Gene Therapy for Alzheimer's Disease

Abstract

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

Description

[Technical Field]

[0001] Claim of priority This application claims the benefits of U.S. Provisional Patent Application No. 62 / 852,716, filed on 24 May 2019. All of the foregoing is incorporated herein by reference.

[0002] Federally funded research or development This invention was made with government assistance under authorization number NS041783 granted by the National Institutes of Health. The government has certain rights to this invention.

[0003] This specification particularly describes compositions and methods for treating Alzheimer's disease (AD) and other neurodegenerative diseases using presenilin gene therapy constructs. [Background technology]

[0004] 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. The rate of progression varies from patient to patient, but the average life expectancy after diagnosis is 3 to 9 years. Currently, there is no cure for Alzheimer's disease. [Overview of the Initiative] [Means for solving the problem]

[0005] This disclosure describes methods and compositions that can be used to treat subjects with Alzheimer's disease (AD) and other neurodegenerative diseases, disorders, or conditions. This disclosure is at least in part based on the finding that supplying codon-optimized wild-type PSEN1 cDNA to cells carrying heterogeneous or allozygous dominant-negative Psen1 mutations, an established familial Alzheimer's disease model, resulted in unexpectedly high expression levels and restoration of impaired γ-secretase activity in such cells. Accordingly, this disclosure provides methods for gene therapy effective for Alzheimer's disease and other neurodegenerative dementias, based on PSEN1 (for PS1 expression) and / or PSEN2 (for PS2 expression), representing a major breakthrough in this disease area.

[0006] This specification provides compositions comprising a human codon-optimized polynucleotide encoding human presenilin 1 protein (PS1), for example, SEQ ID NO: 9, or a polynucleotide containing a sequence at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 9 (with at least one codon optimized for the wild type). Typical human PS1 protein sequences include SEQ ID NOs: 5 and 6, and sequences containing at least a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1). In some embodiments, the compositions are accompanied by exosomes or lipid-based nanoparticles (LNPs) (e.g., formulated using these for delivery).

[0007] This specification also provides compositions comprising a vector for expressing human PSEN1 in cells, comprising a human codon-optimized polynucleotide described herein operably linked to a promoter.

[0008] This specification also provides the use of any of the compositions described herein in a method for treating a neurodegenerative disease, disorder, or condition in a subject.

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

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

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

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

[0013] In some embodiments, 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.

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

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

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

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

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

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

Brief Description of the Drawings

[0020] [Figure 1] (FIGS. 1A-B) Figures showing that the impaired γ-secretase activity is restored by the introduction of WT hPS1 into mutant MEFs. In A, the γ-secretase activity measured by NICD production decreases in a PS dose-dependent manner in mutant MEF cells (WT>PS1 heterozygous KI or KO>homozygous PS1 KI or KO>DKO). In B, the impaired γ-secretase activity is restored by WT hPS1. Increasing amounts of pCI-hPSEN1 plasmid DNA are transfected into MEFs of different genotypes as indicated. Western analysis showed that both PS1 NTF and NICD were restored in various PS mutant MEFs. Heterozygous L435F KI cells are labeled as KI / + or PS1L435F / +. Independent experiments with N = 3. Data are represented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001 (Tukey's post hoc test analysis together with one-way ANOVA). [Figure 2-1] Figure showing the sequence comparison of endogenous human PSEN1 (hPSEN1) cDNA and codon-optimized hPSEN1 cDNA (Opti-hPSEN1). [Figure 2-2]This figure shows a sequence comparison between endogenous human PSEN1 (hPSEN1) cDNA and codon-optimized hPSEN1 cDNA (Opti-hPSEN1). [Figure 3] (Figure 3A-B) This figure shows that the expression level of PS1 NTF was increased with codon-optimized PSEN1 cDNA. A. Psen-null MEFs were transfected with an expansion plasmid expressing either wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1), and Western dysplasia was performed using an antibody specific to the N-terminus of PS1. untrans is an untransfected MEF as a negative control. B. Quantification of PS1 NTF levels in cells transfected with either wild-type endogenous hPSEN1 cDNA (wt_PS1) or codon-optimized hPSEN1 cDNA (opti_PS1). Data are expressed as mean ± SEM (n=3 independent experiments). [Figure 4] This figure shows that codon optimization increased γ-secretase activity. PS DKO MEFs were transfected with increased amounts (12.5, 25, 50, or 100 ng) of pCI-hPS1 or pCI-hPS1opti plasmid DNA and CMV-NΔE, followed by Western blotting analysis of NICDs. MEFs transfected with untransfected or empty vectors were included as negative controls. We found that pCI-hPS1opti resulted in significantly higher γ-secretase activity levels, as measured by NICD generation, compared to pCI-hPS1. Data are presented as mean ± SEM (n=5 independent experiments). Statistical significance was assessed using two-way ANOVA. **p<0.01. [Modes for carrying out the invention]

[0021] definition To facilitate a better understanding of this disclosure, certain terms are first defined as follows. Further definitions of the following terms and other terms are set forth throughout this specification.

