Gene Therapy for Alzheimer's Disease

Abstract

The present disclosure specifically provides human codon-optimized sequences encoding presenilin 1 and methods for using said sequences in gene therapy to treat neurodegenerative diseases, including, but not limited to, Alzheimer's disease, frontotemporal dementia, frontotemporal lobar degeneration, Pick's disease, dementia with Lewy bodies, memory loss, and cognitive impairment, including mild cognitive impairment (MCI).

Description

Technical Field

[0001] Claims of Priority This application claims the benefit of U.S. Provisional Patent Application No. 62 / 852,716, filed May 24, 2019. The entire contents of the foregoing are incorporated herein by reference.

[0002] Research or Development by Federal Support This invention was made with government support under grant number NS041783 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0003] Compositions and methods for treating Alzheimer's disease (AD) and other neurodegenerative diseases using presenilin gene therapy constructs are particularly described herein.

Background Art

[0004] Alzheimer's disease, also known as Alzheimer disease, accounts for the majority of neurodegenerative dementias and is the fourth leading cause of death in the United States after heart disease, cancer, and stroke. It is characterized by the progressive loss of cognitive function, neurodegeneration, neurofibrillary changes, and amyloid plaques in the patient's brain. The rate of progression varies from patient to patient, but the average life expectancy after diagnosis is 3 to 9 years. Currently, there is no treatment for Alzheimer's disease.

Summary of the Invention

Means for Solving the Problems

[0005] Methods and compositions useful for treating subjects having Alzheimer's disease (AD) and other neurodegenerative diseases, disorders or conditions are described herein. The present disclosure is based, at least in part, on the unexpected finding that supplying codon-optimized wild-type PSEN1 cDNA to cells carrying a heterologous or homozygous dominant-negative Psen1 mutation, an established familial Alzheimer's disease model, results in unexpectedly high expression levels and restores impaired γ-secretase activity in such cells. Accordingly, the present disclosure provides methods for gene therapy effective for Alzheimer's disease and other neurodegenerative dementias based on PSEN1 (for PS1 expression) and / or PSEN2 (for PS2 expression), representing a major breakthrough in this disease area.

[0006] Compositions are provided herein that include a human codon-optimized polynucleotide encoding the human presenilin 1 protein (PS1), e.g., SEQ ID NO: 9, or a polynucleotide comprising a sequence that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9 (wherein at least one codon is optimized relative to the wild type). Exemplary human PS1 protein sequences include SEQ ID NOs: 5 and 6, and sequences that include at least therein a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1). In some embodiments, the composition is accompanied by exosomes or lipid-based nanoparticles (LNPs) (e.g., formulated using the same for delivery).

[0007] Compositions are also provided herein that include a vector for expressing human PSEN1 in a cell, the vector including the human codon-optimized polynucleotide described herein operably linked to a promoter.

[0008] Use of any of the compositions described herein in a method for treating a neurodegenerative disease, disorder, or condition in a subject is also provided herein.

[0009] In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector (such as AAV9 or AAVrh10), a lentiviral vector, or a retroviral vector.

[0010] In some embodiments, the promoter is a pan-neural promoter, such as the synapsin I promoter, or a neuron subtype-specific promoter, such as the alpha-calcium / calmodulin kinase 2A promoter.

[0011] Also provided herein is a method for treating a neurodegenerative disease, disorder, or condition, the method comprising administering to a human subject in need of treatment a composition described herein, wherein the subject has one or more mutations in at least one allele of PSEN1, preferably a mutation encoding a dominant-negative PSEN1 protein isoform.

[0012] In some embodiments, the neurodegenerative disease, disorder or condition is Alzheimer's disease.

[0013] In some embodiments, the Alzheimer's disease is familial Alzheimer's disease. In some embodiments, the Alzheimer's disease is late-onset Alzheimer's disease. In some embodiments, the Alzheimer's disease is sporadic Alzheimer's disease. In some embodiments, the Alzheimer's disease is early-onset Alzheimer's disease.

[0014] In some embodiments, the subject has a mutation at E280, Y115, L166, C410, Δex9, G548, D257, R278, L435, G384, L392, N141, G206, H163, A79, S290, A260, A426, A431, R269, L271, C1410, E280, P264, E185, L235, M146, for example, an E280A, Y115H, L166P, C410Y, Δex9, G548, D257A, R278I, L435F, G384A, or L392V mutation in the PSEN1 gene, or an N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V, or M146V mutation in the PSEN1 gene.

[0015] In some embodiments, the neurodegenerative disease, disorder or condition is frontotemporal dementia, memory loss, cognitive decline or cognitive impairment. In some embodiments, the cognitive impairment is mild cognitive impairment (MCI).

[0016] In some embodiments, the composition is administered to the CNS of a subject in need of treatment.

[0017] In some embodiments, a polynucleotide encoding the PSEN1 and / or PSEN2 gene or mRNA is administered to the CNS by intravenous delivery, intrathecal delivery, intracisternal delivery, intraventricular delivery, or stereotactic parenchymal injection into a specific brain region, optionally into the ventricle, or direct injection into the hippocampus or cerebral cortex.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All published documents, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0019] Other features and advantages of the present invention will become apparent from the following detailed description of the invention, the figures, and the claims.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2-1

Figure 2-2

Figure 3

Figure 4

[0021] **DEFINITIONS** To more readily understand the present disclosure, certain terms are first defined as follows. Further definitions of the following terms and other terms are described throughout this specification.

[0022] Administration: As used herein, the term "administration" refers to the delivery or application of a composition to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route. For example, in some embodiments, administration is by bronchial (including by bronchial instillation), buccal, enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by tracheal instillation), transdermal, vaginal, and intravitreal administration.

[0023] Biologically active: As used herein, the phrase "biologically active" refers to the property of any substance that has activity in a biological system (e.g., a cell culture, an organism, etc.). For example, when administered to an organism, a substance that has a biological effect on that organism is considered to be biologically active. Also, biological activity can be determined by in vitro assays (e.g., in vitro enzyme assays). In certain embodiments, when a protein or polypeptide is biologically active, a portion of the protein or polypeptide that shares at least one biological activity of this protein or polypeptide is typically referred to as a "biologically active" portion. In some embodiments, a protein is produced and / or purified from a cell culture system that exhibits biological activity when administered to a subject.

[0024] Control: As used herein, the term "control" has the meaning understood in the art as a standard against which results are compared. Typically, controls are used to isolate variables and to increase the integrity in experiments by drawing conclusions about such variables. In some embodiments, the control is a reaction or assay that is performed concurrently with the test reaction or assay to provide a comparison. In one experiment, a "test" (i.e., the variable to be tested) is applied. In a second experiment, the "control", which is the variable to be tested, is not applied. In some embodiments, the control is a historical control (i.e., a test or assay performed previously, or an amount or result known previously). In some embodiments, the control is, or includes, a printed or stored record. The control can be a positive control or a negative control. In some embodiments, the control can be a "reference control" that is a sample used for comparison with a test sample to examine differences or for the purpose of characterization.

[0025] Gene Therapy: As used herein, the term "gene therapy" refers to any treatment that involves the direct or indirect administration of nucleic acids to a subject. In certain instances, a protein having therapeutic value is expressed from the administered nucleic acid.

[0026] Identity: As used herein, the term "identity" refers to overall relatedness between polymers, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. For example, the calculation of the percentage of identity between two nucleic acid sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., introducing gaps into one or both of the first and second nucleic acid sequences for optimal alignment and ignoring non-identical sequences for comparison purposes). In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at this position. Considering the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences. The comparison of sequences and determination of the percentage of identity between two sequences can be achieved using a mathematical algorithm. For example, the percentage of identity between two nucleotide sequences can be determined using the Meyers and Miller algorithm (CABIOS, 1989, 4: 11-17), which is incorporated into the ALIGN program (version 2.0) and uses the PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage of identity between two nucleotide sequences can be determined using the GAP program of the GCG software package and using the NWSgapdna.CMP matrix. For example, various other sequence alignment programs such as Clustal are available and can be used for the determination of sequence identity.

