 0.05. FIGs. 5A-D. Meis2-dependent suppression of seizures in Cntnap2 KOmice. a, Experimental timeline, AAV injections and ECoG implants (N = 9 for Cntnap2- / -with Ctrl virus; 12 mice for Cntnap2- / -with Meis2 virus). b, Representative seizure recorded in vivo from a Cntnap2 KO mouse (top) and at an expanded timescale (bottom). c, Cntnap2- / -mice injected with AAV-S5E2-Meis2 have significantly fewer seizures in the two-week recording period compared to mice injected with AAV-S5E2-dTom. * P < 0.05, Mann-Whitney Rank Sum. d, Proportion of Cntnap2- / -mice with seizures treated with AAV-S5E2-Meis2 or AAV-S5E2-dTom. GND=ground, REF=reference. FIGs.6A-E. Supporting data for screen for regulators of experience- dependent PV IN plasticity. a, Quality control data: Validation of Ablim3 downregulation in DG of PVCre:Rpl22HAf / fmice injected with lentiviruses expressing Ablim3 shRNA-GFP (vs. non-targeting shRNA, shNT-GFP) into DG (N = 5 mice per group) by qRT-PCR. b, Enrichment of CA3 / CA2 tissue: qRT-PCR for Dsp (DG Attorney Docket No.29539-0804WO1 / MGH 2023-602 enriched) and Bok (CA3 enriched) expression in CA2 / CA3 tissue from PVCre:Rpl22HAf / fmice (N = 2 mice per group). c, Gene Set Enrichment Analysis (GSEA) of ribosome-associated RNAs isolated from CA3 / CA2 PV INs (FDR < 0.05). d, qRT-PCR data for Bcl11a (N = 3 mice for shNT, 4 for shRNA group), Meis2 (N = 3 mice per group), Tbr1 (N = 5 mice per group) and Herc1 (N = 6 mice for shNT, 7 for shRNA group) expression levels in CA2 / CA3 PV INs of PVCre:Rpl22HAf / fmice injected with lentiviruses expressing Ablim3 shRNA-GFP or non-targeting shRNA (shNT-GFP) into DG. e, Left: Experimental design. AAV expressing S5E2 enhancer with dSaCas9 combined with either scrambled gRNA (sagRNA) or Herc1 gRNA were injected into the CA2 / CA3 of 2-month-old wildtype mice 2 weeks prior to perfusion. Right: representative images and quantification of PV+puncta density in CA2 (RGS14+labeling) and CA3 (N = 3 mice for each group). Scale bar, 10 μm. *p < 0.05; **p < 0.01; ***p < 0.001 using two-tailed unpaired t test with Welch’s correction. All experiments were performed in male and female mice, and all data are displayed as mean ± SEM. FIGs.7A-B. Specificity of AAV-S5E2-Meis2-nlsdTomato targeting PV INs. a, 2-month-old wildtype mice were injected with rAAV-S5E2-Meis2-nlsdTomato. Representative images of Meis2-dTom overlapping with PV+cells in CA2 / CA3 subfield. Scale bar, 20 μm. b, Percentage of dTom+PV+ / total dTom+cells (N = 3mice). All data are displayed as mean ± SEM.FIGs.8A-G. Representative traces from ex vivo slice recordings. a-b, Representative traces of sEPSC and sIPSC recordings onto CA2 PNs. c-d, Representative traces of mEPSC and mIPSC recordings onto CA2 PNs. e, Representative traces of mEPSC onto PV INs. f, Representative traces of sEPSC, sIPSC, mEPSC, and mIPSC onto CA2 PNs from Cntnap2+ / +mice injected with AAV- S5E2-Tbr1-P2A-nlsTomato along the stratum lucidum. g, Representative traces depicting EPSC (downward deflections, voltage clamp = -70 mV) and IPSC (upward deflections, voltage clamp = 0 mV) elicited by 10 burst optic stimulation train. FIGs.9A-D. Supporting data for electrophysiological characterization of viral-restoration of Meis2 upregulation in CA3 / CA2 PV INs in Cntnap2- / -or+ / +mice. a, Schematic depicting mossy fiber driven optically evoked EPSC and IPSC recorded from CA2 PNs. Mice were injected with pAAV5-CamKIIa-hChR2-eYFP into the dorsal DG. Line graphs depict paired pulse ratios (2ndevent amplitude / 1st Attorney Docket No.29539-0804WO1 / MGH 2023-602 event amplitude) across the 10-burst optic stimulation train. Two-way RM ANOVA, *p < 0.05, n=8-9 cells, 1-3 cells per mouse, 4-7 mice per group. b, Schematic depicting mossy fiber pathway and whole-cell voltage-clamp onto CA2 PNs. A bipolar stimulating electrode contained in a patch pipette filled with ACSF was placed along the hilus of the DG along the mossy fiber pathway. Plasticity was induced by electrically evoked theta-burst stimulation (TBS) which consisted of bursts of 4 pulses at 100 Hz, interburst interval was 200 ms, and repeated 4 times at 10 sec intervals. IPSCs or EPSCs were optically evoked. Left line graph depicts long-term potentiation (LTP) of optically evoked EPSC in a+ / +mouse. Significance (p < 0.05) was assessed by unpaired t test between baseline (1stbar at -5 to 0 min) and 35 minutes post TBS (2ndbar at 35 to 40 min). The middle line graph depicts long-term depression of IPSC (iLTD) followed by bath application of DCG-IV, group II metabotropic glutamate receptor agonist which suppresses synaptic transmission at mossy fiber pathway. The right line graph depicts optically evoked IPSC without TBS plasticity induction. c, Schematic depicting mossy fiber pathway and whole-cell voltage-clamp onto CA2 PNs. Electrically evoked TBS was delivered along the mossy fiber pathway. Paired pulse ratios (PPR) of the IPSC and EPSC were recorded from CA2 PNs at baseline (- 5 to 0 min) and post TBS (35 to 40 min). Data was analyzed with paired t tests, *p < 0.05, n=9-10 cells, 1 cell per slice, 1-2 slices per mouse, 8-10 mice per group. d,Schematic depicting whole-cell current-clamp onto PV INs along the stratum lucidumadjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Cntnap2- / -or+ / +mice were injected with AAV-S5E2-Meis2-P2A-nlsdTomato or AAV-P2A- nlsdTomato control virus along the stratum lucidum mossy fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Bar graphs depict the resting membrane potential (RMP) and threshold of PV INs. Two-way ANOVA with Uncorrected Fisher’s LSD posthoc, *p < 0.05, n=12-14 cells, 2-3 cells per mouse, 4-5 mice per group. FIGs.10A-D. Boosting Tbr1 in CA3 / CA2 PV INs reduces excitation and increases inhibitory synaptic transmission in Cntnap2+ / +mice. a, Schematic depicting mossy fiber pathway and whole-cell voltage-clamp recording of spontaneous excitatory and inhibitory postsynaptic current (sEPSC / sIPSC) from CA2 pyramidal neurons (PN). Cntnap2+ / +mice were injected with AAV-S5E2-Tbr1-P2A- nlsdTomato or AAV-P2A-nlsdTomato control virus along the stratum lucidum mossy Attorney Docket No.29539-0804WO1 / MGH 2023-602 fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Cumulative probability plots of sEPSC and sIPSC frequency and amplitude from CA2 PNs. Kolmogorov-Smirnov test, n=3-7 cells, 1-2 cells per mouse, 3-7 mice per group.+ / +data from Figure 3 and Figure 3S were used as control comparison group in these experiments. b, Schematic depicting mossy fiber pathway and whole-cell voltage- clamp recording of miniature excitatory and inhibitory postsynaptic current (mEPSC / mIPSC) from CA2 PNs. Bath application of tetrodotoxin (TTX, 1 µM) was done to block voltage-gated sodium channels. Cumulative probability plots of mEPSC and mIPSC frequency and amplitude from CA2 PNs. Kolmogorov-Smirnov test, n=4- 7 cells, 1-2 cells per mouse, 4-7 mice per group. c, Schematic depicting mossy fiber driven optically evoked EPSC and IPSC recorded from CA2 PNs. Mice were injected with pAAV5-CamKIIa-hChR2-eYFP into the dorsal dentate gyrus. Representative traces depicting 10 burst optic stimulation train (473 nm blue light, 1 ms pulse duration, 100 ms inter-stim interval repeated 5 times with 20 sec between trains) delivered above the mossy fiber pathway proximal to the hilus of the DG. Optically evoked EPSCs were recorded by voltage-clamp at -70 mV and IPSCs by voltage- clamp at 0 mV. Line graphs depict EPSC, IPSC, excitation / inhibition ratio and paired pulse ratio (PPR) across optic evoked stimulation train. Graphs were analyzed with Two-way RM ANOVA with Uncorrected Fisher’s LSD posthoc, *p < 0.05, n=2-8cells, 1-2 cells per mouse, 2-5 mice per group. d, Schematic depicting whole-cellcurrent-clamp onto PV INs along the stratum lucidum adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Cntnap2+ / +mice were injected with AAV-S5E2-Tbr1-P2A-nlsdTomato or AAV-P2A-nlsdTomato control virus along the stratum lucidum mossy fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Bar graphs depict PV IN resting membrane potential (RMP), threshold, and rheobase. Bar graphs were analyzed with unpaired t test, *p < 0.05, n=5-9 cells, 2-3 cells per mouse, 3-4 mice per group. The line graph depicts the number of action potential (AP) spike responses to incremental current steps. Two- way RM ANOVA with Tukey posthoc, *p < 0.05, n=2-9 cells, 2-3 cells per mouse, 2- 4 mice per group. FIGs.11A-D. Boosting Meis2 expression in CA2 / CA3 PV INs does not affect locomotion and novel object recognition in Cntnap2 KO mice. a, Schematic of behavior testing schedule. Day 1, OF, open-field task; Day 2, habituation to visual Attorney Docket No.29539-0804WO1 / MGH 2023-602 cue; Day 3, NOL, novel object location and NOR, novel object recognition; Day 4, Social cognition task. b, OF, quantification of total distance traveled, percentage of distance traveled across the center arena, percentage time in center. c, Quantification of the time spent sniffing two identical objects (seconds) during habituation on Day 3. d, NOR, novel object recognition, mouse was exposed to 2 objects over three sessions (5 minutes each session, object locations counterbalanced). Time spent sniffing the objects was quantified. These experiments were performed in male and female mice. All data are displayed as mean ± SEM and analyzed using two-way ANOVA with Bonferroni post hoc test. FIGs.12A-E. Boosting Meis2 expression in CA2 / CA3 PV INs does not affect group social behavior of Cntnap2 KO and wild-type littermates in homecage. a, Pairs of 2-month-old Cntnap2+ / +or- / -mice were injected with either AAV-S5E2-Meis2-nlsdTom or AAV-S5E2-dTom constructs. Following a 2-week incubation period, mice were recorded for 1 hour in their home cages during their night cycle to monitor social interactions. b, Schematic showing node locations (nose, R ear, L ear, center, and rear) used for pose estimation via SLEAP. c, Representative node placements during three types of social interaction: nose-to-nose, nose-to-center, and nose-to-rear. d, Quantification of social interaction data across conditions (3-4 cages per condition, 2 mice per cage), comparing Cntnap2+ / +and- / -groups injectedwith either tdTomato or Meis2 constructs. Nose to nose, nose to center, nose to rearand total Interaction were analyzed (sec). These experiments were performed in male and female mice. e, Representative image of Cal-Light viruses expression and implant in CA2 / CA3 subfield (indicated by asterisk). Scale bar, 250 μm. All data are displayed as mean ± SEM and analyzed using two-way ANOVA with Bonferroni post hoc test. FIGs.13A-C. Analysis of seizure frequency in Cntnap2+ / +mice and seizure duration for all groups. A, ECoG recordings for 2 weeks revealed no seizures in Cntnap2+ / +mice treated with AAV-S5E2-dTom and 2 seizures in a single Cntnap2+ / +mouse treated with AAV-S5E2-Meis2. b. The proportion of Cntnap2+ / +mice with seizures treated with AAV-S5E2-Meis2 or AAV-S5E2-dTom. c. Mean seizure duration for all groups show comparable length of seizures across groups. FIGs.14A-E. Boosting Meis2 in PV INs of Cntnap2 KO mice restores ripple power and duration. a. Experimental design. AAVs expressing dTomato (S5E2-nlsdTom) or Meis2 were injected into the CA2 / CA3 of 2-month-old WT (+ / +) Attorney Docket No.29539-0804WO1 / MGH 2023-602 and KO (- / -) mice 2 weeks before implantation of tetrodes targeting CA1, DG, CA2 and CA3 respectively. LFP and mouse behavior were recorded in the same home cage for 2 hours, before (pre-social interaction) and after (post-social interaction) exposure to a social stimulus (novel juvenile mouse). b. Raw broadband (1-300Hz, black trace line, top plot), filtered (100-250 Hz, brown trace line, middle plot) and ripple power envelope (2-5 z-score cut-off window, orange trace line, middle plot,) and spectrogram from CA1 electrode of a ripple event aligned to its peak, from + / + dTom, - / - dTom, and - / - Meis2. c. Peak amplitude (Z-score) of ripple events (n = 10 mice, 8, 11 for + / + dTom, - / - dTom, and - / - Meis2, respectively; Krukal Wallis test, H = 16.27, P = 0.0003, Post-hoc Dunn's P values indicated on figure). d. Power (Z-score) of ripple frequency band during ripple events (Krukal Wallis test, H = 13.08, P = 0.0014, Post-hoc Dunn's P values). e. Average ripple duration between groups (Krukal Wallis test, H = 17.07, P = 0.0002, Post-hoc Dunn's P values written on figure). The box plot represents the 25th to 75th percentiles of the distribution, while the bold line represents the median of the distribution. Whiskers are the smallest and largest values in the distribution. FIGs.15A-D. Boosting Meis2 in PV INs of Cntnap2 KO mice increases ripple rate and power. A. Tetrode track marks (dotted lines) in an implanted mouse's dorsal hippocampus (DAPI, Iba1, dTomato, Scale 200µm) targeting CA1, DG, CA2,and CA3. b. Total NREM periods during the recording post-social interaction. c.Occurrence of ripple events (Hz) during NREM periods post-social interaction. (n = 10 mice, 8, 11 for + / + dTom, - / - dTom, and - / - Meis2; Krukal Wallis test, H = 10.52, P = 0.0052, Post-hoc Dunn's P values written on the figure). d. Normalized power of ripple frequency band during ripple events (Krukal Wallis test, H = 8.605, P = 0.0135, Post-hoc Dunn's P values). The box plot represents the 25th to 75th percentiles of the distribution, while the bold line represents the median of the distribution. Whiskers are the smallest and largest values in the distribution. DETAILED DESCRIPTION Fast-spiking parvalbumin-expressing inhibitory neurons (PV INs) in the hippocampal dentate gyrus (DG)-CA3 / CA2 circuit influence encoding, storage, retrieval and routing of memories through activity-dependent regulation of CA3 / CA2 principal cell perisomatic inhibition4-12. PV IN mediated feed-forward inhibition of CA3 / CA2 principal cells dictates their spiking, synchronization of principal cell Attorney Docket No.29539-0804WO1 / MGH 2023-602 activity to form ensembles and the generation of network oscillations to mediate intra- hippocampal and inter-regional communication in hippocampal-cortical-subcortical networks3,11,23,24. To exert these effects on circuitry and network properties, PV INs cell-autonomously coordinate experience-dependent changes in their intrinsic properties. These include structural reorganization of axonal arborizations and perisomatic synapses in CA3 / CA2, regulation of feed-forward inhibition of CA3 / CA2, and synaptic plasticity9,10,13-17: collectively referred to here as experience- dependent PV IN plasticity. Loss of PV IN functions in NDDs including ASDs, schizophrenia, sensory disorders like hearing loss, and epilepsies may arise from genetic risk factors that impair experience-dependent refinement of PV IN mediated inhibition during the early postnatal period18-20,25,26. In addition, Alzheimer’s Disease, specifically Mild Cognitive Impairment, is also characterized by loss of PV IN mediated inhibition during aging. Thus, discovery of molecular mechanisms that regulate experience-dependent PV IN plasticity may inform therapeutic strategies to restore PV IN functions and GABAergic inhibition, suppress seizures and rescue cognition in ASDs, sensory disorders, schizophrenia, epilepsy and AD. Transcription factors and epigenetic regulators co-ordinate changes in gene expression underlying synaptic physiology, synaptic and structural plasticity and input-output connectivity to mediate experience-dependent inhibitory and excitatory neuron plasticity. We know a substantial amount about molecular mechanisms that regulate cortical PV IN identity and experience-dependent plasticity14,19,27-29and less about developmental regulators of hippocampal PV IN properties15,30,31. In sharp contrast, evidence for transcription factors and epigenetic regulators that control experience-dependent PV IN plasticity in the adult hippocampus is scarce. PV INs in CA3 / CA2 retain experience-dependent plasticity in adulthood. Specifically, learning6,10and social experience9increase mossy fiber excitatory synaptic inputs onto PV INs to trigger experience-dependent PV IN plasticity– subsequently leading to increased feed-forward inhibition in DG-CA3 and DG-CA29. Increased mossy fiber excitatory drive onto PV INs increases intrinsic excitability, inhibitory synapses onto CA3 and CA2, and perisomatic inhibition of CA3 and CA2 principal cells to support spatial and social memory9,10. At a network level, DG recruitment of PV IN-mediated inhibition of CA3 / CA2 principal cells promotes stable and context-specific neuronal ensembles in hippocampal-cortical networks and network oscillations during memory consolidation10,11. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Impaired experience-dependent refinement of inhibitory circuits contributes to development of NDDs including ASDs18-20,25,26. Loss of hippocampal PV IN functions is thought to increase risk of developing core features of ASDs. These include imbalanced excitation and inhibition, network hyperexcitability, seizures, and cognitive impairment through reduced perisomatic inhibition of principal cells, principal cell recruitment into ensembles, and generation of network oscillations. Exome sequencing has identified ultra-high confidence genes that underlie different NDDs including ASDs and schizophrenia (gene.sfari.org / )34,35but how these different genes converge upon biological mechanisms that govern PV IN functions essential to cognition is poorly understood. Experience-dependent PV IN plasticity is a candidate biological mechanism for NDDs since it underlies the capacity of PV INs to coordinate changes in their intrinsic properties, synaptic physiology, input-output connectivity and synaptic plasticity in response to experiential demands. However, identities of cell-autonomous regulators of experience-dependent PV IN plasticity in adult hippocampus have remained elusive. We designed an input-specific activity sensitive screen for XPGs in CA3 / CA2 PV INs of adult mice that does not rely on imposition of artificial patterns of activity onto PV INs but mimics physiological experience-dependent changes in mossy fiber inputs onto PV INs that trigger a change in PV IN cell state properties. We discovereda suite of XPGs including transcription factors and epigenetic regulators whoseexpression in PV INs is increased in response to increased mossy fiber excitatory drive. A significant proportion of upregulated, but not downregulated, XPGs in CA3 / CA2 PV INs in our unbiased screen exhibit loss-of-function mutations in ASDs and epilepsies, and several encode ultra-high confidence risk genes for schizophrenia. Based on the directionality of expression change in XPGs in our screen and loss-of- function mutations in these genes in NDDs, we propose that impaired experience- dependent PV IN plasticity is a convergent mechanism for NDDs including ASDs and schizophrenia. To test the hypothesis that loss of experience-dependent PV IN plasticity in CA3 / CA2 is a substrate for cognitive impairments and seizures in NDDs, we chose the Cntnap2 KO model of NDD risk. This was not to model a specific NDD, but because it exhibits a plethora of developmental brain-wide deficits including neuronal migration, myelination, neuronal excitability, loss of GABAergic inhibition and PV Attorney Docket No.29539-0804WO1 / MGH 2023-602 INs, cognitive impairments and seizures40-43. We characterized MEIS2 as a transcriptional regulator of experience-dependent PV IN plasticity in CA3 / CA2 and demonstrated that Meis2-dependent restoration of experience-dependent PV IN plasticity in CA3 / CA2 in adulthood in Cntnap2 KO mice is sufficient to reverse developmental deficits in excitation-inhibition balance, cognitive impairments and seizure risk. MEIS2-dependent suppression of seizures may reflect coordinated reduction in excitatory and increased inhibitory synaptic transmission in DG-CA247and potentially, also in DG-CA3. Further, we used PV IN enhancer-driven AAV-Meis2 viral vectors to test the hypothesis that restoration of experience-dependent PV IN plasticity in the adult hippocampus in a prototypical NDD risk model is sufficient to reverse developmental deficits in circuitry, ensemble specificity, network excitability, seizures and cognition. The effects of increasing levels of MEIS2 in CA3 / CA2 PV INs on physiology, synaptic connectivity and plasticity reflect an amalgam of previously identified roles for MEIS2 in regulating GABAergic projection neuron identity in development and maintenance of sensory neuron physiology and terminal specialization21,38,39. Without wishing to be bound by theory, MEIS2 may function as a permissive coactivator that acts in concert with other transcription factors to instruct different facets of experience-dependent PV IN plasticity. The observation that Meis2 expression is notdetected in the medial ganglionic eminence8,21, the embryonic source for hippocampalPV INs, suggests an evolutionarily parsimonious repurposing of Meis2 based on function rather than PV IN identity specification, to regulate experience-dependent programs within PV INs in the adult brain. In conclusion, we identify experience-dependent PV IN plasticity as a convergent biological mechanism for NDDs and demonstrate that restoring experience-dependent PV IN plasticity in adulthood offers an opportunity for therapeutic intervention in adulthood to alleviate cognitive impairments and reduces seizures in NDDs. Since the principal cell-PV IN-principal cell feed-forward inhibition motif is found in brain circuits supporting memory, cognitive flexibility, decision making and attention, targeting experience-dependent PV IN plasticity in these brain regions can be used to reduce network excitability and impact different domains of cognition in NDDs. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Methods of Treatment Described herein are genetic therapy methods and compositions that can be used to restore PV IN mediated GABAergic inhibition, to reduce or reverse cognitive impairment and reduce seizures in disorders associated with excitation / inhibition imbalance; this includes NDDs such as ASD, epilepsies (e.g., Dravet syndrome, Scn2a-associated epilepsy, or seizures associated with NDDs), and schizophrenia (SCZ); and to reduce or reverse cognitive impairment in aging and Alzheimer's disease (AD). PV dysfunction has been implicated as causal in NDDs such as ASD and SCZ, AD, mild cognitive impairment (MCI), and aging-associated cognitive decline, and hearing loss, and restoration of PV function is likely to yield Tx impact. See, for example, Jimenez-Balado and Eich, Semin Cell Dev Biol.2021 Aug:116:146- 159 (GABAergic dysfunction, neural network hyperactivity and memory impairments are involved in human aging and Alzheimer's disease); Lam et al., Nat Med.2017 Jun;23(6):678-680 (Alzheimer’s disease patients have silent seizures, as a result of network hyperexcitability). Impairment in fast-spiking PV INs has been shown to affect cognitive decline in Alzheimer's disease (Hijazi et al., Mol Psychiatry.2023 Dec;28(12):4954-4967). Hippocampal hyperactivity in MCI is a robust, reproducible finding (see, e.g., Reagh et al., Neuron.2018 Mar 7;97(5):1187-1198.e4; Quiroz et al., Ann Neurol.2010 Dec;68(6):865-75; Hujibers et al., Brain.2015 Apr;138(Pt 4):1023-35; Giorgio et al., Neuron. 