Compositions and methods for the treatment of christianson syndrome

Abstract

The disclosure provides methods and compositions for treating Christianson syndrome and diseases and disorders associated with mutations in NHE6 using gene therapy.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF CHRISTIANSON SYNDROMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] This disclosure claims priority to U.S. Provisional Patent App. No.: 63 / 651 ,289, filed 23-May-2024, the entire contents of which are hereby incorporated by reference.FIELD OF THE INVENTION

[0002] The embodiments of the present invention relate to compositions and methods for the treatment of Christianson Syndrome, namely gene therapy for preventing or reversing the symptoms of Christianson Syndrome.BACKGROUND OF THE INVENTION

[0003] Christianson syndrome (CS) is an X-linked disorder characterized by impairments in cognitive development, e.g., intellectual disability (ID) / developmental delay (DD), motor function (e.g., ataxia), behavior (e.g., autism, Angelman syndrome, and hyperactivity), and neurologic symptoms (e.g., epilepsy, postnatal microcephaly, and eye movement abnormalities) (Kavanaugh, et al., 2024; Pescosolido, et al., 2014; Morrow & Pescosolido 2018). Progressive and neurodegenerative features have been reported in aging males and females such as regression (e.g., loss of previously acquired adaptive and / or motor skills), cerebellar atrophy, neuronal loss and gliosis, tau deposition, and Parkinsonian-related disorders (Kavanaugh, et al., 2024; Pescosolido, et al., 2014; Garbern, et al., 2010; Pescosolido, et al., 2019). Other clinical symptoms may include high pain tolerance, hypotonia, gastroesophageal reflux, feeding difficulties, and poor weight gain (Kavanaugh, et al., 2024; Pescosolido, et al., 2014). Premature mortality has been reported in a few CS patients; however, it is premature to conclude whether CS patients are at-risk without a natural history study in a large CS cohort (Kavanaugh, et al., 2024; Pescosolido, et al., 2014).

[0004] CS is caused by a range of mutations in SLC9A6 that encodes the endosomal Na+ / H+ exchanger 6 (NHE6) (Gilfillan, et al., 2008). The NHE6 protein consists of a twelvemembrane spanning motif with a Na+ / H+ exchanger, and a carboxyl domain. There are nine mammalian NHEs and NHE6 localizes throughout the endocytic pathway. The SLC9A6 gene is~59 kb and has three transcripts (e.g., SLC9A6.1 (NM_001042537), SLC9A6.2 (NM_006359), and SLC9A6.3 (NM_001177651)). The largest and most highly expressed transcript, SLC9A6.1 , contains 16 coding exons.

[0005] Pathogenic CS mutations include nonsense, splice, missense, indels, and copy number variants (CNVs) (Kavanaugh, et al., 2024; Morrow & Pescosolido 2018). Importantly, a vast majority of CS mutations lead to complete loss of protein. Therefore, gene replacement is a plausible treatment. It is among the most common causes of X-linked-ID as it was found in ~1% of pedigrees suspected of X-linked-ID (Tarpey, et al., 2009). Although originally described as an inherited disorder in which the mother is a carrier of the pathogenic SLC9A6 variant (Christianson, et al., 1999), CS can also be caused by de novo SLC9A6 variants. CS is estimated to affect between 1 in 16,000 to 100,000 individuals worldwide (Pescosolido, et al., 2014). NHE6 dysfunction also has broader implications for other neurodevelopmental and neurodegenerative disorders. The Morrow lab reported decreased cortical NHE6 expression in a postmortem transcriptome study of patients with idiopathic autism (Schwede, et al., 2014). Brain transcriptome data from 2 large normal and pathological aging cohorts revealed that decreased NHE6 expression is correlated with greater tau deposition (Pescosolido, et al., 2019).

[0006] To date, there is no treatment for CS. CS patients typically receive standard medical care consistent with individuals with severe ID and epilepsy. A range of anti-seizure medications have been prescribed to treat seizures in CS patients; however, no seizure treatment guidelines exist (Pescosolido, et al., 2014). Accordingly, there is an urgent need for new treatments for patients afflicted with CS.BRIEF SUMMARY OF THE INVENTION

[0007] The embodiments of the present invention provide compositions and methods for treating a subject afflicted with Christianson syndrome (CS).

[0008] In one aspect of any of the embodiments described herein are gene therapybased methods for promoting expression of human NHE6 protein in a patient afflicted with Christianson syndrome (CS). The present disclosure relates to gene therapy technology including, but not limited to, recombinant adeno-associated virus (rAAV) treatments. The present disclosure provides rAAV viral vectors comprising sequences encoding human NHE6 to treat CS. The rAAV vector comprises, in the 5’ to 3’ direction: (a) a first AAV ITR sequence, (b), a promoter sequence, (c) a transgene nucleic molecule encoding human NHE6, (d) a regulatorysequence, and (e) a second AAV ITR sequence.

[0009] In some embodiments of any of the aspects, the rAAV vectors comprise an AAV capsid protein. An AAV capsid protein can be an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh capsid protein, or an AAVrg capsid protein.

[0010] In some embodiments of any of the aspects, the rAAV vectors comprise of a promoter sequence. A promoter sequence be an EF1a promoter, a U1A promoter, a CAG promoter, a ChAT promoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter.

[0011] In some embodiments of any of the aspects, the DNA sequence encoding the human NHE6 further encodes an HA or V5 / His sequence, wherein human NHE6 produced thereby is tagged with HA or V5 / His.

[0012] In some embodiments of any of the aspects, the DNA sequence encoding human NHE6 corresponds to an RNA transcript selected from SLC9A6.1, SLC9A6.2 and SLC9A6.3.

[0013] In some embodiments of any of the aspects, the DNA sequence encoding human NHE6 corresponds to codon-optimized SLC9A6.1 RNA transcripts.

[0014] In another aspect of any of the embodiments, described herein is a method for treating a patient afflicted with CS by administering to the patient an rAAV vector described herein.

[0015] In some embodiments of any of the aspects, the rAAV vector is administered to patient afflicted CS by intraparenchymal (IPa), intrathecal (IT), intracerebral (IC), intracerebroventricular (ICV), or intravenous (IV) administration.

[0016] In some embodiments, Christianson syndrome is a rare neurodevelopmental disorder that can be characterized by intellectual disability, epilepsy, and ataxia, among other symptoms. Traditional approaches to managing Christianson syndrome have primarily focused on symptomatic treatment, such as the use of antiepileptic drugs to control seizures and supportive therapies to address developmental delays and behavioral issues. These treatments, however, do not address the underlying genetic cause of the disorder, which is linked to mutations in the SLC9A6 gene encoding the sodium / hydrogen exchanger 6 (NHE6) protein.

[0017] According to some aspects, gene therapy is disclosed herein as a promising approach for treating genetic disorders by delivering functional copies of defective genes toaffected cells. In the context of Christianson syndrome, the use of viral vectors, such as lentiviruses and adenoviruses, are examples to deliver the SLC9A6 gene. These vectors can have limitations, including potential immunogenicity and integration into the host genome, which can lead to insertional mutagenesis. The necessary efficiency of gene delivery and expression in target tissues, such as the central nervous system, is provided in various embodiments herein.

[0018] Recombinant adeno-associated virus (rAAV) vectors have gained attention due to their ability to transduce non-dividing cells and their relatively low immunogenicity. Various serotypes of AAV have been investigated for their tropism and efficiency in delivering therapeutic genes to specific tissues. For instance, AAV9 has been shown to cross the bloodbrain barrier, making it a candidate for central nervous system disorders. The development of an effective rAAV-based gene therapy for Christianson syndrome that ensures stable and sufficient expression of NHE6 in relevant tissues is a novel and much needed technology provided by this disclosure.

[0019] In an example, the present disclosure relates to a method for treating Christianson syndrome by administering a recombinant adeno-associated virus (rAAV) vector to a subject in need. The rAAV vector comprises a nucleic acid with a first AAV inverted terminal repeat (1st ITR), a promoter linked to a nucleic acid sequence encoding NHE6, and a second AAV inverted terminal repeat (2nd ITR). The viral vector is encapsulated within a capsid composed of an AAV capsid protein, selected from a group including AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, and AAVrg capsid proteins. This method provides a targeted approach to deliver therapeutic genetic material to treat Christianson syndrome effectively.

[0020] As such, keeping in mind possible combination embodiments and the above discussion, as an additional brief summary or to provide discussion points for a brief summary, some example features of the technology disclosed herein can be briefly summarized by the following list of features, any of which can be inter-combined or discussed optionally with any other feature, Figure, Drawing, detail, embodiment, aspect, or example disclosed herein:

[0021] Feature 1 : A method for the treatment of Christianson syndrome in a subject in need thereof, the method comprising administering to the subject a recombinant adeno- associated virus (rAAV) vector comprising a nucleic acid, wherein the nucleic acid comprises: a first nucleic acid sequence comprising a first AAV inverted terminal repeat (1st ITR); a promoter operably linked to a nucleic acid sequence encoding NHE6; and a second nucleic acid sequence comprising a second AAV inverted terminal repeat (2nd ITR); wherein the viral vectoris enclosed in a capsid comprising an AAV capsid protein selected from the group consisting of AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, and AAVrg capsid proteins.

[0022] Feature 2: The method of feature 1 , wherein the rAAV vector further comprises a promoter sequence including an EF1a promoter, a U1A promoter, a CAG promoter, a ChAT promoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter, wherein the promoter sequence is selected based on its ability to drive high levels of NHE6 expression in the brain.

[0023] Feature 3: The method of feature 1 , wherein the promoter is a tissue-specific promoter that is selected to target NHE6 expression to specific cell types in the brain affected by Christianson syndrome.

[0024] Feature 4: The method of feature 3, wherein the tissue-specific promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain.

[0025] Feature 5: The method of feature 1 , wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression.

[0026] Feature 6: The method of feature 1 , wherein the rAAV vector is administered by intravenous injection, intramuscular injection, intracranial injection, intrathecal injection, intranasal administration, intraparenchymal (I Pa) administration, intracerebral (IC) administration, or intracerebroventricular (ICV) administration, wherein the route of administration is selected based on its ability to deliver the rAAV vector to the brain regions affected by Christianson syndrome.

[0027] Feature 7: The method of feature 1 , wherein the subject is a human patient diagnosed with Christianson syndrome based on clinical symptoms and genetic testing.

[0028] Feature 8: The method of feature 1 , wherein the subject has a mutation in the NHE6 gene that is known to cause Christianson syndrome, such as a missense, nonsense, splice site, or frameshift mutation.

[0029] Feature 9: The method of feature 1 , wherein administering the rAAV vector results in expression of functional NHE6 protein in the brain of the subject, as demonstrated by immunohistochemistry, Western blotting, or other protein detection methods.

[0030] Feature 10: The method of feature 1, wherein administering the rAAV vector improves one or more symptoms of Christianson syndrome in the subject, such as seizures, intellectual disability, ataxia, or autistic behaviors, as measured by standardized clinical assessments.

[0031] Feature 11: The method of feature 1 , further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells.

[0032] Feature 12: The method of feature 5, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells.

[0033] Feature 13: The method of feature 1 , further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0034] Feature 14: The method of feature 5, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0035] Feature 15: A recombinant adeno-associated virus (rAAV) vector for the treatment of Christianson syndrome, the rAAV vector comprising a nucleic acid, wherein the nucleic acid comprises: a first nucleic acid sequence comprising a first AAV inverted terminal repeat (1st ITR); a promoter operably linked to a nucleic acid sequence encoding NHE6; and a second nucleic acid sequence comprising a second AAV inverted terminal repeat (2nd ITR); wherein the rAAV vector is enclosed in a capsid comprising an AAV capsid protein selected from the group consisting of AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, and AAVrg capsid proteins.

[0036] Feature 16: The rAAV vector of feature 15, wherein the rAAV vector further comprises a promoter sequence including an EF1a promoter, a U1A promoter, a CAG promoter, a ChAT promoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter, wherein the promoter sequence is selected based on its ability to drive high levels of NHE6 expression in the brain.

[0037] Feature 17: The rAAV vector of feature 15, wherein the promoter is a tissuespecific promoter that is selected to target NHE6 expression to specific cell types in the brain affected by Christianson syndrome.

[0038] Feature 18: The rAAV vector of feature 17, wherein the tissue-specific promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain.

[0039] Feature 19: The rAAV vector of feature 15, wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression.

[0040] Feature 20: The rAAV vector of feature 15, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells.

[0041] Feature 21: The rAAV vector of feature 19, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells.

[0042] Feature 22: The rAAV vector of feature 15, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0043] Feature 23: The rAAV vector of feature 19, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0044] Feature 24: A pharmaceutical composition for the treatment of Christianson syndrome, the pharmaceutical composition comprising the rAAV vector of feature 15 and a pharmaceutically acceptable carrier.

[0045] Feature 25: The pharmaceutical composition of feature 24, wherein the pharmaceutically acceptable carrier is suitable for intravenous injection, intramuscular injection, intracranial injection, intrathecal injection, intranasal administration, intraparenchymal (IPa) administration, intracerebral (IC) administration, or intracerebroventricular (ICV) administration,wherein the carrier is selected based on its ability to stabilize the rAAV vector and facilitate delivery to the brain.

[0046] Feature 26: The pharmaceutical composition of feature 24, wherein the AAV capsid protein is an AAV9 capsid protein, which has been shown to efficiently cross the bloodbrain barrier and transduce neurons and glial cells in the brain.

[0047] Feature 27: The pharmaceutical composition of feature 24, wherein the promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain affected by Christianson syndrome.

[0048] Feature 28: The pharmaceutical composition of feature 24, wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression in the brain after administration.

[0049] Feature 29: The pharmaceutical composition of feature 24, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 2 is a wild-type human NHE6 protein sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0050] Feature 30: The pharmaceutical composition of feature 28, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 2 is a wild-type human NHE6 protein sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity.

[0051] Other implementations are also described and recited herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] For the purpose of illustration, certain embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and instruments shown. In the drawings:

[0053] FIG. 1 shows the expression levels of SLC9A6 transcripts: 1 (NM_001042537), 2 (NM_006359), and 3 (NM_001177651). RNA-seq performed on induced neurons derived frominduced pluripotent stem cells (iPSCs) in human controls.

[0054] FIG. 2A-H shows the hNHE6 AAV vector maps with the following promoters: EF1a (FIG. 2A-C), U1A (FIG. 2D), CAG (FIG. 2E), synapsin 1 (FIG. 2F), CaMKII (FIG. 2G), and L7 (FIG. 2H).

[0055] FIGs. 3A-3D show the biodistribution of the AAV9-EF1a-hNHE6-V5 in the brain of an NHE6-null mouse at 1-month post-injection. Mouse brain was stained for DAPI (nuclei), an NHE6 antibody, and V5-tagged hNHE6 (from the AAV9 construct) and imaged using confocal microscopy.

[0056] FIG. 4 shows the biodistribution of the AAV9-U1 A-hNHE6-V5 in the brains of NHE6-null and WT male rats at 2 months post-injection. Mouse brains were stained for DAPI (nuclei), an NHE6 antibody, and V5-tagged hNHE6 (from the AAV9 construct) and imaged using confocal microscopy.

[0057] FIGs. 5A-5D show the biodistribution of the AAV9-CAG-hNHE6-V5 in the brain of an NHE6-null mouse at 1-month post-injection. Mouse brain was stained for DAPI (nuclei), an NHE6 antibody, and V5-tagged hNHE6 (from the AAV9 construct) and imaged using confocal microscopy.

[0058] FIGs. 6A-6E show the biodistribution of the AAV9-SYN-hNHE6-V5 in the brain of a WT male mouse at 1-month post-injection. FIGs. 6A-6D each show a representative image exogenous hNHE6 expression in the mouse brain stained for DAPI (nuclei), an NHE6 antibody, and V5-tagged hNHE6 (from the AAV9 construct) and imaged using confocal microscopy.FIG. 6E shows the biodistribution of exogenous hNHE6 in the primary motor cortex or hippocampal region stained for NeuN (neuron-specific nuclear marker), GFAP (astrocytes), IBA1 (microglia), and V5-tagged hNHE6 (from the AAV9 construct).

[0059] FIGs. 7A-7D show the biodistribution of the AAV9-CaMKII-hNHE6-V5 in the brain of a WT male mouse at 1-month post-injection. Mouse brain was stained for DAPI (nuclei), an NHE6 antibody, and V5-tagged hNHE6 (from the AAV9 construct) and imaged using confocal microscopy.

[0060] FIG. 8 shows hNHE6 gene therapy using AAV9-EF1a-hNHE6 improves motor activity in OS rats, as measured by total distance travelled (meters) in the open field test.DETAILED DESCRIPTION OF THE INVENTION

[0061] The subject innovation is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the followingdescription, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. In other instances, well-known structures and devices are shown in block diagram form in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.DEFINITIONS

[0062] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. 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. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0063] As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0064] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0065] As used herein, the term “or” means “and / or.” The term "and / or" as used in a phrase such as "A and / or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0066] As used herein, the term "comprising" means that other elements can also bepresent in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0067] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0068] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0069] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two-standard deviation (2SD) or greater difference.

[0070] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex.

[0071] As used herein, the terms "effective amount" and “therapeutically effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as Christianson syndrome (CS). It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0072] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally“effective” if one or more symptoms or clinical parameters are improved. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or clinical parameters, but also a cessation or at least slowing down of progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized ( / .e., not worsening) state of e.g., impairments in cognitive development (e.g., intellectual disability (ID) / developmental delay (DD)), motor function (e.g., ataxia), behavior (e.g., autism, Angelman syndrome, and hyperactivity), and neurologic symptoms (e.g., epilepsy, postnatal microcephaly, and eye movement abnormalities), delay or slowing down of the symptoms of CS, and an increased lifespan as compared to that expected in the absence of treatment.

[0073] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more.

[0074] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intra-lymphatic, intra-lymph node, intravenous, intraportal vein, intrahepatic arterial, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, intranasal, intratracheal, intrathecal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0075] The terms: “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example,a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0076] The terms: “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.BlOPHARMACEUTICAL DEFINITIONS:

[0077] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, sumoylated, farnesylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0078] In the various embodiments described herein, it is further contemplated thatvariants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions, insertions, or suppressor mutations to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0079] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wildtype reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.

[0080] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions, substitutions, or suppressor mutations. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more insertions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity or function. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.

[0081] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or artificial nucleic acid analogues (e.g., peptide nucleic acid, morpholino- and locked nucleic acid, glycol nucleic acid, threose nucleic acid and hexitol nucleic acid), or any analogs thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double- strandedDNA. Alternatively, it can be a single-stranded nucleic acid not derived from any doublestranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA.

[0082] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell is typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.

[0083] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., an antibody or antibody reagent) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, oncolytic virus-like vesicle (VLV), virion, extracellular vesicles, etc.

[0084] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA, peptides and proteins and as appropriate, secreting peptides or proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification, degradation, shuttling, shuffling, and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro, ex vivo or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR, "trailer" or “leader” sequences, as well as intervening sequences (introns) or spacers between individualcoding segments (exons).

[0085] The term “isolated” or “partially purified” as used herein refers, in the case of a nucleic acid or polypeptide, to a nucleic acid or polypeptide separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that would be present with the nucleic acid or polypeptide when expressed by a cell or secreted in the case of secreted polypeptides. A chemically synthesized nucleic acid or polypeptide or one synthesized using in vitro transcription / translation is considered “isolated.” The terms “purified” or “substantially purified” refer to an isolated nucleic acid or polypeptide that is at least 95% by weight the subject nucleic acid or polypeptide, including, for example, at least 96%, at least 97%, at least 98%, at least 99% or more. In some embodiments, the antibody, antigen-binding portion (e.g., scFv or nanobody) thereof, bispecific or tri-specific T cell engager, bispecific or tri-specific NK cell engager, immune cell recruiter, or chimeric antigen receptor (CAR) described herein is isolated. In some embodiments, the antibody, antibody reagent, antigen-binding portion thereof, engager, recruiter or CAR described herein is purified.

[0086] As used herein, “engineered” refers to the aspect of having been manipulated by the hand of man. For example, an antibody, antibody reagent, antigen-binding portion (e.g., scFv or nanobody) thereof, or tri-specific T cell engager, bispecific or tri-specific NK cell engager, immune cell recruiter (such as Bifunctional checkpoint-inhibitory T cell engager (CiTE), simultaneous multiple interaction T cell engager (SMITE), immune-mobilizing monoclonal TCRs against cancer (ImmTACs)), CAR or is considered to be “engineered” when the sequence of the antibody, antibody reagent, antigen-binding portion thereof, engager, recruiter or CAR is manipulated by the hand of man to differ from the sequence of an antibody as it exists in nature. As is common practice and is understood by those in the art, progeny and copies of an engineered polynucleotide and / or polypeptide are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.PHARMACEUTICAL COMPOSITIONS

[0087] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example,aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive corn, sunflower, grapeseed, vegetable, fish oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration ( / .e., routes, such as injection, pump infusion, transplantation or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen- free. The excipients can be chosen, for example, to effectuate delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, gel, spray, aerosol (e.g., pressurized intraperitoneal aerosol), radioactive isotope, intervention (e.g., trans-arterial embolization, radiofrequency ablation), injection, infusion or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0088] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, decrease degradation, rejection or clearance, increase solubility, increase immunogenicity, or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans; antioxidants, such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; adjuvants (e.g., Alum, MF59, AS01 / 03 / 04, CpG1018) or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0089] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, metals, radioactive isotope / radiation particles, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, rejection, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0090] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, vegetable oil, cottonseed oil, safflower oil, sesame oil, olive oil, sunflower oil, grapeseed oil, fish oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar, matrigel or hydrogel; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline;(18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) nanoparticles such as liposomes, polymers, micelles, metal nanoparticles, carbon nanotubes, solid lipid nanoparticles, noisomes, and dendrimers; (22) extracellular vesicles; and (23) other non-toxic compatible substances employed in pharmaceutical formulations.