[0022] Administration: As used herein, the term “administration” refers to the delivery or application of a composition to a subject or system. Administration to an animal subject (e.g., human) may be by any suitable route. For example, in some embodiments, administration may be by bronchial (including by bronchial drip), buccal, enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, subarachnoid, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (including by intratracheal infusion), transdermal, vaginal, and intravitreous administration.

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

[0024] Contrast: As used herein, the term “control” has the meaning understood in the art of being a standard for comparing results. Typically, a control is used to isolate a variable and enhance the completeness of an experiment by drawing conclusions about such variable. In some embodiments, the control is a reaction or assay that is performed concurrently with the test reaction or assay to provide a comparator. In one experiment, the “test” (i.e., the variable being tested) is applied. In a second experiment, the “control,” which is the variable being tested, is not applied. In some embodiments, the control is a historical control (i.e., a previously performed test or assay, or a previously known quantity or result). In some embodiments, the control is or includes a printed or stored record. The control may be a positive control or a negative control. In some embodiments, the control may be a “reference control,” which is a sample used for comparison with a test sample to examine differences, or for characterization purposes.

[0025] Gene therapy: As used herein, the term “gene therapy” refers to any treatment involving the direct or indirect administration of nucleic acids to a target. In certain cases, a therapeutically valuable protein is expressed from the administered nucleic acid.

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

[0027] Improvement, increase / rise, or decrease / decrease: As used herein, the terms “improvement,” “increase,” or “reduce,” or grammatically equivalent terms, refer to values ​​related to baseline measurements, e.g., measurements in the same individual before the initiation of the treatment described herein, or measurements in a control individual (or more control individuals) in the absence of the treatment described herein. A “control individual” is an individual with the same type and approximately the same severity of, for example, Alzheimer’s disease as the individual being treated, and is approximately the same age as the individual being treated (to ensure that the disease stages of the individual being treated and the control individual(s) are comparable).

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

[0029] subject: As used herein, the term “subject” means a human or any non-human animal (e.g., a mammal such as a mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may also be a patient, meaning a human being who visits a medical institution for the diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient.” The subject may have or be susceptible to a disease or disorder, but it is unknown whether or not they exhibit symptoms of the disease or disorder.

[0030] To be suffering from: An individual “suffering” from a disease, disorder, and / or condition (e.g., Alzheimer’s disease) is diagnosed with or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0031] ~sensitive to: Individuals who are "susceptible" to a disease, disorder, and / or condition may not have been diagnosed with, and / or exhibit, symptoms of, the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition (e.g., Alzheimer's disease) may be characterized by one or more of the following: (1) gene mutations associated with the development of the disease, disorder, and / or condition; (2) gene polymorphisms associated with the development of the disease, disorder, and / or condition; (3) increased and / or decreased expression and / or activity of proteins associated with the disease, disorder, and / or condition; (4) habits and / or lifestyles associated with the development of the disease, disorder, and / or condition; (5) family history of the disease, disorder, and / or condition; (6) a reaction to a particular bacterium or virus; or (7) exposure to a particular chemical. In some embodiments, individuals susceptible to a disease, disorder, and / or condition develop the disease, disorder, and / or condition. In some embodiments, individuals susceptible to a disease, disorder, and / or condition do not develop the disease, disorder, and / or condition.

[0032] Therapeutic effective dose: As used herein, the term “therapeutic dose” refers to the amount of therapeutic protein that confers a therapeutic effect to the treated subject in a reasonable benefit-risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by certain tests or markers) or subjective (i.e., the subject exhibits signs of or feels the effect). In particular, “therapeutic dose” refers to the amount of therapeutic protein or composition effective in treating, restoring, or preventing a desired disease or condition, or in exhibiting a detectable therapeutic or preventive effect, for example, by restoring symptoms associated with the disease, preventing or delaying the onset of the disease, and / or similarly, by reducing the severity or frequency of symptoms of the disease. Therapeutic doses are generally administered in dosing regimens that may contain multiple unit doses. For any particular therapeutic protein, the therapeutic dose (and / or appropriate unit dose in an effective dosing regimen) may vary depending, for example, the route of administration, and combinations with other pharmaceuticals. Furthermore, a specific therapeutically effective dose (and / or unit dose) for any particular patient may depend on a variety of factors, including the disorder being treated and its severity; the activity of the specific drug being used; the specific composition being used; the patient's age, weight, overall health, sex, and diet; the timing of administration, the route of administration, and / or the excretion or metabolic rate of the specific fusion protein being used; the duration of treatment; and similar factors well known in the medical technology.