[0027] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or grammatical equivalents thereof, refer to a value relative to a baseline measurement, e.g., a measurement in the same individual prior to the start of treatment described herein, or a measurement in a control individual (or individuals) in the absence of the treatment described herein. A “control individual” is of the same type as the individual being treated, is an individual suffering from, e.g., Alzheimer's disease, of approximately the same severity, and is approximately the same age as the individual being treated (to ensure that the disease stages of the individual being treated and the control individual(s) are comparable).

[0028] Neurodegeneration: As used herein, the term “neurodegeneration” means a process in which one or more nerve cells are damaged, lose function, become dysfunctional, and / or are lost through cell death. Neurodegeneration encompasses both rapid and progressive forms and intermediate forms. Thus, neurodegenerative diseases, conditions, or symptoms are characterized in that the disease is typically associated with nerve cell damage and / or cell death.

[0029] Subject: As used herein, the term “subject” refers to a human or any non-human animal (e.g., a mammal such as a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Humans include both prenatal and postnatal forms. In many embodiments, the subject is a human. A subject may be a patient, referring to a human who visits a medical institution for diagnosis or treatment of a disease. The term “subject” is used interchangeably herein with “individual” or “patient.” A subject may have or be susceptible to a disease or disorder, but it is not known whether the subject exhibits symptoms of the disease or disorder.

[0030] suffering from~ An individual “suffering from” a disease, disorder, and / or condition (e.g., Alzheimer's disease) has been diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0031] sensitive to ~ An individual who is "sensitive to" a disease, disorder, and / or condition may not have been diagnosed with and / or may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is sensitive to a disease, disorder, and / or condition (e.g., Alzheimer's disease) may be characterized by one or more of: (1) a genetic mutation associated with the onset of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the onset of the disease, disorder, and / or condition; (3) an increase and / or decrease in the expression and / or activity of a protein associated with the disease, disorder, and / or condition; (4) a habit and / or lifestyle associated with the onset of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; (6) a reaction to a particular bacterium or virus; (7) exposure to a particular chemical substance. In some embodiments, an individual who is sensitive to a disease, disorder, and / or condition develops the disease, disorder, and / or condition. In some embodiments, an individual who is sensitive to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0032] therapeutically effective amount As used herein, the term "therapeutically effective amount" refers to the amount of a therapeutic protein that confers a therapeutic effect on a treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect can be objective (i.e., measurable by some test or marker) or subjective (i.e., the subject exhibits signs of, or feels, the effect). In particular, a "therapeutically effective amount" is effective to treat, cure, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventive effect, for example, by reversing symptoms associated with the disease, preventing or delaying the onset of the disease, and / or similarly, reducing the severity or frequency of symptoms of the disease. A therapeutically effective amount is generally administered by a dosing regimen that may include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and / or the appropriate unit dose in an effective dosing regimen) can vary, for example, depending on the route of administration, combination with other pharmaceuticals, etc. Also, the particular therapeutically effective amount (and / or unit dose) for any particular patient depends on various factors including the disorder being treated and the severity of the disorder; the activity of the particular pharmaceutical being utilized; the particular composition being utilized; the patient's age, weight, general health, sex and diet; the time of administration, route of administration, and / or the excretion or metabolic rate of the particular fusion protein being utilized; the duration of the treatment; and similar factors well known in the medical arts.

[0033] Treatment: As used herein, the term "treatment" (also "treat" or "treating") in its broad sense refers to any administration of a substance (e.g., the provided composition) that partially or completely alleviates, restores, mitigates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment can be administered to a subject who does not exhibit signs of the associated disease, disorder and / or condition, and / or to a subject who exhibits only initial signs of the disease, disorder, and / or condition. Alternatively, or in addition, in some embodiments, treatment can be administered to a subject who exhibits one or more established signs of the associated disease, disorder and / or condition. In some embodiments, treatment can be treatment of a subject diagnosed as having the associated disease, disorder, and / or condition. In some embodiments, treatment can be treatment of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the associated disease, disorder, and / or condition.

[0034] Generally speaking, "PS1" refers to the presenilin 1 protein and "PS2" refers to the presenilin 2 protein, although in some instances, PS1 or PS2 is used to refer to the mRNA or gene.

[0035] Detailed Description The present disclosure particularly provides compositions and methods for treating subjects having Alzheimer's disease and other neurodegenerative diseases, disorders and conditions, based on delivery of functional presenilin-1 (PS1) protein to subjects in need thereof. In particular, the present disclosure contemplates gene therapy by providing a human codon-optimized polynucleotide encoding presenilin-1 (PS1) to a subject in need of treatment having a PSEN1 or PSEN2 mutation, such as a dominant negative mutation, associated with AD, such as early onset familial AD (FAD) or late onset sporadic AD.

[0036] Various aspects of the present invention are described in detail in the following sections. The use of these sections is not meant to limit the present invention. Each section can be applied to any aspect of the present invention. In such application, the use of "or" means "and / or" unless stated otherwise.

[0037] Methods of treatment As a non-limiting example, the methods of the present invention include, for example, gene therapy for expressing wild-type human presenilin 1 in a subject (e.g., a familial AD patient or a sporadic AD patient carrying a PSEN1 or PSEN2 mutation) suffering from or susceptible to a neurodegenerative disease associated with a dominant-negative mutation in PSEN1 or PSEN2, such as Alzheimer's disease. The purpose of such gene therapy is, in particular, to promote the expression of PS1 in the brains of familial or sporadic AD patients and correct or overcome defects in the expression and / or activity of PS1 or PS2. In FAD patients, it is expected that the gene therapy methods described herein will result in an increase in the expression of wild-type PS1 in the brain and the restoration of the functional impairment of γ-secretase activity associated with the PS1 or PS2 mutation.

[0038] Mutations in the presenilin genes, PSEN1 and PSEN2, are highly penetrant and account for approximately 90% of all mutations identified in familial AD (FAD), underscoring their importance in AD pathogenesis. Over 260 distinct mutations have been reported in PSEN1, which are autosomal dominant and mostly missense mutations. Dominant-negative mutations in the PSEN1 and PSEN2 genes are known to be associated with early-onset familial Alzheimer's disease. The PS1 and presenilin 2 (PS2) proteins are part of the γ-secretase complex, and mutations in the PSEN1 and PSEN2 genes are generally thought to contribute to the accumulation of amyloid-beta (Aβ) protein in Alzheimer's disease patients. Pathogenic PSEN1 mutations act in cis to disrupt mutant PS1 function and in trans to inhibit wild-type presenilin 1 (PS1) function (Heilig et al. J Neurosci 33:11606-717 (2013), Zhou et al. Proc Natl Acad Sci USA 114:12731-12736 (2017)). Typically, by their very nature, dominant-negative mutations cannot be rescued by the expression of the wild-type protein (Herskowitz, I. Nature, 329:219-222 (1987)). However, surprisingly, as shown herein, transfection of immortalized MEFs carrying heterozygous or homozygous PS1 mutations with codon-optimized hPSEN1 cDNA can much more readily restore γ-secretase activity impaired in such cells than the wild-type human sequence (see the following examples), indicating that the dominant-negative action of the mutant presenilin protein can be overcome by the expression of wild-type PS1 from the codon-optimized exogenous sequence. Without wishing to be bound by any particular theory, the expression of PS1 may achieve this purpose by raising the total level of wild-type PS1, restoring the functional impairment of γ-secretase expression and / or activity in AD patients.

