Gene therapy for Alzheimer's disease

Abstract

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

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 852,716, filed May 24, 2019. The entire contents of the foregoing document are incorporated herein by reference.

[0003] Federally sponsored research or development

[0004] This invention was made with U.S. government support under Grant No. NS041783 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention. Technical Field

[0005] Described herein are, inter alia, compositions and methods for treating Alzheimer's disease (AD) and other neurodegenerative diseases using presenilin gene therapy constructs. Background Art

[0006] Alzheimer's disease, also known as Alzheimer's 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 progressive loss of cognitive function, neurodegeneration, neurofibrillary tangles, and amyloid plaques in the patient's brain. Although the rate of progression varies among different patients, the average life expectancy after diagnosis is 3 to 9 years. Currently, there is no treatment for Alzheimer's disease. Summary of the invention

[0007] Described herein are methods and compositions useful for treating subjects with Alzheimer's disease (AD) and other neurodegenerative diseases, disorders or conditions. The present disclosure is based, at least in part, on the discovery that providing codon-optimized wild-type PSEN1 cDNA to cells carrying heterozygous or homozygous dominant negative Psen1 mutations, a well-established familial Alzheimer's disease model, provides unexpectedly high expression levels and rescues impaired γ-secretase activity in these cells. Thus, the present disclosure provides methods for effective gene therapy based on PSEN1 (expressing PS1) and / or PSEN2 (expressing PS2) for Alzheimer's disease and other neurodegenerative dementias, representing a major breakthrough in this disease area.

[0008] Provided herein are compositions comprising human codon-optimized polynucleotides encoding human presenilin 1 protein (PS1), e.g., polynucleotides comprising SEQ ID NO: 9 or a sequence having at least 80%, 90%, 95% or 99% identity to SEQ ID NO: 9 (at least one codon is optimized relative to the wild type). Exemplary human PS1 protein sequences include SEQ ID NO: 5 and 6, and may include the following sequence, which is a sequence comprising at least a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1). In some embodiments, the composition is associated with exosomes or lipid-based nanoparticles (LNPs) (e.g., formulated for delivery using exosomes or lipid-based nanoparticles (LNPs)).

[0009] Also provided herein is a composition comprising a vector for expressing human PSEN1 in a cell, the vector comprising the human codon-optimized polynucleotide described herein, the human codon-optimized polynucleotide being operably linked to a promoter.

[0010] Also provided herein is use of any of the compositions described herein in a method of treating a neurodegenerative disease, disorder, or condition in a subject.

[0011] In some embodiments, the vector is a viral vector, such as an adeno-associated virus (AAV) vector (eg, AAV9 or AAVrh10); a lentiviral vector; or a retroviral vector.

[0012] In some embodiments, the promoter is a pan-neuronal promoter, such as the synapsin I promoter, or a neuronal subtype-specific promoter, such as the α-calcium / calmodulin kinase 2A promoter.

[0013] Also provided herein are methods for treating a neurodegenerative disease, disorder or condition comprising administering a composition described herein to a human subject in need of treatment, wherein the subject has more than one mutation in at least one allele of PSEN1, preferably a mutation encoding a dominant negative PSEN1 protein isoform.

[0014] In some embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease.

[0015] In some embodiments, Alzheimer's disease is familial Alzheimer's disease. In some embodiments, Alzheimer's disease is late-onset Alzheimer's disease. In some embodiments, Alzheimer's disease is sporadic Alzheimer's disease. In some embodiments, Alzheimer's disease is early-onset Alzheimer's disease.

[0016] 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, e.g., E280 in the PSEN1 gene. A, Y115H, L166P, C410Y, Δex9, G548, D257A, R278I, L435F, G384A, or L392V mutations, or N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V, or M146V mutations in the PSEN1 gene.

[0017] In some embodiments, the neurodegenerative disease, disorder or condition is frontotemporal dementia, memory loss, cognitive decline or cognitive impairment. In some embodiments, cognitive impairment is mild cognitive impairment (MCI).

[0018] In some embodiments, the composition is administered to the CNS of a subject in need of treatment.

[0019] In some embodiments, polynucleotides encoding PSEN1 and / or PSEN2 genes or mRNA are administered to the CNS by intravenous delivery, by intrathecal delivery, by intracisternal delivery, by intraventricular delivery, or by stereotactic injection into a specific area of ​​the brain, optionally into the ventricles, or by direct injection into the hippocampus or cortical areas.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification including definitions will prevail.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1A-B. The introduction of WT hPS1 rescues the impaired γ-secretase activity in mutant MEFs. A, γ-secretase activity measured by NICD production is reduced in mutant MEF cells in a PS dose-dependent manner (WT>PS1 heterozygous KI or KO>homozygous PS1 KI or KO>DKO). B, impaired γ-secretase activity is restored by WT hPS1. As shown in the figure, increasing amounts of pCI-hPSEN1 plasmid DNA were transfected into MEFs of different genotypes. Protein immunoblot analysis showed that both PS1 NTF and NICD were restored in various PS mutant MEFs. Heterozygous L435F KI cells were labeled as KI / + or PS1L435F / +. N=3 independent experiments. Data are expressed as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 (one-way ANOVA and Tukey post hoc analysis).

[0023] Figure 2 . Sequence comparison of endogenous human PSEN1 (hPSEN1) cDNA and codon-optimized hPSEN1 cDNA (Opti-hPSEN1).

[0024] FIG3A-B . Increased expression levels of PS1 NTFs with codon-optimized PSEN1 cDNA. A, Psen-null MEFs were transfected with increasing amounts of plasmids expressing wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1) and subjected to western blot analysis using an antibody specific for the PS1 N-terminus. Untransfected (Untrans): Untransfected MEFs served as negative control. B, Quantification of PS1 NTF levels in cells transfected with wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1). Data are presented as mean ± SEM (n = 3 independent experiments).

[0025] Figure 4 .Codon optimization results in increased γ-secretase activity. PS DKO MEFs were transfected with increasing amounts (12.5, 25, 50 or 100 ng) of pCI-hPS1 or pCI-hPS1opti plasmid DNA and CMV-NΔE, followed by protein immunoblot analysis of NICD. MEFs that were not transfected or transfected with an empty vector were included as negative controls. We found that pCI-hPS1opti resulted in significantly higher levels of γ-secretase activity relative to pCI-hPS1, as measured by NICD production. Data are expressed as mean ± SEM (n = 5 independent experiments). Two-way ANOVA was used to evaluate statistical significance. **p<0.01. DETAILED DESCRIPTION

[0026] definition

[0027] In order to more easily understand the present disclosure, certain terms are first defined below. Additional definitions for the following terms and other terms are recorded throughout the specification.

[0028] Application:

[0029] As used herein, the term "administering" refers to delivering or applying a composition to a subject or system. It can be administered to an animal subject (e.g., to a human) by any appropriate route. For example, in some embodiments, administration can be intrabronchial (including by bronchial instillation), oral, enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal instillation), transdermal, vaginal, and vitreous.

[0030] Biological activity:

[0031] As used herein, the phrase "biological activity" refers to the characteristics of any substance (e.g., cell culture, organism, etc.) that is active in a biological system. For example, a substance that has a biological effect on an organism when applied to the organism is considered to be biologically active. Biological activity can also be determined by in vitro detection (e.g., in vitro enzyme detection). In specific embodiments, when a protein or polypeptide is biologically active, a portion of the protein or polypeptide that shares at least one biological activity of the protein or polypeptide is generally 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.

[0032] Comparison:

[0033] As used herein, the term "control" has the meaning of its field understanding, as a standard for the compared results. Generally, the control is used to draw a conclusion about such variables by separating variables and increase the integrity of the experiment. In some embodiments, the control is a reaction or detection carried out simultaneously with the test reaction or detection to provide a comparator (comparator). In an experiment, "test" (that is, the tested variable) is applied. In the second experiment, the "control" as the tested variable is not applied. In some embodiments, the control is a historical control (that is, a historical control of the test or detection previously performed or a previously known amount or result). In some embodiments, the control is or includes a record printed or otherwise preserved. The control can be a positive control or a negative control. In some embodiments, the control can be a "reference control", which is a sample for finding a difference or for characterization purposes compared with a test sample.

[0034] Gene therapy:

[0035] As used herein, the term "gene therapy" refers to any treatment that involves administering a nucleic acid directly or indirectly to a subject. In a specific example, a protein of therapeutic value is expressed from the administered nucleic acid.

[0036] Identity:

[0037] As used herein, the term "identity" refers to the overall correlation between polymeric molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. The calculation of the identity percentage of two nucleic acid sequences, for example, can be performed by comparing two sequences for the best comparison purpose (e.g., for the best comparison, a room can be introduced in one or both of the first and second nucleic acid sequences and non-identical sequences can be ignored for the purpose of comparison). In certain embodiments, the length of the sequence compared for the purpose of comparison 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 the corresponding nucleotide positions are then compared. When the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecule is identical at this position. Considering the need to introduce the number of spaces and the length of each space to achieve the best comparison of the two sequences, the identity percentage between the two sequences is a function of the number of identical positions shared by the sequences. Mathematical algorithms can be used to complete the comparison of sequences and the determination of the identity percentage between the two sequences. For example, the percent identity between two nucleotide sequences can be determined by using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix. Various other sequence alignment programs are available and can be used to determine sequence identity, such as Clustal, etc.

[0038] To improve, increase, or decrease:

[0039] As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to a value relative to a baseline measurement, e.g., a measurement in the same individual before the start of a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein. A "control individual" is an individual with the same kind or nearly the same severity of disease, e.g., Alzheimer's disease, as the individual being treated, who is approximately the same age as the individual being treated (to ensure that the stages of the disease in the treated individual and the control individual(s) are comparable).

[0040] Neurodegenerative Diseases:

[0041] As used herein, the term "neurodegeneration" refers to a process in which one or more neurons are damaged, have reduced function, become dysfunctional, and / or are lost due to cell death. Neurodegeneration includes both rapid and gradual, as well as intermediate forms. Therefore, neurodegenerative diseases, conditions, or symptoms are generally characterized as diseases associated with neuronal damage and / or cell death.

[0042] Subjects:

[0043] 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 prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, which refers to a person who meets with a medical professional in order to diagnose or treat a disease. The term "subject" is used interchangeably with "individual" or "patient" herein. A subject may suffer from or be susceptible to a disease or condition, but may or may not show symptoms of the disease or condition.

[0044] suffer:

[0045] An individual "suffering from" a disease, disorder, and / or condition (eg, Alzheimer's disease) has been diagnosed with or displays one or more symptoms of the disease, disorder, and / or condition.

[0046] Susceptible to:

[0047] An individual who is "susceptible to" a disease, disorder, and / or condition has not been diagnosed with and / or may not display symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition (e.g., Alzheimer's disease) may be characterized by one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder, and / or condition; (2) a genetic polymorphism associated with the development 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) habits and / or lifestyle associated with the development of the disease, disorder, and / or condition; (5) a family history of the disease, disorder, and / or condition; (6) a reaction to certain bacteria or viruses; (7) exposure to certain chemicals. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0048] Therapeutically effective amount:

[0049] As used herein, the term "therapeutically effective amount" refers to a therapeutic protein that imparts a therapeutic effect to the 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 gives a sign or feeling of the effect). In particular, a "therapeutically effective amount" refers to an amount of a therapeutic protein or composition that effectively treats, improves, or prevents a desired disease or condition, or that shows a detectable therapeutic or preventive effect, such as by improving symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also alleviating the severity or frequency of the symptoms of the disease. The therapeutically effective amount is usually administered in a dosage regimen that may include multiple unit doses. For any particular therapeutic protein, the therapeutically effective amount (and / or a suitable unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, in combination with other pharmaceutical preparations. Furthermore, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on a variety of factors including: the condition to be treated and the severity of the condition; the activity of the specific pharmaceutical formulation employed; the specific composition employed; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and / or rate of secretion or metabolism of the specific fusion protein employed; the duration of treatment; and other factors well known in the medical arts.