[0033] Treatment: As used herein, the term “treatment” (also “treat” or “treating”) means, in its broadest sense, any administration of a substance (e.g., a composition provided) that partially or completely reduces, restores, alleviates, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of one or more symptoms, characteristics, and / or causes of a particular disease, disorder, and / or condition. In some embodiments, such treatment may be administered to subjects who do not show signs of the disease, disorder, and / or condition in question, and / or subjects who show only initial signs of the disease, disorder, and / or condition. Alternatively, or in addition, in some embodiments, treatment may be administered to subjects who show one or more established signs of the disease, disorder, and / or condition in question. In some embodiments, treatment may be administered to subjects diagnosed with the disease, disorder, and / or condition in question. In some embodiments, treatment may be administered to subjects who are known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the disease, disorder, and / or condition in question.

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

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

[0036] Various aspects of the present invention are described in detail in the following sections. Use of these sections is not intended to limit the present invention. Each section may be applied to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise specified.

[0037] Treatment methods In non-limiting examples, the present invention includes gene therapy to express wild-type human presenilin 1 in subjects suffering from or susceptible to neurodegenerative diseases associated with dominant-negative mutations in PSEN1 or PSEN2, such as Alzheimer's disease (e.g., familial AD or sporadic AD patients carrying PSEN1 or PSEN2 mutations). The objective of such gene therapy is, in particular, to promote PS1 expression in the brains of familial or sporadic AD patients to correct or overcome defects in PS1 or PS2 expression and / or activity. In FAD patients, the gene therapy methods described herein are expected to result in increased expression of wild-type PS1 in the brain and restoration of γ-secretase activity dysfunction associated with PS1 or PS2 mutations.

[0038] Mutations in the presenilin genes, PSEN1 and PSEN2, are highly penetrating, accounting for approximately 90% of all identified mutations in familial Alzheimer's disease (FAD), highlighting their importance in the pathogenesis of AD. More than 260 distinct mutations have been reported in PSEN1, which are dominant heritable and mostly missense mutations. Dominant-negative mutations in the PSEN1 and PSEN2 genes are known to be associated with early-onset familial Alzheimer's disease. PS1 and presenilin 2 (PS2) proteins are part of the γ-secretase complex, and mutations in the PSEN1 and PSEN2 genes are generally thought to contribute to the accumulation of amyloid-beta (Aβ) protein in Alzheimer's disease patients. Pathogenic PSEN1 mutations act in the cis state to impair mutant PS1 function and in the trans state 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, these properties themselves prevent dominant-negative mutations from being restored by wild-type protein expression (Herskowitz, I. Nature, 329:219-222 (1987)). Surprisingly, however, as shown herein, transfecting immortalized MEFs harboring heterologous or homogeneous PS1 mutations with codon-optimized hPSEN1 cDNA can restore impaired γ-secretase activity in such cells far more effectively than with wild-type human sequences (see examples below), demonstrating that expression of wild-type PS1 from codon-optimized exogenous sequences can overcome the dominant-negative effects of mutant presenilin protein. While we do not wish to be bound by any particular theory, PS1 expression may achieve this objective by increasing the total level of wild-type PS1, thereby restoring the dysfunction of γ-secretase expression and / or activity in AD patients.