[0039] The methods and compositions described herein can similarly be used to treat other neurodegenerative diseases, disorders or conditions.

[0040] Alzheimer's disease The methods described herein can be used to treat or reduce the risk of developing subjects having any type of Alzheimer's disease, including but not limited to familial and sporadic Alzheimer's, early-onset or late-onset Alzheimer's disease. In some embodiments, the methods of the invention can be used to treat or reduce the risk of developing early-onset familial Alzheimer's disease (AD) associated with mutations in presenilin 1 (PS1) and / or presenilin 2 (PS2) (Sherrington, et al., Nature 375:754-760 (1995); Rogaev, et al., Nature 376:775-778 (1995); Levy-Lahad, et al., Science 269:970-973 (1995); Hiltunen, et al., Eur. J. Hum. Genet. 8:259-266 (2000); Jonghe, et al., Hum. Mol. Genet. 8:1529-1540 (1999); Tysoe, et al., Am. J. Hum. Genet. 62:70-76 (1998); Crook, et al., Nat. Med. 4:452-455 (1998), all of which are incorporated herein by reference).

[0041] In some embodiments, the methods of the invention can be used to treat a subject having a mutation in the PSEN1 or PSEN2 allele, e.g., a mutation having a dominant negative effect on the wild-type PS protein. Exemplary mutations include C410Y, Δex9, G548, D257A, L166P, R278I, L435F, G384A, Y115H, and L392V, as well as N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V, and M146V mutations (see, e.g., Heilig et al., J. Neurosci., 33(28):11606-11617 (2013); Watanabe et al., J. Neurosci. 32(15):5085-5096 (2012); Brouwers et al., 2008 Ann Med 40 (8): 562-83); Watanabe and Shen, PNAS November 28, 2017 114 (48) 12635-12637; Zhou et al., PNAS November 28, 2017 114 (48) 12731-12736; Hsu et al., Alzheimers Res Ther. 2018 Jul 18;10(1):67). Further exemplary mutations that can have a dominant negative effect on the wild-type PS protein include, in PSEN1: N32N; R35Q; D40del (delGAC); D40del (delACG); E69D; A79V; V82L; I83_M84del (DelIM, ΔI83 / M84, ΔI83 / ΔM84); I83T; M84V; L85P; P88L; V89L (G>T); V89L (G>C); C92S; V94M; V96F; V97L; T99A; F105C; F105I; F105L; F105V; R108Q; L113_I114insT (Intron4, InsTAC, p.113+1delG, splice5); L113P; L113Q; Y115C; Y115D; Y115H; T116I; T116N; T116R; P117A; P117L; P117R; P117S; E120D (A>C);E120D(A>T); E120G; E120K; E123K; Q127_R128del(CAGA); InsG(G)(c.379_382delXXXXinsG); H131R; S132A; L134R; N135D; N135S; N135Y; A136G; M139I(G>C); M139I(G>A); M139K; M139L; M139T; M139V; V142F; I143F; I143M; I143N; I143T; I143V; M146I(G>C); M146I(G>T); M146I(G>A); M146L(A>C); M146L(A>T); M146V; T147I; T147P; L150P; L153V; Y154C; Y154N; Y156F; Y156_R157insIY; R157S; H163P; H163R; H163Y; A164V; W165C(G>C); W165C(G>T); W165G; L166H; L166P; L166R; L166V; L166del; I167del(TTAdel); I167del(TATdel); I168T; S169del(ΔS169、Ser169del、ΔS170); S169L; S169P; S170F; S170P; L171P; L173F(G>C); L173F(G>T); L173W; L174del; L174M; L174R; F175S; F176L; F177L; F177S; S178P; G183V; E184D; E184G; V191A; I202F; G206A; G206D; G206S; G206V; G209A; G209E; G209R; G209V; S212Y; I213F; I213L; I213T; H214D; H214N; H214Y; G217D; G217R; L219F; L219P; L219R; R220P; Q222H; Q222P; Q222R; Q223R; L226F; L226R; I229F; S230I; S230N; S230R; A231P; A231T; A231V; L232P; M233I(G>A); M233I(G>C); M233L(A>T); M233L(A>C); M233T; M233V; L235P; L235R; L235V; F237I; F237L; I238M; K239N; T245P; A246E; A246P; L248P; L248R; L250F; L250S; L250V; Y256S; A260V; V261F; V261L;L262F; L262V; C263F; C263R; P264L; G266S; P267A; P267L; P267S; R269G; R269H; L271V; V272A; E273A; E273G; T274R; A275V; R278I; R278K; R278S; R278T; E280A; (Paisa); E280G; E280K; L282F; L282R; L282V; F283L; P284L; P284S; A285V; L286P; L286V; T291A; T291P; K311R; E318G; D333G; R352C; R352_S353insR; T354I; R358Q; S365A; S365Y; R377M; R377W; G378E; G378V; G378fs; L381F; L381V; G384A; F386I; F386S; F388L; S390I; S390N; V391F; V391G; L392P; L392V; G394V; A396T; N405S; I408T; A409T; C410Y; V412I; I416T; G417S; L418F; L420R; L424F; L424H; L424R; L424V; A426P; A431E; (Jalisco); A431V; A434C; A434T; L435F; P436Q; P436S; I437V; I439S; I439V; T440del; 869-2A>G; 869-22_869-23ins18(ΔE9, Δ9, deltaE9); I238_K239insI; S290C; T291_S319del(ΔE9Finn, Δ9Finn, Δ9); S290C; T291_S319del(ΔE9, Δ9); S290C; T291_S319del A>G(ΔE9, Δ9); S290C; T291_S319del G>A(ΔE9, Δ9); S290C; T291_S319del G>T(ΔE9, Δ9); or S290W; S291_R377del(Δ9-10, Delta9-10, p.Ser290_Arg377delinsTrp, g.73671948_73682054del)(the mutation is named relative to Uniprot P49768.1 / GenBank accession number NM_000021.4), and in PSEN-2: T18M; R29H; G34S; R62C; R62H; P69A; R71W; K82R; A85V; V101M; K115Efs*;Mutations include, but are not limited to, T122P; T122R; P123L; E126fs; E126K; S130L; V139M; N141I (Volga German); N141Y; L143H; V148I; K161R; R163H; H169N; M174V; S175C; G212V; V214L; Q228L; Y231C; I235F; A237V; L238F; L238P; M239I; M239V; A252T; A258T; T301M; K306fs; P334A; P334R; P348L; A377V; V393M; T430M; or D439A (the mutations are named relative to Uniprot P49810.1 / GenBank accession number NP_000438.2). See, for example, Sun et al., Proc Natl Acad Sci USA. 2017;114:E476-E485; Heilig et al., J Neurosci. 2013 Jul 10;33(28):11606-17; Zhou et al., PNAS November 28, 2017 114 (48) 12731-12736. In some embodiments, the method can include determining that the subject has such a mutation, for example, using methods known in the art. In some embodiments, the subject has the mutations described herein (e.g., identified as having the mutations described herein using methods known in the art) and optionally has a family history of AD and / or one or more symptoms of AD, and the subject is treated using the methods described herein. In some embodiments, the subject does not yet have full-blown AD.;

[0042] Typically, increased forgetfulness or mild confusion are early symptoms of Alzheimer's disease. Gradually, the cognitive impairment associated with Alzheimer's disease results in memory loss, particularly short-term memory, disorientation and misunderstanding of spatial relationships; difficulties in language, description, thought, and reasoning; changes in personality and behavior resulting in depression, anxiety, withdrawal, mood swings, distrust of others, irritability and aggression; changes in sleep habits, wandering, loss of inhibition, delusions, and ultimately death.