[0050] treat:

[0051] As used herein, the term "treatment" (noun, verb or gerund form), in its broadest sense, refers to partial or complete relief, improvement, regeneration, inhibition, delay of onset, reduction of severity, and / or reduction of incidence of one or more symptoms, features, and / or causes of a specific disease, condition, and / or condition. In some embodiments, such treatment may be applied to subjects who do not show signs of the relevant disease, disorder, and / or condition and / or subjects who only show early signs of the disease, disorder, and / or condition. Alternatively or additionally, in some embodiments, treatment may be applied to subjects who show one or more determined signs of the relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who have been diagnosed with a relevant disease, disorder, and / or condition. In some embodiments, treatment may be directed to subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of the development of the relevant disease, disorder, and / or condition.

[0052] Although, generally "PS1" refers to the Presenilin-1 protein, and "PS2" refers to the Presenilin-2 protein, in some cases PS1 or PS2 is used to refer to the mRNA or gene.

[0053] Detailed description

[0054] The present disclosure provides, among other aspects, compositions and methods for treating subjects with Alzheimer's disease and other neurodegenerative diseases, disorders and conditions based on delivering functional presenilin-1 (PS1) to subjects in need thereof. In particular, the present disclosure contemplates gene therapy by providing human codon-optimized polynucleotides encoding presenilin-1 (PS1) to subjects in need thereof, wherein the subjects in need thereof have a PSEN1 or PSEN2 mutation associated with AD, such as a dominant negative mutation, for example, associated with early-onset familial Alzheimer's disease (FAD) or with late-onset sporadic AD.

[0055] Various aspects of the present invention are described in detail in the following sections. The use of these sections is not intended to limit the present invention. Each section can be applied to any aspect of the present invention. In this application, unless otherwise stated, the use of "or" means "and / or".

[0056] Treatment

[0057] As a non-limiting example, the method includes gene therapy expressing wild-type human presenilin-1 in a subject suffering from or susceptible to a neurodegenerative disease, such as Alzheimer's disease, associated with a dominant negative mutation in PSEN1 or PSEN2 (e.g., a familial AD patient or a sporadic AD patient carrying a PSEN1 or PSEN2 mutation). Among other aspects, such gene therapy aims to enhance the expression of PS1 in the brain of a familial or sporadic AD patient to correct or overcome deficiencies in PS1 or PS2 expression and / or activity. In FAD patients, it is expected that the gene therapy methods described herein cause an increase in the expression of wild-type PS1 in the brain, rescuing the impairment of γ-secretase activity associated with PS1 or PS2 mutations.

[0058] Mutations in the presenilin genes – PSEN1 and PSEN2 – are highly penetrant and account for ~90% of all mutations identified in familial AD (FAD), highlighting their importance in the pathogenesis of AD. More than 260 different mutations in PSEN1 have been reported, which are dominantly inherited and are mainly missense mutations. Dominant-negative mutations in the PSEN1 and PSEN2 genes are known to be associated with early-onset familial Alzheimer's disease. It is generally believed that the PS1 and presenilin-2 (PS2) proteins are part of the γ-secretase complex and that mutations in the PSEN1 and PSEN2 genes contribute to the accumulation of amyloid β (Aβ) in Alzheimer's patients. Pathogenic PSEN1 mutations act in cis to impair 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, dominant-negative mutations, by their nature, cannot be rescued by expressing wild-type protein (Herskowitz, I. Nature, 329: 219-222 (1987)). However, surprisingly, as shown herein, codon-optimized hPSEN1 Transfection of cDNA into immortalized MEFs carrying heterozygous and homozygous PS1 mutations can rescue the impaired γ-secretase activity in these cells, even better than the wild-type human sequence (see Examples below), indicating that expression of wild-type PS1 from codon-optimized exogenous sequences can overcome the dominant-negative effects of mutant presenilin proteins. Without wishing to be bound by any particular theory, expression of PS1 can achieve this by increasing the total level of wild-type PS1, rescuing the impairment of γ-secretase expression and / or activity in AD patients.

[0059] The methods and compositions described herein are equally useful for treating other neurodegenerative diseases, disorders, or conditions.

[0060] Alzheimer's disease

[0061] The methods described herein can be used to treat subjects with all types of Alzheimer's disease, including but not limited to familial and sporadic Alzheimer's disease, early-onset or late-onset Alzheimer's disease, or reduce the risk of their development. In some embodiments, the method can be used to treat early-onset familial type of Alzheimer's disease (AD) associated with mutations in presenilin-1 (PS1) and / or presenilin-2 (PS2) or reduce the risk of its development (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), the entire contents of which are incorporated herein by reference).

[0062] In some embodiments, the present methods can be used to treat a subject having a mutation in a PSEN1 or PSEN2 allele, such as a mutation that has a dominant negative effect on the wild-type PS protein. Exemplary mutations include C410Y, Δex9, G548, D257A, L166P, R278I, L435F, G384A, Y115H and L392V, and 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 2017 Nov 28; 114(48)12635-12637; Zhou et al., PNAS 2017 Nov 28; 114(48)12731-12736; Hsu et al., Alzheimers Res Ther. 2018 Jul 18;10(1):67). Additional exemplary mutations that may have a dominant negative effect on wild-type PS protein include, but are not limited to, in PSEN-1: 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 (intron 4, I nsTAC,p.113+1delG,spliceon 5);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; T29 1P; K311R; E318G; D333G; R352C; R352_S353insR; T354I; R358Q; S365A; S365Y; R377 M; R377W; G378E; G378V; G378fs; L381F; L381V; G384A; F386I; F386S; F388L; S390I; S390N; V391F; V391G; L392P; L392V; G394V; A396T; N405S; I408T; A409T; C410Y; V41 2I; I416T; G417S; L418F; L420R; L424F; L424H; L424R; L424V; A426P; A431E; (Jalis co); A431V; A434C; A434T; L435F; P436Q; P436S; I437V; I439S; I439V; T440del; 869 -2A>G;869-22_869-23ins18(ΔE9,Δ9,deltaE9);I238_K239insI;S290C;T291_S31 9del(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) (mutations relative to Uniprot P49768.1 / GenBank Ref. No. NM_000021.4), and in PSEN-2; T18M; R29H; G34S; R62C; R62H; P69A; R71W; K82R; A85V; V101M; K115Efs*; 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 Ref. No. NP_000438.2). See, e.g., 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 2017 Nov 28 114(48) 12731-12736. In some embodiments, the method can include determining that the subject has such a mutation, e.g., using methods known in the art. In some embodiments, the subject has a mutation as described herein (e.g., identified as having a mutation as described herein using methods known in the art), and optionally has a family history of AD and / or more than one symptom of AD, and the subject is treated using the methods described herein. In some embodiments, the subject does not yet have full-blown AD. ;

[0063] Typically, increased forgetfulness or mild confusion are early symptoms of Alzheimer's disease. Gradually, the cognitive impairment associated with Alzheimer's disease leads to loss of memory, especially recent memory, disorientation and distortion of spatial relationships, difficulty speaking, writing, thinking, and reasoning, changes in personality and behavior leading to depression, anxiety, social withdrawal, mood swings, distrust of others, irritability and aggression, changes in sleep habits, confusion, loss of self-control, delusions, and eventually death.

[0064] Other neurodegenerative diseases, disorders or conditions

[0065] In addition to Alzheimer's disease, the present methods can also be used to treat other neurodegenerative diseases, disorders or conditions, including frontotemporal dementia, various types of memory loss, including but not limited to cognitive impairment such as mild cognitive impairment (MCI), or other conditions associated with loss of PS1 or PS2, for example due to mutations in PSEN1 or PSEN2 such as those that produce dominant negative isoforms.

[0066] Codon-optimized presenilin-1 (PSEN1)

[0067] Codon-optimized presenilin-1 (polynucleotides encoding PSEN1 suitable for use in the compositions and methods described herein may include full-length cDNAs or portions or fragments thereof that encode proteins that retain substantial γ-secretase activity of the wild-type protein, such as at least 50% of the γ-secretase activity, or at least 60, 70, 80, 90, or 95%, or more than 100% of the activity of the wild-type protein as determined by (e.g., in an in vitro γ-secretase assay including those described in the Examples section, see also Watanabe et al., J. Neurosci. 32(15):5085–5096 (2012)). In some embodiments, suitable codon-optimized PSEN1 encodes a protein that is substantially identical to the full-length cDNA or a portion or fragment thereof. A wild-type PS1 or PS2 protein sequence is a protein sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical. Exemplary wild-type genomic, cDNA or protein sequences of human PSEN1 / PS1 or PSEN2 / PS2 are shown in Table 1 and below. PS1 is typically cleaved into N- and C-terminal fragments that are the active form. PS-1 is processed into two fragments: an N-terminal 28 kDa fragment, and a C-terminal 18 kDa fragment; the major endoproteolytic cleavage occurs at and near Met298 in the proximal portion of the 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). Sequences comprising or encoding these cleaved forms can also be used in the methods and compositions described herein, such as 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 NO:6-8.

[0068] Table 1: GenBank accession numbers

[0069]

[0070] >NM_000021.3 Homo sapiens presenilin 1 (PSEN1), transcript variant 1, mRNA (SEQ ID NO: 1)

[0071]

[0072] >NM_007318.2 Homo sapiens presenilin 1 (PSEN1), transcript variant 2, mRNA (SEQ ID NO: 2)

[0073]

[0074] >NM_000447.2 Homo sapiens presenilin 2 (PSEN2), transcript variant 1, mRNA (SEQ ID NO: 3)

[0075]

[0076] >NM_012486.2 Homo sapiens presenilin 2 (PSEN2), transcript variant 2, mRNA (SEQ ID NO: 4)

[0077]

[0078] >NP_000012.1 Presenilin-1 isoform I-467 [Homo sapiens] (SEQ ID NO: 5)

[0079]

[0080] >NP_015557.2 Presenilin-1 isoform I-463 [Homo sapiens] (SEQ ID NO: 6)

[0081]

[0082] >NP_000438.2 Presenilin-2 isoform 1 [Homo sapiens] (SEQ ID NO: 7)

[0083]

[0084] >NP_036618.2 Presenilin-2 isoform 2 [Homo sapiens] (SEQ ID NO: 8)

[0085]

[0086] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are compared for the best comparison purpose (for example, for the best comparison, a room can be introduced in one or both of the first and second amino acid or nucleic acid sequences and non-homologous sequences can be ignored for the purpose of comparison). The length of the reference sequence compared for the purpose of comparison is at least 80% of the length of the reference sequence, and in some embodiments at least 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecule is identical at that position. Considering the need to introduce the number of spaces and the length of each space to achieve the best comparison of the two sequences, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. In other embodiments, the percent identity of two amino acid sequences can be a function of the conservation of amino acid residues within the same amino acid family (e.g., positively charged, negatively charged, polar and uncharged, hydrophobic) at corresponding positions in the two amino acid sequences (e.g., a substitution of an alanine residue for a valine residue at a particular position in the two sequences shows high conservation, but a substitution of an arginine residue for an aspartic acid residue at a particular position in the two sequences shows low conservation).

[0087] For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm, which has been incorporated into the GAP program in the GCG software package, using, for example, a Blossum scoring matrix with default gap penalties, a gap extension penalty of 4, and a frameshift gap penalty.