2024 Feb 21;112(4):676-686.e4). GABAergic PV INs wereshown to be lost in an APP knock-in mouse model of AD (Petrache et al., Cereb Cortex.2019 Apr 1;29(4):1834-1850). PV IN dysfunction has also been shown to play a role in neurodevelopmental disorders including ASD (Contractor et al., Nat Neurosci.2021 Dec;24(12):1648- 1659) and SCZ (Glausier and Lewis, Handb Clin Neurol.2018:150:389-417). Chemogenetic enhancement of PV IN function reverse circuit and behavioral impairments associated with Dyrk1a heterozygosity (Shih et al., Neuron.2023 Oct 4;111(19):3084-3101.e5). PV neurons are responsible for cognitive flexibility and necessary for prefrontal functions, which are lost in schizophrenia (Sohal, Curr Opin Neurobiol.2024 Feb;84:102820; Cho, Nature.2023 May;617(7961):548-554 (Dravet Syndrome)). Attorney Docket No.29539-0804WO1 / MGH 2023-602 PV IN dysfunction has also been shown to be implicated in epilepsies (Dudok et al., Epilepsy Curr.2021 Oct 31;22(1):54-60; Kaneko et al., Cell Rep.2022 Mar 29;38(13):110580). A loss of experience-dependent regulation of PV IN-mediated inhibition has been demonstrated in aging (Guo et al., Nat Med.2018 May;24(4):438-449; Villanueva-Castillo et al., Neurobiol Aging.2017 Jan;49:119-137). Further, a loss of PV IN mediated inhibition has been shown to play a role in hearing loss (Kotak et al., PLoS One.2013;8(1):e53438). For example, tinnitus, loudness perception, and auditory hypersensitivity have been shown to be due to PV IN hypofunction (see Kujawa et al., bioRxiv [Preprint].2024 May 30:2024.05.30.596691; Masri et al., J Neurosci.2021 Oct 20;41(42):8848-8857). Methods that are able to restore PV IN mediated inhibition can be used in the above conditions as well as epilepsy and psychiatric comorbidities (see, e.g., Godoy et al., Front Integr Neurosci.2022 Feb 17:16:765324). AAV and other virally-mediated gene therapies are one avenue to treat these disorders (Ling et al. Nat Rev Drug Discov.2023 Oct;22(10):789-806). The genes identified as XP transgenes or XPGs have been associated with these conditions; see, e.g., Singh et al., Nature volume 604, pages509–516 (2022)(Herc1 is associated with SCZ); Satterstrom et al., 2020, Cell 180, 1–17(Bcl11a, Meis2 and Tbr1 are ASD genes).The present methods use upregulation of experience-dependent PV IN plasticity (XP) transgenes Tbr1, Bcl11a, Meis2 or Herc1; as shown herein, increased expression of these genes resulted in an increase in PV IN synapses in CA3 and CA2 pyramidal neurons (Fig.1d, Fig.6e). Thus, the methods include administering a therapeutically effective amount of a nucleic acid comprising a sequence encoding a human XP transgene, e.g., a Tbr1, Bcl11a, Meis2 or Herc1 transgene, preferably in an expression vector comprising an S5E2 enhancer that restricts transgene expression to PV INs (Vormstein-Schneider et al., Nat Neurosci.2020 Aug 17;23(12):1629–1636). Alternatively, the methods can include administering a dCas9 activator fusion protein with a gRNA targeting the activator to TBR1, BCL11A, MEIS2, or HERC1. Subjects who have these disorders and can be treated using the present methods can be identified by a skilled health care provider. Attorney Docket No.29539-0804WO1 / MGH 2023-602 The subjects may present, e.g., with mild-to-severe developmental delay (DD) and / or intellectual disability (ID); epilepsy, including absence or atypical absence seizures, epilepsy with myoclonic-atonic seizures, and / or generalized tonic-clonic seizures, e.g., Dravet syndrome, Scn2a-associated epilepsy, or seizures associated with NDDs; autism spectrum disorder, or age- or Alzheimer’s disease-related cognitive decline. Generally, the methods include administering a composition comprising a therapeutically effective amount of a nucleic acid encoding a human XP transgene, e.g., as described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, the methods include administering the composition directly to the brain of the subject. For example, the gene therapy construct can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection, e.g., optionally into the cisterna magna, cerebral ventricles, or lumbar intrathecal space (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)). In some embodiments, delivery methods of XP transgene expression constructs as described herein include intravenous, intrathecal, intracerebroventricular, intracisternal, intranerve, subcutaneous, intradermal, epidural, transforaminal, selective nerve root, and stereotactic intraparenchymal administration, e.g., to target the hippocampus. Preferably intrathecal, intracerebroventricular, or intracisternaladministration is used.As used in this context, to “treat” means to ameliorate at least one symptom of the disorder. A treatment comprising or consisting of administration of a therapeutically effective amount of a composition described herein can result in a reduction in rate, frequency, severity, or extent of cognitive impairment, symptoms of autism, neurodevelopmental disorders-ASD and SCZ, Neurological disorders such as AD, and seizures, and a return or approach to normal cognition, including improvements in memory, cognitive flexibility, social cognition, working memory, and spatial memory. XP Gene Therapy Constructs The present compositions can include naked mRNA or DNA encoding an XP transgene as described herein, as well as expression constructs comprising sequences encoding an XP transgene as provided herein, e.g., sequences that are at least 60%, 75%, 80%, 90%, 95%, 97%, or 99% identical to a human coding sequence, or code Attorney Docket No.29539-0804WO1 / MGH 2023-602 for a protein that is at least 80%, 90%, 95%, 97%, or 99% identical to a human protein sequence; Table A provides exemplary sequences and additional exemplary sequences are provided herein. The compositions, e.g., expression constructs, can be administered to a subject in need thereof. Table A. Exemplary sequences of human XP transgenes For HERC1, which is too large to be packaged in AAV, a CRISPR activator gene therapy strategy was used that relied on delivery of a catalytically inactive Cas9 (dCas9) fused to an activator, e.g., VP64, delivered by AAV, e.g., AAV S5E2dCas9a, with guide RNAs (gRNAs) targeting the dCAs9a to the endogenous Herc1 gene, e.g., S5E2-dSaCas9-VP64 +AAV-U6>mHerc1[msSagRNA#2]- U6>mHerc1[msSagRNA#12]. This CRISPR activation approach involves dCas9activator +gRNAs that target a dCAs9activator to the endogenous Herc1 locus to boost transcription. dCas9a +gRNA gene therapy can be used as an alternative method to increase expression of an endogenous XP locus. See, e.g., Tamura et al., Nature.2025 Sep 17. doi: 10.1038 / s41586-025-09522-w. Thus, the methods can include administering a dCas9 activator fusion protein with a gRNA targeting TBR1, BCL11A, MEIS2, or HERC1. See, e.g., Maeder et al., Nat Methods.2013 Jul 25;10(10):977–979. Expression constructs comprising sequences encoding an XP transgene can include viral vectors, including recombinant retroviruses, adenovirus, adeno- associated virus, lentivirus, and herpes simplex virus-1, or recombinant bacterial or eukaryotic plasmids. An exemplary approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing the nucleic acid, e.g., a cDNA encoding the protein. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within Attorney Docket No.29539-0804WO1 / MGH 2023-602 the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid. Viral vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and in some cases the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. Protocols for producing recombinant viruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et al., eds., Gene Therapy Protocols Volume 1: Production and In Vivo Applications of Gene Transfer Vectors, Humana Press, (2008), pp.1-32 and other standard laboratory manuals. A preferred viral vector system useful for delivery of nucleic acids is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al., Curr. Topics in Micro and Immunol.158:97-129 (1992); see also Domenger and Grimm, Human Molecular Genetics, 28(R1):R3–R14 (October 2019)). AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. Although AAV vector genomes can persist within cells as episomes, vector integration has been observed (see for example Deyle andRussell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442–447; Asokan et al., Mol Ther.2012 April; 20(4): 699–708; Flotte et al., Am. J. Respir. Cell. Mol. Biol.7:349-356 (1992); Samulski et al., J. Virol.63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989)). AAV vectors, particularly AAV2, have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther.2009 Aug; 11(4): 442–447; Asokan et al., Mol Ther.2012 April; 20(4): 699–708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. Space for exogenous DNA is limited to about 4.5 kb. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example the references cited above and those cited in Asokan et al., Molecular Therapy (2012); 204, 699–708; and Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol.4:2072-2081 (1985); Attorney Docket No.29539-0804WO1 / MGH 2023-602 Wondisford et al., Mol. Endocrinol.2:32-39 (1988); Tratschin et al., J. Virol.51:611- 619 (1984); and Flotte et al., J. Biol. Chem.268:3781-3790 (1993); Hammond et al., PLoS One.2017 Dec 15;12(12):e0188830; Haggert et al., Mol Ther Methods Clin Dev.2019 Nov 26;17:69-82. In some embodiments, preferred viral vectors are adeno- associated virus type 1, 2, 5, 7, 8, 9, rh.10, AAV2 / 1, AAVDJ8, PHP.B, PHP.eB, PhP.S or Anc80. See, e.g., US PGPub 20190100560; Hammond et al., PLoS One.2017 Dec 15;12(12):e0188830; Haggert et al., Mol Ther Methods Clin Dev.2019 Nov 26;17:69- 82; Jackson et al., Frontiers in Molecular Neuroscience (9) (2016); doi.org / 10.3389 / fnmol.2016.00116; Choi et al., Curr. Gene Ther.2005; 5: 299-310; Chan et al., Nat Neurosci.2017 Jun 26;20(8):1172–1179. In some embodiments, a self-complementary AAV is used, which contains an inverted repeat genome that folds to make double-stranded DNA, and wherein the right (3’) Inverted Terminal Repeat (ITR) contains a deletion of the D-sequence (the packaging signal). See, e.g., Hirata and Russell and Russell, J. Virol. (2000) 74:4612- 4620; Raj et al., Expert Rev Hematol.2011 Oct; 4(5): 539–549; US 20170362608; McCarty et al., Gene Therapy.8 (16): 1248–54 (2001); McCarty et al., Mol. Ther. 2008; 16: 1648-1656; McCarty et al., Gene Ther.2003; 10: 2112-2118; US20020006664, US20030153519, US 20030139363; US20040029106; U.S. Pat. Nos.9,783,824; 6,547,099; 6,506,559; and 4,766,072; PCT Application Nos. WO01 / 92551, WO 01 / 68836, and WO 03 / 010180.Other viruses, e.g., retroviruses or adenovirus-derived vectors, can also be used. Suitable expression constructs can include: a coding region; a promoter sequence, e.g., a promoter sequence that restricts expression to a selected cell type as described herein; an enhancer sequence, e.g., an S5E2 enhancer; untranslated regulatory sequences, e.g., a 5' untranslated region (UTR), a 3'UTR; a polyadenylation site; and / or an insulator sequence. Such sequences are known in the art, and the skilled artisan would be able to select suitable sequences. See, e.g., Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14; Vancura (ed.), Transcriptional Regulation: Methods and Protocols (Methods in Molecular Biology (Book 809)) Humana Press; 2012 edition (2011) and other standard laboratory manuals. In some embodiments, the Attorney Docket No.29539-0804WO1 / MGH 2023-602 expression construct is capable of directing expression of the XP transgene nucleic acid preferentially in the brain, e.g., in PV INs. Modifications of these sequences may be possible or desirable in certain applications, and such modifications are within the scope of this disclosure. The vectors preferably include inverted terminal repeats (ITRs); promoters, enhancers (e.g., CMV enhancer or S5E2 enhancer), other cis-regulatory elements, and / or capsid serotype variants that control and drive expression of the XP transgene protein. With regard to promoters, vectors can include promoters that drive expression in many cell types (e.g., human β-actin, beta-actin minimal promoter, human elongation factor-1α, chicken β-actin combined with cytomegalovirus early enhancer, cytomegalovirus (CMV), simian virus 40 (SC40), herpes simplex virus thymidine kinase (HSVTK), JeT (US20020098547), PGK, CAG, sCAG, or CASI) or specifically in neurons (e.g., synapsin I (Syn1), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase and platelet-derived growth factor beta chain promoters, and hybrid promoters created by fusing cytomegalovirus enhancer (E) to those neuron- specific promoters (Hioki et al., Gene Therapy 14:872–882 (2007)). Promoters specific for PV INs can be used, e.g., PV promoter or GAD65 promoter; see, e.g., Duba-Kiss et al., Front Neurol.2021 Oct 4:12:745159; Tkatch et al., Sci Rep.2022 Oct 25;12(1):17851 (which describes both a short parvalbumin promoter and aspecialized poly(A) sequence for expression in PV INs). Other cis-regulatoryelements can include posttranscriptional regulatory elements; 2A enhancers; polyadenylation sequences; and / or an intron, e.g., a chimeric intron. Posttranscriptional regulatory elements can include HBV Posttranscriptional Regulatory Element (HPRE), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or variants thereof (e.g., WPRE2, WPRE3 see, e.g., Kalev-Zylinska ML, During MJJ Neurosci.2007 Sep 26; 27(39):10456-67; Zanta- Boussif et al., Gene Therapy (16): 605–619 (2009); Choi et al., Mol Brain.7:17 (2014); US6136597). One or more 2A sequences can be used, e.g., 18–22 aa-long peptides that share a core sequence motif of DxExNPGP (SEQ ID NO:1) and induce ribosomal skipping during translation of a protein in a cell. Exemplary polyadenylation sequences, which include SV40, human growth hormone (hGH), bovine growth hormone (bGH), synthetic polyadenylation (spA), and rbGlob, preferably include the sequence motif AAUAAA that promotes both polyadenylation Attorney Docket No.29539-0804WO1 / MGH 2023-602 and termination (Buck and Wijnholds, Int J Mol Sci.2020 Jun; 21(12): 4197). Commonly used 2A sequences include P2A, E2A, F2A and T2A. F2A is derived from foot-and-mouth disease virus 18; E2A is derived from equine rhinitis A virus; P2A is derived from porcine teschovirus-12A; T2A is derived from thosea asigna virus 2A. See, e.g., Lewis et al., J Neurosci Methods.2015 Dec 30; 256: 22–29; Liu et al., Sci Rep.2017 May 19;7(1):2193. In some embodiments, the AAV also includes a furin cleavage sequence (see Fang et al., Nat Biotechnol 23: 584–590). Introns can include SV40 intron, F.IX truncated intron 1; β-globin SD / immunoglobin heavy chain SA; Adenovirus SD / immunoglobulin SA; SV40 late SD / SA (19S / 16S); Hybrid adenovirus SD / IgG SA; or minute virus of mice (MVM) intron (see Powell and Rivera-Soto, Discov Med.2015 Jan; 19(102): 49–57. Capsid variants can include PHP.eB, PHP.B and those described in Castle et al., Methods Mol. Biol.2016; 1382: 133-149; Davidsson et al., PNAS.116 (52) 27053-27062 (2019); Lee et al., Current Opinion in Biomedical Engineering (7):58-63 (2018). In some embodiments, the AAV capsid includes a cell penetrating peptide (CPP), e.g., CPP.16 or CPP.21 (Yao et al., Nat Biomed Eng.2022 Nov;6(11):1257-1271); see also Liu et al., Mol Ther Methods Clin Dev.2014; 1: 12; Tan et al., Nature Communications 10:3733 (2019). AAV-F is an AAV9 with a capsid variant that increases brain transduction (Hanlon et al., Mol Ther Methods Clin Dev.2019 Oct 23:15:320-332), For additional disclosure regarding viralvectors, see also Li and Samulski, Nature Reviews Genetics 21:255–272 (2020); Nairet al., iScience.2020 Mar 27;23(3):100888; Haery et al., Front Neuroanat.2019; 13: 93; Domenger and Grimm, Human Molecular Genetics, 28(R1):R3–R14 (October 2019); and WO / 2019 / 200286, and references cited therein. The constructs can include, e.g., a viral delivery vector, e.g., preferably an adeno-associated virus (AAV) vector that comprises sequences encoding an XP transgene. Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka, N., Curr Top Microbiol Immunol, 1992.158: p.97-129. AAV vectors efficiently transduce various cell types and can produce long-term expression of transgenes in vivo. AAV vectors have been extensively used for gene augmentation or replacement and have shown therapeutic efficacy in a range of animal models as well as in the clinic; see, e.g., Mingozzi and High, Nat Rev Genet, 2011.12(5): p.341-55; Deyle and Russell, Attorney Docket No.29539-0804WO1 / MGH 2023-602 Curr Opin Mol Ther, 2009.11(4): p.442-7; Asokan et al., Mol Ther, 2012.20(4): p. 699-708). AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. In some embodiments, the AAV vector can include (or include a sequence encoding) an AAV capsid polypeptide described in WO 2015 / 054653; for example, a virus particle comprising an AAV capsid polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, and 17 of WO 2015 / 054653, and a sequence encoding an XP transgene as described herein. In some embodiments, the AAV capsid polypeptide is an Anc80 polypeptide, e.g., Anc80L27; Anc80L59; Anc80L60; Anc80L62; Anc80L65; Anc80L33; Anc80L36; or Anc80L44. Alternatively, AAV.CPP.21 or AAV.CPP.16 can be used, as described in Yao et al., Nat Biomed Eng.2022 Nov;6(11):1257-1271. In some embodiments, the AAV incorporates inverted terminal repeats (ITRs), e.g., derived from the AAV2 or AAV9 serotype. It should be noted, however, that numerous modified versions of the AAV2 or AAV9 ITRs are used in the field. Modifications of these sequences are known in the art, or will be evident to skilled artisans, and are thus included in the scope of this disclosure. In some embodiments, the expression vector comprises a nucleic acid sequence from 5’ -3’: ITR – S5E2 enhancer - promoter– beta-actin minimal or CMV promoter - XP transgene – optional WPRE - pA – ITR, optionallywherein the nucleic acid sequence is at least 90%, 95%, 97%, or 99%, or is 100%identical to a sequence provided herein; in some embodiments, differences from can be present in the sequences between the elements recited above, e.g., different restriction enzyme sites. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses are known in the art, e.g., can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. The use of AAV vectors to deliver constructs for expression in the brain has been described, e.g., in Iwata et al., Sci Rep.2013;3:1472; Hester et al., Curr Gene Ther.2009 Oct;9(5):428-33; Doll et al., Gene Therapy 1996, 3(5):437-447; and Foley et al., J Control Release.2014 Dec 28;196:71-8. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Thus, in some embodiments, the XP transgene encoding nucleic acid is present in a vector for gene therapy, such as an AAV vector. In some instances, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, and AAV12. A vector as described herein can be a pseudotyped vector. Pseudotyping provides a mechanism for modulating a vector’s target cell population. For instance, pseudotyped AAV vectors can be utilized in various methods described herein. Pseudotyped vectors are those that contain the genome of one vector, e.g., the genome of one AAV serotype, in the capsid of a second vector, e.g., a second AAV serotype. Methods of pseudotyping are well known in the art. For instance, a vector may be pseudotyped with envelope glycoproteins derived from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains), rabies virus (e.g., various Evelyn–Rokitnicki–Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus, a rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein B type (FuG-B), a variant of FuG-B (FuG- B2) or Moloney murine leukemia virus (MuLV). A virus may be pseudotyped for transduction of one or more neurons or groups of cells. In addition, the capsid can be altered to include one or more peptides that increase expression in the CNS, see, e.g.,Yao et al., Nat Biomed Eng. 2022 Oct 10; Chatterjee et al., Gene Ther. 2022Jun;29(6):390-397; Meng et al., Mol Ther Methods Clin Dev.2021 Feb 27;21:28-41; Zhang et al., Biomaterials.2022 Feb;281:121340; Gray, Cell Gene Ther. Insights 5, 1361–1368 (2019); Nonnenmacher et al., Mol. Ther. Methods Clin. Dev.20, 366–378 (2021). Without limitation, illustrative examples of pseudotyped vectors include recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrh10, AAV11, and AAV12 serotype vectors. It is known in the art that such vectors may be engineered to include a transgene encoding a human protein or other protein. In particular instances, the present disclosures can include a pseudotyped AAV9 or AAVrh10 viral vector including a nucleic acid as disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003. In some instances, a particular AAV serotype vector may be selected based upon the intended use, e.g., based upon the intended route of administration. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Various methods for application of AAV vector constructs in gene therapy are known in the art, including methods of modification, purification, and preparation for administration to human subjects (see, e.g., Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, AAV based gene therapy targeted to cells of the CNS has been described (see, e.g., U.S. patents 6,180,613 and 6,503,888). High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. No.5,658,776 A vector construct refers to a polynucleotide molecule including all or a portion of a viral genome and a transgene. In some instances, gene transfer can be mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful in methods of gene therapy are known in the art. For example, a construct as disclosed herein can include an alphavirus, herpesvirus, retrovirus, lentivirus, or vaccinia virus. Adenoviruses are a relatively well characterized group of viruses, including over 50 serotypes (see, e.g., WO 95 / 27071, which is herein incorporated by reference). Adenoviruses are tractable through the application of techniques of molecular biology and may not require integration into the host cell genome. Recombinant Ad-derived vectors, including vectors that reduce the potential for recombination and generation of wild-type virus, have been constructed (see, e.g.,international patent publications WO 95 / 00655 and WO 95 / 11984, which are hereinincorporated by reference). Wild-type AAV has high infectivity and is capable of integrating into a host genome with a high degree of specificity (see, e.g., Hermonat and Muzyczka 1984 Proc. Natl. Acad. Sci., USA 81:6466-6470 and Lebkowski et al. 1988 Mol. Cell. Biol.8:3988-3996). The viral vectors, e.g., AAV, e.g., packaged in AAV capsids, can be included in compositions (such as pharmaceutical compositions) and / or administered to subjects. An exemplary pharmaceutical composition comprising a viral vector, e.g., an AAV, as described herein can include a pharmaceutically acceptable carrier such as balanced saline solution (BSS) and one or more surfactants; exemplary formulations are described in Grossen et al., Eur J Pharm Biopharm.2023 Sep:190:1-23. Other pharmaceutical formulation elements known in the art may also be suitable for use in the compositions described herein. Attorney Docket No.29539-0804WO1 / MGH 2023-602 For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection, e.g., optionally into the cisterna magna, cerebral ventricles, lumbar intrathecal space, direct injection into hippocampus (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)) and / or the entorhinal cortex. In some embodiments, delivery methods of XP transgene-expressing virus include intravenous, intrathecal, intracerebroventricular, intracisternal, and stereotactic intraparenchymal administration. The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is embedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can comprise one or more cells, which produce the gene delivery system. Exemplary Sequences and Constructs In some embodiments, the sequence of a protein or nucleic acid used in a composition or method described herein is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to an exemplary or reference sequence set forth herein. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second amino acid or nucleic acid sequencefor optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In some embodiments, the sequence of a protein or nucleic acid used Attorney Docket No.29539-0804WO1 / MGH 2023-602 in a composition or method described herein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 differences from a reference sequence described herein. In some embodiments, the sequence of a protein used in a composition or method described herein lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids from the N terminus or the C terminus. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol.48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5. Human TBR1 nucleic acid sequence - NM_006593.4 Coding region: 115-2163 (atg bolded) 1 atttgctggt tgaagtgctt tctgtctagt gagggggtct gtggatttct agtttatgat 61 aaataggact ttaaaaacca gggacgggag ggcgagtgtt caggttctag agctatgcag 121 ctggagcact gcctttctcc ttctatcatg ctctccaaga aatttctcaa tgtgagcagc 181 agctacccac attcaggcgg atccgagctt gtcttgcacg atcatcccat tatctcgacc 241 actgacaacc tggagagaag ttcacctttg aaaaaaatta ccagggggat gacgaatcag 301 tcagatacag acaattttcc tgactccaag gactcaccag gggacgtcca gagaagtaaa 361 ctctctcctg tcttggacgg ggtctctgag cttcgtcaca gtttcgatgg ctctgctgca 421 gatcgctacc tcctctctca gtccagccag ccacagtctg cggccactgc tcccagtgcc 481 atgttcccgt accccggcca gcacggaccg gcgcaccccg ccttctccat cggcagccct 541 agccgctaca tggcccacca cccggtcatc accaacggag cctacaacag cctcctgtcc 601 aactcctcgc cgcagggata ccccacggcc ggctacccct acccacagca gtacggccac 661 tcctaccaag gagctccgtt ctaccagttc tcctccaccc agccggggct ggtgcccggc 721 aaagcacagg tgtacctgtg caacaggccc ctttggctga aatttcaccg gcaccaaacg 781 gagatgatca tcaccaaaca gggaaggcgc atgtttcctt ttttaagttt taacatttct 841 ggtctcgatc ccacggctca ttacaatatt tttgtggatg tgattttggc ggatcccaat 901 cactggaggt ttcaaggagg caaatgggtt ccttgcggca aagcggacac caatgtgcaa 961 ggaaatcggg tctatatgca tccggattcc cccaacactg gggctcactg gatgcgccaa 1021 gaaatctctt ttggaaaatt aaaacttacg aacaacaaag gagcttcaaa taacaatggg 1081 cagatggtgg ttttacagtc cttgcacaag taccagcccc gcctgcatgt ggtggaagtg 1141 aacgaggacg gcacggagga cactagccag cccggccgcg tgcagacgtt cactttccct 1201 gagactcagt tcatcgccgt caccgcctac cagaacacgg atattacaca actgaaaata 1261 gatcacaacc cttttgcaaa aggatttcgg gataattatg acacgatcta caccggctgt 1321 gacatggacc gcctgacccc ctcgcccaac gactcgccgc gctcgcagat cgtgcccggg 1381 gcccgctacg ccatggccgg ctctttcctg caggaccagt tcgtgagcaa ctacgccaag 1441 gcccgcttcc acccgggcgc gggcgcgggc cccgggccgg gtacggaccg cagcgtgccg 1501 cacaccaacg ggctgctgtc gccgcagcag gccgaggacc cgggcgcgcc ctcgccgcaa 1561 cgctggtttg tgacgccggc caacaaccgg ctggacttcg cggcctcggc ctatgacacg 1621 gccacggact tcgcgggcaa cgcggccacg ctgctctctt acgcggcggc gggcgtgaag 1681 gcgctgccgc tgcaggctgc aggctgcact ggccgcccgc tcggctacta cgccgacccg 1741 tcgggctggg gcgcccgcag tcccccgcag tactgcggca ccaagtcggg ctcggtgctg 1801 ccctgctggc ccaacagcgc cgcggccgcc gcgcgcatgg ccggcgccaa tccctacctg 1861 ggcgaggagg ccgagggcct ggccgccgag cgctcgccgc tgccgcccgg cgccgccgag Attorney Docket No.29539-0804WO1 / MGH 2023-602 1921 gacgccaagc ccaaggacct gtccgattcc agctggatcg agacgccctc ctcgatcaag 1981 tccatcgact ccagcgactc ggggatttac gagcaggcca agcggaggcg gatctcgccg 2041 gccgacacgc ccgtgtccga gagttcgtcc ccgctcaaga gcgaggtgct ggcccagcgg 2101 gactgcgaga agaactgcgc caaggacatt agcggctact atggcttcta ctcgcacagc 2161 taggccgccc ctgcccgccc ggccccgccg cggcccggac ccccagccag cccctcacag 2221 ctcttcccca gctccgcctc cccacactcc tccttgcgca cccactcatt ttatttgacc 2281 ctcgatggcc gtctgcagcg aataagtgca ggtctccgag cgtgatttta accttttttg 2341 cacagcagtc tctgcaatta gctcaccgac cttcaacttt gctgtaaacc ttttggtttt 2401 cctacttact cttcttctgt ggagttatcc tcctacaatt cccctccccc tcgtctttct 2461 cttacctcct acttctcttt cttgtaatga aactcttcac ctttaggaga cctgggcagt 2521 cctgtcaggc agcagcgatt ccgacccgcc aagtctcggc ctccacatta accataggat 2581 gttgactcta gaacctggac ccacccagcg cgtcctttct tatccccgag tggatggatg 2641 gatggatgga tggtagggat gttaataatt ttagtggaac aaagcctgtg aaatgattgt 2701 acatagtgtt aatttattgt aacgaatggc tagtttttat tctcgtcaag gcacaaaacc 2761 agttcatgct taaccttttt ttcctttcct ttctttgctt ttctttctct cctctcatac 2821 tttctcttct ctctctttta attttcttgt gagataatat tctaagaggc tctagaaaca 2881 tgaaatactc agtagtgatg ggtttcccac ttctcctcaa tccgttgcat gaaataatta 2941 ctatgtgccc taatgcacac aaatagctaa ggagaatcca cccaaacacc tttaaaggat 3001 aggtgtctgt tcataggcaa gtcgattaag tggcatgatg cctgcaaagc aaagtcaact 3061 ggagttgtat gttcccccca ccttctaaat agaatagctc gacatcagca atattatttt 3121 gccttatttg tttttcccca aagtgccaaa tccattactg gtctgtgcag gtgccaaata 3181 tgctgacaaa ctgtttctga atatctttca gtaccccttc acctttatat gctgtaaatc 3241 tttgtaatga atactctatt aatgatatag atgactgaat tgttggtaac tatagtgtag 3301 tctagtgaag atgaattgtg tgagttgtat attttactgc attttagttt tgaaaatgac 3361 ttccccacca cctagaaaca gctgaaattt gacttccttg ggagaacact agcattaatg 3421 caagtaagac tgattttccc ctaagtcttg ttatatttga taaggagcat taatccccct 3481 ggaaatagat tagtaggatt tctaatgttg tgtagcaaac ctatactttt ttgtatttaa 3541 aaattaatgt gaaatatgca tcatacacaa tattcaatct agattccagt ccatgggggg 3601 atttttccta ataggaattc agggtctaaa cgtgtgtata ttttggctct tctgtaaatc 3661 taatgttgtg atttttatat ttgtttcgtt ttgtctgtga actgaataat ttatacaaga 3721 acacactcca ttgagaaacg ttttgttttt tgctcgtttg tatcgtctgt gtataacaag 3781 taaaataaac ctggtaaaaa cgctaa SEQ ID NO:18 Human TBR1 protein sequence NP_006584.1 1 mqlehclsps imlskkflnv sssyphsggs elvlhdhpii sttdnlerss plkkitrgmt 61 nqsdtdnfpd skdspgdvqr sklspvldgv selrhsfdgs aadryllsqs sqpqsaatap 121 samfpypgqh gpahpafsig spsrymahhp vitngaynsl