[0091] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, pills, suppository, patch, paste, infusion pumps, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral, nasal, urinary, rectal or vaginal mucosa {e.g., sublingually, aerosol); subcutaneously; intradermally, transdermally (for example as a patch applied to the skin); intramuscularly; intravenously; and topically (for example, as a cream, lotion, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731 ,000, 5,541 ,231 , 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0092] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0093] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0094] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0095] To prepare solid dosage forms for oral administration, capsules, including sprinkle capsules and gelatin capsules, (coated and uncoated, bi-layer, mini-) tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, gel, hydrogel, matrigel, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as modified and unmodified cyclodextrins; (11) flavoring agents; (12) thermoregulation (coolingor heating) agents; and (13) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0096] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfaceactive or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0097] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient(s) therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, nanoparticles such as liposomes and / or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the respiratory, urogenital, gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, extracellular vesicle form, if appropriate, with one or more of the above-described excipients.

[0098] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, microcapsules, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (e.g., fish, cottonseed, groundnut, corn, germ, vegetable,grapeseed, sunflower, olive, castor and sesame), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0099] Besides inert diluents, the oral compositions can also include adjuvants such as Alum, MF59, AS01 / 03 / 04, CpG1018, wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0100] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0101] Dosage forms for the topical or transdermal administration include powders, sprays, creams, lotions, ointments, gels, hydrogel, matrigel, solutions, patches, pastes, microneedles (for transdermal, intraocular, vaginal, transungual, cardiac, vascular, gastrointestinal and intracochlear delivery) and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, adjuvant, and with any preservatives, buffers, or propellants that may be required.

[0102] The ointments, pastes, creams, lotions, patches, and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0103] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0104] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0105] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intra-articular, intra-lymph node, intra-lymphatic, intratumoral,intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intracochlear, intraperitoneal, intravaginal, transdermal, transtracheal, transungual, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0106] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Proper fluidity (flow / adhesion ratio) can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0107] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial, antiviral, and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0108] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0109] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in nanoparticles such as liposomes, extracellular vesicles, microencapsulations or microemulsions that are compatible with body tissue.

[0110] For use in the methods of this invention, active compounds can be given perse or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0111] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow-release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0112] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0113] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or clearance of the particular compound(s) being employed, the duration or frequency of the treatment, other drugs that may interact with or affect the metabolism or efficacy of the compound of the invention, compounds and / or materials (e.g., vaccines, antibodies and derivatives) used in combination with the particular compound(s) employed, other therapeutic approaches (e.g., surgery, cell-based therapy, chemotherapy, radiotherapy, interventional therapy), the age, sex, body weight, conditions or comorbidities, diet, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0114] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required.For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the body weight, sex, age, comorbidities, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, therapeutic approaches, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher, et al., (1996).

[0115] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0116] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0117] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

[0118] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent or combined with a therapeutic approach (e.g., surgery, chemotherapy, radiotherapy, interventional therapy).

[0119] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1 H-imidazole, lithium,L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, and undecylenic acid salts.

[0120] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0121] Wetting agents, emulsifiers, dispersants and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, cooling or heating agents, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0122] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alphatocopherol, polyunsaturated fatty acids (PUFAs) such as Omega-3 fatty acids, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, succimer (dimercaptonol), dimercaprol (BAL), and the like.

[0123] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, animal models, (engineered or genetically modified) cells, organoids, constructs, vectors, carriers, adjuvants, compounds, drug delivery system, antibodies and derivatives, vaccines, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy; The Encyclopedia of Molecular Cell Biology and Molecular Medicine; Molecular Biology and Biotechnology: a Comprehensive Desk Reference; Immunology; Janeway's Immunobiology; Lewin's Genes XI; Molecular Cloning: A Laboratory Manual.; Basic Methods in Molecular Biology; Laboratory Methods in Enzymology; Current Protocols in Molecular Biology (CPMB); Current Protocols in Protein Science (CPPS); and Current Protocols in Immunology (CPI).

[0124] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.

[0125] Other terms are defined herein within the description of the various aspects of the invention.CHRISTIANSON SYNDROME AND NHE6 HUMAN STUDIES

[0126] As discussed above, CS is an X-linked disorder characterized by impairments in cognitive development (e.g., intellectual disability (ID) / developmental delay (DD)), motor function (e.g., ataxia), behavior (e.g., autism, Angelman syndrome, and hyperactivity), and neurologic symptoms (e.g., epilepsy, postnatal microcephaly, and eye movement abnormalities) (Pescosolido, et al., 2014; Morrow & Pescosolido 2018). Progressive and neurodegenerative features have been reported in aging males and females such as regression (e.g., loss of previously acquired adaptive and / or motor skills), cerebellar atrophy, neuronal loss and gliosis, tau deposition, and Parkinsonian-related disorders (Garbern, et al., 2010; Pescosolido, et al., 2014; Pescosolido, et al., 2019). Other clinical symptoms may include high pain tolerance,hypotonia, gastroesophageal reflux, feeding difficulties, and poor weight gain (Pescosolido, et al., 2014). Premature mortality has been reported in a few CS patients. However, it is premature to conclude whether CS patients are at-risk without a natural history study in a large CS cohort (Pescosolido, et al., 2014).

[0127] CS is caused by a range of mutations in SLC9A6 that encodes the endosomal Na+ / H+exchanger 6 (NHE6) (Gilfillan, et al., 2008). The NHE6 protein consists of a twelvemembrane spanning motif with an Na+ / H+exchanger, and a carboxyl domain. There are nine mammalian NHEs and NHE6 localizes throughout the endocytic pathway. The SLC9A6 gene is ~59 kb and has three transcripts (e.g., SLC9A6.1 (NM_001042537), SLC9A6.2 (NM_006359), and SLC9A6.3 (NM_001177651)). The largest and most highly expressed transcript, SLC9A6.1 , contains 16 coding exons. See, FIG. 1.

[0128] Pathogenic CS mutations include nonsense, splice, missense, indels, and copy number variants (CNVs) (Morrow & Pescosolido 2018). Importantly, a vast majority of CS mutations lead to complete loss of protein. Therefore, gene replacement is a plausible treatment. It is among the most common causes of X-linked-ID, as it was found in ~1% of pedigrees suspected of X-linked-ID (Tarpey, et al., 2009). Although originally described as an inherited disorder in which the mother is a carrier of the pathogenic SLC9A6 variant (Christianson, et al., 1999), CS can also be caused by de novo SLC9A6 variants (Pescosolido, et al., 2014). CS is estimated to affect between 1 in 16,000 to 100,000 individuals worldwide (Pescosolido, et al., 2014). NHE6 dysfunction also has broader implications for other neurodevelopmental and neurodegenerative disorders. We reported decreased cortical NHE6 expression in a postmortem transcriptome study of patients with idiopathic autism (Schwede, et al., 2014). Brain transcriptome data from two large normal and pathological aging cohorts revealed that decreased NHE6 expression is correlated with greater tau deposition (Pescosolido, et al., 2019).

[0129] In 2014, we reported the range of NHE6 mutations and clinical phenotypes in the largest CS cohort to date (Pescosolido, et al., 2014). At the time, our cohort consisted of twelve independent pedigrees with a total of 14 affected males (ages 4-19). Prior to this publication, the CS literature only reported findings from <3 pedigrees. Since this publication, our cohort has expanded to 68 pedigrees and 62 affected males (ages 1.8-30 years). This work established the diagnostic criteria for Christianson syndrome. Specifically, we proposed core CS symptoms (>85% of probands) as nonverbal status, ID, epilepsy, ataxia, postnatal microcephaly, and hyperkinesis. Secondary symptoms (>35% of probands) include autism features, Angelman syndrome feature, eye movement problems like strabismus, hypotonia, gastroesophageal refluxdisease, regressions (especially in 1st decade of life), low height / weight, and cerebellar vermal atrophy (particularly after first decade). Given NHE6 mutations were among the most common in a sequencing study of 208 pedigrees suspected of X-linked ID and found in 1-2% of cases (Tarpey, et al., 2009), we estimated the prevalence of CS to affect 1 in 16,000 to 100,000 individuals ( / .e., 1-3% of world’s population diagnosed with ID and 10-20% of ID is X-linked).

[0130] In this study, we recruited and enrolled families with a confirmed or suspected CS diagnosis. A CS diagnosis was suspected if: (1) it occurred in boys, (2) it involved ID, (3) it involved seizures, (4) it involved ataxia, and (5) there was a plausible deleterious NHE6 mutation. The identified proband as well as extended family (e.g., all available parents, grandparents, aunts, uncles, and siblings) were then enrolled. Once enrolled, families were evaluated by a standardized neuromedical history of proband(s) and their family pedigree as well as behavioral assessments.

[0131] We identified a spectrum of NHE6 mutations: nine single nucleotide variants (SNVs), two indels, and one copy number variation (CNV) deletion (Pescosolido, et al., 2014). Most of these mutations were protein-truncating (83%), affecting exons 1, 3, 11 , 12,13, and 14. The rest were splice mutations (17%). CS was originally considered an inherited genetic disorder (Christianson, et al., 1999). However, we found that 7 of 12 mutations (58%) were de novo. Two mutations were recurrent and found in unrelated pedigrees (c.1498 ot, p.R500X and c.1710 g>a, p.W570X). We did not observe any significant genotype-phenotype correlations between early vs. late truncating or de novo vs. inherited mutations.

[0132] To identify the pathogenic NHE6 variants, we screened all exons in the proband and then tested relatives for the presence / absence of that specific variant. We amplified all 16 coding exons of the NHE6 gene and exon / intron junctions (including >50 base pairs into introns) by polymerase chain reaction (PCR) and sequenced via Sanger sequencing. We developed PCR primers for each NHE6 exon. Variants were determined by chromatogram using Chromas Lite software.

[0133] We assessed the range of clinical phenotypes encompassing neurological, medical, and behavioral features in our CS cohort. All CS probands exhibited profound developmental delays across multiple domains such as motor (gross and fine), social, language, and cognition. All males were diagnosed with intellectual disability (ID) / developmental delay (DD) and were either nonverbal or produced minimal words. A subset was reported by parents to have regression or the loss of a previously acquired skill: walking (57%), eating (14%), eye contact / facial expressions (14%), fine / gross motor skills (14%), and words / sounds (57%). Regressions occurred across a range of ages: 15 months, 2-3 years, 4 years, 5 years, 9 years,and 16 years. Notably, regression typically followed a severe medical illness and / or seizures. CS participants had hypotonia or decreased muscle tone (79%). Walking was delayed in most probands and ranged from 1 to 3 years of age (mean age = 20.2 months). All males demonstrated an unsteady gait consistent with truncal ataxia.

[0134] We were able to perform the Leiter-R on 2 boys to measure nonverbal cognitive function. Both boys had a Brief IQ of 36, which is in the deficient range (<1%). We also performed the Vineland II on 3 males (ages 7-17 years), to measure adaptive functioning. Composite adaptive function was low range (<1%) consistent with severe to profound ID in all cases. Receptive language equivalents were between 1 to 8 months while expressive language equivalents were between <1 to 5 months. Daily living skills, such as personal, domestic, and community, age equivalents spanned <1 month to 1 year 8 months. Socialization was in the low range (<1 percentile) with subdomains like interpersonal relationships and coping skills ranging from <1 month to 1 year 2 months. Motor skills (assessed in 2 probands) were in the low range (<1 percentile) with subdomains like gross and fine motor skills age equivalents ranging from 7 months to 1 year 6 months.

[0135] All CS participants were diagnosed with epilepsy. Seizure onset ranged between 4 months to 3 years (mean = 16. + 7.9 months). Seizure types were infantile spasms, tonic, tonic-clonic, myoclonic, drop, and staring spells. Most cases were consistent with general seizures, although there were examples of focal seizures (e.g., left face grimacing and focal eye deviation). Seizure frequency ranged from daily seizure clusters to being seizure-free for over 1 year. We reviewed electroencephalograms (EEGs) in four males as well as EEG clinical reports in four other males. The majority of EEGs were abnormal with background abnormalities (e.g., generalized slowing and absence of normal sleep features) and epileptiform abnormalities (e.g., frequent generalized spike-wave complexes, irregular generalized spike-wave pattern, and multifocal and sometimes synchronous spikes). Epilepsy disorders included epileptic encephalopathy (n=4), Lennox-Gastaut syndrome (n=4), and infantile spasms (n=1). For example, Lennox-Gastaut syndrome features include multiple seizure types (e.g., tonic, tonic- clonic, myoclonic, absence, drop, complex partial) and abnormal EEG findings (e.g., slow spikewave complexes and 2 to 4 Hz slow spike-wave). Seizures have been treated with a range of antiepileptic medications (e.g., carbamazepine, levetiracetam, lamotrigine, and phenobarbital) as well as non-medical treatments (ketogenic diet and vagus nerve stimulator).

[0136] CS symptoms overlap with the neurogenetic disorder Angelman syndrome (AS). In fact, many older CS males were originally diagnosed with Angelman syndrome by clinicians, prior to more recent advances in genetic testing. Using clinical diagnostic criteria for AS, wefound that almost all CS participants demonstrated AS features (93%). For example, CS probands exhibited major AS criteria: ID (100%), limited speech (100%), happy disposition (100%), unprovoked laughter (64%). Minor AS criteria included: seizures (100%), microcephaly (92%), sleep problems (64%), and fascination with water (29%).

[0137] Most CS participants exhibited autistic behaviors. Almost half of the participants were clinically diagnosed with autism (43%). We evaluated three probands with the Autism Diagnostic Interview-Revised (ADI-R) to determine whether they met criteria for an autism diagnosis. All met criteria for autism by displaying autistic symptoms such as absence of social play, lack of a variety of facial expressions to communicate, and poor eye contact. We also used the Social Communication Questionnaire (SCQ) in nine probands to screen for autism. Almost all participants met autism criteria (89%) with scores ranging from 14 to 28 (mean=23.2, autism cutoff score=15). Autism behaviors frequently noted were poor eye contact, use of caregiver’s body parts as tools, unusual sensory interests, and lack of reciprocal play and / or social interest.

[0138] Most CS probands had microcephaly ( / .e., small head circumference) with impaired postnatal head growth (92%). Cerebellar atrophy was documented in 33% of participants. Notable pathogenic magnetic resonance imaging (MRI) findings include moderate to severe cerebellar atrophy, bilateral lesions in the inferior cerebellum, and enlarged ventricles.

[0139] Most CS participants had problems with eye movement (79%) and visual acuity (54%). Some (29%) required eye surgery, mainly for treating strabismus-associated problems. Other ophthalmic diagnoses in individual probands include cortical visual impairment, refractive error, and right V1 nerve palsy. Another CS male was reported by another group to have retinitis pigmentosa (Mignot, et al., 2013). Most families reported sleep problems (64%) and described males as having no sleep pattern and rarely sleeping through the night.

[0140] CS probands had a range of gastrointestinal (Gl)-related problems such as gastroesophageal reflux disease (GERD, 50%) with some needing Nissen fundoplication (14%), feeding difficulties (e.g., difficulty chewing), and swallowing difficulties (29%). Although most males were a normal weight at birth, some parents reported that males were unable to gain weight despite normal caloric intake. Many parents also reported that males had a high pain threshold. CS probands exhibited a range of other medical symptoms such as hyperkinesis (100%), failure to thrive (21%), cyanosis (21%), and eczema (21 %). Medical diagnoses noted in a single CS participant included: osteopenia, anemia, 5th toenail hypoplasia, hemangioma, prominent aortic root, pancreatic enzyme insufficiency, apraxia, pseudobulbar palsy, Todd’s paralysis, and cerebral palsy. Some CS participants had lengthy hospitalizations (21 %) due to pneumonia, status epilepticus, and possible encephalitis.

[0141] A significant proportion of female carriers are unaffected. Female carriers display heterogeneous phenotypes including deficits in cognition (e.g., ID / DD, language delay, executive function, and visuospatial), motor (e.g., ataxia and hypotonia), neurologic (e.g., microcephaly and neuropathy), neuropsychiatric / behavior (e.g., attention, behavior problems) (Pescosolido, et al., 2014; Pescosolido, et al., 2019). We performed neuropsychological testing in this individual at 10 years and 10 months of age. Because females are high functioning, this enhance feasibility of the gene therapy treatment as this means that the cells are mosaic, and we would not need infect all cells of the brain.

[0142] We further examined the phenotypic spectrum in CS female carriers in 20 female carriers from nine pedigrees (Pescosolido, et al., 2019). CS female carriers were identified from CS pedigrees in our international CS cohort as described in Pescosolido et al 2014. All NHE6 mutations were considered loss-of-function. Female carrier’s mean age was 30.3±17.6 years and spanning 2 to 65 years. We also enrolled five non-carrier females from 4 CS pedigrees as a familial control. Their mean age was 29.7±21.1 years and spanning 10 to 75 years. We performed in-depth neuropsychological testing in a subset of enrolled participants: 13 carriers (mean=30.1±15.9 years, range: 3 to 55 years) and three non-carriers (mean=21.7±18.5 years, range: 10 to 45 years) from seven CS pedigrees. Neurocognition was measured in adults (>18 years) using the following assessments: Wechsler Abbreviated Scales of Intelligence-Second Edition (WASI-II), Repeatable Battery for the Assessment of Neuropsychological Status (RBANS), Trail Making Test A&B (TMT A&B), Controlled Oral Word Association Test (COWAT), and Mini-Mental State Exam (MMSE). Neurocognition was measure in children ( / .e., < 18 years old) using the following assessments: A Developmental Neuropsychological Assessment- Second Edition (NEPSY-II), Wechsler Preschool Primary Scales of Intelligence-Fourth Edition (WPPSI-IV), WASI-II, Children’s Memory Scales (CMS), TMT A&B, Wechsler Intelligence Scale for Children-Fourth Edition (WISC-IV), Rey Complex Figure Test (RCFT), Beery-Buktenica Developmental Test of Visual-Motor Integration (Beery), and COWAT. We also calculated domain composite scores to measure IQ (RBANS), language (RBANS, WPPSI-IV), visuospatial function (RBANS, NEPSY-II, RCFT, Beery), attention (RBANS, WPPSI-IV, WISC-IV), memory (RBANS, NEPSY-II, CMS), and executive function (TMT A&B, COWAT, NEPSY-II). Pearson correlations were performed to examine the association between age (i.e., child or adult) and neurocognition variables.

[0143] The majority of female carriers (85%) exhibited a deficit (i.e., standardized, age- normed z-scores <1.5) in at least one neurocognitive domain. Most female carriers exhibited deficits in at least two domains (69%). A neuropsychological profile emerged in female carrierswith deficits in visuospatial function (62%), attention (62%), and executive function (54%). Almost all female carriers had a deficit in one of these three domains (92%, 12 / 13 female carriers) and 39% (5 / 13) had deficits in all three domains. A subset of female carriers had a full- scale IQ <70 (31%, 4 / 13). Domains less likely to be impaired in female carriers included immediate memory (31%), delayed memory (31 %), and language (25%). We examined whether there were age-dependent effects on neurocognition. There were no statistically significant association between pediatric or adult female carriers and neurocognition.

[0144] We also observed a range of neuropsychiatric symptoms in female carriers (Pescosolido, et al., 2019). A subset were diagnosed with ID / DD (20%) while 31% who were not diagnosed with ID / DD were notable for learning difficulties. CS female carriers were also diagnosed with speech / language delays (30%), ADHD (20%), eye problems such as esotropia (20%), ataxia (10%), ASD (10%), schizoaffective disorder, hypotonia (10%), and neuropathy (10%).

[0145] Aging CS female carriers ( / .e., >50 years old) exhibited features consistent with neurodegeneration. Two female carriers from different CS pedigrees were clinically diagnosed with corticobasal degeneration (CBD) and atypical parkinsonism. We examined the female carrier diagnosed with CBD around age 65. CBD symptoms appeared 1-2 years before our exam due to multiple forward falling episodes and gait disturbance. A mental status exam found rapid cognitive decline including profound loss of daily living activities, meaningful language, and communication skills. Dysautonomic symptoms were noted such as constipation and temperature dysregulation. A neurological exam noted masked facies, no upward gaze, limited downward gaze, intact horizontal eye movements, bradykinesia with abnormal tone including cogwheeling and right-side rigidity, myoclonic jerks, hyperreflexia, and sustained clonus in both ankles. She had a resting tremor at 3-6 Hz. Although mostly non-ambulatory, her gait was remarkable for freezing, bradykinesia, and shuffling. Although originally diagnosed with progressive supranuclear palsy given her eye movement problems, her diagnosis was changed to CBD following brain MRI results (age 65). Notable MRI findings include asymmetric cortical atrophy, particularly affecting the left frontal and parietal regions as well as generalized parenchymal volume loss and increased ventricular size. We directly evaluated another female carrier with parkinsonism who was diagnosed around age 55. Tremors in her right hand and upper limbs have become more pronounced over time. Cranial nerve examination revealed mild left eye hypophoria, reduced convergence, hypomimia, and decreased blink frequency. Motor examination was positive for bradykinesia and abnormal tone (e.g., cogwheeling and gegenhalten). This female carrier had clonus in her right ankle and her gait was remarkable forshuffling with decreased arm swing and difficulty turning. A brain MRI in her early 50s revealed generalized brain atrophy, particularly in the cerebellum. Cerebellar atrophy occurred in both the vermis and hemispheres, with severe atrophy in the vermis. We also identified two other likely female carriers over the age of 50 from our CS cohort of >30 families who were diagnosed with CBD or multiple systems atrophy (MSA). Unfortunately, these individuals were unable for enrollment in our study.

