Compositions and methods for treatment and / or prophylaxis of neurological and psychological disorders

Abstract

Systems, methods and compositions are provided for the prophylaxis and / or treatment of autism spectrum disorder (ASD) (genetic, environmental and / or infection-induced) and other neuropsychological conditions by improving or correcting Aβ42 proteinopenia (low levels) and improving or restoring normal α7 nicotinic acetylcholine receptor signaling in patients. Therapeutic agents include Aβ polypeptide replacement therapy and neprilysin inhibitors.

Description

Docket No. P321663.WO.01COMPOSITIONS AND METHODS FOR TREATMENT AND / OR PROPHYLAXIS OFNEUROLOGICAL AND PSYCHOLOGICAL DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Appl. No. 63 / 708,690, filed October 17, 2024, and entitled ‘COMPOSITIONS AND METHODS FOR TREATMENT AND / OR PROPHYLAXIS OF NEUROLOGICAL AND PSYCHOLOGICAL DISORDERS.” The aforementioned application is incorporated herein by reference, in its entirety, for any purpose.

[0002] This application is related to international patent applicationNos. PCT / US2024 / 042811, filed 16 August 2024. PCT / EP2022 / 064374, filed 26 May 2022. PCT / US2024 / 042811, filed 16 August 2024, PCT / US2024 / 042960. filed 19 August 2024 and PCT / US2021 / 052997, filed 30 September 2021, each of which is incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] This disclosure relates generally to polypeptide replacement therapy for polypeptide depletion and associated neurological disorders, and more specifically to systems, methods and compositions for the prophylaxis and / or treatment of autism spectrum disorder (ASD).BACKGROUND

[0004] Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors. They are constructed from five constituent polypeptide subunits that share a common structural motif, including four transmembrane hydrophobic domains. The M2 transmembrane domain from each of the five subunits create a channel, whose opening is dependent on the binding of the endogenous full agonists, acetylcholine (ACh) and choline (Ch), to the receptor’s orthosteric binding site (also known as its agonist recognition site). The homopentameric a7nAChR binds five molecules of ACh / Ch, whereas heteropentameric receptors bind only two (Deutsch & Burket, 2020). A transmembrane site exists that binds allosteric agonists and positive allosteric modulatory ligands (PAMs). PAMs preserve the spatial and temporal specificity of endogenous ligands but lack intrinsic efficacy of their own; a7nAChR-selective PAMs act only where and when endogenous ACh or Ch is released to increase the likelihood the channelDocket No. P321663.WO.01 assumes an open configuration. a7nAChR-selective PAMs can also influence the deactivation kinetics of these ligand-gated ion channel receptors. a7nAChR is unique among nAChRs. because of its high Ca2+ permeability, relatively low sensitivity to acetylcholine (ECso = 30 pM), full activation by choline as a full agonist (EC50 = 0.4- 1.6 mM), high-affinity for a-bungarotoxin (from the snake Bungarus multi cinctus), relatively low affinity for nicotine (EC50 = 18-91 pM), and fast desensitization as determined by the submillisecond time scale following exposure to agonists (Albuquerque et al., 2009).

[0005] In the brain, high levels of a7nAChR are found in regions implicated in cognitive function and memory, including the hypothalamus, geniculate nuclei, colliculi, hippocampus, medial habenula, thalamus, cortex, and amygdala, a-bungarotoxin-binding assays yield abundant a7nAChR protein expression in hippocampal interneuron populations. Levels of a7nAChR are low in the striatum, forebrain, medulla, and various brain nuclei. a7nAChRs are expressed in presynaptic and postsynaptic locations that facilitate neurotransmitter release and in perisynaptic locations that activate signaling pathways through volume transmission. Presynaptic and postsynaptic a7nAChRs modulate neurotransmitter release in the brain through Ca2+-dependent mechanisms, and a7nAChRs regulate neuronal growth and differentiation in the developing CNS. In nonneuronal cells, including astrocytes, microglia, dendritic cells, lymphocytes, macrophages, and endothelial cells, a7nAChRs play a role in immunity, inflammation, and neuroprotection. The expression of a7nAChRs in the immune system is of particular interest, as this system plays a crucial role in regulating the cholinergic antiinflammatory’ pathway. In addition, the pathophysiology of autism spectrum disorders (ASD) has been linked to abnormalities in genes related to a7nAChR expression.BRIEF SUMMARY

[0006] A method of treating an autism spectrum disorder (ASD) in a subject, can include administering to the subject one or both of: a therapeutically effective amount of an amyloid beta 42 (A|342) peptide analogue, and a therapeutically effective amount of a neprilysin inhibitor. The the A(342 peptide analogue may have a decreased propensity for beta-sheet stacking and amyloid formation relative to wild type A 42 peptide. The A[342 peptide analogue may include a C-terminal domain modification that includes one or more of a deletion of 1-13 amino acids, a C-terminus chemical modification, and a substitution including an amino acid, an amino acid analogue, or both. In someDocket No. P321663.WO.01 variations, the C-terminal modification confers on the A(142 peptide analogue one or more enhancements over wild type A(342 peptide, where said enhancements are selected from: increased solubility in a liquid medium, increased protease resistance, reduced administration volume, increased circulation time, and increased half-life in cerebrospinal fluid (CSF). In some variations the Ap42 peptide analogue retains a function native to wild type Ap42 peptide, such as facilitating a7 nicotinic acetylcholine receptor (a7nAChR) signaling in the subject.

[0007] The amount of the Ap42 peptide analogue administered may be about 0.01 to about 30 mg, or about 0.1 to about 20 mg, or about 1 to about 10 mg per kg of the subject's body weight. The method may also include where the administered amount of the AP42 peptide analogue is sufficient to provide a concentration of the analogue in the CSF of a subject of about 200 to about 600 pg / mL. Administering the Ap42 peptide analogue may be performed via intrathecal injection or infusion, or more particularly via a cerebrospinal fluid (CSF) exchange using a fluid that includes the Ap42 peptide analogue.

[0008] In some variations, the neprilysin inhibitor can be a biaryl substituted derivative of an aminopropionic acid or of an aminobutyric acid. In some variations, the neprilysin inhibitor is a compound of Formula 1:biaryl

[0009] or a pharmaceutically acceptable ester or salt thereof, where each occurrence of X independently represents hydrogen or derivatization with a pharmaceutically acceptable ester or amide, Ri represents hydrogen, lower alkyl, C3-C7-cycloalkyl-lower alkyl, ary l-lower alkyl, biaryl-low^er alkyl, lower alkoxy, aryl-lower alkoxy, aryloxy, N- lower alkylamino, N,N-di-lower alkylamino, N-aryl-lower alkylamino, N,N-di-aryl-lower alkylamino, N-arylamino. N,N-diarylamino. lower alkanoylamino, aryl-lower alkanoylamino or aroylamino, R2 represents hydrogen, hydroxy, lower alkoxy, low?er alkyl, aryl-lower alkyl, Cs-Cv-cycloalkyl-lower alkyl, amino-lower alkyl, hydroxy-lower alkyl, lower alkylthio-lower alkyl, lower alkoxy-lower alkyl, aryl-lower alkylthio-lower alkyl or aryl-lower alkoxy-lower alkyl, biaryl represents phenyl substituted byDocket No. P321663.WO.01 carbocyclic or heterocyclic ary l, A represents a direct bond, lower alkylene, phenylene or cyclohexylene, n represents 1 or zero, and m represents 1 or zero, provided that m represents 1 when A is a direct bond.

[0010] In other variations, the neprilysin inhibitor is a compound of Formula III:

[0011] or a pharmaceutically acceptable salt or ester thereof, where: R1is phenyl optionally substituted by alkyl, R2is hydrogen or alkyl-S(0)o-2(CH2)q, R3is hydrogen or lower alkyl, Q is hydrogen or alkyl-CO-, n is 0-2, and q is 1-4.

[0012] In certain variations the neprilysin inhibitor is selected from sacubitril. sacubitrilat, thiorphan, retro-thiorphan, acetyl thiorphan, phosphoramidon, 3-{[(2S)-l- {3'-chloro-[l,r-biphenyl]-4-yl}-4-ethoxy-4-oxobutan-2-yl]carbamoyl [propanoic acid, and (3R)-3-(3-carboxypropanamido)-4-{3'-chloro-[l,l'-biphenyl]-4-yl}butanoic acid, and pharmaceutically acceptable salts and esters thereof.

[0013] The method may include administering both the A(342 peptide analogue and the neprilysin inhibitor. In various embodiments, the A[342 peptide analogue and the neprilysin inhibitor may be administered over differing time periods, such as overlapping time periods or sequential time periods. In some variations, the neprilysin inhibitor is administered over a time period at least part of which is later than that over which the A(342 peptide analogue is administered. The subject may be under about 30 years of age, under about 25 years of age, under about 20 years of age, under about 15 years of age, under about 10 years of age, or under about 5 years of age.

[0014] The method may also include a prior step of identifying the subject as a candidate for treatment based on assessing at least one of reduced A|342 level, neurological phenotype, a cognitive or behavioral symptom, a genotype, and the presence of beta- amyloid plaques. The method may further include modulating empathy in the subject, by modulating type 2 theta oscillations in a brain region of the subject.

[0015] In some variations the patient is a pediatric patient. The method may also include providing sacubitril in a monotherapy formulation, or in a combination formulation with an ACE receptor blocker such as valsartan. The method may alsoDocket No. P321663.WO.01 include administering an initial daily dose based on patient weight, and further may include titrating the dose upward even' 3 to 4 weeks until a final daily dose is reached.

[0016] Use of the above compositions is also provided for the manufacture of a medicament for the treatment of an autism spectrum disorder (ASD) in a subject.

[0017] A method of restoring and / or improving a7 nicotinic acetylcholine receptor (a7nAChR) activity in a subject in need thereof, can include administering to the subject an agent configured to increase expression of a7nAChR or a variant thereof in the central nervous system of the subject. In some variations the agent includes a nuclease selected from Cas9. a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and a meganuclease. In some variations the agent includes a nucleic acid encoding a nuclease selected from Cas9, a TALEN, a ZFN, and a meganuclease. The nuclease can be specific to a target sequence that includes a mutation in the CHRNA7 gene. The method may also include further including administering a nucleic acid molecule configured for homologous recombination with the target sequence. In some variations, said nucleic acid molecule includes a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to wild-ty pe CHRNA7 gene. In some variations, the a7nAChR variant includes a protein sequence having at least 70%, 75%, 80%. 85%. 90%, 95%, 98%, or 99% identity to SEQ ID NO:2. In some variations, the variant includes a modified a7 protein sequence including at least one substitution selected from D64A, D66A and K68A, or more particularly is one of SEQ ID NOS:3-9. In some variations, the variant includes a modified a7 protein sequence including an L270T substitution, or more particularly is SEQ ID NO: 10.