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

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

[0041] In some embodiments, the methods of the present invention may be used to treat subjects having mutations in the PSEN1 or PSEN2 allele, for example, mutations that have a dominant-negative effect on the wild-type PS protein. Examples of mutations include C410Y, Δex9, G548, D257A, L166P, R278I, L435F, G384A, Y115H, and L392V, as well as N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V, and M146V mutations (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. al., 2008 Ann Med 40 (8): 562-83);Watanabe and Shen, PNAS November 28, 2017 114 (48) 12635-12637;Zhou et al., PNAS November 28, 2017 114 (48) 12731-12736;Hsu et al., Alzheimers Res Ther. 2018 Jul 18;10(1):67). Further examples of mutations that may have a dominant-negative effect on wild-type PS protein include: in PSEN1: N32N;R35Q;D40del(delGAC);D40del(delACG);E69D;A79V;V82L;I83_M84del(DelIM, ΔI83 / M84, ΔI83 / ΔM84);I83T;M84V;L85P;P88L;V89L(G>T);V89L(G>C) ;C92S;V94M;V96F;V97L;T99A;F105C;F105I;F105L;F105V;R108Q;L113_I114insT(Intron4, InsTAC, p.113+1de lG, splice5);L113P;L113Q;Y115C;Y115D;Y115H;T116I;T116N;T116R;P117A;P117L;P117R;P117S;E120D(A>C);E120D(A>T);E120G;E120K;E123K;Q127_R128del(CAGA);InsG(G)(c.379_382delXXXXinsG);H131R;S132A;L134R;N135D;N135S;N135Y;A136G;M139I(G>C);M139I(G>A);M139K;M139L;M139T;M139V;V142F;I143F;I143M;I143N;I143T;I143V;M146I(G>C);M146I(G>T);M146I(G>A);M146L(A>C);M146L(A>T);M146V;T147I;T147P;L150P;L153V;Y154C;Y154N;Y156F;Y156_R157insIY;R157S;H163P;H163R;H163Y;A164V;W165C(G>C);W165C(G>T);W165G;L166H;L166P;L166R;L166V;L166del;I167del(TTAdel);I167del(TATdel);I168T;S169del(ΔS169、Ser169del、ΔS170);S169L;S169P;S170F;S170P;L171P;L173F(G>C);L173F(G>T);L173W;L174del;L174M;L174R;F175S;F176L;F177L;F177S;S178P;G183V;E184D;E184G;V191A;I202F;G206A;G206D;G206S;G206V;G209A;G209E;G209R;G209V;S212Y;I213F;I213L;I213T;H214D;H214N;H214Y;G217D;G217R;L219F;L219P;L219R;R220P;Q222H;Q222P;Q222R;Q223R;L226F;L226R;I229F;S230I;S230N;S230R;A231P;A231T;A231V;L232P;M233I(G>A);M233I(G>C);M233L(A>T);M233L(A>C);M233T;M233V;L235P;L235R;L235V;F237I;F237L;I238M;K239N;T245P;A246E;A246P;L248P;L248R;L250F;L250S;L250V;Y256S;A260V;V261F;V261L;L262F;L262V;C263F;C263R;P264L;G266S;P267A;P267L;P267S;R269G;R269H;L271V;V272A;E273A;E27 3G;T274R;A275V;R278I;R278K;R278S;R278T;E280A;(Paisa);E280G;E280K;L282F;L282R;L282V;F283 L;P284L;P284S;A285V;L286P;L286V;T291A;T291P;K311R;E318G;D333G;R352C;R352_S353insR;T354I ;R358Q;S365A;S365Y;R377M;R377W;G378E;G378V;G378fs;L381F;L381V;G384A;F386I;F386S;F388L;S3 90I;S390N;V391F;V391G;L392P;L392V;G394V;A396T;N405S;I408T;A409T;C410Y;V412I;I416T;G417S ;L418F;L420R;L424F;L424H;L424R;L424V;A426P;A431E;(Jalisco);A431V;A434C;A434T;L435F;P436 Q;P436S;I437V;I439S;I439V;T440del;869-2A>G;869-22_869-23ins18(ΔE9, Δ9, deltaE9);I238_K239 insI;S290C;T291_S319del(ΔE9Finn, Δ9Finn, Δ9);S290C;T291_S319del(ΔE9, Δ9);S290C;T291_S319del A>G(ΔE9, Δ9);S290C;T291_S319del G>A(ΔE9, Δ9);S290C;T291_S319del G>T(ΔE9, Δ9);or S290W;S291_R377del(Δ9-10, Delta9-10, p.Ser290_Arg377delinsTrp, g.73671948_73682054del) (the mutation is named for Uniprot P49768.1 / GenBank reference number 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 variant is Uniprot Examples of mutations include, but are not limited to, the mutation named P49810.1 / GenBank reference number NP_000438.2. See, for example, Sun et al., Proc Natl Acad Sci USA. 2017;114:E476-E485;Heilig et al., J Neurosci. 2013 Jul 10;33(28):11606-17;Zhou et al., PNAS November 28, 2017 114 (48) 12731-12736. In some embodiments, the method may include the step of determining whether a subject has such a mutation, for example, using a method known in the art. In some embodiments, the subject has the mutation described herein (for example, identified as having the mutation described herein using a method known in the art), and optionally has a family history of AD and / or one or more symptoms of AD, and the subject is treated using the method described herein. In some embodiments, the subjects do not yet have full-symptomatic AD.

[0042] Typically, increased amnesia or mild confusion is an early symptom of Alzheimer's disease. Gradually, cognitive impairment associated with Alzheimer's disease progresses, including memory loss, particularly recent memory, disorientation and misinterpretation of spatial relationships; difficulty with language, writing, thinking and reasoning; changes in personality and behavior resulting in depression, anxiety, withdrawal, mood swings, distrust of others, irritability and aggression; changes in sleep patterns, wandering, loss of inhibition, delusions, and ultimately death.