[0043] Other neurodegenerative diseases, disorders or conditions In addition to Alzheimer's disease, the methods of the invention can be used to treat other neurodegenerative diseases, disorders or conditions, including frontotemporal dementia, various types of memory loss, cognitive disorders including, but not limited to, mild cognitive impairment (MCI), or other conditions associated with deletion of PS1 or PS2, such as those caused by mutations in PSEN1 or PSEN2, which generate, for example, a dominant negative isoform.

[0044] Codon-optimized presenilin 1 (PSEN1) Polynucleotides encoding codon-optimized presenilin 1 (PSEN1) suitable for use in the compositions and methods described herein can include full-length cDNA or portions or fragments thereof that encode a protein that retains the gamma-secretase activity of the wild-type protein, e.g., at least 50% of the gamma-secretase activity, or at least 60, 70, 80, 90, or 95%, or greater than 100% of the activity of the wild-type protein, as determined by an in vitro gamma-secretase assay, including, for example, the assays described in the Examples section (see also Watanabe et al., J. Neurosci. 32(15):5085-5096 (2012)). In some embodiments, a suitable codon-optimized PSEN1 encodes a protein sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the full-length wild-type PS1 or PS2 protein sequence, respectively. Examples of wild-type genomic, cDNA or protein sequences for human PSEN1 / PS1 or PSEN2 / PS2 are provided in Table 1 and the following As shown. PS1 is usually cleaved into active N- and C-terminal fragments. PS1 is processed to yield two fragments, an N-terminal fragment of 28 kDa and a C-terminal fragment of 18 kDa, and major intracellular proteolytic cleavage occurs at and near Met298 in the proximal part of a large hydrophilic loop (Podlisny et al., Neurobiol Dis. 1997;3(4):325-37; Marambaud et al., EMBO J. 2002 Apr 15;21(8):1948-56). Also, sequences that contain or encode such cleaved forms, for example, the sequences encoding amino acids 1-291, 1-292, 1-293, 1-294, 1-295, 1-296, 1-297, 1-298 or 1-299 of SEQ ID NO: 5, or the corresponding fragments of SEQ ID NOs: 6-8, can be used in the methods and compositions described herein.

[0045]

Table 1

[0046] >NM_000021.3 Homo sapiens presenilin 1 (PSEN1), transcript variant 1, mRNA (SEQ ID NO: 1)

[0047]

Chemical

[0048]

Chemical

[0049]

Chemical

[0050] >NM_007318.2 Homo sapiens presenilin 1 (PSEN1), transcript variant 2, mRNA (SEQ ID NO: 2)

[0051]

Chem.

[0052]

Chem.

[0053]

Chem.

[0054] >NM_000447.2 Homo sapiens presenilin 2 (PSEN2), transcript variant 1, mRNA (SEQ ID NO: 3)

[0055]

Chem.

[0056] >NM_012486.2 Homo sapiens presenilin 2 (PSEN2), transcript variant 2, mRNA (SEQ ID NO: 4)

[0057]

Chem.

[0058] >NP_000012.1 Presenilin 1 isoform I-467 [Homo sapiens] (SEQ ID NO: 5)

[0059]

Chem.

[0060] >NP_015557.2 Presenilin 1 isoform I-463 [Homo sapiens] (SEQ ID NO: 6)

[0061] [Chemical]

[0062] >NP_000438.2 Presenilin 2 Isoform 1 [Homo sapiens] (Accession No. 7)

[0063] [Chemical]

[0064] >NP_036618.2 Presenilin 2 Isoform 2 [Homo sapiens] (Accession No. 8)

[0065] [Chemical]

[0066] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, for purposes of optimal comparison, the sequences are aligned (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for purposes of comparison). The length of the reference sequence to be aligned for purposes of comparison is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100% of the length. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position. Considering the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences. In another embodiment, the percentage of identity between two amino acid sequences can be evaluated as a function of the conservation of amino acid residues within the same amino acid family at the corresponding positions of both amino acid sequences (e.g., positive charge, negative charge, polarity and uncharged, hydrophobicity) (e.g., the presence of an alanine residue in place of a valine residue at a particular position in both sequences indicates a high level of conservation, while the presence of an arginine residue in place of an aspartic acid residue at a particular position in both sequences indicates a low level of conservation).

[0067] For example, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:444-453), which is incorporated into the GAP program of the GCG software package, using the BLOSUM scoring matrix and, for example, an initial value for the gap penalty, a gap extension penalty of 4, and a frameshift gap penalty.

[0068] Codon-optimized presenilin 1 Codon optimization is desirable for expressing proteins in specific host cells, such as bacterial, mouse, and human cells. As a result of the degeneracy of the genetic code, those skilled in the art will understand that it is possible to generate a number of cDNAs encoding human presenilin 1, some of which have minimal similarity to the cDNA of any known and natural gene. Accordingly, the present invention contemplates each and every possible cDNA variation that can result from the selection of combinations based on possible codon selections. These combinations are made according to the standard triplet genetic code as applied to polynucleotides encoding natural human presenilin variants, and it is contemplated that all such variations are specifically disclosed. Representative codon-optimized human PSEN1 nucleotide sequences are disclosed herein, for example, SEQ ID NO: 9. See Figure 2. This codon-optimized human PSEN1 nucleotide sequence was generated by replacing codons within the natural PSEN1 nucleotide sequence that occur at low frequency in human cells with codons that occur at high frequency in human cells. Codon-optimized human PSEN1 nucleotide sequences include sequences in which less than 100% of the codons are optimized, for example, sequences in which only 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the wild-type non-optimized codons are optimized. The frequency of occurrence of codons can be computationally determined by methods known in the art. Representative calculations of these codon frequencies are disclosed in Table 1.

[0069] Representative codon-optimized human PSEN1 sequences are as follows. > Codon-optimized Homo sapiens presenilin 1 (PSEN1) cDNA (SEQ ID NO: 9)

[0070]

Chemical Formula

[0071]

Table 2-1

[0072]

Table 2-2

[0073] Mutant presenilin 1 In some embodiments, the PS1 protein comprises a mutation. In some embodiments, the mutation is a conservative substitution. Such mutations include substitutions of any of isoleucine (I), valine (V), and leucine (L) with any of the other of such hydrophobic amino acids, substitution of aspartic acid (D) with glutamic acid (E) and vice versa, substitution of glutamine (Q) with asparagine (N) and vice versa, and substitution of serine (S) with threonine (T) and vice versa. Also, other substitutions can be considered conservative depending on the environment and role of the particular amino acid in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can often be exchanged with leucine and isoleucine, and optionally valine. Lysine (K) and arginine (R) are often interchangeable at positions where the prominent feature of the amino acid residue is this charge and the different pKs of such two amino acid residues are not prominent. Still other mutations can be considered “conservative” in a particular environment (see, e.g., Table III of US20110201052; pages 13-15 “Biochemistry” 2nd ED. Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

[0074] In some embodiments, the method includes introducing one or more additional mutations into the human PS1 sequence (SEQ ID NO: 5 or 6). Thus, in some embodiments, the sequence can be at least 60%, 70%, 80%, 90% or 100% identical to human PS1 and at least 80%, 85%, 90%, 95% or 99% identical.

[0075] To determine the percentage identity between two amino acid sequences or two nucleic acid sequences, for purposes of optimal comparison, the sequences are aligned (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for purposes of comparison). The length of the reference sequence aligned for purposes of comparison is typically at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100% of the length. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at this position (as used herein, "identity" of an amino acid or nucleic acid is equivalent to "homology" of an amino acid or nucleic acid). Considering the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap, the percentage identity between the two sequences is a function of the number of identical positions shared by the sequences. In another embodiment, the percentage identity between two amino acid sequences can be evaluated as a function of the conservation of amino acid residues within the same amino acid family at corresponding positions in both amino acid sequences (e.g., positive charge, negative charge, polarity and uncharged, hydrophobicity) (e.g., the presence of an alanine residue instead of a valine residue at a particular position in both sequences indicates a high level of conservation, while the presence of an arginine residue instead of an aspartic acid residue at a particular position in both sequences indicates a low level of conservation).