[0018] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.TERMS AND DEFINITIONS

[0019] Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise / ’ and variations such as “comprises” and “comprising,” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. By “consisting of is meant including, and limited to, whatever follows the phrase “consisting of’ Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. ByDocket No. P321663.WO.01“consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that no other elements are present that materially affect the activity or action of the listed elements.

[0020] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0021] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and / or” should be understood to mean either one, or both of the alternatives.

[0022] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In one embodiment, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0023] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment.” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in a particular embodiment.

[0024] “Modified” or “engineered” as used herein to describe a peptide or nucleic acid refers to the aspect of having been manipulated by human intervention. Disclosed hereinDocket No. P321663.WO.01 are engineered peptides, polypeptides, proteins, genes, etc. In one example, a protein is considered to be “engineered” when at least one aspect of the polypeptide, e.g.. its sequence, has been intentionally manipulated by human intervention (directly or indirectly) to differ from the aspect as it exists in a patient / subject or in nature. Artificial manipulation may be accomplished by chemical synthesis or by the editing (insertion, deletion, mutation, etc.) of isolated segments of nucleic acids, e.g., by genetic engineering techniques. In contrast, “native” or “wild-type” as used herein refers to unengineered and / or un-modified genes, peptides, proteins, nucleic acid sequences, amino acid sequences, and portions thereof.

[0025] The terms “treat,” “treating,” and “treatment” refer to eliminating, reducing, suppressing, or ameliorating, either temporarily or permanently, either partially or completely, a clinical symptom, manifestation or progression of an event, disease or condition associated with proteinopenia and diseases described herein. As is recognized in the pertinent field, methods and compositions employed as therapies may reduce the severity of a given disease state but need not abolish every manifestation of the disease to be regarded as useful. Similarly, a prophylactically administered treatment need not be completely effective in preventing the onset of a condition to constitute a viable prophylactic method or agent. Simply reducing the impact of a disease and / or reducing the number or severity’ of associated symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect, or reducing the likelihood that the disease will occur or worsen in a subject, is sufficient.

[0026] “Therapeutically effective amount” means an amount of a drug, composition, compound, treatment, or therapy of the present disclosure that alone, or in combination with other therapies, (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays, the onset of one or more symptoms of the particular disease, condition, or disorder described herein.DETAILED DESCRIPTION

[0027] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools, and methods which are meant to be exemplary and illustrative, not limiting in scope.Docket No. P321663.WO.01

[0028] This disclosure includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0029] A number of publications are cited herein in order to more fully describe and disclose the subject matter and the state of the art to which the subject matter pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

[0030] This disclosure relates generally to polypeptide replacement therapy for polypeptide depletion and associated neurological disorders, and more specifically to systems, methods and compositions for the prophylaxis and / or treatment of autism spectrum disorder (ASD) whether genetic, environmental and / or infection-induced, and other neuropsychological conditions by improving or correcting amyloid beta proteinopenia (low levels) and thus improving or restoring normal a7nAChR signaling in patients.

[0031] The pathophysiology of autism spectrum disorders (ASD), seizures, intellectual disability’, and schizophrenia has occasionally been linked to sequence variants in the promoter region of CHRNA7 (the gene encoding the a7nACh receptor subunit) and copy number variants, specifically microdeletions and, more rarely, microduplications on chromosome 15ql3.3. the genetic locus containing CHRNA7 (Deutsch & Burket. 2020). Mutations of chromosome 15ql 3.3 have been reported in both preclinical and clinical studies to increase the risk of schizophrenia and ASD and linked with cognitive deficits and ASD. Additionally, Rett syndrome, which is an ASD, is caused by mutations in the X-linked gene. methyl-CpG binding protein 2 (MeCP2), which is a positive modulator of CHRNA7 expression (Zhang et al., 2016). In animal models, microdeletion chromosome 15ql 3.3 syndrome demonstrated neurological and behavioral features associated with ASD, reduced expression of a7nACh receptor, and altered function of hippocampal GABAergic inhibitory’ circuits. These findings collectively suggest that dysregulation of a7nAChR signaling contributes to the pathogenesis of ASD and related disorders including seizures, intellectual disability’, and schizophrenia. The a7nACh receptor is crucial for controlling neural activity’ including firing of GABAergic projections expressing parvalbumin (PV), which are necessary for coordinating the oscillatory outputDocket No. P321663.WO.01 of pyramidal neuron assemblies involved in higher executive functions such as learning, memory, problem-solving, reasoning, and functional connections between distinct brain regions. In some subsets of GABAergic interneurons in rodent ASD models, the a7nAChR has been suggested to regulate PV expression and functions in concert with the co-expressed NMDA receptor (Deutsch & Burket, 2020; Oz et aL, 2024).

[0032] Amyloid precursor protein (APP) is also widely expressed in several interneuron subtypes, both in hippocampus and cortex. When sequentially cleaved by f> and y secretases (encoded by BAC1 and presenilin genes, respectively), APP produces amyloid beta (A(3) peptides including amyloid beta peptide 40 and 42 (A(340 and A(342). A 42 plays an important role in neurogenesis, synaptic plasticity, memory, and cognition. Many of these functions are related to the well-established interaction of A|342 with the a7nAChR. In animal models, knocking down APP and its homologue APLP2 in GABAergic forebrain neurons impairs synaptic plasticity and cognition (Mehr et al., 2020). Additionally, knocking down all three APP family members (APP / APLP1 / APLP2) during development impairs synaptic function and plasticity, disrupts learning, and causes an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction (Steubler et al., 2021). Moreover, age-dependent reduction of PV- and somatostatin-immunoreactive interneurons is more pronounced in the neocortex and hippocampus of presenilin knockout mice (Kang & Shen, 2020). In patients with severe autism, while soluble APPa levels are increased, the levels of Ap40, A|342, and soluble APPp levels are significantly decreased (Ray et al., 2011). Lower Ap levels have been reported across different cohorts of autistic patients (Al-Ayadhi et aL. 2012; Banik et al., 2015). Moreover, Ap amyloid plaques were found with higher prevalence in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects (Wegiel et al., 2012). These result supports the concept of mechanistic and functional links between autism, epilepsy, and alterations of APP processing especially in the relation to the role of AP42 in a7 nAChR signaling.

[0033] The present disclosure describes approaches to treating ASD or other proteinopenic diseases of the CNS in a subject by employing one or more therapeutic agents selected to improve or correct a state of A 42 depletion. More specifically, the agents may be selected to restore normal concentration and function of AP42. In various aspects of the present disclosure such approaches can comprise replacing A(342 and / orDocket No. P321663.WO.01 reducing the further loss of functional A042 resulting from degradation or sequestration into amyloid plaques. The A0 depletion state may be selected from a group consisting of autism spectrum disorder (ASD) (mild or severe). Rett syndrome, epilepsy. schizophrenia, bipolar disease, attention deficit hyperactivity disorder (ADHD), post- infectious neurological sequelae induced by viral, bacterial, protozoan, or helminthic infections. In various embodiments, ASD may be selected from classical autism, Asperger's syndrome, pervasive developmental disorder-not otherwise specified (PDD- NOS), Rett's disorder and childhood disintegrative disorder, or may arise from genetic conditions including fragile X syndrome, tuberous sclerosis, maternal duplication of 15ql l-ql3, and duplications of 16pl l.

[0034] In an aspect of the present disclosure, a method of enhancing a7nAChR activity can comprise contacting the receptor with one or more therapeutic agents selected to improve or correct Ap42 proteinopenia, or increasing a7nAChR channel expression or improving a7nAChR channel function.

[0035] In some embodiments, a method of treating ASD or other proteinopenic diseases of the CNS in a subject can comprise administering to the subject a therapeutically effective amount of an A042 peptide analogue having a decreased propensity for beta-sheet stacking and amyloid formation relative to wild type A042 peptide. Peptide analogues suitable for this purpose were previously described in patent applications PCT / EP2022 / 064374 and PCT / US2024 / 042811 which are incorporated herein by reference in their entirety. As described therein, the A042 peptide analogue can include one or more modifications to the A042 C-terminal domain, including deletions, insertions, substitutions comprising amino acid and / or amino acid analogues, and C-terminus chemical modifications.

[0036] Thus modified, these analogues may exhibit enhancements over wild type A042. In some embodiments, the A042 peptide analogue has a lower propensity for beta-sheet stacking and amyloid formation relative to wild type A042 peptide. In some embodiments, the A042 peptide analogue includes increased peptide solubility and reduced administration volume relative to wild type A 42 peptide. In some embodiments, the A 42 peptide analogue includes an increased post-administration circulation time relative to wild type A042 peptide. In some embodiments, the A042 peptide analogue includes an increased protease resistance relative to wild ty pe A042 peptide. In some embodiments, the A042 peptide analogue includes an increased CSF half-life relative toDocket No. P321663.WO.01 wild ty pe A 42 peptide. In some embodiments, the A042 peptide analogue retains a native function of the A(342 peptide. More particularly, the native function may comprise facilitating a7nAChR signaling.

[0037] In some embodiments, the method comprises administering a pharmaceutical composition comprising an A(342 peptide analogue as described herein and one or more pharmaceutically acceptable carriers or diluents, such as for example buffers, solubilizers (e.g. DMSO), preservatives, binders, fillers, antioxidants, humectants, disintegrants, lubricants, glidants, coatings, sweeteners or other pharmaceutical excipients that are suitable for different dosage forms for example: injection, tablet, capsule, inhalation powder / solution, topical cream / ointment, suppository or other.

[0038] Still other embodiments provide methods of preparing a composition comprising an Ap42 peptide analogues, comprising the step of mixing an Ap42 peptide analogue as described herein with a pharmaceutically acceptable carrier or diluent. Still other embodiments are directed to the use of an A[342 peptide analogue as described herein in the manufacture of a medicament for use, for example, in a method of treatment of an Ap depletion state and / or proteinopenic diseases of the CNS such as ASD.