[0043] Other neurodegenerative diseases, disorders, or conditions In addition to Alzheimer's disease, the methods of the present invention may be used to treat other neurodegenerative diseases, disorders, or conditions, including frontotemporal dementia, various types of memory loss, cognitive impairments including but not limited to mild cognitive impairment (MCI), or other conditions associated with deletions of PS1 or PS2, for example, due to mutations in PSEN1 or PSEN2 that produce dominant-negative isoforms.

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

[0045] [Table 1]

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

[0047] [ka]

[0048] [ka]

[0049] [ka]

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

[0051] [ka]

[0052] [ka]

[0053] [ka]

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

[0055] [ka]

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

[0057] [ka]

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

[0059] [ka]

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

[0061] [ka]

[0062] >NP_000438.2 Presenilin 2-Isoform 1 [Human (Homo sapiens)] (SEQ ID NO: 7)

[0063] [ka]

[0064] >NP_036618.2 Presenilin 2 isoform 2 [Human (Homo sapiens)] (SEQ ID NO: 8)

[0065] [ka]

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

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

[0068] Codon-Optimized Presenilin 1 Codon optimization is desirable for expressing proteins in specific host cells, such as bacteria, mice, and human cells. Those skilled in the art will understand that, as a result of genetic coding degeneracy, it is possible to generate a large number of cDNAs encoding human presenilin 1, some of which have minimal similarity to the cDNAs of any known and native gene. Therefore, the present invention examines each and all of the possible cDNA variations that may arise from the selection of combinations based on possible codon selection. These combinations are constructed according to standard triplet genetic coding, such as that applied to polynucleotides encoding native human presenilin variants, and all such variations are considered to be specifically disclosed. A typical codon-optimized human PSEN1 nucleotide sequence is disclosed herein, for example, SEQ ID NO: 9. See Figure 2. This codon-optimized human PSEN1 nucleotide sequence was generated by substituting codons in the native PSEN1 nucleotide sequence that occur infrequently in human cells with codons that occur frequently in human cells. Examples of codon-optimized human PSEN1 nucleotide sequences include sequences in which less than 100% of codons are optimized, such as sequences in which only 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the wild-type unoptimized codons are optimized. The codon frequencies can be computationally determined by methods known in the art. Typical calculations of these codon frequencies are disclosed in Table 1.

[0069] A typical codon-optimized human PSEN1 sequence is as follows: >Codon-optimized Homo sapiens presenilin 1 (PSEN1) cDNA (SEQ ID NO: 9)

[0070] [ka]

[0071] [Table 2-1]

[0072] [Table 2-2]

[0073] Mutant presenilin 1 In some embodiments, the PS1 protein contains mutations. In some embodiments, the mutations are conserved substitutions. Such mutations include substitutions of any of isoleucine (I), valine (V), and leucine (L) with any other of these hydrophobic amino acids, substitutions of aspartic acid (D) and glutamic acid (E) and vice versa, substitutions of glutamine (Q) and asparagine (N) and vice versa, and substitutions of serine (S) and threonine (T) and vice versa. Other substitutions can also be considered conserved depending on the environment and role of the particular amino acid in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A), as well as alanine (A) and valine (V), may often be interchangeable. Relatively hydrophobic methionine (M) can often be exchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are amino acid residues whose prominent feature is this charge, and the different pKs of such two amino acid residues are often interchangeable at non-prominent positions. Further other mutations can be considered "conserved" in certain environments (see, for example, Table III of US20110201052; pages 13-15 “Biochemistry” 2nd ED. Stryer ed (Stanford University); Henikoff et al., PNAS 1992 Vol 89 10915-10919; Lei et al., J Biol Chem 1995 May 19; 270(20):11882-6).

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

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

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

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

[0078] Highly efficient viral vectors capable of transduction into CNS neurons can be utilized, including rAAV (e.g., AAV1-AAV12) vectors of any serotype, recombinant or chimeric AAV vectors, and lentiviruses or other suitable viral vectors. In some embodiments, the codon-optimized polynucleotide encoding PS1 is operably bound to a promoter suitable for expression in the CNS. For example, a neuronal subtype-specific promoter, such as the alpha-calcium / calmodulin kinase 2A promoter, may be used to target excitatory neurons. Alternatively, a panneuronal promoter, such as the synapsin I promoter, may be used to induce PS1 expression. Other examples of promoters include, but are not limited to, the cytomegalovirus (CMV) early enhancer / promoter; the hybrid CMV enhancer / chicken β-actin (CBA) promoter; a promoter containing the CMV early enhancer element, the first exon and first intron of the chicken β-actin gene, and the splice receptor site of the rabbit β-globulin gene (commonly called the "CAG promoter"); or a 1.6kb hybrid promoter consisting of the CMV pre-early enhancer and CBA intron 1 / exon 1 (commonly called the CAGGS promoter; Niwa et al. Gene, 108:193-199 (1991)). The CAGGS promoter (Niwa et al., 1991) has been shown to result in ubiquitous and long-term expression in the brain (Klein et al., Exp. Neurol. 176:66-74 (2002)). One approach to introducing nucleic acids into cells in vivo involves the use of viral vectors containing codon-optimized cDNA encoding nucleic acids, such as PS1. In particular, infection of cells with viral vectors has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Furthermore, the cDNA contained within the viral vector allows molecules encoded within the viral vector to be efficiently expressed in cells that have received the viral vector nucleic acid.