[0076] For the method of the present invention, comparison of sequences and determination of the percentage of identity between two sequences can be achieved using the Blossum 62 score matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0077] Delivery vector The codon-optimized nucleic acid encoding the PS1 polypeptide or a therapeutically active fragment thereof can be incorporated into a gene construct and used as part of a gene therapy protocol. For example, targeting of an expression vector for in vivo delivery and expression of the codon-optimized polynucleotide encoding the PS1 polypeptide or an active fragment thereof in specific cell types, particularly cortical neurons, are described herein. Expression constructs of such components can be administered by any carrier effective, e.g., any formulation or composition capable of effectively delivering a genetic component to cells in vivo. The method includes inserting the gene into a viral vector, preferably an adeno-associated virus. The viral vector typically transduces cells directly.

[0078] It is possible to utilize viral vectors capable of highly efficient transduction into CNS neurons, including rAAV (e.g., AAV1 - AAV12) vectors of any serotype, recombinant or chimeric AAV vectors, as well as lentiviral or other suitable viral vectors. In some embodiments, the codon - optimized polynucleotide encoding PS1 is operably linked to a promoter suitable for expression in the CNS. For example, a neuron subtype - specific promoter, such as the alpha - calcium / calmodulin kinase 2A promoter, can be used to target excitatory neurons. Alternatively, a pan - neuronal promoter, such as the synapsin I promoter, can be used to induce PS1 expression. Other exemplary promoters include the cytomegalovirus (CMV) immediate - early enhancer / promoter; the hybrid CMV enhancer / avian beta - actin (CBA) promoter; a promoter comprising a CMV immediate - early enhancer element, the first exon and the first intron of the avian beta - actin gene, and the splice acceptor site of the rabbit beta - globin gene (commonly referred to as the "CAG promoter"); or a 1.6 kb hybrid promoter consisting of the CMV immediate - early enhancer and the CBA intron 1 / exon 1 (commonly referred to as the CAGGS promoter; Niwa et al. Gene, 108:193 - 199 (1991)), but is not limited thereto. The CAGGS promoter (Niwa et al., 1991) has been found to provide ubiquitous and long - term expression in the brain (Klein et al., Exp. Neurol. 176:66 - 74 (2002)). One approach for introducing nucleic acids into cells in vivo is by the use of viral vectors having a nucleic acid, such as a codon - optimized cDNA encoding PS1. In particular, infection of cells with viral vectors has the advantage that a majority of the targeted cells can receive the nucleic acid. Additionally, for example, due to the cDNA contained in the viral vector, the molecule encoded within the viral vector is efficiently expressed in the cells that receive the viral vector nucleic acid.

[0079] A viral vector system that is particularly useful for nucleic acid delivery is the adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or herpesvirus, as a helper virus for efficient replication and a productive life cycle (see for review, Muzyczka et al., Curr. Topics in Micro and Immunol. 158:97-129 (1992)). AAV vectors can efficiently transduce various cell types and can result in long-term expression of the transgene in vivo. The AAV vector genome can persist intracellularly as an episome, although vector integration has been observed (see, for example, Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April;20(4): 699-708; Flotte et al., Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al., J. Virol. 63:3822-3828 (1989); and McLaughlin et al., J. Virol. 62:1963-1973 (1989)). AAV vectors, such as AAV2, have been widely used for gene augmentation or replacement, and therapeutic efficacy has been demonstrated in a wide range of animal models as well as in the clinic. See, for example, Mingozzi and High, Nature Reviews Genetics 12, 341-355 (2011); Deyle and Russell, Curr Opin Mol Ther. 2009 Aug; 11(4): 442-447; Asokan et al., Mol Ther. 2012 April; 20(4): 699-708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can result in recombinant protein expression.Protocols for the production of recombinant retroviruses and for the infection of cells in vitro or in vivo with such viruses are known in the art and can be found, for example, in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard manuals. The use of AAV vectors to deliver constructs for expression in the brain is described, for example, in Iwata et al., Sci Rep. 2013;3:1472; Hester et al., Curr Gene Ther. 2009 Oct;9(5):428-33; Doll et al., Gene Therapy 1996, 3(5):437-447; and Foley et al., J Control Release. 2014 Dec 28;196:71-8.

[0080] Thus, in some embodiments, codon-optimized PSEN1 encoding a nucleic acid is present in a vector for gene therapy, such as an AAV vector. In some examples, the AAV vector is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAV11, and AAV12. In a preferred embodiment, the AAV is AAV9 or AAVrh10.

[0081] The vectors described herein can be pseudotyped vectors. Pseudotyping provides a mechanism for modulating the target cell population of the vector. For example, pseudotyped AAV vectors can be utilized in the various methods described herein. A pseudotyped vector is a vector that contains the genome of one vector, e.g., the genome of one AAV serotype, within the capsid of a second vector, e.g., a second AAV serotype. Methods of pseudotyping are well known in the art. For example, the vector can be pseudotyped with an envelope glycoprotein derived from a rhabdovirus vesicular stomatitis virus (VSV) serotype (Indiana and Chandipura strains), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus, rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein B type (FuG-B), a variant of FuG-B (FuG-B2) or Moloney murine leukemia virus (MuLV). The virus can be pseudotyped for transduction of one or more nerve cells or cell groups.

[0082] Exemplary examples of pseudotype vectors include, but are not limited to, vectors of the recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrh10, AAV11, and AAV12 serotypes. It is known in the art that such vectors can be modified to contain a transgene encoding a human protein or other protein. In certain examples, the present disclosure may include a pseudotype AAV9 or AAVrh10 viral vector containing the nucleic acids disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0083] In some examples, vectors of a particular AAV serotype can be selected based on the intended use, e.g., based on the intended route of administration.

[0084] Various methods for the application of AAV vector constructs in gene therapy are known in the art and include methods of modification, purification, and preparation for administration to human subjects (see, e.g., Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, AAV-based gene therapy targeting CNS cells has been described (see, e.g., U.S. Pat. Nos. 6,180,613 and 6,503,888). The preparation of high-titer AAV can be carried out using techniques known in the art, such as those described in U.S. Pat. No. 5,658,776.

[0085] A vector construct refers to a polynucleotide molecule that contains all or a part of a viral genome and a transgene. In some examples, gene transfer can be mediated by a DNA viral vector, such as an adenovirus (Ad) or an adeno-associated virus (AAV). Other vectors useful in methods of gene therapy are known in the art. For example, the constructs disclosed herein can include an alphavirus, a herpesvirus, a retrovirus, a lentivirus, or a vaccinia virus.

[0086] Adenoviruses are a relatively well-characterized group of viruses that include over 50 serotypes (see, e.g., International Publication No. WO 95 / 27071, which is incorporated herein by reference). Adenoviruses are amenable to manipulation by the techniques of molecular biology and may not require integration into the host cell genome. Recombinant Ad-derived vectors that contain vectors that reduce the likelihood of recombination and production of wild-type virus have been constructed (see, e.g., International Patent Publications Nos. WO 95 / 00655 and WO 95 / 11984, which are incorporated herein by reference). Wild-type AAV has high infectivity and can integrate into the host genome with a high degree of specificity (see, e.g., Hermonat and Muzyczka 1984 Proc. Natl. Acad. Sci., USA 81:6466-6470 and Lebkowski et al. 1988 Mol. Cell. Biol. 8:3988-3996).