[0146] We further investigated the association between NHE6 and neurodegeneration by examining NHE6 expression in postmortem brain samples from two aging / dementia cohorts (Pescosolido, et al., 2019). Specifically, we examined brain transcriptome data from 740 participants in the Religious Orders Study (ROS, Bennett, et al., 2012a) and the Rush Memory and Aging Project (MAP, Bennett, et al., 2012b) studies. Specimen collection and methods have been extensively documented elsewhere (Yu, et al., 2015). RNA-Seq expression was performed on dorsolateral prefrontal cortex gray matter. Alzheimer’s disease (AD)-associated neuropathology results have also been extensively reported elsewhere (Yu, et al., 2015) and include neurofibrillary tangles, diffuse plaques, and neuritic plaques. Using SAS, we performed a linear regression on brain transcriptome expression in order to assess the relationship between NHE6 and AD pathology variables. As part of our modeling, A|3 load and tau accumulation were continuous measures and covariates included age, gender, and study (e.g., ROS or MAP). Benjamini-Hochberg procedure was used to correct for multiple comparisons. We found that decreased NHE6 expression was significantly associated with greater tau tangle density using a strict transcriptome-wide corrections. Decreased NHE6 expression was associated with greater Ap load and decreased cognitive functioning when not adjusting for multiple corrections. NHE6 expression was not associated with some disease variables such as neurofibrillary tangles, neuropathological severity, or AD severity. We also examined whether these disease variables were correlated with the three main NHE6 isoforms: SLC9A6-001 (Transcript ID: ENST00000370698), SLC9A6-002 (Transcript ID: ENST00000370695), and SLC9A6-003 (Transcript ID: ENST00000370701). Tau deposition was correlated with the SLC9A6-003 isoform.

[0147] Finally, we sequenced NHE6 in 156 females diagnosed with idiopathic Parkinson-related disorders such as PSP (n=150) and CBD (n=6) to determine whether NHE6 mutations could be identified (Pescosolido, et al., 2019). We requested samples from the National Institute of Neurological Disorders and Stroke (NINDS) neurodegeneration biorepository. All NHE6 exons and 15-20 bp into exon / intron junctions (in order to identify splice variants) were sequenced using the MiSeq platform (Illumina). Any potentially pathogenicvariant was confirmed via Sanger sequencing. We did not detect any deleterious mutation (e.g., nonsense, missense, or indel) in this cohort.

[0148] The Morrow lab has reported altered NHE6 brain expression in autism (Schwede, et al., 2014). This analysis was performed using microarray data collected from postmortem cerebral cortex samples in autism and control individuals (n=29 per group, Voineagu, et al., 2011). NHE6 expression was significantly downregulated in the autism cortex. A Pearson correlation revealed that NHE6 expression was associated with synapse-related gene expression (n=21). Notably, decreased cortical NHE6 expression was further validated in two other similar autism microarray data sets.STUDIES OF CHRISTIANSON SYNDROME AND NHE6 HUMAN STUDIES

[0149] Study 1 : Christianson Syndrome Patient-Derived Induced Pluripotent Stem Cells (iPSCS). In one study, we generated patient-derived iPSCs that reflect the NHE6 mutation spectrum, with the explicit goal to aid therapeutic interventions. We investigated how these mutations affect NHE6 in iPSC-derived CS neurons and whether gene transfer strategies successfully ameliorate CS pathobiology (Lizarraga, et al., 2021). iPSCs were developed from the following five CS patients (as well as an unaffected brother without an NHE6 mutation for each patient): (1) c.1414dupA, p.R472fsX4; (2) c.1568G>A, p.W523X; (3) c.1148G>A, P.G383D; (4) c.540_547dupAGAAGTAT, p.F183fsX1 ; and (5) c.1710G>A, p.W570X. Further, for each CS line, genome-corrected iPSC lines were generated to serve as isogenic controls. Like the majority of CS patients, most of these frameshift / nonsense mutations appear to be loss-of-function. It was hypothesized that the recurrent missense mutation (j.e., p.G383D) alters mRNA splicing. For research design, experiments were replicated at least three times with multiple subclones from patient and control iPSC lines. Most statistical analyses employed unpaired Student’s t-test or two-tailed Welch’s t test, unless otherwise specified.

[0150] First, we evaluated how these pathogenic mutations affect gene and protein expression. Northern blot and NanoString nCounter analyses confirmed that NHE6 mRNA is missing in all four protein-truncating mutations. It was hypothesized that this was due to nonsense-mediated decay (NMD). Therefore, to test whether NHE6 mRNA is degraded by NMD, iPSCs were treated with an NMD-inhibitor cycloheximide (CHX, 100 pg / mL for 3 hours). NHE6 mRNA expression was enhanced in CHX-treated nonsense mutation CS lines, but not in the G383D missense mutation line.

[0151] These results were further corroborated using small interfering RNA (siRNA) of NMD targets UPF1 and UPF3 (compared to a scrambled siRNA control). However, NHE6mRNA expression was not completely lost in the G383D missense mutation and had between 56-58% of control NHE6 mRNA (as measured by both NanoString nCounter and Northern blot, respectively). Although CHX-treated G383D did display increased NHE6 mRNA expression ( / .e., about 78% of control), consistent with some NMD. Western blot analysis of NHE6 immunoprecipitates confirmed the complete loss of NHE6 protein in nonsense mutations, consistent with these being loss-of-function mutations. Thus, truncated NHE6 proteins are highly unlikely to be translated in sufficient amounts. However, the G383D mutation exhibited significantly decreased yet not absent NHE6 protein levels relative to controls ( / .e., NHE6 monomer ~20% and dimer ~3% of control expression). While this missense mutation produces a coding change, it occurs adjacent to a 3’ splice acceptor site at the first base pair of exon 9. It was hypothesized this mutation alters splicing by skipping exon 9 and splicing exon 8 to exon 10, which would cause a frameshift and premature stop codon two codons downstream of the exon 8 / 10 junction. Reverse transcription-polymerase chain reaction (RT-PCR) and sequencing of cDNA collected from iPSCs confirmed the alternative splicing event that was hypothesized.

[0152] Thus, the G383D missense mutation leads to two mRNAs: (1) exon skipping leading to a nonsense mutation (and susceptible to NMD given our CHX studies showing transcript recovery) and (2) mRNA that produces a protein with the G383D missense mutation. Further, it was modeled how NHE6 structure was perturbed by the G383D missense mutation based on the crystal structure of the Escherichia coli NHE Na+ / H+ antiporter A (NhaA). This model predicted the G383D residue faces the core of the transport on transmembrane 8 (TM8) that likely regulates ion transport in the NHE family. Mutating this nonpolar glycine residue to acidic aspartate is likely to disrupt the transport’s structure via the helix packing between TM7 and TM8. It is also possible that this mutation creates a salt bridge with R500 on TM11. Taken together, structural modeling supports G383D impairs the structure and function of the transmembrane and is predicted to be loss-of-function by complex mechanisms j.e., splicing and protein missense).

[0153] Given the postnatal microcephaly observed in CS males, it was hypothesized this could be due, in part, to deficits in neuronal arborization in the neocortex rather than progenitor neurogenesis, cell fate determination or cell death mechanisms found in primary microcephaly. Therefore, differentiated CS and control iPSCs were generated to excitatory cortical neurons using a dual SMAD inhibition monolayer differentiation protocol. To test whether progenitor neurogenesis, cell fate determination or cell death account for CS microcephaly neuronal cultures were examined by immunocytochemistry and RNA techniques. There were no differences in the percentage of cells expressing markers for neuronal progenitors (e.g., PAX6),neurons (e.g., MAP2) and deep-layer cortical projection neurons (TBR1 , COUP-TF and CTIP2). Then mRNA expression of genes involved in these cellular processes was examined by NanoString. There were no notable differentially expressed genes between CS and control iPSC-derived neurons across any of these markers. Taken together, it is unlikely that mechanisms mediating primary microcephaly (e.g., progenitor neurogenesis, cell fate determination or cell death) are involved in CS-associated microcephaly.

[0154] Next, we examined whether impaired neuronal arborization mediated CS postnatal microcephaly. It was hypothesized arbor deficits were the most likely mechanism given we previously identified arborization dysfunction in a CS mouse model (Ouyang, et al., 2013). Therefore, neurite outgrowth and arborization in iPSC-differentiated neurons were measured from CS patients and controls. Time-lapse live-imaging of neurons on laminin stripes revealed that CS neurons had decreased elongation and retraction rates compared to their controls. It was corroborated these deficits in arborization in CS neurons by using Sholl analysis of GFP-transfected neurons. Scholl analyses were analyzed using two-way ANOVA with Bonferroni correction for multiple comparisons. Taken together, these CS mutations disrupt proper neuronal arborization that likely mediate postnatal microcephaly symptoms found in CS.

[0155] This raised the question whether transfecting CS neurons with NHE6 cDNA would rescue neuronal arborization deficits. Neurons were grown to 32 days following neuronal induction and transfected with either (1) a single construct encoding GFP as a control or (2) the GFP construct plus a construct encoding full-length NHE6 with an HA-tag. NHE6 gene transfer cell-autonomously rescued arborization deficits in all nonsense CS mutations as measured by Sholl analysis. Specifically, NHE6 re-expression significantly increased neurite length and the number of intersections compared to CS iPSC lines transfected with GFP only. However, this NHE6 gene transfer did not rescue arbor dysfunction in the complex missense / splice mutation G383D. This finding suggests endogenously expressed G383D NHE6 protein acts as a dominant-negative mutation, possibly by forming non-functional heterodimers with exogenous NHE6. It is critical to determine whether this is the case as NHE6 gene therapy would be unsuccessful in these mutation categories.

[0156] Then the function of the NHE6 G383D protein in human HEK-293T cells was examined. An HEK-293T cell line with the G383D mutation was generated using CRISPR-Cas9 technology. Endosomal pH was measured using a transferrin-based assay. While exogenous NHE6 expression in NHE6-null cells alkalinized endosomes, exogenous expression of NHE6 G383D in NHE6-null cells was unable to alkalinize endosomes. This data supports the conclusion that the G383D missense mutations impairs cation exchange, consistent with loss-of-function of the NHE6 protein. Further, co-expressing wild-type and G383D NHE6 is able to ameliorate endosomal over-acidification. It was found that the NHE6 G383D protein forms homodimers with itself as well as heterodimers with exogenously expressed wild-type NHE6. Super-resolution structured illumination microscopy revealed altered subcellular distribution of G383D protein with less colocalization with the early endosomal marker transferrin compared to control cells. Thus, our endosomal pH and dimerization studies confirm the NHE6 G383D protein functions as a dominant-negative, likely through inhibiting wild-type NHE6 protein function by forming nonfunctional heterodimers.

[0157] These CS patient-derived iPSCs offer the opportunity to utilize cell-based assays in order to test the efficacy of potential therapeutics. While it was demonstrated NHE6 gene therapy represents a potential treatment option for CS patients with loss-of-function mutations, this raised the question whether other alternative treatments could rescue CS pathobiology. Specifically, therapeutics were considered that the inventors have previously shown to ameliorate CS pathology such as (1) drugs that alkalinize the endosomal lumen and (2) growth factors (Ouyang, et al., 2013).

[0158] It was tested whether alkalinizing agents could normalize intraendosomal pH in CS iPSCs by two methods - ratiometric fluorescently-conjugated transferrin and ratiometric VAMP3-pHluorin2 (a recycling endosome marker). For the transferrin-based method, iPSCs were loaded with pH sensitive FITC-conjugated transferrin and pH insensitive Alexa Fluor- 546- conjugated transferrin. A ratio was calculated for mean fluorescence intensity of FITC to Alexa- 546. VAMP3-pHluorin2 constructs were transfected into iPSCs. Fluorescence intensity was measured, and a ratio was calculated for mean fluorescence intensity of excitation at 488 nm to 407 nm. Fluorescence intensity was measured in both assays by flow cytometry.

[0159] Gene-edited mutation correction by CRISPR-Cas9 rescued endosomal overacidification. Endosomal pH was rescued ( / .e., no statistically significant difference compared to controls) in R472fsX4 (transferrin), W523X (transferrin), G383D (transferrin and VAMP3- pHluorin) and F183fsX1 (transferrin and VAMP3-pHluorin) lines. However, both pH assays revealed that gene-correction of the W570X line did not ameliorate endosomal overacidification. Notably, the degree of pH normalization was variable across CS families with some families displaying strong effects (e.g., R472fsX4 and w523x), while others displayed milder effects (e.g., W570X). Therefore, alkalinizing drugs (e.g., quinolines, weak bases, or V-ATPase inhibitors) were not further investigated as a rescue strategy.

[0160] We then investigated whether exogenous BDNF and / or IGF-1 treatment would rescue neuronal arborization in CS patient-derived neurons. Previously, it was demonstratedthat exogenous BDNF treatment improved neuronal arborization in a CS mouse model (Ouyang, et al., 2013). Neuronal cultures were treated with media containing BDNF (10 ng / ml), IGF- 1 (20 ng / ml), or vehicle for 72 hours prior to fixation for arborization analysis and staining with MAP2 and TAU1 antibodies. Both BDNF and IGF-1 treatments rescued neuronal arborization deficits in all CS mutations. For example, BDNF significantly increased length per neurite in the following CS mutations compared to its untreated mutations: R472fsX4 (untreated=24, BDNF=19), W523X (untreated=53 cells, BDNF=37 cells) and G383D (untreated=32 cells, BDNF=31 cells). BDNF treatment also significantly increased the number of branch points per neurite in CS mutations compared to its untreated mutations (same sample size as length per neurite). IGF-1 treatment significantly increased length per neurite in the following CS mutations compared to its untreated mutations: R472fsX4 (untreated=24 cells, IGF=36 cells), W523X (untreated=53 cells, IGF=37 cells) and G383D (untreated=32 cells, IGF=35 cells). IGF-1 also significantly increased the number of branch points per neurite in CS mutations compared to its untreated mutations (same sample size as length per neurite). In conclusion, these findings support growth factor treatment (e.g., BDNF and IGF-1) in an in vitro patient iPSC model across all types of CS mutations.

[0161] To spur molecular, cellular and translational research in CS using the iPSC model, iPSC lines were generated from a CS patient with an NHE6 nonsense mutation c.1569G>A (p.W523X), an unaffected brother control iPSC line and two CRISPR-Cas9- mediated gene-corrected control iPSC lines (Ma, et al., 2021). Peripheral blood mononuclear cells (PBMCs) collected from the affected male and his unaffected brother who does not carry the NHE6 mutation were reprogrammed to create these lines. CRISPR-Cas9 technology was employed to correct the c.1569G>A (p.W523X) mutation using homology directed repair (HDR) knock-in methodology. This mutation is located on exon 12 so gRNA to target exon 12 as well as a single-stranded oligodeoxyribonucleotide (ssODN) with the targeted A>G mutation at the exact center for HDR were designed. The ssODN included three silent mutations in each protospacer adjacent motif (PAM, i.e., PAM1-3) in order to block re-targeting and recutting by Cas9. A BtsIMutl restriction site (CAGTGTG) in PAM2 was introduced to identify successfully targeted clones. It was confirmed c.1569G>A (p.W523X) mutation correction by Sanger sequencing NHE6 after selecting colonies transfected with the ribonucleoprotein (RNP) / Cas9 complex and the ssODN. Two clones (out of 24 sequenced polyclonal colonies) were found with the A>G correction and one of these clones was expanded in order to generate clones from a single cell. Then, two gene-corrected CS iPSC lines (out of 14 total lines), 403-S7-KI-5 and 403- S7-KI-17, were characterized.

[0162] Morphological analysis of all four iPSC lines (e.g., CS, biologically-related control and two gene-corrected isogenic controls) confirmed these cell lines exhibited pluripotent stem cell morphology that grew in high-density monolayers (Ma, et al., 2021). These iPSC lines expressed pluripotent protein markers as measured by antibodies such as NANOG, OCT3 / 4, and SOX2. They also differentiate into three germ layers (i.e., SOX17, BRACHYURY, and NEUN). Western blot confirmed both monomer and dimer forms of NHE6 protein in the biological control and gene-corrected control iPSC lines, but not in the CS iPSC line. No mycoplasma was found in any of the four iPSC lines. In conclusion, these iPSC cell lines represent an important resource for preclinical and clinical CS research.

[0163] Cortical neurons were generated from CS iPSC lines to study neurodegeneration mechanisms underlying CS (Fernandez, et al., 2022). Two NHE6 knockout (KO) (c.346_424del (p.L116YfsX3) and c.[336_352del; 423_427del] (p.G113WfsX7)) and isogenic control iPSC cell lines (that were previously characterized by Lizarraga, et al., (2021)) were generated. These iPSC lines were differentiated into layer 2 / 3 cortical neurons by NGN2 expression for 21 days, where they express neuronal markers (e.g., Tau NeuN, pl I l-tubulin) by microscopy techniques. Statistical analyses included Student’s f-test and one-way ANOVA with multiple comparison tests (e.g., Sidak’s or Dunnet’s post test).

[0164] After confirming successful generation of iPSC-derived neurons, we investigated how loss of NHE6 affects tau pathology (Fernandez, et al., 2022). First, TBS-soluble, 1 % sarkosyl-soluble and 1 % sarkosyl-insoluble fractions were collected via sequential protein extraction. Then, the total (K9JA antibody) and phosphorylated (AT8) tau protein levels of each fraction were measured by Western blot. The AT8 antibody recognizes phosphorylated tau at Ser202 and Thr205, which is one of the earliest phosphorylation events in Alzheimer’s disease (AD) pathogenesis. Both NHE6 KO clones displayed a statistically significant increase (~1 .5-fold) in the ratio of phosphorylated tau to total tau compared to the isogenic control line across all three fractions.

[0165] Next, total tau and phospho-tau levels in each fraction were measured without using a loading control via the Revert total protein staining (Licor) technology. Tau and phosphor-tau levels were significantly increased in both NHE6 KO clones compared to the isogenic control. It was also found that other phosphor-tau epitopes (e.g., Thr181 (p181) and Ser404 (p404)) were significantly increased in NHE6 KO clones as well. The analysis was extended to another aggregation-prone protein associated with neurodegeneration, a-synuclein. There was not any difference detected in both monomer and dimer forms of a-synuclein in 1 % sarkosyl-soluble fractions by Western blot. The analysis was then extended to iPSC-derivedneurons from a CS patient (c.1569G>A, (p.W523X)) and 2 CRISPR-corrected isogenic control lines that were previously published as a resource for the CS community (Ma, et al., 2021). As with our NHE6 KO lines, the CS iPSC-derived neuron line displayed elevated levels of the ratio of phosphorylated tau to total tau compared to both isogenic controls. These findings support the conclusion that loss of NHE6 leads to specific accumulation of tau, and not a-synuclein, in human iPSC neurons in vitro.

[0166] Amyloid precursor protein (APP), which is sequentially cleaved by 3- and y-secretases to generate Ap, is associated with AD pathogenesis. Notably, APP is cleaved following endocytosis in early endosomes in which membrane-anchored p-secretase is proteolytically active in the mildly acidic early endosome environment. This cleavage produces a C-terminal product (CTFP), which is then cleaved by y-secretase to release Ap into the endosomal lumen. Knockdown of NHE6 in non-neuronal cell lines leads to over-acidified endosomes and has been found to increase [3-secretase activity and Ap levels (Prasad & Rao, 2015). Therefore, APP in our NHE6 KO iPSC-derived neurons was investigated. No differences in full-length APP or APP CTFs in NHE6 KO cells was detected compared to control cells by Western blot. To measure p- and a-secretase cleavage of APP, APPsa and APPsp levels in conditioned media of iPSC-derived neurons was quantified by enzyme-linked immunoassay (ELISA). There was a small but significant increase in the ratio between APPsp to APPsa in NHE6 KO neurons. This finding suggests loss of NHE6 increases APP processing by p- secretase rather than a-secretase. Next, levels of Ap 37, 38, 40, and 42 were quantified. No differences were detected Ap peptide levels or their ratios in cell lysates or conditioned media. Therefore, loss of NHE6 slightly increases p-secretase-processed APP but does not increase Ap levels in iPSC-derived neurons.

[0167] Given the extensive findings of endolysosome dysfunction in NHE6-null rodent models (described below), it was sought to determine whether human NHE6 KO iPSC-derived neurons also exhibit similar endolysosome pathology. First, high-content image analysis was utilized using an IN Cell analyzer and Cell Profiler to assess the endocytic pathway. iPSC- derived neurons were stained with markers associated with early endosomes (EEA1), late endosomes (RAB7), and lysosomes (LAMP1).

[0168] NHE6 KO neurons had greater EEA1 puncta per cell and decreased average area of EEA1 puncta. NHE6 KO neurons had significantly greater average area of puncta for RAB7 compared to control cells. Although there was no difference in average area of LAMP1 puncta, there were significantly less Lysotracker puncta (a low pH dye used as a lysosomal marker) in NHE6 KO neurons. To measure lysosome function, the activity of the lysosomalenzyme cathepsin B was measured by a fluorescence readout whereby greater fluorescence reflects greater cathepsin B enzyme activity. NHE6 KO neurons displayed lower fluorescence, consistent with lower cathepsin B activity, compared to control neurons. Since degradation of intracellular material requires proper delivery of cargo to lysosomes via the autophagy pathway, levels of p62, a marker for autophagic degradation was measured. NHE6 KO neurons had significantly elevated p62 protein levels via Western blot, autophagic flux, which consists of autophagosome formation, maturation, fusion with lysosomes and degradation was then measured. Using a well-established assay, cells are treated with lysosome inhibitors and LC3II (autophagosome marker) protein levels are measured via Western blot to compare treated versus untreated LC3II levels. Neurons were treated with either bafilomycin A or chloroquine. NHE6 KO neurons exhibited reduced autophagic flux compared with control neurons in both bafilomycin A and chloroquine conditions. Thus, these findings suggest loss of NHE6 leads to impaired endolysosomal and autophagic functioning in iPSC-derived neurons.

[0169] This raised the question whether treating NHE6 KO iPSC-derived neurons with various treatments would ameliorate tau, endolysosomal, and / or autophagic dysfunction in these neurons. First, it was hypothesized trehalose, a non-reducing disaccharide that enhances autophagy in an mTOR-independent manner, would improve pathogenic tau accumulation. Trehalose has been shown to increase degradation of proteins associated with neurodegeneration such as tau. Trehalose treatment (50 and 100 mM) partially reduced the ratio of phosphorylated tau to total tau in NHE6 KO neurons compared to vehicle treated NHE6 KO neurons. Thus, trehalose treatment specifically reduced phosphorylated tau protein levels. Trehalose treatment also increased LC3II protein levels in NHE6 KO neurons. Since LC3II levels increased in NHE6 KO neurons treated with trehalose and chloroquine, this suggests trehalose promoted autophagic flux, resulting in an overall improvement of the autophagy pathway.