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

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

[0081] The vectors described herein may be pseudotype vectors. Pseudotyping provides a mechanism for modulating the target cell population of the vector. For example, pseudotype AAV vectors can be used in the various methods described herein. A pseudotype vector is a vector that contains the genome of one vector, for example, the genome of one AAV serotype, in a second vector, for example, the capsid of a second AAV serotype. Methods for pseudotyping are well known in the art. For example, vectors can be pseudotyped using envelope glycoproteins derived from rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipla strains), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and attack virus standards (CVS)), Lyssavirus Mokola virus, rabies-related viruses, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein type B (FuG-B), variants of FuG-B (FuG-B2), or Moloney murine leukemia virus (MuL V). Viruses can pseudotype to transduce one or more nerve cells or groups of cells.

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

[0083] In some cases, a specific AAV serotype vector may be selected based on its intended use, for example, based on the intended route of administration.

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

[0085] A vector construct refers to a polynucleotide molecule containing all or part of a viral genome and a transgene. In some cases, gene transfer may be mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV). Other vectors useful in gene therapy methods are known in the art. For example, constructs disclosed herein may include alphaviruses, herpesviruses, retroviruses, lentiviruses, or vaccinia viruses.

[0086] Adenoviruses are a relatively well-characterized group of viruses, including more than 50 serotypes (see, for example, International Publication 95 / 27071, incorporated herein by reference). Adenoviruses are readily available through the application of molecular biology techniques and may not require integration into the host cell genome. Recombinant Ad-derived vectors, including vectors that reduce the likelihood of recombination and generation of wild-type viruses, have been constructed (see, for example, International Patent Publications 95 / 00655 and 95 / 11984, incorporated herein by reference). Wild-type AAV is highly infectious and can be integrated into the host genome with high specificity (see, for example, Hermonat and Muzyczka 1984 Proc. Natl. Acad. Sci., USA 81:6466-6470 and Lebkowski et al. 1988 Mol. Cell. Biol. 8:3988-3996).

[0087] Non-natural regulatory sequences, gene regulatory sequences, promoters, non-coding sequences, introns, or coding sequences may be included in the nucleic acids disclosed herein. The inclusion of nucleic acids encoding nucleic acid tags or signaling sequences, or protein tags or protein signaling sequences, is further discussed herein. Typically, coding regions are operably bound to one or more regulatory nucleic acid components.

[0088] Promoter characteristics of nucleic acids disclosed herein may include tissue- or cell-type specific promoters, promoters specific to multiple tissues or cell types, organ-specific promoters, promoters specific to multiple organs, systemic or ubiquitous promoters, or nearly systemic or ubiquitous promoters. Promoter characteristics that result in stochastic expression, inducible expression, conditional expression, or conversely, discontinuous, irregular, or unpredictable expression are also included within the scope of this disclosure. Promoter characteristics may include any of the above characteristics or other promoter characteristics known in the art.

[0089] In clinical settings, gene delivery systems for therapeutic genes can be introduced into a target by one of several methods, each of which is known in the art. For example, a pharmaceutical preparation of a gene therapy system can be introduced systemically, for example, by intravenous injection, and specific transduction of the protein in target cells is primarily due to the specificity of transfection, which is brought about by the gene delivery medium, cell-type or tissue-type expression, or a combination thereof, via transcriptional regulatory sequences that regulate the expression of the receptor gene. In other embodiments, the initial delivery of the recombinant gene is further restricted, and introduction into the target becomes highly localized. For example, the gene delivery medium can be introduced by catheter (see U.S. Patent No. 5,328,470) or, optionally, by stereotactic injection into the cisterna magna, ventricles, lumbar spinal cavity, or direct injection into the hippocampus (e.g., Chen et al., PNAS USA 91: 3054-3057 (1994)). In preferred embodiments, methods for delivering a virus expressing presenilin include intravenous, subarachnoid, ventricular, cisterna magna, and stereotactic intraparenchymal administration.

[0090] The method can be further optimized through preclinical studies to achieve the best possible recovery from neurodegeneration, dementia, synaptic dysfunction, and molecular degeneration in presenilin conditional dual knockout mice and presenilin 1 knock-in mice expressing FAD mutations.