[0087] Non-native regulatory sequences, gene regulatory sequences, promoters, non-coding sequences, introns, or coding sequences can be included in the nucleic acids disclosed herein. The inclusion of nucleic acid tags or signaling sequences, or nucleic acids encoding protein tags or protein signaling sequences, is further contemplated herein. Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0088] The promoters contained in the nucleic acids disclosed herein can be tissue- or cell-type specific promoters, promoters specific for multiple tissues or cell types, organ-specific promoters, promoters specific for multiple organs, systemic or ubiquitous promoters, or nearly systemic or ubiquitous promoters. Also included within the scope of the present disclosure are promoters having stochastic expression, inducible expression, conditional expression, or conversely, discontinuous, irregular, or unpredictable expression. The promoter can include any of the above characteristics or other promoter characteristics known in the art.

[0089] In a clinical setting, a gene delivery system for a therapeutic gene can be introduced into a subject by any of several methods, each of which is known in the art. For example, a pharmaceutical preparation of a gene therapy system can be introduced systemically, e.g., by intravenous injection, and specific transduction of a protein in target cells is effected primarily by the specificity of transfection brought about by a transcriptional control sequence that regulates the expression of a receptor gene, cell-type or tissue-type expression, or combinations thereof. In other embodiments, the initial delivery of the recombinant gene is more restricted and the introduction into the subject is highly localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent No. 5,328,470) or, e.g., optionally, by stereotactic injection into a cisterna magna, ventricle, lumbar intrathecal space, or direct injection into the hippocampus (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)). In a preferred embodiment, delivery methods for viruses expressing presenilin include intravenous, intrathecal, intraventricular, intracisternal, and stereotactic parenchymal administration.

[0090] The methods can be further optimized by preclinical studies to achieve the best recovery of neurodegeneration, dementia, synaptic dysfunction, and molecular degeneration in presenilin conditional double knockout mice and presenilin 1 knockin mice expressing FAD mutations.

[0091] The pharmaceutical preparation of the gene therapy construct may essentially comprise a gene delivery system in an acceptable diluent or may comprise a sustained release matrix that encapsulates the gene delivery vehicle. Alternatively, if a complete gene delivery system, such as a retroviral vector, can be produced intact from recombinant cells, the pharmaceutical preparation may contain one or more cells by which the gene delivery system can be produced.

[0092] Delivery formulations and pharmaceutical compositions In some embodiments, the polynucleotides described herein for in vivo delivery to a target tissue are encapsulated or bound to nanoparticles.Methods for packaging into nanoparticles are well known in the art, for example, as described in Bose S, et al (Role of Nucleolin in Human Parainfluenza Virus Type 3 Infection of Human Lung Epithelial Cells. J. Virol. 78:8146. 2004); Dong Y et al. Poly(d,l-lactide-co-glycolide) / montmorillonite nanoparticles for oral delivery of anticancer drugs. Biomaterials 26:6068. 2005); Lobenberg R. et al (Improved body distribution of 14C-labelled AZT bound to nanoparticles in rats determined by radioluminography. J Drug Target 5:171.1998); Sakuma S R et al (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int J Pharm 177:161. 1999); Virovic L et al. Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2:707.2005); and Zimmermann E et al, Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52:203. 2001).In some embodiments, one or more polynucleotides are delivered in vivo to target tissues by vesicles, such as liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.). In some embodiments, lipid-based nanoparticles (LNPs) are used. See, for example, Robinson et al., Mol Ther. 2018 Aug 1;26(8):2034-2046; U.S. Patent No. 9,956,271.

[0093] The methods and compositions of the present invention include microvesicles or preparations thereof, which may contain one or more therapeutic molecules described herein, such as polynucleotides or RNAs. "Microvesicles", as the term is used herein, refers to membrane-derived microvesicles that include exosomes, microparticles, and shed microvesicles secreted by many cell types under both normal physiological and pathological conditions, including a broad range of extracellular vesicles. See, for example, European Patent No. 2010663. The methods and compositions described herein can be applied to microvesicles of any size, and in one embodiment are 30-200 nm, in one embodiment are 30-800 nm, and in one embodiment are up to 2 μm. Also, the methods and compositions described herein can be widely applied to any extracellular vesicles, and this term includes exosomes, shed microvesicles, oncosomes, ectosomes, and retrovirus-like particles. Such microvesicles or preparations are produced by the methods described herein. When using this term herein, a microvesicle preparation refers to a population of microvesicles obtained / prepared from the same cell source. Such preparations are produced, for example, in vitro by culturing cells expressing the nucleic acid molecules of the present invention and isolating the microvesicles produced by the cells. Methods for isolating such microvesicles are known in the art (Thery et al., Isolation and characterization of exosomes from cell culture supernatants and biological fluids, in Current Protocols Cell Biology, Chapter 3, 322, (John Wiley, 2006); Palmisano et al., (Mol Cell Proteomics. 2012 August; 11(8):230-43) and Waldenstrom et al., ((2012) PLoS ONE 7(4): e34653.doi: 10.1371 / journal.pone.0034653)), and some examples of this are described herein.Such techniques for isolating microvesicles from cells in culture include, but are not limited to, sucrose gradient purification / separation and differential centrifugation, and may be adapted for use in the methods or compositions described herein. See, for example, European Patent No. 2010663.

[0094] In some embodiments, microvesicles are isolated by gently centrifuging (e.g., at about 300 g) the culture medium of donor cells for a period sufficient to separate the cells from the medium (e.g., about 15 minutes). This leaves the microvesicles in the supernatant, thus yielding a microvesicle preparation. In one embodiment, the culture medium or supernatant from the gentle centrifugation is further centrifuged more strongly (e.g., at about 16,000 g) for a period sufficient to precipitate cell debris (e.g., about 30 minutes). This leaves the microvesicles in the supernatant, thus yielding a microvesicle preparation. In one embodiment, the culture medium, gently centrifuged preparation, or strongly centrifuged preparation is subjected to filtration (e.g., through a 0.22 μm filter or a 0.8 μm filter), whereby the microvesicles pass through the filter. In one embodiment, the filtrate is subjected to a final ultracentrifugation (e.g., at about 110,000 g) for a period sufficient to fully precipitate the microvesicles (e.g., about 80 minutes). The resulting pellet contains the microvesicles and is resuspended in a large volume of buffer to obtain a useful concentration for further use, thereby yielding a microvesicle preparation. In one embodiment, the microvesicle preparation is generated by sucrose density gradient purification. In one embodiment, the microvesicles are further treated with DNAse (e.g., DNAseI) and / or RNAse and / or protease to remove any externally contaminating DNA, RNA, or protein, respectively. In one embodiment, the microvesicle preparation contains one or more RNAse inhibitor substances.

[0095] The molecules contained within the microvesicle preparation include therapeutic molecules. Typically, the microvesicles in the preparation are a heterogeneous population, and each microvesicle contains a complement of molecules that may or may not differ from the molecules of other microvesicles in the preparation. The content of therapeutic molecules in the microvesicle preparation can be expressed either quantitatively or qualitatively. One such method is to express the content as a percentage of the total molecules in the microvesicle preparation. By way of example, if the therapeutic molecule is mRNA, the content can be expressed as a percentage of the total RNA content of the microvesicle preparation or as a percentage of the total mRNA content. Similarly, if the therapeutic molecule is a protein, the content can be expressed as a percentage of the total protein in the microvesicle. In one embodiment, the therapeutic microvesicles or preparations thereof generated by the methods described herein contain a detectable and statistically significant increased amount of the therapeutic molecule when compared to microvesicles obtained from control cells (cells obtained from the same source that have not been scientifically engineered to increase the expression of the therapeutic molecule). In one embodiment, the therapeutic molecule is present in an amount that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% more than the amount in microvesicles obtained from control cells. Even higher levels of enrichment can be achieved. In one embodiment, the therapeutic molecule is present in the microvesicles or preparations thereof in an amount that is at least 2-fold more than in control cell microvesicles. Even higher fold enrichments can be obtained (e.g., 3, 4, 5, 6, 7, 8, 9 or 10-fold).