[0039] Still other embodiments provide a kit comprising an A 42 peptide analogue as described herein, preferably provided as a pharmaceutical composition and in a suitable container and / or with suitable packaging, and instructions for use, for example, comprising written instructions on how to dose and / or administer the composition.

[0040] In various embodiments, therapeutic compositions comprising the A 42 peptide analogue or nucleic acids encoding such peptides as described herein are administered in any suitable manner now known or developed in future, including direct administration, genetic engineering techniques, liposome-mediated delivery including lipid nanoparticle delivery, viral vectors or the like. Modes of liposome-mediated delivery' can include direct delivery' of the engineered peptide or a nucleic acid (e.g., mRNA) encoding the engineered peptide for expression by a cell, or DNA encoding the engineered peptide together with suitable mechanisms (e.g. CRISPR-Cas gene editing systems) to integrate the DNA into the genome of the cell to facilitate expression of the engineered peptide by the cell, or using a viral vector as an expression module for the desired peptide.

[0041] Modes of direct administration can include subcutaneous, intravenous, intracerebroventricular, intracerebral, intrathecal, intraperitoneal, intramuscular or intravenous injection, infusion, or topical, nasal, oral (including sublingual or buccal),Docket No. P321663.WO.01 rectal, ocular or otic, or other form of delivery', including pumping or direct injection into the brain of a subject. The analogues are preferably administered using delivery systems that facilitate delivery of agents to the central nervous system. For administration by routes other than intrathecal, various blood-brain barrier (BBB) permeability enhancers can be used to transiently and reversibly increase the permeability7of the blood brain barrier.

[0042] The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity7of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein can include a single treatment or, preferably, can include a series of treatments. In some embodiments the amount of analogue to be administered or caused to be expressed is sufficient to provide a concentration of the analogue in the CSF of a subject of about 200 to about 600 pg / mL, including any value or subrange therebetween, e.g. 250, 300, 350, 400, 450, 500 or 550 pg / mL. In some embodiments, the amount of analogue to be administered or caused to be expressed is less than an amount of native A042 peptide that would be administered to achieve the same therapeutic effect. In various embodiments, a subject is treated with A042 peptide analogue in the range of between about 0.01 to about 30 mg / kg body weight, or about 0. 1 to about 20 mg / kg body weight, or about 1 to about 10 mg / kg body weight. It will also be appreciated that the effective dosage of A042 peptide analogue used for treatment may be higher or lower depending on the administration route and associated formulation. For example, the effective dosage when administered by a route that involves crossing the BBB may be lower for a formulation that includes a permeability enhancer than for a formulation that does not. It will also be appreciated that the effective dosage may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as known in the art.

[0043] In certain embodiments the treatment of patients in severe disease states may comprise a total CSF infusion to replace the CSF with a proper composition of needed proteins, including A042. The infused fluid can include other proteins such as such as a- syn, SOD1, C90rf72, TDP43, PrP. MeCP2, P53, progranulin, GLP-1. This can be via artificial CSF exchange and self-irrigating systems, where CSF or interstitial fluid (ISF) is replaced with artificial CSF or other non-autologous fluids with added A042 or non-Docket No. P321663.WO.01 aggregating analogues. CSF replacement can be autologous, i.e., where CSF is replaced with CSF from the same subject, or heterologous, where CSF is replaced with healthy CSF from a different subject. In either case, the CSF may be treated with A042 analogues before infusion. In some embodiments, after extraction the CSF is treated to remove inflammatory agents and / or infective particles such as bacterial, viral, or fungal matter before infusing the CSF into the recipient subject.

[0044] Such CSF exchange / replacement / liquorpheresis can either be continuous (CSF is exchanged continuously for days, weeks, or months), intermittent (CSF is exchanged intermittently in separate sessions), or punctual (CSF is exchanged in one single session).

[0045] The device / system for CSF exchange can be implantable, where the subject can move freely during the procedure, or extracorporeal, where a component of the system (typically a catheter) is temporarily implanted while other parts of the system are external and connected to the implanted component. Infusion pressure can be provided and controlled via electromechanical pumps, or generated naturally, where CSF is moved by the natural gradient of pressures between cavities in the organism or between a cavity and the exterior.

[0046] In an aspect of the present disclosure, approaches to treating ASD or other proteinopenic diseases of the CNS in a subject by restoring normal concentration and function of A042 can comprise the use of inhibitors of neutral endopeptidase EC 3.4.24.11. Neutral endopeptidase EC 3.424.11, also called neprily sin (NEP), is a transmembrane, zinc-activated endopeptidase, which cleaves peptides up to 40 to 50 amino acids. Its target peptides include atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), C-type natriuretic peptide (CNP), bradykinin, adrenomedullin, substance P; angiotensin I and II, endothelin, neurotensin, A04O, A042, enkephalins, endomorphins. corticotropin, neuropeptide Y, gastrin, cholecystokinin, somatostatin, glucagon, vasoactive intestinal peptide, and oxytocin (Bozkurt et al., 2023). It has a broad role in cardiovascular, endocrine, pulmonary, gastrointestinal, renal, and neurologic functions. It is widely distributed across different tissues, including the renal tubules, lung, endothelial cells, adrenal gland, cardiac myocytes, gut, fibroblasts, smooth muscle cells, and the brain. NEP is found in animal and human cerebrospinal fluids. In humans, NEP is distributed heterogeneously among brain regions with the highest levels being found in the globus pallidus and pars reticulata of the substantia nigra. It is primarily located on neuronal cells, especially in the striatonigral pathway and in muchDocket No. P321663.WO.01 lower amounts in the hippocampus. The enzyme has also been found on Schwann cells in the peripheral nervous system. The ability of NEP to catabolize Ap at multiple sites was first demonstrated in vitro (Howell et al., 1995) which was later confirmed in vivo with intracerebrally administered A into the rat brain parenchyma (Iwata et al., 2000). Further, the correlation between the levels of Ap peptide in the brain and NEP activity7was demonstrated both in NEP knockout mice (Iwata et al., 2001) and in rats chronically administered with the NEP inhibitor thiorphan (Zou et al., 2006).

[0047] Based on, but not intended to be bound by, the proteinopenia hypothesis of neurocognitive disorders, neprilysin inhibitors may be used in accordance with the present disclosure to restore, prolong, and potentiate the neuronal effects of amyloid P peptides in mammals, particularly humans, by inhibiting the degradation amyloid P peptides to less active metabolites.

[0048] In some embodiments, the neprilysin inhibitor is a compound of Formula I:biaryl or a pharmaceutically acceptable ester or salt thereof, wherein:• each occurrence of X independently represents hydrogen or derivatization with a pharmaceutically acceptable ester or amide;• Ri represents hydrogen, lower alkyl, Cs-Cy-cycloalkyl-lower alkyl, aryl-lower alkyl, biaryl-lower alkyl, lower alkoxy, ary l-lower alkoxy, aryloxy, N-lower alkylamino, N.N-di-lower alkylamino, N-aryl-lower alkylamino. N,N-di-aryl-lower alkylamino, N-arylamino, N,N-diarylamino, lower alkanoylamino, ary l-lower alkanoylamino or aroylamino;• R.2 represents hydrogen, hydroxy, lower alkoxy, lower alkyl, aryl-lower alkyl. C3- C?-cycloalkyl-lower alkyl, amino-lower alkyl, hydroxy-lower alkyl, lower alkylthiolower alkyl, lower alkoxy-lower alkyl, aryl-lower alkylthio-lower alkyl or aryl-lower alkoxy-lower alkyl;• biaryl represents phenyl substituted by carbocyclic or heterocyclic aryl;• A represents a direct bond, lower alkylene, phenylene or cyclohexylene;• n represents 1 or zero; andDocket No. P321663.WO.01• tn represents 1 or zero, provided that m represents 1 when A is a direct bond.

[0049] In some embodiments, the neprilysin inhibitor is a compound of Formula II:or a pharmaceutically acceptable salt or ester thereof, wherein:• R1is Ci-7alkyl;• for each occurrence, R2is independently Ci-?alkyl, NO2, CN, halo. C3-?cycloalkyl, hydroxy, Ci-7alkoxy, halo-Ci-7alkyl, NRbRc, Ce-ioaryl, heteroaryl or heterocyclyl; wherein Rband Rcfor each occurrence, are independently H or Ci-7alkyl;• R3is A1C(O)X1or A2-R4;• R4is Cg-ioaryl or a heteroaryl, which can be monocyclic or bicyclic and which can be optionally substituted with one or more substituents independently selected from hydroxy, hydroxy-Ci-7alkyl, NRbRc, nitro, Ci-7alkoxy, halo, Ci-7alkyl, halo-Ci-7alkyl, C2-7alkenyl, Cg-ioaryl, heteroaryl, — C(O)Ci-7alkyl, — NHS(O)2-Ci-7alkyl, — SO2C1- 7alkyl and benzyl;• R5is H, halo, hydroxy, Ci-ralkoxy, halo, Chalky! or halo-Ci.7alkyl; and• X and X1are independently OH, — O — Ci-7alkyl, — NRbRc, — NHS(O)2 — Ci- 7alkyl, — NHS(O)2-benzyl or — O — Cg-ioaryl; wherein alkyl is optionally substituted with one or more substituents independently selected from the group consisting of aryl, heteroaryl, heterocyclyl, — C(O)NH2, — C(O)NH — Ci-ealkyl, and — C(O)N(Ci- 6alkyl)2;• A1is: o a bond or a linear Ci-4alkylene substituted with one or more substituents independently selected from the group consisting of halo, O-acetate, C1-7 alkylDocket No. P321663.WO.01 and C3-7cycloalkyl; in which two geminal alkyl can optionally combine to form a C?-7cycloalkyl; or o a linear or branched C2-ealkenylene; or o a linear C1-4 alkylene wherein one or more carbon atom(s) is / are replaced with an heteroatom selected from O, NRa; and A1is optionally substituted with one or more substituents independently selected from the group consisting of halo and Ci-7alkyl; in which Rafor each occurrence, is independently H. Ci- ralkyl or CH2C(O)OH; or o a Cs-7cycloalkyl, a heterocyclyl, a phenyl or a heteroaryl in which phenyl and heteroaryl are optionally substituted with one or more substituents independently selected from the group consisting of Ci-7alkyl, Cs-rcycloalkyl. halo-Ci-7alkyL hydroxy, Ci-7alkoxy, halo, NRbRc, OCH2CO2H, and OCH2C(O)NH2; or o — Ci-4alkylene-C6-io-aryl-, — Ci-4alkylene-heteroaryl- or — Ci-4alkylene- heterocyclyl-, wherein A1may be in either direction;• A2is a bond or a linear or branched Ci-7alkylene which is optionally substituted with one or more substituents independently selected from the group consisting of halo, Ci-7alkoxy, hydroxy, O-acetate and Cs-7cycloalkyl; and• n is 0, 1, 2, 3, 4 or 5; wherein each heteroaryl is a monocyclic or bicyclic aromatic ring comprising 5-10 ring atoms selected from carbon atoms and 1 to 5 heteroatoms, and each heterocyclyl is a monocyclic saturated or partially saturated but non-aromatic moiety comprising 4-7 ring atoms selected from carbon atoms and 1-5 heteroatoms, wherein each heteroatom of a heteroaryl or a heterocyclyl is independently selected from O, N and S.