[0091] A pharmaceutical preparation of a gene therapy construct may essentially contain a gene delivery system in an acceptable diluent, or it may contain a sustained-release matrix that embeds a gene delivery medium. Alternatively, if a complete gene delivery system, such as a retroviral vector, can be generated intact from recombinant cells, the pharmaceutical preparation may contain one or more cells that thereby generate the gene delivery system.

[0092] Delivery formulations and pharmaceutical compositions In some embodiments, the polynucleotides described herein for in vivo delivery to target tissue are encapsulated or bound to nanoparticles.Methods for packaging into nanoparticles are well known in this art, 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 in rats determined by radioluminography. J Drug Target 5:171. 1998); Sakuma SR et al (Mucoadhesion of polystyrene nanoparticles having surface hydrophilic polymeric chains in the gastrointestinal tract. Int J Pharm 177:161. This is described in Virovic L et al. (1999); Novel delivery methods for treatment of viral hepatitis: an update. Expert Opin Drug Deliv 2:707.2005); and Zimmermann E et al., Electrolyte- and pH-stabilities of aqueous solid lipid nanoparticle (SLN) dispersions in artificial gastrointestinal media. Eur J Pharm Biopharm 52:203.2001).In some embodiments, one or more polynucleotides are delivered in vivo to target tissues via vesicles, such as liposomes (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; refer to the same book in general). In some embodiments, lipid-based nanoparticles (LNPs) are used. See, for example, Robinson et al., Mol Ther. 2018 Aug 1;26(8):2034-2046; U.S. Patent No. 9956271.

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

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

[0095] The molecules contained within the microvesicle preparation include therapeutic molecules. Typically, the microvesicles in the preparation are a heterogeneous population, and each microvesicle contains complement molecules that are different from or not different from the molecules of other microvesicles in the preparation. The contents of therapeutic molecules in the microvesicle preparation can be expressed either quantitatively or qualitatively. One such method is to express the contents as a percentage of the total molecules in the microvesicle preparation. For example, if the therapeutic molecule is mRNA, the contents can be expressed as a percentage of the total RNA contents in the microvesicle preparation, or as a percentage of the total mRNA contents. Similarly, if the therapeutic molecule is a protein, the contents can be expressed as a percentage of the total protein in the microvesicle. In one embodiment, therapeutic microvesicles or preparations produced by the method described herein contain a detectable and statistically significant increase in therapeutic molecules when compared to microvesicles obtained from control cells (cells obtained from the same source that have not been chemically manipulated to increase the expression of therapeutic molecules). In one embodiment, the therapeutic molecule is present in amounts at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% greater than in the microvesicles obtained from control cells. Even higher levels of concentration can be achieved. In one embodiment, the therapeutic molecule is present in at least twice as many microvesicles or their preparations as in control cell microvesicles. Even higher concentrations can be obtained (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 times).

[0096] In one embodiment, a relatively high proportion of the vesicular contents is a therapeutic molecule (achieved, for example, by high expression of the molecule or specific targeting of the microvesicular contents). In one embodiment, the vesicular contents of the therapeutic molecule constitute at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the total (allogeneic) molecular contents (for example, the therapeutic molecule is mRNA and constitutes about 10% of the total mRNA contents of the microvesicular contents). Even higher levels of enrichment can be achieved. In one embodiment, the therapeutic molecule is present in the microvesicular contents or preparations at least twice as much as all other such (allogeneic) molecules. Even higher enrichment ratios can be obtained (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 times). [Examples]

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

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

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

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

[0101] Western blotting Cell lysates were subjected to SDS-PAGE, and proteins were transferred to a nitrocellulose membrane. After blocking with TBST / 5% skim milk powder, the membrane was incubated overnight with the primary antibody. To control loading, the membrane was peeled off and reprobed with an anti-β-actin antibody. Band intensity was quantified using ImageJ software, and the results were normalized to β-actin levels. Antibodies used included rat anti-PS1-NTF, rabbit anti-cleavage type Notch (Val1744) (NICD), and mouse anti-β-actin.

[0102] result First, the inventors expressed wt_hPS1 or opti_hPS1 in Psen-null MEFs to eliminate any influence from endogenous PS1 or PS2. The inventors detected a gradual increase in PS1 NTF levels when Psen-null MEFs were transfected with an augmentation vector encoding wt_hPS1 or opti_hPS1. Optimized hPS1 consistently expressed more PS1 than wt_hPS1 (Figures 3A-3B).

[0103] We directly measured γ-secretase activity by co-expressing NotchΔE, a substrate for γ-secretase-mediated cleavage, with wt_hPS1 or opti_hPS1. Notch is cleaved by γ-secretase, releasing the Notch intracellular domain (NICD). We evaluated the dose-response relationship for γ-secretase-mediated NotchΔE cleavage to determine NICD generation in response to γ-secretase activity. NICD generation increased linearly among lower PS1 vector transfections but approached saturation at higher levels. More importantly, at each point, optimized hPS1 exhibited higher γ-secretase activity (Figure 3).