[0088] Codon-optimized presenilin 1

[0089] Codon optimization is desirable for expression of proteins in specific host cells - e.g., bacteria, mice, humans. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, a large number of cDNAs encoding human Presenilin 1 can be produced, some of which have minimal similarity to the cDNA of any known and naturally occurring gene. Therefore, the present invention contemplates each and every possible cDNA variation produced by selecting combinations based on possible codon usage. These combinations are generated according to the standard triplet genetic code applicable to polynucleotides encoding naturally occurring human Presenilin variants, and all such variations are considered to be specifically disclosed. Exemplary codon-optimized human PSEN1 nucleotide sequences are disclosed herein - e.g., SEQ ID NO:9; see Figure 2 . The codon-optimized human PSEN1 nucleotide sequence is generated by replacing codons that occur at a higher frequency in human cells with codons in a naturally occurring PSEN1 nucleotide sequence that occur at a lower frequency in human cells. The codon-optimized human PSEN1 nucleotide sequence may include a sequence in which less than 100% of the codons are optimized, for example, 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 determined by calculation by methods known in the art. Table 1 discloses exemplary calculations of these codon frequencies.

[0090] An exemplary codon-optimized human PSEN1 sequence is as follows:

[0091] >Codon-optimized Homo sapiens presenilin 1 (PSEN1) cDNA (SEQ ID NO: 9)

[0092]

[0093] Table 1. Frequency of codon usage in humans (Source: Gen GenScript Codon Usage Frequency Table Tool)

[0094]

[0095]

[0096] Mutated presenilin 1

[0097] In some embodiments, the PS1 protein comprises a mutation. In some embodiments, the mutation is a conservative substitution. Such changes include the substitution of any isoleucine (I), valine (V), and leucine (L) for any other amino acid of these hydrophobic amino acids; the substitution of aspartic acid (D) for glutamic acid (E), and vice versa; the substitution of glutamine (Q) for asparagine (N), and vice versa; and the substitution of serine (S) for threonine (T), and vice versa. Other substitutions may also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) may often be interchanged, and alanine (A) and valine (V) may also often be interchanged. Relatively hydrophobic methionine (M) may often be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) may often be interchanged at positions where the important feature of the amino acid residue is its charge and the difference in the pK of the two amino acid residues is not significant. Other changes may still be considered “conservative” under certain circumstances (see, e.g., Table III of US20110201052; pp. 13-15 of “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).

[0098] In some embodiments, the method comprises introducing one or more additional mutations into the human PS1 sequence (SEQ ID NO: 5 or 6). Thus, in some embodiments, the sequence may be at least 80%, 85%, 90%, 95% or 99% identical to at least 60%, 70%, 80%, 90% or 100% of human PS1.

[0099] To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence compared for comparison purposes is typically at least 80% of the length of the reference sequence, and in some embodiments at least 90% or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identical" is equivalent to amino acid or nucleic acid "homologous"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced to achieve optimal alignment of the two sequences, and the length of each space. In other embodiments, the percent identity of two amino acid sequences can be a function of the conservation of amino acid residues within the same amino acid family (e.g., positively charged, negatively charged, polar and uncharged, hydrophobic) at corresponding positions in the two amino acid sequences (e.g., a substitution of an alanine residue for a valine residue at a particular position in the two sequences shows high conservation, but a substitution of an arginine residue for an aspartic acid residue at a particular position in the two sequences shows low conservation).

[0100] For the purposes of this method, the comparison of sequences and determination of percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0101] Delivery vehicle

[0102] Codon-optimized nucleic acids encoding PS1 polypeptides or therapeutically active fragments thereof can be incorporated into gene constructs for use as part of a gene therapy regimen. For example, targeted expression vectors are described herein for in vivo delivery and expression of codon-optimized polynucleotides encoding PS1 polypeptides or active fragments thereof in specific cell types, particularly cerebral cortical neuronal cells. Such component expression constructs can be administered in any effective vector, such as any formulation or composition that can effectively deliver component genes to cells in vivo. Methods include inserting the gene into a viral vector, preferably an adeno-associated virus. Viral vectors typically directly transduce cells.

[0103] Viral vectors capable of efficiently transducing CNS neurons can be used, including any serotype of rAAV (e.g., AAV1-AAV12) vectors, recombinant or chimeric AAV vectors, and lentivirus 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, neuronal subtype-specific specific promoters such as α-calcium / calmodulin kinase 2A promoters can be used to target stimulatory neurons. Alternatively, pan-neuronal promoters, such as synapsin I promoters, can be used to drive PS1 expression. Other exemplary promoters include, but are not limited to, cytomegalovirus (CMV) early enhancer / promoter; hybrid CMV enhancer / chicken β-actin (CBA) promoter; a promoter including CMV early enhancer elements, the first exon and first intron of chicken β-actin, and the splice acceptor of the rabbit β-globin gene (commonly referred to as "CAG promoter"); or a 1.6 kb hybrid promoter consisting of the CMV immediate early enhancer and CBA intron 1 / exon 1 (commonly referred to as the CAGGS promoter; Niwa et al. Gene, 108: 193-199 (1991)). The CAGGS promoter (Niwa et al., 1991) has been shown to provide ubiquitous and long-term expression in the brain (Klein et al., Exp. Neurol. 176: 66-74 (2002)). One method for introducing nucleic acids into cells in vivo is by using a viral vector containing a nucleic acid such as a codon-optimized cDNA encoding PS1. Among other aspects, infection of cells with viral vectors has the advantage that a large fraction of target cells can receive the nucleic acid. In addition, molecules encoded within viral vectors, such as cDNAs contained within viral vectors, are efficiently expressed in cells that have taken up the viral vector nucleic acid.

[0104] A particularly useful viral vector system for the delivery of nucleic acids is adeno-associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires other viruses such as adenovirus or herpes virus as helper viruses for efficient replication and productive life cycle. (For review, see Muzyczka et al., Curr. Topics in Micro and Immunol. 158: 97-129 (1992)). AAV vectors effectively transduce various cell types and can produce long-term expression of transgenes in vivo. Although the AAV vector genome can be maintained as an episome within the cell, vector integration has been observed (see, e.g., Deyle and Russell, Curr Opin Mol Ther. 2009 Aug;11(4):442-447; Asokan et al., Mol Ther. 2012 Apr;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 amplification or replacement and have shown therapeutic efficacy in 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 August; 11(4): 442–447; Asokan et al., Mol Ther. 2012 April; 20(4): 699–708. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can produce recombinant protein expression. Protocols for producing recombinant retroviruses and for infecting cells with such viruses in vitro or in vivo 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 laboratory manuals.The use of AAV vectors to deliver constructs for expression in the brain has been 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.

[0105] Thus, in some embodiments, the codon-optimized PSEN1 encoding nucleic acid is present in a vector for gene therapy, such as an AAV vector. In some cases, 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.

[0106] The vectors described herein can be pseudotyped vectors. Pseudotyping provides a mechanism for regulating the target cell population of the vector. For example, pseudotyped AAV vectors can be used in the various methods described herein. Pseudotyped vectors are those containing the genome of a vector, such as a genome of an AAV serotype, in the capsid of a second vector, such as a second AAV serotype. Pseudotyping methods are well known in the art. For example, the vector can be pseudotyped with an envelope glycoprotein derived from the rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Jantipula strains), rabies virus (e.g., various Evelyn–Rokitnicki–Abelseth ERA strains and challenge virus standard strain (CVS)), lyssavirus, rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein type B (FuG-B), variant of FuG-B (FuG-B2), or Moloney murine leukemia virus (MuLV). The virus can be pseudotyped for transduction of one or more neurons or cell populations.

[0107] Without limitation, illustrative examples of pseudotyped vectors include recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV9, AAVrh10, AAV11, and AAV12 serotype vectors. It is known in the art that such vectors can be engineered to include transgenes encoding human proteins or other proteins. In specific examples, the disclosure may include pseudotyped AAV9 or AAVrh10 viral vectors containing nucleic acids as disclosed herein. See Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0108] In some cases, a particular AAV serotype vector may be selected based on intended use, for example, based on intended route of administration.

[0109] Various methods for applying AAV vector constructs in gene therapy are known in the art, including methods for 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 cells of the CNS has been described (see, e.g., U.S. Pat. Nos. 6,180,613 and 6,503,888). High titer AAV preparations can be produced using techniques known in the art, such as described in U.S. Pat. No. 5,658,776.

[0110] Vector construct refers to a polynucleotide molecule that includes all or part of a viral genome and a transgene. In some cases, gene transfer can be mediated by DNA viral vectors such as adenovirus (Ad) or adeno-associated virus (AAV). Other vectors for gene therapy methods are known in the art. For example, the construct disclosed herein may include alphavirus, herpes virus, retrovirus, lentivirus, or vaccinia virus.

[0111] Adenovirus is a relatively well-characterized group of viruses, including more than 50 serotypes (see, e.g., WO 95 / 27071, which is incorporated herein by reference). Adenovirus is tractable by applying molecular biology techniques and may not require integration into the host cell genome. Recombinant Ad-derived vectors have been constructed, including vectors that reduce the potential for recombination or production of wild-type viruses (see, e.g., International Patent Publications WO 95 / 00655 and WO 95 / 11984, which are incorporated herein by reference). Wild-type AAV is highly infective and can be integrated into the host genome with high 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).

[0112] Non-natural regulatory sequences, gene control sequences, promoters, non-coding sequences, introns, or coding sequences may be included in nucleic acids as disclosed herein. Further contemplated herein are nucleic acids comprising nucleic acid tags or signal sequences, or encoding protein tags or protein signal sequences. Typically, the coding region is operably linked to one or more regulatory nucleic acid components.

[0113] The promoter contained in the nucleic acids as disclosed herein can be a tissue or cell type specific promoter, a promoter specific to multiple tissues or cell types, an organ specific promoter, a promoter specific to multiple organs, a systemic or ubiquitous promoter, or a nearly systemic or ubiquitous promoter. Promoters with stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, variable, or unpredictable expression are also included in the scope of the present disclosure. The promoter can include any of the above features or other promoter features known in the art.

[0114] In clinical settings, the gene delivery system for treating genes can be introduced into the subject by any one of a variety of methods, and each method is familiar in the art. For example, the pharmaceutical preparation of the gene delivery system can be introduced systemically, such as by intravenous injection, and the specific transduction of the protein in the target cell occurs mainly by the transfection specificity provided by the gene delivery vehicle, the cell type or tissue type expression caused by the transcriptional regulatory sequence of the control receptor gene expression, or its combination. In other embodiments, the initial delivery of recombinant genes is more limited, and the introduction into the subject is quite local at the same time. For example, the gene delivery vehicle can be introduced by a catheter (see U.S. Patent No. 5,328,470) or by stereotactic injection, for example, optionally injected into the cerebellomedullary cistern, cerebral ventricle, lumbar intrathecal space, directly injected into the hippocampus (for example, Chen et al., PNAS USA91:3054-3057 (1994)). In a preferred embodiment, the delivery method of the presenilin expression virus includes intravenous, intrathecal, intraventricular, intracistern and stereotactic intraparenchymal administration.

[0115] The methods can be further optimized through preclinical trials to achieve optimal rescue of neurodegeneration, dementia, synaptic dysfunction and molecular alterations in presenilin conditional double knockout mice and presenilin-1 knockin mice expressing FAD mutations.

[0116] The pharmaceutical preparation of gene therapy construct can be basically composed of the gene delivery system in an acceptable diluent, or can include a slow-release matrix wherein embedded with a gene delivery vehicle. Alternatively, a complete gene delivery system (complete gene delivery system) can be completely produced in recombinant cells such as retroviral vectors, and pharmaceutical preparation can include one or more cells that produce a gene delivery system.