lsnsspqgyp tagypypqqy 181 ghsyqgapfy qfsstqpglv pgkaqvylcn rplwlkfhrh qtemiitkqg rrmfpflsfn 241 isgldptahy nifvdvilad pnhwrfqggk wvpcgkadtn vqgnrvymhp dspntgahwm 301 rqeisfgklk ltnnkgasnn ngqmvvlqsl hkyqprlhvv evnedgtedt sqpgrvqtft 361 fpetqfiavt ayqntditql kidhnpfakg frdnydtiyt gcdmdrltps pndsprsqiv 421 pgaryamags flqdqfvsny akarfhpgag agpgpgtdrs vphtngllsp qqaedpgaps 481 pqrwfvtpan nrldfaasay dtatdfagna atllsyaaag vkalplqaag ctgrplgyya 541 dpsgwgarsp pqycgtksgs vlpcwpnsaa aaarmaganp ylgeeaegla aersplppga 601 aedakpkdls dsswietpss iksidssdsg iyeqakrrri spadtpvses ssplksevla 661 qrdcekncak disgyygfys hs SEQ ID NO:19 Human BCL11A nucleic acid sequence NM_022893.4 Coding sequence 385-2892 (start codon bolded) 1 gtctctgtcc atccagactc ctgacgttca agttcgcagg gacgtcacgt ccgcacttga 61 acttgcagct caggggggct tttgccattt ttttcatctc tctctctctc tctccctcta 121 tctctcttct ctctctctcc ctcttttttt tttttttttt tttttttttt ttgcttaaaa Attorney Docket No.29539-0804WO1 / MGH 2023-602 181 aaaagccatg acggctctcc cacaattcat cttccctgcg ccatctttgt attatttcta 241 atttattttg gatgtcaaaa ggcactgatg aagatatttt ctctggagtc tccttctttc 301 taacccggct ctcccgatgt gaaccgagcc gtcgtccgcc cgccgccgcc gccgccgccg 361 ccgccgcccg ccccgcagcc caccatgtct cgccgcaagc aaggcaaacc ccagcactta 421 agcaaacggg aattctcgcc cgagcctctt gaagccattc ttacagatga tgaaccagac 481 cacggcccgt tgggagctcc agaaggggat catgacctcc tcacctgtgg gcagtgccag 541 atgaacttcc cattggggga cattcttatt tttatcgagc acaaacggaa acaatgcaat 601 ggcagcctct gcttagaaaa agctgtggat aagccacctt ccccttcacc aatcgagatg 661 aaaaaagcat ccaatcccgt ggaggttggc atccaggtca cgccagagga tgacgattgt 721 ttatcaacgt catctagagg aatttgcccc aaacaggaac acatagcaga taaacttctg 781 cactggaggg gcctctcctc ccctcgttct gcacatggag ctctaatccc cacgcctggg 841 atgagtgcag aatatgcccc gcagggtatt tgtaaagatg agcccagcag ctacacatgt 901 acaacttgca aacagccatt caccagtgca tggtttctct tgcaacacgc acagaacact 961 catggattaa gaatctactt agaaagcgaa cacggaagtc ccctgacccc gcgggttggt 1021 atcccttcag gactaggtgc agaatgtcct tcccagccac ctctccatgg gattcatatt 1081 gcagacaata acccctttaa cctgctaaga ataccaggat cagtatcgag agaggcttcc 1141 ggcctggcag aagggcgctt tccacccact ccccccctgt ttagtccacc accgagacat 1201 cacttggacc cccaccgcat agagcgcctg ggggcggaag agatggccct ggccacccat 1261 cacccgagtg cctttgacag ggtgctgcgg ttgaatccaa tggctatgga gcctcccgcc 1321 atggatttct ctaggagact tagagagctg gcagggaaca cgtctagccc accgctgtcc 1381 ccaggccggc ccagccctat gcaaaggtta ctgcaaccat tccagccagg tagcaagccg 1441 cccttcctgg cgacgccccc cctccctcct ctgcaatccg cccctcctcc ctcccagccc 1501 ccggtcaagt ccaagtcatg cgagttctgc ggcaagacgt tcaaatttca gagcaacctg 1561 gtggtgcacc ggcgcagcca cacgggcgag aagccctaca agtgcaacct gtgcgaccac 1621 gcgtgcaccc aggccagcaa gctgaagcgc cacatgaaga cgcacatgca caaatcgtcc 1681 cccatgacgg tcaagtccga cgacggtctc tccaccgcca gctccccgga acccggcacc 1741 agcgacttgg tgggcagcgc cagcagcgcg ctcaagtccg tggtggccaa gttcaagagc 1801 gagaacgacc ccaacctgat cccggagaac ggggacgagg aggaagagga ggacgacgag 1861 gaagaggaag aagaggagga agaggaggag gaggagctga cggagagcga gagggtggac 1921 tacggcttcg ggctgagcct ggaggcggcg cgccaccacg agaacagctc gcggggcgcg 1981 gtcgtgggcg tgggcgacga gagccgcgcc ctgcccgacg tcatgcaggg catggtgctc 2041 agctccatgc agcacttcag cgaggccttc caccaggtcc tgggcgagaa gcataagcgc 2101 ggccacctgg ccgaggccga gggccacagg gacacttgcg acgaagactc ggtggccggc 2161 gagtcggacc gcatagacga tggcactgtt aatggccgcg gctgctcccc gggcgagtcg 2221 gcctcggggg gcctgtccaa aaagctgctg ctgggcagcc ccagctcgct gagccccttc 2281 tctaagcgca tcaagctcga gaaggagttc gacctgcccc cggccgcgat gcccaacacg 2341 gagaacgtgt actcgcagtg gctcgccggc tacgcggcct ccaggcagct caaagatccc 2401 ttccttagct tcggagactc cagacaatcg ccttttgcct cctcgtcgga gcactcctcg 2461 gagaacggga gtttgcgctt ctccacaccg cccggggagc tggacggagg gatctcgggg 2521 cgcagcggca cgggaagtgg agggagcacg ccccatatta gtggtccggg cccgggcagg 2581 cccagctcaa aagagggcag acgcagcgac acttgtgagt actgtgggaa agtcttcaag 2641 aactgtagca atctcactgt ccacaggaga agccacacgg gcgaaaggcc ttataaatgc 2701 gagctgtgca actatgcctg tgcccagagt agcaagctca ccaggcacat gaaaacgcat 2761 ggccaggtgg ggaaggacgt ttacaaatgt gaaatttgta agatgccttt tagcgtgtac 2821 agtaccctgg agaaacacat gaaaaaatgg cacagtgatc gagtgttgaa taatgatata 2881 aaaactgaat agaggtatat taatacccct ccctcactcc cacctgacac cccctttttc 2941 accactcccc ttccccatcg ccctccagcc ccactccctg taggattttt ttctagtccc 3001 atgtgattta aacaaacaaa caaacaaaca gaagtaacga agctaagaat atgagagtgc 3061 ttgtcaccag cacacctgtt ttttttcttt ttctttttct tttttctttt tccttttttt 3121 tttttttcct ttatgttctc accgtttgaa tgcatgatct gtatggggca atactattgc 3181 attttacgca aactttgagc ctttctcttg tgcaataatt tacatgttgt gtatgttttt 3241 ttttaaactt agacagcatg tatggtatgt tatggctatt ttaaattgtc cctaattcgt 3301 tgctgagcaa acatgttgct gtttccagtt ccgttctgag agaaaaagag agagagagag 3361 aaaaagacca tgctgcatac attctgtaat acatatcatg tacagtttta ttttataacg 3421 tgaggaggaa aaacagtctt tggattaacc ctctatagac agaatagata gcactgaaaa 3481 aaaatctcta tgagctaaat gtctgtctct aaagggttaa atgtatcaat tggaaaggaa 3541 gaaaaaaggc cttgaattga caaattaaca gaaaaacaga acaagtttat tctatcattt 3601 ggttttaaaa tatgagtgcc ttggatctat taaaaccaca tcgatggttc tttctacttg 3661 ttataaactt gtagcttaat tcagcattgg gtgaggtaat aaaccttagg aactagcata 3721 taattctata ttgtatttct cacaacaatg gctacctaaa aagatgaccc attatgtcct 3781 agttaatcat catttttcct ttagtttaat tttataaaca aaactgatta taccagtata Attorney Docket No.29539-0804WO1 / MGH 2023-602 3841 aaagctactt tgctcctggt gagagcttaa aagaaatggg ctgttttgcc caaagtttta 3901 ttttttttaa acaatgatta aattgaatgt gtaatgtgca aaagccctgg aacgcaatta 3961 aatacactag taaggagttc attttatgaa gatatttgct ttaataatgt ctttttaaaa 4021 atactggcac caaaagaaat agatccagat ctacttggtt gtcaagtgga caatcaaatg 4081 ataaacttta agaccttgta taccatattg aaaggaagag gctgacaata aggtttgaca 4141 gaggggaaca gaagaaaata atatgattta ttagcacaac gtggtactat ttgccattta 4201 aaactagaac aggtatataa gctaatattg atacaatgat gattaactat gaattcttaa 4261 gacttgcatt taaatgtgac attcttaaaa aaagaagaga aagaatttta agagtagcag 4321 tatatatgtc tgtgctccct aaaagttgta cttcatttct tttccataca ctgtgtgcta 4381 tttgtgttaa catggaagag gattcattgt ttttattttt atttttttaa ttttttcttt 4441 tttattaagc tagcatctgc cccagttggt gttcaaatag cacttgactc tgcctgtgat 4501 atctgtatct tttctctaat cagagataca gaggttgagt ataaaataaa cctgctcaga 4561 taggacaatt aagtgcactg tacaattttc ccagtttaca ggtctatact taagggaaaa 4621 gttgcaagaa tgctgaaaaa aaattgaaca caatctcatt gaggagcatt ttttaaaaac 4681 taaaaaaaaa aaaactttgc cagccattta cttgactatt gagcttactt acttggacgc 4741 aacattgcaa gcgctgtgaa tggaaacaga atacacttaa catagaaatg aatgattgct 4801 ttcgcttcta cagtgcaagg atttttttgt acaaaacttt tttaaatata aatgttaaga 4861 aaaatttttt ttaaaaaaca cttcattatg tttagggggg aactgcattt tagggttcca 4921 ttgtcttggt ggtgttacaa gacttgttat ccatttaaaa atggtagtgg aaattctatg 4981 ccttggatac acaccgctct tcaggttgta aaaaaaaaaa acatacattg gggaaaggtt 5041 taagattata tagtacttaa atataggaaa atgcacactc atgttgattc ctatgctaaa 5101 atacatttat ggtctttttt ctgtatttct agaatggtat ttgaattaaa tgttcatcta 5161 gtgttaggca ctatagtatt tatattgaag cttgtatttt taactgttgc ttgttctctt 5221 aaaaggtatc aatgtacctt ttttggtagt ggaaaaaaaa aagacaggct gccacagtat 5281 atttttttaa tttggcagga taatatagtg caaattattt gtatgcttca aaaaaaaaaa 5341 aaagagagaa acaaaaaagt gtgacattac agatgagaag ccatataatg gcggtttggg 5401 ggagcctgct agaatgtcac atggatggct gtcatagggg ttgtacatat ccttttttgt 5461 tcctttttcc tgctgccata ctgtatgcag tactgcaagc taataacgtt ggtttgttat 5521 gtagtgtgct ttttgtccct ttccttctat caccctacat tccagcatct taccttcata 5581 tgcagtaaaa gaaagaaaga aaaaaaaagg aaaaaaaaaa aaaaaccaat gttttgcagt 5641 ttttttcatt gccaaaaact aaatggtgct ttatatttag attggaaaga atttcatatg 5701 caaagcatat taaagagaaa gcccgcttta gtcaatactt ttttgtaaat ggcaatgcag 5761 aatattttgt tattggcctt ttctattcct gtaatgaaag ctgtttgtcg taacttgaaa 5821 ttttatcttt tactatggga gtcactattt attattgctt atgtgccctg ttcaaaacag 5881 aggcacttaa tttgatcttt tatttttctt tgtttttatt ttttttttta tttagatgac 5941 caaaggtcat tacaacctgg ctttttattg tatttgtttc tggtctttgt taagttctat 6001 tggaaaaacc actgtctgtg tttttttggc agttgtctgc attaacctgt tcatacaccc 6061 attttgtccc tttattgaaa aaataaaaaa aattaaagta ca SEQ ID NO:20 Human BCL11a protein sequence NP_075044.2 1 msrrkqgkpq hlskrefspe pleailtdde pdhgplgape gdhdlltcgq cqmnfplgdi 61 lifiehkrkq cngslcleka vdkppspspi emkkasnpve vgiqvtpedd dclstssrgi 121 cpkqehiadk llhwrglssp rsahgalipt pgmsaeyapq gickdepssy tcttckqpft 181 sawfllqhaq nthglriyle sehgspltpr vgipsglgae cpsqpplhgi hiadnnpfnl 241 lripgsvsre asglaegrfp ptpplfsppp rhhldphrie rlgaeemala thhpsafdrv 301 lrlnpmamep pamdfsrrlr elagntsspp lspgrpspmq rllqpfqpgs kppflatppl 361 pplqsappps qppvksksce fcgktfkfqs nlvvhrrsht gekpykcnlc dhactqaskl 421 krhmkthmhk sspmtvksdd glstasspep gtsdlvgsas salksvvakf ksendpnlip 481 engdeeeeed deeeeeeeee eeeelteser vdygfglsle aarhhenssr gavvgvgdes 541 ralpdvmqgm vlssmqhfse afhqvlgekh krghlaeaeg hrdtcdedsv agesdriddg 601 tvngrgcspg esasgglskk lllgspssls pfskriklek efdlppaamp ntenvysqwl 661 agyaasrqlk dpflsfgdsr qspfassseh ssengslrfs tppgeldggi sgrsgtgsgg 721 stphisgpgp grpsskegrr sdtceycgkv fkncsnltvh rrshtgerpy kcelcnyaca 781 qsskltrhmk thgqvgkdvy kceickmpfs vystlekhmk kwhsdrvlnn dikte SEQ ID NO:21 Attorney Docket No.29539-0804WO1 / MGH 2023-602 Human MEIS2 nucleic acid sequence NM_170675.5 Coding sequence 1084-2517 (start codon bolded) 1 gcactcgttg catatcagag gctcggcgcg gcgcgctcct cctcgctccc gctccccact 61 cccgggatgt gtctccgccg tacgacgggc tatggccacc acgacttccg ggttccgtca 121 tttcgttctc ccgccgccga cccgcgccgc caaactgagg ctcttcaata agccaggcag 181 cagccaacct gccaacacct actgacactc actcatctcc cagagagaga aagagagcga 241 gagagagcga gcgcgagaga gcgagcgcga gtgagagcga gcgagcgagc gagaaagaga 301 gagagggaga gacaaaatac ctaccaggaa agggggggag gaagtccaat ttttgcaaac 361 tattcatttt tttttcttga tttttctcac tgctttcttt gaacaatact ttaaagagag 421 aggatcgtat tatagatacc gcgggggcaa agctaaaaaa ggggggaggg gggaggaaaa 481 aattcaagaa gcagaaaccc ctcgcggagt tttactggaa gaaaaaaacg ggtctgaaag 541 attcctcctc ttcatcatca tcaaccatca ttcattcact accttgacat tccgggcttt 601 gattgacagc tggagtggca aaaagccatg aaacacgaca gttcggttac atgtgggctg 661 ctgacgggcc gctcgtaacc ttcagttcgg gggcttgaca atttttttct tctttttctt 721 ctttttcttt tctttctttt tttttccaac tgaggggaag agaagagaaa gagggggaaa 781 ggaggaccga agaggaggag gaggggaggg gggaggagga ggaggtggag gaggaggagg 841 aagatcagga ggaggaggaa gaagaggaaa aaagagaaaa agaagaaata tcacagaaaa 901 aaaaattctt cgttgtctag actgggcttt ttttcccccc taaaaaatag catattggag 961 aattgggaga agtctctttg gtttggaaaa aaaaaaaagg aatcttcagc ctagatcact 1021 ttcttatccg gactgggata ttaaatatac gacacatcca ggagtttatt ggagcgcaga 1081 ctgatggcgc aaaggtacga tgagctgccc cattacggcg ggatggacgg agtaggggtt 1141 cccgcttcca tgtacggaga ccctcacgcg ccgcggccga tccccccggt tcaccacctg 1201 aaccacgggc cgccgctcca cgccacacag cactacggcg cgcacgcccc gcaccccaat 1261 gtcatgccgg ccagtatggg atccgctgtc aacgacgcct tgaagcggga caaggacgcg 1321 atctatgggc acccgttgtt tcctctgtta gctctggtct ttgagaagtg cgagctggcg 1381 acctgcactc cccgggaacc tggagtggct ggcggagacg tctgctcctc cgactccttc 1441 aacgaggaca tcgcggtctt cgccaagcag gttcgcgccg aaaagccact tttttcctca 1501 aatccagagc tggacaattt gatgatacaa gcaatacaag tactaaggtt tcatcttttg 1561 gagttagaaa aggtccacga actgtgcgat aacttctgcc accgatacat tagctgtttg 1621 aaggggaaaa tgcccatcga cctcgtcatt gatgaaagag acggcagctc caagtcagat 1681 catgaagaac tttcaggctc ctccacaaat ctcgctgacc ataacccttc ttcttggcga 1741 gaccacgatg atgcaacctc aacccactca gcaggcaccc cagggccctc cagtgggggc 1801 catgcttccc agagcggaga caacagcagt gagcaagggg atggtttaga caacagtgta 1861 gcttcacctg gtacaggtga cgatgatgat ccggataagg acaaaaaacg ccagaagaaa 1921 agaggcattt tccccaaagt agcaacaaat atcatgagag catggctctt ccagcatctc 1981 acacatccgt acccttccga agagcagaag aaacagttag cgcaagacac aggacttaca 2041 attctccaag taaacaactg gtttattaat gccagaagaa gaatagtaca gcccatgatt 2101 gaccagtcaa atcgagcagg ttttcttctt gatccttcag tgagccaagg agcagcatat 2161 agtccagagg gtcagcccat ggggagcttt gtgttggatg gtcagcaaca catggggatc 2221 cggcctgcag gtttgcagag catgccaggg gactacgttt ctcagggtgg tcctatggga 2281 atgagtatgg cacagccaag ttacactcct ccccagatga ccccacaccc tactcaatta 2341 agacatggac ccccaatgca ttcatatttg ccaagccatc cccaccaccc agccatgatg 2401 atgcacggag gaccccctac ccaccctgga atgactatgt cagcacagag ccccacaatg 2461 ttaaattctg tagatcccaa tgttggcgga caggttatgg acattcatgc ccaatagtat 2521 aagggaactc aagggaaaag gaaacacacg caaaaactat tttaagactt tctgaacttt 2581 gaccagatgt tgacacttaa tatgaaattc cagacagctg tgattatttt ttacttttgt 2641 catttttcat caagcaacag aggaccaatg caacaagaac acaaatgtga aatcatgggc 2701 tgactgagac aattctgtcc atgtaaagat cctctggaaa aagactccga gagttataac 2761 tactgtagta taaatatagg aactaagtta aacttgtaca tttctgttga tcacgccgtt 2821 atgttgcctc aaatagtttt agaagagaaa aaaaaatata tccttgtttt ccacactatg 2881 tgtgttgttc ccaaaagaat gactgttttg gttcatcagt gaattcacca tccaggagag 2941 actgtggtat atattttaaa cctgttgggc caatgagaaa agaaccacac tggagatcat 3001 gatgaacttt tggctgaacc tcatcactcg aactccagct tcaagaatgt gttttcatgc 3061 ccggcctttg ttcctccata aatgtgtcct ttagtttcaa acagatcttt atagttcgtg 3121 cttcataagc caattcttat tattattttt gggggactct tcttcaaaga gcttgccaat 3181 gaagatttaa agacagagca ggagcttctt ccaggagttc tgagccttgg ttgtggacaa 3241 aacaatctta agttgggcag ctttcctcaa cacaaaaaaa agttattaat ggtcattgaa 3301 ccataactag gactttatca gaaactcaaa gcttggggga taaaaaggag caagagaata 3361 ctgtaacaaa cttcgtacag agttcggtct attaattgtt tcatgttaga tattctatgt Attorney Docket No.29539-0804WO1 / MGH 2023-602 3421 gtttacctca attgaaaaaa aaaagaatgt ttttgctagt atcagatctg ctgtggaatt 3481 ggtattgtat gtccatgaat tcttcttttc tcagcacgtg ttcctcacta gaagaaaatg 3541 ctgttacctt taagctttgt caaatttaca ttaaaatact tgtatgagga ctgtgacgtt 3601 atgttaaaaa aaaaaaggtg ttaagtcaca aaaagcggta ataaatattt catttttgat 3661 tttttgttag gcttttgcgt ttttaatggt tttaggacaa gactgcacaa tgccaactct 3721 atacagtgtt aaaagtaggt ttttcccccc agcagttgag tatattttaa actcatcaat 3781 aatttttaag gtaatcatct atagcctttc aatgtctatt tgacctataa gttccaccag 3841 cagaaaactt ctaaaggtac gaaactatgt gtacatgatg agccaggttg aaagatcccc 3901 ttgaacatgc tcaacctcct ttttagatgc atgacgttaa aaaatatata ctttcagata 3961 aagcttttca tgggcatcct ctgccagaat aaaggacttt tcagggttaa ttttacaaca 4021 taagaaataa tgtctgtgct caaaggtaat ttatctgcct gtgtttttcc tctgcctgtc 4081 ttactacaat attgttggat atgtaaatga aaatttacat atgggtgttc atagataagt 4141 aaacaacttc tatggagttt gaaattctga ctttaaaaga ttacttaatc attaatattt 4201 tataaattat attgtggtat atttttattt cagacacatc catgtgcagg gatgctagtg 4261 tgatgatttg ataactaaac ttaaactttt ttctgaaaac aaaacaaaac aaaatggacg 4321 gtataatgcc attttcaaaa ttttatgcaa cattcctgga aatctatacc attagcttat 4381 tgatactgca caaacaatgt ctgctaaagt gtgcaaaaag ggtgaggcat gtcccctcaa 4441 ttttcaaata aatatactat cctggaattt tttaactctt aaattttgaa tctttgttta 4501 gaaagaataa tgtcatatac tttggctaga tatgtgatgt caaccggcta aggaaattat 4561 tcatcatctc tgcaaactta aacttggaag aaatccatag ttattatttg gggctcttcc 4621 atctttccat ttcagacagc atttatggag gttctatata aatgaacgag gaaatacact 4681 tctattgttt tgtgttatat ttagtcaggt agaattttta caaatacatt tcagtttaag 4741 tgttgtgttg ctgtttaaac atagaaataa tgcttctgac acaaactaac ttaatagaga 4801 aattcagtgc cacttgggaa agagagatag cttccctctc atttttgcta ttctatttta 4861 catgaagtct ttttgtaaat tttaaatatt tgcctatttt tactttaaat acttttatgt 4921 aaactatgaa aattccccat tataaatcaa ctctcacatc aggtgggaaa aagaaccttt 4981 ttttctttcc actcagtaaa ttctgaaggg tttccttggc aattggctcg tgtttaggat 5041 gtgttttatg caattatcaa gttatctcat tgaagtttag agaccagagc tctcaactct 5101 tggttttaat gaataaagtc atctcatgct tagtttattc gtcaaaccca cgtcctatca 5161 tgttaacttt aaccttctta aatatttgga gatgaatgat aattgtaata tttgtttgca 5221 ttaccttgct ctcccccacc cccaacccca tgataacaca gagaagtgat agatttgtcc 5281 aatttttata atgctttttt tacaccaccc cggaaggcac ttgctcctat ctcgggttta 5341 ctatttagtg taaagagttc cgatttgggt atatttaaga aagactcagc tgtcaaaagc 5401 aaagaaactg ggaatggtgt tttgacaacc atatagtgtt aaaggaaata ctgtagtctg 5461 aataaaattg cttatgttct ccaaaa SEQ ID NO:22 Human MEIS2 protein sequence NP_733775.1 1 maqrydelph yggmdgvgvp asmygdphap rpippvhhln hgpplhatqh ygahaphpnv 61 mpasmgsavn dalkrdkdai yghplfplla lvfekcelat ctprepgvag gdvcssdsfn 121 ediavfakqv raekplfssn peldnlmiqa iqvlrfhlle lekvhelcdn fchryisclk 181 gkmpidlvid erdgssksdh eelsgsstnl adhnpsswrd hddatsthsa gtpgpssggh 241 asqsgdnsse qgdgldnsva spgtgddddp dkdkkrqkkr gifpkvatni mrawlfqhlt 301 hpypseeqkk qlaqdtglti lqvnnwfina rrrivqpmid qsnragflld psvsqgaays 361 pegqpmgsfv ldgqqhmgir paglqsmpgd yvsqggpmgm smaqpsytpp qmtphptqlr 421 hgppmhsylp shphhpammm hggppthpgm tmsaqsptml nsvdpnvggq vmdihaq SEQ ID NO:23 Human HERC1 nucleic acid sequence NM_003922.4 Coding sequence 149-14734 (start codon bolded) 1 gtctttccct ttgacaagtc gctgctgctg cagcaaaaat aaaggacgcg accgccgcag 61 cctctgcgga gccgggctcg ggtcgccgga gccgcgcccc accccgccag ctccagagcc 121 acgactaatg gctgaaggat aaatcaacat ggcaactatg attccaccag tgaagctgaa 181 atggcttgaa cacttgaaca gctcctggat tacagaggac agtgaatcta ttgctacaag 241 agagggagtt gctgttctgt attctaaact ggttagcaat aaggaagtag tacctttgcc 301 ccaacaagtt ttatgcctca aaggaccaca gttgccagac tttgaacgtg agtctctttc 361 aagtgatgag caggaccact atttggatgc ccttcttagc agccagctag cattggcaaa Attorney Docket No.29539-0804WO1 / MGH 2023-602 421 gatggtatgt tcagattccc catttgccgg ggcacttaga aaacgactgc ttgtactcca 481 gcgtgtcttt tatgcacttt ctaataaata ccatgacaaa ggcaaggtga agcagcagca 541 gcattctccg gagagcagtt ctggttcagc agatgtccat tctgttagtg aacgcccccg 601 gtcaagcact gatgcactta tagaaatggg tgttcgaact ggtctaagtt tattatttgc 661 gcttctaaga caaagttgga tgatgcctgt gtcaggacct ggtctcagtc tttgcaacga 721 tgtcattcat actgcaattg aagttgtgag ctctttgcca ccattatcat tagcaaatga 781 aagcaagatt cctcctatgg gcttggactg cttatcgcaa gtaacaacat ttcttaaagg 841 agtcactatt cctaattctg gggcagacac tttaggtcgt agattagctt ctgagttgct 901 gcttggtttg gcagctcaac gaggctcatt gcgatatctt cttgaatgga tagaaatggc 961 tttgggggct tcggcagttg tacacaccat ggagaaaggc aaactactct caagccagga 1021 aggaatgatc agctttgact gctttatgac catattaatg cagatgaggc gttctttggg 1081 ttcatctgct gatcggagtc agtggagaga accaaccaga acatcggatg gcttgtgctc 1141 cctttacgag gcagcattat gtctctttga agaggtttgc agaatggctt ctgattattc 1201 gagaacatgt gctagcccag atagcattca gactggtgat gctcccattg tctccgaaac 1261 ctgtgaggtt tatgtttggg ggagcaatag cagccatcag ttggtagaag gtacacagga 1321 gaaaatactg caacccaaac tggctcctag tttctctgat gcacagacca ttgaagctgg 1381 acagtactgc acttttgtca tttctacgga tggctctgtt agagcttgcg ggaaaggcag 1441 ctatgggaga ctgggccttg gagactccaa taatcagtca actttaaaaa agttaacatt 1501 cgagcctcac agatccatta aaaaggtttc atcttctaaa ggatctgatg gtcacacttt 1561 agcctttacg acagaaggag aagtcttcag ttggggagat ggtgattatg ggaaactggg 1621 gcatggaaat agttcaacac agaaatatcc caagcttatt cagggacctc tacaaggaaa 1681 ggtagttgtt tgtgtgtcag ctggatacag acatagtgct gctgtcacag aggatgggga 1741 attatacaca tggggtgaag gagactttgg aagattaggt catggtgaca gcaatagtcg 1801 taacattcca acattagtaa aagacatcag caatgtagga gaggtttctt gtggcagttc 1861 acatactatt gctctgtcta aagatgggag aactgtatgg tcttttggag gaggagacaa 1921 tggtaaactt ggtcatggtg ataccaacag agtgtataaa cctaaagtta ttgaagcttt 1981 acaaggaatg ttcattcgca aagtttgtgc tgggagccag tcttcacttg ctttgacatc 2041 aacagggcag gtctatgctt ggggctgtgg agcttgtcta ggttgtggtt cttcagaagc 2101 tactgctttg agacccaagc ttattgaaga actggctgcc acaagaatag ttgatgtttc 2161 tattggagac agtcattgtt tggctctttc tcatgataat gaagtttatg cctggggcaa 2221 taactcaatg gggcaatgtg gtcagggaaa ttccacaggt cctattacta aaccaaagaa 2281 agtgagtggc ttagatggca tagctattca gcagatttcg gctggaacat cacatagtct 2341 ggcatggact gctcttccta gggacagaca agttgttgca tggcaccgac cttattgtgt 2401 agatcttgaa gagagtacct tctcacacct gcgttctttt cttgagagat actgtgataa 2461 aataaacagt gagattcccc cactcccttt cccttcatca agagaacacc acagttttct 2521 caagctgtgc ctgaagctac tttcaaatca ccttgctctt gcacttgcgg gaggggtagc 2581 taccagcatt ctcgggaggc aggcaggtcc acttcgaaat ttgctcttca gactgatgga 2641 ctcaactgtc ccagatgaaa tccaagaggt ggtaattgaa actttatcag tgggagcaac 2701 catgctgtta cctccattac gagaacggat ggaattactt cattctcttt tacctcaagg 2761 acctgataga tgggaaagct tatctaaagg acagagaatg caactggata tcatcctgac 2821 aagtttgcaa gatcataccc acgtagcctc cctacttggc tatagttcac cctctgatgc 2881 tgctgaccta tcttctgtgt gtactggcta cggaaatctg tcagatcaac cttacggcac 2941 tcagagctgc catccagata cccacctggc tgaaattttg atgaagaccc tcttaagaaa 3001 tttaggattt tatacagatc aagcatttgg agagctagaa aagaatagtg ataaatttct 3061 acttggaaca tcatcatcag aaaacagtca gcctgctcat cttcatgaac tgctatgttc 3121 actacagaaa cagctgctgg cattttgcca tatcaataac attagtgaga actcaagcag 3181 tgtggcattg cttcataaac atcttcagct tttgttgcct catgccacag atatttattc 3241 acgttctgca aatttgctca aagaaagtcc ttggaatggc agtgttggag aaaaattaag 3301 agatgtgata tacgtctcag ctgctggcag tatgctctgc cagattgtta actccctgct 3361 gttactccct gtgtcagtgg ctcggccttt attgagttac ctcctcgact tgttgccacc 3421 tcttgattgc cttaatagac tcctgccagc tgctgatctt ttagaagacc aggagttaca 3481 gtggcctctt catggagggc cagaactaat tgatcctgct ggtctgccat tacctcagcc 3541 agctcagtcc tgggtatggc ttgtggatct agaaagaaca attgctctcc ttattgggcg 3601 gtgtcttggt ggcatgcttc agggctcccc tgtgtctcca gaggaacagg acactgcata 3661 ttggatgaaa acgccactgt tcagtgacgg tgtagaaatg gacactcctc aattggataa 3721 atgtatgagt tgcctgttag aagtagcact ttctggaaat gaagaacaga agccttttga 3781 ttataaattg cggcctgaaa ttgctgtcta tgtagacttg gcattgggtt gttctaaaga 3841 gcctgcccga agcctttgga tcagcatgca ggactatgct gttagtaaag attgggacag 3901 tgcaacttta agtaatgagt cactcttgga cactgtgtct agatttgttc ttgcagctct 3961 tctgaaacac acaaatttac ttagtcaagc atgtggagaa agccgatatc aacctggtaa 4021 acacttatca gaagtgtacc gttgtgtata caaagttcga agtcgtttac ttgcttgcaa Attorney Docket No.29539-0804WO1 / MGH 2023-602 4081 gaaccttgaa cttattcaaa caaggtcatc atcacgggac agatggatat cagaaaacca 4141 ggactctgca gatgttgatc ctcaggagca ttcatttact cgaactattg atgaagaagc 4201 tgaaatggaa gaacaggctg agagagaccg ggaagagggg catccggagc cagaggatga 4261 agaggaggaa cgggaacatg aagtgatgac agctggcaaa atctttcagt gtttcctctc 4321 agcccgtgaa gtagctcgta gccgagaccg agatagaatg aacagtgggg cagggtctgg 4381 ggctcgagct gatgatccac ctcctcagtc tcagcaagag cgaagggtca gcacagacct 4441 tcctgagggt caggatgtgt acactgctgc atgcaactcc gtgatccatc ggtgtgccct 4501 gttaatatta ggagtaagtc ctgtgataga tgagcttcag aagcgaagag aagaaggaca 4561 gttgcagcaa ccttcaacaa gtgcctctga agggggtgga cttatgacca ggagtgaaag 4621 tcttactgca gagagccggc tagtccacac aagcccaaat tatagactga tcaaatcgag 4681 gagtgaatct gatttgtctc agcctgaatc agatgaagag ggttacgcac tgagtggcag 4741 acgaaatgtt gatttggatt tggcagcatc tcacagaaag agaggtccta tgcacagtca 4801 attggaatcc ctgagtgact cttgggctcg cctgaaacat agcagagact ggttatgcaa 4861 ctcctcctat tcctttgagt cagattttga tcttaccaag tctttgggag ttcacacttt 4921 gattgaaaat gttgtaagct ttgtgagtgg agatgtgggg aatgccccag gttttaaaga 4981 gccagaggaa agtatgtcta caagtcccca ggcctccatc attgcaatgg aacagcagca 5041 gttaagggca gaacttcgtt tagaggcact tcatcagatc ctcgttctat tgtctgggat 5101 ggaagaaaaa ggtagcatct cactggcagg aagcagattg agttcaggct tccagtcctc 5161 cacactactc acgtctgtga ggctgcagtt cctagcaggg tgttttggtt taggcactgt 5221 tggacacaca ggaggcaagg gagagagtgg ccgattgcat cactatcagg atgggatcag 5281 agcagctaag agaaatattc agattgaaat ccaggtagct gtgcataaaa tttatcaaca 5341 gttgtctgct accctggaaa gagccctgca agcaaacaag catcacattg aagcccagca 5401 acgtctgctt ctggttacag tttttgccct aagtgttcat tatcaaccag tagatgtttc 5461 tttggcaatt tccactggtc tgctaaacgt attgtcacag ttgtgtggta cagacaccat 5521 gctaggacag cccctgcagt tgttgccaaa gacgggtgtt tcccagctta gcacagcttt 5581 gaaagtggcc agtacaaggt tgctccagat tctagccatc actactggga cctatgctga 5641 taaactgagt cccaaagtag ttcaatcctt gttggatcta ctctgtagtc agttgaagaa 5701 tttattgtcc caaactggtg tactacatat ggcctctttc ggagaagggg agcaagaaga 5761 cggtgaagaa gaagaaaaaa aagttgactc cagtggagaa actgagaaga aagatttcag 5821 agctgctctt aggaaacaac atgcagccga actccatcta ggggattttt tagtttttct 5881 tcgcagagtt gtatcttcaa aagcaattca atcaaaaatg gcttccccaa agtggaccga 5941 agtgcttcta aatatagcat ctcagaaatg ttcttcaggt atccctctgg ttggtaactt 6001 aagaacaagg ctccttgcac ttcatgtcct tgaagctgtg ctgccagctt gtgaatctgg 6061 tgtagaagat gatcaaatgg cccagattgt tgagcgctta ttttcccttc tctctgattg 6121 tatgtgggag acacccattg ctcaggccaa acatgctatt cagataaagg aaaaagaaca 6181 agaaataaaa ctacagaagc agggcgagtt ggaagaagaa gatgagaatc ttcctatcca 6241 agaagtatcc tttgacccgg agaaagctca gtgttgccta gtggagaatg gacagatttt 6301 aactcacggc agtggaggga aaggatatgg attggcatct acaggagtaa cttctgggtg 6361 ctatcagtgg aagttttata ttgtgaagga aaacagaggt aatgaaggca cgtgtgttgg 6421 agtttctcgc tggccagtac atgactttaa tcaccgcact acctcggata tgtggctcta 6481 tagggcctac agtggtaacc tctatcacaa tggagaacag actctcacat tgtccagctt 6541 tactcaagga gatttcatta cctgtgtgtt agacatggaa gccaggacca tttcttttgg 6601 gaaaaatgga gaggaaccca aattagcttt tgaagatgtg gatgcagcag agttgtaccc 6661 atgtgtgatg ttctatagta gcaatccagg ggaaaaggtg aaaatttgtg atatgcagat 6721 gcgtggcaca ccccgagact tacttccagg agaccctatt tgtagtccag tagcagcagt 6781 gctggctgag gccactattc agctcatccg tatccttcac cgaacagacc gttggactta 6841 ctgcattaac aaaaaaatga tggaaaggct tcacaaaatt aagatatgta ttaaagagtc 6901 aggtcagaag ctaaagaaaa gccgctcggt tcagagccga gaggaaaatg aaatgagaga 6961 ggagaaggag agcaaagagg aagagaaagg taaacatact aggcatggcc tcgctgacct 7021 ctcagagctg cagctgagga ctctttgcat agaggtgtgg cccgtgctgg ctgtgatagg 7081 aggagttgat gctggtctta gagttggagg tcggtgtgtt cacaagcaaa ctgggcgcca 7141 tgccacgctg ctgggagtgg tcaaagaggg cagcacgtct gccaaggtcc aatgggatga 7201 agcagaaatt actatcagct tcccaacttt ttggtcgcct agtgatactc cattgtataa 7261 tctggaaccc tgtgaaccat tgccgtttga tgtggcgcga ttccgaggcc tgacggcttc 7321 tgtgctgctg gacctaacat atctcactgg cgttcatgaa gacatgggca aacagagcac 7381 caaacgacat gaaaagaaac accgacatga atccgaggag aaaggggatg ttgagcagaa 7441 acctgagagt gaatccgctt tagatatgcg aacaggccta acatctgatg acgtcaaaag 7501 tcagagtacc acaagctcca aatcagaaaa tgaaatcgct tcattttctt tagatccaac 7561 actgccaagt gtggaatccc aacatcaaat aacagaaggg aaaagaaaaa atcatgaaca 7621 catgtccaaa aaccatgatg tagcccagtc agaaatcaga gcagtccagc tgtcctatct 7681 ttacctcggt gctatgaagt cacttagtgc ccttcttggc tgtagtaaat atgctgagct Attorney Docket No.29539-0804WO1 / MGH 2023-602 7741 gttgctgata ccaaaagttc tggctgaaaa tggccacaac tcagactgtg caagttctcc 7801 agttgttcat gaagacgtgg agatgcgagc agccctgcag ttcttgatgc gacacatggt 7861 gaagcgagca gtcatgcggt cacccataaa gagagcattg ggattagctg atctggaacg 7921 agcgcaagcc atgatctata aattagtggt tcatgggctt ttggaagacc agtttggggg 7981 caaaattaag caagagattg atcaacaagc tgaagaaagt gaccctgccc agcaggcaca 8041 gacaccagtt actactagcc catcagcctc aagcacgacc tcctttatga gcagctctct 8101 ggaggacacc acaactgcca ccactccagt cactgacaca gaaacagtgc ctgcatccga 8161 gtccccggga gtgatgcctc ttagtcttct caggcaaatg ttctctagtt acccaactac 8221 cactgtactt cccacacgtc gggcacagac tcctccaata tcttcgttac caacctctcc 8281 ttctgatgaa gtaggaagga ggcaaagttt aacttctcct gattcccagt cagcaaggcc 8341 agctaaccgc acagccttgt cagacccaag cagtagactt tcaacttctc ctcctcctcc 8401 agcaattgca gttcccttgc tggaaatggg gttctctctt cggcagattg ccaaagccat 8461 ggaagctaca ggtgctaggg gagaggctga tgcccagaat atcactgtcc ttgccatgtg 8521 gatgatagag caccctgggc atgaggatga agaggagccc cagtcgggca