[0096] In one embodiment, a relatively high percentage of the microvesicle content is the therapeutic molecule (e.g., achieved by high expression of the molecule or specific targeting to the microvesicle). In one embodiment, the microvesicle content of the therapeutic molecule is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the total (homologous) molecule content (e.g., the therapeutic molecule is mRNA and is about 10% of the total mRNA content of the microvesicle). Even higher levels of enrichment can be achieved. In one embodiment, the therapeutic molecule is present in the microvesicles or preparations thereof in an amount that is at least 2-fold more than all other such (homologous) molecules. Even higher fold enrichments can be obtained (e.g., 3, 4, 5, 6, 7, 8, 9 or 10-fold).

Example

[0097] The present invention is further described in the following examples, which do not limit the scope of the present invention described in the claims.

[0098] [Example 1] Different PS genotypes: PS1 + / + , PS1 L435F / + , PS1 + / - , PS1 L435F / L435F , PS1 - / - and PS1 - / - ; PS2 - / - Dose-dependent recovery of γ-secretase activity in MEF having To determine whether the reduced γ-secretase activity associated with PSEN1 mutations can be corrected by the introduction of wild-type (WT) hPS1, different PS genotypes, PS1 + / + , PS1 L435F / + , PS1 + / - , PS1 L435F / L435F , PS1 - / - and PS1 - / - ; PS2 - / - (DKO)-bearing primary MEFs derived from embryos were obtained. Immortalized MEFs were transiently transfected with CMV-NΔE, and γ-secretase activity was evaluated by measuring the levels of NICD and PS1 NTF / CTF. The NICD level was reduced in a PS1 dose-dependent manner and undetectable in DKO cells (Figure 1A). The NICD level was reduced but detectable in PS1 L435F / L435F MEF (“L435F KI / KI” MEF) and PS1 - / - MEF (Figure 1A), but de novo NICD generation was undetectable by in vitro γ-secretase assay using embryonic brains from L435F KI / KI and PS1 - / - (Xia et al., Neuron. 2015 Mar 4;85(5):967-81). Without wishing to be bound by any particular theory, the applicant believes that this is because PS2, which is normally expressed in embryonic brains, is present at low levels compared to MEF, and L435F KI / KI and PS1 - / -This could be due to a lower total PS activity in the brain compared to MEF. To test this hypothesis, PS1 L435F / + ;PS2 - / - γ-secretase activity was measured in MEF and compared to PS1 L435F / + MEF. γ-secretase activity was lower in PS1 L435F / + ;PS2 - / - MEF compared to PS1 L435F / + MEF (Figure 1B).

[0099] To determine whether the impaired γ-secretase activity in various PS mutant MEFs could be restored by the introduction of WT hPS1, different amounts (0, 20, 40, 80 ng) of wild-type hPS1 cDNA (pCI-hPS1) were transfected into MEF together with CMV-NΔE. Notably, the increased amount of pCI-hPS1 transfected into MEF resulted in the accumulation of PS1 protein and the restoration of PS1 NTF and NICD levels in mutant (PS1 L435F / + 、PS1 L435F / + ;PS2 - / - and DKO) MEF (Figure 1B). These results indicate that the impaired γ-secretase activity can be restored in various PS mutant MEFs by exogenous WT hPS1.

[0100] [Example 2] Development of an in vitro expression system for optimized wild-type human PS1 Method Cell culture and transfection Psen-null mouse embryonic fibroblasts (MEF) lacking endogenous PS1 and PS2 were maintained in DMEM supplemented with 10% FBS and transiently transfected with a plasmid expressing either wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1) using Lipofectamine 3000 according to the instructions, in the presence or absence of the γ-secretase reporter CMV-NΔE. Cell lysates were collected at the 24-hour time point.

[0101] Western blotting The cell lysates were subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane. After blocking with TBST / 5% non-fat dry milk, the membrane was incubated with the primary antibody overnight. To control the loading, the membrane was stripped and reprobed with an anti-β-actin antibody. The intensity of the bands was quantified using ImageJ software and the results were normalized against the β-actin levels. The antibodies used were rat anti-PS1-NTF, rabbit anti-cleaved Notch (Val1744) (NICD), and mouse anti-β-actin.

[0102] Results First, the inventors expressed wt_hPS1 or opti_hPS1 in Psen-null MEFs to exclude any influence of endogenous PS1 or PS2. The inventors detected a progressive increase in PS1 NTF levels when transfecting Psen-null MEFs with increasing amounts of vectors encoding wt_hPS1 or opti_hPS1. Optimized hPS1 consistently expressed more PS1 than wt_hPS1 (Figures 3A - 3B).

[0103] NotchΔE, as a substrate for cleavage mediated by γ-secretase, was co-expressed with wt_hPS1 or opti_hPS1 to directly measure γ-secretase activity. Notch is cleaved by γ-secretase to release the Notch intracellular domain (NICD). The inventors evaluated the dose-response relationship for NotchΔE cleavage mediated by γ-secretase to generate NICD in response to γ-secretase activity. The generation of NICD increased linearly during transfection with a small amount of the PS1 vector but approached saturation at higher levels. More importantly, at each point, optimized hPS1 had higher γ-secretase activity (Figure 4 ).

[0104] The levels of secreted endogenous Aβ40 and 42 are measured in the medium using ELISA. γ-secretase activity is evaluated by transiently transfecting each of the MEF cell lines with CMV-C99, another γ-secretase substrate, together with increasing amounts of pCI-hPS1opti. Primary and immortalized C410Y, E280A and D385A KI / + and KI / KI MEF as well as PS1 KI / +;PS2- / - MEF are established. It is determined whether the KI / + and KI / KI MEF recapitulate a phenotype similar to that of KI / + and KI / KI brains, and whether the introduction of WT hPS1 can restore reduced γ-secretase activity in a dose-dependent manner, as measured by the generation of NICD and AICD. The experiments are repeated in multiple independent MEF cell lines for each genotype. A power analysis is performed to determine the sample size and the number of independent experiments required to complete this study.

[0105] [Example 3] Development of an in vivo expression system for optimized wild-type human PS1 Transgenic mice are developed that constitutively or inducibly express human PSEN1 wild-type cDNA under the control of the CAMK2A promoter. To maximize the production and activity of PS1, hPS1 is codon-optimized (hPS1opti), and then the PS1 levels and γ-secretase activity are compared between the endogenous hPS1 cDNA and the hPS1opti cDNA by co-transfecting increasing amounts of either pCI-hPS1 or pCI-hPS1opti and CMV-NΔE into PS DKO MEF. As measured by NICD generation, the hPS1opti cDNA resulted in higher levels of PS1 NTF and higher γ-secretase activity compared to the endogenous hPS1 cDNA (Figure 4 ).

[0106] [Example 4] Determine whether postnatal delivery of hPS1opti can restore the phenotype in PS cDKO mice Vectors listed in Table 2 were prepared. Identify the vector that results in the highest GFP staining at 4 and 8 weeks after injection. For this purpose, inject into PS cDKO offspring mice (10 breeding cages of Cre / Cre; - / - mated with lot - F / F; - / -; F / F) at P0 - 2.

[0107] [Table 3]

[0108] Select AAV, such as AAV9 / hCaMKII - intron - hPS1opti - T2A - EGFP - SV40pA or AAV9 / hCaMKII - intron - EGFP - SV40pA, and inject into PS cDKO mice by ICV at P0 - P2. Perform Western analysis at 4 and 8 weeks of age to determine the levels of PS1, APP, nicastrin, and PEN - 2. Include control and PS cDKO mice at 4 and 8 weeks as additional controls. Measure gamma - secretase activity, NICD production, and Aβ levels (by ELISA of cortical lysates) at 8 weeks of age. Furthermore, at 2 months of age, perform electrophysiological analyses, such as Schaffer collateral, PPF, FF, and LTP, and perform behavioral analyses, such as water maze, at 2 - 3 months of age. Perform neuropathological analysis at 6 months of age.