[0050] In some embodiments, the neprilysin inhibitor is a compound of Formula III:or a pharmaceutically acceptable salt or ester thereof, wherein:• R1is phenyl optionally substituted by alkyl;• R2is hydrogen or alkyl-S(0)o-2(CH2)q;Docket No. P321663.WO.01• R3is hydrogen or lower alkyl;• Q is hydrogen or alkyl-CO-;• n is 0-2; and• q is 1-4.

[0051] Examples of compounds that can be utilized for this invention include but are not limited to, the compounds shown in Table 1 and esters or salts thereof.Docket No. P321663.WO.01Docket No. P321663.WO.01

[0052] In some variations, the synthesis of Compounds 5 and 6 may be performed via the processes described in W02008031567A1, W02008083967A2 and US10479753, which are hereby incorporated by reference in their entirety. In some variations, the synthesis of Compounds 7 and 8 may be performed via the processes described in Kawanami et al., 2020), which is hereby incorporated by reference in its entirety. Compounds of the present disclosure are either obtained in the free form, as a salt thereof, or as prodrug derivatives thereof.

[0053] In particular embodiments, the neprilysin inhibitor is selected from thiorphan, retro-thiorphan, acetyl thiorphan. and acetorphan.

[0054] In various embodiments, the method of treatment includes administering a pharmaceutical composition comprising a compound of the present disclosure or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers. In some variations, a pharmaceutical formulation comprises a repurposed compound according to any compound mentioned herein. In another variation, aDocket No. P321663.WO.01 pharmaceutical formulation is provided, comprising a therapeutically effective amount of sacubitril without an angiotensin converting enzyme receptor blocker.

[0055] The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration. More particularly the composition may be formulated for parenteral routes such as intravenous, intrathecal, topical and intranasal. In addition, the pharmaceutical compositions of the present disclosure can be made up in a solid form including capsules, tablets, pills, granules, powders or suppositories, or in a liquid form including solutions, suspensions or emulsions. In certain embodiments, the suspension or emulsion is a liposomal suspension or emulsion. The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, etc.

[0056] In some embodiments the pharmaceutical compositions are tablets and gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and / or polyethylene glycol, for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; if desired, d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and / or e) absorbents, colorants, flavors and sweeteners. Tablets may be either film coated or enteric coated according to methods known in the art.

[0057] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and / or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-75%, or contain about 1-50%, of the active ingredient.

[0058] Suitable compositions for transdermal application include an effective amount of a compound of the disclosure with carrier. Carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example.Docket No. P321663.WO.01 transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound of the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

[0059] Suitable compositions for topical application, e.g.. to the skin and eyes, include aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, e.g., for delivery by aerosol or the like. Such topical delivery systems will in particular be appropriate for dermal application. They are thus particularly Suited for use in topical, including cosmetic, formulations well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0060] As used herein a topical application may also pertain to an inhalation or to an intranasal application. They are conveniently delivered in the form of a dry powder (either alone, as a mixture, for example a dry blend with lactose, or a mixed component particle, for example with phospholipids) from a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, atomizer or nebulizer, with or without the use of a suitable propellant.

[0061] The present disclosure further provides anhydrous pharmaceutical compositions and dosage forms comprising the compounds of the present disclosure as active ingredients, since water may facilitate the degradation of certain compounds.

[0062] Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0063] The pharmaceutical composition can be in therapeutically effective unit dosage of about 0.001-2000 mg of active ingredient(s) for a subject of about 10-80 kg, or about 0.001-500 mg, or about 0.001-250 mg, or about 0.001-150 mg, or about 0.001-100 mg, or about 0.001-50 mg of active ingredients.

[0064] The therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, theDocket No. P321663.WO.01 body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease. In some variations where sacubitril is the neprilysin inhibitor, the total daily dosage may be in the range of about 20 to about 500 mg / day, or about 40 to about 300 mg / day, or about 40 to about 200 mg / day. In some variations, the total daily dosage may be about 0.3 mg / kg / day to about 5 mg / kg / day, about 1 mg / kg / day to about 4 mg / kg / day. In further variations, sacubitril may be dosed twice per day, with each dose in the range of about 10 to about 250 mg / dose, about 20 to about 150 mg / dose, about 20 to about 100 mg / dose, about 0.15 mg / kg / dose to 2.5 mg / kg / dose, about 0.5 mg / kg / dose to about 2 mg / kg / dose, about 1 mg / kg / dose to about 2 mg / kg / dose. or about 1.4 mg / kg / dose to about 1.8 mg / kg / dose.

[0065] In some variations for pediatric dosing of sacubitril for treatment of autism spectrum disorder or other pediatric neurocognitive conditions, dose titration regimens may be for sacubitril monotherapy or dual-therapy with an ACE inhibitor or ACE receptor blocker may used. Pediatric patients may include patients with an age of less than 15 years, 16 years, 18 years or 21 years at the time of initial therapy.

[0066] For pediatric patients prescribed sacubitril monotherapy or combination therapy with a sacubitril component and have a weight of less than 40 kg, the initial starting dose of the sacubitril or sacubitril component may be 0.4 mg / kg to 0.8 mg / kg twice per day, with use of the lower starting dose for pediatric patient that have not previously taken an ACE inhibitor or ACE receptor blocker for another condition, or whether they have estimated glomerular filtration rate of less than 60 ml / min / 1.73 m2 body surface area, or who have hepatic impairment. Sacubitril monotherapy may be titrated upward every' 3-4 weeks to the next highest dose of 0.8 mg / kg or 1.2 mg / kg, and then to the target dosing of 1.6 mg / kg. For pediatric patients prescribed sacubitril monotherapy or combination therapy with a sacubitril component, and have a weight of between 40 kg to 50 kg, the initial starting dose may be 0.4 mg / kg or 0.8 mg / kg twice per day, but subsequent upward dose titrations after 0.8 mg / kg may utilize fixed doses of 24 mg, 49 mg, with a target dose of 72 mg twice daily. For pediatric patients greater than 50 kg weight, starting dose of the sacubitril or sacubitril component can be 24 mg twice daily with titration every 3-4 weeks with 49 mg and then 72 mg, to a target dose of 97 mg. In other variations, for target dosing at 0.4 mg / kg, 0.8 mg / kg, 1.2 mg / kg or 1.6 mg / kg, prescribed initial dosesDocket No. P321663.WO.01 may be in the range of 0.3 mg / kg to 0.5 mg / kg, 0.6 mg / kg to 1 mg / kg, 1.1 mg / kg to 1.4 mg / kg or 1.5 mg / kg to 1.8 mg / kg, respectively.

[0067] For pediatric patients prescribed dual therapy with sacubitril / valsartan for the treatment of autism spectrum and have a weight of less than 40 kg, the initial starting dose may be 0.8 mg / kg (referring to the total mg dose of sacubitril and valsartan) or 1.6 mg / kg. with use of the lower starting dose for pediatric patient that have not previously taken an ACE inhibitor or ACE receptor blocker for another condition, or whether they have estimated glomerular filtration rate of less than 60 ml / min / 1.73 m2 body surface area, or who have hepatic impairment. The dual therapy may then be titrated upward every 3-4 weeks to the next highest dose of 1.6 or 2.3 mg / kg, and then to the target dosing of 3.1 mg / kg. For pediatric patients prescribed dual therapy and have a weight of between 40 kg to 50 kg, for those with indications to use a lower starting dose 0.8 mg / kg (total mg dose of sacubitrtil and valsartan) may be used, while pediatric patients not requiring the lower starting dose may initiate therapy with a fixed dose of 24 mg / 26 mg sacubitril / valsartan. Subsequent upward dose titrations after 0.8 mg / kg may utilize fixed doses of 24 mg / 26 mg, 49 mg / 51 mg, with a target dose of 72 mg / 78 mg twice daily. For pediatric patients weighing at least 50 kg and who meet the criteria for a lower starting dose, 24 mg / 26 mg of sacubitril / valsartan twice daily may be used as the starting dose, while those not indicated for the lower starting dose may initiate therapy at 49 mg / 51 mg. Subsequent upward titration is performed with fixed doses of 24 mg / 26 mg, 49 mg / 51 mg, 72 mg / 78 mg until reaching a target dose of 97 mg / 103 mg twice daily.

[0068] For adult patients initiating sacubitil monotherapy or dual-therapy for treatment of autism spectrum disorder or other neurocognitive disorder recited herein, the sacubitril or sacubitril component of the dual therapy may have an initial dose of 49 mg twice daily may be used, or optionally 24 mg if the patient has a systolic blood pressure lower than 110 mm Hg. The dosage should be titrated upward every 2 to 4 weeks, or after 2 to weeks, to 49 mg, or to the target dose of 97 mg twice daily. Titration and / or therapy may be stopped if the patient’s potassium level exceeds 5.4 mmol / 1 or if their systolic blood pressure goes below 100 mm Hg. Similarly, patients should undergo blood pressure and serum potassium level screening using the same criteria and be excluded from therapy accordingly.

[0069] For adult patients initiating sacubitil / valsartan dual therapy for treatment of autism spectrum disorder or other neurocognitive disorder recited herein, an initial doseDocket No. P321663.WO.01 of 49 mg / 51 mg of sacubitril / valsartin twice daily may be used, or optionally 24 m / 26 mg g if the patient has a systolic blood pressure lower than 110 mm Hg. The dosage should be titrated upward even- 2 to 4 weeks, or after 2 to weeks, to 49 mg / 51 mg. or to the target dose of 97 mg / 103 mg twice daily. Titration and / or therapy may be stopped if the patient’s potassium level exceeds 5.4 mmol / 1 or if their systolic blood pressure goes below 100 mm Hg. Similarly, patients should undergo blood pressure and serum potassium level screening using the same criteria and be excluded from therapy accordingly.