gcacagcaga 8581 ctctaggcct ggagcagccg ttctaggcag tggcgggaag tcaaatgatc cctgttattt 8641 gcagtcacct ggagacatac catcagctga tgctgctgaa atggaggaag gttttagtga 8701 aagccctgat aatttggatc atacagagaa tgcagcttct ggaagtggac catcagctag 8761 aggtcgctca gcggtaacaa gaagacacaa gtttgactta gctgctcgca cactgctagc 8821 aagagcagcg ggattatacc gctctgtgca ggcccacagg aatcaaagtc ggagagaagg 8881 aatatctttg cagcaagacc caggggcgtt gtatgacttt aatttagatg aggaattgga 8941 aattgatctt gatgatgagg cgatggaagc tatgtttgga caagacctga ccagtgacaa 9001 tgatattctg ggaatgtgga tcccagaggt actggattgg cctacctggc atgtttgtga 9061 gtctgaagac agggaagaag tggtggtgtg tgaactgtgt gaatgcagcg tcgtcagctt 9121 caatcagcac atgaagagaa accatccagg ctgtgggcgc agtgcaaacc gccagggcta 9181 tcgcagcaat ggttcctatg tggatggctg gtttggcggt gaatgtggga gtggaaatcc 9241 gtactacctg ttatgtggca cctgcaggga gaagtactta gccatgaaga ccaaatctaa 9301 gtcaacaagt tctgaaaggt acaagggaca agctccagat ctaattggca agcaagacag 9361 tgtgtatgaa gaagactggg acatgttgga tgttgatgaa gatgaaaagc taactggtga 9421 agaagaattt gaattacttg ctggaccgct tggtttaaat gaccggcgca ttgtaccaga 9481 accagttcag ttccctgaca gcgatccact gggagcatca gtagcaatgg tcacagccac 9541 caacagtatg gaagagactc tgatgcaaat aggttgccat ggctccgtag aaaagagctc 9601 ctctgggaga ataacgttag gagagcaggc agctgcccta gcaaaccctc atgaccgtgt 9661 ggtggcttta aggagagtga ctgctgctgc tcaggttctt ctggccagaa ccatggtcat 9721 gagagcgctg tctcttctct cagtcagtgg ttccagttgt agcctggctg ctggtcttga 9781 gtctctgggg ctaacagata tccgaacgct agttcgatta atgtgcttgg cagcagcagg 9841 gagagctggc ctctccacca gcccttctgc catggctagc acctcagaac gatcacgagg 9901 tgggcatagc aaggctaaca agcctatctc ttgcctggcc tatttgagca cagcagtggg 9961 atgtctggca tcaaatgctc ctagtgctgc caaactgctt gtacagttgt gtacacagaa 10021 cttgatttct gctgcaacag gtgtaaatct aaccacagtt gatgactcaa ttcagcgaaa 10081 gtttctaccc agctttctcc gaggaattgc tgaagagaac aagcttgtga cctccccaaa 10141 ctttgttgta acacaggccc ttgtggcatt gctagcagac aaaggggcca aactaagacc 10201 taactatgat aagtcagaag ttgaaaagaa aggccctctg gagttggcta atgccctggc 10261 agcctgctgc ctctcctcca ggctgtcctc acagcatcgg caatgggcag ctcagcaact 10321 cgtgcgcact cttgctgcac acgaccgtga caaccaaact actctgcaga cacttgctga 10381 tatgggagga gatcttagaa aatgctcctt tatcaaattg gaggctcatc agaacagagt 10441 aatgacatgt gtttggtgta ataaaaaagg tcttttggct acaagtggca atgatggcac 10501 catccgcgta tggaatgtta ccaagaagca atattcactg caacagacct gtgtgttcaa 10561 cagattggaa ggggatgctg aggaaagcct gggatcaccc agtgatccaa gtttctcacc 10621 agtttcctgg agtatcagtg gcaaatatct agcaggcgct ttggaaaaga tggtgaatat 10681 ctggcaagtt aatggaggaa aaggattagt agatattcag cctcattggg tatctgccct 10741 ggcttggcca gaagagggtc cggctacagc ctggtcagga gagtctccag aattgttgtt 10801 ggtgggacgg atggatggat ctctgggact gattgaagtt gttgatgtgt ccaccatgca 10861 ccgtcgagaa ttggagcatt gctatcgaaa ggatgtgtct gttacttgca ttgcatggtt 10921 cagtgaagac agaccatttg cagtgggata ttttgatgga aaactgttac tgggaacaaa 10981 ggaaccactt gagaaaggag gcattgttct aattgatgca cataaggata ctcttattag 11041 catgaagtgg gaccctacag gtcatattct tatgacatgt gccaaagaag acagtgtgaa 11101 actctggggc tctatttcgg gatgctggtg ctgtctacat tcactctgcc atccatctat 11161 tgtaaatggc attgcttggt gccgccttcc agggaaagga tccaagttgc agttactgat 11221 ggctactggc tgtcagagtg gcttagtatg tgtttggcgc attcctcaag atactacaca 11281 gaccaatgtg actagtgcag aaggatggtg ggagcaggaa tcaaattgcc aggatggata 11341 taggaaatca tcaggagcca agtgtgttta tcagctgcgg ggacacatca ctcctgttcg Attorney Docket No.29539-0804WO1 / MGH 2023-602 11401 gactgttgcc tttagttctg atgggttggc cctggtgtct ggtggactag gtgggctcat 11461 gaacatttgg tctttaaggg atggctctgt cttgcaaact gttgtgatag gctctggagc 11521 tattcagacc acagtatgga ttccagaagt tggagtagct gcttgctcaa atagatcaaa 11581 ggatgttttg gtcgtgaatt gtacagcaga atgggcagct gccaatcatg ttttggcaac 11641 ctgtaggaca gcattgaaac agcagggtgt tctgggattg aacatggctc cctgcatgag 11701 agcatttttg gagcggctcc ccatgatgct tcaggagcag tatgcctatg aaaagcctca 11761 tgtggtttgt ggtgaccaac ttgttcatag cccctatatg caatgcttgg cttcccttgc 11821 tgtgggactt catctggatc agctgttgtg taaccctcca gtgccaccac accaccagaa 11881 ctgtctccct gaccctgcat cctggaatcc aaatgaatgg gcctggttag aatgtttctc 11941 aaccactata aaagctgccg aagccctgac caatggagcc cagtttccag aatcttttac 12001 cgttccagat ctagaacctg ttccagagga tgaacttgta tttctaatgg ataacagtaa 12061 atggattaac ggcatggatg aacaaattat gtcttgggca acttccagac ctgaggactg 12121 gcacctggga ggtaaatgtg atgtctactt atggggtgct ggtaggcatg gacagctggc 12181 agaagctgga agaaatgtaa tggtacctgc agcagctccc tcattctcac aggcccaaca 12241 ggtcatttgt ggtcagaatt gtacctttgt catccaggcc aatggcacag tgttggcttg 12301 tggggaagga agttatggca gattaggaca aggaaattca gatgaccttc atgtgctgac 12361 agttatttca gccttacaag gctttgtggt gacccagctg gtgacttcct gtggttctga 12421 tgggcactct atggccctaa ctgaaagtgg tgaggtcttt agctggggag atggtgacta 12481 tggtaaactt ggccatggga acagcgacag gcagcggcgg cccaggcaga tcgaggcctt 12541 acaaggagaa gaagtggtgc agatgtcttg tggcttcaag cactcagcag tggtcacttc 12601 agatggcaaa ctgttcacct ttgggaatgg tgactatggt cgtctgggtc ttggaaatac 12661 ctctaacaaa aaacttccag agagagtgac tgcactggag ggatatcaga ttggacaggt 12721 ggcctgtgga ttaaaccaca ctttggcagt gtcagcagat ggttccatgg tgtgggcttt 12781 tggagatgga gactatggaa aactaggctt aggaaattcc actgcaaaat cttcacctca 12841 gaaaattgac gtcctttgtg gaattggaat aaaaaaggtt gcttgtggaa ctcagttttc 12901 tgttgctttg accaaagatg gtcatgtgta tacctttggt caagatcgcc tgataggctt 12961 gccagagggg cgtgctcgca atcacaatcg accgcaacaa atccctgtcc tggctggagt 13021 aatcatagaa gatgtggcag ttggagctga acacacactt gctttggcat caaatggaga 13081 tgtgtatgcc tgggggagca attcagaagg gcagctcggc ttaggccata ccaaccatgt 13141 tcgagaacca accctggtaa caggtctgca agggaaaaat gttcggcaga tctcggctgg 13201 ccgctgccac agtgctgcat ggacagcacc acctgtccca ccaagagcac caggtgtgtc 13261 agtacctctg cagctgggcc tgcctgacac agtgcccccc cagtatgggg cgctgagaga 13321 agtcagcatt cacacggtgc gggccaggct ccggctgctc taccacttct ctgacctcat 13381 gtactcatcc tggagactgc tgaaccttag ccccaacaac cagaacagca catcccatta 13441 taatgctgga acttggggca ttgtacaggg acaacttcgg cctttgttag ccccaagagt 13501 ctacactctg ccaatggtgc gctccatagg aaaaaccatg gttcaaggca aaaactatgg 13561 acctcagata actgtaaaga ggatatcaac cagaggacgg aagtgtaagc ctatttttgt 13621 ccaaatagcg agacaagtag ttaagctgaa tgcttcagac ctccgcctgc cttcccgagc 13681 gtggaaggtt aagctggttg gagaaggggc tgatgatgct ggaggagtgt ttgatgacac 13741 catcacagag atgtgccagg aacttgaaac tggtattgtt gaccttctta taccctctcc 13801 caatgccacc gcagaagtgg gttacaatag ggacaggttc ctttttaacc cttctgcctg 13861 cctcgatgaa cacttaatgc agtttaagtt tttaggaatt ttaatggggg ttgccattcg 13921 cacaaagaag cctctggacc tccacttggc ccctctggtg tggaagcagc tgtgctgtgt 13981 cccactcacc ctagaggacc tggaggaggt ggatctgctc tacgtgcaga ctctcaacag 14041 cattcttcac attgaagaca gtgggattac cgaggagagt ttccatgaga tgattcctct 14101 tgattctttt gttggccaga gtgctgatgg caaaatggtt cctataatcc ctggtggaaa 14161 tagtatccca ctcacatttt ccaacaggaa ggaatatgtg gagagggcca ttgaatatcg 14221 acttcatgag atggacagac aggtggctgc agtccgagaa gggatgtcct ggattgttcc 14281 tgtgccgctg ctgtccctcc tcacagcaaa acaactggag cagatggtgt gtgggatgcc 14341 cgagatctct gtggaagtct tgaagaaagt ggtgcggtac cgtgaggtgg atgagcagca 14401 tcagctggtg cagtggttct ggcacacgct ggaagagttc tccaatgagg agcgggtgct 14461 tttcatgagg tttgtgtcag gaagatctcg actaccagcc aacactgctg acatttctca 14521 gagatttcaa atcatgaagg ttgataggcc ttacgacagt ctgcctacct cacagacctg 14581 cttcttccag ctgaggctgc ccccgtactc cagccagctg gtcatggccg agcgcctgcg 14641 ctatgccatc aacaactgcc gctcaatcga catggacaac tacatgctct cgagaaacgt 14701 ggacaacgcc gagggctccg acactgacta ctgaccgtgc gggtgctctc accctccctt 14761 ctctccctca ataatgctca cttctgattt gatgttgata tacttttatg gtaactacat 14821 agatgttata agaacataaa ccaacattat aaacaatggc cacatttagt tactctaaat 14881 gtaacaaaga aattagatgt ttttattttt ctgtgattgt acaaaaacaa caaaaacgaa 14941 gtgctctcag tcaggttttt ccctccatat ttttggtcac ttttgataag tttgcatgaa 15001 accattttgg tgcattttta gttgggaatg gtacattttt gtaaatccac ccagtgaaca Attorney Docket No.29539-0804WO1 / MGH 2023-602 15061 tgaaattgta cattgtgtat aattgttcat tagaaaggac agttttacat gaatattcat 15121 atatttattt tgttttaatt tgaattgcct gttcagggtt ccttatgcag agaaataaag 15181 cagattcagg aattgga SEQ ID NO:24 Human HERC1 protein sequence NP_003913.3 1 matmippvkl kwlehlnssw itedsesiat regvavlysk lvsnkevvpl pqqvlclkgp 61 qlpdferesl ssdeqdhyld allssqlala kmvcsdspfa galrkrllvl qrvfyalsnk 121 yhdkgkvkqq qhspesssgs advhsvserp rsstdaliem gvrtglsllf allrqswmmp 181 vsgpglslcn dvihtaievv sslpplslan eskippmgld clsqvttflk gvtipnsgad 241 tlgrrlasel llglaaqrgs lryllewiem algasavvht mekgkllssq egmisfdcfm 301 tilmqmrrsl gssadrsqwr eptrtsdglc slyeaalclf eevcrmasdy srtcaspdsi 361 qtgdapivse tcevyvwgsn sshqlvegtq ekilqpklap sfsdaqtiea gqyctfvist 421 dgsvracgkg sygrlglgds nnqstlkklt fephrsikkv ssskgsdght lafttegevf 481 swgdgdygkl ghgnsstqky pkliqgplqg kvvvcvsagy rhsaavtedg elytwgegdf 541 grlghgdsns rniptlvkdi snvgevscgs shtialskdg rtvwsfgggd ngklghgdtn 601 rvykpkviea lqgmfirkvc agsqsslalt stgqvyawgc gaclgcgsse atalrpklie 661 elaatrivdv sigdshclal shdnevyawg nnsmgqcgqg nstgpitkpk kvsgldgiai 721 qqisagtshs lawtalprdr qvvawhrpyc vdleestfsh lrsflerycd kinseipplp 781 fpssrehhsf lklclkllsn hlalalaggv atsilgrqag plrnllfrlm dstvpdeiqe 841 vvietlsvga tmllpplrer mellhsllpq gpdrweslsk gqrmqldiil tslqdhthva 901 sllgysspsd aadlssvctg ygnlsdqpyg tqschpdthl aeilmktllr nlgfytdqaf 961 geleknsdkf llgtsssens qpahlhellc slqkqllafc hinnisenss svallhkhlq 1021 lllphatdiy srsanllkes pwngsvgekl rdviyvsaag smlcqivnsl lllpvsvarp 1081 llsylldllp pldclnrllp aadlledqel qwplhggpel idpaglplpq paqswvwlvd 1141 lertiallig rclggmlqgs pvspeeqdta ywmktplfsd gvemdtpqld kcmsclleva 1201 lsgneeqkpf dyklrpeiav yvdlalgcsk eparslwism qdyavskdwd satlsnesll 1261 dtvsrfvlaa llkhtnllsq acgesryqpg khlsevyrcv ykvrsrllac knleliqtrs 1321 ssrdrwisen qdsadvdpqe hsftrtidee aemeeqaerd reeghpeped eeeerehevm 1381 tagkifqcfl sarevarsrd rdrmnsgags garaddpppq sqqerrvstd lpegqdvyta 1441 acnsvihrca llilgvspvi delqkrreeg qlqqpstsas eggglmtrse sltaesrlvh 1501 tspnyrliks rsesdlsqpe sdeegyalsg rrnvdldlaa shrkrgpmhs qleslsdswa 1561 rlkhsrdwlc nssysfesdf dltkslgvht lienvvsfvs gdvgnapgfk epeesmstsp 1621 qasiiameqq qlraelrlea lhqilvllsg meekgsisla gsrlssgfqs stlltsvrlq 1681 flagcfglgt vghtggkges grlhhyqdgi raakrniqie iqvavhkiyq qlsatleral 1741 qankhhieaq qrlllvtvfa lsvhyqpvdv slaistglln vlsqlcgtdt mlgqplqllp 1801 ktgvsqlsta lkvastrllq ilaittgtya dklspkvvqs lldllcsqlk nllsqtgvlh 1861 masfgegeqe dgeeeekkvd ssgetekkdf raalrkqhaa elhlgdflvf lrrvvsskai 1921 qskmaspkwt evllniasqk cssgiplvgn lrtrllalhv leavlpaces gveddqmaqi 1981 verlfsllsd cmwetpiaqa khaiqikeke qeiklqkqge leeedenlpi qevsfdpeka 2041 qcclvengqi lthgsggkgy glastgvtsg cyqwkfyivk enrgnegtcv gvsrwpvhdf 2101 nhrttsdmwl yraysgnlyh ngeqtltlss ftqgdfitcv ldmeartisf gkngeepkla 2161 fedvdaaely pcvmfyssnp gekvkicdmq mrgtprdllp gdpicspvaa vlaeatiqli 2221 rilhrtdrwt ycinkkmmer lhkikicike sgqklkksrs vqsreenemr eekeskeeek 2281 gkhtrhglad lselqlrtlc ievwpvlavi ggvdaglrvg grcvhkqtgr hatllgvvke 2341 gstsakvqwd eaeitisfpt fwspsdtply nlepceplpf dvarfrglta svlldltylt 2401 gvhedmgkqs tkrhekkhrh eseekgdveq kpesesaldm rtgltsddvk sqsttsskse 2461 neiasfsldp tlpsvesqhq itegkrknhe hmsknhdvaq seiravqlsy lylgamksls 2521 allgcskyae lllipkvlae nghnsdcass pvvhedvemr aalqflmrhm vkravmrspi 2581 kralgladle raqamiyklv vhglledqfg gkikqeidqq aeesdpaqqa qtpvttspsa 2641 ssttsfmsss ledtttattp vtdtetvpas espgvmplsl lrqmfssypt ttvlptrraq 2701 tppisslpts psdevgrrqs ltspdsqsar panrtalsdp ssrlstsppp paiavpllem 2761 gfslrqiaka meatgargea daqnitvlam wmiehpghed eeepqsgsta dsrpgaavlg 2821 sggksndpcy lqspgdipsa daaemeegfs espdnldhte naasgsgpsa rgrsavtrrh 2881 kfdlaartll araaglyrsv qahrnqsrre gislqqdpga lydfnldeel eidlddeame 2941 amfgqdltsd ndilgmwipe vldwptwhvc esedreevvv celcecsvvs fnqhmkrnhp 3001 gcgrsanrqg yrsngsyvdg wfggecgsgn pyyllcgtcr ekylamktks kstsserykg 3061 qapdligkqd svyeedwdml dvdedekltg eeefellagp lglndrrivp epvqfpdsdp 3121 lgasvamvta tnsmeetlmq igchgsveks ssgritlgeq aaalanphdr vvalrrvtaa Attorney Docket No.29539-0804WO1 / MGH 2023-602 3181 aqvllartmv mralsllsvs gsscslaagl eslgltdirt lvrlmclaaa graglstsps 3241 amastsersr gghskankpi sclaylstav gclasnapsa akllvqlctq nlisaatgvn 3301 lttvddsiqr kflpsflrgi aeenklvtsp nfvvtqalva lladkgaklr pnydksevek 3361 kgplelanal aacclssrls sqhrqwaaqq lvrtlaahdr dnqttlqtla dmggdlrkcs 3421 fikleahqnr vmtcvwcnkk gllatsgndg tirvwnvtkk qyslqqtcvf nrlegdaees 3481 lgspsdpsfs pvswsisgky lagalekmvn iwqvnggkgl vdiqphwvsa lawpeegpat 3541 awsgespell lvgrmdgslg lievvdvstm hrrelehcyr kdvsvtciaw fsedrpfavg 3601 yfdgklllgt keplekggiv lidahkdtli smkwdptghi lmtcakedsv klwgsisgcw 3661 cclhslchps ivngiawcrl pgkgsklqll matgcqsglv cvwripqdtt qtnvtsaegw 3721 weqesncqdg yrkssgakcv yqlrghitpv rtvafssdgl alvsgglggl mniwslrdgs 3781 vlqtvvigsg aiqttvwipe vgvaacsnrs kdvlvvncta ewaaanhvla tcrtalkqqg 3841 vlglnmapcm raflerlpmm lqeqyayekp hvvcgdqlvh spymqclasl avglhldqll 3901 cnppvpphhq nclpdpaswn pnewawlecf sttikaaeal tngaqfpesf tvpdlepvpe 3961 delvflmdns kwingmdeqi mswatsrped whlggkcdvy lwgagrhgql aeagrnvmvp 4021 aaapsfsqaq qvicgqnctf viqangtvla cgegsygrlg qgnsddlhvl tvisalqgfv 4081 vtqlvtscgs dghsmaltes gevfswgdgd ygklghgnsd rqrrprqiea lqgeevvqms 4141 cgfkhsavvt sdgklftfgn gdygrlglgn tsnkklperv talegyqigq vacglnhtla 4201 vsadgsmvwa fgdgdygklg lgnstakssp qkidvlcgig ikkvacgtqf svaltkdghv 4261 ytfgqdrlig lpegrarnhn rpqqipvlag viiedvavga ehtlalasng dvyawgsnse 4321 gqlglghtnh vreptlvtgl qgknvrqisa grchsaawta ppvpprapgv svplqlglpd 4381 tvppqygalr evsihtvrar lrllyhfsdl mysswrllnl spnnqnstsh ynagtwgivq 4441 gqlrpllapr vytlpmvrsi gktmvqgkny gpqitvkris trgrkckpif vqiarqvvkl 4501 nasdlrlpsr awkvklvgeg addaggvfdd titemcqele tgivdllips pnataevgyn 4561 rdrflfnpsa cldehlmqfk flgilmgvai rtkkpldlhl aplvwkqlcc vpltledlee 4621 vdllyvqtln silhiedsgi teesfhemip ldsfvgqsad gkmvpiipgg nsipltfsnr 4681 keyveraiey rlhemdrqva avregmswiv pvpllsllta kqleqmvcgm peisvevlkk 4741 vvryrevdeq hqlvqwfwht leefsneerv lfmrfvsgrs rlpantadis qrfqimkvdr 4801 pydslptsqt cffqlrlppy ssqlvmaerl ryainncrsi dmdnymlsrn vdnaegsdtd 4861 y SEQ ID NO:25 S5E2 enhancer AATCTAACATGGCTGCTATAGCTTACTGACTAGAAGTTAAGTGCACACTTCCTAAAA GAAGGCTTTGACACAAGCCACTTCAGTTCCCTCCTCATTTTCTTGTCCCCATTCCTC TCTCTGTAGAATTCTGAGATTTCAATTCAGTTTTATACAGAAACCACATTACTGTAA GCCCTACAAAGTTATGGCAATATAGCTATATGGAGTCAAGTAATGTAGGTTATTTTT TTCCCAATGGTGCTGGTGAAGGTGGCAATTATGTAGCTATACTTAGCAGACTGAGGA AATTCTGCTAGAGTCAGCATTTGTCTCTTCATTGCTATGAAACAGTAATGGAAAAAT AAACAAAAACAAAAGGCAAACACTATGCATAATTCCCTCAGATCATATTAACATGTG ATGTTGGAGTAAATTGTTATAACCCCATTTTGGAAATACTTACCTTAATTAACTATG ATTTCCTTAAAATAATGCAGTATTTACAATCTATATGAAAGCACTATATGGGACACA TGGTATGATGGAACAGTGCACCCAAGAGACACCAAGAACATTCCTGTCTGTGGCAGT CTTTTCTCTATACAGAGGCATTTAGTCTCAATTGCTCAGAGTTATTTT SEQ ID NO:26 S5E2-Meis2-nlsdTom construct CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAACTAGTGGTTTAATCTAAC ATGGCTGCTATAGCTTACTGACTAGAAGTTAAGTGCACACTTCCTAAAAGAAGGCTT TGACACAAGCCACTTCAGTTCCCTCCTCATTTTCTTGTCCCCATTCCTCTCTCTGTA GAATTCTGAGATTTCAATTCAGTTTTATACAGAAACCACATTACTGTAAGCCCTACA Attorney Docket No.29539-0804WO1 / MGH 2023-602 AAGTTATGGCAATATAGCTATATGGAGTCAAGTAATGTAGGTTATTTTTTTCCCAAT GGTGCTGGTGAAGGTGGCAATTATGTAGCTATACTTAGCAGACTGAGGAAATTCTGC TAGAGTCAGCATTTGTCTCTTCATTGCTATGAAACAGTAATGGAAAAATAAACAAAA ACAAAAGGCAAACACTATGCATAATTCCCTCAGATCATATTAACATGTGATGTTGGA GTAAATTGTTATAACCCCATTTTGGAAATACTTACCTTAATTAACTATGATTTCCTT AAAATAATGCAGTATTTACAATCTATATGAAAGCACTATATGGGACACATGGTATGA TGGAACAGTGCACCCAAGAGACACCAAGAACATTCCTGTCTGTGGCAGTCTTTTCTC TATACAGAGGCATTTAGTCTCAATTGCTCAGAGTTATTTTAAACTTCCTGCAGCCCG GGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTAGCCTG CAAGTCGAGGAGCGCAGCCTTCCAGAAGCAGAGCGCGGCGCCTTAAGCTGCAGAAGT TGGTCGTGAGGCACTGGGCACGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGAC CAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCT ATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGCGAGCTCGCTAGCGCGA TCGCTGCCACCATGTTTCTGTACGATGAGCTGCCCCATTACGGCGGGATGGACGGAG TAGGGGTTCCCGCTTCCATGTACGGAGACCCTCACGCGCCGCGGCCGATCCCCCCGG TTCACCACCTAAACCACGGGCCGCCGCTCCACGCCACGCAGCACTACGGCGCGCACG CCCCGCACCCCAATGTCATGCCAGCCAGCATGGGATCTGCTGTCAACGACGCCTTGA AAAGAGACAAGGACGCAATCTATGGGCACCCGTTGTTTCCTCTGTTAGCTCTGGTTT TTGAGAAGTGCGAGCTGGCGACCTGCACTCCCCGGGAACCCGGAGTGGCCGGCGGAG ACGTCTGTTCCTCTGACTCCTTCAACGAGGACATCGCGGTCTTCGCCAAGCAGGTTC GCGCCGAAAAGCCTCTTTTTTCCTCAAACCCAGAGCTGGATAATTTGATGATACAAG CAATTCAAGTACTAAGGTTTCATCTTCTGGAGTTAGAAAAGGTCCACGAACTATGTG ATAACTTCTGCCACCGGTACATTAGCTGTTTGAAGGGAAAAATGCCCATTGACCTCG TGATTGATGAGAGAGATGGAAGCTCCAAGTCAGATCATGAAGAACTTTCAGGCTCCT CCACAAATCTCGCCGACCACAACCCTTCATCCTGGCGAGACCACGATGACGCAACCT CAACGCACTCCGCAGGCACCCCAGGACCCTCCAGTGGGGGCCATGCTTCCCAGAGTG GAGACAACAGCAGTGAGCAAGGCGCTTCGGAGACTGAGGCTGACCATTTACTATTGT GGAAGCCCATGACTGATCGCACAGGATGCGTTTCAGTGCAAATGTCAAGAGAAGATA GAGAAGAAAGCAGAGAGGGAAAACACTCTCCACAACCACTTGAAAACATCATCATTC CCTGTCAGCCGCCCTTTCGTGGGGAAAGCGAAGCTGGAGGCGATGGGTTAGACAACA GCGTAGCTTCACCTGGCACAGGTGATGACGACGATCCAGACAAGGACAAAAAACGCC AGAAGAAAAGAGGCATATTCCCCAAAGTCGCGACAAATATCATGAGAGCGTGGCTCT TCCAGCATCTCACACACCCGTACCCTTCAGAAGAACAGAAGAAACAGTTAGCGCAAG ACACGGGACTGACAATTCTGCAAGTGAACAACTGGTTTATCAATGCCAGAAGAAGAA TAGTGCAGCCCATGATTGACCAGTCAAATCGAGCAGGTTTTCTTCTTGATCCTTCAG TGAGCCAAGGAGCAGCGTATAGTCCAGAGGGTCAGCCCATGGGGAGCTTTGTGTTGG ATGGTCAGCAACACATGGGGATCCGGCCTGCAGGTTTGCAGAGCATGCCAGGGGACT ACGTTTCTCAGGGTGGTCCAATGGGAATGGGTATGGCCCAGCCAAGTTACACTCCTC CCCAGATGACCCCACACCCTACTCAGTTAAGACATGGACCCCCAATGCATTCATATT TGCCAAGCCATCCCCACCACCCAGCCATGGTGATGCACGGAGGACCCCCTACCCACC CTGGAATGACTATGTCAGCACAGAGCCCCACAATGTTAAATTCTGTAGATCCCAATG TTGGCGGACAGGTTATGGACATTCATGCCCAATCCGGAGATAACGGAAGCGGAGCCA CTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCATCTA GAATGGCCCCCAAGAAGAAGAGGAAGGTGGTGAGCAAGGGCGAGGAGGTCATCAAAG AGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGA TCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGG TGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCATGT ACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGT CCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGG TGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGA TGCGCGGCACCAACTTCCCCCCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCT Attorney Docket No.29539-0804WO1 / MGH 2023-602 GGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCC ACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCT ACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCTGG ACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGG GCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTAAGTCGACGGCG CGCCCCACCCCTGCAGGGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATT ACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTAT GTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCA TTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCG TTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTT GGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTA TTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGC TGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGC TGCTCGCCTATGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTT CGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTC TTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCC CGCATCGATACCGAGCGCTGCTCGAGAGATCTGTGATAGCGGCCATCAAGCTGGCCG CGACTCTAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAA AACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTT AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTC ACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCAGCTTATCGATACCGCATGCACGTGCGGACCGAGCGGCCGCAGGAACCCCTA GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCG CGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGT ATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAA GCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAG CGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCC GTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACC TCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGAT AGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGT TCCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGA TTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACG CGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCT GCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGC CCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCG GGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGG GCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGA CGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT AAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAAT AATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCT TTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAA AAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACA GCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAAC TCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAG AAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCA TGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGC TAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAAC CGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAA TGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC Attorney Docket No.29539-0804WO1 / MGH 2023-602 AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGG CCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTC GCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCT ACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG GTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTT AGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACC CCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCT GCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGC TACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTG TTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGT GTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCT GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGA GATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGG ACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAG GGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGC GTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACG CGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT SEQ ID NO: 27 S5E2-Bcl11a-nlsdTom construct CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGG CGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCA ACTCCATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAACTAGTGGTTTAAT CTAACATGGCTGCTATAGCTTACTGACTAGAAGTTAAGTGCACACTTCCTAAAAGAA GGCTTTGACACAAGCCACTTCAGTTCCCTCCTCATTTTCTTGTCCCCATTCCTCTCT CTGTAGAATTCTGAGATTTCAATTCAGTTTTATACAGAAACCACATTACTGTAAGCC CTACAAAGTTATGGCAATATAGCTATATGGAGTCAAGTAATGTAGGTTATTTTTTTC CCAATGGTGCTGGTGAAGGTGGCAATTATGTAGCTATACTTAGCAGACTGAGGAAAT TCTGCTAGAGTCAGCATTTGTCTCTTCATTGCTATGAAACAGTAATGGAAAAATAAA CAAAAACAAAAGGCAAACACTATGCATAATTCCCTCAGATCATATTAACATGTGATG TTGGAGTAAATTGTTATAACCCCATTTTGGAAATACTTACCTTAATTAACTATGATT TCCTTAAAATAATGCAGTATTTACAATCTATATGAAAGCACTATATGGGACACATGG TATGATGGAACAGTGCACCCAAGAGACACCAAGAACATTCCTGTCTGTGGCAGTCTT TTCTCTATACAGAGGCATTTAGTCTCAATTGCTCAGAGTTATTTTAAACTTCCTGCA GCCCGGGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTA GCCTGCAAGTCGAGGAGCGCAGCCTTCCAGAAGCAGAGCGCGGCGCCTTAAGCTGCA GAAGTTGGTCGTGAGGCACTGGGCACGTAAGTATCAAGGTTACAAGACAGGTTTAAG GAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGG CACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGCGAGCTCGCTAG CGCGATCGCTGCCACCATGTCTCGCCGCAAGCAAGGCAAACCCCAGCACTTAAGCAA ACGGGAATTCTCGCCCGAACCTCTTGAAGCCATTCTTACAGATGATGAACCAGACCA TGGCCCGTTGGGAGCTCCAGAAGGGGACCACGACCTTCTCACCTGTGGGCAGTGCCA GATGAATTTCCCACTGGGGGACATTCTTATTTTTATCGAGCACAAACGGAAACAATG CAATGGCAGCCTCTGCTTAGAAAAAGGTGTGGATAAGCCGCCTTCCCCTTCTCCCAT CGAGATGAAAAAGGCATCCAATCCTGTGGAGGTTGGCATCCAGGTCACGCCAGAGGA TGACGATTGTTTATCAACGTCATCTAGAGGAATTTGCCCCAAACAGGAACACATAGC AGATAAACTTCTGCACTGGAGGGGCCTGTCCTCTCCTCGGTCTGCACACGGAGCTCT AATCCCCACGCCCGGGATGAGTGCAGAATATGCCCCGCAGGGTATTTGTAAAGATGA Attorney Docket No.29539-0804WO1 / MGH 2023-602 GCCCAGCAGCTACACATGTACAACTTGCAAACAGCCATTCACCAGTGCATGGTTTCT CTTGCAACACGCACAGAACACTCATGGATTAAGAATCTACTTAGAAAGTGAACACGG AAGTCCCCTGACCCCGCGGGTTGGTATCCCTTCAGGACTAGGTGCAGAATGTCCTTC CCAGCCACCTCTCCATGGGATTCATATTGCAGACAATAACCCCTTTAACCTGCTAAG AATACCAGGATCAGTATCGAGAGAGGCTTCCGGCCTGGCAGAAGGGCGCTTTCCACC CACTCCCCCCCTGTTTAGTCCACCACCGAGACATCACTTGGACCCCCACCGCATAGA GCGCCTGGGGGCGGAAGAGATGGCCCTGGCCACCCATCACCCGAGTGCCTTTGACAG GGTGCTGCGGTTGAATCCAATGGCTATGGAGCCTCCCGCCATGGATTTCTCTAGGAG ACTTAGAGAGCTGGCAGGGAACACGTCTAGTCCACCGCTGTCCCCAGGCCGGCCCAG TCCTATGCAAAGGTTACTGCAACCATTCCAGCCAGGTAGCAAGCCACCCTTCCTGGC GACGCCCCCCCTCCCTCCTCTGCAATCCGCCCCTCCTCCCTCCCAACCCCCGGTCAA GTCCAAGTCATGCGAGTTCTGCGGCAAGACGTTCAAATTTCAGAGCAACTTGGTGGT TCACCGACGCAGCCATACTGGTGAGAAGCCCTATAAGTGCAACCTGTGCGACCACGC GTGCACACAGGCCAGCAAGCTGAAGCGTCACATGAAGACACACATGCACAAATCGTC CCCCATGACAGTCAAGTCCGACGATGGCCTCTCCACAGCCAGCTCCCCGGAACCTGG TACCAGCGACCTGGTGGGCAGCGCCAGCAGTGCGCTCAAGTCAGTGGTGGCCAAGTT CAAGAGTGAGAACGACCCCAACTTGATCCCAGAGAACGGGGATGAGGAGGAAGAGGA GGACGACGAGGAAGAAGAAGAAGAGGAGGAAGAGGAGGAGGAGGAGCTGACGGAGAG CGAGAGGGTGGACTACGGCTTCGGGCTGAGCCTGGAGGCTGCACGCCACCATGAGAA CAGCTCTCGGGGCGCAGTGGTGGGCGTGGGCGACGAGGGCCGCGCCCTGCCCGATGT CATGCAGGGCATGGTGCTCAGCTCCATGCAGCACTTCAGCGAGGCCTTCCACCAGGT CCTGGGCGAAAAGCATAAGCGTAGCCACCTGGCCGAGGCCGAGGGCCATAGGGACAC TTGTGATGAAGACTCGGTGGCCGGTGAGTCAGACCGCATAGACGATGGCACTGTTAA TGGTCGTGGCTGCTCCCCCGGCGAATCGGCTTCGGGGGGTCTGTCCAAAAAGCTGCT GCTGGGTAGCCCCAGCTCGCTGAGCCCCTTCTCCAAGCGCATCAAGCTGGAGAAGGA GTTTGACCTGCCCCCGGCCGCGATGCCTAACACGGAGAACGTGTATTCGCAGTGGCT CGCTGGCTATGCGGCCTCCAGGCAGCTCAAAGATCCCTTCCTTACTTTCGGAGACTC CAGACAATCGCCTTTTGCCTCCTCATCAGAGCACTCCTCGGAGAACGGGAGCTTGCG CTTCTCCACACCGCCCGGGGAGCTGGACGGAGGGATCTCAGGGCGCAGCGGCACAGG AAGTGGAGGGAGCACGCCCCATATTAGTGGTCCGGGCCCGGGCAGGCCCAGCTCAAA AGAGGGCAGACGCAGCGACACTTGTCCTTCACACACCCCCGTTCGGCGTAGTACCCC GCGAGCTCAAGATGTGTGGCAGTTTTCGGATGGAAGCTCAAGAACCCTTAAGTTCTC CGGAGATAACGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGT CGAAGAGAATCCCGGGCCATCTAGAATGGCCCCCAAGAAGAAGAGGAAGGTGGTGAG CAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTC CATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGG CACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGA CATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGA CATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGAT GAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGG CACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCCCCGT AATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGA CGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTA CCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTA CTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCATCGT GGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGA GCTGTACAAGTAAGTCGACGGCGCGCCCCACCCCTGCAGGGAATTCGATATCAAGCT TATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAA CTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGC TATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTC TCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTT Attorney Docket No.29539-0804WO1 / MGH 2023-602 TGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGG GACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGG GAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCGG GACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGG CCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCG GATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGAGCGCTGCTCGAGAGATCTGT GATAGCGGCCATCAAGCTGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTG TAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATA AAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAAT AAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTT GTGGTTTGTCCAAACTCATCAATGTATCAGCTTATCGATACCGCATGCACGTGCGGA CCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGG CGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATT TTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTA CGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGAC CGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCT CGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTT CCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTC ACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCAC GTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGG CTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGA GCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTT ATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACA CCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTA CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC ACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGT CATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACA ATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACA TTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGG TTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGA ACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG TATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGA ATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGT AACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCG TGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGA ACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGT TGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATC TGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAA GCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACG AAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAG GATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT TTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCC TTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGT GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG Attorney Docket No.29539-0804WO1 / MGH 2023-602 CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACC GGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCAC GCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGG AGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGG GTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAG CCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCC TTTTGCTCACATGT SEQ ID NO: 28 S5E2-Tbr1-nlsdTom construct CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAACTAGTGGTTTAATCTAAC ATGGCTGCTATAGCTTACTGACTAGAAGTTAAGTGCACACTTCCTAAAAGAAGGCTT TGACACAAGCCACTTCAGTTCCCTCCTCATTTTCTTGTCCCCATTCCTCTCTCTGTA GAATTCTGAGATTTCAATTCAGTTTTATACAGAAACCACATTACTGTAAGCCCTACA AAGTTATGGCAATATAGCTATATGGAGTCAAGTAATGTAGGTTATTTTTTTCCCAAT GGTGCTGGTGAAGGTGGCAATTATGTAGCTATACTTAGCAGACTGAGGAAATTCTGC TAGAGTCAGCATTTGTCTCTTCATTGCTATGAAACAGTAATGGAAAAATAAACAAAA ACAAAAGGCAAACACTATGCATAATTCCCTCAGATCATATTAACATGTGATGTTGGA GTAAATTGTTATAACCCCATTTTGGAAATACTTACCTTAATTAACTATGATTTCCTT AAAATAATGCAGTATTTACAATCTATATGAAAGCACTATATGGGACACATGGTATGA TGGAACAGTGCACCCAAGAGACACCAAGAACATTCCTGTCTGTGGCAGTCTTTTCTC TATACAGAGGCATTTAGTCTCAATTGCTCAGAGTTATTTTAAACTTCCTGCAGCCCG GGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTAGCCTG CAAGTCGAGGAGCGCAGCCTTCCAGAAGCAGAGCGCGGCGCCTTAAGCTGCAGAAGT TGGTCGTGAGGCACTGGGCACGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGAC CAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCT ATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGCGAGCTCGCTAGCGCGA TCGCTGCCACCATGCAGCTGGAGCATTGCCTCTCTCCTTCTATCATGCTCTCCAAGA AATTTCTCAATGTGAGCAGCAGCTACCCACATTCGGGCGGATCTGAGCTTGTCTTGC ATGATCATCCCATTATCTCGACCACTGACAACCTGGAGAGAAGTTCACCTTTGAAAA AAATTACCAGGGGGATGACGAATCAGTCAGATACAGACAATTTTCCTGACTCCAAGG ACTCACCAGGGGACGTCCAGAGAAGTAAACTCTCTCCTGTCTTGGACGGGGTCTCTG AGCTTCGTCACAGTTTCGATGGCTCTGCTGCAGATCGTTACCTACTCTCTCAGTCCA GCCAGCCACAGTCTGCGGCCACCGCTCCCAGTGCCATGTTCCCGTACCCCAGCCAGC ACGGACCGGCGCATCCCGCCTTCTCCATCGGCAGCCCCAGTCGCTACATGGCCCACC ACCCGGTCATTACCAACGGAGCTTACAACAGCCTGCTGTCCAACTCTTCGCCGCAGG GCTACCCCACGGCCGGCTACCCCTACCCACAGCAGTACGGCCACTCCTACCAAGGAG CCCCTTTCTACCAGTTCTCCTCCACCCAGCCCGGGTTGGTGCCCGGCAAGGCGCAAG TATACCTGTGCAACAGGCCACTTTGGCTGAAATTTCATCGGCATCAAACGGAGATGA TCATCACTAAACAGGGAAGGCGCATGTTTCCCTTTTTGAGTTTTAACATTTCTGGTC TCGATCCCACCGCTCATTACAATATTTTTGTGGATGTGATTTTGGCGGATCCCAATC ACTGGAGGTTTCAAGGAGGCAAATGGGTTCCTTGTGGCAAAGCGGACACCAATGTGC AAGGAAACCGGGTCTATATGCATCCGGATTCCCCCAACACTGGGGCTCACTGGATGC GGCAAGAAATCTCTTTTGGAAAATTAAAACTTACCAACAACAAGGGAGCATCAAACA ACAATGGGCAGATGGTGGTTTTACAGTCCCTGCACAAGTACCAGCCCCGTCTGCACG TGGTGGAAGTGAATGAGGATGGCACAGAGGACACCAGCCAGCCAGGCCGAGTCCAGA Attorney Docket No.29539-0804WO1 / MGH 2023-602 CGTTCACTTTTCCGGAGACTCAGTTCATCGCTGTCACCGCCTACCAGAACACGGATA TTACACAACTAAAAATAGATCATAACCCCTTTGCAAAAGGATTTCGAGATAACTATG ACACGATCTACACGGGCTGCGACATGGACCGCTTGACCCCGTCGCCCAACGACTCTC CGCGCTCGCAGATCGTGCCCGGCGCCCGCTACGCCATGGCCGGCTCTTTCCTGCAAG ACCAGTTCGTGAGCAACTACGCCAAGGCCCGCTTCCACCCGGGCGCCGGCGCGGGTC CCGGGCCGGGCACGGACCGCAGCGTGCCGCACACCAACGGGCTGCTGTCCCCGCAGC AGGCCGAGGACCCGGGCGCGCCGTCGCCGCAGCGCTGGTTCGTCACGCCGGCCAACA ACCGGCTGGACTTCGCGGCCTCGGCCTACGACACGGCCACGGACTTCGCCGGCAACG CGGCCACGCTGCTGTCGTATGCGGCCGCGGGCGTGAAGGCGCTGCCCTTGCAGGCCG CGGGCTGCACGGGCCGCCCGCTCGGCTACTACGCCGACCCTTCGGGCTGGGGCGCGC GCAGCCCCCCGCAGTACTGCGGCGCCAAGTCGGGCTCCGTGCTCCCCTGCTGGCCCA ACAGCGCCGCGGCCGCCGCGCGCATGGCCGGCGCCAACCCCTATCTGGGCGAGGAGG CCGAGGGCCTGGCGGCCGAGCGCTCGCCGCTGGCGCCCGCCGCCGAGGACGCCAAGC CCAAGGACCTGTCCGACTCCAGCTGGATCGAGACGCCCTCCTCCATCAAATCCATCG ACTCCAGCGACTCGGGGATTTACGAGCAGGCCAAGCGGAGGCGGATCTCGCCGGCTG ACACGCCGGTGTCTGAGAGCTCGTCCCCGCTCAAGAGCGAGGTGCTGGCCCAGCGGG ACTGCGAGAAGAACTGCGCCAAGGACATAGGCGGCTACTATGGCTTCTACTCGCACA GCTCCGGAGATAACGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGG ATGTCGAAGAGAATCCCGGGCCATCTAGAATGGCCCCCAAGAAGAAGAGGAAGGTGG TGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGG GCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACG AGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCT GGGACATCCTGTCCCCCCAGTTCATGTACGGCTCCAAGGCGTACGTGAAGCACCCCG CCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCG TGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGG ACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGACGGCC CCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCC GCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCC ACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCG GCTACTACTACGTGGACACCAAGCTGGACATCACCTCCCACAACGAGGACTACACCA TCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGG ACGAGCTGTACAAGTAAGTCGACGGCGCGCCCCACCCCTGCAGGGAATTCGATATCA AGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTC TTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATC ATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGC TGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTG TGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTT CCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCC TTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC GCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGA GTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGAGCGCTGCTCGAGAGAT CTGTGATAGCGGCCATCAAGCTGGCCGCGACTCTAGATCATAATCAGCCATACCACA TTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAA CATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTAC AAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCT AGTTGTGGTTTGTCCAAACTCATCAATGTATCAGCTTATCGATACCGCATGCACGTG CGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCC CGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGG Attorney Docket No.29539-0804WO1 / MGH 2023-602 TATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCAT AGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCG TGACCGCTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCT TTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAG GGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGT CCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCT CGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAA ATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAA TTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCC GACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCG CTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGT CATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTA ATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGC GCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGA GACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTC AACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTG CTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCG AAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTAT CCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAA GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTC TGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACG AGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTG GCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATA AAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATA AATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG AACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGT CAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTA AAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTG AGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAG ATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAG CGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCT TCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACC ACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAG TGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGT TACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCT TGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCG CCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAA CAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGC GGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCT GGCCTTTTGCTCACATGT SEQ ID NO: 29 S5E2-dSaCas9-VP64 construct CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC Attorney Docket No.29539-0804WO1 / MGH 2023-602 ATCACTAGGGGTTCCTGCGGCCGCACGCGTTTAATTAAACTAGTGGTTTAATCTAAC ATGGCTGCTATAGCTTACTGACTAGAAGTTAAGTGCACACTTCCTAAAAGAAGGCTT TGACACAAGCCACTTCAGTTCCCTCCTCATTTTCTTGTCCCCATTCCTCTCTCTGTA GAATTCTGAGATTTCAATTCAGTTTTATACAGAAACCACATTACTGTAAGCCCTACA AAGTTATGGCAATATAGCTATATGGAGTCAAGTAATGTAGGTTATTTTTTTCCCAAT GGTGCTGGTGAAGGTGGCAATTATGTAGCTATACTTAGCAGACTGAGGAAATTCTGC TAGAGTCAGCATTTGTCTCTTCATTGCTATGAAACAGTAATGGAAAAATAAACAAAA ACAAAAGGCAAACACTATGCATAATTCCCTCAGATCATATTAACATGTGATGTTGGA GTAAATTGTTATAACCCCATTTTGGAAATACTTACCTTAATTAACTATGATTTCCTT AAAATAATGCAGTATTTACAATCTATATGAAAGCACTATATGGGACACATGGTATGA TGGAACAGTGCACCCAAGAGACACCAAGAACATTCCTGTCTGTGGCAGTCTTTTCTC TATACAGAGGCATTTAGTCTCAATTGCTCAGAGTTATTTTAAACTTCCTGCAGCCCG GGCTGGGCATAAAAGTCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTAGCCTG CAAGTCGAGGAGCGCAGCCTTCCAGAAGCAGAGCGCGGCGCCTTAAGCTGCAGAAGT TGGTCGTGAGGCACTGGGCACGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGAC CAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCT ATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGCGAGCTCGCTAGCGCGA TCGCTGCCACCATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAG CAGCCAAGCGGAACTACATCCTGGGCCTGGCCATCGGCATCACCAGCGTGGGCTACG GCATCATCGACTACGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAG AGGCCAACGTGGAAAACAACGAGGGCAGGCGGAGCAAGAGAGGCGCCAGAAGGCTGA AGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTTCGACTACAACC TGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTGAAGG GCCTGAGCCAGAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCA AGAGAAGAGGCGTGCACAACGTGAACGAGGTGGAAGAGGACACCGGCAACGAGCTGT CCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAATACGTGGCCG AACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGAT TCAAGACCAGCGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCT ACCACCAGCTGGACCAGAGCTTCATCGACACCTACATCGACCTGCTGGAAACCCGGC GGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTCGGCTGGAAGGACATCAAAG AATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAACTGCGGAGCG TGAAGTACGCCTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAATCTCG TGATCACCAGGGACGAGAACGAGAAGCTGGAATATTACGAGAAGTTCCAGATCATCG AGAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCAAAGAAATCC TCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGT TCACCAACCTGAAGGTGTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTA TTGAGAACGCCGAGCTGCTGGATCAGATTGCCAAGATCCTGACCATCTACCAGAGCA GCGAGGACATCCAGGAAGAACTGACCAATCTGAACTCCGAGCTGACCCAGGAAGAGA TCGAGCAGATCTCTAATCTGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGG CCATCAACCTGATCCTGGACGAGCTGTGGCACACCAACGACAACCAGATCGCTATCT TCAACCGGCTGAAGCTGGTGCCCAAGAAGGTGGACCTGTCCCAGCAGAAAGAGATCC CCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAAGCTTCATCC AGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCA TTATCGAGCTGGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGA TGCAGAAGCGGAACCGGCAGACCAACGAGCGGATCGAGGAAATCATCCGGACCACCG GCAAAGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACATGCAGGAAG GCAAGTGCCTGTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTGAACAACCCCT TCAACTATGAGGTGGACCACATCATCCCCAGAAGCGTGTCCTTCGACAACAGCTTCA ACAACAAGGTGCTCGTGAAGCAGGAAGAAGCCAGCAAGAAGGGCAACCGGACCCCAT TCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAACCTTCAAGAAGCACA TCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAGTATCTGC Attorney Docket No.29539-0804WO1 / MGH 2023-602 TGGAAGAACGGGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACC TGGTGGATACCAGATACGCCACCAGAGGCCTGATGAACCTGCTGCGGAGCTACTTCA GAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCACCAGCTTTC TGCGGCGGAAGTGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCG AGGACGCCCTGATCATTGCCAACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGG ACAAGGCCAAAAAAGTGATGGAAAACCAGATGTTCGAGGAAAAGCAGGCCGAGAGCA TGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCATCACCCCCCACCAGA TCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGAAGC CTAATAGAGAGCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGCA ACACCCTGATCGTGAACAATCTGAACGGCCTGTACGACAAGGACAATGACAAGCTGA AAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACGACCCCCAGA CCTACCAGAAACTGAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGT ACAAGTACTACGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACG GCCCCGTGATCAAGAAGATTAAGTATTACGGCAACAAACTGAACGCCCATCTGGACA TCACCGACGACTACCCCAACAGCAGAAACAAGGTCGTGAAGCTGTCCCTGAAGCCCT ACAGATTCGACGTGTACCTGGACAATGGCGTGTACAAGTTCGTGACCGTGAAGAATC TGGATGTGATCAAAAAAGAAAACTACTACGAAGTGAATAGCAAGTGCTATGAGGAAG CTAAGAAGCTGAAGAAGATCAGCAACCAGGCCGAGTTTATCGCCTCCTTCTACAACA ACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGAACAACGACC TGCTGAACCGGATCGAAGTGAACATGATCGACATCACCTACCGCGAGTACCTGGAAA ACATGAACGACAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGA GCATTAAGAAGTACAGCACAGACATTCTGGGCAACCTGTATGAAGTGAAATCTAAGA AGCACCCTCAGATCATCAAAAAGGGCAAAAGGCCGGCGGCCACGAAAAAGGCCGGCC AGGCAAAAAAGAAAAAGGGATCCGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGG ACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACA TGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCC TTGATGATTTCGACCTGGACATGCTGATTAACTCAAGATGACTCGAGAGATCTGTGA TAGCGGCCATCAAGCTGGCCGCGACTCTAGATCATAATCAGCCATACCACATTTGTA GAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAA ATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAA AGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCAGCTTATCGATACCGCATGCACGTGCGGACC GAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGC TCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCG GCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTT CTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACG CGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCG CTACACTTGCCAGCGCCTTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCG CCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCC GATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCAC GTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGT TCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACTCTATCTCGGGCT ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGC TGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTAT GGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACC CGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACA GACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCA TGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAA CCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAAT AACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATT Attorney Docket No.29539-0804WO1 / MGH 2023-602 TCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTT ACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAAC GTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTA TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGG TTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAAT TATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAA CGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAA CTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTG ACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAAC TACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTG GAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGC CCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAA ATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGA TCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTT CGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTT TTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGG TTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCA GAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCA AGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTG CTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGG ATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGC TTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAG AGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGT TTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCC TATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTT TTGCTCACATGT SEQ ID NO: 30 AAV-U6>mHerc1[msSagRNA#2]-U6>mHerc1[msSagRNA#12] construct CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCC ATCACTAGGGGTTCCTTCTAGACAACTTTGTATAGAAAAGTTGGAGGGCCTATTTCC CATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAAT TAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATA ATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCT TACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACG AAACACCGCAGGAAAAAGCCTGGTCTTCAGTTTTAGTACTCTGGGCCAACATGAGGA TCACCCATGTCTGCAGGGCCCAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTAT CTCGTCAACTTGTTGGCGAGATTTTTTCAAGTTTGTACAAAAAAGCAGGCTTCTAGA GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGA GAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGA CGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATG GACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATC TTGTGGAAAGGACGAAACACCGAAAACAAATTCATGTGTATGTGTTTTAGTACTCTG GGCCAACATGAGGATCACCCATGTCTGCAGGGCCCAGAATCTACTAAAACAAGGCAA AATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTTGTCTAGACTGCAGAGG GCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGC Attorney Docket No.29539-0804WO1 / MGH 2023-602 CTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAAT TGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGC ACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCA CCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCC CCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCA CGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGA CTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGC GCAGACCCAGCTTTCTTGTACAAAGTGGGCCACCATGGCTTCAAACTTTACTCAGTT CGTGCTCGTGGACAATGGTGGGACAGGGGATGTGACAGTGGCTCCTTCTAATTTCGC TAATGGGGTGGCAGAGTGGATCAGCTCCAACTCACGGAGCCAGGCCTACAAGGTGAC ATGCAGCGTCAGGCAGTCTAGTGCCCAGAAGAGAAAGTATACCATCAAGGTGGAGGT CCCCAAAGTGGCTACCCAGACAGTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAG GTCCTACCTGAACATGGAGCTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGA ACTCATCGTGAAGGCAATGCAGGGGCTCCTCAAAGACGGTAATCCTATCCCTTCCGC CATCGCCGCTAACTCAGGTATCTACAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGG AAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGATC CCCTTCAGGGCAGATCAGCAACCAGGCCCTGGCTCTGGCCCCTAGCTCCGCTCCAGT GCTGGCCCAGACTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGC TCCAGCCCCTGTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAGTGCCCAA GTCTACACAGGCCGGCGAGGGGACTCTGAGTGAAGCTCTGCTGCACCTGCAGTTCGA CGCTGATGAGGACCTGGGAGCTCTGCTGGGGAACAGCACCGATCCCGGAGTGTTCAC AGATCTGGCCTCCGTGGACAACTCTGAGTTTCAGCAGCTGCTGAATCAGGGCGTGTC CATGTCTCATAGTACAGCCGAACCAATGCTGATGGAGTACCCCGAAGCCATTACCCG GCTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGAACCAG CGGCCTGCCTAATGGGCTGTCCGGAGATGAGGACTTCTCAAGCATCGCTGATATGGA CTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAGGGAGGAGGTGGAAGCGG CTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCGGTGACCGTGCC CGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAAGAGCTCCTGTC TCCCCAGGAGCCCCCCAGGCCTCCCGAGGCAGAGAACAGCAGCCCGGATTCAGGGAA GCAGCTGGTGCACTACACAGCGCAGCCGCTGTTCCTGCTGGACCCCGGCTCCGTGGA CACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACTTCTC CGAAGGGGACGGCTTCGCCGAGGACCCCACCATCTCCCTGCTGACAGGCTCGGAGCC TCCCAAAGCCAAGGACCCCACTGTCTCCGGAAGCGGAGCCACGAACTTCTCTCTGTT AAAGCAAGCAGGAGATGTTGAAGAAAACCCCGGGCCTATGGTGAGCAAGGGCGAGGA GGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTC CGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGG CACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGA CATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGA CATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGAT GAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGG CGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGT AATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGA CGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTA CGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGC CTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGT GGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTA CAAGTAACAACTTTATTATACATAGTTGGAATTCCTAGAGCTCGCTGATCAGCCTCG ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTG ACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCG CATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGGGCCGCAGGAACCCCTAG Attorney Docket No.29539-0804WO1 / MGH 2023-602 TGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAC CAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGC GCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTA TTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAG CGCGGCGGGGGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCTTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCG TCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCT CGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA GACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTT CCAAACTGGAACAACACTCAACTCTATCTCGGGCTATTCTTTTGATTTATAAGGGAT TTTGCCGATTTCGGTCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC GAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTG CTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCC CTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGG GAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGG CCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGAC GTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTA AATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATA ATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTT TTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAG CGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGA AAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCAT GAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACC GGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAAT GGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA ACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGAAGCCG CGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTA CACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGG TGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTA GATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGA TAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCC CGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCT ACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTG TCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTG AACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAG ATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGG GGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCG TCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCA G SEQ ID NO: 31 Attorney Docket No.29539-0804WO1 / MGH 2023-602 EXAMPLES The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. Methods The following materials and methods were used in the Examples below. Mice. All mice were group housed and experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committees at the Massachusetts General Hospital and Tufts University School of Medicine and NIH guidelines. All mice were housed in a 12-h (7:00 a.m. to 7:00 p.m.) light–dark colony room at 22–24 °C with ad libitum access to food and water. Cntnap2- / -mice, Pvalb-cre mice, and Rpl22HAmice were obtained from the Jackson Laboratories (strain #017482, 017320, and 011029, respectively). Viruses and virus constructs. AAV-S5E2-dTom-nlsdTom plasmid and AAV PHP.eB virus were purchased from Addgene (plasmid #135630). AAV-S5E2-Meis2- nlsdTom virus was generated by subcloning mouse Meis2 into AAV-S5E2-dTom- nlsdTom plasmid (VectorBuilder). AAV-S5E2-dSACas9 / VP64 was generated by subcloning mouse dSACas9 / VP64 into AAV-S5E2-dTom-nlsdTom plasmid (VectorBuilder). AAV-U6-sagRNA#1#2-Syn1-P2A-mcherry and AAV- Herc1gRNA#1#2-Syn1-P2A-mcherry were generated by VectorBuilder. Cal-Light viruses: AAV-pCMV-Myc-TM-KA2-CaM-NES-TEV-N-AsLOV2-TEVseq-tTA, pAAV-hSYN-M13-TEV-C-P2A-tdTomato, pAAV-TRE-EGFP (gifts from Hyun Lab). AAV.PHP.eB viruses were produced by Boston Children’s Hospital Viral Core. Coding sequences used included mMeis2-NM_001346036.1; mBcl11a- NM_016707.3; and mTbr1-NM_009322.3. Immunohistochemistry. Mice were anaesthetized with ketamine and xylazine (10 mg / ml and 1.6 mg / ml, IP), transcardially perfused with 4% PFA, and brains were incubated in 4% PFA at 4°C overnight. Brains were placed in 30% sucrose / PBS for 2 days and then embedded in medium (OCT, Fisher HealthCare).35 μm