[0170] Next, iPSC-derived neurons were treated with another drug known to enhance autophagy via the mTOR pathway, rapamycin (1 pM). Like the trehalose treatment, NHE6 KO neurons treated with rapamycin reduced the ratio of phosphorylated tau to total tau in NHE6 KO neurons compared to vehicle treated NHE6 KO neurons. These therapeutic interventions in NHE6 KO iPSC-derived human neurons provide mechanistic insight into new drug targets for neurodegeneration in patients afflicted with CS.SUMMARY

[0171] We generated patient-derived induced pluripotent stem cells (iPSCs) from fiveCS families with different NHE6 mutations as well as genetically-related and isogenic controls (Lizarraga, et al., 2021 ; Ma, et al., 2021 ; Fernandez, et al., 2022). By creating these lines, we were able to test whether a range of treatments ameliorated a range of cellular CS phenotypes in human neurons. Importantly, we have shown treating iPSC-derived neurons from CS patients with loss-of-function mutation with full length NHE6 ( / .e., human NHE6 cDNA) rescues arborization defects as measured by Sholl analysis. Additionally, treating CS iPSC-derived neurons pharmacologically with BDNF or IGF-1 rescued arborization defects. We have also generated a range of non-neuronal CS cell lines including HAP1 and HEK293T (Lizarraga, et al., 2021).

[0172] To date, there is no treatment for Christianson syndrome. CS patients typically receive standard medical care consistent with individuals with severe ID and epilepsy. A range of anti-seizure medications have been prescribed to treat seizures in CS patients; however, no seizure treatment guidelines exist (Pescosolido, et al., 2014). There are a range of critical target symptoms including motor symptoms, cognitive symptoms as well as epilepsy (seizures). We have established animal models that have assays for all of these symptoms. Prior research has emphasized the role of the cerebellum in a gene therapy model (Figueroa, et al., 2022). However, we have substantial data that gene therapy targets are much broader than cerebellum alone. For example, we have evidence that corticospinal tract is involved in motor deficits. We have further evidence that hippocampus is involved in the seizure phenotype. Ultimate gene therapy treatment may require a combination of virus with distinct promoter systems.

[0173] Study 2: Preclinical CS Models of Christianson Syndrome - Mouse Model. In this present study, we created and used CS mouse models to illuminate cellular mechanisms underlying CS. Early postnatal neurodevelopment was investigated to identify mechanisms underlying CS (Ouyang, et al., 2013). In order to visualize NHE6 protein expression, a rabbit anti-NHE6 polyclonal antibody that reacts in human, mouse, and rat was developed and validated. Using this antibody, notable NHE6 protein staining was found in mice in growing axon tracts ( / .e., colocalizes with L1 staining) in the cortical plate, thalamus, and striatum of mice at embryonic day 15.5 (E15.5) as well as major fiber tracts (e.g., corpus callosum, anterior commissure, fimbria, and other fiber tracts of hippocampus) at postnatal day 0 (P0). Although present, NHE6 staining was less pronounced in earlier embryonic periods (E12.5) of embryonic development in the cortical plate, the ventral telencephalon, and medial ganglionic eminence. NHE6 was also found in glial cells ( / .e., colocalized with glial marker GFAP) in vitro.

[0174] In order to determine the subcellular distribution of NHE6, immunocytochemistry (ICC) in mouse primary hippocampal neurons was performed. NHE6 staining patterns werepunctate and predominantly localized to the prenuclear region near the Golgi apparatus (i.e., GM130 antibody) at 4 days in vitro (DIV). Additionally, NHE6 was found in both growing axons (phospho-Tau1) and dendrites (MAP2) at 9 DIV. NHE6 was further observed in in both pre- (SV2) and post-synapse (PSD95) in mature primary neurons at 21 DIV, and significantly enriched in the presynaptic region compared to the post-synapse (SV2: n=10 cells, PSD95: n=11 cells, p=0.014). NHE6 protein was localized to endosomes. Using a common pulse-chase protocol to label early endosomes, neurons were incubated with fluorescently-tagged transferrin for 10 minutes and stained with NHE6. About 80% of transferrin-positive early endosomes contained NHE6, while approximately 30% of NHE6 punctae contained fluorescent transferrin. Other endosome-associated markers were also used to more precisely characterize the distribution of NHE6 throughout the endocytic pathway. About 80% of Rab5-positive early endosomes overlapped with NHE6, while approximately 35% of NHE6 punctae colocalized with Rab5. These findings are consistent with our transferrin-early endosome experiment. About 85% of Rab11 -positive recycling endosomes overlapped with NHE6, while approximately 40% of NHE6 colocalized with Rab11. About 50% of Rab7-positive late endosomes overlapped with NHE6, while approximately 35% of NHE6 colocalized with Rab7.

[0175] Ouyang, et al., (2013) reported that neurons missing NHE6 exhibit impaired axon and dendrite branching. An NHE6-null mouse line was generated in which a LacZ-Neo cassette was inserted into exon 6 to inactivate the Nhe6 gene. To investigate neuronal morphology, mouse primary hippocampal neurons from NHE6-null and wild-type littermates were transfected with green fluorescent protein (GFP) and analyzed at 2 and 5 divisions (DIV). At both 2 and 5 DIV, NHE6-null neurons had significantly decreased number of primary dendrites and dendritic branch points. At 5 DIV, it was possible to discern axons from dendrites and it was found that NHE6-null neurons had reduced axon branch points. Neuronal morphogenesis was then investigated in vivo by examining mouse hippocampus and neocortex tissue. To visualize dendrites and axons in CA3 and CA1 hippocampal pyramidal neurons, Golgi-Cox staining at P21 was performed. NHE6-null mice had significantly less dendritic branching for both basal and apical dendrites. Cortical pyramidal axons (layer III) that connect with layer V had significantly less branch points in NHE6-null mice. Cortical deficits in dendrite branching (layer V) were also observed in NHE6-null mice expressing YFP with a Thy-1 promoter.

[0176] These neuronal morphology deficits were rescued by expressing full-length NHE6 (e.g., NHE6.0 transcript) in NHE6-null neurons. Mutant neurons transfected with GFP- only (i.e., transfection control) had a 50% reduction in axonal branching and 43% reduction in dendritic branching. Transfecting with full-length restored to wild-type levels. Importantly,transfecting mutant neurons with a functionally incompetent cation-exchanger NHE6 construct is unable to rescue neuron morphology deficits. This finding suggests NHE6’s function as an endosomal proton leak is critical for neuronal arborization.

[0177] To determine whether loss of NHE6 alters neuronal function, a set of electrophysiologic experiments were performed. This compared extracellular synaptic field potentials from acute hippocampal slides in NHE6-null and wild-type littermates. NHE6-null mice had approximately 23% less extracellular synaptic potential compared to wild-type mice. This finding could reflect either (1) decreased functional synapses or (2) less axon and / branch activation by presynaptic stimuli. To test these hypotheses, presynaptic function in acute hippocampal slices was assessed. By measuring paired-pulse facilitation, a form of short-term presynaptic plasticity, it was found that there were no differences between mutant and wild-type synapses. This finding suggests NHE6-null synapses are functional. To determine whether this is due to less axons, fiber volley amplitude while inhibiting AMPARs, which correlates to the amount of action potentials in stimulated axons was measured. NHE6-null fiber volley amplitude was significantly decreased by 25% compared to wild-type littermates. Taken together, these findings suggest loss of NHE6 leads to circuit defects likely due to impaired axonal and dendritic branching. Fewer axonal branching could potentially result in fewer synapses. Therefore, the number of synapses along dendrites was measured using the synaptic marker SV2. NHE6-null primary hippocampal neurons (10 DIV) had about 42% less SV2-positive presynapses per 10 |u.m of dendrites compared to wild-type neurons. These findings were further extended in vivo. CA1 hippocampal pyramidal neurons were stained using Golgi-Cox methods to label spines. NHE6-null neurons had significantly less dendritic spines per 10 .m compared to wildtype spines. It was also possible to compare the maturity of dendritic spines based on their morphology, whereby immature spines appeared like lines while mature spines were rounded. NHE6-null mice had fewer mature spines as well as greater immature spines in both apical and basal dendrites. Taken together, these in vitro and in vivo findings support the conclusion that loss of NHE6 reduces the number of synapses.

[0178] The major function of Na7H+exchangers is to move cations (e.g., Na+and K+) against their concentration gradient while leaking acidic protons (H+), which ultimately alkalinizes the endosomal lumen. Therefore, it was hypothesized that loss of NHE6 would cause neuronal endosomes to become over-acidified (i.e., lower pH). Endosomal lumen pH was quantified using multiple strategies.

[0179] First, primary hippocampal neurons were loaded with fluorescein-conjugated (pH-sensitive) and Alexa-Fluor- 546 (pH-insensitive) transferrin to calculate endosomal pH usinga fluorescent ratio intensity. Live cells were imaged after incubating with these transferrin constructs for 10 minutes to label early endosomes / the early endocytic pathway (WT n=57 cells, MUT=53 cells). Soma endosomal pH was 6.2 in wild-type and 5.8 in NHE6 mutant cells while endosomal pH in processes (e.g., dendrites / axons) was 7.2 in wild-type and 6.5 in mutant cells. This experiment reveals that loss of NHE6 hyper-acidifies the early endosomal lumen throughout the neuron (i.e., soma and processes). An alternative ratio metric pH measurement LysoSensor DND-160 was used to measure endosomal pH in primary hippocampal neurons fluorescently-tagged exchanger-deficient NHE6 construct. Endosomal pH in wild-type neurons with exchanger-deficient NHE6 was 6.57 and 5.88 in mutant neurons with exchanger-deficient NHE6. Finally, the amount of highly acidic endosomes and lysosomes was quantified using the low-pH dye LysoTracker DND-99 in primary hippocampal neurons (DIV 5). NHE6 mutant neurons had significantly more distal LysoTracker puncta compared to wild-type neurons. This result is interesting since acidic endosomes / lysosomes are usually only found near the soma and not in processes. In conclusion, these critical experiments show that loss of total NHE6 or a functional exchanger over-acidifies endosomes in neurons.

[0180] This raised the question whether loss of NHE6, that leads to hyper-acidified endosomes, further disrupts endosomal signaling mechanisms mediating neuronal arborization, such as the BDNF / TrkB pathway. When the BDNF ligand binds with the TrkB receptor on the plasma membrane, it is endocytosed wherein it triggers signaling pathways for arborization in signaling endosomes (Cosker, et al., 2008). First, it was found that TrkB receptors colocalized with endogenous NHE6 (i.e., our lab-generated NHE6 antibody) in the perinuclear regions as well as growing axons / dendrites in vitro. This raised the question whether precocious acidification of endosomes would lead to premature degradation of TrkB and, therefore, TrkB- dependent signaling. TrkB and phosphorylated Trk protein levels was first measured by Western blot following a 30-minute incubation of BDNF (50 ng / mL) in primary hippocampal neurons at 4 DIV. TrkB protein levels were reduced by 76.6% in NHE6-null neurons compared to wild-type controls and this reduction occurred for 2 hours post-BDNF treatment. Phosphorylated Trk (phospho-Trk) levels, a marker of Trk activity, was significantly reduced by about 48% in NHE6-null neurons compared to wild-type neurons throughout BDNF treatment. Taken together, these experiments suggest that loss of NHE6 alters TrkB protein levels and endosomal signaling, which likely mediate neuronal arborization deficits found in NHE6 mutants.

[0181] We investigated whether TrkB signaling was impaired due to TrkB degradation by treating primary neurons with leupeptin, a protease inhibitor that impairs degradation in the endocytic pathway. Leupeptin treatment rescued TrkB and phospho-Trk protein levels in NHE6-null cells. In fact, 30 minutes post-treatment, mutant TrkB levels were nearly identical to wildtype (95% of wild-type) and phospho-Trk levels were 128.5% of wild-type levels. Given these findings, it was concluded that impaired TrkB levels / signaling are mediated by increased protein degradation due to: (1) endosomal hyper-acidification; (2) early protease activity in acidified endosomes; and / or (3) enlargement of degradative lysosomes.

[0182] Next, we examined whether exogenous BDNF treatment would rescue neuronal arborization deficits in NHE6-null primary neurons. Primary hippocampal cultures were treated with BDNF (50 ng / mL) at 2 DIV and axonal / dendritic branching was measured at 5 DIV. Importantly, BDNF treatment boosted axonal and dendritic branching in NHE6-null neurons. BDNF-treated mutant neurons had significant increases in axonal branching (from 16.7 untreated to 27.7 treated), dendritic branching (from 20.3 to 34.4) and primary dendrites (from 6.5 to 8.7). Based on the findings from Ouyang, et al., (2013), the following model was proposed to explain the cellular pathobiology underlying impaired neuronal arborization in CS. Loss of NHE6 leads to the following cascade of cellular events: (1) over-acidification of the endosomal lumen pH: (2) premature degradation of TrkB and Trk-related signaling in endosomes; and (3) impaired neuronal arborization mediated by decreased Trk signaling.

[0183] The progressive and neurodegenerative features of NHE6-null mice were investigated in order to characterize the pathobiology of CS brain conditions (Xu, et al., 2018). NHE6-null mice were compared with wild-type littermates. Statistical analyses were performed as followed: group comparisons tested by Students’ t-test, longitudinal changes in brain measurements tested by two-way ANOVA followed by Tukey’s multiple comparison test, and cerebellar experiments tested by one-way ANOVA followed by Tukey’s multiple comparison test.

[0184] First, it brain volume trajectories were assessed across the lifespan by measuring brains at P0, 1- month, 2-months, 6 months and ~2-year-old male mice. Gross brain size was similar at the earlier developmental stages {e.g., P0 and 1 month). However, NHE6- null mice had significantly smaller whole brain volume compared to wild-type controls at later time points (e.g., 2-months, 6-months and 26-months). Specific brain regions also had a similar trajectory. While there were no differences early in development, the NHE6-null neocortex, cerebellum, and cerebellum + midbrain regions were significantly smaller compared to wild-type controls as they aged (e.g., 2-months, 6-months and 26-months). These postnatal brain trajectories are consistent with undergrowth as well as reduced cerebral volume during aging ( / .e., decrease in volume from 6 to 26-months).

[0185] Tissue volume of specific brain regions were then examined usingimmunohistological techniques in NHE6-null mice at 22 months old. Nissl staining revealed NHE6-null mice had significantly reduced tissue volume thickness in the cortex, striatum, hippocampus, cerebellum, and spinal cord compared to control male littermates. In order to better characterize the onset and trajectory of brain volume, our immunohistochemical examination was extended to 2-month old mice. A significant reduction in tissue volume of 2-month old NHE6-null mice was observed in the cerebellum compared to littermate controls. Two-way ANOVA with Tukey’s multiple comparison tests were employed to statistically analyze brain volume differences between genotype and age. There was a significant effect of both genotype ( / .e., NHE6-null) and age across all brain regions. Linear regression analysis of the slope ( / .e., rate of change from 2 to 22 months) found significantly lower brain area in the striatum, hippocampus, and cerebellum, but not the cortex. Thus, we conclude distinct trajectories of different brain regions. For example, the cortex, striatum and hippocampus reflect some undergrowth while the cerebellum had a notable downward slope consistent with degeneration.

[0186] Given the cerebellar pathology found in OS, the temporal and anatomical patterns of Purkinje cell (PC) loss was characterized in two different NHE6-null mouse lines. Hematoxylin and eosin (H&E) staining of midsagittal cerebellar sections (vermal primary fissure) in 5- and 11-13-month-old male mice revealed NHE6-null mice had significantly decreased PC density and calbindin signal (a PC marker) compared to control male littermates at both 5 and 11-13 months. Further, 11-13-month-old NHE6-null mice had significantly decreased PC density and calbindin signal compared to 5-month-old NHE6-null mice. PC density analysis was then extended to other cerebellar regions other than the primary fissure to include the anterior and flocculonodular lobes at the same time points. NHE6-null mice had significantly less PC density in the primary fissure and anterior lobe, but not in the flocculonodular lobe at both 5 and 11-13 months compared to control male littermates. PC density was only affected in the vermis in NHE6-null mice, while the periphery was largely unaffected.

[0187] While NHE6-null mouse studies to date have utilized the mutant model that introduces a lacZ / Neo cassette into exon 6 that produces a stop codon after the protonexchanger domain, another NHE6-null line that inserts a lacZ / Neo cassette into exon 2 / 3 that produces a stop codon before the proton-exchanger domain was generated. It was confirmed that this NHE6-null mouse line leads to loss of NHE6 mRNA expression (PCR) and NHE6 protein (Western blot). These NHE6-null male mice also exhibit significantly decreased PC density in the primary fissure and anterior lobe, but not the flocculonodular lobe at 6 months of age. Hemizygous males and homozygous female NHE6-null (exon 2 / 3) mice had significantlydecreased PC density and calbindin signal at 5 months compared to sex-matched control littermates. Taken together, loss of NHE6 in multiple CS mouse models leads to PC loss that affects certain cerebellar regions and worsens over time.

[0188] Since neurodegenerative features have been reported in CS patients, cellular markers of neurodegeneration in NHE6-null mice (exon 6) such as neuroinflammatory mechanisms were investigated. Using immunohistochemical techniques, 22-month-old NHE6- null mice with markers of astrocytes (GFAP) and microglia (I ba 1 ) were labelled. NHE6-null mice displayed enhanced GFAP and Iba1 staining throughout the brain, particularly in major axonal tracts like the corpus collosum. NHE6-null mice exhibited significantly greater Iba1-positive cells in the cortex, striatum, and hippocampus compared to wildtype littermates. NHE6-null mice also show a heightened microglial response, as well as GFAP staining, in cells that morphologically resemble Bergmann glia in the molecular layer of the cerebellum as well as the spinal cord. Microglial activity was then examined by staining for CD68, a lysosome marker that labels actively phagocytic microglia. NHE6-null exhibited enhanced CD68 staining in the corpus collosum, cortex, striatum, hippocampus, and cerebellum. Evidence was also found of gliosis in other axonal tracts such as the anterior commissure, medial septum, and spinal cord via CD68 staining. Taken together, these findings are consistent with neurodegenerative pathogenesis in aging NHE6-null mice involving innate immunity featuring microglia.

[0189] We investigated how endolysosomal mechanisms may lead to neurodegeneration in NHE6-null mice (Pescosolido, et al., 2021). NHE6-null male mice were compared to wild-type male littermates. In vitro experiments typically included key developmental time points like axonal outgrowth (3 DIV), dendritic outgrowth (5 DIV), and synaptic maturation (14 DIV). Statistical analysis for two group comparisons were performed by Student’s t-test (data normally distributed) or Mann-Whitney U test (data not normally distributed).

[0190] The degradative functioning in lysosomes in vitro was first examined using confocal microscopy. NHE6-null and wild-type male neurons were treated with DQ-BSA ( / .e., BSA conjugated with fluorophores that fluoresce when degraded) overnight to measure how much endocytosed material is degraded. NHE6-null neurons had significantly less mean fluorescence intensity per cell throughout all time points ( / .e., 3, 5, and 14 DIV). Importantly, there were no differences in the amount of fluorescent BSA endocytosed / internalized between NHE6-null and wild-type neurons in any of these time points. These findings are consistent with impaired lysosomal proteolysis. Hydrolases are enzymes that carry out degradation in the lysosome. The lysosome enzyme cathepsin D (CatD), which is associated withneurodegenerative disorders was analyzed. First, active cathepsin D was visualized using the BODIPY FL pepstatin A probe that fluoresces when the CatD-inhibitor pepstatin A binds to active CatD. While there were no differences at 3 DIV, NHE6-null neurons displayed significantly less BODIPY-pepstatin A mean fluorescence intensity as well as the number of puncta per cell compared to wild-type neurons at both 5 and 14 DIV.

[0191] Next, the amount of active and inactive CatD protein was measured in hippocampal tissue at 8 weeks old using Western blot analysis. While there were no differences in inactive pro-CatD, NHE6-null mice exhibited significantly less active cleaved-CatD. This analysis of lysosome enzymes was extended to -NAG and acid phosphatase in 8-week old mouse brain tissue (e.g., cerebellum, cortex, and hippocampus) and primary hippocampal neurons at 14 DIV. [3-NAG activity was significantly decreased in NHE6-null hippocampal tissue and primary hippocampal neurons compared to wild-type male littermates, whereas acid phosphatase was unaffected. These differences in lysosome enzymes may be due to their intracellular trafficking routes. Many newly-synthesized enzymes, including CatD and [3-NAG, are trafficked from the trans-Golgi network to the endocytic pathway by binding to mannose-6- phosphate receptors (M6PRs). On the other hand, acid phosphatase is trafficked in a M6PR- independent pathway. Therefore, loss of NHE6 leads to impaired proteolysis and lysosome enzyme activity like CatD and p-NAG, which is trafficked in a M6PR-dependent pathway.

[0192] Since it was previously shown that NHE6-null neurons exhibit endosomal overacidification in vitro (Ouyang, et al., 2013), pH analysis was extended to the lysosome lumen. A ratio-metric fluorescence microscopy technique using dextrans to primary neurons was adapted. Specifically, primary hippocampal neurons (8 DIV) were treated with pH-sensitive (OG-488) and pH-insensitive (TMR) dextran and chased overnight to ensure proper trafficking to lysosomes. The fluorescence ratio, which was measured by a high-content imaging system, was converted to pH levels via a calibration curve. NHE6-null neurons had significantly lower lysosomal pH ( / .e., more acidic) in both the soma and processes. As a positive control, we treated primary neurons with bafilomycin A1 that alkalinizes the lysosomal lumen. It was confirmed that confirmed lysosomal lumen pH increased ( / .e., less acidic) in both NHE6-null and wild-type neurons. Finally, the endosome pH findings were corroborated using this method as NHE6-null neurons had hyper-acidified endosomes in both the soma and processes.In summary, it was demonstrated that loss of NHE6 over-acidifies the lysosomal lumen.