[0070] Pre-screening of pediatric patients prior to neprilysin inhibitor use may exclude patients with a potassium level of greater than 5.3 mmol / 1 or a systolic blood pressure that is less than 100 mm Hg.

[0071] Some embodiments are directed to the use of a neprilysin inhibitor as described herein, in the manufacture of a medicament, for example, for use in a method of treatment of an A(3 depletion state and / or proteinopenic diseases of the CNS such as ASD. Still other embodiments provide a kit comprising a neprilysin inhibitor and optionally further comprising an A(342 peptide analogue, preferably provided as one or more pharmaceutical compositions and in a suitable container and / or with suitable packaging, and instructions for use, for example, comprising written instructions on how to dose and / or administer the composition(s).

[0072] An aspect of the present disclosure provides methods of treating an ASD or other A[3 depletion state in a subject that comprise administering a combination of therapeutic agents to the subject. More particularly, in some embodiments such a method can comprise administering an A 42 peptide analogue as described herein in combination with a neprilysin inhibitor as described herein. In various embodiments, these two agents may be administered via different routes, where each agent is provided in a pharmaceutical composition formulated for its respective administration route. Accordingly, each agent in this combination therapy may be formulated for and administered by any route appropriate for said agent as discussed above. By way of nonlimiting example, a combination therapy may comprise CSF infusion of an A 42 peptide analogue in combination with oral administration of a neprilysin inhibitor, or CSF infusion of an A 42 peptide analogue in combination with intravenous injection of a neprilysin inhibitor. In other embodiments, the A 42 peptide analogue and neprilysin inhibitor may be formulated for and administered via the same route.Docket No. P321663.WO.01

[0073] In various embodiments, the combination therapy can comprise concurrent, overlapping, or sequential administration of the therapeutic agents. It will be understood that the respective dosing regimes for each agent may be selected by a skilled person based upon a number of factors such as administration route, pharmacological response, patient tolerance, and observed symptoms. Therefore, the sequence, time period, and total number of doses of each therapeutic agent may differ. For example, a method of treatment can comprise administering an A 42 peptide analogue to the subject one to five times in combination with daily, weekly, or monthly administration of a neprilysin inhibitor over a number of days, weeks, months, or years.

[0074] In some embodiments, the dosing regime may be based upon the status and progression of a particular diagnostic indicator. In some embodiments, the method may comprise serially measuring A0 level during treatment and adjusting the dosing of the neprilysin inhibitor and / or the A(342 peptide analogue based upon an A(3 depletion state. In some such embodiments, the concentration of A(342 in the cerebrospinal fluid of the subject is determined using an immunoassay or liquid chromatography -tandem mass spectrometry. The A|3 depletion state may be a concentration of A|342 in a sample of the plasma that is less than 50 pg / ml, or less than 40 pg / ml, or less than 30 pg / ml, or less than 20 pg / ml, less than 10 pg / ml, or less than 5 pg / ml, or less than 2 pg / ml, or less than 1 pg / ml. The A(3 depletion state may be a concentration of A(342 in a sample of cerebrospinal fluid of the subject that is less than 800 pg / ml, or less than 700 pg / ml, or less than 600 pg / ml, or less than 500 pg / ml, or less than 400 pg / ml, or less than 300 pg / ml, or more less than 200 pg / ml, or less than 100 pg / ml.

[0075] In some variations, the method of treatment may comprise administration of one or a combination of therapeutic agents to raise a subject's A(3 level above a depletion state or to a normal level, followed by a different dosing regimen selected to maintain said A(3 level. By way of nonlimiting example, the method may comprise administering to a subject an amount of a A 42 peptide analogue selected to raise the subject's Ap level to a target plasma concentration, followed by regular administration of an amount of a neprilysin inhibitor selected to maintain Ap levels at or near said target concentration. In some variations, the administration of Ap42 peptide analogue is performed, for example by total CSF infusion, to substantially replace native Ap peptide with the analogue. The method may further comprise obtaining an AP level of the patient prior to treatment.Docket No. P321663.WO.01

[0076] In some embodiments, the dosing regime may be based on an assessment of a neurological function facilitated by A0 peptides, such as a7nAChR activity. For example, the method may comprise serially assessing the ct7nAChR activity level during treatment, and adjusting the dosing of the neprilysin inhibitor or Af> analogue based on the a7nAChR activity level. The method may further comprise obtaining a baseline a7nAChR activity level prior to treatment. The a7nAChR activity level may be determined using EEG or MRI or indirectly inferred from pathology measurements utilizing PET. The method may comprise where MRI- or EEG-based spatial and temporal brain activity patterns associated with a7nAChR-expressing cholinergic neurons are assessed.

[0077] In some embodiments, treatments using therapeutic agents as described above may be performed in combination with an adjunct treatment selected to improve one or more neurological symptoms in a subject. In some variations, the adjunct treatment is selected to improve a symptom of ASD or other proteinopenic disease of the CNS that is not reversed by correction of the A0 depletion state. In some variations, the symptom is suboptimal empathy and the adjunct treatment comprises modulating oscillatory patterns in the subject brain. Methods of improving empathy in a subject by modulating type 2 theta oscillations in a brain region of the subject were previously described in patent application PCT / US2021 / 052997 which is incorporated herein by reference in its entirety’. As described therein, such methods can comprise optogenetic treatment, electric stimulation of a brain region, administration of a pharmaceutical drug, or a combination thereof.

[0078] An aspect of the present disclosure provides methods of identifying a subject as a candidate for therapy according to the methods described above. ASD and other proteinopenic diseases of the CNS often manifest early in the life of subjects, and the symptoms can cause particular difficulties in social and intellectual development. It is hypothesized that there may be therapeutic windows during neurodevelopment where the use of therapy may induce greater and / or more persistent changes in therapeutic effect. Accordingly, in various embodiments, the methods described herein are initiated or performed on a subject under about 30 years of age, under about 25 years of age, under about 20 years of age. under about 15 years of age, under about 10 years of age, or under about 5 years of age. In some variations, therapy may begin as early as 12 months, 18 months, 24 months, 30 months or 36 months of age.Docket No. P321663.WO.01

[0079] The patients selected for therapy may be further selected based on one or more of a reduced Ap level, neurological phenotype, a cognitive or behavioral symptom, a genotype, and the presence of beta-amyloid plaques or other confirmed biomarkers of ASD or another proteinopenic disease of the CNS. In some embodiments, identifying can comprise obtaining an A level of the subject, where the subject is a candidate to determine if an Ap depletion state is present as described above. It is hypothesized that in some embodiments, the Ap depletion state may not require a change in beta-amyloid plaques for indicating therapy. Pre-treatment confirmation of the presence of betaamyloid plaques in the brain may not be required. Pre-treatment confirmation of the presence of beta-amyloid plaques may be performed using PET imaging, CSF analysis or fMRI. In some embodiments, neurological phenotypes such as macrocephaly, hypoactivation of the fusiform face area, and hypoactivation of the superior temporal sulcus may be used to identify a candidate for treatment.

[0080] In some embodiments, identifying can comprise observing and assessing symptoms associated with a disorder believed to include Ap depletion, such as ASD. For example, a candidate for treatment may be identified based on the presence of one or more symptoms of ASD symptom domains: socialization, communication, restricted / stereotyped / repetitive behavior patterns. Specific symptoms include attentional difficulties, hyperactivity, affective difficulties such as anxiety and depression, interfering repetitive activity, irritability, aggression, self-injurious behavior, and sleep disruption.

[0081] In some embodiments, identifying can comprise performing a genetic test to detect the presence of mutations associated with a disorder believed to include Ap depletion, such as ASD. For example, the presence of mutations to genes including, but not limited to, CHD8, FMRI, MECP2, neuroligin 4 NLGN4X, and neuroligin 3 NLGN3 may be used to identify a candidate for treatment.

[0082] An aspect of the present disclosure provides for employing treatment described herein to prevent disorder in high-risk patients. Such preventative measures may be taken following infection, injury, or for patients with genetic risk factors. In some variations, a genetic test may be used to identify a candidate for preventative treatment before symptoms and / or loss of function manifest themselves.

[0083] Additionally, the present disclosure also provides methods of making an agent for restoring and / or increasing the activity and / or expression of a7nAChR in aDocket No. P321663.WO.01 mammalian subject. In some embodiments, the agent comprises a nucleic acid molecule encoding a7nAChR or an expression product thereof. The nucleic acids encoding a7nAChR protein can be natural or synthetic nucleic acids, including DNA and RNA, such as cDNA, splice-switching oligonucleotides, and mRNA.

[0084] Methods for restoring and / or increasing the activity and / or expression of a7nAChR in a mammalian subject can include using nuclease agents to help modify a target gene locus, such as a sequence comprising a mutation to the CHRNA7 gene. Such nuclease agents can promote homologous recombination between a donor nucleic acid molecule (e.g., a DNA repair template) and the target gene locus. In some embodiments, the nuclease agent can comprise an endonuclease agent. In some embodiments, the method further comprises administering a nucleic acid molecule configured to facilitate repair or replacement of the target sequence. In certain embodiments, the nucleic acid molecule can comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to wild-type CHRNA7 gene (SEQ ID NO: 1). In some embodiments, the edited gene encodes a variant of the a7 subunit of nAChR. In particular embodiments, the variant protein can comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identify to wild-type a7 sequence (SEQ ID NO:2). In certain embodiments, the variant protein sequence comprises edits for modulating the calcium ion permeability of the a7nAChR. For example, the variant protein sequence can comprise one, two, or all of the following substitutions: D64A, D66A, K68A, such as, for example, SEQ ID NOS:3-9. In certain embodiments, the variant protein sequence comprises a L270T substitution, for example SEQ ID NOTO.