[0117] Delivery formulations and pharmaceutical compositions

[0118] In some embodiments, a polynucleotide as disclosed herein for in vivo delivery to a target tissue is encapsulated in or associated with a nanoparticle. Methods for nanoparticle packaging are known in the art and are described in, for example, 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 inrats determined by radioluminography. J Drug Target 5: 171. 1998); Sakuma SR et al. (Mucoadhesion of polystyrene nanoparticles having surface hydrophilicpolymeric chains in the gastrointestinal tract. Int J Pharm 177:161.1999); Virovic L, et al. 52:203.2001).In some embodiments, one or more polynucleotides are delivered to a target tissue in vivo in a vesicle, such as in a liposome (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 as above). In some embodiments, lipid-based nanoparticles (LNPs) are used; see, e.g., Robinson et al., Mol Ther. 2018 Aug 1; 26(8): 2034-2046; US9956271B2.

[0119] The present methods and compositions may include microvesicles or preparations thereof containing more than one therapeutic molecule as described herein, such as polynucleotides or RNA. As used herein, "microvesicles" refer to membrane-derived microvesicles, which include a series of extracellular vesicles, including exosomes, microparticles, and shed microvesicles secreted by many cell types under normal physiological and pathological conditions. See, for example, EP2010663B1. The methods and compositions described herein can be applied to microvesicles of all sizes; in one embodiment, 30 to 200nm, in one embodiment, 30 to 800nm, in one embodiment, up to 2um. The methods and compositions described herein can also be more widely applied to all extracellular vesicles, which are terms including exosomes, shed microvesicles, tumor secretions (oncosomes), ectosomes, and retrovirus-like particles. Such microvesicles or preparations are produced by the methods described herein. As used herein, microvesicle preparations refer to microvesicle groups 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. Such microvesicle isolation methods 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 et al., ((2012) PLoS ONE 7(4):e34653.doi:10.1371 / journal.pone.0034653)), some examples of which 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, e.g., EP2010663B1.

[0120] In some embodiments, microvesicles are separated by slowly centrifuging (e.g., with about 300 g) the culture medium of donor cells with a time (e.g., about 15 minutes) sufficient to separate cells from the culture medium. This leaves microvesicles in the supernatant, thereby producing a microvesicle preparation. In one embodiment, more vigorously centrifuge (e.g., with about 16,000 g) from the culture medium or supernatant of slow centrifugation with a time (e.g., about 30 minutes) sufficient to precipitate cell debris. This leaves microvesicles in the supernatant, thereby producing a microvesicle preparation. In one embodiment, the culture medium, the slowly centrifuged preparation, or the vigorously centrifuged preparation is filtered (e.g., through a 0.22 μm filter or a 0.8 μm filter, thereby passing the microvesicles through the filter. In one embodiment, the filtrate is subjected to a final ultracentrifugation (e.g., at about 110,000 g) for a time sufficient to precipitate the microvesicles (e.g., about 80 minutes). The resulting pellet contains the microvesicles and can be resuspended in a volume of buffer that produces a concentration useful for further use, thereby producing a microvesicle preparation. In one embodiment, the microvesicle preparation is prepared by sucrose concentration gradient purification. In one embodiment, the microvesicles are further treated with DNAse (e.g., DNAse I) and / or RNAse and / or protease to eliminate contaminating DNA, RNA, or protein from the outside, respectively. In one embodiment, the microvesicle preparation contains more than one RNAse inhibitor.

[0121] The molecule included in the microvesicle preparation will include therapeutic molecules.Typically, the microvesicle in the preparation will be a heterologous colony, and each microvesicle will contain the complement of the molecule that can be the same or different with the molecule of other microvesicles in the preparation.The content of the therapeutic molecules in the microvesicle preparation can be expressed quantitatively or qualitatively.A kind of such method is that content is expressed as the percentage of the total molecule in the microvesicle preparation.For example, if the therapeutic molecule is mRNA, content can be expressed as the percentage of the total RNA content of the microvesicle preparation, or alternatively expressed as the percentage of the total mRNA content of the microvesicle preparation.Similarly, if the therapeutic molecule is protein, content can be expressed as the percentage of total protein in the microvesicle.In one embodiment, the therapeutic microvesicle or its preparation produced by the method described herein, compared with the microvesicle obtained from the control cell (obtained from the cell of the same source that does not carry out scientific operation to increase the expression of the therapeutic molecule), contains the therapeutic molecules of the amount that can be detected, statistically significantly increased. In one embodiment, therapeutic molecules are present in an amount of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% more than that obtained from the microvesicles of control cells. It is also possible to achieve a higher level of enrichment. In one embodiment, therapeutic molecules are present in microvesicles or their preparations at least 2 times more than control cell microvesicles. It is also possible to obtain a higher enrichment (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 times).

[0122] In one embodiment, the microvesicle content of relatively high percentage is therapeutic molecule (for example, by overexpression or by molecular specific targeting microvesicle to realize). In one embodiment, the microvesicle content of therapeutic molecule is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (for example, therapeutic molecule is mRNA and is about 10% of the total mRNA content of microvesicle) of total (similar) molecule content. It is also possible to achieve higher level of enrichment. In one embodiment, therapeutic molecule is present in microvesicle or its preparation with at least 2 times more than all other such (similar) molecules. It is also possible to obtain higher multiple enrichment (for example, 3,4,5,6,7,8,9 or 10 times).

[0123] Example

[0124] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0125] Example 1: Dose-dependent rescue of γ-secretase activity in MEFs with various PS genotypes: PS1 + / + 、PS1 L435F / + 、PS1 + / - 、PS1 L435F / L435F 、PS1 - / - and PS1 - / - ;PS2 - / -

[0126] To determine whether the reduced γ-secretase activity associated with PSEN1 mutations could be corrected by introduction of wild-type (WT) hPS1, primary MEFs were derived from embryos carrying various PS genotypes: PS1 + / + 、PS1 L435F / + 、PS1 + / - 、PS1 L435F / L435F 、PS1 - / - and PS1 - / - ;PS2 - / - (DKO). Immortalized MEFs were transiently transfected with CMV-NΔE, and γ-secretase activity was assessed by measuring the levels of NICD and PS1 NTF / CTF. NICD levels were reduced in a PS1 dose-sensitive manner and were undetectable in DKO cells ( Figure 1A NICD levels in PS1 L435F / L435F MEF ("L435F KI / KI" MEF) and PS1 - / - Reduced but detectable in MEFs ( Figure 1A ), however, by using L435F KI / KI and PS1 - / -In vitro γ-secretase assays of embryonic brain failed to detect de novo NICD production (Xia et al. Neuron. 2015 Mar 4; 85(5): 967-81). Without wishing to be bound by any particular theory, the applicants propose that this may be due to the low level of PS2 normally expressed in embryonic brain relative to MEFs, resulting in the low expression of PS2 in L435F KI / KI and PS1 - / - Overall PS activity in brain is lower than in MEFs. To test this hypothesis, L435F / + ;PS2 - / - γ-Secretase activity in MEFs and PS1 L435F / + Compared with MEF. L435F / + Compared with MEF, γ-secretase activity in PS1 L435F / + ;PS2 - / - In MEF, the lower Figure 1B ).

[0127] To determine whether the impaired γ-secretase activity in various PS mutant MEFs could be rescued by the introduction of WT hPS1, various amounts (0, 20, 40, 80 ng) of wild-type hPS1 cDNA (pCI-hPS1) were transfected into MEFs together with CMV-NΔE. Notably, transfection of increasing amounts of pCI-hPS1 into MEFs resulted in the mutant (PS1 L435F / + 、PS1 L435F / + ;PS2 - / - The accumulation of PS1 protein in DKO and DKO MEFs and the restoration of PS1 NTF and NICD levels ( Figure 1B ). These results indicate that exogenous WT hPS1 can rescue the impaired γ-secretase activity in various PS mutant MEFs.

[0128] Example 2: Development of an optimized wild-type human PS1 in vitro expression system

[0129] method

[0130] Cell culture and transfection

[0131] Psen-null mouse embryonic fibroblasts (MEFs) lacking endogenous PS1 and PS2 were maintained in DMEM supplemented with 10% FBS and transiently transfected with plasmids expressing wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1) with or without the γ-secretase reporter gene CMV-NΔE using Lipofectamine 3000 as indicated. Cell lysates were collected at the 24 hour time point.

[0132] Western blotting

[0133] Cell lysates were subjected to SDS-PAGE and proteins were transferred to nitrocellulose membranes. After blocking in TBST / 5% skim milk powder, the membranes were incubated overnight with primary antibodies. To control for loading, the membranes were stripped and re-probed with anti-β-actin antibodies. Band intensities were quantified using ImageJ software and the results were normalized to β-actin levels. Antibodies used included rat anti-PS1-NTF, rabbit anti-cleaved Notch (Val1744) (NICD), and mouse anti-β-actin.

[0134] result

[0135] First, we expressed wt_hPS1 or opti_hPS1 in Psen-null MEFs to eliminate any contribution from endogenous PS1 or PS2. When Psen-null MEFs were transfected with increasing amounts of vectors encoding wt_hPS1 or opti_hPS1, we detected a gradual increase in the levels of PS1 NTFs. Relative to wt_hPS1, optimized hPS1 consistently expressed more PS1 ( Figure 3A-3B ).

[0136] NotchΔE as a substrate for γ-secretase-mediated cleavage was co-expressed with wt_hPS1 or opti_hPS1 to directly measure γ-secretase activity. Notch was cleaved by γ-secretase to release Notch intracellular domain (NICD). We evaluated the dose-response relationship of γ-secretase-mediated NotchΔE cleavage producing NICD as a function of γ-secretase activity. The production of NICD increased linearly when the amount of transfected PS1 vector was low, but approached saturation at higher levels. More importantly, for each point, the optimized hPS1 had higher γ-secretase activity (Figure 3).

[0137] Secreted endogenous Aβ40 and 42 levels were determined in the culture medium using ELISA. Another γ-secretase substrate, APP C99, was used to evaluate γ-secretase activity by transiently transfecting CMV-C99 into each MEF cell line, together with increasing amounts of pCI-hPS1opti. Primary and immortalized C410Y, E280A and D385A KI / + and KI / KI MEFs and PS1KI / +; PS2- / -MEFs were established. It was determined whether KI / + and KI / KI MEFs reproduced a phenotype similar to that of KI / + and KI / KI brains, and whether the introduction of WT hPS1 could restore the reduced γ-secretase activity in a dose-dependent manner was determined by NICD and AICD production. The experiment was repeated with multiple independent MEF cell lines of each genotype. Efficacy analysis was performed to determine the sample size and number of independent experiments required to complete the study.

[0138] Example 3: Development of an optimized wild-type human PS1 in vivo expression system

[0139] Transgenic mice expressing human PSEN1 wild-type cDNA constitutively or inducibly under the control of the CAMK2A promoter were developed. To maximize the production and activity of PS1, hPS1 was codon optimized (hPS1opti), and then PS1 levels and γ-secretase activity between endogenous hPS1 and hPS1opti cDNA were compared by cotransfecting increasing amounts of pCI-hPS1 or pCI-hPS1opti and CMV-NΔE to PS DKO MEFs. Relative to endogenous hPS1 cDNA, hPS1opti cDNA caused higher levels of PS1 NTF and higher γ-secretase activity, as determined by NICD production (Figure 3).

[0140] Example 4: Determining whether postnatal delivery of hPS1opt can rescue the phenotype in PScDKO mice

[0141] The vectors listed in Table 2 were prepared. The vectors that conferred the highest GFP staining at 4 and 8 weeks after injection were identified. For this purpose, PS cDKO pups (batches - 10 breeding cages: F / F; - / -; Cre / Cre crossed with F / F; - / -) were injected at P0-2.