[0193] Newly synthesized CatD is transported in an enzymatically inactive form ( / .e., pro-CatD) from the Golgi complex to endosomes. Once delivered to lysosomes, CatD is converted to its active form ( / .e., cleaved-CatD) by the highly acidic lysosomal lumen. Since itwas found NHE6-null neurons have deficits in enzymatically active CatD, we examined whether this was caused by impaired trafficking and / or distribution of CatD in the endocytic pathway. It was hypothesized that loss of NHE6 in neurons would become prematurely active in endosomes due to our previous findings of endosomal hyper-acidification. Therefore, the colocalization of active CatD was measured by staining with BODIPY FL pepstatin A with endosomal and lysosomal markers. First, primary neurons were treated at 5 and 14 DIV with fluorescent dextran overnight to label lysosomes. NHE6-null neurons displayed significantly less CatD-dextran colocalization at both 5 and 14 DIV compared to wild-type littermates. The degree of colocalization with another lysosome-associated marker, LAMP1 was then measured. Although it should be noted that LAMP1 can also label endosomes. NHE6-null neurons also exhibited significantly less active CatD-LAMP1 colocalization compared to wild-type littermate controls. Taken together, these results suggest that NHE6-null neurons have less active CatD in lysosomes.

[0194] Next, colocalization with the late endosome marker, RAB7 was measured. NHE6-null neurons had significantly greater active CatD-RAB7 colocalization compared to wildtype littermate controls. Other late (LBPA) and early (RAB5) endosome markers had greater active CatD colocalization in NHE6-null neurons at 5 DIV, but not at 14 DIV, compared to wildtype controls. Therefore, loss of NHE6 leads to premature CatD activation in endosomes, likely due to endosomal over-acidification. To further corroborate the confocal microscopy data that CatD is less active in lysosomes in NHE6 neurons, lysosome-enriched fractions were collected from 4-month-old NHE6-null mouse brain tissue (neocortex and hippocampus). CatD protein levels (pro and cleaved forms) were measured by Western blot, which were normalized to the protein level of the lysosome marker LAMP1. NHE6-null mice had significantly less inactive and active CatD protein levels in lysosome-enriched fractions compared to wild-type littermates.

[0195] To understand how the loss of NHE6 affects the endocytic pathway, endolysosomal features were investigated using molecular biology and confocal microscopy techniques. We measured protein levels via Western blot in both mouse hippocampal tissue (8-weeks old) and primary hippocampal neurons (14 DIV). 3D volumetric reconstruction of confocal microscopy images primary neurons (5 and 14 DIV) were also performed using Imaris 5.1 software (Bitplane). Neurons were labeled with endolysosomal markers to measure the following cellular features: number of puncta per cell, average puncta volume per cell, and total puncta volume per cell. To more precisely label lysosomes, primary neurons were treated with dextran overnight like previously described experiments. Compared to wild-type littermate neurons, NHE6-null neurons had larger average dextran puncta volume at 14 DIV. Lysosomeswere also labeled with a LAMP1 antibody. There were no differences in LAMP1 protein levels as measured by Western blot. Confocal microscopy revealed NHE6-null neurons have more LAMP1 puncta per cell and greater total puncta volume per cell compared to wild-type littermate neurons at 5 DIV. However, at 14 DIV, NHE6-null neurons have less puncta and larger average puncta volume compared to control neurons. Taken together, these lysosome marker findings are consistent with lysosome dysfunction as lysosomes with impaired degradation can become enlarged as they accumulate excessive amounts of materials for degradation. Late endosomes were then labeled with a RAB7 antibody. NHE6-null neurons had significantly larger RAB7 average puncta volume per cell and greater total puncta volume per cell at 5 DIV compared to wild-type littermate neurons. Early endosomes were labeled with a RAB5 antibody yet no significant differences between NHE6-null and littermate controls were detected. Finally, M6PRs were labeled with a M6PR antibody. NHE6-null neurons had significantly greater total M6PR puncta volume per cell at 14 DIV compared to control neurons. Thus, the loss of NHE6 in neurons led to altered endolysosomal composition.

[0196] Since these findings support altered activity of M6PR-dependent lysosomal enzymes, the steady-state distribution of M6PRs in vitro were investigated using confocal microscopy. M6PRs alternate between the trans-Golgi network (TGN) and the endocytic pathway, and back to the TGN by the retromer complex prior to reaching lysosomes. First, M6P6-TGN colocalization was measured using a TGN46 antibody. NHE6-null neurons at both 5 and 14 DIV had significantly less colocalization compared to wild-type control littermates. Alternatively, NHE6-null neurons exhibited significantly greater M6PR-RAB7 and M6PR-LAMP1 colocalization at both 5 and 14 DIV. Additionally, NHE6-null neurons displayed greater M6PR- RAB5 colocalization at 5 DIV, but not 14 DIV. Therefore, these findings suggest that loss of NHE6 in neurons disrupts M6PR trafficking, likely by impairing retrograde trafficking of M6PRs from endosomes to the TGN.

[0197] Loss of the NHE6 homolog in yeast, Nhx1 , impairs late endosome fusion with the yeast equivalent of lysosomes, vacuoles (Karim & Brett, 2018). Therefore, a microscopy method was developed to measure endosome-lysosome fusion in vitro. Primary hippocampal neurons at 4 DIV were incubated with TMR-dextran overnight to traffic to lysosomes. The next day, at 5 DIV, neurons were incubated with another fluorescent dextran, AlexaFluor-647-dextran, for 10 minutes to be internalized into neurons via endocytosis. time-lapse images were then collected every 20 minutes over a 2-hour span to visualize endosome-lysosome fusion events. It was found that NHE6-null neurons exhibited significantly less endosome-lysosome fusion ( / .e., AlexaFluor-647-dextran colocalization with lysosomal TMR-dextran) across all time pointscompared to wild-type littermate controls. As a positive control, primary neurons were treated with bafilomycin A, which impairs endosome-lysosome fusion. As expected, bafilomycin treatment led to less endosome-lysosome fusion in both wild-type and NHE6-null neurons. Thus, this demonstrates, for the first time in mammalian cells, that loss of NHE6 diminishes endosome-lysosome fusion.

[0198] The loss of NHE6 disrupts endosome to lysosome trafficking raised the question whether late endosomes were more likely to fuse with the plasma membrane (PM) and release their contents extracellularly, which are referred to as exosomes. A CD63-pHluorin construct was used that specifically visualizes individual late endosome fusion with the plasma membrane / exosome release events by total internal reflection fluorescence (TIRF) microscopy. Live-imaging of primary hippocampal neurons at 14 DIV revealed NHE6-null neurons had significantly greater late endosome-PM fusion / exosome release compared to wild-type controls. As a positive control, wild-type hippocampal neurons treated with bafilomycin A1 increased late endosome-PM fusion / exosome release compared to untreated wild-type neurons. CD63 protein levels in primary neurons were also measured at 14 DIV. NHE6-null neurons had significantly less CD63 compared to wild-type neurons. This may be due to greater extracellular release of CD63 in NHE6-null neurons. This raised the question whether loss of NHE6 led to greater lysosome fusion with the PM, which is known as lysosomal exocytosis. The activity of lysosomal enzymes P-Hex and CatD collected in neuronal media was measured. As a positive control, primary neurons were treated with ionomycin, a calcium ionophore, that increases lysosomal exocytosis. No differences between control and NHE6-null neurons in extracellularly released P-Hex and CatD were detected, although ionomycin treatment did increase activity in both genotypes.

[0199] Based on these results, the following model of endolysosomal dysfunction in NHE6-null neurons is proposed (Pescosolido, et al., 2021). The primary effect caused by the loss of NHE6 is the over-acidification of endosomal compartments that ultimately leads to lysosome dysfunction. This is largely mediated by impaired endosome maturation and trafficking of the endocytic pathway. Endosome-lysosome fusion is disrupted, leading to inefficient delivery of cargo required for proper lysosome functioning as well as endocytic cargo for degradation. M6PR-dependent trafficking, a key intracellular pathway for delivering inactive lysosomal hydrolases from the TGN to endosomes, is also obstructed. Specifically, M6PRs accumulate on late endosomes and are unable to be trafficked back to the TGN in order to replenish the endosomes with hydrolases. Further, lysosomal hydrolases are prematurely active in hyperacidified endosomes and, concomitantly, less active in lysosomes due to impaired endosome-lysosome fusion. While late endosomes exhibit delayed fusion with lysosomes, they show enhanced fusion with the plasma membrane and release of their contents extracellularly which are known as exosomes. Our study provides the most comprehensive understanding of how loss of NHE6 affects the endolysosomal system in the nervous system and proposes novel mechanistic insight into how NHE6 dysfunction may lead to neurodegeneration.SUMMARY

[0200] We developed a range of preclinical CS models to dissect CS pathobiology, such as generating multiple NHE6 mouse models (Ouyang, et al., 2013; Xu, et al., 2018; Ouyang, et al., 2019). Using these mouse models, we have found loss of NHE6 causes both neurodevelopmental and neurodegenerative pathology. For example, NHE6-null mice exhibit developmental defects including impaired neuronal arborization, reduced synapse number, and decreased mature spines (Ouyang, et al., 2013). Treating NHE6-null neurons with exogenous brain-derived neurotrophic factor (BDNF), a secretory growth factor, rescued these branching defects (Ouyang, et al., 2013). NHE6-null mice also display features consistent with neurodegeneration like brain volume loss, cerebellar degeneration, and glial activation (Xu, et al., 2018).

[0201] Mainly using primary neurons from these models, we have identified cell pathology that likely underlie CS pathology. Loss of NHE6 leads to over-acidification of endosomes and lysosomes, consistent with NHE6’s role regulating luminal pH (Ouyang, et al., 2013, Pescosolido, et al., 2021). Hyper-acidification of the endocytic pathway in NHE6-null neurons disrupts endosome-lysosome trafficking that ultimately leads to lysosome dysfunction (Pescosolido, et al., 2021). Specifically, lysosome enzymes, such as cathepsin D, are prematurely active in over-acidified endosomes and less likely to be delivered to the lysosome. These cellular pathologies are also observed in some lysosomal storage disorders (LSDs), which exhibit overlapping neurological symptoms with CS.

[0202] Study 3: Preclinical CS Models of Christianson Syndrome - Rat Model. An NHE6-null rat model was generated on a Sprague-Dawley background using CRISPR / Cas9 technology (Lee, et al., 2022). Cas9-mediated cleavage of Slc9a6 at exon 7 led to a 2 bp (TT) insertion, which caused a frameshift and premature stop codon. Guide RNA (5 -CGGCTGTGTAACCCTGATGA-3', SEQ ID NO: 5) was microinjected into the pronucleus. The rat Slc9a6 gene is found on the X chromosome and has 18 exons with ATG initiation codons at exons 1 , 3, and 4. Therefore, exon 7 was targeted to impair expression of all isoforms. Two independent, mosaic male founders were generated for establishing our NHE6rat colony. Wild-type and knock-out status was confirmed at DNA, mRNA and protein levels. Genomic DNA sequencing from tail biopsy confirmed (1) wild-type and (2) null 2-bp insertion sequences, and germ line transmission was successful. Next, it was confirmed that mutant mRNA brain expression was decreased due to nonsense mediated decay by quantitative realtime PCR. Finally, it was confirmed NHE6-null rat brains did not express the NHE6 protein by immunoprecipitation and immunohistochemistry. Therefore, there is confidence that this CRISPR-Cas9 generated NHE6 rat model is a valid model for studying CS.

[0203] After confirming the validity of this genetically engineered CS rat model, the neurodegenerative gross pathology as well as the cellular neuropathogenic cascade that leads to neurodegeneration was characterized. All experiments used male NHE6-null and wild-type littermates. Given CS patients exhibit postnatal microcephaly, CS rat brain morphology was measured throughout the lifespan. The anterior-posterior (A-P) axis of wild-type and NHE6-null littermates was compared at 3, 9, and 12 months of age. Across all time points, NHE6-null A-P’s were significantly reduced compared to wild-type littermates. Further, NHE6-null A-P at 12-months was less than at 9-months, suggestive of neurodegeneration. The size of the cerebral cortex (CTX) and cerebellum was further investigated. NHE6-null CTX was significantly reduced at 12-months while the cerebellar area was reduced across 3, 9 and 12-months. In summary, the data is consistent with neurodegeneration that affects the cerebellum earlier than the cerebral cortex.

[0204] Cerebellar pathology in sagittal cerebellar sections at 2-months was then further examined. There was significantly less calbindin signal, a marker for Purkinje cells, in NHE6-null rats compared to wild-type littermates. Pathogenic GM2 ganglioside accumulation, which has previously been reported in NHE6-null mice (Stromme, et al., 2011) was also measured. NHE6- null rats had much greater GM2 ganglioside staining than wild-type littermates, and GM2 colocalized with the PC marker calbindin. Additionally, it was found that NHE6-null rats had greater amounts of p62 and ubiquitin, consistent with deficits in autophagy. Decreased calbindin and increased GM ganglioside staining were found as early as 1-month in NHE6-null rats, but no significant differences were detected for p62 and ubiquitin. These findings are consistent with lysosome dysfunction preceding autophagy dysfunction in NHE6-null cerebellar pathology. Next, cerebellar degeneration at 12-months was investigated using immunohistochemistry techniques. Bielschowsky’s silver staining revealed decreased staining in NHE6-null rats, consistent with axonal loss. Nissl staining revealed decreased staining of Purkinje cell bodies in NHE6-null rats, likely reflecting Purkinje cell degeneration. Haematoxylin and eosin (H&E) staining revealed gliosis as well as multifocal vacuolization in the molecular layer (paramedianlobule), consistent with endolysosomal pathology.

[0205] While the cerebellum displays hastened neurodegeneration, the NHE6-null rat cerebrum exhibits a prolonged neurodegenerative timeline allowing for extensive characterization of the neuropathological cascade. First, GM2 ganglioside accumulation in the hippocampus and basolateral amygdala (BLA), which was previously reported in a CS mouse model (Stromme, et al., 2011) was quantified. Coronal brain sections encompassing the hippocampal (e.g., CA1 and CA3) and BLA regions, were stained for GM2 and NeuN (neuronal marker) at 3-months. While GM2 ganglioside staining was mostly absent in wild-type males, NHE6-null males had significantly greater GM2 accumulation at 3-months in the CA1 , CA3, and BLA regions. GM2 accumulation was largely confined to neurons ( / .e., GM2 colocalizing with NeuN). This finding was also observed at 18-months in CA1 and CA3, and GM2 accumulation was significantly greater in NHE6-null rats at 18-months relative to 3-months. Similar findings were observed in the lysosomal marker Lampl in the CA1 at 3 and 18-months. Next, autophagic dysfunction in the hippocampus and neocortex was investigated. No differences were detected in p62 and ubiquitin between NHE6-null and wild-type rat brains at 3-months. NHE6-null rats demonstrated increased p62, ubiquitin and LC3 staining at 18-months in the hippocampus and neocortex. Taken together, these findings support the conclusion that lysosomal dysfunction predates autophagic dysfunction in NHE6-null rat brains.

[0206] The dynamics of neuronal loss and axonal pathology in NHE6-null rat cerebrum was investigated. Rat brain sections were Nissl stained. Neuronal loss, atrophy, and enlarged lateral ventricles were noted in the hippocampus, piriform / entorhinal CTX, and amygdala at 12-months. H&E staining of the hippocampus (CA3) at 12-months revealed multifocal vacuolizations and less neurons in the NHE6-null rat. In order to resolve whether the observation that NHE6-null rats have fewer neurons reflects a neurodevelopmental phenotype or progressive loss, brain section with NeuN at 3 and 12-months was stained. There were no significant differences in the number of CA1 NeuN+cells between NHE6-null and wild-type littermates (n=10 litters) at 3-months. However, NHE6-null rats had significantly less NeuN+cells compared to wild-type littermates at 12-months (n=10 litters). Therefore, these results are consistent with loss of NHE6 leading to progressive neuronal loss. Next, axonal pathology was examined by Bielschowsky’s silver staining in the CTX and corpus callosum (CC, i.e., axonal tracts). At 12-months, NHE6-null rats had significantly less silver staining in the CTX and CC compared to wild-type littermates consistent with loss of axonal tracts. There were no indications of neurofibrils and senile plaques. Additionally, H&E staining uncovered multifocal vacuolizations in white matter (e.g., CC) and grey matter surrounding major fiber tracks inNHE6-null rats, which were not found in wild-type littermates.

[0207] The above-described finding of neuronal pathology in NHE6-null rats raised the question whether this also extended to non-neuronal cells in the brain such as astrocytes and microglia. Gliosis and glial activation are hallmarks of neurodegeneration, and it has been previously reported these glial processes in NHE6-null mice (Xu, et al., 2018). Therefore, the onset and location of gliosis and glial activation in NHE6-null rats was examined. Specifically, this raised the question whether glial pathology occurred before or after other pathologic events (e g., lysosome dysfunction and neuronal loss). Cerebellar sections were stained with IBA1 and GFAP antibodies. We did not observe any differences in either marker were observed at 1-month. However, NHE6-null rats had significantly greater IBA1 and GFAP staining in NHE6- null rats at 2-months compared to both wild-type littermates at 2-months and NHE6-null rats at 1-month. At 3-months, NHE6-null rats had significantly greater IBA1 and GFAP staining in the cerebrum (CA1 , CA3, CC and piriform cortex) compared to wild-type littermates. Thus, glial pathology is a relatively early neurodegenerative feature in NHE6-null rats along with lysosome dysfunction ( / .e., compared to autophagic dysfunction and neuronal loss).

[0208] Tau pathology has been reported post-mortem in brain examination of CS patients in their 40s-50s (Garbern, et al., 2010). It has been previously reported that aging CS female carriers diagnosed with neurodegenerative disorders associated with tau deposition (Pescosolido, et al., 2019). It was further found that decreased NHE6 expression is correlated with tau accumulation in post-mortem human brains from two large aging studies (Pescosolido, et al., 2019). Since NHE6-null mice do not recapitulate this tau pathology, it is not possible to study tau pathogenesis in the CS mouse model. Therefore, it was investigated whether tau pathology was present in the NHE6-null rat. Tau accumulation was measured by sequentially extracted brain tissue at 3 and 18-months. Sequential extraction involved the collection of: TBS- soluble, TBS-insoluble Sarkosyl-soluble, and Sarkosyl-insoluble fractions. Western blot analysis measured the following protein markers: AT8 (phosphorylated tau at serine 202 and 205) and TAU5 (phosphorylated and non-phosphorylated tau).

[0209] NHE6-null samples had significantly greater phosphorylated tau levels relative to total tau ( / .e., AT8 levels / TAU5 levels) in Sarkosyl-insoluble fractions at 18-months compared to wild-type samples at 18 months as well as NHE6-null samples at 3 months. There were no differences in phosphorylated tau levels in TBS-soluble and Sarkosyl-insoluble fractions (although increased phosphorylated tau in NHE6-null samples at 18-months were trending toward significance, p=0.07). Next, tau inclusions throughout the brain were quantified in NHE6- null rats at 3, 12, and 18-months. To visualize phosphorylated tau, staining was done with PHF1and AT8 antibodies. NHE6-null rats exhibited increased AT8 staining in the hippocampus, CTX, corpus callosum, and substantia nigra at 18-months. Further, AT8-positive inclusions were present in glia (i.e., GFAP-positive astrocytes) in the CC. These inclusions were not found in wild-type littermates. Also, NHE6-null rats at 18-months displayed PHF1 staining in NeuN- positive cells throughout the brain, such as the substantia nigra. No or low signal was detected in wild-type littermates. Brain sections were further stained for ThioflavinS (ThioS), a marker for 0-pleated sheets within aggregating proteins like tau / amyloid, at 18-months. NHE6-null rats displayed a statistically significant increase in ThioS staining in the hippocampus (CA1), CC, and substantia nigra compared to wild-type littermates. In summary, aging NHE6-null rats demonstrate a robust, widespread tau phenotype that has not been observed in other preclinical CS models.

[0210] Although amyloid-0 plaque were not found in a post-mortem brain examination in two CS patients (Garbern, et al., 2010), hyper-acidification of the endocytic pathway is speculated to enhance 0-secretase, which is a critical stage in amyloid-0 generation (Ouyang, et al., 2013; Prasad & Rao 2015). Therefore, amyloid- accumulation in both CS mouse and rat brains was investigated using ELISA assays for multiple amyloid-0 forms. NHE6-null mice at 24-months exhibited a statistically significant 6% increase in total amyloid-0 ( / .e., sum of amyloid-042, amyloid-040, and amyloid-038), which is largely driven by amyloid-040. On the other hand, there were no differences in total amyloid-0 in rats at 18-months. However, amyloid-0 species (e.g., total, amyloid-042, amyloid-040, and amyloid-0 42 / 40 ratio) were significantly increased in NHE6-null rats at 18-months in GuHCI soluble fractions. Pathogenic amyloid-0 accumulation was then visualized using well-known amyloid-0 antibodies - clones 6E10 (extracellular marker) and OC (oligomeric amyloid fibril marker) - at 18-months. NHE6- null rats displayed significantly greater signal of both amyloid-0 antibodies in the CC and CTX compared to wild-type rats. These findings suggest that loss of NHE6 can mediate amyloid-0 pathology, which is reported for the first time in a mammalian CS model in vivo.