[0109] [Example 5] Determine whether postnatal delivery of hPS1opti can restore the phenotypes caused by FAD mutations Generate the mice listed in Table 3 and analyze using Western blot analysis for measuring PS1 and APP levels; gamma - secretase activity assay in vitro; ELISA, electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; neuropathological analysis at 6, 12, and 18 months of age.

[0110] [Table 4]

[0111] [Example 6] Determine whether AAV9 / hPS1opti delivered to the adult brain can restore in vivo the phenotypes (PS cDKO, FAD KI) caused by the loss of PS function As listed in Table 2, vectors were prepared and a 50 ul Hamilton syringe was used to deliver 10 ul of the injection solution into the cisterna magna via the CSF (1×10 12 vg) via the ICM, via parenchymal delivery, or 10 ul of 3.3×10 11 vg of lumbar puncture was used for intrathecal delivery to inject mice. The mice under study include (i) PS cDKO, (ii) KI / +, (iii) KI / +; PS2- / -, and (iv) KI / fPS1; PS2- / -, Cre. The inventors perform the following analyses in mice: Western analysis to measure PS1 and APP levels; γ-secretase assay in vitro; ELISA measurement of Aβ peptide; electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; neuropathological analysis at 6, 12, and 18 months of age.

[0112] Other embodiments The present invention has been described together with the forms for carrying out the invention, but it is understood that the foregoing description is intended to be illustrative and does not limit the scope of the present invention, and the present invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The present invention includes the following embodiments. [1] A composition comprising a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1). [2] The composition according to [1] above, wherein the human codon-optimized polynucleotide encoding human PSEN1 is at least 95% identical to SEQ ID NO: 9 and at least one codon is optimized relative to the wild type. [3] The composition according to [1] above, wherein the human codon-optimized polynucleotide encoding human PSEN1 comprises SEQ ID NO: 9. [4] The composition according to any one of [1] to [3] above, accompanied by exosomes or lipid-based nanoparticles (LNPs) (e.g., formulated using the same for delivery). [5] A composition comprising a vector for expressing human PSEN1 in cells, the vector comprising a human codon-optimized polynucleotide according to any one of [1] to [3] above, operably linked to a promoter. [6] The composition according to [5] above, wherein the vector is a viral vector. [7] The composition according to [6] above, wherein the viral vector is an adeno-associated virus (AAV) vector. [8] The composition according to [7] above, wherein the AAV vector is AAV9 or AAVrh10. [9] The composition according to [6] above, wherein the viral vector is a lentiviral vector or a retroviral vector.

[10] The composition according to [5] above, wherein the promoter is a pan-neural promoter.

[11] The composition according to

[10] above, wherein the pan-neural promoter is a synapsin I promoter.

[12] The composition according to [5] above, wherein the promoter is a neuron subtype-specific promoter.

[13] The composition according to

[12] above, wherein the neuron subtype-specific promoter is an alpha-calcium / calmodulin kinase 2A promoter.

[14] A method for treating a neurodegenerative disease, disorder, or condition, the method comprising administering a composition according to any one of [1] to

[13] above to a human subject in need thereof, wherein the subject has one or more mutations in at least one allele of PSEN1, preferably a mutation encoding a dominant-negative PSEN1 protein isoform.

[15] The method according to

[14] above, wherein the neurodegenerative disease, disorder, or condition is Alzheimer's disease.

[16] The method according to

[15] above, wherein the Alzheimer's disease is familial Alzheimer's disease.

[17] The method according to

[15] or

[16] above, wherein the subject has a mutation of E280A, Y115H, L166P, C410Y, Δex9, G548, D257A, R278I, L435F, G384A or L392V in the PSEN1 gene, or a mutation of N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V or M146V in the PSEN1 gene.

[18] The method according to

[15] above, wherein the Alzheimer's disease is sporadic Alzheimer's disease.

[19] The method according to

[15] above, wherein the Alzheimer's disease is late-onset or early-onset Alzheimer's disease.

[20] The method according to

[14] above, wherein the neurodegenerative disease, disorder or condition is frontotemporal dementia, memory loss, cognitive decline or cognitive impairment.

[21] The method according to

[20] above, wherein the cognitive impairment is mild cognitive impairment (MCI).

[22] The method according to any one of

[14] to

[20] above, wherein the composition is administered to the CNS of the subject in need of treatment.

[23] The method according to

[22] above, wherein the polynucleotide encoding the PSEN1 and / or PSEN2 gene or mRNA is administered to the CNS by intravenous delivery, intrathecal delivery, intracisternal delivery, intraventricular delivery, or stereotactic parenchymal injection into a specific brain region, optionally into the cistern, ventricle, lumbar intrathecal space, or direct injection into the hippocampus or cerebral cortex.

Claims

1. A composition comprising a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1), wherein the human codon-optimized polynucleotide encoding human PSEN1 comprises the nucleotide sequence set forth in SEQ ID NO:

9.

2. The composition according to claim 1, accompanied by exosomes or lipid-based nanoparticles (LNP).

3. A composition comprising a vector for expressing human PSEN1 in a cell, the vector comprising a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1) operably linked to a promoter, wherein the human codon-optimized polynucleotide encoding human PSEN1 comprises the nucleotide sequence set forth in SEQ ID NO:

9.

4. The composition according to claim 3, wherein the vector is a viral vector.

5. The composition according to claim 4, wherein the viral vector is an adeno-associated virus (AAV) vector.

6. The composition according to claim 5, wherein the AAV vector is AAV9 or AAVrh10.

7. The composition according to claim 4, wherein the viral vector is a lentiviral vector or a retroviral vector.

8. The composition according to claim 3, wherein the promoter is a pan-neural promoter.

9. The composition according to claim 8, wherein the pan-neural promoter is a synapsin I promoter.

10. The composition according to claim 3, wherein the promoter is a neuron subtype-specific promoter.

11. The composition according to claim 10, wherein the neuron subtype-specific promoter is an alpha-calcium / calmodulin kinase 2A promoter.

12. A composition according to any one of claims 1 to 11 for treating a neurodegenerative disease, disorder, or condition in a human subject, wherein the subject has one or more mutations in at least one allele of PSEN1.

13. The composition according to claim 12, wherein the neurodegenerative disease, disorder or condition is Alzheimer's disease.

14. The composition according to claim 13, wherein the Alzheimer's disease is familial Alzheimer's disease.

15. The composition according to claim 13 or 14, wherein the subject has a mutation of E280A, Y115H, L166P, C410Y, Δex9, D257A, R278I, L435F, G384A or L392V in the PSEN1 gene, or a mutation of N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, E280G, P264L, E185D, L235V or M146V in the PSEN1 gene.

16. The composition according to claim 13, wherein the Alzheimer's disease is sporadic Alzheimer's disease.

17. The composition according to claim 13, wherein the Alzheimer's disease is late-onset or early-onset Alzheimer's disease.

18. The composition according to claim 12, wherein the neurodegenerative disease, disorder or condition is frontotemporal dementia, memory loss, cognitive decline or cognitive impairment.

19. The composition according to claim 18, wherein the cognitive impairment is mild cognitive impairment (MCI).

20. The composition according to any one of claims 12 to 18, which is administered to the CNS of the subject.

21. The composition according to claim 20, which is administered to the CNS by intravenous delivery, intrathecal delivery, intracisternal delivery, intraventricular delivery, stereotactic parenchymal injection into the cistern, ventricle or lumbar spinal canal, or direct injection into the hippocampus or cerebral cortex.

Images