[0142] Table 2. Vector list

[0143] UID describe 01 AAV9 / mCaMKII-intron-hPS1opti-T2A-EGFP-SV40pA 02 AAV9 / hCaMKII-intron-hPS1opti-T2A-EGFP-SV40pA 03 AAV9 / hCaMKII-intron-EGFP-SV40pA 04 AAV9 / hSynI-intron-hPS1opti-T2A-EGFP-SV40pA 05 AAV9 / hCaMKII-intron-hPS1opti-SV40pA 06 AAV9 / hSynI-intron-hPS1opti-SV40A

[0144] AAVs were selected—e.g., AAV9 / hCaMKII-intron-hPS1opti-T2A-EGFP-SV40pA or AAV9 / hCaMKII-intron-EGFP-SV40pA—and injected into PS cDKO mice by ICV at P0-P2. Western blot analysis was performed at 4 and 8 weeks of age to determine the levels of PS1, APP, Nicastrin, PEN-2; control and PS cDKO mice were included as additional controls at 4 and 8 weeks. γ-secretase activity, NICD production, and Aβ levels (by ELISA of cortical lysates) were determined at 8 weeks of age. In addition, electrophysiological analyses—e.g., Schaffer collaterals, PPF, FF, and LTP—were performed at 2 months of age, and behavioral analyses—e.g., water maze—were performed at 2-3 months of age. Neuropath analysis was performed at 6 months of age.

[0145] Example 5: Determining whether postnatal delivery of hPS1opt can rescue phenotypes caused by FAD mutations

[0146] The mice listed in Table 3 were generated and analyzed using western blot analysis to determine PS1 and APP levels; in vitro γ-secretase activity assay; ELISA, electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; and neuropathy analysis at 6, 12, and 18 months of age.

[0147] Table 3. List of mouse genotypes

[0148]

[0149] Example 6: Determining whether AAV9 / hPS1opti delivered in the adult brain can rescue phenotypes caused by loss of PS function in vivo (PS cDKO, FAD KI)

[0150] The vectors were prepared as listed in Table 2 and a 10 ul injection was delivered via intra-CSF, via ICM using a 50 ul Hamilton syringe into the cisterna magna 1x10 12 vg), intraparenchymal delivery, or intrathecal delivery using 10ul 3.3x10 11vg lumbar puncture to inject it into mice. The mice studied will include (i) PS cDKO; (ii) KI / +; (iii) KI / +; PS2- / -; and (iv) KI / fPS1; PS2- / -; Cre. The mice will be subjected to the following analyses: Western blot analysis to measure PS1 and APP levels; in vitro γ-secretase assay; ELISA for Aβ peptide, electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; and neuropathy analysis at 6, 12, and 18 months of age.