[0211] CS patients display gross motor abnormalities such as ataxia. This raised the question whether our CS rat model also exhibits motor dysfunction. First, we assessed motor exploration using the open field test at P20 and P54 by measuring movement and time spent in the arena using beams breaks for 30-minutes. Beam breaks were detected by horizontal and vertical infrared beams to quantify horizontal / vertical movements, total distance travelled, and time spent in center. No differences in motor function were detected at P20. Yet NHE6-null rats exhibited decreased horizontal and vertical activity, less total distance travelled, and decreased time spent in the center compared to wild-type littermates at P54. Next, gross motor behaviorwas assessed using the rotorod assay in rats at 1 and 2-months. While there were no differences in performance at 1 month, 2-month old NHE6-null rats had a lower latency to fall (in seconds) compared to wild-type littermate rats, consistent with greater motor dysfunctional. Importantly, NHE6-null rats could not be tested on the rotorod due to an inability to balance on the rod. Motor learning was at 2-months also measured using the rotorod by testing the latency to fall across three successive days. Over these three days, wild-type rats increased their latency to fall time, reflecting an ability to learn this task and improve their performance. However, NHE6-null rats’ latency to fall was significantly reduced compared to wild-type rats across all three days. Therefore, these results suggest that motor learning is impaired in NHE6- null rats. Gait performance was then assessed using DigiGait whereby rats were put on a treadmill with a transparent belt and a video on their ventral side records their stride and gait at 2, 3 and 4-months. A hindlimb ataxia coefficient was calculated based on stride variability (MAX stride length - MIN stride length / MEAN stride length). NHE6-null rats had a greater ataxia coefficient, reflecting ataxia, at 4-months compared to wild-type littermates as well NHE6-null rats at 2- months. These findings are consistent with ataxic behaviors that progressively worsen over time, which is reported in CS patients. Finally, there were no differences in forelimb or hindlimb grip strength at 1 and 2-months indicating that motor dysfunction is unlikely due to grip strength.

[0212] In conclusion, this is the first CS rat model to study NHE6-associated pathology (Lee, et al., 2022). Our CS rat model faithfully recapitulates key CS features that are not as prominent in CS mouse models such as severe motor dysfunction and ataxia. The cerebellum exhibits cellular pathology before the cerebrum. It was also found that lysosomal and glial dysfunction are early pathologic feature in the NHE6-null rat brain. Autophagy deficits occur much later, suggesting they are mediated by the primary lysosome dysfunction. Importantly, it is shown that NHE6-null rats exhibit abnormal endogenous tau accumulation using two tau antibodies that recognize phosphorylated tau. Tau accumulated in both neuron and glia throughout the brain such as CC, hippocampus, and substantia nigra. Thus, this CS rat provides a novel model for studying CS neurodegeneration as well as test novel therapeutics.SUMMARY

[0213] In this study, we created an NHE6-null rat model that recapitulates many of the CS pathobiology features observed in our CS mouse model, while also providing a more robust preclinical model to study neurodegeneration (Lee, et al., 2022). Consistent with our prior findings in mice, NHE6-null rats exhibit progressive cerebellar atrophy, Purkinje cell loss, andlysosome dysfunction. Notably, our NHE6-null rat represents a superior animal model to study neurodegeneration in CS since they exhibit tau and amyloid-[3 pathology, as well as increased astrogliosis and microgliosis. Severe motor dysfunction, a prominent feature in CS, was observed in young NHE6-null rats ( / .e., ~2 months old).MATERIALS AND METHODS FOR STUDIES 1 & 2

[0214] Animals: A CS mouse model ( / .e. NHE6-null) and wildtype male mice were used for rAAV expression studies (Ouyang, et al., 2013). A CS rat model was used for rAAV expression (AAV9-U1A-NHE6) as well as a motor behavior study. The Morrow lab used CRISPR-Cas9 technology to generate NHE6-null rats on a Sprague-Dawley background (Lee, et al., 2022). Animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Brown University.

[0215] Vectors: The following V5-tagged hNHE6 rAAV vectors were used for expression and efficacy studies: AAV9-EF1a-hNHE6 (FIG. 2A), AAV9-U1A-hNHE6 (FIG. 2D), AAV9-CAG- hNHE6 (FIG. 2E), AAV9-SYN1-hNHE6 (FIG. 2F), and AAV9-CaMKII-hNHE6 (FIG. 2G).

[0216] rAAV Administration: Bilateral ICV injections were performed on CS and WT rodents according to the protocols reported in Taylor, et al., (2021) and Donsante, et al., (2016). Rodents received 1.5x10A9 GC / kg of rAAV hNHE6 vectors (4 ul total, 2 ul per ventricle).

[0217] rAAV Expression: Rodents were injected via ICV with hNHE6 rAAVs at 7-8 weeks old, except for AAV9-CAG-hNHE6 which was performed at 10 weeks old. Brains were harvested at 1 -month post-injection for immunofluorescence analysis, except for the rat administered the AAV9-U1A-hNHE6 vector which was harvested at 2-months post-injection.

[0218] Immunofluorescence & Confocal Microscopy: Immunofluorescence analysis was performed according to previously published methods by the Morrow lab (Lee, et al., 2022, Ouyang, et al., 2013, Xu, et al., 2018). The following antibodies were used: DAPI (nuclei), NHE6 antibody, V5 (to tag hNHE6 from the AAV9 construct), NeuN (neuron-specific nuclear marker), GFAP (astrocytes), and IBA1 (microglia). Brain sections were imaged by an Olympus FV3000 microscope using a 20X and 60X objectives.

[0219] Treatment: CS and male WT littermate rats were injected via ICV with rAAVs at 7-8 weeks old. CS and WT rats were assigned to one of the following groups: NHE6 gene (AAV9-EF1a-hNHE6), a GFP control vector (AAV9-EF1a-eGFP), or sham surgery ( / .e. mice undergoing same surgery for AAV delivery without injecting AAV vectors).

[0220] Open Field Test: Rats were examined using a battery of motor assays adapted from Lee, et al., (2022). Each rat was placed in an open field arena (80x80 cm). ANY-mazesoftware (Stoelting Inc. USA) was used for the tracking and video acquisition for 15 minutes. Ethovision XT50 software was used for analyzing the spontaneous locomotor activity as total distance travelled was recorded in meters (m). The open field test was performed monthly through 8 months post-injection. Baseline measurements were recorded 1-week prior to rAAV administration ( / .e. 6-7 weeks old). Each treatment group had between 4-10 rats.

[0221] Statistical Analysis: Data are reported as mean±SEM. Statistical analyses were performed using GraphPad. Two-way ANOVA with repeated measures was used for analysis of time course data. All genotypes / treatment groups were compared with NHE6-null mice treated with the GFP control vector ( / .e. AAV9-EF1a-eGFP). To correct for multiple comparisons, Dunnett’s test was performed. Asterisks represent p values as follows: *p < 0.05, **p < 0.01 , ***p < 0.001.NHE6

[0222] In some embodiments, the methods described herein treat diseases or disorders caused by NHE6 mutations. In some embodiments, an rAAV vector can comprise an NHE6 coding sequence for SLC9A6.1, SLC9A6.2, or SLC9A 6.3 transcript (FIG. 1). In some embodiments, NHE6 is encoded by the SLC9A6.1 nucleic acid sequence of SEQ ID NO:1. In some embodiments, a nucleic acid described herein comprises the nucleic acid sequence of SEQ ID NO: 1 , or a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, NHE6 is human NHE6.

[0223] In some embodiments an NHE6 polypeptide described herein consists of an amino acid sequence SEQ ID NO: 2, or an amino acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one portion of the amino acid sequence put forth in SEQ ID NO: 2, or a fragment thereof.

[0224] In some embodiments, NHE6 may be encoded by a codon-optimized nucleic acid sequence. In some embodiments, the codon-optimized sequence comprises the nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the codon- optimized sequence comprises the nucleic acid sequence of SEQ ID NO: 4, or a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the codon-optimized nucleic acid sequence encoding an NHE6 polypeptide, such as those listed in SEQ ID NO. 3 and 4, can have a GC content that differs from the GC content of the WT human nucleic acid sequence encoding the NHE6 polypeptide. In some embodiments, the optimized nucleic acid sequence encoding an NHE6 polypeptide,such as those listed in SEQ ID NO. 3 and 4, exhibits at least 5%, at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 500%, or at least 1000% increased expression in a human subject relative to wildtype or noncodon-optimized nucleic acid sequence encoding an NHE6 polypeptide.ITR SEQUENCES

[0225] In some embodiments, an rAAV vector can comprise an AAV inverted terminal repeat (ITR) sequence. In some embodiments, an rAAV vector can comprise two AAV ITR sequences which flank an NHE6 coding sequence. In some embodiments, the AAV ITR is an AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, or AAVrg. In some embodiments, the AAV ITR is derived from AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, or AAVrg. In some embodiments, the AAV ITR sequence is truncated. In some embodiments, the AAV ITR sequence is mutated relative to the wild-type sequence.PROMOTERS

[0226] In some embodiments, an rAAV vector can comprise a promoter operably linked to the NHE6 coding sequence. In some embodiments, the promoter is a human promoter. Nonlimiting examples of promoter sequences include, but are not limited to, an EF1a promoter, a U1A promoter, a CAG promoter, a ChAT promoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin 1 promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter.

[0227] In some embodiments, an rAAV can comprise a polyadenylation (polyA) sequence. Non-limiting examples of polyA sequences include, but are not limited to, a bovine growth hormone (bGH) polyA sequence, a human growth hormone (hGH) polyA sequence, a rabbit globin polyA sequence, a beta globin polyA sequence, or an SV40 polyA sequence.

[0228] In some embodiments, an rAAV can comprise a hybrid or chimeric intron, e.g. an intron having sequences from two or more sources.

[0229] In some embodiments, an rAAV can comprise a post-translational regulatory element. A non-limiting example of a post-translational regulatory element include, but are not limited, to Woodchuck Post-transcriptional Regulatory Element (WPRE).

[0230] In some embodiments, an rAAV vector can comprise a protein tag. Without wishing to be bound by theory, inclusion of a protein tag can permit detection and visualization of the exogenous NHE6 polypeptide. Non-limiting examples include, but are not limited to, HA, V5, HIS, or a combination thereof. In some embodiments, the NHE6 rAAV vector lacks a tag.Tags are removed from therapeutic vectors. In some embodiments, the NHE6 rAAV vector lacks an HA tag. In some embodiments, the NHE6 rAAV vector lacks a V5 tag. In some embodiments, the NHE6 rAAV vector lacks a HIS tag.AAV VIRAL VECTORS

[0231] In some embodiments, the isolated polypeptides containing the nucleic acid molecule described herein can be an rAAV viral vector. These rAAV viral vectors can be used to treat Christianson syndrome.

[0232] AAV capsid proteins: In some embodiments, a nucleic acid described herein is packaged in an rAAV viral vector comprising a capsid protein. In some embodiments, the AAV capsid protein is an AAV9 capsid protein. The AAV9 capsid targets delivery to the central nervous system. In some embodiments, the AAV capsid protein is an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh capsid protein, or an AAVrg capsid protein. In some embodiments, the AAV capsid protein has tropism to the central nervous system (CNS) and / or the nervous system. In some embodiments, the capsid protein has a wild-type capsid protein sequence. In some embodiments, the capsid protein comprises mutations relative to the wildtype capsid protein sequence, but is capable of forming a capsid. The AAV capsid proteins described herein include the wild-type sequence and mutants (e.g., functional mutants) thereof.METHODS OF USE

[0233] The present disclosure provides the methods of preventing and treating a disorder consisting of administering to a subject a therapeutically effective amount of one of the rAAV vectors disclosed herein. In some embodiments, the subject can be a human, a mouse, a rat, a pig, a dog, a cat, or a non-human primate. In some embodiments, the rAAV viral vector is targeted to neurons, the nervous system, and / or other affected organs. In some embodiments, the rAVV viral vector targets a host cell. In some embodiments, the host cell is in vitro, in vivo, or ex vivo.

[0234] In some embodiments, an rAAV viral vector is used in a method of treating Christianson syndrome. In some embodiments, an rAAV viral vector is used in a method of treating a disease or disorder caused by a mutation in NHE6. A mutation in an NHE6 gene can be any mutation that is known in the art. Non-limiting examples include, but are not limited to, single nucleotide variants (SNVs), nonsense mutations, insertions, deletions, duplications,frameshift mutations, repeat expansions, insertions and deletions (INDELs), alternative splicing, alternative initiation of translation, and proteomic cleavage. In some embodiments, a disease can be a disease characterized by a loss-of-function NHE6 mutation in the genome of the subject. In some embodiments, a disease can be a disease characterized by a dominantnegative NHE6 mutation in the genome of the subject. In some embodiments, a disease can be a disease characterized by a gain-of-function NHE6 mutation in the genome of the subject.

[0235] In some embodiments, a disease can be a disease that is characterized by a decrease in expression of the NHE6 gene in a subject as compared to a control subject that does not have the disease. In some embodiments, the decrease in expression can be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%.

[0236] In some embodiments, treatment with the rAAV viral vector described herein alleviates and / or ameliorates one or more symptom associated with Christianson syndrome. CS symptoms can include, but are not limited to, intellectual disability / developmental delay, nonverbal status, microcephaly, epilepsy / seizures, ataxia / motor dysfunction, hyperkinesia, high pain tolerance, autism, unprovoked laughter, contractures, visual acuity problems, eye movement abnormalities (e.g. strabismus), low body weight, swallowing problems, gastroesophageal reflux disease (GERD), constipation, osteopenia, regressions (e.g. social, eating, speech, motor), cerebellar atrophy, and neurodegenerative features such as neuronal / glial loss, neuroinflammation, and pathogenic tau deposition. In some embodiments, treatment with the rAAV viral vector described herein can prevent or delay CS-associated symptoms.

[0237] In some embodiments, standard AAV titers are administered. In some embodiments, the rAAV viral vector or pharmaceutical composition of the present disclosure can be administered to a subject at a dose ranging from about 1010to about 1019viral vector particles, e.g., about 101°, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, or 1019viral particles.

[0238] In some embodiments, rAAV viral vectors or pharmaceutical compositions are administered using the following methods: intraparenchymal (IPa), intrathecal (IT), intracerebroventricular (ICV), intravenous (IV), intranasally, orally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intranervally, intrapleurally, or topically. In some embodiments, rAAV viral vectors or pharmaceutical compositions are administered using methods that cross the blood-brain barrier (BBB).

[0239] In a discussion, study or a reading of the details, features, embodiments, aspects, any figure or any part of any figure, and / or examples of the technology disclosed herein, any of the features, embodiments, aspects, and / or examples herein can be optionally inter-combined (or inter-discussed) with the example details listed below, and any portion (or aspect) of any detail below can be inter-combined with any portion of any feature or example disclosed herein:

[0240] Detail 1 : A method for the treatment of Christianson syndrome in a subject in need thereof, the method comprising: administering to the subject a viral vector comprising a nucleic acid sequence encoding NHE6 operably linked to a promoter, wherein the viral vector is enclosed in a capsid from an AAV serotype or variant, and wherein the subject is diagnosed with Christianson syndrome based on the presence of a mutation in the SLC9A6 gene encoding NHE6.

[0241] Detail 2: The method of detail 1 , wherein the subject is a human male, as Christianson syndrome is an X-linked disorder primarily affecting males, and / or wherein the subject is further diagnosed based on the presence of one or more symptoms of Christianson syndrome, such as intellectual disability, developmental delay, seizures, ataxia, autistic behaviors, hyperactivity, aggression, and sleep disturbances.

[0242] Detail 3: The method of detail 1 , wherein the nucleic acid sequence encoding NHE6 is a human NHE6 sequence, and wherein the sequence encodes a full-length, functional NHE6 protein, and / or wherein the sequence is codon-optimized for expression in human cells.

[0243] Detail 4: The method of detail 1 , wherein the promoter is a tissue-specific promoter, selected to drive expression of NHE6 in the tissues most affected by Christianson syndrome, such as the brain, spinal cord, and peripheral nerves, and / or wherein the promoter is a constitutive promoter, such as the CMV or EF1a promoter, to drive high levels of NHE6 expression in all transduced cells.

[0244] Detail 5: The method of detail 4, wherein the tissue-specific promoter is a brainspecific promoter, such as the human synapsin 1 promoter or the human glial fibrillary acidic protein (GFAP) promoter, to target expression of NHE6 to neurons or glial cells in the brain, and / or wherein the promoter is a neuronal-specific promoter, such as the human neuronspecific enolase (NSE) promoter or the human platelet-derived growth factor p-chain (PDGF-P) promoter.

[0245] Detail 6: The method of detail 1 , wherein the AAV serotype is selected from the group consisting of AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, and AAV13, based on their ability to efficiently transduce cells of the central nervoussystem, and / or wherein the AAV serotype is selected based on its ability to transduce specific cell types in the brain, such as neurons (AAV2, AAV9), astrocytes (AAV5, AAV8), or oligodendrocytes (AAV1 , AAV6).

[0246] Detail 7: The method of detail 1 , wherein the AAV variant is selected from the group consisting of AAVPHP.B, AAVrh, and AAVrg, based on their enhanced ability to cross the blood-brain barrier and transduce cells in the brain, and / or wherein the AAV variant is selected based on its reduced immunogenicity compared to wild-type AAV serotypes.

[0247] Detail 8: The method of detail 1 , wherein the viral vector further comprises a first nucleic acid sequence comprising a first AAV inverted terminal repeat (ITR) and a second nucleic acid sequence comprising a second AAV ITR, which are required for AAV vector packaging and replication, and / or wherein the viral vector further comprises a post- transcriptional regulatory element, such as a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), to enhance the stability and expression of the NHE6 transgene.

[0248] Detail 9: The method of detail 8, wherein the first nucleic acid sequence and the second nucleic acid sequence flank the nucleic acid sequence encoding NHE6, to ensure proper packaging of the NHE6 transgene into the AAV vector, and / or wherein the nucleic acid sequence encoding NHE6 is inserted in a single-stranded or self-complementary AAV vector genome.

[0249] Detail 10: The method of detail 1 , wherein the viral vector is administered systemically, to allow widespread distribution of the vector throughout the body and brain, and / or wherein the viral vector is administered locally to the brain, such as by stereotactic injection into specific brain regions affected by Christianson syndrome.

[0250] Detail 11 : The method of detail 10, wherein the viral vector is administered intravenously, as a minimally invasive route of administration that allows efficient delivery of the vector to the brain, and / or wherein the viral vector is administered through the carotid artery or vertebral artery, to preferentially target the vector to the brain while minimizing systemic exposure.

[0251] Detail 12: The method of detail 1 , wherein the viral vector is administered directly to the central nervous system, to achieve high levels of transduction in the brain and spinal cord while minimizing systemic exposure, and / or wherein the viral vector is administered by intracranial injection, to deliver the vector directly to the brain parenchyma.

[0252] Detail 13: The method of detail 12, wherein the viral vector is administered intracerebroventricularly or intrathecally, to deliver the vector directly into the cerebrospinal fluid surrounding the brain and spinal cord, and / or wherein the viral vector is administered by lumbarpuncture or cisternal puncture, to deliver the vector into the subarachnoid space.

[0253] Detail 14: The method of detail 1 , wherein the viral vector is administered in a single dose, to provide long-term expression of NHE6 following a single treatment, and / or wherein the viral vector is administered as a single injection or infusion.

[0254] Detail 15: The method of detail 1 , wherein the viral vector is administered in multiple doses, to achieve higher levels of NHE6 expression or to provide repeated treatments over time, and / or wherein the viral vector is administered as multiple injections or infusions, either at the same site or at different sites in the brain or spinal cord.

[0255] Detail 16: The method of detail 1 , wherein the viral vector is administered at a dose of about 1x10A11 to about 1x10A16 vector genomes per kilogram of body weight, which is expected to result in therapeutically relevant levels of NHE6 expression based on preclinical studies in animal models, and / or wherein the viral vector dose is adjusted based on the age, weight, or body surface area of the subject.

[0256] Detail 17: The method of detail 1 , wherein administration of the viral vector results in expression of NHE6 in the brain of the subject, particularly in regions that are most affected by Christianson syndrome, such as the hippocampus, cerebral cortex, and cerebellum, and / or wherein administration of the viral vector results in expression of NHE6 in the spinal cord and peripheral nerves of the subject.

[0257] Detail 18: The method of detail 17, wherein expression of NHE6 in the brain of the subject reduces or ameliorates one or more symptoms of Christianson syndrome, by restoring the proper function of endosomes and lysosomes in neurons and glial cells, and / or wherein expression of NHE6 in the brain of the subject improves the subject's cognitive function, behavior, motor skills, and / or quality of life.

[0258] Detail 19: The method of detail 18, wherein the one or more symptoms of Christianson syndrome are selected from the group consisting of intellectual disability, developmental delay, seizures, ataxia, autistic behaviors, hyperactivity, aggression, and sleep disturbances, which are the most common and debilitating features of the disorder, and / or wherein the one or more symptoms of Christianson syndrome are measured using standardized tests or scales, such as the Bayley Scales of Infant and Toddler Development, the Vineland Adaptive Behavior Scales, the Aberrant Behavior Checklist, or the Quality of Life Inventory- Disability.

[0259] Detail 20: A method for the treatment of Christianson syndrome, the method comprising: administering to a subject in need thereof a viral vector comprising a nucleic acid sequence encoding NHE6 operably linked to a promoter and flanked by AAV inverted terminalrepeats, wherein the viral vector is enclosed in a capsid from an AAV serotype or variant, and wherein administration of the viral vector results in expression of NHE6 in the brain of the subject, thereby reducing or ameliorating one or more symptoms of Christianson syndrome, and wherein the subject is diagnosed with Christianson syndrome based on the presence of a mutation in the SLC9A6 gene, and wherein the one or more symptoms of Christianson syndrome are selected from the group consisting of intellectual disability, developmental delay, seizures, ataxia, autistic behaviors, hyperactivity, aggression, and sleep disturbances.

[0260] Detail 21 : The method of detail 1 , wherein the subject is administered an immunosuppressive agent prior to, concurrently with, or following administration of the viral vector, to prevent or reduce an immune response against the viral vector or the expressed NHE6 protein, and / or wherein the immunosuppressive agent is administered systemically or locally to the brain.