cryosections were obtained (Leica) and stored in PBS (0.01% sodium azide) at 4°C. For immunostaining, floating sections were permeabilized, blocked in blocking solution for 2 h (PBS containing 0.3 % Triton X-100 and 10% normal donkey serum, NDS), Attorney Docket No.29539-0804WO1 / MGH 2023-602 and then incubated with primary antibodies (PBS containing 10% NDS) at 4°C overnight. Sections were then washed with PBS 3 times, 10 min each, then incubated with secondary antibodies in PBS for 2 h at room temperature (RT). Sections were then washed with PBS 3 times, 10 min each, mounted on glass slides and coverslipped with mounting medium containing DAPI. Image analysis. For PV puncta and synaptotagmin-2 puncta: Images were obtained from 3 sections per mouse hippocampus blind to treatment and genotype. A Leica SP8 confocal laser microscope and LAS software were used to capture images in the stratum lucidum at high-resolution (2,048). Single confocal plane images were captured in the CA2 and CA3ab subfields using a 63X oil objective plus 43X digital zoom. Quantification sample size: PV+puncta or Syt2+puncta, densities were averaged from 18 images per mouse. Puncta was analyzed using the StarDist 2D plugin and particle analysis tools in FIJI ImageJ. Threshold values were held constant across images. For Cal-Light, dCA3a cells were manually counted. To reduce contribution of background fluorescence, a threshold based on maximizing Yen entropy (ieeexplore.ieee.org / document / 366472) was applied to GFP images during quantification. Antibodies. PV (rabbit, Swant PV25, 1 / 5000; goat, Swant PVG213, 1 / 1000 );RGS14 (mouse, NeuroMab 75-170, 1 / 500; rabbit, Proteintech 16258-1-AP, 1 / 500); Synaptotagmin 2 (mouse, Abcam AB154035-1001, 1 / 250); RFP (rabbit, Rockland 600-401-370, 1 / 1000; goat, Sicgen AB1140-100, 1 / 500); cFOS (guinea pig, Synaptic Systems 226-004, 1 / 3000); GFP (chicken, Invitrogen A10262, 1 / 500). Fluorescent- label-coupled secondary antibodies (Alexa-Fluor-488-conjugated donkey anti-rabbit IgG, 711-545-152, 1:500; Cy3-conjugated donkey anti-rabbit-IgG, 711-165-152, 1:500; Alexa-Fluor-488-conjugated donkey anti-mouse-IgG, 715-545-151, 1:500; Cy3-conjugated donkey anti-mouse- IgG, 715-165-151, 1:500; Alexa-Fluor-488- conjugated donkey anti-chicken-IgG, 703-545-155, 1:500; Cy3-conjugated donkey anti-goat-IgG, 705-165-147, 1:500; 647-conjugated donkey anti-guinea pig-IgG, 706- 605-1481; Cy3-conjugated donkey anti-guinea-pig-IgG, 706- 165-148, Jackson ImmunoResearch, in 1 / 500 dilution. Attorney Docket No.29539-0804WO1 / MGH 2023-602 RNA in situ hybridization and immunofluorescence imaging. RNAscope in situ hybridization was performed following the RNAScope Multiplex Fluorescent Reagent Kit v2 protocol (323100) (Advanced Cell Diagnostics, ACD). Briefly, mice were perfused with PBS and followed by 4% PFA. Brain slices (10 um) were mounted and dried at -20 °C for 2 hours. The slides were washed in PBS, then dehydrated in a gradient of ethanol (50%, 70%, 100%, and 100%) for 5 min each and air-dried for 5 min. RNAscope Hydrogen Peroxide was applied to tissues and incubated at RT for 10 min, followed by treatment with RNAScope 1X Target Retrieval Reagent. Tissues were incubated for 10 min at 95 °C followed by washed in ddH2O and 100% ethanol. Tissues were dried at RT for 3 min. RNAScope Protease III was applied and incubated for 30 min at 40 °C, then washed with water. The target probes were prepared (MmRgs14, 416651; Mm-Pvalb-C2, 421931-C3; Mm-Meis2- C3, 436371-C3). Target probes were applied and incubated at 40 °C for 2 h, after which tissues were washed with 1X wash buffer and placed in 5X SSC buffer overnight. Tissues were washed with wash buffer twice followed by incubation with AMP1, AMP2, and AMP3 (30 min for AMP1 and AMP2, 15 min for AMP3, at 40 °C). Tissues were washed with wash buffer twice in between each amplification step, then incubated with HRP specific to each channel (15 min 40 °C). Tissues were washed twice, after which the TSA fluorophore specific to each channel / probe (Opal520, 570, and 690, Akoya Biosciences)was added and incubated for 30 min at 40 °C).Tissues were washed twice and incubated with RNAScope HRP Blocker for 15 min at 40 °C. Fluorescent images were captured using an SP8 Leica confocal microscope. All Pvalb+cells were outlined and Meis2 intensity were quantified. RNA sequencing Tissue collection and RNA isolation Hippocampal CA2 / CA3 regions were harvested following injections of Ablim3 shRNA or non-target RNA-expressed lentiviruses into the DG of 2-month old PV-Cre:Rpl22HAf / fmice for 2 weeks. Brain tissues were snap-frozen and pooled from 6 mice (male and female) for every sample. Samples were immunoprecipitated with anti-HA magnetic beads (Pierce: 88836) for 3 hours at 4 °C51 32. After elution with RNeasy Plus Micro Kit (Quiagen), purified ribosome-associated RNAs were stored at -80 °C. RNA quality was assessed on a TapeStation (Agilent) and RNA amounts were Attorney Docket No.29539-0804WO1 / MGH 2023-602 quantified using the Qubit 4.0 Fluorometer (Life Tech.). Only RNA samples with RIN above 8.0 were used for library preparation and sequencing. RNA-seq RNA-seq libraries were constructed from total RNA using Clontech SMARTer v4 kit (Takara), followed by sequencing on an Illumina HiSeq 2500 instrument, resulting in 20-30 million 50 bp reads per sample. The STAR aligner52was used to map sequencing reads to transcriptome in the mouse mm9 (GRCm37) reference genome. Read counts for individual genes were produced using the unstranded count function in HTSeq v.0.6.053, followed by the estimation of expression values and detection of differentially expressed transcripts using EdgeR54and included only the genes with count per million reads (CPM) 1 for one or more samples55. Differentially expressed genes were defined by at least 1.5-fold change with FDR< 0.05. qRT-PCR analysis. Hippocampal DG or CA2 / CA3 regions were harvested and snap-frozen (jove.com / video / 1543 / dissection-of-hippocampal-dentate-gyrus- from-adult-mouse). Briefly, cDNA samples reverse transcribed from RNA collected from PVCre:Rpl22HA / HAmice with either lenti-shNT or shRNA (Ablim3) injection into DG. Total RNA was quantified using a NanoDrop spectrophotometer (ThermoScientific) and then equal amounts of RNA were used for reverse transcription(SuperScript IV First-strand synthesis system, Invitrogen). qRT-PCR was carried out with SYBR green (BioRad) and primers (Primer bank)56with following sequences: Attorney Docket No.29539-0804WO1 / MGH 2023-602 GSEA. Gene Set Enrichment Analysis was performed using the GSEA package (gsea-msigdb.org / gsea / index.jsp) against mouse MSigDB v2024.1.Mm collection of pathways57,58, with default settings and the cutoff of false discovery rate (FDR) < 0.05. Stereotactic viral injection. Mice were administered carprofen (5 mg / kg, SQ) before surgery and were then anaesthetized with ketamine and xylazine (10 mg / ml and 1.6 mg / ml, IP). Mice were placed in a stereotaxic frame, and a small hole was drilled at each injection site (Foredom K.1070 High Speed Rotary Micromotor Kit). Bilateral injections were performed using Hamilton microsyringes (Hamilton, Neuros Syringe 7001) or Nanoject digital microinjectors that were slowly lowered into target sites and remained in place for 8 min prior to viral infusion at a rate of 50 nl / min. The coordinates relative to bregma: dorsal DG: –1.8 mm (AP), ±1.35 mm (ML), –2.25 mm (DV) and dorsal CA2 / CA3: –1.8 mm (AP), ±2.45 mm (ML), –2.35 mm (DV).Recombinant AAVs (titre: 1x1013) were injected for a total volume of 100 nl perinjection site.10 minutes after infusion, the microsyringes were slowly withdrawn and the skin above the incision was sutured with coated vicryl sutures. For Cal-Light46, viruses were mixed (1:1:1 ratio) and injected for a total volume of 300 nl. One week later, AAV-control or AAV-Meis2 virus were injected for a total volume of 100 nl per injection site. The coordinates relative to bregma: AP -1.80, ML ±2.28, DV - 2.20 for virus injection; AP -1.80, ML ±2.28, DV -1.80 for optical fiber probe implant). Post-surgery, mice were placed in a clean empty cage on top of a heating pad with ambient temperature to 36°C until full recovery from anesthesia. Mice were monitored and received a daily injection of carprofen (5 mg / kg, IP) for 3 days following surgery9. Behavioral procedures. Two weeks after viral injections, mice were handled for 3 days prior to behavioral experiments to habituate them to human handling and transportation from vivarium to behavioral testing rooms. The behavioral assays were performed in the following order: open-field (OF, day 1), visual cue habituation (day 2), novel object location followed by novel object recognition (NOR, day 3), and social recognition and discrimination (day 4). All the behavioral assays were Attorney Docket No.29539-0804WO1 / MGH 2023-602 performed in the same chambers (40 × 40 cm, MazeEngineers). Videos were recorded and exported from Freezeframe (Actimetrics) and analyzed with EthoVision XT 15 (Noldus). Center point tracking was used to record movement and nose point tracking was used to evaluate object and social interaction. An interaction was registered when the test mouse’s nose position to object or stimulus mouse was within 1 cm9. Behavioral paradigm for PV IN synapse / Syt2 / puncta analysis: Mice were handled for 7 days prior to behavioral experiments to habituate them to human handling, transportation from vivarium to behavioral testing rooms and the context for one hour. On Day 8, mice were either exposed to context only or with one stimulus mouse in one pencil cup for 10 min. Mice were returned to their home cage and subsequently perfused 90 minutes later. Behavioral paradigm for Meis2 intensity analysis: Mice were handled for 3 days prior to behavioral experiments to habituate them to human handling, transportation from vivarium to behavioral testing rooms and the context for one hour. On Day 4, mice were either exposed to context only or with one stimulus mouse in one pencil cup for 10 min. Mice were returned to their home cage and subsequently perfused 10 minutes later. Open-field paradigm. Mice were transported into a holding room andhabituated for one hour prior to testing. Total distance traveled and the time spent inthe center of the arena were quantified over 30 min9. Novel object location and recognition. Two identical objects (2 x 4 x 6 cm) were placed in the OF chamber along one side (5 cm distance from the wall). Mice were placed in the opposite side of the object and allowed to explore freely for 5 min and then returned to their home cage for 2 hrs. Then one object was moved to the opposite side, and mice were placed in the middle of objects and allowed to explore for 5 min. Mice were returned to home cage for 10 min. Next, one object was replaced with a novel object (4 x 4 x 6 cm, 5 cm distance from the wall), and mice were placed in the middle of the objects and allowed to explore freely for 5 min. The time spent exploring the objects (noise point within 2 cm) was quantified9. Social recognition and social discrimination. Stimulus mice (strain, age, and sex- matched) were habituated to being placed in a pencil wire cup in the OF chamber Attorney Docket No.29539-0804WO1 / MGH 2023-602 prior to the task day for 3 days, 15 min per day. The task consisted of 3 trials: habituation (empty cup vs empty cup), recognition (empty cup vs stimulus), and discrimination (novel vs familiar), with 5 min intertrial intervals. Subjects were placed in the center of the chamber for 10 min. The locations of the familiar and novel stimuli were counterbalanced across trials. The time spent exploring the stimulus mouse was quantified (nose point within 1 cm)9. Tagging socially active neurons by soma-targeted Cal-light. We used the soma-targeted Cal-Light system46to tag active cells in the CA3a region of the hippocampus during social interaction. Mice were bilaterally injected in CA2 / 3 with a CAL-light viral mix three weeks prior to behavioral testing. One week after injection, animals were implanted with an optical fiber probe above CA2 / 3 and allowed a two- week recovery before behavioral testing. Behavioral paradigm and tagging protocol: Before testing, animals were placed in holding cages for a minimum of one hour, using the same cage each day. Subject mice underwent a three-day habituation protocol, during which they explored an open-field chamber with two empty cups placed in opposite corners while attached to lightless fiberoptic cables. One day following habituation, subject mice were exposed to a novel stimulus mouse (stimulus 1) placed under one of the cups andallowed to explore the chamber for 10 minutes. When subjects approached within 5cm of the stimulus mouse, blue light was delivered through fiber optic cables to mediate Cal-Light tagging of the engram. Immediately after the session, subjects were returned to their holding cages. Four hours after initial exposure, subjects were reintroduced to the chamber for a second 10-minute session, with a familiar mouse under the same cup as before. Following this, subjects were returned to holding cages and subsequently perfused 90 minutes later. Optical fibers and laser delivery: Optical fibers were constructed using a 200 μm core, 0.37 numerical aperture multimode fiber from Thorlabs. Fibers were inserted through a 230 μm core zirconia ferrule 701 (Precision Fiber Products) and glued in place before thoroughly polishing for a smooth connection between fiber and optical cable from the laser. For CAL-Light activation, a 100 mW 475 nm blue laser diode was used (OEM Laser Systems). When the subject animal was within a predefined zone, the light was delivered at a frequency of 1 KHz and an intensity of Attorney Docket No.29539-0804WO1 / MGH 2023-602 5-7 mW using an external arbitrary waveform generator (Agilent) attached to the signal port of the laser. Ex vivo electrophysiology. Mice were unilaterally injected with 0.3 µL pAAV5-CamKIIa-hChR2-eYFP into the dorsal DG, and bilaterally injected with 0.3 µL AAV PhP.eB-S5E2-Meis2-P2A-nlsTdtomato or AAV-P2A-nlsTdtomato control virus along the stratum lucidum mossy fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus. Mice were 2-3 months old prior to viral transfection (see above for stereotactic coordinates). At 2-3 weeks after viral infusion, mice were anaesthetized with ketamine and xylazine (10 mg / ml and 1.6 mg / ml, IP) then transcardially perfused with ice-cold (4 ºC) choline chloride-based artificial cerebrospinal fluid (ACSF) composed of (in mM): 92 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 glucose, and 10 MgSO4·7H2O. Their brains were rapidly extracted following decapitation. Coronal slices (300 µm thick) containing the dorsal hippocampus were cut in ice-cold (4 ºC) choline chloride ACSF using a Leica VT1000 vibratome (Leica Biosystems) and transferred to warm (33 ºC) normal ACSF for 30 min. Normal ACSF contained (in mM): 124 NaCl, 2.5 KCl, 1.25 NaH2PO4, 24 NaHCO3, 5 HEPES, 12.5 glucose, 2 MgSO4·7H2O, 2 CaCl2·2H2O. All ACSF solutions were adjusted to a pH of 7.4, mOsm of 305, andwere continuously saturated with carbogen (95% O2 and 5% CO2). Slices wereallowed to cool to room temperature (20-22 ºC) for 1 hour before recordings. Whole-cell patch-clamp recordings were amplified, low-pass filtered at 1.8 kHz with a four-pole Bessel filter, and digitized (Muliclamp 700B, Digidata 1550B, Molecular Devices). Slices were placed in a polytetrafluoroethylene submersion chamber and continually perfused with normal ACSF (>2 mL / min). Neurons were visually identified by infrared differential interference contrast imaging combined with epifluorescence using LED illumination (pE-300 white, CoolLED). Pyramidal neurons in CA2 and CA3ab were distinguished by their anatomical location and distinct electrophysiological properties. Borosilicate patch pipettes had a resistance of 4-5 MΩ and filled with an internal solution containing (in mM): 120 CsMeS, 4 MgCl2, 1 EGTA, 10 HEPES, 5 QX-314, 0.4 Na3GTP, 4 MgATP, 10 phosphocreatine, 2.6 biocytin, pH 7.3, 290 mOsm. For current-clamp recordings, patch pipettes were filled with 130 mM potassium gluconate in place of CsMeS and Attorney Docket No.29539-0804WO1 / MGH 2023-602 QX-314 excluded. Once GΩ seal was obtained, neurons were held in voltage-clamp configuration at -70 mV and the input resistance, resting membrane potential, and capacitance were measured. Series resistance (<30 MΩ) was monitored throughout recordings and recordings were discarded if series resistance changed by >20% from baseline. Excitatory and inhibitory postsynaptic current (EPSC and IPSC) were optically evoked with 1 ms 473 light pulses delivered above the mossy fiber pathway – the hilus of the DG. Current responses were recorded at 1.5 × threshold, defined as the minimum stimulation intensity required to produce a consistent current response beyond baseline noise. Isolation of EPSC was done by voltage clamp at -70 mV and IPSC at 0 mV. Optical 10 pulse stimulation trains were evoked 5 times at an interval of 20 sec between trains. The interevent interval between pulse stimulation was 100 ms. Long-term potentiation (LTP) of the EPSC and long-term depression of the IPSC (iLTD) was induced by electrically evoked theta-burst stimulation (TBS) which consisted of bursts of 4 pulses at 100 Hz, interburst interval was 200 ms, and repeated 4 times at 10 sec intervals. A bipolar tungsten electrode in a patch pipette with a resistance of 1-2 MΩ and filled with ACSF was placed on the mossy fiber pathway – the hilus of the DG. Stimulation was set to 1.5 × threshold of the EPSC. Therecording consisted of a 10 min baseline of the optically evoked EPSC or IPSC, TBS,then 40 min of the optically evoked postsynaptic current. Plasticity was calculated by the percent change from the last 5 min of the baseline current amplitude to the last 5 min of the 40 min recording. Current clamp configuration was used to record intrinsic membrane properties of PV INs. To assess action potential spiking activity, the neuron was clamped at -70 mV and 10 pA ascending step currents were delivered at 500 ms durations. Bursting neurons were identified by their asynchronous rapid firing within the first 50 ms of the current step followed by failure to fire and were excluded from analyses. Spontaneous excitatory and inhibitory postsynaptic current (sEPSC and sIPSC) were recorded by voltage clamp at -70 mV for sEPSCs and 0 mV for sIPSCs. Pharmacological validation were conducted to ensure no sIPSCs were visible during sEPSC recordings and vice versa (data not shown). Briefly, no inward or outward events were detected after bath application of cyanquixaline (CNQX, 20 µM), an Attorney Docket No.29539-0804WO1 / MGH 2023-602 AMPA / kainite receptor competitive antagonist while voltage clamped at -70 mV. Likewise, no events were detected after bath application of gabazine (10 µM), GABAA receptor antagonist while voltage clamped at 0 mV. Miniature EPSC and IPSC (mEPSC and mIPSC) were recorded after bath application of tetrodotoxin (TTX, 1 µM). Isolation of mEPSC was done by voltage clamp at -70 mV and mIPSC at 0 mV. Autodetection parameters for inclusion of spontaneous and miniature events was determined by calculating minimum threshold: root mean square (RMS)2 × 1.5. Data acquisition was performed using Clampex and analyzed with Clampfit (Molecular Devices) and EasyElectrophysiology (V2.5.2) software. Electrocorticography (ECoG) surgical implant. All animal surgeries and subsequent experiments were conducted in accordance with Tufts University's Institutional Animal Care and Use Committee guidelines and animal use protocols.6– 7-month-old Cntnap2- / - and + / +mice that had previously undergone CA2 / CA3 AAV injection were anesthetized with isoflurane (3% induction, 1.5% for maintenance, 2 l / min O2 flow rate) and given systemic analgesic (buprenorphine, 0.1 mg / kg, subcutaneous [SC]) as well as local analgesia near the incision site (bupivacaine, 4 mg / kg, SC). Mice were then placed in a stereotactic frame and the dorsal side of theskull was shaved and disinfected with iodine and ethanol. A scalp incision was made,and the skull surface cleaned and dried. Four burr holes were drilled into the skull without puncturing the dura mater. Drill bit tip diameter = 0.7 mm (Item No.19007- 07, Fine Science Tools, CA). Stereotaxic coordinates of the four drill holes: Anterior burr holes were −0.6 mm (bregma, AP axis) and 2 mm left (ECoG) and right (ground) of the midline (ML), posterior burr holes were – 2.6 mm (bregma, AP) and 2.5 mm left (reference) and right (ECoG) to ML. Four 0.1’’ stainless steel screw electrodes with attached silver wires (Part # 8403, Pinnacle Technologies, Wyckoff, NJ, USA) were gently screwed into the burr holes and fixed with dental cement and superglue to increase implant stability for chronic ECoG recordings. The silver wires of the electrode screws were then soldered to the headmount (Part # 8402, Pinnacle Technologies, Wyckoff, NJ, USA). The headmount / electrode construct was then secured to the skull with dental cement. After surgery, animals were allowed to recover for at least seven days before chronic ECoG recordings started and were Attorney Docket No.29539-0804WO1 / MGH 2023-602 given analgesia (Buprenorphine, 0.1 mg / kg, SC) as needed for the first 3 days after surgery. Chronic ECoG recording. After recovering from surgery, a preamplifier (100 x gain, 1 Hz high-pass filter, Part # 8213, Pinnacle Technologies, Wyckoff, NJ, USA) was plugged into the implanted headmount and preamplifiers were attached to a commutator in the ECoG recording system (Pinnacle Technologies). Mice were housed in a round, acrylic ECoG recording chamber with access to food and water and kept on a standard light / dark cycle. ECoG data was recorded using LabChart Pro software (AD instruments, Colorado Springs, CO, USA) with a 1 kHz sampling rate. ECoG data was collected continuously 24 hours a day for at least 14 days. After recording, ECoG signals were filtered (100 Hz low-pass filter) and recordings were manually reviewed manual by an experienced, blinded investigator. When seizures were identified, their duration was quantified, and their time of incidence was noted. SLEAP. Mice were pair-housed for over two weeks prior to recording. On the recording day, the cage lid was removed, and the cage was placed inside a recording chamber overnight with access to food and water. Behavior was continuously recorded for 8 hours under infrared lighting, with a one hour segment, captured onehour after the cage was placed in the chamber, selected for analysis. We used SLEAP(Pereira et al., Nature Methods 19, 486–495 (2022)) to track and estimate the poses of freely behaving mice in their home cages. To analyze social interactions between mice, a multi-instance, bottom-up model with a U-Net architecture was trained on 2,751 instances across 917 frames, with 306 validation instances from 102 frames. Six body parts were labeled as nodes: nose, right ear, left ear, head, body center, and tail base. Our average distance between the predicted and ground truth nodes was 4.3 pixels. Training frames were randomly selected from videos containing different mice, with slight variations in contrast between subjects and background. Recordings were captured continuously during a 1-hour session, and the frame rate was 23.97 frames per second (fps). A batch size of 6 was used for training, with data augmentation through rotation (360°). For inference, identity tracking across frames was performed using a flow tracker, with centroid similarity and the Hungarian matching algorithm applied over Attorney Docket No.29539-0804WO1 / MGH 2023-602 an elapsed frame window of 4 frames. Instances were manually inspected for identity switches and corrected before data export. Data were analyzed using a Python script modified from SLEAP analysis examples (sleap.ai / notebooks / Analysis_examples.html) was used to analyze social interactions based on the distance between nodes. First, Missing nodes were linearly interpolated, and predictions were smoothed using a Savitzky-Golay filter to reduce jitter. We then measured nose-to-nose, nose-to-center, and nose-to-rear distances. Interactions were defined by pixel thresholds: 61 pixels (nose-to-nose), 100 pixels (nose-to-center), and 70 pixels (nose-to-rear). Thresholds were verified against manually scored data to ensure accuracy. Sex as a biological variable. Sex of all mice was factored into design and analyses. If no statistical difference or interaction between sex were observed, then mice were grouped and analyzed according to experimental condition. Statistics, rigor, and reproducibility. All experimenters were blind to treatment conditions throughout data collection, scoring, and analyses. Statistical analyses were conducted using Prism v10 (GraphPad) and the minimum sample size was determined based on prior experience of experimental paradigms, existingliterature, and power analyses. Statistical significance was defined as p < 0.05.Appropriate nonparametric tests were used when data sets failed to meet parametric assumptions. Grubbs’ or ROUT tests were used to identify outliers with α or Q = 0.05. Example 1. Screen for experience-dependent PV IN plasticity genes in adult CA2 / CA3 To identify candidate regulators of experience-dependent PV IN plasticity genes or “XPGs” in CA3 / CA2 PV INs, we designed an in vivo functional screen in adult mice to isolate translated mRNAs from CA3 / CA2 PV INs that receive increased mossy fiber inputs, a trigger for experience-dependent PV IN plasticity (Fig.1a). To this end, we virally downregulated a molecular brake of mossy fiber filopodia-PV IN connectivity, Ablim3 (Non-target shRNA (shNT) vs. shAblim3-RNA), in DG mossy fibers9-11, to reproduce the effects of experience on mossy fiber filopodial inputs onto PV INs9-11. We used PV-Cre; Rpl22HA / HAmice32, in which expression of the Attorney Docket No.29539-0804WO1 / MGH 2023-602 hemagglutinin (HA) epitope-tagged ribosomal protein L22 (Rpl22) is restricted to PV INs10. Following optimization of signal-to-noise for mRNA isolation from this extremely sparse population of PV INs33(Fig.6 a-b), we compared mRNAs biochemically isolated from genetically tagged PV INs in the naïve state versus an experience-induced activated state in CA3 / CA2. Using this approach we identified differentially expressed candidate XPGs associated with the PV IN activated state (Fig.1b-c). Significantly