[0151] Other Implementations

[0152] It should be understood that even though the invention has been described in conjunction with the detailed description of the invention, the foregoing description is intended to be illustrative and is not intended to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims. Sequence Listing <110> The Brigham and Women's Hospital, Inc. The General Hospital Corporation <120> Gene therapy for Alzheimer's disease <130> 29618-0234WO1 <150> 62 / 852,716 <151> 2019-05-24 <160> 11 <170> PatentIn Version 3.5 <210> 1 <211> 6107 <212> DNA <213> Homo sapiens <400> 1 aaatgacgac aacggtgagg gttctcgggc ggggcctggg acaggcagct ccggggtccg 60 cggtttcaca tcggaaacaa aacagcggct ggtctggaag gaacctgagc tacgagccgc 120 ggcggcagcg gggcggcggg gaagcgtata cctaatctgg gagcctgcaa gtgacacag 180 cctttgcggt ccttagacag cttggcctgg aggacac atgaaagaa gaacctcaag 240 aggctttgtt ttctgtgaaa cagtatttct attackagttgc tccaatgaca gagttacctg 300 caccgttgtc ctacttccag atgcacaga tgtctgagga caccacctg agcaatactg 360 tacgtagcca gatgacaat agagaacggc aggagcacaa cgacagacgg agccttggcc 420 accctgagcc attatctaat ggacgacccc agggtactc ccggcaggtg gtggagcaag 480 atgaggaag agatgaggag ctgacattga atatggcgc caagcatgtg atcatgctct 540 ttgtccctgt gactctgc atggtggtgg tcgtggctac cattaagtca gtcagctttt 600 atacccggaa ggatgggcag ctaatctata ccccattcac agaagatacc gagactgtgg 660 gccagagagc cctgcactca attctgaatg ctgccatcat gatcagtgtc attgttgtca 720 tgactatcct cctggtggtt ctgtataaat acaggtcta taagttcatc catgcctggc 780 ttattatatc atctctattg tgctgttct ttttcatt catttacttg ggggaagtgt 840 900. ttaaaaccta taacgttgct gtggactaca ttactgttgc actcctgatc tggaattttg gtgtggtggg aatgatttcc attcactgga aaggtccact tcgactccag caggcatatc 960 tcattatgat tagtgccctc atggccctgg tgtttatcaa gtacctccct gaatggactg cgtggctcat cttggctgtg atttcagtat atgatttagt ggctgttttg tgtccgaaag gtccacttcg tatgctggtt gaaacagctc aggagagaa tgaaacgctt tttccagctc tcatttactc ctcaacaatg gtgtggttgg tgaattggc agaaggagac ccggaagctc aaaggagagt atccaaaaat tccaagtata atgcagaaag cacagaaagg gagtcacaag acactgttgc agagaatgat gatggcgggt tcagtgagga atgggaagcc cagagggaca 1320. gtcatctagg gcctcatcgc tctacacctg agtcacgagc tgctgtccag gaactttcca gcagtatcct cgctggtgaa gacccagagg aaaggggagt aaaacttgga ttgggagatt tcattttcta cagtgttctg gttggtaaag cctcagcaac agccagtgga gactggaaca 1560. ctgtttcgta gccatta ttggtttgtg ccttacatta ttactccttg ccattttcaa gaagcattg ccagctctc caatctccat caccttggg cttgttttct 1620 actttgccac agatttactt gtacagcctt ttatggacca attagcattc catcatttt 1680 atatctagca tatttgcggt tagaatccca tggatgttc ttctttgact atacaaat 1740 ctggggagga aaggtgat ttcctgtgt ccacatctaa caaagtcaag attcccggct 1800 ggactttgc agctccttc caagtcttcc tgaccacctt gcactttgg acttggaag 1860 gaggtgccta taggaaacga ttttgaacat acttcatcgc agtggactgt gtccctcggt 1920 gcagaaacta ccagatttga gggacgaggt caggagata tgataggccc ggaagttgct 1980 gtgccccatc agcagcttga cgcgtggtca caggacgatt tcactgacac tgcgaacct 2040 caggactacc gttaccaga gttaggtga agtggtttta accaaacgga actctcatc 2100 ttaaactaca cgttgaaaat caacccaata attctgtatt aactgaattc tgaactttc 2160 aggaggtact gtgaggaaga gcaggcacca gcagcagaat ggggaatgga gaggtgggca 2220 ggggttccag cttccctttg atttttgct gcagactcat cctttttaaa tgagacttgt 2280 tttcccctct ctttgagtca agtcaaatat gtagattgcc tttggcaatt cttcttctca 2340 agcactgaca ctcattaccg tctgtgattg ccatttcttc ccaaggccag tctgaacctg 2400 aggttgcttt atcctaaaag ttttaacctc aggttccaaa ttcagtaaat tttggaaaca 2460 gtacagctat ttctcatcaa ttctctatca tgttgaagtc aaatttggat tttccaccaa 2520 attctgaatt tgtagacata cttgtacgct cacttgcccc agatgcctcc tctgtcctca 2580 ttcttctctc ccacacaagc agtctttttc tacagccagt aaggcagctc tgtcgtggta 2640 gcagatggtc ccattatct agggtcttac tctttgtatg atgaaaagaa tgtgttatga 2700 atcggtgctg tcagccctgc tgtcagacct tcttccacag caaatgagat gtatgcccaa 2760 2820 gtttttgctt ttgccacaca gtagctcaga atttgaacaa atagccaaaa gctggtggtt 2880 gatgaattat gaactagttg tatcaacaca aagcaagagt tgggaaagc catatttaac 2940 ttggtgagct gtgggagaac ctggtggcag aaggagaacc aactgccaag gggaaagaga 3000 aggggcctcc agcagcgaag gggatacagt gagctaatga tgtcaaggag gagtttcagg 3060 ttattctcgt cagctccaca aatgggtgct ttgtggtctc tgcccgcgtt acctttcctc 3120 tcaatgtacc tttgtgtgaa ctgggcagtg gaggtgcctg ctgcagttac catggagttc 3180 aggctctggg cagctcagtc aggcaaaaca cacaaacagc catcagcctg tgtgggctca 3240 gggcacctct ggacaaaggc ttgtggggca taaccttctt taccacagag agcccttagc 3300 tatgctgatc agaccgtaag cgtttatgag aaacttagtt tcctcctgtg gctgaggagg 3360 ggccagcttt ttcttctttt gcctgctgtt ttctctccca atctatgata tgatatgacc 3420 tggtttgggg ctgtctttgg tgtttagaat atttgttttc tgtcccagga tatttcttat 3480 aagaacctaa cttcaagagt agtgtgcgag tactgatctg aatttaaatt aaaattggct 3540 tatattaggc agtcacagac aggaaaaata agagctatgc aaagaaaggg ggatttaaag 3600 tagtaggttc tatcatctca attcattttt ttccatgaaa tcccttcttc caagattcat 3660 tccctctctc agacatgtgc tagcatgggt attatcattg agaaagcaca gctacagcaa 3720 agccacctga atagcaattt gtgattggaa gcattcttga gggatcccta atctagagta 3780 atttatttgt gtaaggatcc caaatgtgtt gcacctttca tgatacattt cttctctgaa 3840 gagggtacgt ggggtgtgtg tatttaaatc catcctatgt attactgatt gtcctgtgta 3900 gaaagatggc aattatctg tctctttctc caagtttgag ccacatctca gccacattgt 3960 tagacagtgt acagagaacc tatctttcct tttttttttt ttaaaggaca ggattttgct 4020 gtgttgccca ggctagactt gaactcctgg gctcaagtaa tccacctcag cctgagtagc 4080 tgagactaca gcccatctta tttctttaaa tcattcatct caggcagaga acttttccct 4140 caaacattct tttagaatt agttcagtca ttcctaaaac atccaaatgc tagtcttcca 4200 ccatgaaaaa tagattgtca ctggaaagaa cagtagcaat ttccataagg atgtgccttc 4260 actcacacgg gacaggcggt ggttatagag tcgggcaaaa ccagcagtag agtatgacca 4320 gccaagccaa tctgcttaat aaaaagatgg aagacagtaa ggaaggaaag tagccactaa 4380 gagtctgagt ctgactgggc tacagaataa agggtattta tggacagaat gtcattacat 4440 gcctatggga ataccaatca tatttggaag atttgcagat ttttttcag agaggaaaga 4500 ctcaccttcc tgtttttggt tctcagtagg ttcgtgtgtg ttcctagaat cacagctctg 4560 actccaaatg actcaatttc tcaattagaa aaagtagaag cttcttaagc aacttggaag 4620 aaaacagtca taagtaagca atttgttgat tttactacag aagcaacaac tgaagaggca 4680 gtgtttttac tttcagactc cgggattccc attctgtagt ctctctgctt ttaaaaaccc 4740 tccttttgca atagatgccc aaacagatga tgtttattac ttgttattta cgtggcctca 4800 gacagtgtat gtattctcga tataacttgt agagtgtgaa atataagtttt aactaccaaa 4860 taaggtctcc cagggttaga tgactgcggg aagcctttga tcccaacccc caaggctttg 4920 tatatttgat catttgtgat ctaaccctgg aagaaaaaga gctcagaaac cactatgaaa 4980 aaatttgttc agtgttttct gtgttcccgt aggttctgga gtctgaggat gcaaagatga 5040 ataagataaa ttctcagaat gtagttataa tctcttgttt tctggtatat gccatctttc 5100 tttaacttct ctaaaatatt gggtatttgt caaataacca cttttaacag ttaccattac 5160 tgagggctta tacattggtg ttaaaaagt gacttgattc agaaatcaat ccattcagta aagtactcct tctctaaatt tgctgttatg tctaagga acagtttgac ctgcccttct cctcacctcc tcacctgcct tccaacattg aatttggag gagacgtga aattggacat 5340. ttggttttgc ccttgggctg fathers agtttgagcc tags ccttgtgatc ttctcacctc tttaaattcc cacaacacaa cacaacacaa acagaggttt cagctcttca tagtgcgttg tgaatggct ggccagagtg taccaacaaa gctgtcatcg ggctcacagc tcagagacat ctgcatgtga tcatctgcat agtcctctcc tctaacggga aacacctcag atttgcatat aaaaaagcac cctggtgctg aaatgaaccc ctttcttgaa catcaaagct gtctcccaca gccttgggca gcagggtgcc tcttagtgga tgtgctgggt 5700. ccaccctgag ccctgacatg tggtggcagc attgccagtt ggtctgtgtg tctgtgtagc 5760 agggacgatt tcccagaaag caatttcct tttgaaatac gtaattgttg agactaggca gtttcaaagt cagctgcata tagtagcaag tacaggactg tcttgttttt ggtgtccttg gaggtgctgg ggtgagggtt tcagtgggat catttactct cacatgttgt ctgccttctg 5940 cttctgtgga cactgctttg tacttaattc agacagactg tgaatacacc ttttttataa 6000 atacctttca aattcttggt aagatataat tttgatagct gattgcagat tttctgtatt 6060 tgtcagatta ataaagactg catgaatcca aaaaaaaaaa aaaaaaa 6107 <210> 2 <211> 6095 <212> DNA <213> Homo sapiens <400> 2 aaatgacgac aacggtgagg gttctcgggc ggggcctggg acaggcagct ccggggtccg 60 cggtttcaca tcggaaacaa aacagcggct ggtctggaag gaacctgagc tacgagccgc 120 ggcggcagcg gggcggcggg gaagcgtata cctaatctgg gagcctgcaa gtgacaacag 180 cctttgcggt ccttagacag cttggcctgg aggagaacac atgaaagaaa gaacctcaag 240 aggctttgtt ttctgtgaaa cagtatttct atacagttgc tccaatgaca gagttacctg 300 caccgttgtc ctacttccag aatgcacaga tgtctgagga caaccacctg agcaatacta 360 atgacaatag agaacggcag gagcacaacg acagacggag ccttggccac cctgagccat 420 <h2 style=";text-align:left;direction:ltr">tatctaatgg acgaccccag ggtaactccc ggcaggtggt ggagcaagat gaggaagaag 480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgaggagct gacattgaaa tatggcgcca agcatgtgat catgctcttt gtccctgtga 540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctctctgcat ggtggtggtc gtggctacca ttaagtcagt cagcttttat accggaagg 600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgggcagct aatctatacc ccattcacag aagataccga gactgtggggc cagagagccc 660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgcactcaat tctgaatgct gccatcatga tcagtgtcat tgttgtcatg actatcctcc 720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tggtggttct gtataaatac aggtgctata aggtcatcca tgcctggctt attatatcat 780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctctattgtt gctgttcttt ttttcattca tttacttggg ggaagtgttt aaaacctata 840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acgttgctgt ggactacatt actgttgcac tcctgatctg gaattttggt gtggtgggaa 900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgatttccat tcactggaaa ggtccacttc gactccagca ggcatatctc attatgatta 960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtgccctcat ggccctggtg tttatcaagt acctccctga atggactgcg tggctcatct 1020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tggctgtgat ttcagtatat gatttagtgg ctgttttgtg tccgaaaggt ccacttcgta 1080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgctggttga aacagctcag gagagaaatg aaacgctttt tccagctctc atttactcct 1140<h2 style=";text-align:left;direction:ltr"> caacaatggt gtggttggtg aatatggcag aaggagacccc ggaagctcaa aggagagtat 1200 ccaaaaattc caagtataat gcagaaagca cagaaaggga gtcacaagac actgttgcag 1260 agaatgatga tggcgggttc agtgaggaat gggaagccca gagggacagt catctagggc 1320 ctcatcgctc tacacctgag tcacgagctg ctgtccagga actttccagc agtatcctcg 1380 ctggtgaaga cccagaggaa aggggagtaa aacttggatt gggagatttc atttctaca 1440 gtgttctggt tggtaaagcc tcagcaacag ccagtggaga ctggaacaca accatagcct 1500 gttcgtagc catattaatt ggtttgtgcc ttacattatt actccttgcc attttcaaga 1560 aagcattgcc agctcttcca atctccatca cctttgggct tgttttctac tttgccacag 1620 attatcttgt acagcctttt atggaccaat tagcattcca tcaattttat atctagcata 1680 tttgcggtta gaatcccatg gatgtttctt cttgactat aaaaaatct ggggaggaca 1740 aaggtgattt tcctgtgtcc acatctaaca aagtcaagat tcccggctgg acttttgcag 1800 cttccttcca agtcttcctg accaccttgc actattggac tttggaagga ggtgcctata 1860 gaaaacgatt ttgaacatac ttcatcgcag tggactgtgt ccctcggtgc agaaactacc 1920 agatttgagg gacgaggtca aggagatatg ataggcccgg aagttgctgt gccccatcag 1980 cagcttgacg cgtggtcaca ggacgatttc actgacactg cgaactctca ggactaccgt 2040 taccaagagg ttaggtgaag tggtttaaac caaacggaac tcttcatctt aaactacacg 2100 ttgaaaatca acccaataat tctgtattaa ctgaattctg aacttttcag gaggtactgt 2160 gaggaagagc aggcaccagc agcagaatgg ggaatggaga ggtgggcagg ggttccagct 2220 tccctttgat tttttgctgc agactcatcc tttttaaatg agacttgttt tcccctctct 2280 ttgagtcaag tcaaatatgt agattgcctt tggcaattct tcttctcaag cactgacact 2340 cattaccgtc tgtgattgcc atttcttccc aaggccagtc tgaacctgag gttgctttat 2400 cctaaaagtt ttaacctcag gttccaaatt cagtaaattt tggaaacagt acagctattt 2460 ctcatcaatt ctctatcatg ttgaagtcaa atttggattt tccaccaaat tctgaatttg 2520 tagacatact tgtacgctca cttgccccag atgcctcctc tgtcctcatt cttctctccc 2580 acacaagcag tctttttcta cagccagtaa ggcagctctg tcgtggtagc agatggtccc 2640 attattctag ggtcttactc tttgtatgat gaaaagaatg tgttatgaat cggtgctgtc 2700 agccctgctg tcagaccttc ttccacagca aatgagatgt atgcccaaag acggtagaat 2760 taaagaagag taaaatggct gttgaagcac tttctgtcct ggtattttgt ttttgctttt 2820 gccacacagt agctcagaat ttgaacaaat agccaaaagc tggtggttga tgaattatga 2880 actagttgta tcaacacaaa gcaagagttg gggaaagcca tatttaactt ggtgagctgt 2940 gggagaacct ggtggcagaa ggagaaccaa ctgccaaggg gaaagagaag gggcctccag 3000 cagcgaaggg gatacagtga gctaatgatg tcaaggagga gtttcaggtt attctcgtca 3060 gctccacaaa tgggtgcttt gtggtctctg cccgcgttac ctttcctctc aatgtacctt 3120 tgtgtgaact gggcagtgga ggtgcctgct gcagttacca tggagttcag gctctgggca 3180 gctcagtcag gcaaaacaca caaacagcca tcagcctgtg tgggctcagg gcacctctgg 3240 acaaaggctt gtggggcata accttcttta ccacagagag cccttagcta tgctgatcag 3300 accgtaagcg tttatgagaa acttagtttc ctcctgtggc tgaggagggg ccagcttttt 3360 cttctttgc ctgctgtttt ctctcccaat ctatgatatg atatgacctg gtttggggct 3420 gtctttggtg tttagaatat ttgttttctg tcccaggata tttcttataa gaacctaact 3480 tcaagagtag tgtgcgagta ctgatctgaa tttaaattaa aattggctta tattaggcag 3540 tcacagacag gaaaaataag agctatgcaa agaaaggggg atttaaagta gtaggttcta 3600 tcatctcaat tcattttttt ccatgaaatc cctcttcca agattcattc cctctctcag 3660 acatgtgcta gcatgggtat tatcattgag aaagcacagc tacagcaaag ccacctgaat 3720 agcaatttgt gattggaagc attcttgagg gatccctaat ctagagtaat ttatttgtgt 3780 aaggatccca aatgtgttgc acctttcatg atacatttct tctctgaaga gggtacgtgg 3840 ggtgtgtgta tttaaatcca tcctatgtat tactgattgt cctgtgtaga aagatggcaa 3900 ttattctgtc tctttctcca agtttgagcc acatctcagc cacattgtta gacagtgtac 3960 agagaaccta tctttcctttt tttttttt aaaggacagg atttgctgt gttgcccagg 4020 ctagacttga actcctgggc tcaagtaatc cacctcagcc tgagtagctg agactacagc 4080 ccatcttatt tctttaatc attcatctca ggcagagaac ttttccctca aacattctt 4140 ttagaattag ttcagtcatt cctaaaacat ccaatgcta gtctccacc atgaaaaata 4200 gattgtcact ggaaagaaca gtagcaattt ccataggat gtgccttcac tcacacggga 4260 caggcggtgg ttatagagtc gggcaaacc agcagtag tatgaccagc caagccaatc 4320 tgcttataa aagatggaa gaagtagg aaggaaagta gccactaaga gtctgagtct 4380 gactgggcta cagaataag ggtatttatg gagaatgt cattacatgc ctatgggaat 4440 accaatcata ttggaagat ttgcagattt ttttcagag aggaagact caccttccctg 4500 tttttgttc tcagtaggtt cgtgtgtgtt cctagaatca cagctctgac tccaaatgac 4560 tcaatttctc aattagaaaa agtagaagct ttctaagcaa cttggaagaa aacagtcata 4620 agtaagcaat ttgttgatt tactacagaa gcaacactg agaggcagt gttttactt 4680 tcagactccg ggattcccat tctgtagtct ctctgcttt aaaaaccctc cttttgcaat 4740 agatgcccaa acagatgatg tttattactt gttatttacg tggcctcaga cagtgtatgt 4800 attctcgata taacttgtag agtgtgaaat ataagtttaa ctaccaaata aggtctccca 4860 gggttagatg actgcgggaa gcctttgatc ccaaccccca aggctttgta tatttgatca 4920 tttgtgatct aaccctggaa gaaaaagagc tcagaaacca ctatgaaaaa atttgttcag 4980 tgttttctgt gttcccgtag gttctggagt ctgaggatgc aaagatgaat aagataaatt 5040 ctcagaatgt agttataatc tcttgttttc tggtatatgc catctttctt taacttctct 5100 aaaatattgg gtatttgtca aataaccact tttaacagtt accattactg agggcttata 5160 cattggtgtt ataaaagtga cttgattcag aaatcaatcc attcagtaaa gtactcttc 5220 tctaaatttg ctgttatgtc tataaggaac agtttgacct gcccttctcc tcacctctc 5280 acctgccttc caacattgaa tttggaagga gacgtgaaaa ttggacattt ggttttgcc 5340 ttgggctgga aactatcata taatcataag tttgagccta gaagtgatcc ttgtgatctt 5400 ctcacctctt taaattccca caacacaaga gattaaaaac agaggtttca gctcttcata 5460 gtgcgttgtg aaatggctgg ccagagtgta ccaacaaagc tgtcatcggg ctcacagctc 5520 agagacatct gcatgtgatc atctgcatag tcctctcctc taacgggaaa cacctcagat 5580 ttgcatataa aaaagcaccc tggtgctgaa atgaacccct ttcttgaaca tcaaagctgt 5640 ctcccacagc cttgggcagc agggtgcctc ttagtggatg tgctgggtcc accctgagcc 5700 ctgacatgtg gtggcagcat tgccagttgg tctgtgtgtc tgtgtagcag ggacgatttc 5760 ccagaaagca attttccttt tgaaatacgt aattgttgag actaggcagt ttcaaagtca 5820 gctgcatata gtagcaagta caggactgtc ttgtttttgg tgtccttgga ggtgctgggg 5880 tgagggtttc agtgggatca tttactctca catgttgtct gccttctgct tctgtggaca 5940 ctgctttgta cttaattcag acagactgtg aatacacctt ttttataaat acctttcaaa 6000 ttcttggtaa gatataattt tgatagctga ttgcagattt tctgtatttg tcagattaat 6060 aaagactgca tgaatccaaa aaaaaaaaaa aaaaa 6095 <210> 3 <211> 2302 <212> DNA <213> Homo sapiens <400> 3 ggggcctggg ccggcgccgg gtccggccgg gcgctcagcc agctgcgtaa actccgctgg 60 agcgcggcgg cagagcaggc atttccagca gtgaggagac agccagaagc aagcttttgg 120 agctgaagga acctgagaca gaagctagtc ccccctctga attttactga tgaagaaact 180 gaggccacag agctaaagtg acttttccca aggtcgccca gcgaggacgt gggacttctc 240 agacgtcagg agagtgatgt