[0261] Detail 22: The method of detail 21 , wherein the immunosuppressive agent is selected from the group consisting of corticosteroids, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin, and / or wherein the immunosuppressive agent is administered orally, intravenously, intramuscularly, or intrathecally.

[0262] Detail 23: The method of detail 1 , wherein the viral vector is administered in combination with one or more additional therapeutic agents for the treatment of Christianson syndrome or its associated symptoms, such as antiepileptic drugs, antipsychotics, or behavioral therapies, and / or wherein the one or more additional therapeutic agents are administered before, during, or after administration of the viral vector.

[0263] Detail 24: The method of detail 23, wherein the one or more additional therapeutic agents are selected from the group consisting of valproic acid, levetiracetam, lamotrigine, risperidone, aripiprazole, and clonidine, and / or wherein the one or more additional therapeutic agents are administered orally, intravenously, intramuscularly, or intrathecally.

[0264] Detail 25: The method of detail 1 , wherein the effectiveness of the treatment is monitored by assessing the subject's cognitive function, behavior, seizure frequency, motor skills, or quality of life before and after administration of the viral vector, and / or wherein the effectiveness of the treatment is monitored using neuroimaging techniques, such as magnetic resonance imaging (MRI) or positron emission tomography (PET), to assess changes in brain structure or function.

[0265] Detail 26: The method of detail 25, wherein the assessment is performed using standardized tests or scales, such as the Bayley Scales of Infant and Toddler Development, the Vineland Adaptive Behavior Scales, the Aberrant Behavior Checklist, or the Quality of LifeInventory-Disability, and / or wherein the assessment is performed at multiple time points following administration of the viral vector, such as 1 month, 3 months, 6 months, and 12 months post-treatment.

[0266] Detail 27: The method of detail 1 , wherein the safety of the treatment is monitored by assessing the subject for adverse events, such as injection site reactions, immune responses, or off-target effects of NHE6 expression, and / or wherein the safety of the treatment is monitored by assessing the subject for signs of toxicity or inflammation in the brain or other organs.

[0267] Detail 28: The method of detail 27, wherein the assessment for adverse events includes clinical evaluations, laboratory tests, imaging studies, or histological analyses of tissue biopsies, and / or wherein the assessment for adverse events is performed at multiple time points following administration of the viral vector, such as 1 week, 1 month, 3 months, and 6 months post-treatment.

[0268] Detail 29: The method of detail 1 , wherein the viral vector is manufactured under Good Manufacturing Practices (GMP) conditions to ensure its quality, safety, and consistency for clinical use, and / or wherein the viral vector is purified and formulated for administration to humans.

[0269] Detail 30: The method of detail 29, wherein the GMP conditions include strict control of the manufacturing process, extensive quality control testing, and detailed documentation of all steps and materials used, and / or wherein the viral vector is tested for sterility, purity, potency, and absence of adventitious agents prior to administration to the subject.

[0270] Detail 31 : A method for the treatment of Christianson syndrome in a subject in need thereof, the method comprising administering to the subject a recombinant adeno- associated virus (rAAV) vector comprising a nucleic acid, wherein the nucleic acid comprises: a first nucleic acid sequence comprising a first AAV inverted terminal repeat (1st ITR); a promoter operably linked to a nucleic acid sequence encoding NHE6; and a second nucleic acid sequence comprising a second AAV inverted terminal repeat (2nd ITR); wherein the viral vector is enclosed in a capsid comprising an AAV capsid protein selected from the group consisting of AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, and AAVrg capsid proteins, and wherein the AAV capsid protein is selected based on its tropism for brain tissue and ability to cross the blood-brain barrier.

[0271] Detail 32: The method of detail 31 , wherein the rAAV vector further comprises a promoter sequence including an EF1a promoter, a U1A promoter, a CAG promoter, a ChATpromoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter, wherein the promoter sequence is selected based on its ability to drive high levels of NHE6 expression in the brain and its specificity for neurons, glial cells, or other cell types affected by Christianson syndrome.

[0272] Detail 33: The method of detail 31 , wherein the promoter is a tissue-specific promoter that is selected to target NHE6 expression to specific cell types in the brain affected by Christianson syndrome, such as neurons in the hippocampus, cortex, or cerebellum.

[0273] Detail 34: The method of detail 33, wherein the tissue-specific promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain, and is chosen based on its strength, specificity, and ability to drive long-term expression of NHE6.

[0274] Detail 35: The method of detail 31 , wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression, as well as purification and characterization of the expressed protein.

[0275] Detail 36: The method of detail 31 , wherein the rAAV vector is administered by intravenous injection, intramuscular injection, intracranial injection, intrathecal injection, intranasal administration, intraparenchymal (I Pa) administration, intracerebral (IC) administration, or intracerebroventricular (ICV) administration, wherein the route of administration is selected based on its ability to deliver the rAAV vector to the brain regions affected by Christianson syndrome, while minimizing systemic exposure and off-target effects.

[0276] Detail 37: The method of detail 31 , wherein the subject is a human patient diagnosed with Christianson syndrome based on clinical symptoms and genetic testing, and who has a confirmed mutation in the NHE6 gene.

[0277] Detail 38: The method of detail 31 , wherein the subject has a mutation in the NHE6 gene that is known to cause Christianson syndrome, such as a missense, nonsense, splice site, or frameshift mutation, and wherein the specific mutation is identified by genetic sequencing of the patient's NHE6 gene.

[0278] Detail 39: The method of detail 31 , wherein administering the rAAV vector results in expression of functional NHE6 protein in the brain of the subject, as demonstrated by immunohistochemistry, Western blotting, or other protein detection methods, and wherein the expressed NHE6 protein is properly localized to the endosomal compartment and restoresnormal endosomal pH and function.

[0279] Detail 40: The method of detail 31 , wherein administering the rAAV vector improves one or more symptoms of Christianson syndrome in the subject, such as seizures, intellectual disability, ataxia, or autistic behaviors, as measured by standardized clinical assessments, and wherein the improvement is sustained over time and correlates with the level of NHE6 expression in the brain.

[0280] Detail 41 : The method of detail 31 , further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells by replacing rare codons with more frequently used codons, while avoiding sequence motifs that may impair gene expression or stability.

[0281] Detail 42: The method of detail 35, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells by replacing rare codons with more frequently used codons, while avoiding sequence motifs that may impair gene expression or stability, and wherein the HA or V5 / His tag is also codon-optimized for efficient translation.

[0282] Detail 43: The method of detail 31 , further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics.

[0283] Detail 44: The method of detail 35, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics, and wherein the HA or V5 / His tag is also optimized for the chosen NHE6 sequence.

[0284] Detail 45: The method of detail 31 , wherein the rAAV vector is administered at a dose of between 1e11 and 1e16 vector genomes per kilogram of body weight, and wherein thedose is determined based on the patient's age, weight, and disease severity, as well as the specific rAAV vector and route of administration used.

[0285] Detail 46: The method of detail 31 , wherein the rAAV vector is administered in a single dose or in multiple doses over time, and wherein the dosing schedule is determined based on the patient's response to treatment, as well as the duration and stability of NHE6 expression in the brain.

[0286] Detail 47: The method of detail 31 , further comprising administering an immunosuppressive agent before, during, or after administration of the rAAV vector, wherein the immunosuppressive agent is selected from the group consisting of corticosteroids, cyclosporine, tacrolimus, mycophenolate mofetil, and rapamycin, and wherein the choice and dose of immunosuppressive agent is based on the patient's immune status and risk of developing anti- AAV antibodies.

[0287] Detail 48: The method of detail 31 , further comprising monitoring the patient for adverse events and clinical outcomes after administration of the rAAV vector, wherein the monitoring includes regular assessments of neurological function, cognitive development, and quality of life, as well as laboratory tests for immune responses and vector persistence.

[0288] Detail 49: A recombinant adeno-associated virus (rAAV) vector for the treatment of Christianson syndrome, the rAAV vector comprising a nucleic acid, wherein the nucleic acid comprises: a first nucleic acid sequence comprising a first AAV inverted terminal repeat (1st ITR); a promoter operably linked to a nucleic acid sequence encoding NHE6; and a second nucleic acid sequence comprising a second AAV inverted terminal repeat (2nd ITR); wherein the rAAV vector is enclosed in a capsid comprising an AAV capsid protein selected from the group consisting of AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, and AAVrg capsid proteins, and wherein the AAV capsid protein is selected based on its tropism for brain tissue and ability to cross the blood-brain barrier.

[0289] Detail 50: The rAAV vector of detail 49, wherein the rAAV vector further comprises a promoter sequence including an EF1a promoter, a U1A promoter, a CAG promoter, a ChAT promoter, a cytomegalovirus (CMV) promoter, a hybrid chicken beta-actin promoter, a ubiquitous chicken actin hybrid (CBh) promoter, a synapsin promoter, an MeCP2 promoter, a CaMKII promoter, or an L7 promoter, wherein the promoter sequence is selected based on its ability to drive high levels of NHE6 expression in the brain and its specificity for neurons, glial cells, or other cell types affected by Christianson syndrome.

[0290] Detail 51 : The rAAV vector of detail 49, wherein the promoter is a tissue-specificpromoter that is selected to target NHE6 expression to specific cell types in the brain affected by Christianson syndrome, such as neurons in the hippocampus, cortex, or cerebellum.

[0291] Detail 52: The rAAV vector of detail 51 , wherein the tissue-specific promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain, and is chosen based on its strength, specificity, and ability to drive long-term expression of NHE6.

[0292] Detail 53: The rAAV vector of detail 49, wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression, as well as purification and characterization of the expressed protein.

[0293] Detail 54: The rAAV vector of detail 49, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells by replacing rare codons with more frequently used codons, while avoiding sequence motifs that may impair gene expression or stability.

[0294] Detail 55: The rAAV vector of detail 53, further comprising a nucleic acid sequence encoding NHE6 that corresponds to a codon-optimized SLC9A6.1 RNA transcript, wherein the codon optimization is designed to improve NHE6 expression in human cells by replacing rare codons with more frequently used codons, while avoiding sequence motifs that may impair gene expression or stability, and wherein the HA or V5 / His tag is also codon- optimized for efficient translation.

[0295] Detail 56: The rAAV vector of detail 49, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics.

[0296] Detail 57: The rAAV vector of detail 53, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered humanNHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics, and wherein the HA or V5 / His tag is also optimized for the chosen NHE6 sequence.

[0297] Detail 58: The rAAV vector of detail 49, wherein the rAAV vector is produced using a triple transfection method in HEK293 cells, and wherein the rAAV vector is purified by column chromatography and formulated in a sterile, isotonic buffer for in vivo administration.

[0298] Detail 59: The rAAV vector of detail 49, wherein the rAAV vector is characterized by a high titer of at least 1e13 vector genomes per milliliter, a low empty capsid ratio of less than 10%, and a high purity of at least 90% as measured by silver staining or other analytical methods.

[0299] Detail 60: A pharmaceutical composition for the treatment of Christianson syndrome, the pharmaceutical composition comprising the rAAV vector of detail 49 and a pharmaceutically acceptable carrier, wherein the carrier is selected based on the route of administration and the desired pharmacokinetic profile of the rAAV vector.

[0300] Detail 61 : The pharmaceutical composition of detail 60, wherein the pharmaceutically acceptable carrier is suitable for intravenous injection, intramuscular injection, intracranial injection, intrathecal injection, intranasal administration, intraparenchymal (IPa) administration, intracerebral (IC) administration, or intracerebroventricular (ICV) administration, wherein the carrier is selected based on its ability to stabilize the rAAV vector and facilitate delivery to the brain, while minimizing systemic exposure and off-target effects.

[0301] Detail 62: The pharmaceutical composition of detail 60, wherein the AAV capsid protein is an AAV9 capsid protein, which has been shown to efficiently cross the blood-brain barrier and transduce neurons and glial cells in the brain, and which has a favorable safety profile in humans.

[0302] Detail 63: The pharmaceutical composition of detail 60, wherein the promoter is a brain-specific promoter that is selected from promoters known to be active in neurons, glial cells, or other cell types in the brain affected by Christianson syndrome, and which has been optimized for strong and sustained expression of NHE6 in the target cells.

[0303] Detail 64: The pharmaceutical composition of detail 60, wherein the nucleic acid sequence encoding NHE6 comprises a nucleic acid sequence encoding a human NHE6 protein, and wherein the nucleic acid sequence further encodes an HA or V5 / His sequence, such that the human NHE6 protein produced thereby is tagged with HA or V5 / His to facilitate detection and quantification of NHE6 expression in the brain after administration, as well as purificationand characterization of the expressed protein.

[0304] Detail 65: The pharmaceutical composition of detail 60, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 2 is a wild-type human NHE6 protein sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics.

[0305] Detail 66: The pharmaceutical composition of detail 64, further comprising a nucleic acid sequence encoding NHE6 selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, wherein SEQ ID NO: 1 is a wild-type human NHE6 sequence, SEQ ID NO: 2 is a wild-type human NHE6 protein sequence, SEQ ID NO: 3 is a codon-optimized human NHE6 sequence, and SEQ ID NO: 4 is an engineered human NHE6 sequence with enhanced stability and activity, and wherein the choice of sequence is based on the desired level and duration of NHE6 expression, as well as the specific patient population and disease characteristics, and wherein the HA or V5 / His tag is also optimized for the chosen NHE6 sequence.

[0306] Detail 67: The pharmaceutical composition of detail 60, wherein the pharmaceutical composition is formulated as a sterile, isotonic solution with a pH between 7.0 and 8.0, and wherein the composition is stable for at least 6 months when stored at -80°C and for at least 24 hours when thawed and stored at room temperature.

[0307] Detail 68: The pharmaceutical composition of detail 60, wherein the pharmaceutical composition is packaged in a single-use vial or syringe, and wherein the vial or syringe is labeled with the lot number, expiration date, and storage conditions of the composition, as well as the dose and route of administration for the intended patient population.

[0308] Detail 69: The pharmaceutical composition of detail 60, wherein the pharmaceutical composition is administered at a dose of between 1e11 and 1e16 vector genomes per kilogram of body weight, and wherein the dose is determined based on the patient's age, weight, and disease severity, as well as the specific rAAV vector and route of administration used.

[0309] Detail 70: The pharmaceutical composition of detail 60, wherein the pharmaceutical composition is administered in a single dose or in multiple doses over time, and wherein the dosing schedule is determined based on the patient's response to treatment, aswell as the duration and stability of NHE6 expression in the brain, and wherein the composition is administered in combination with an immunosuppressive agent to minimize the risk of immune-mediated toxicity or loss of efficacy.

[0310] The detailed description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0311] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0312] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.EXAMPLES

[0313] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention.EXAMPLE 1. CS GENE THERAPY EXPRESSES EXOGENOUS HUMAN NHE6 PROTEIN IN CS RODENTMODELS IN VIVO

[0314] The following non-limiting example demonstrates administration of hNHE6 rAAVs results in exogenous expression of hNHE6 protein in the brains of CS rodent models.

[0315] rAAV constructs were developed comprising the AAV9 capsid, human NHE6 nucleic acid sequence (SEQ ID NO.1), V5 / His tag, and one of a range of promoters (FIG. 2A- H). To determine whether these rAAV constructs successfully expressed NHE6 in vivo, these constructs were tagged with V5 / His. rAAV constructs were generated with different promoters for ubiquitous delivery (e.g., EF1a, U1A, and CAG promoters), neuronal delivery (e.g., Synapsinl and CaMKII promoters), and cerebellar delivery (L7).

[0316] In these experiments, rodents were administered hNHE6 rAAVs via bilateral ICV injections (1.5x10A9 GC / kg) at 7-8 weeks old (except for AAV9-CAG-hNHE6 which was injected at 10 weeks old). These NHE6 rAAVs contained a V5-His tag to visualize expression of the human NHE6 construct. Brain expression was examined at 1-2 months post-injection (PI) using immunohistochemistry and brain slices were imaged by confocal microscopy. Specifically, fluorescence intensity and distribution of V5 was used as a readout for human NHE6 expression. The Morrow lab developed an NHE6 antibody (Ouyang, et al., 2013) to further visualize NHE6 expression. Exogenous human NHE6 was present in the following NHE6 rAAVs: AAV9-EF1a-hNHE6-V5 (FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D), AAV9-U1A-hNHE6-V5 (FIG. 4), AAV9-CAG-hNHE6-V5 (FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D), AAV9-SYN-hNHE6-V5 (FIGs. 6A-6D), and AAV9-CaMKII-hNHE6-V5 (FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D). Human NHE6 expression was enriched in brain tissue near injections sites. The NHE6 rAAV with the neuron-specific synapsin 1 promoter (AAV9-SYN-hNHE6-V5) expressed exogenous hNHE6 in the perinuclear region of neurons (NeuN, i.e. neuronal nuclear marker) and had minimal, if any, colocalization with astrocytes (GFAP) and microglia (I BA1 ) (FIGs. 6A-6D). These results demonstrate the SYN-NHE6-AAV targets NHE6 expression to neuronal cells in the CNS.

[0317] Taken together, these results demonstrate that administration of an rAAV viral vector comprising a nucleic acid encoding human NHE6 protein effectively expresses NHE6 in rodent brains. Therefore, these proof-of-principle results suggest AAV-based therapy is feasible for CS.EXAMPLE 2. CS GENE THERAPY AMELIORATES MOTOR DYSFUNCTION IN CS RAT MODEL

[0318] The following non-limiting example demonstrates administration of hNHE6 rAAVs can effectively ameliorate motor abnormalities in a CS rat model.

[0319] CS rats recapitulate motor abnormalities observed in CS (Lee, et al.,2022). Specifically, CS rats exhibit motor behavior deficits around 2 months of age. To test motor activity, the open field test was performed. CS rats exhibit significantly less distance travelled in the open field at P54, but not P20 compared to WT male littermates (Lee, et al., 2022). CS rats treated with AAV9-EF1a-hNHE6 had significantly increased motor activity, as measured by total distance travelled, compared to CS rats receiving a control AAV (i.e. AAV9- EF1a-eGFP) from 1- to 8-months PI (FIG. 8). There were no significant differences between CS rats receiving a control AAV and a sham surgery. The data provided herein demonstrate that it is possible to deliver enough therapeutic NHE6 to improve a CS-associated symptom (e.g. motor activity) in a rat CS model. Further, these results show NHE6 gene therapy treatment can ameliorate a CS-associated symptom in the long-term (i.e. through 8 months PI).EXAMPLE 3. EXAMPLE SEQUENCES

[0320] LENGTH: 2103; TYPE: DNA; ORGANISM: Homo sapiens; NHE6; “SEQUENCE: 1”SEQ ID NO : 1 i s as fol lows :ATGGCTCGGCGCGGCTGGCGGCGGGCACCCCTCCGCCGTGGCGTCGGCAGCAGTCCCCGAGCCC GCAGGCTCATGCGGCCCCTTTGGTTGCTCCTCGCAGTGGGCGTCTTTGACTGGGCAGGGGCTTC GGACGGCGGCGGCGGAGAGGCTAGAGCCATGGACGAGGAGATCGTGTCCGAGAAGCAAGCCGAG GAGAGCCACCGGCAGGACAGCGCCAACCTGCTCATCTTCATCCTGCTGCTCACCCTCACCATTC TCACAATCTGGCTCTTCAAGCACCGCCGGGCCCGCTTCCTGCACGAAACCGGCCTGGCTATGAT TTATGGTCTTTTGGTGGGCCTTGTGCTTCGGTATGGCATTCATGTTCCGAGTGATGTAAATAAT GTGACCCTGAGCTGTGAAGTGCAGTCAAGTCCAACTACCTTACTGGTAAATGTTAGTGGAAAAT T T T AT GAG T AT AT G C T GAAAGGAGAGAT T AG T T GAG AT GAAC T C AAT AAT G T T C AAGAT AAT GA AAT GC T T AGAAAG GTTACTTTTGATC C AGAAG T AT T T T T GAAC AT AT TACTTCCTCCTATCATA TTTTATGCAGGTTATAGCCTGAAAAGGAGACATTTTTTTCGAAATCTTGGGTCTATCCTAGCAT ACGCTTTTCTTGGAACAGCAATTTCTTGTTTCGTTATTGGGTCAATAATGTATGGCTGTGTAAC GCTGATGAAGGTAACGGGACAACTTGCAGGAGATTTTTACTTTACAGATTGCCTACTGTTTGGT GCCATTGTATCAGCAACTGATCCAGTGACTGTTCTTGCTATATTCCACGAGCTTCAAGTTGATG TTGAACTCTATGCACTTCTTTTTGGTGAAAGTGTCCTCAATGATGCTGTTGCCATAGTGCTGTC CTCCTCAATAGTGGCATACCAGCCAGCTGGAGACAACAGTCACACCTTTGATGTCACAGCGATG TTCAAGTCTATTGGGATCTTCCTTGGAATCTTCAGTGGATCTTTTGCAATGGGTGCTGCTACTG GAGTGGTGACAGCTTTAGTGACAAAGTTCACCAAATTACGGGAGTTCCAGTTGTTGGAGACAGG CCTGTTCTTCTTGATGTCCTGGAGTACCTTCCTCTTGGCTGAAGCATGGGGCTTCACAGGTGTA G T T GC AG TATTGTTTTGTGGCAT C AC AC AAG CAC AT TAT AC G T AT AAT AAT T T G T C AAC G GAG TCTCAGCATAGAACTAAACAGTTGTTTGAGCTTCTCAATTTCTTGGCAGAGAATTTCACTTTCTC CTACATGGGGCTGACACTGTTCACCTTCCAGAACCATGTCTTTAACCCAACATTTGTAGTAGGA GCATTTGTTGCTATTTTCTTGGGAAGAGCTGCCAATATTTACCCCTTGTCCCTCTTACTTAATT TGGGTAGAAGAAGTAAGATTGGATCAAATTTTCAACACATGATGATGTTTGCTGGCCTTCGTGG TGCAATGGCATTTGCCTTGGCCATTCGAGATACTGCCACTTATGCACGGCAAATGATGTTCAGCACCACGCTTCTGATTGTGTTTTTTACCGTGTGGGTATTTGGTGGTGGCACCACTGCAATGCTGT CATGCTTGCATATCAGGGTTGGTGTTGATTCAGACCAAGAACACTTGGGTGTTCCTGAAAATGA AAGGAGAACTACCAAAGCAGAGAGTGCTTGGCTTTTCCGGATGTGGTACAACTTTGATCATAAC TATCTGAAGCCTCTGCTGACCCACAGCGGGCCTCCGCTGACAACAACACTCCCTGCCTGCTGTG GACCCATCGCCAGGTGCCTCACCAGCCCCCAGGCTTACGAAAACCAGGAACAGTTGAAAGATGA T GAT T C T GAT C T TAT T C T CAAT GAT GGT GACAT GAG T T T GACAT AT GGAGAT T C TAG T G T GAAC ACTGAACCGGCCACATCCAGCGCCCCAAGGAGATTTATGGGAAACAGTTCTGAAGATGCCTTGG ATCGGGAGCTTGCATTTGGGGACCATGAACTGGTCATTCGAGGAACACGCCTGGTTCTTCCAAT GGATGATTCTGAACCCCCGCTAAATTTGTTAGATAATACGAGACATGGTCCAGCC