upregulated genes included 67 Category 1, ASD SFARI genes (gene.sfari.org / )34, and 3 genes causally implicated in schizophrenia through exome sequencing via Schizophrenia Exome Sequencing Meta-analysis (SCHEMA)35. In sharp contrast, we identified only 3 category 1, syndromic ASD, genes in the list of significantly downregulated XPGs. Therefore, we focused on upregulated XPGs in this study.18 out of 67 ASD genes encode histone methyltransferase, histone demethylases, ATP-dependent chromatin remodeling factors, transcription factors and co-activators whose functions in PV INs are poorly defined. Gene set enrichment analysis of all genes upregulated in experience-induced PV IN activated state suggests a role for these XPGs in synaptic physiology, structural remodeling and presynaptic terminal specialization (Fig.6c). Half of the ASD-linked upregulated XPGs are implicated in epilepsies, consistent with the high incidence of seizures in ASD individuals (Fig.1c)(gene.sfari.org / ). Since ASD and SCZ individualsprimarily carry loss-of-function mutations in these genes, the transcripts of which areupregulated in PV INs in response to increased mossy fiber inputs, our findings suggest that loss of PV IN experience-dependent plasticity is a convergent mechanism for different NDDs including ASD, epilepsies and schizophrenia. After validating upregulation of select candidate ASD-linked XPGs (Meis2, Bcl11a, Tbr1) (gene.sfari.org / ) and the SCHEMA gene, Herc135, in activated CA3 / CA2 PV INs by qPCR (Fig.6d), we asked whether boosting expression of these XPGs in PV INs using PV IN enhancer “S5E2” driven AAV viral vectors36promotes formation of PV IN-CA3 / CA2 synapses in adult mice. Viral upregulation of Tbr1, Bcl11a, Meis2 or Herc1 resulted in an increase in PV IN synapses in CA3 and CA2 pyramidal neurons (Fig.1d, Fig.6e). Meis2 is expressed at negligibly low levels in a subpopulation of hippocampal PV INs in the hippocampus37where its function is not known. MEIS2 is a homeodomain containing transcription factor that functions as a cellular context-dependent co-activator to regulate developmental specification of Attorney Docket No.29539-0804WO1 / MGH 2023-602 GABAergic projection neurons21,38and maintenance of intrinsic excitability and terminal arborization in postmitotic mechanosensitive sensory neurons39. Based on these observations we prioritized Meis2 as a candidate XPG to restore experience- dependent PV IN plasticity in NDDs. Since many NDDs including ASD are characterized by a plethora of developmental deficits, intellectual disability, seizures and impaired social cognition, we selected Cntnap2 KO (Cntnap2- / -) mice to test our hypothesis. Specifically, Cntnap2- / -mice exhibit an array of developmental deficits including loss of hippocampal and cortical PV INs, social and spatial cognitive impairments and seizures by around 4-6 months of age40-43. Example 2. Cntnap2 KO mice exhibit loss of social experience-dependent induction of PV IN synapses and Meis2 upregulation in CA3 / CA2 PV INs We asked whether CA3 / CA2 PV INs in adult (2 months old)Cntnap2 KO (Cntnap2- / -) mice retain competence to increase their perisomatic synapses in response to social experience. Our analysis revealed that Cntnap2 KO mice had fewer PV IN perisomatic synapses (PV puncta) in CA3 as compared to wild-type littermates (Cntnap2+ / +)(Fig.2a-b). Additionally, exposure to a social stimulus following habituation to a context resulted in an increase in PV IN perisomatic synapses in Cntnap2+ / +mice but not in Cntnap2- / -littermates (Fig.2a-b). Next, we asked whether Meis2 expression levels in CA3 / CA2 PV INs in adult Cntnap2- / -mice and Cntnap2+ / +littermates change in response to social experience. Multiplex fluorescent in situhybridization for Meis2, PV and RGS14 in hippocampal sections obtained from mice habituated to a context (social experience naïve) and mice exposed to a social stimulus in the habituated context revealed a social experience-dependent increase in Meis2 transcripts (Meis2 intensity per PV IN and proportion of PV INs that express Meis2) in Cntnap2+ / +mice but not in Cntnap2- / -mice (Fig.2c-d). Example 3. Meis2-dependent restoration of CA3 / CA2 PV IN synapses, inhibitory and excitatory synaptic transmission and PV IN synaptic plasticity in adult Cntnap2 KO mice To acutely boost Meis2 levels in CA3 / CA2 PV INs, we generated a S5E2 PV IN enhancer driven Meis2-P2A-nlsdTomato AAV that preferentially targets expression of Meis2 in PV INs including basket cells and axo-axonic cells in CA3 / CA2 (Fig.7a-b). Virally boosting Meis2 in CA3 / CA2 of adult Cntnap2- / -mice Attorney Docket No.29539-0804WO1 / MGH 2023-602 was sufficient to restore PV IN perisomatic synapses in CA2 as quantified using synaptotagmin 2 immunohistochemistry8(Fig.2e-f). We next determined if anatomical restoration of PV IN synapses in CA2 of adult Cntnap2- / -mice was accompanied by changes in PV IN properties, physiology and synaptic transmission in the DG-CA2 circuit. We targeted expression of Meis2 or dTomato in PV INs along the stratum lucidum mossy fiber pathway adjacent to CA2 / CA3a pyramidal cell layer of the dorsal hippocampus in adult Cntnap2- / -mice or+ / +littermates using AAV- S5E2-Meis2-P2A-nlsdTomato or AAV-S5E2-dTom-P2A-nlsdTomato control viruses. Ex vivo whole-cell patch-clamp recordings from PV INs and CA2 pyramidal neurons (PN) revealed that Cntnap2- / -mice exhibit increased spontaneous excitatory postsynaptic current (sEPSC) in CA2 PNs compared to Cntnap2+ / +mice and boosting Meis2 in PV INs reduced sEPSC frequency and amplitude in Cntnap2- / -and Cntnap2+ / +mice (Fig.3a, Fig.8a). Spontaneous inhibitory postsynaptic current (sIPSC) frequency in CA2 PNs from Cntnap2- / -was reduced compared to Cntnap2+ / +littermates, and boosting Meis2 in PV INs reversed this reduction in Cntnap2- / -mice (Fig.3b, Fig.8b). To assess action potential-independent spontaneous vesicle release, we recorded miniature excitatory and inhibitory postsynaptic current (mEPSC / mIPSCs) from CA2 PNs. We found that Cntnap2- / -mice have higher mEPSC frequency than Cntnap2+ / +littermates, and boosting Meis2 in PV INsreversed this increase (Fig.3c, Fig. 8c). There was no difference in the frequency oramplitude of mIPSCs from CA2 PN between Cntnap2- / -and Cntnap2+ / +mice, however boosting Meis2 in PV INs increased mIPSC frequency and amplitude in CA2 PNs of Cntnap2- / -mice (Fig.3d, Fig.8d). We assessed mEPSC in PV INs and found that boosting Meis2 in these interneurons increased mEPSC amplitude in both Cntnap2- / -and Cntnap2+ / +mice (Fig.3e, Fig.8e). To assess mossy fiber driven evoked current onto CA2 PNs, we injected pAAV5-CamKIIa-hChR2-eYFP into the dorsal DG and AAV-S5E2 driven Meis2 or dTtomato expressing viruses into CA3 / CA2 and elicited optically evoked stimulation trains targeting the mossy fiber pathway (a train of 10 optic stimulations, 1 ms pulse duration, 100 ms inter-stim interval repeated 5 times with 20 sec between trains) (Fig.3f). We found that Cntnap2- / -mice exhibit higher amplitude optically-evoked EPSCs and lower amplitude IPSCs across the stim train compared to Cntnap2+ / +mice. Boosting Meis2 in PV INs attenuated evoked EPSC and increased evoked IPSC Attorney Docket No.29539-0804WO1 / MGH 2023-602 amplitude to responses comparable to those observed in Cntnap2+ / +mice (Fig.3g, Fig.8g). Compared to all other groups, Cntnap2- / -mice exhibit an overall higher excitation to inhibition ratio (Fig.3g). We observed no differences in vesicle release probability across the stimulation train, indicating the stimulation frequency does not induce short-term plasticity (Fig.9a). These results suggest Cntnap2- / -mice have higher frequency of excitatory transmission and reduced inhibition onto CA2 PNs and that boosting Meis2 in PV INs reverses these developmental phenotypes. The reduction in excitatory synaptic transmission in CA2 may arise in response to sustained increases in MEIS2-dependent inhibition of pyramidal neurons. Next, we asked whether boosting Meis2 in PV INs influences mossy fiber driven, inhibitory-dependent synaptic plasticity (iLTD) which is thought to facilitate routing of information44. To elicit inhibitory-dependent plasticity, we stimulated the mossy fiber pathway by electrical theta burst stimulation (TBS) and recorded from CA2 PNs44. TBS along the mossy fiber pathway induces long-term depression of the IPSC (iLTD) and long-term potentiation (LTP) of the EPSC upon CA2 PNs (Fig.3h, Fig.9b). We found that Cntnap2- / -mice exhibit reduced occurrence of iLTD compared to Cntnap2+ / +littermates, and boosting Meis2 in PV INs restored the occurrence of iLTD in both Cntnap2- / -and Cntnap2+ / +mice (Fig.3h). We assessed vesicle release probability of IPSC and EPSC at baseline (5 min before TBS) and postiLTD (between 35 to 40 min post TBS). We found that the vesicle release probabilityof IPSC was reduced from baseline to post iLTD in all other groups except Cntnap2- / -mice. There were no changes in vesicle release probability in the EPSC across all groups (Fig.9c). This suggests that mossy fiber driven iLTD and subsequent LTP of the EPSC upon CA2 PNs is a presynaptic mechanism in inhibitory synapses. Next, we assessed how boosting Meis2 in PV INs influences PV IN passive membrane properties in the four groups. Current injections in PV INs revealed reduced excitability in Cntnap2- / -mice and boosting Meis2 in these interneurons restored excitability and reduced rheobase in both Cntnap2- / -and+ / +mice (Fig.3i). We found no differences in the resting membrane potential in any of the groups, however PV INs from Cntnap2- / -mice exhibit a more depolarized threshold. Interestingly, boosting Meis2 did not affect the threshold in Cntnap2- / -or in Cntnap2+ / +mice, suggesting that the depolarized threshold arises from a Meis2- independent compensatory mechanism (Fig.9d). Attorney Docket No.29539-0804WO1 / MGH 2023-602 To determine if another XPG, the T-brain-1 (Tbr1), a T-box transcription factor, and Meis2 have overlapping or distinct roles in regulating PV IN physiology, we injected AAV-S5E2-Tbr1- P2A-nlsdTomato along the stratum lucidum adjacent to CA2 / CA3a pyramidal cell layer in Cntnap2+ / +mice and performed recordings from PV INs and CA2 pyramidal neurons (Fig.10, Fig.8f). We found that boosting Tbr1 in PV INs results in reduced sEPSC frequency and amplitude, reduced mEPSC frequency, and increased mIPSC frequency upon CA2 PNs (Fig.10a-b, Fig.8f). Mossy fiber driven evoked current onto CA2 PNs with a 10 burst optic stimulation train revealed an increase in IPSC amplitude, however there were no significant differences in excitation / inhibition ratio compared to Cntnap2+ / +mice (Fig.10c). Boosting Tbr1 in PV INs did not result in any differences in intrinsic excitability and passive membrane properties from PV INs (Fig.10d). Together, these observations demonstrate that boosting Meis2 expression in CA3 / CA2 PV INs of adult Cntnap2- / -mice reverses developmental deficits in PV IN intrinsic excitability, input-output synaptic connectivity, excitatory and inhibitory synaptic transmission and inhibitory-dependent synaptic plasticity. Additionally, our analysis of Meis2 and Tbr1 suggests that these two XPGs independently and differentially contribute to experience-dependent PV IN plasticity. Example 4. Meis2-dependent rescue of cognition in adult Cntnap2 KOmiceWe next asked whether Meis2-dependent restoration of experience-dependent PV IN plasticity in CA3 / CA2 PV INs of adult Cntnap2- / -mice rescues spatial and social cognition impairments. Following viral upregulation of Meis2 in PV INs, adult (2-3 months) Cntnap2+ / +and- / -mice were tested in the novel object location task that probes the capacity of the hippocampus to bind object and spatial information into conjunctive representations, or “contexts”(Fig.4a-b). Consistent with a role for DG- CA3 / CA2 circuitry in encoding configural relationships between objects and their locations within contexts, Cntnap2+ / +but not Cntnap2- / -mice spent more time exploring the displaced object over the non-displaced object in the habituated context. Increasing Meis2 in CA3 / CA2 PV INs of Cntnap2- / -mice rescued cognitive behavioral impairment in this task, matching behavior of Cntnap2+ / +littermates (Fig. 4c). In the social recognition memory paradigm, mice are habituated to a context in trial 1 (T1) to two empty inverted cups, encode a social stimulus in one cup in T2 Attorney Docket No.29539-0804WO1 / MGH 2023-602 (counter balanced, familiarization phase), and are then challenged to discriminate between the familiar social stimulus (from T2) and a new social stimulus in trial 3 (Fig.4d). Trial T2 probes social recognition (often referred to as sociability) and is dependent on DG recruitment of feed-forward inhibition of CA29. All four groups of mice equivalently explored the context and both empty cups in the habituation phase (Fig.4e-f). However, Cntnap2+ / +but not Cntnap2- / -mice, spent more time exploring the social stimulus over the empty cup in trial 2 and discriminated between the novel social stimulus and the familiar social stimulus in trial 3. Boosting Meis2 in CA3 / CA2 PV INs of Cntnap2- / -mice reversed these impairments in social recognition and social discrimination (Fig.4f). Unlike in Cntnap2- / -mice, elevating Meis2 levels in CA3 / CA2 PV INs of Cntnap2+ / +mice impaired social and spatial cognition. Thus, when GABAergic inhibition is intact, increasing MEIS2 dosage does not enhance cognition (Fig.4c, 4f). Analysis of this same cohort of mice in assays for anxiety-like behavior (open field), novel object recognition and homecage group social behavior using Social LEAP Estimates Animal Poses or SLEAP45did not detect differences between these groups (Fig.11a-d, Fig.12a-d). Increased MEIS2 levels in CA3 / CA2 PV INs may rescue social recognition in Cntnap2- / -by promoting specificity of neuronal ensembles encoding social stimuli. To test this hypothesis, we used a dual light and calcium-dependent neuronal activitytagging system, soma-targeted Cal-Light 46, to tag active neurons (with induction ofGFP) during exploration of social stimulus in a habituated context and then quantify reactivation of the social stimulus-associated ensemble upon re-exposure to same social stimulus. Higher reactivation [% (cFOS+GFP+) / (GFP+ cells)] suggests increased specificity of the neuronal ensemble (Fig.4g, Fig.12e). In both Cntnap2+ / +and Cntnap2- / -mice, we detected equivalent numbers of active cells in recognition and retrieval phases, however, boosting Meis2 in CA3 / CA2 PV INs of Cntnap2- / -mice significantly enhanced ensemble reactivation of the social stimulus-associated ensemble compared with control virus infected Cntnap2- / -mice (Fig.4h-i). Together, these observations demonstrate that boosting Meis2 expression in CA3 / CA2 PV INs of adult Cntnap2- / -mice reverses developmental deficits in hippocampal dependent cognition and promotes ensemble specificity. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Example 5. Meis2-dependent suppression of seizures in Cntnap2 KO mice Cntnap2- / -mice develop seizures between 4-6 months of age40,42,43.4-5 months following viral-mediated upregulation of Meis2 in CA3 / CA2 PV INs of 2 months old Cntnap2+ / +and Cntnap2- / -mice, electrocorticography (ECoG) electrodes were implanted, and 24 / 7 chronic ECoG recordings were performed for 2 weeks after recovery from surgery (Fig.5a). Seizure frequency and duration were quantified in all experimental groups. Two-thirds of Cntnap2- / -mice (6 of 9 mice) treated with a control AAV (AAV-S5E2-dTom, 6 of 9 mice) displayed spontaneous electrographic seizures with a mean frequency of 2.79 (± 6.64 SD, ± 2.21 SEM) per day. These seizures displayed stereotypical preictal spiking, high amplitude synchronous activity, and a post-ictal depression, and they were associated with convulsive motor seizures. One-third of Cntnap2- / -mice (4 of 12) treated with Meis2 AAV (AAV-S5E2-Meis2, 4 of 12 mice) displayed spontaneous seizures with a mean frequency of 0.08 (± 0.16 SD, ± 0.04 SEM) per day. Boosting Meis2 in CA3 / CA2 PV INs significantly reduced seizure frequency in Cntnap2- / -mice (Fig.5b-d). We also examined Cntnap2+ / +mice and found no seizures in any Cntnap2+ / +mice treated with the control virus (AAV- S5E2-dTom, 0 of 3 mice) but we did identify one Cntnap2+ / +mouse treated with AAV-Meis2 (AAV-S5E2-Meis2) that had 2 seizures during the 2-week recording window (1 of 6 mice)(Fig.13a-b). Seizure duration was similar across all groups(Fig. 13c). These findings demonstrate that boosting Meis2 in CA3 / CA2 PV INsreduces the likelihood of seizures in Cntnap2- / -mice. Example 6. Viral expression of Meis2 in CA3 / CA2 PV INs in Cntnap2 KOs restores CA1 SWR power and duration Methods The following methods and materials were used in Example 6. Surgery. Subjects were 2 month-old male and female mice. Mice were anesthetized with 1.75% isoflurane and placed in a stereotaxic frame, and a burr hole was drilled for injection site on the right hemisphere (Foredom K.1070 High Speed Rotary Micromotor Kit). AAV injections were performed using glass pipette filled with AAVs attached to Nanoject digital microinjectors that was slowly lowered into target sites and remained in place for 8 min before infusion (50 nl / min). The coordinates relative to bregma : dorsal CA2 / CA3: –1.8 mm (AP), +2.45 mm (ML), – Attorney Docket No.29539-0804WO1 / MGH 2023-602 2.35 mm (DV). Recombinant AAVs (titre: 1x1013) were injected for a total volume of 100 nl . The Cntnap2+ / + mice were injected with AAV-S5E2-dTom-P2A- nlsTdtomato (+ / +dTom) . Moreover, the Cntnap- / - mice were injected either with AAV-S5E2-dTom-P2A-nlsTdtomato (- / -dTom) or with AAV-S5E2-Meis2-P2A- nlsTdtomato (- / -Meis2).Ten minutes after infusion, the pipettes were slowly withdrawn and the skin above the incision was sutured with coated vicryl sutures. Mice were allowed to recover and two weeks later went through second surgery of tetrode implantation. Again the mice were placed under stereotaxic frame post anesthesia (1.75% Isoflurane) to implant three bone screws and a 16-channel, custom- built, twisted wire electrode bundle. A four-wire electrode bundle was created by twisting together four 75-µm diameter nichrome wires (California Fine Wire). The bundle was cut at an angle that spans 0.5 mm. Four different sites were chosen for twisted wire implantation: the tips of the twisted wire were located in the dorsal hippocampus (DG: –1.8 mm, +1.35 mm, –2.25 mm (AP, ML, DV); CA1:–1.8 mm, +1.35 mm, –2.18 mm; CA3: –1.8 mm, +2.45 mm, –2.35 mm and CA2: –1.8 mm, +2.45 mm, –2.31 mm). The twisted electrodes were each attached to a pin in a Mill- Max connector. After confirming that the recording electrode array extended through CA1, the wires and connectors were secured in place with dental cement. The Omnetics connector of the recording electrode and the Mill-Max connector of theelectrode bundle were anchored to the skull along with bone screws using dentalcement mixture (C&B Metabond, Parkel, and TEETs Denture Material, Cooralite Dental Mfg). Recording setup. The implanted mice were attached to the headstage (RHD 32 ch, #C3314, Intan Technologies) through a custom-made Omnetics to Mill-Max adaptor (Mill-Max model 852-10-100-10-001000). Behavior was recorded using a monochromatic camera (30 fps, Flea3 USB3, FLIR). Electrophysiological signals were sampled at 30 kHz and recorded using the Open Ephys Acquisition system via an ultra-thin SPI interface cable connected to the headstage board. Electrophysiological recordings were synchronized with recorded video using a TTL trigger pulse and by recording camera frame strobes. Mice movement was analyzed based on markerless tracking of the snout using DLC (Mathis et al., 2018). A snout speed of less than 1.5 cm / s in the home cage was considered an immobility period. Attorney Docket No.29539-0804WO1 / MGH 2023-602 Histology. Upon completion of the recordings, all mice were anesthetized and further transcardially perfused with 4 % PFA, and the brains were sectioned as described in the Immunohistochemistry section above. To visualize the electrode locations in mice brain, these sections were stained using antibodies to visualize IBA1 immunoreactive cells around electrode mark (rabbit anti-mouse IBA1, 1:500, #019- 19741, FujiFilm and Alexa-Fluor-488-conjugated donkey anti-rabbit IgG, 1:2000, 711-545-152, Jackson ImmunoResearch). After washing secondary antibody with PBS, the sections were mounted over slides dried for 2 hours at room temperature. The sections were covered with DAPI filled mounting media and cover-slipped. The slides were scanned at 10× with an Olympus VS120 microscope, and the images were subsequently examined for electrode tracks to verify the recording locations. Extracting NREM periods. Local Field Potential (LFP) data processing and ripple analysis were conducted using a custom Python script. Initially, the LFP data from the selected CA1 channel was scaled to microvolts and downsampled to 1000 Hz. The non-rapid eye movement (NREM) sleep periods were identified in the downsampled CA1 LFP by first computing a spectrogram (10-second window, 1- second step, 0-300 Hz). Features, including the first principal component (PC1, derived from the z-transformed 0-300 Hz spectral power) and theta dominance (5-10 Hz power / 2-16 Hz power), were then extracted. NREM states were defined asperiods where PC1 exceeded its 75th percentile for at least 5 seconds, accompaniedby a low theta dominance ratio (Yang et al., 2024). Ripple analysis. Ripple events were subsequently detected within these NREM epochs from the downsampled CA1 LFP. The LFP was bandpass filtered (100-250 Hz, 3rd-order Butterworth), and z-scored. Further this z-scored data’s power envelope was derived by rectifying it and then filtering it (1-20 Hz, 3rd-order Butterworth) (Tingley & Buzsáki, 2020; Zutshi & Buzsáki, 2023). Ripple events were marked when this envelope exceeded 2, required a peak greater than 5, had a duration between 20-150 ms, and were merged if the inter-event interval was less than 20 ms (Abbaspoor & Hoffman, 2024; Oliva et al., 2016). An artifact rejection was performed when the DG channel's Z-scored envelope concurrently exceeded the high threshold, coinciding with the detected ripple event in the CA1 channel. For each valid ripple event, the following properties were extracted: event start time, end time, Attorney Docket No.29539-0804WO1 / MGH 2023-602 peak power time, and peak LFP time, providing precise timestamps for the event's boundaries and its peak activity points. The duration of each ripple was calculated in milliseconds, representing its total temporal extent. The peak power quantified the maximum deviation of the ripple's power envelope from the baseline, normalized by its median and median absolute deviation. Further, the peak LFP amplitude was determined as the maximum absolute value within each ripple's detected boundaries. For visualization of each ripple, time-frequency power was calculated by applying a continuous complex Morlet wavelet transform to the raw LFP signal (0-300Hz) surrounding the event using PyWavelets (Lee et al., 2019). Power was estimated for frequencies from 100 to 250 Hz, and the resulting spectrograms were generated on a decibel (dB) scale. Statistical methods. Statistical tests were performed using GraphPad Prism (10.3.1). The differences between multiple groups were assessed using the Kruskal- Wallis test. Upon observing a main effect, corrections using Dunn's test were employed for multiple comparisons. Results Sharp-wave ripples (SWR, 100-250Hz) are a neural biomarker of memory in mammals. SWRs are high frequency oscillations that occur during rest and non-REM sleep and are believed to be important neural substrate for memory consolidation inmammals: rodents, monkeys and humans (Colgin, 2016). PV IN play a key role inSWR generation, impacting the power, amplitude and duration of ripples (Huang et al., 2024; Schlingloff et al., 2014). To determine if boosting Meis2 expression in PV INs restores SWRs in Cntnap2 KO mice, we injected AAVs targeting PV neurons in the dorsal CA2 / CA3 of Cntnap2- / - (Meis2 or dTomato) and wildtype (dTomato) littermates and, two weeks later, implanted tetrodes into the dorsal hippocampus of these mice. We recorded local field potentials (LFPs) for two hours before (Pre) and after (Post) a ten-minute social interaction with a sex-matched juvenile. We probed the changes in ripple properties from the CA1 region of the three groups, as this region is considered a major output of the hippocampus to other brain regions (Basu and Siegelbaum, 2015). We found that the three cohorts of mice did not differ in the total duration of NREM periods. However, the rate of ripple events during NREM sleep was lower in Cntnap- / - mice (KO) after post-social interaction compared to Attorney Docket No.29539-0804WO1 / MGH 2023-602 Cntnap+ / + mice, but was significantly increased in the Meis2 boosted KO group. Furthermore, Meis2 expression increased peak amplitude and power of these events in Cntnap- / - mice (KO) after social interaction and even when compared to the baseline period before social interaction. Meis2 substantially increased the duration of ripples in KO mice. Overall, these results suggest that boosting XPGs, such as Meis2 in PV INs, improves memory consolidation after social experience by strengthening the local inhibitory feed-forward network in KO mice, thereby enhancing the power of SWR and extending ripple duration similar to that seen in wildtype littermates. 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Hippocampal sharp-wave ripples and their spike assembly content are regulated by the medial entorhinal cortex. Current Biology, 33(17), 3648–3659. 69 Abbaspoor, S., & Hoffman, K. L. (2024). Circuit dynamics of superficial and deep CA1 pyramidal cells and inhibitory cells in freely moving macaques. Cell Reports, 43(8). 70 Oliva, A., Fernández-Ruiz, A., Buzsáki, G., & Berényi, A. (2016). Role of hippocampal CA2 region in triggering sharp-wave ripples. Neuron, 91(6), 1342–1355. 71 Lee, G., Gommers, R., Waselewski, F., Wohlfahrt, K., & O’Leary, A. (2019). PyWavelets: A Python package for wavelet analysis. Journal of Open Source Software, 4(36), 1237. Attorney Docket No.29539-0804WO1 / MGH 2023-602 OTHER EMBODIMENTS It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No.29539-0804WO1 / MGH 2023-602 WHAT IS CLAIMED IS:

1. A method of treating a subject to reduce or reverse cognitive impairment and reduce seizures in disorders associated with excitation / inhibition imbalance, the method comprising administering a therapeutically effective amount of an expression vector comprising a promoter that directs expression of a transgene sequence encoding an experience-dependent parvalbumin-expressing inhibitory neuron (PV IN) plasticity (XP) protein selected from human Meis2, Tbr1, Bcl11a, and Herc1 in parvalbumin-expressing inhibitory neurons (PV INs), and optionally an SE52 enhancer.

2. The method of claim 1, wherein the disorder is a neurodevelopmental disorder (NDD).

3. The method of claim 2, wherein the NDD is autism spectrum disorder (ASD), epilepsy, or schizophrenia (SCZ).

4. The method of claim 1, wherein the disorder is Alzheimer's disease (AD), cognitive impairment in aging, or hearing loss.

5. The method of claim 1, wherein the promoter comprises a parvalbumin promoter, a GAD65 promoter, or a beta actin promoter.

6. The method of claim 1, wherein the vector is a viral vector.

7. The method of claim 6, wherein the viral vector is a recombinant adeno-associated virus (AAV), retroviruses, adenovirus, lentivirus, and herpes simplex virus-1.

8. The method of claim 7, wherein the viral vector is an AAV, optionally selected from AAV2, AAV9, AAV-F, AAV.CPP.16, and AAVPhP.eB, and capsid and serotype variants thereof.

9. The method of claim 8, wherein the expression vector comprises from 5’ -3’: inverted terminal repeat (ITR) – an optional S5E2 enhancer - promoter– XP protein coding transgene - woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) - pA – ITR.Attorney Docket No.29539-0804WO1 / MGH 2023-602 10. The method of claim 1 or 8, wherein the XP protein is human Meis 2.

11. The method of claim 10, wherein the human Meis 2 protein sequence is at least 95% identical to SEQ ID NO:

23.

12. The method of any of claims 1 to 11, wherein the expression vector is administered by intravenous, intrathecal, intracerebroventricular, intracisternal, intranerve, subcutaneous, intradermal, epidural, transforaminal, selective nerve root, or stereotactic intraparenchymal administration.

13. An expression vector comprising a promoter, an optional SE52 enhancer, and a transgene sequence encoding an experience-dependent parvalbumin-expressing inhibitory neuron (PV IN) plasticity (XP) protein selected from human Meis2, Tbr1, Bcl11a, and Herc1.

14. The expression vector of claim 13, wherein the promoter comprises a parvalbumin promoter, a GAD65 promoter, or a beta actin promoter.

15. The expression vector of claims 13 or 14, wherein the vector is a viral vector.

16. The expression vector of claim 15, wherein the viral vector is a recombinant adeno-associated virus (AAV), retroviruses, adenovirus, lentivirus, and herpes simplex virus-1.

17. The expression vector of claim 16, wherein the viral vector is an AAV, optionally selected from AAV2, AAV9, AAV-F, AAV.CPP.16, and AAVPhP.eB, and capsid and serotype variants thereof.

18. The expression vector of claim 17, comprising from 5’ -3’: inverted terminal repeat (ITR) – optional S5E2 enhancer - promoter– XP transgene - woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) - pA – ITR.

19. The expression vector of claim 13 or 18, wherein the XP protein is human Meis 2.

20. The expression vector of claim 19, wherein the human Meis 2 protein sequence is at least 95% identical to SEQ ID NO:23.Attorney Docket No.29539-0804WO1 / MGH 2023-602 21. The expression vector of any of claims 13 to 20, for use in a method of treating a subject to reduce or reverse cognitive impairment and reduce seizures in disorders associated with excitation / inhibition imbalance.

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