gagggagctg tgtgaccata gaaagtgacg tgttaaaaac 300 cagcgctgcc ctctttgaaa gccagggagc atcattcatt tagcctgctg agaagaagaa 360 accaagtgtc cgggattcag acctctctgc ggccccaagt gttcgtggtg cttccagagg 420 cagggctatg ctcacattca tggcctctga cagcgaggaa gaagtgtgtg atgagcggac 480 gtccctaatg tcggctgaga gccccacgcc gcgctcctgc caggagggca ggcagggccc 540 agaggatgga gagaacactg cccagtggag aagccaggag aacgaggagg acggtgagga 600 ggaccctgac cgctatgtct gtagtggggt tcccgggcgg ccgccaggcc tggaggaaga 660 gctgaccctc aaatacggag cgaagcacgt gatcatgctg tttgtgcctg tcactctgtg 720 catgatcgtg gtggtagcca ccatcaagtc tgtgcgcttc tacacagaga agaatggaca 780 gctcatctac acgccattca ctgaggacac accctcggtg ggccagcgcc tcctcaactc 840 cgtgctgaac accctcatca tgatcagcgt catcgtggtt atgaccatct tcttggtggt 900 gctctacaag taccgctgct acaagttcat ccatggctgg ttgatcatgt cttcactgat 960 gctgctgttc ctcttcacct atatctacct tggggaagtg ctcaagacct acaatgtggc 1020 catggactac cccaccctct tgctgactgt ctggaacttc ggggcagtgg gcatggtgtg 1080 catccactgg aagggccctc tggtgctgca gcaggcctac ctcatcatga tcagtgcgct 1140 catggcccta gtgttcatca agtacctccc agagtggtcc gcgtgggtca tcctgggcgc 1200 catctctgtg tatgatctcg tggctgtgct gtgtcccaaa gggcctctga gaatgctggt 1260 agaaactgcc caggagagaa atgagcccat attccctgcc ctgatatact catctgccat 1320 ggtgtggacg gttggcatgg cgaagctgga cccctcctct cagggtgccc tccagctccc 1380 ctacgacccg gagatggaag aagactccta tgacagtttt ggggagcctt cataccccga 1440 agtctttgag cctcccttga ctggctaccc aggggaggag ctggaggaag aggaggaaag 1500 gggcgtgaag cttggcctcg gggacttcat cttctacagt gtgctggtgg gcaaggcggc 1560 tgccacgggc agcggggact ggaataccac gctggcctgc ttcgtggcca tcctcattgg 1620 cttgtgtctg accctcctgc tgcttgctgt gttcaagaag gcgctgcccg ccctccccat 1680 ctccatcacg ttcgggctca tcttttactt ctccacggac aacctggtgc ggccgttcat 1740 ggacaccctg gcctcccatc agctctacat ctgagggaca tggtgtgcca caggctgcaa 1800 gctgcaggga attttcattg gatgcagttg tatagtttta cactctagtg ccatatattt 1860 ttaagacttt tctttcctta aaaaataaag tacgtgttta cttggtgagg aggaggcaga 1920 accagctctt tggtgccagc tgtttcatca ccagactttg gctcccgctt tggggagcgc 1980 ctcgcttcac ggacaggaag cacagcaggt ttatccagat gaactgagaa ggtcagatta 2040 gggcggggag aagagcatcc ggcatgaggg ctgagatgcg caaagagtgt gctcgggagt 2100 ggcccctggc acctgggtgc tctggctgga gaggaaaagc cagttcccta cgaggagtgt 2160 tcccaatgct ttgtccatga tgtccttgtt attttattgc ctttagaaac tgagtcctgt 2220 tcttgttacg gcagtcacac tgctgggaag tggcttaata gtaatatcaa taaatagatg 2280 agtcctgtta gaatcttgaa aa 2302 <210> 4 <211> 2299 <212> DNA <213> Homo sapiens <400> 4 ggggcctggg ccggcgccgg gtccggccgg gcgctcagcc agctgcgtaa actccgctgg 60 agcgcggcgg cagagcaggc atttccagca gtgaggagac agccagaagc aagcttttgg 120 agctgaagga acctgagaca gaagctagtc ccccctctga attttactga tgaagaaact 180 gaggccacag agctaaagtg acttttccca aggtcgccca gcgaggacgt gggacttctc 240 agacgtcagg agagtgatgt gagggagctg tgtgaccata gaaagtgacg tgttaaaaac 300 cagcgctgcc ctctttgaaa gccagggagc atcattcatt tagcctgctg agaagaagaa 360 accaagtgtc cgggattcag acctctctgc ggccccaagt gttcgtggtg cttccagagg 420 cagggctatg ctcacattca tggcctctga cagcgaggaa gaagtgtgtg atgagcggac 480 gtccctaatg tcggctgaga gccccacgcc gcgctcctgc caggagggca ggcagggccc 540 agaggatgga gagaacactg cccagtggag aagccaggag aacgaggagg acggtgagga 600 ggaccctgac cgctatgtct gtagtggggt tcccgggcgg ccgccaggcc tggaggaaga 660 gctgaccctc aaatacggag cgaagcacgt gatcatgctg tttgtgcctg tcactctgtg 720 catgatcgtg gtggtagcca ccatcaagtc tgtgcgcttc tacacagaga agaatggaca 780 gctcatctac acgccattca ctgaggacac accctcggtg ggccagcgcc tcctcaactc 840 cgtgctgaac accctcatca tgatcagcgt catcgtggtt atgaccatct tcttggtggt 900 gctctacaag taccgctgct acaagttcat ccatggctgg ttgatcatgt cttcactgat 960 gctgctgttc ctcttcacct atatctacct tggggaagtg ctcaagacct acaatgtggc 1020 catggactac cccaccctct tgctgactgt ctggaacttc ggggcagtgg gcatggtgtg 1080 catccactgg aagggccctc tggtgctgca gcaggcctac ctcatcatga tcagtgcgct 1140 catggcccta gtgttcatca agtacctccc agagtggtcc gcgtgggtca tcctgggcgc 1200 catctctgtg tatgatctcg tggctgtgct gtgtcccaaa gggcctctga gaatgctggt 1260 agaaactgcc caggagagaa atgagcccat attccctgcc ctgatatact catctgccat 1320 ggtgtggacg gttggcatgg cgaagctgga cccctcctct cagggtgccc tccagctccc 1380 ctacgacccg gagatggaag actcctatga cagttttggg gagccttcat accccgaagt 1440 ctttgagcct cccttgactg gctacccagg ggaggagctg gaggaagagg aggaaagggg 1500 cgtgaagctt ggcctcgggg acttcatctt ctacagtgtg ctggtgggca aggcggctgc 1560 cacgggcagc ggggactgga ataccacgct ggcctgcttc gtggccatcc tcattggctt 1620 gtgtctgacc ctcctgctgc ttgctgtgtt caagaaggcg ctgcccgccc tccccatctc 1680 catcacgttc gggctcatct tttacttctc cacggacaac ctggtgcggc cgttcatgga 1740 caccctggcc tcccatcagc tctacatctg agggacatgg tgtgccacag gctgcaagct 1800 gcagggaatt ttcattggat gcagttgtat agttttacac tctagtgcca tatattttta 1860 agacttttct ttccttaaaa aataaagtac gtgtttactt ggtgaggagg aggcagaacc 1920 agctctttgg tgccagctgt ttcatcacca gactttggct cccgctttgg ggagcgcctc 1980 gcttcacgga caggaagcac agcaggttta tccagatgaa ctgagaaggt cagattaggg 2040 cggggagaag agcatccggc atgagggctg agatgcgcaa agagtgtgct cgggagtggc 2100 ccctggcacc tgggtgctct ggctggagag gaaaagccag ttccctacga ggagtgttcc 2160 caatgctttg tccatgatgt ccttgttatt ttattgcctt tagaaactga gtcctgttct 2220 tgttacggca gtcacactgc tgggaagtgg cttaatagta atatcaataa atagatgagt 2280 cctgttagaa tcttgaaaa 2299 <210> 5 <211> 467 <212> PRT <213> Homo sapiens <400> 5 Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met 1 5 10 15 Ser Glu Asp Asn His Leu Ser Asn Thr Val Arg Ser Gln Asn Asp Asn 20 25 30 Arg Glu Arg Gln Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu 35 40 45 Pro Leu Ser Asn Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu 50 55 60 Gln Asp Glu Glu Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys 65 70 75 80 His Val Ile Met Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val 85 90 95 Val Ala Thr Ile Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln 100 105 110 Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg 115 120 125 Ala Leu His Ser Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val 130 135 140 Val Met Thr Ile Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys 145 150 155 160 Val Ile His Ala Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe 165 170 175 Phe Ser Phe Ile Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala 180 185 190 Val Asp Tyr Ile Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val 195 200 205 Gly Met Ile Ser Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala 210 215 220 Tyr Leu Ile Met Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr 225 230 235 240 Leu Pro Glu Trp Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr 245 250 255 Asp Leu Val Ala Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val 260 265 270 Glu Thr Ala Gln Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr 275 280 285 Ser Ser Thr Met Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu 290 295 300 Ala Gln Arg Arg Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr 305 310 315 320 Glu Arg Glu Ser Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe 325 330 335 Ser Glu Glu Trp Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg 340 345 350 Ser Thr Pro Glu Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile 355 360 365 Leu Ala Gly Glu Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly 370 375 380 Asp Phe Ile Phe Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala 385 390 395 400 Ser Gly Asp Trp Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile 405 410 415 Gly Leu Cys Leu Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu 420 425 430 Pro Ala Leu Pro Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala 435 440 445 Thr Asp Tyr Leu Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln 450 455 460 Phe Tyr Ile 465 <210> 6 <211> 463 <212> PRT <213> Homo sapiens <400> 6 Met Thr Glu Leu Pro Ala Pro Leu Ser Tyr Phe Gln Asn Ala Gln Met 1 5 10 15 Ser Glu Asp Asn His Leu Ser Asn Thr Asn Asp Asn Arg Glu Arg Gln 20 25 30 Glu His Asn Asp Arg Arg Ser Leu Gly His Pro Glu Pro Leu Ser Asn 35 40 45 Gly Arg Pro Gln Gly Asn Ser Arg Gln Val Val Glu Gln Asp Glu Glu 50 55 60 Glu Asp Glu Glu Leu Thr Leu Lys Tyr Gly Ala Lys His Val Ile Met 65 70 75 80 Leu Phe Val Pro Val Thr Leu Cys Met Val Val Val Val Ala Thr Ile 85 90 95 Lys Ser Val Ser Phe Tyr Thr Arg Lys Asp Gly Gln Leu Ile Tyr Thr 100 105 110 Pro Phe Thr Glu Asp Thr Glu Thr Val Gly Gln Arg Ala Leu His Ser 115 120 125 Ile Leu Asn Ala Ala Ile Met Ile Ser Val Ile Val Val Met Thr Ile 130 135 140 Leu Leu Val Val Leu Tyr Lys Tyr Arg Cys Tyr Lys Val Ile His Ala 145 150 155 160 Trp Leu Ile Ile Ser Ser Leu Leu Leu Leu Phe Phe Phe Ser Phe Ile 165 170 175 Tyr Leu Gly Glu Val Phe Lys Thr Tyr Asn Val Ala Val Asp Tyr Ile 180 185 190 Thr Val Ala Leu Leu Ile Trp Asn Phe Gly Val Val Gly Met Ile Ser 195 200 205 Ile His Trp Lys Gly Pro Leu Arg Leu Gln Gln Ala Tyr Leu Ile Met 210 215 220 Ile Ser Ala Leu Met Ala Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp 225 230 235 240 Thr Ala Trp Leu Ile Leu Ala Val Ile Ser Val Tyr Asp Leu Val Ala 245 250 255 Val Leu Cys Pro Lys Gly Pro Leu Arg Met Leu Val Glu Thr Ala Gln 260 265 270 Glu Arg Asn Glu Thr Leu Phe Pro Ala Leu Ile Tyr Ser Ser Thr Met 275 280 285 Val Trp Leu Val Asn Met Ala Glu Gly Asp Pro Glu Ala Gln Arg Arg 290 295 300 Val Ser Lys Asn Ser Lys Tyr Asn Ala Glu Ser Thr Glu Arg Glu Ser 305 310 315 320 Gln Asp Thr Val Ala Glu Asn Asp Asp Gly Gly Phe Ser Glu Glu Trp 325 330 335 Glu Ala Gln Arg Asp Ser His Leu Gly Pro His Arg Ser Thr Pro Glu 340 345 350 Ser Arg Ala Ala Val Gln Glu Leu Ser Ser Ser Ile Leu Ala Gly Glu 355 360 365 Asp Pro Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile Phe 370 375 380 Tyr Ser Val Leu Val Gly Lys Ala Ser Ala Thr Ala Ser Gly Asp Trp 385 390 395 400 Asn Thr Thr Ile Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys Leu 405 410 415 Thr Leu Leu Leu Leu Ala Ile Phe Lys Lys Ala Leu Pro Ala Leu Pro 420 425 430 Ile Ser Ile Thr Phe Gly Leu Val Phe Tyr Phe Ala Thr Asp Tyr Leu 435 440 445 Val Gln Pro Phe Met Asp Gln Leu Ala Phe His Gln Phe Tyr Ile 450 455 460 <210> 7 <211> 448 <212> PRT <213> Homo sapiens <400> 7 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu 1 5 10 15 Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln 20 25 30 Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg 35 40 45 Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80 Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85 90 95 Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr 100 105 110 Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr 115 120 125 Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile 130 135 140 Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr 145 150 155 160 Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser 165 170 175 Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu 180 185 190 Lys Thr Tyr Asn Val Ala Met Asp Tyr Pro Thr Leu Leu Leu Thr Val 195 200 205 Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro 210 215 220 Leu Val Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala 225 230 235 240 Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu 245 250 255 Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly 260 265 270 Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile 275 280 285 Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met 290 295 300 Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp 305 310 315 320 Pro Glu Met Glu Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr 325 330 335 Pro Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu 340 345 350 Glu Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile 355 360 365 Phe Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp 370 375 380 Trp Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys 385 390 395 400 Leu Thr Leu Leu Leu Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu 405 410 415 Pro Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn 420 425 430 Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile 435 440 445 <210> 8 <211> 447 <212> PRT <213> Homo sapiens <400> 8 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu 1 5 10 15 Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln 20 25 30 Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg 35 40 45 Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80 Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85 90 95 Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr 100 105 110 Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr 115 120 125 Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile 130 135 140 Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr 145 150 155 160 Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser 165 170 175 Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu 180 185 190 Lys Thr Tyr Asn Val Ala Met Asp Tyr Pro Thr Leu Leu Leu Thr Val 195 200 205 Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro 210 215 220 Leu Val Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala 225 230 235 240 Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu 245 250 255 Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly 260 265 270 Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile 275 280 285 Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met 290 295 300 Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp 305 310 315 320 Pro Glu Met Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr Pro 325 330 335 Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu Glu 340 345 350 Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile Phe 355 360 365 Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp Trp 370 375 380 Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys Leu 385 390 395 400 Thr Leu Leu Leu Leu Ala Val Phe Lys Lys Ala Leu Pro Ala Leu Pro 405 410 415 Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn Leu 420 425 430 Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile 435 440 445 <210> 9 <211> 1414 <212> DNA <213> Artificial sequence <220> <223> Codon-optimized Homo sapiens presenilin 1 (PSEN1) cDNA <400> 9 cgacgccacc atgacagaac tgcctgcccc cctgagctac ttccagaacg cccagatgag 60 cgaggacaac cacctgagca acaccgtgcg gagccagaac gacaacagag agcggcagga 120 acacaacgac aggcggagcc tgggaccccc tgagcccctg tctaatggca gaccccaggg 180 caacagcaga caggtggtgg aacaggacga ggagaggac gaaactga ccctgaagta 240 cggcgccaag cacgtgatca tgctgttcgt gcccgtgacc ctgtgcatgg tcgtggtggt 300 ggccacaatc aagagcgtgt ccttctacac ccggaggac ggccagctga tctacacccc 360 cttcaccgag gacaccgaga cagtgggaca gagagccctg cacagcatcc tgaacgccgc 420 catcatgatc agcgtgatcg tcgtgatgac catcctgctg gtggtgctgt acaagtaccg 480 gtgctacaaa gtgatccacg cctggctgat catcagcagc ctgctgctgc tgttcttctt 540 tagcttcatc tacctgggcg aggtgttcaa gacctacaac gtggccgtgg actacatcac 600 cgtggccctg ctgatctgga acttcggcgt cgtgggcatg atctccatcc actggaaggg 660 ccccctgaga ctgcagcagg cctacctgat tatgatctcc gccctgatgg ccctggtgtt 720 catcaagtac ctgcccgagt ggaccgcttg gctgatcctg gccgtgatct ccgtgtacga 780 cctggtggcc gtgctgtgcc ctaagggacc tctgcggatg ctggtggaaa ccgcccagga 840 acggaacgag acactgttcc ctgccctgat ctactccagc acaatggtgt ggctcgtgaa catggccgag ggcgatcctg aggcccagcg gagagtgtcc aagaactcca agtacaacgc 960 cgagagcacc gagcgcgaga gccaggatac agtggccgag aatgacgacg gcggcttcag 1020 cgaggaatgg gaggcccaga gagatagcca cctgggccct cacagaagca cccctgaatc tagagccgcc gtgcaggaac tgagcagctc cattctggcc ggcgaggacc ccgaagaaag aggcgtgaaa ctgggcctgg gcgacttcat cttctacagc gtgctcgtgg gcaaggccag cgccacagct agcggcgact ggacaccac aatcgcctgc ttcgtggcca tcctgatcgg cctgtgtctg acacttctgc tgctggccat cttcaagaag gccctgcccg ccctgcctat 1320 cagcatcacc ttcggcctgg tgttttactt cgccaccgac tacctggtgc agcccttcat 1380 ggaccagctg gccttccacc agttctacat ctga <210> 10 <211> 10 <212> DNA <213> The snowstorm <220> <223> The Kozak snowstorm <220> <221> misc_feature <222> (4)..(4) <223> n=g or a <400> 10 gccnccatgg 10 <210> 11 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> hPSEN1opt Kozak seq <400> 11 cgacgccacc atgacagaac tgc 23