[0321] LENGTH: 701 ; TYPE: PRT; ORGANISM: Homo sapiens; NHE6; “SEQUENCE: 2"SEQ ID NO : 2 i s as fol lows :MARRGWRRAPLRRGVGSSPRARRLMRPLWLLLAVGVFDWAGASDGGGGEARAMDEE IVSEKQAE ESHRQDSANLLI FILLLTLT ILT I LFKHRRARFLHETGLAMIYGLLVGLVLRYGIHVPSDVNN VTLSCEVQSSPTTLLVNVSGKFYEYMLKGE I SSHELNNVQDNEMLRKVTFDPEVFFNILLPPI I FYAGYSLKRRHFFRNLGS ILAYAFLGTAI SCFVIGS IMYGCVTLMKVTGQLAGDFYFTDCLLFG AIVSATDPVTVLAI FHELQVDVELYALLFGESVLNDAVAIVLSSS IVAYQPAGDNSHTFDVTAM FKS IGI FLGI FSGS FAMGAATGWTALVTKFTKLREFQLLETGLFFLMSWSTFLLAEAWGFTGV VAVLFCGI TQAHYTYNNLSTESQHRTKQLFELLNFLAENFI FSYMGLTLFTFQNHVFNPTFWG AFVAI FLGRAANI YPLSLLLNLGRRSKIGSNFQHMMMFAGLRGAMAFALAIRDTATYARQMMFS TTLLIVFFTVWVFGGGTTAMLSCLHIRVGVDSDQEHLGVPENERRTTKAESAWLFRMWYNFDHN YLKPLLTHSGPPLTTTLPACCGPIARCLTSPQAYENQEQLKDDDSDLILNDGDI SLTYGDSTVNTEPATSSAPRRFMGNSSEDALDRELAFGDHELVIRGTRLVLPMDDSEPPLNLLDNTRHGPA

[0322] LENGTH: 2103; TYPE: DNA; ORGANISM: Artificial Sequence; Codon-optimizedNHE6; “SEQUENCE: 3”SEQ ID NO : 3 i s as fol lows :ATGGCAAGGAGAGGGTGGAGAAGAGCACCGCTGAGAAGAGGGGTCGGTAGTAGTCCAAGAGCAC GCAGGTTGATGAGGCCTCTCTGGCTTTTGCTCGCCGTGGGAGTTTTCGATTGGGCAGGCGCATC TGATGGGGGAGGGGGCGAAGCTCGGGCCATGGATGAAGAGATCGTTTCCGAAAAACAGGCAGAG GAGTCACATCGGCAGGATTCCGCCAATCTGCTTATTTTTATACTTCTGCTTACATTGACCATCC TGACCATTTGGCTGTTTAAACACCGGAGGGCACGGTTCTTGCATGAAACAGGACTGGCGATGAT CTACGGTCTTCTGGTGGGATTGGTTCTCAGATATGGCATTCATGTTCCTTCCGACGTTAATAAC GTCACCCTGAGCTGTGAAGTCCAGTCATCCCCTACCACTCTCTTGGTTAACGTGAGCGGGAAGT TTTATGAGTATATGCTGAAAGGGGAAATTAGCTCCCACGAATTGAACAATGTGCAGGACAACGA GATGTTGCGCAAGGTGACCTTTGACCCCGAAGTCTTTTTCAATATTCTGCTGCCACCCATAATT TTTTATGCTGGGTATTCCCTCAAAAGAAGGCACTTTTTTAGGAACTTGGGATCCATTCTGGCCT ATGCTTTTCTCGGGACCGCAATTAGCTGTTTTGTTATCGGATCAATCATGTATGGCTGCGTCAC TCTTATGAAGGTGACAGGCCAACTGGCCGGCGACTTCTATTTCACCGACTGTCTTCTCTTCGGT GCTATCGTGTCTGCTACAGATCCTGTGACAGTGCTCGCTATTTTTCACGAGCTGCAGGTGGACG TTGAACTGTACGCCTTGCTGTTTGGAGAGAGCGTGCTGAATGATGCCGTCGCTATAGTGCTTAG TAGCAGCATAGTTGCTTATCAGCCAGCAGGTGACAATTCCCACACATTCGATGTAACAGCTATGTTCAAGTCCATTGGTATCTTCCTGGGCATATTCTCCGGTTCCTTCGCAATGGGAGCAGCGACTG GCGTTGTGACTGCCCTGGTGACCAAATTTACCAAACTGAGGGAGTTTCAGCTCCTGGAGACTGG GCTCTTCTTTCTTATGAGCTGGTCCACTTTTCTCTTGGCCGAGGCCTGGGGCTTTACAGGCGTT GTTGCCGTTCTGTTCTGCGGCAT T AC AC AAG CAC AT T AC AC T T AT AAT AAT C T C T C AAC AGAG T C C CAACACAGGAC GAAGCAAC T G T T C GAAC T G T T GAAT T T C T T GGC C GAGAAC T T CAT C T T CAG CTACATGGGTCTGACTCTCTTCACATTCCAGAATCATGTGTTCAACCCCACTTTCGTGGTGGGG GCTTTTGTCGCGATTTTCCTTGGCAGGGCAGCAAATATCTACCCCCTGTCCCTGCTTCTTAATC TTGGCAGGAGATCCAAGATCGGCAGTAACTTCCAGCATATGATGATGTTCGCAGGCCTGAGAGG GGCAATGGCTTTTGCCCTGGCCATCAGGGATACTGCCACGTATGCCAGACAGATGATGTTCTCC ACAACTCTGCTGATCGTGTTCTTCACAGTCTGGGTGTTTGGTGGAGGTACAACCGCCATGCTGT CTTGCCTGCACATTAGGGTCGGGGTGGATTCCGACCAAGAACATTTGGGGGTGCCAGAAAATGA GCGGCGGACGACAAAGGCAGAATCAGCCTGGCTGTTTAGAATGTGGTATAACTTCGACCACAAC TACCTGAAGCCTTTGCTCACGCACTCTGGGCCCCCTTTGACTACAACTCTGCCCGCATGCTGTG GCCCAATCGCCCGCTGTCTCACAAGTCCACAGGCCTACGAAAATCAGGAGCAGCTGAAGGATGA CGACTCAGACCTGATCCTGAATGACGGCGACATATCCCTGACTTATGGAGACAGCACGGTGAAC ACAGAACCCGCTACTAGTAGCGCTCCCCGCCGGTTTATGGGTAACTCTTCTGAGGACGCCTTGG ACCGGGAACTGGCCTTTGGCGACCACGAACTCGTTATCCGCGGAACTCGGCTGGTGCTGCCTATGGATGATAGCGAACCCCCTCTGAACCTGCTTGACAACACCAGGCATGGCCCTGCT

[0323] LENGTH: 2106; TYPE: DNA; ORGANISM: Artificial Sequence; Codon-optimized NHE6; “SEQUENCE: 4”SEQ ID NO : 4 i s as fol lows :ATGGCCCGGAGAGGCTGGCGCAGGGCCCCTCTTCGCAGGGGCGTGGGCTCCTCACCCAGAGCTC GGCGGCTGATGAGGCCCCTGTGGCTCCTGCTGGCCGTGGGAGTGTTCGATTGGGCCGGAGCCAG CGATGGAGGCGGCGGCGAGGCCAGGGCCATGGACGAGGAGATCGTTAGCGAAAAGCAGGCAGAA GAGTCTCATAGACAGGATTCCGCCAATCTCCTGATTTTTATCCTGCTGCTCACACTGACAATTC TGACCATCTGGCTGTTCAAACACCGCCGCGCTAGGTTTCTGCATGAGACAGGCCTGGCCATGAT CTATGGCCTGCTGGTGGGCCTGGTGCTGAGGTACGGAATCCACGTGCCCAGCGACGTGAACAAT GTGACTCTGTCTTGCGAGGTGCAGAGCAGCCCTACTACACTGCTGGTGAATGTGTCTGGCAAGT TCTATGAGTACATGCTCAAGGGCGAGATCAGCTCCCACGAACTGAACAATGTGCAGGACAACGA GATGCTGAGAAAAGTGACCTTCGATCCCGAGGTGTTCTTCAATATCCTGCTGCCACCCATCATC TTCTACGCTGGCTACTCCCTGAAACGGAGACATTTTTTCAGGAATCTCGGATCCATCCTGGCCT ACGCCTTTCTGGGGACCGCCATCAGCTGCTTCGTTATCGGCTCCATCATGTACGGATGCGTGAC ACTTATGAAAGTGACAGGCCAGCTGGCCGGGGATTTCTACTTCACCGATTGCCTGCTGTTCGGA GCCATCGTGTCCGCCACTGATCCTGTGACCGTGCTGGCAATTTTCCACGAGCTGCAGGTGGACG TGGAGCTGTACGCCCTGCTGTTTGGGGAATCTGTGCTGAATGACGCCGTGGCTATCGTGCTGTC ATCTTCAATCGTGGCCTACCAGCCTGCCGGCGACAACTCTCACACCTTTGACGTGACCGCCATG TTCAAGTCTATCGGGATCTTCCTGGGAATCTTCTCCGGCTCTTTCGCCATGGGGGCCGCTACAG GCGTGGTGACCGCCCTGGTGACCAAGTTCACAAAGCTGAGGGAATTCCAGCTGCTGGAGACAGG CCTGTTCTTTCTGATGAGCTGGTCTACCTTCCTGCTGGCCGAAGCCTGGGGCTTTACCGGCGTG GTCGCCGTGCTGTTCTGCGGGATCACACAGGCCCACTACACTTACAACAATCTGTCCACCGAGT CCCAGCACCGGACCAAGCAGCTGTTCGAGCTGCTGAACTTTCTGGCAGAAAACTTTATCTTCTC ATACATGGGACTGACCCTGTTCACATTTCAGAACCACGTGTTCAACCCCACCTTTGTGGTGGGC GCCTTCGTGGCCATCTTTCTGGGCCGGGCTGCAAACATTTATCCACTGAGCCTGCTGCTGAATC TGGGAAGGAGGAGCAAGATCGGCAGCAATTTCCAGCACATGATGATGTTTGCAGGGCTGAGAGGCGCAATGGCATTCGCTCTGGCCATTAGAGATACCGCCACCTACGCCCGACAGATGATGTTCTCA ACCACTCTGCTGATTGTGTTTTTCACCGTCTGGGTGTTCGGCGGCGGAACAACCGCTATGCTGT CCTGCCTGCACATTCGCGTCGGCGTGGATTCAGACCAGGAGCATCTGGGTGTGCCTGAAAACGA GCGGAGGACAACCAAAGCCGAGAGCGCCTGGCTGTTCAGGATGTGGTACAATTTTGATCACAAT TACCTGAAACCACTGCTGACCCACTCAGGACCCCCACTTACCACCACACTGCCAGCTTGCTGCG GCCCAATTGCCAGATGCCTGACCTCCCCCCAGGCCTATGAGAATCAGGAGCAGCTGAAGGATGA T GAT T C C GAG C T GAT C C T GAAT GAG G GC GAT AT GAG C C T GAG AT AC G GAGAC T C T AC C G T C AAC ACCGAACCCGCCACATCCTCTGCCCCCCGCCGGTTTATGGGCAACAGCAGCGAGGACGCCCTGGATCGAGAGCTGGCCTTTGGAGATCACGAGCTCGTGATCAGGGGGACAAGGCTGGTGCTGCCAAT GGACGACAGTGAGCCTCCCCTGAACCTGCTGGACAACACCAGGCACGGGCCTGCCTGAREFERENCES:Anderson C., et al., (2021). “Gene therapy in a rat model of Christianson syndrome, an epileptic encephalopathy with ataxia (2113)." 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(2018). “The Na+(K+) / H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion." Mol. Biol. Cell. 29: 317-325.Kavanaugh, B.C., et al., (2024). “Christianson syndrome across the lifespan: genetic mutations and longitudinal study in children, adolescents, and adults." J. Med. Genet. 61 : 1031-1039.Lee, Y., et al., (2022). “Early lysosome defects precede neurodegeneration with amyloid-fi and tau aggregation in NHE6-null rat brain." Brain. 145: 3187-3202.Lizarraga, S.B., et al., (2021). “Human neurons from Christianson syndrome iPSCs reveal allele-specific responses to rescue strategies." Sci. Transl. Med. 13: 1-39.Ma, L., et al., (2021). “Generation of pathogenic TPP1 mutations in human stem cells as a model for CLN2 disease " Stem Cell Res. 53: 102323.Morrow, E.M. and Pescosolido, M.F. (2018). “Christianson Syndrome." GeneReviews, In: M.P. Adam,, et al.,, eds. GENEREVIEWS [Internet], Seattle, WA: University of Washington, Seattle (PMID: 29334451 ; Bookshelf ID: NBK475801).Ouyang, Q., et al., (2013). “Christianson syndrome protein NHE6 modulates TrkB endosomal signaling required for neuronal circuit development." Neuron. 80: 97-112.Ouyang, Q., et al., (2019). “Functional assessment in vivo of the mouse homolog of the human Ala-9-Ser NHE6 variant" eNeuro. 6: 0046-19.2019.Pescosolido, M.F., et al., (2014). “Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome." Ann. Neurol. 76: 581-593.Pescosolido, M.F.„ et al., (2019). “Complex neurological phenotype in female carriers of NHE6 mutations." Mol. Neuropsychiatry. 5: 98-108.Pescosolido M.F., et al., (2021). “Loss of Christianson syndrome Na+ / H+ Exchanger 6 (NHE6) causes abnormal endosome maturation and trafficking underlying lysosome dysfunction in neurons." J. Neurosci. 41 : 9235-9256.Prasad, H. & Rao, R. (2015). “The Na+ / H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease ." J. Biol. Chem. 290: 5311-5327.Schwede, M.„ et al., (2014). “Genes for endosomal NHE6 and NHE9 are misregulated in autism brains." Mol. Psychiatry. 19: 277-279.Stromme, P., et al., (2011). “X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal-lysosomal dysfunction.” Brain. 134: 3369-3383.Tarpey, P.S., et al., (2009). “A systematic, large-scale resequencing screen of X-chromosome coding exons in mental retardation.” Nat. Genet. 41 : 535-543.Taylor, Z.V., ef al., (2021). “An optimized intracerebroventricular injection of CD4+T cells into mice." STAR. Protoc. 2: 100725.Voineagu, L, et al., (2011). “Transcriptomic analysis of autistic brain reveals convergent molecular pathology." Nature. 474: 380-384.Xu, M., et al., (2018). “Mixed Neurodevelopmental and Neurodegenerative Pathology in Nhe6-Null Mouse Model of Christianson Syndrome." eNeuro. 4: 0388-17.2017.Yu, L., et al., (2015). “Association of Brain DNA methylation in SORL1, ABCA7, HLA-DRB5, SLC24A4, and BIN1 with pathological diagnosis of Alzheimer disease." JAMA Neurol. 72: 15-24.Isselbacher,, et al., (1996). HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, 13 ed., 1814-1882.THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, (2011). 19thEdition, published by Merck Sharp & Dohme Corp., (ISBN 978-0-911910-19-3).THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE, Robert s. Porter, et al., (eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908).MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, (1995). Robert A. Meyers (ed.), published by VCH Publishers, Inc. (ISBN 1-56081-569-8).IMMUNOLOGY, (2006). Werner Luttmann, published by Elsevier.JANEWAY'S IMMUNOBIOLOGY, (2014). 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[0324] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0325] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

[0326] In some examples, the invention provides a method for treating Christianson syndrome by administering a viral vector containing a nucleic acid sequence encoding NHE6. This sequence is operably linked to a promoter and enclosed in a capsid from an AAV serotype or variant. The method targets subjects diagnosed with Christianson syndrome due to a mutation in the SLC9A6 gene. The treatment aims to express NHE6 in the brain, particularly in regions affected by the syndrome, such as the hippocampus, cerebral cortex, and cerebellum, to reduce or ameliorate symptoms like intellectual disability, developmental delay, seizures, and autistic behaviors. The viral vector can be administered systemically or directly to the central nervous system, with options for single or multiple doses. The method also considers the use of immunosuppressive agents and additional therapeutic agents to enhance treatment efficacy and monitor safety and effectiveness through standardized assessments. The claims are nonlimiting in view of the goal of the Invention which is to save human lives.

Claims

1. CLAIMS14 / e claim:1 . A nucleic acid comprising:(a) a first nucleic acid sequence comprising a first AAV inverted terminal repeat (ITR);(b) a promoter;(c) a nucleic acid sequence encoding NHE6, wherein (b) is operably linked to (c); and(d) a second nucleic acid sequence comprising a second AAV inverted terminal repeat (ITR).

2. The nucleic acid of claim 1 , wherein the first ITR is 5’ of the nucleic acid sequence encoding NHE6.

3. The nucleic acid of claim 1 or 2, wherein the first ITR is 5’ of the promoter.

4. The nucleic acid of claims 1-3, wherein the second ITR is 3’ of the nucleic acid sequence encoding NHE6.

5. The nucleic acid of claims 1-4, which further comprises a poly(A) signal.

6. The nucleic acid of claim 5, wherein the poly(A) signal is a bGH, an hGH, a rabbit globin, a beta globin, or an SV40 polyA signal.

7. The nucleic acid of any of claims 1-6, wherein (a), (b), (c), and (d) are arranged in a 5’-3’ direction.

8. The nucleic acid of any of claims 1-7, wherein (a), (b), (c), and (d) are arranged in a 3’-5’ direction.

9. The nucleic acid of claims 1-8, wherein the promoter is the EF1a promoter, the U1A promoter, the CAG promoter, the synapsin 1 promoter, the CaMKII promoter, or the L7 promoter.

10. The nucleic acid of any of claims 1-9, wherein the NHE6 is human NHE6.

11. The nucleic acid of any of claims 1-10, wherein the nucleic acid sequence encoding NHE6 is codon optimized.

12. The nucleic acid of any of claims 1-11, wherein the nucleic acid sequence encoding NHE6 is SEQ ID NO:1 , SEQ ID NO: 3, or 4, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

13. The nucleic acid of any of claims 1-12, wherein the AAV ITR is derived from AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, or AAVrg.

14. The nucleic acid of any of claims 1-13, wherein the AAV is an ITR from AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, or AAVrg.

15. The nucleic acid of any of claims 1-14, which encodes an intron.

16. The nucleic acid of any of claims 1-15, which comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

17. The nucleic acid of any of claims 1-16, wherein the nucleic acid is linear.

18. The nucleic acid of any of claims 1-17, wherein the nucleic acid is single stranded.

19. The nucleic acid of any of claims 1-18, wherein the nucleic acid is DNA.

20. An AAV viral vector comprising the nucleic acid of any of claims 1-19, enclosed in a capsid.

21. The AAV viral vector of claim 20, wherein the capsid comprises an AAV2, AAV9, AAV1 , AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh, or AAVrg capsid protein.

22. A pharmaceutical composition comprising the AAV viral vector of claim 20 or 21.

23. A method of treating Christianson syndrome in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 22 or the AAV viral vector of any of claims 20 or 21 .

24. A method of treating a disease or disorder caused by a mutation in NHE6 in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 22 or the AAV viral vector of any of claims 20 or 21.

25. A method of treating a disease that is characterized by a decrease in expression of the NHE6 gene in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 22 or the AAV viral vector of any of claims 20 or 21 .

26. The method of any of claims 23-25, wherein the subject is a mammal.

27. The method of any of claims 23-25, wherein the subject is a human.

28. The method of any of claims 23-25, wherein the dose for rAAV administration is in a range of 1010to about 1019viral vector particles.

29. The method of any of claims 23-25, wherein the rAAV is administered using the followingmethods: intraparenchymal (IPa), intrathecal (IT), intracerebroventricular (ICV), intravenous (IV), intranasally, orally, transmucosally, inhalationally, transdermally, parenterally, subcutaneously, intradermally, intramuscularly, intranervally, intrapleurally, or topically.

30. The method of any of claims 23-29, wherein the subject has, or is identified as having, intellectual disability / developmental delay, nonverbal status, microcephaly, epilepsy / seizures, ataxia / motor dysfunction, hyperkinesia, high pain tolerance, autism, unprovoked laughter, contractures, visual acuity problems, eye movement abnormalities (e.g. strabismus), low body weight, swallowing problems, gastroesophageal reflux disease (GERD), constipation, osteopenia, regressions (e.g. social, eating, speech, motor), and / or cerebellar atrophy.

31. The method of any of claims 23-30, wherein the subject has, or is identified as having, neurodegenerative features such as neuronal / glial loss, neuroinflammation, and pathogenic tau deposition.

32. The method of any of claims 23-32, wherein the subject has, or is identified as having, neurodegenerative disorders such as dementia, Parkinson’s disease, parkinsonism, atypical parkinsonism, corticobasal degeneration (CBD), multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and / or tauopathy.