[0085] As used herein, the term "recognition site for a nuclease agent" generally refers to a DNA sequence in which a nick or double-strand break can be induced by a nuclease agent. The recognition site for the nuclease agent can be endogenous to the cell (or native), or the recognition site can be exogenous to the cell. In some embodiments, the recognition site may be exogenous to the cell, and thus not naturally present in the genome of the cell. In other embodiments, the exogenous or endogenous recognition site may be present only once in the genome of the host cell. In particular embodiments, endogenous or native sites that occur only once within the genome may be identified. Such sites can then be used to design nuclease reagents that will create nicks or double strand breaks at the endogenous recognition sites.Docket No. P321663.WO.01

[0086] The length of the recognition site can vary7, and includes, for example, recognition sites that are at least 4, 6. 8, 10, 12, 14, 16, 18, 19, 20. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32. 33. 34. 35. 36. 37. 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length. In one embodiment, each monomer of the nuclease agent can recognize a recognition site having at least 9 nucleotides. In other embodiments, the recognition site can be about 9 to about 12 nucleotides, about 12 to about 15 nucleotides, about 15 to about 18 nucleotides, or about 18 to about 21 nucleotides in length and any combination of the sub-ranges (e.g., 9-18 nucleotides). The recognition site may be palindromic, that is to say the sequence on one strand is read identically to the complementary7strand in the opposite direction. It will be recognized that a given nuclease agent can bind to a recognition site and cleave that binding site, or alternatively, a nuclease agent can bind to a sequence different from the recognition site. Furthermore, the term recognition site may include both nuclease agent binding sites and nicking / cleavage sites regardless of whether the nicking / cleavage site is inside or outside of the nuclease agent binding site. In another variation, cleavage by nuclease agents may occur at nucleotide positions that are closely opposed to each other to create blunt end cuts (or in other cases, the cuts may be staggered to create single-stranded overhangs also known as "sticky ends," which may be 5'-end overhangs or 3'-end overhangs.

[0087] Any nuclease agent that induces a nick or double strand break in the desired recognition site can be used in the methods of the present application. Naturally occurring or natural nuclease agents can be employed, so long as the nuclease agent induces a nick or double-strand break in the desired recognition site. Alternatively, modified or engineered nuclease agents may be employed. An "engineered nuclease agent" includes a nuclease that is engineered (modified or derived) from its native form to achieve specific recognition and induce nicks or double strand breaks in the desired recognition sites. Thus, the engineered nuclease agent can be derived from a naturally occurring nuclease agent, or it can be artificially created or synthesized. The nuclease agent can be modified by as little as one amino acid in a protein cleavage agent or one nucleotide in a nucleic acid cleavage agent. In some embodiments, the engineered nuclease may induce nicks or double-strand breaks in a recognition site, wherein the recognition site is not a sequence that would have been recognized by a native (non-engineered or non-modified) nuclease agent. Creating nicks or double-strand breaks in a recognition site or other DNA may be referred to herein as "cleaving" the recognition site or other DNA.Docket No. P321663.WO.01

[0088] In some embodiments, the nuclease agent can be a Transcription Activator-Like Effector Nuclease (TALEN). TALENs are a class of sequence-specific nucleases that can be used to generate double-strand breaks at specific target sequences in the genome of prokaryotic or eukaryotic organisms. TALENs can be created by fusing a natural or engineered transcription activator-like (TAL) effector, or functional portion thereof, to an endonuclease, such as, for example, a catalytic domain like Fokl. Unique modular TAL effector DNA binding domains allow for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domain of TAL effector nucleases can be engineered to recognize specific DNA target sites, thus serving to create a doublestrand break at a desired target sequence.

[0089] In some embodiments, the nuclease agent can be a Zinc Finger Nuclease (ZFN). For example, each monomer of a ZFN may comprise 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain may bind a 3 base pair (bp) subsite. In other embodiments, the ZFNs may be chimeric proteins comprising a zinc finger-based DNA binding domain operably linked to an independent nuclease. In some embodiments, the independent endonuclease can be a Fokl endonuclease. In some embodiments, the nuclease agent can comprise a first ZFN and a second ZFN, wherein the first ZFN and the second ZFN are each operably linked to a Fokl nuclease, wherein the first ZFN and the second ZFN recognize two consecutive target DNA sequences separated by about 6 base pairs to about 40 base pair cleavage sites or about 5 base pairs to about 6 base pair cleavage sites in each strand of the target DNA sequence, and wherein the Fokl nucleases dimerize and create a double-strand break.

[0090] In some embodiments, the nuclease agent can be a megabase nuclease (meganuclease). Meganucleases have been classified into four families based on conserved sequence motifs, the families being the LAGLID ADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in coordination of metal ions and hydrolysis of phosphodiester bonds. Such enzymes are known for their long recognition sites and for allowing some sequence polymorphisms (sequence polymorphisms) in their DNA substrates.

[0091] In some embodiments, the nuclease agent employed in the methods of the present application can employ a CRISPR / Cas system. The system may include, for example, Cas9 nuclease, which in some cases may be codon-optimized for the desired cell type in which it is to be expressed. The system may further employ a fusion crRNA-tracrRNA constructDocket No. P321663.WO.01 that functions with a codon-optimized Cas9. This single RNA may often be referred to as a single guide RNA or sgRNA. Briefly, a short DNA fragment containing the target sequence can be inserted into the sgRNA expression plasmid. The sgRNA expression plasmid may comprise a target sequence (in some embodiments, about 20 nucleotides), some form of tracrRNA sequence (backbone), and a suitable promoter active in the cell and elements necessary for proper processing in eukaryotic cells. The sgRNA expression cassette and Cas9 expression cassette can then be introduced into the cell.

[0092] In some embodiments, a method for restoring and / or increasing the activity and / or expression of a7nAChR may comprise administering to a mammalian subject in need thereof a therapeutically effective amount of an agent capable of restoration and / or increasing the activity and / or expression of a7nAChR in said subject. The agents (and any additional therapeutic agents) used in the methods of the invention may be administered by any suitable means, including parenterally, intrapul monary and intranasally, and if desired for topical treatment, intralesional administration. Parenteral infusion includes intramuscular, intrathecal, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, the agents (and any additional therapeutic agents) used in the methods of the invention may be administered via suitable delivery vector including compositions for passive and / or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and / or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and lipid nanoparticles (LNPs)), and delivery by virus-like particles.EXAMPLES

[0093] Example 1. A 20 year-old male (weight: 85 kg) has a diagnosis of ASD after presenting with attentional difficulties, self-injurious behavior and interfering repetitive activity7. A()42 depletion (CSF level of 720 pg / ml) is confirmed by liquid chromatography - tandem mass spectrometry. The patient is selected for A|3 peptide analogue therapy in tandem with sacubitril therapy. A(342 depletion (CSF level of 720 pg / ml) is confirmed by liquid chromatography -tandem mass spectrometry.

[0094] An Ap peptide analogue is administered to the patient through heterologous CSF replacement using donor CSF pretreated with 850 mg of the analogue. The postreplacement CSF level of A 42 is measured at 950 pg / ml one week post-procedure. Oral sacubitril therapy is commenced at that time with an initial dose of 49 mg given twiceDocket No. P321663.WO.01 daily. The dosage is increased every' two weeks until a target dose of 97 mg twice daily is reached. CSF A(342 level is monitored biweekly during the ramp-up period and monthly afterward for one year. The minimum A 42 level measured during this period is 910 pg / ml.

[0095] Example 2. A 12 year-old male (weight: 41 kg) has a diagnosis of ASD after presenting with attentional difficulties, hyperactivity and interfering repetitive activity. The patient is selected for A|3 peptide analogue therapy in tandem with sacubitril / valsartan therapy.

[0096] An AP peptide analogue solution containing 820 mg of the analogue is administered to the patient by intrathecal infusion. The post-replacement CSF level of Ap42 is measured at 1050 pg / ml one week post-procedure. Oral sacubitril / valsartan therapy is commenced at that time with a fixed dose of 24 mg / 26 mg sacubitril / valsartan given twice daily. The dosage is increased every’ two weeks until a target dose of 72 mg / 78 mg twice daily is reached. CSF A 42 level is measured biweekly during the ramp-up period and monthly afterward for one year. The minimum Ap42 level measured during this period is 1010 pg / ml.

[0097] The claims following this written disclosure are hereby expressly incorporated into the present written disclosure, with each claim standing on its own as a separate embodiment. This disclosure includes all permutations of the independent claims with their dependent claims. Moreover, additional embodiments capable of derivation from the independent and dependent claims that follow are also expressly incorporated into the present written description.

[0098] Without further elaboration, it is believed that one skilled in the art can use the preceding description to utilize the invention to its fullest extent. The claims and embodiments disclosed herein are to be construed as merely illustrative and exemplary, and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having ordinary skill in the art, with the aid of the present disclosure, that changes may be made to the details of the above-described embodiments without departing from the underlying principles of the disclosure herein. In other words, various modifications and improvements of the embodiments specifically^ disclosed in the description above are within the scope of the appended claims. Moreover, the order of the steps or actions of the methods disclosed herein may be changed by those skilled in the art without departing from the scope of the present disclosure. In other words, unless a specific order of steps orDocket No. P321663.WO.01 actions is required for proper operation of the embodiment, the order or use of specific steps or actions may be modified. The scope of the invention is therefore defined by the following claims and their equivalents.REFERENCES

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Claims

Docket No. P321663.WO.01CLAIMSWhat is claimed is:

1. A method of treating an autism spectrum disorder (ASD) in a subject, comprising administering to the subject one or both of: a therapeutically effective amount of an amyloid beta 42 (AP42) peptide analogue; and a therapeutically effective amount of a neprilysin inhibitor.

2. The method of claim 1. wherein the AP42 peptide analogue has a decreased propensity for beta-sheet stacking and amyloid formation relative to wild type AP42 peptide.

3. The method of claim 1 or 2, wherein the Ap42 peptide analogue comprises a C- terminal domain modification comprising one or more of: a deletion of 1-13 amino acids; a C-terminus chemical modification; and a substitution comprising an amino acid, an amino acid analogue, or both.

4. The method of claim 3, wherein the C-terminal domain modification confers on the Ap42 peptide analogue one or more enhancements over wild type Ap42 peptide, where said enhancements are selected from: increased solubility in a liquid medium, increased protease resistance, reduced administration volume, increased circulation time, and increased half-life in cerebrospinal fluid (CSF).

5. The method of any one of claims 1 to 4, wherein the Ap42 peptide analogue retains a function native to wild type Ap42 peptide.

6. The method of claim 5, wherein the native function comprises facilitating a7 nicotinic acetylcholine receptor (a7nAChR) signaling in the subject.

7. The method of any one of claims 1 to 6. wherein the amount of the AP42 peptide analogue per kg of the subject's body weight is about 0.01 mg to about 30 mg, or about 0.1 mg to about 20 mg, or about 1 mg to about 10 mg.Docket No. P321663.WO.