Claims

1. A composition comprising a human codon-optimized polynucleotide encoding human PSEN1, wherein the human codon-optimized polynucleotide encoding human PSEN1 is SEQ ID NO:

9.

2. The composition of claim 1, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO:

5.

3. The composition of claim 1, wherein upon expression, the human codon-optimized polynucleotide results in increased levels of PSEN-1 and / or γ-secretase activity compared to the polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 2, respectively.

4. The composition according to any one of claims 1 to 3, which is associated with exosomes or lipid-based nanoparticles. 5 . A composition comprising a vector for expressing human PSEN1 in a cell, the vector comprising the human codon-optimized polynucleotide according to any one of claims 1 to 4 operably linked to a promoter.

6. The composition of claim 5, wherein the vector is a viral vector.

7. The composition of claim 6, wherein the viral vector is an adeno-associated virus (AAV) vector.

8. The composition of claim 7, wherein the AAV vector is AAV9 or AAVrh10.

9. The composition of claim 6, wherein the viral vector is a lentiviral vector or a retroviral vector.

10. The composition of claim 5, wherein the promoter is a pan-neuronal promoter.

11. The composition of claim 10, wherein the pan-neuronal promoter is the synapsin I promoter.

12. The composition of claim 5, wherein the promoter is a neuronal subtype-specific promoter.

13. The composition of claim 12, wherein the neuronal subtype-specific promoter is the α-calcium / calmodulin kinase 2A promoter.

14. Use of a composition according to any one of claims 1 to 13 in the preparation of a medicament for treating Alzheimer's disease by the following method, the method comprising administering the composition to a human subject in need of treatment, wherein the subject has more than one mutation encoding a dominant negative PSEN1 protein isoform in at least one allele of PSEN1.

15. The use according to claim 14, wherein the Alzheimer's disease is familial Alzheimer's disease.

16. The use according to claim 14, wherein the subject has E280A, Y115H, L166P, C410Y, D257A, R278I, L435F, G384A or L392V mutations in the PSEN1 gene, or has N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, E280G, P264L, E185D, L235V or M146V mutations in the PSEN1 gene.

17. The use according to claim 14, wherein the Alzheimer's disease is sporadic Alzheimer's disease.

18. The use according to claim 14, wherein the Alzheimer's disease is late-onset or early-onset Alzheimer's disease.

19. Use according to any one of claims 14 to 18, wherein the composition is administered to the CNS of a subject in need of treatment.

20. The use of claim 19, wherein the administration to the CNS is by intravenous delivery, by intrathecal delivery, by intracisternal delivery, by intraventricular delivery, or by stereotactic injection into the cisterna magna, cerebral ventricles, or lumbar intrathecal space, or by direct injection into the hippocampus or cortical area.

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