018. The method of any one of claims 1 to 7, wherein the amount of the A042 peptide analogue is sufficient to provide a concentration of the analogue in the CSF of a subject of about 200 pg / ml to about 600 pg / mL.

9. The method of any one of claims 1 to 8, wherein administering the Ap42 peptide analogue is performed via a route selected from subcutaneous, intravenous, intracerebroventricular, intracerebral, intrathecal, intraperitoneal, intramuscular or intravenous injection, infusion, or topical, nasal, oral, rectal, ocular and otic.

10. The method of claim 9, wherein administering the Ap42 peptide analogue is performed via intrathecal injection or intrathecal infusion.

11. The method of claim 10, wherein administering is performed via a cerebrospinal fluid (CSF) exchange using a fluid comprising the Ap42 peptide analogue.

12. The method of claim 11, wherein the fluid is selected from autologous CSF, heterologous CSF, and artificial CSF.

13. The method of claim 1, wherein the neprilysin inhibitor is a biaryl substituted derivative of an aminopropionic acid or of an aminobutyric acid.

14. The method of claim 1, wherein the neprilysin inhibitor is a compound of Formula I:biaryl or a pharmaceutically acceptable ester or salt thereof, wherein: each occurrence of X independently represents hydrogen or derivatization with a pharmaceutically acceptable ester or amide;Ri represents hydrogen, lower alkyl, C3-C7-cycloalkyl-lower alkyl, ary l-lower alkyl, biaryl-lower alkyl, lower alkoxy, aryl-lower alkoxy, aryloxy, N-lower alkylamino, N,N-di-lower alkylamino, N-aryl-lower alkylamino, N,N-di-aryl-lower alkylamino, N- arylamino, N,N-diarylamino, lower alkanoylamino, ary l-lower alkanoylamino or aroyl amino;Docket No. P321663.WO.01R.2 represents hydrogen, hydroxy, lower alkoxy, lower alkyl, aryl-lower alkyl, C3- C?-cycloalkyl-lower alkyl, amino-lower alkyl, hydroxy-lower alkyl, lower alkylthiolower alkyl, lower alkoxy-lower alkyl, aryl-lower alkylthio-lower alkyl or aryl-lower alkoxy-lower alkyl; biaryl represents phenyl substituted by carbocyclic or heterocyclic ary l;A represents a direct bond, lower alkylene, phenylene or cyclohexylene; n represents 1 or zero; and m represents 1 or zero, provided that m represents 1 when A is a direct bond.

15. The method of claim 1, wherein the neprilysin inhibitor is a compound of Formula II:or a pharmaceutically acceptable salt or ester thereof, wherein:R1is Ci-7alkyl; for each occurrence, R2is independently Ci-?alkyl, NO2, CN, halo, C3-?cycloalkyl, hydroxy, Ci-7alkoxy, halo-Ci-7alkyl, NRbRc, Ce-ioaryl, heteroaryl or heterocyclyl; wherein Rband Rcfor each occurrence, are independently H or Ci-?alkyl;R3is A1C(0)X1or A2-R4;R4is Ce-ioaryl or a heteroaryl, which can be monocyclic or bicyclic and which can be optionally substituted with one or more substituents independently selected from hydroxy, hydroxy-Ci-7alkyl, NRbRc, nitro, Ci-7alkoxy, halo, Ci-7alkyl, halo-Ci-7alkyl, C2- 7alkenyl, Ce-ioaryl, heteroaryl, — C(0)Ci-7alkyl, — NHS(O)2-Ci-7alkyl, — SO2Ci-7alkyl and benzyl;R5is H. halo, hydroxy, Ci-7alkoxy, halo, Ci-7alkyl or halo-Ci-7alkyl; andX and X1are independently OH, — O — Ci-7alkyl, — NRbRc, — NHS(O)2 — Cu ?alkyl, — NHS(O)2-benzyl or — O — Ce-ioaryl; wherein alkyl is optionally substituted with one or more substituents independently selected from the group consisting of aryl, heteroaryl, heterocyclyl, — C(0)NH2, — C(0)NH — Ci-ealkyl, and — C(O)N(Ci-6alkyl)2;Docket No. P321663.WO.01A1is: a bond or a linear Ci-4alkylene substituted with one or more substituents independently selected from the group consisting of halo. O-acetate. C1-7 alkyl and C3-7cycloalkyl; in which two geminal alkyl can optionally combine to form a C3- 7cycloalkyl; or a linear or branched Cz-ealkenylene; or a linear C1-4 alkylene wherein one or more carbon atom(s) is / are replaced with an heteroatom selected from O, NRa; and A1is optionally substituted with one or more substituents independently selected from the group consisting of halo and Ci-?alkyl; in which Rafor each occurrence, is independently H, Ci-?alkyl or CH2C(O)OH; or a C -7Cycloalkyl. a heterocyclyl, a phenyl or a heteroaryl in which phenyl and heteroaryl are optionally substituted with one or more substituents independently selected from the group consisting of Ci-?alkyl, C3-7cycloalkyl, halo-Ci-7alkyl, hydroxy, Ci-?alkoxy, halo, NRbRc, OCH2CO2H. and OCH2C(O)NH2; or— Ci-4alkylene-C6-io-aryl-, — Ci-ralkylene-heteroaryl- or — Ci-4alkylene- heterocyclyl-, wherein A1may be in either direction;A2is a bond or a linear or branched Ci-7alkylene which is optionally substituted with one or more substituents independently selected from the group consisting of halo, Ci-?alkoxy, hydroxy, O-acetate and C -7cycloalkyl; and n is 0, 1, 2, 3, 4 or 5; wherein each heteroaryl is a monocyclic or bicyclic aromatic ring comprising 5-10 ring atoms selected from carbon atoms and 1 to 5 heteroatoms, and each heterocyclyl is a monocyclic saturated or partially saturated but non-aromatic moiety comprising 4-7 ring atoms selected from carbon atoms and 1-5 heteroatoms, wherein each heteroatom of a heteroaryl or a heterocyclyl is independently selected from O, N and S.

16. The method of claim 1, wherein the neprilysin inhibitor is a compound of FormulaIII:Docket No. P321663.WO.01or a pharmaceutically acceptable salt or ester thereof, wherein:R1is phenyl optionally substituted by alkyl;R2is hydrogen or alkyl-S(0)o-2(CH2)q;R3is hydrogen or lower alkyl;Q is hydrogen or alkyl-CO-; n is 0-2; and q is 1-4.

17. The method of claim 1, wherein the neprilysin inhibitor is selected from sacubitril, sacubitrilat, thiorphan, retro-thiorphan, acetyl thiorphan, phosphoramidon, 3-{[(2S)-l- {3'-chloro-[l,T-biphenyl]-4-yl}-4-ethoxy-4-oxobutan-2-yl]carbamoyl (propanoic acid, and (3R)-3-(3-carboxypropanamido)-4-{3'-chloro-[l, l'-biphenyl]-4-yl}butanoic acid, and pharmaceutically acceptable salts and esters thereof.

18. The method of claim 17, wherein the neprilysin inhibitor is sacubitril.

19. The method of any one of claims 1 to 18, wherein administering the neprilysin inhibitor is performed daily and the amount is about 20 mg / day to about 500 mg / day, about 40 mg / day to about 300 mg / day. or about 40 mg / day to about 200 mg / day.

20. The method of any one of claims 1 to 19, wherein administering the neprilysin inhibitor is performed via a route selected from subcutaneous, intravenous, intracerebroventricular, intracerebral, intrathecal, intraperitoneal, intramuscular, topical, nasal, oral, rectal, ocular and otic.

21. The method of any one of claims 1 to 20, comprising administering both the A042 peptide analogue and the neprilysin inhibitor.

22. The method of claim 21, wherein the A042 peptide analogue and the neprilysin inhibitor are administered over differing time periods.Docket No. P321663.WO.0123. The method of claim 21 or 22, wherein the Ap42 peptide analogue and the neprilysin inhibitor are administered over overlapping time periods.

24. The method of claim 21 or 22, wherein the A042 peptide analogue and the neprilysin inhibitor are administered over sequential time periods.

25. The method of any one of claims 21 to 24, wherein the neprilysin inhibitor is administered over a time period at least part of which is later than that over which the Ap42 peptide analogue is administered.

26. The method of any one of claims 1 to 25, wherein the subject is under about 30 years of age, under about 25 years of age, under about 20 years of age, under about 15 years of age, under about 10 years of age, or under about 5 years of age.

27. The method of any one of claims 1 to 26, comprising a prior step of identifying the subject as a candidate for treatment based assessing at least one of reduced AP42 level, neurological phenotype, a cognitive or behavioral symptom, a genotype, and the presence of beta-amyloid plaques.

28. The method of claim 27, wherein the cognitive or behavioral symptom is selected from attentional difficulties, hyperactivity, affective difficulties such as anxiety and depression, interfering repetitive activity, irritability, aggression, self-injurious behavior, and sleep disruption.

29. The method of claim 27, wherein the genotype comprises a mutation to a gene selected from CHD8, FMRI, MECP2, NLGN4X, and NLGN3.

30. The method of claim 27, wherein the neurological phenotype comprises brain activity measured by EEG, MRI or PET.

31. The method of claim 27, wherein the neurological phenotype comprises a7nAChR activity.

32. The method of claim 27, wherein the neurological phenotype comprises one or more of macrocephaly, hypoactivation of the fusiform face area, and hypoactivation of the superior temporal sulcus.Docket No. P321663.WO.0133. The method of any one of claims 1 to 32, further comprising modulating empathy in the subject, by modulating type 2 theta oscillations in a brain region of the subject.

34. The method of any one of claims 1 to 33, wherein the subject is a pediatric patient.

35. The method of claim 34, wherein the sacubitril is a monotherapy formulation.

36. The method of claim 34 or 35, wherein the sacubitril is in a combination formulation with an ACE receptor blocker.

37. The method of claim 36, wherein the ACE receptor blocker is valsartan.

38. The method of any one of claims 34 to 37, wherein the pediatric patient has a weight of less than 40 kg and is treated with an initial starting sacubitril dose of 0.4 mg or 0.8 mg by mouth twice per day.

39. The method of claim 38, wherein the sacubitril dose is titrated upward every 3 to 4 weeks to the next highest dose of 0.8 mg / kg, 1.2 mg / kg and to a final dose of 1.6 mg / kg by mouth twice per day.

40. The method of claim 39, wherein the pediatric patient has a weight more than 40 kg but less than 50 kg, and is treated with an initial starting sacubitril dose of 0.8 mg / kg or 24 mg by mouth twice per day.

41. The method of claim 40, wherein the dose is titrated upward every' 3 to 4 weeks to the next highest sacubitril dose of 24 mg, 49 mg, 72 mg and to a final dose of 97 mg by mouth twice per day.

42. The method of any one of claims 34 to 37, wherein the pediatric patient has a weight of at least 50 kg, and is treated with an initial starting sacubitril dose of 24 mg or 49 my by mouth twice daily.

43. The method of claim 42, wherein the dose is titrated upward every' 3 to 4 weeks to the next highest sacubitril dose of 49 mg, 72 mg and to a final dose of 97 mg by mouth twice per day.Docket No. P321663.WO.0144. Use of an amyloid beta 42 (Ap42) peptide analogue, a neprilysin inhibitor, or both for the manufacture of a medicament for the treatment of an autism spectrum disorder (ASD) in a subject.

45. The use of claim 44, wherein the Ap42 peptide analogue has a decreased propensity for beta-sheet stacking and amyloid formation relative to wild type Ap42 peptide.

46. The use of claim 44 or 45, wherein the Ap42 peptide analogue comprises a C- terminal domain modification comprising one or more of: a deletion of 1-13 amino acids; a C-terminus chemical modification; and a substitution comprising an amino acid, an amino acid analogue, or both.

47. The use of claim 46, wherein the C-terminal modification confers on the Ap42 peptide analogue one or more enhancements over wild type Ap42 peptide, where said enhancements are selected from: increased solubility in a liquid medium, increased protease resistance, reduced administration volume, increased circulation time, and increased half-life in cerebrospinal fluid (CSF).

48. The use of any one of claims 44 to 47, wherein the AP42 peptide analogue retains a function native to wild type Ap42 peptide.

49. The use of claim 48, wherein the native function comprises facilitating a7 nicotinic acetylcholine receptor (a7nAChR) signaling in the subject.

50. The use of claim 44, wherein the neprilysin inhibitor is a biaryl substituted derivative of an aminopropionic acid or of an aminobutyric acid.

51. The use of claim 44, wherein the neprilysin inhibitor is a compound of Formula I:biaryl or a pharmaceutically acceptable ester or salt thereof, wherein:Docket No. P321663.WO.01 each occurrence of X independently represents hydrogen or derivatization with a pharmaceutically acceptable ester or amide;Ri represents hydrogen, lower alkyl, Cs-Cr-cycloalkyl-lower alkyl, aryl-lower alkyl, biaryl-lower alkyl, lower alkoxy, aryl-lower alkoxy, aryloxy, N-lower alkylamino, N,N-di-lower alkylamino, N-aryl-lower alkylamino, N,N-di-aryl-lower alkylamino, N- arylamino, N,N-diarylamino, lower alkanoylamino, aryl-lower alkanoylamino or aroyl amino;R.2 represents hydrogen, hydroxy, lower alkoxy, lower alkyl, aryl-lower alkyl, C?- C?-cycloalkyl-lower alkyl, amino-lower alkyl, hydroxy-lower alkyl, lower alkylthiolower alkyl, lower alkoxy -lower alkyl, aryl-lower alkylthio-lower alkyl or ary l-lower alkoxy-lower alkyl; biaryl represents phenyl substituted by carbocyclic or heterocyclic aryl;A represents a direct bond, lower alkylene, phenylene or cyclohexylene; n represents 1 or zero; and m represents 1 or zero, provided that m represents 1 when A is a direct bond.

52. The use of claim 44, wherein the neprilysin inhibitor is a compound of Formula II:or a pharmaceutically acceptable salt or ester thereof, wherein:R1is Ci-7alkyl; for each occurrence, R2is independently Ci-7alkyl, NO2, CN, halo, Cs-vcycloalkyl, hydroxy, Ci-7alkoxy, halo-Ci-7alkyl, NRbRc. Ce-ioaryl, heteroaryl or heterocyclyl; wherein Rband Refor each occurrence, are independently H or Ci-7alkyl;R3is A1C(O)X1or A2-R4;R4is Ce-ioaryl or a heteroaryl, which can be monocyclic or bicyclic and which can be optionally substituted with one or more substituents independently selected from hydroxy. hydroxy-Ci-7alkyl, NRbRc, nitro. Cnralkoxy, halo, Ci-7alkyl. halo-Ci-7alkyl, C2-Docket No. P321663.WO.01 valkenyl, Ce-ioaryl, heteroaryl, — C(O)Ci-?alkyl, — NHS(O)2-Ci-7alkyl, — SO2Ci-7alkyl and benzyl;R5is H. halo, hydroxy, Ci.7alkoxy, halo, Ci-7alkyl or halo-Ci.7alkyk andX and X1are independently OH, — O — Ci-7alkyl, — NRbRc, — NHS(O)2 — Ci- 7alkyl, — NHS(O)2-benzyl or — O — Ce-ioaryl; wherein alkyl is optionally substituted with one or more substituents independently selected from the group consisting of aryl, heteroaryl, heterocyclyl, — C(O)NH2, — C(O)NH — Ci-ealkyl, and — C(O)N(Ci-6alkyl)2;A1is: a bond or a linear Chalky lene substituted with one or more substituents independently selected from the group consisting of halo, O-acetate, C 1-7 alkyl and C3-7cycloalkyl; in which two geminal alkyl can optionally combine to form a C3- 7cycloalkyl; or a linear or branched C2-6alkenylene; or a linear C1-4 alkylene wherein one or more carbon atom(s) is / are replaced with an heteroatom selected from O, NRa; and A1is optionally substituted with one or more substituents independently selected from the group consisting of halo and Ci-7alkyl; in which Rafor each occurrence, is independently H, Ci.7alkyl or CH2C(O)OH; or a C3-7Cycloalkyl, a heterocyclyl, a phenyl or a heteroaryl in which phenyl and heteroaryl are optionally substituted with one or more substituents independently selected from the group consisting of Ci.7alkyl, C3-7cycloalkyl, halo-Ci-7alkyl, hydroxy, Ci-7alkoxy, halo, NRbRc, OCH2CO2H, and OCH2C(O)NH2; or— Ci-4alkylene-C6-io-aryl-, — Ci-4alkylene-heteroaryl- or — Ci-4alkylene- heterocyclyl-, wherein A1may be in either direction;A2is a bond or a linear or branched Ci.7alkylene which is optionally substituted with one or more substituents independently selected from the group consisting of halo, Ci-valkoxy, hydroxy, O-acetate and C3-7Cycloalkyl; and n is 0, 1, 2. 3, 4 or 5; wherein each heteroaryl is a monocyclic or bicyclic aromatic ring comprising 5-10 ring atoms selected from carbon atoms and 1 to 5 heteroatoms, and each heterocyclyl is a monocyclic saturated or partially saturated but non-aromatic moiety comprising 4-7 ring atoms selected from carbon atoms and 1-5 heteroatoms,Docket No. P321663.WO.01 wherein each heteroatom of a heteroaryl or a heterocyclyl is independently selected from O, N and S.

53. The use of claim 44, wherein the neprilysin inhibitor is a compound of Formula III:or a pharmaceutically acceptable salt or ester thereof, wherein:R1is phenyl optionally substituted by alkyl;R2is hydrogen or alkyl-S(0)o-2(CH2)q;R3is hydrogen or lower alkyl;Q is hydrogen or alkyl-CO-; n is 0-2; and q is 1-4.

54. The use of claim 44, wherein the neprilysin inhibitor is selected from sacubitril, sacubitrilat, thiorphan, retro-thiorphan, acetyl thiorphan, phosphoramidon, 3-{[(2S)-l- {3'-chloro-[l,l'-biphenyl]-4-yl}-4-ethoxy-4-oxobutan-2-yl]carbamoyl}propanoic acid, and (3R)-3-(3-carboxypropanamido)-4-{3'-chloro-[l, l'-biphenyl]-4-yl}butanoic acid, and pharmaceutically acceptable salts and esters thereof.

55. The use of claim 54, wherein the neprilysin inhibitor is sacubitril.

56. A method of restoring and / or improving a7 nicotinic acetylcholine receptor (a7nAChR) activity in a subject in need thereof, comprising administering to the subject an agent configured to increase expression of a7nAChR or a variant thereof in the central nervous system of the subject.

57. The method of claim 56, wherein the agent comprises a nuclease selected from Cas9, a Transcription Activator-Like Effector Nuclease (TALEN), a Zinc Finger Nuclease (ZFN), and a meganuclease.Docket No. P321663.WO.0158. The method of claim 56, wherein the agent comprises a nucleic acid encoding a nuclease selected from Cas9, a TALEN, a ZFN, and a meganuclease.

59. The method of claim 57 or 58, wherein said nuclease is specific to a target sequence that includes a mutation in the CHRNA7 gene.

60. The method of claim 59, further including administering a nucleic acid molecule configured for homologous recombination with the target sequence.

61. The method of claim 60, wherein the nucleic acid molecule comprises a sequence having at least 70%. 75%, 80%, 85%, 90%, 95%, 98%. or 99% identity to SEQ ID NO: 1.

62. The method of any one of claims 56 to 61, wherein the variant comprises a protein sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO 2.

63. The method of any one of claims 56 to 61, wherein the variant comprises a modified a7 protein sequence including at least one substitution selected from D64A, D66A and K68A.

64. The method of claim 63, wherein the modified a7 protein sequence is one of SEQ ID NOS:3-9.

65. The method of any one of claims 56 to 61, wherein the variant comprises a modified a7 protein sequence including an L270T substitution.

66. The method of claim 65, wherein the modified a7 protein sequence is SEQ ID NOTO.

67. The method of any one of claims 56 to 66, wherein administration is performed via a delivery' vector selected from compositions for passive and / or active transport to cells such as plasmids, virus-based recombinant vectors such as AAV and lentivirus vectors, non-virus-based systems such as liposomes and lipid nanoparticles, and viruslike particles.

68. The method of any one of claims 56 to 67, wherein a route of administration is selected from parenteral, intrapulmonary and intranasal, and intralesional.Docket No. P321663.WO.0169. The method of claim 68, wherein the parenteral route is selected from intramuscular, intravenous, intrathecal, intraarterial, intraperitoneal and subcutaneous.

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