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

[0105] [Example 3] Development of an optimized in vivo expression system for wild-type human PS1 We developed transgenic mice that constitutively or inductively express human PSEN1 wild-type cDNA under the control of the CAMK2A promoter. To maximize PS1 generation and activity, hPS1 was codon-optiminated (hPS1opti), and then PS1 levels and γ-secretase activity were compared between endogenous hPS1 cDNA and hPS1opti cDNA by cotransfecting PS DKO MEFs with either augmented pCI-hPS1 or pCI-hPS1opti and CMV-NΔE. As measured by NICD generation, hPS1opti cDNA yielded higher levels of PS1 NTF and higher γ-secretase activity compared to endogenous hPS1 cDNA (Figure 3).

[0106] [Example 4] Determining whether postpartum delivery of hPS1opti can restore the phenotype in PS cDKO mice. The vectors listed in Table 2 were prepared. The vectors that yielded the highest GFP staining at 4 and 8 weeks post-injection were identified. For this purpose, P0-2 were injected into PS cDKO offspring mice (10 housing cages of Cre / Cre;- / - mated with lot-F / F;- / -;F / F).

[0107] [Table 3]

[0108] Select an AAV, e.g., AAV9 / hCaMKII-intron-hPS1opti-T2A-EGFP-SV40pA or AAV9 / hCaMKII-intron-EGFP-SV40pA, and inject it into PS cDKO mice via ICV in phases P0-P2. Western blotting analysis is performed at 4 and 8 weeks of age to determine the levels of PS1, APP, nicatrin, and PEN-2. Control and PS cDKO mice are included at 4 and 8 weeks as further controls. Gamma-secretase activity, NICD production, and Aβ levels (by ELISA of cortical lysates) are measured at 8 weeks of age. Furthermore, electrophysiological analysis, e.g., Schaefer collateral, PPF, FF, and LTP, is performed at 2 months of age, and behavioral analysis, e.g., water maze, is performed at 2-3 months of age. Neurological disorder analysis is performed at 6 months of age.

[0109] [Example 5] To determine whether postpartum delivery of hPS1opti can restore the phenotype caused by the FAD mutation. Mice listed in Table 3 were generated and analyzed using Western blot analysis to measure PS1 and APP levels; in vitro gamma-secretase activity assay; ELISA; electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; and neurological analysis at 6, 12, and 18 months of age.

[0110] [Table 4]

[0111] [Example 6] To determine whether AAV9 / hPS1opti delivered to the brain of an adult can restore in vivo phenotypes (PS cDKO, FAD KI) resulting from loss of PS function. The vectors were prepared as listed in Table 2, and a 50 µl Hamilton syringe was used to deliver 10 µl of the injection solution to the large vat via the CSF (1 × 10 12 vg) Via ICM, via substantial intra-delivery, or 3.3 × 10 of 10µl 11 The drug is injected into mice via subarachnoid delivery using lumbar puncture. The mice studied include (i) PS cDKO, (ii) KI / +, (iii) KI / +; PS2- / -, and (iv) KI / fPS1; PS2- / -; Cre. The inventors perform the following analyses on the mice: Western blotting to measure PS1 and APP levels; in vitro γ-secretase assay; ELISA measurement of Aβ peptide; electrophysiological analysis at 6 months of age; behavioral analysis at 6 and 12 months of age; neurological analysis at 6, 12, and 18 months of age.

[0112] Other Embodiments Although the present invention has been described along with embodiments for carrying it out, the foregoing description is intended to be illustrative and does not limit the scope of the invention, and it is understood that the present invention is defined by the appended claims. Other embodiments, advantages, and modifications are contained in the following claims.

Claims

1. A composition comprising a human codon-optimized polynucleotide encoding human presenilin 1 (PSEN1).

2. The composition of claim 1, wherein the human codon-optimized polynucleotide encoding human PSEN1 is at least 95% identical to SEQ ID NO: 9 and at least one codon is optimized relative to the wild type.

3. The composition of claim 1, wherein the human codon-optimized polynucleotide encoding human PSEN1 comprises SEQ ID NO:

9.

4. 4. The composition of any one of claims 1 to 3, associated with (e.g., formulated for delivery using) exosomes or lipid-based nanoparticles (LNPs).

5. A composition comprising a vector for expressing human PSEN1 in a cell, the vector comprising the human codon-optimized polynucleotide of any one of claims 1 to 3 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 viral (AAV) vector.

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

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 a 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 alpha-calcium / calmodulin kinase 2A promoter.

14. A method for treating a neurodegenerative disease, disorder, or condition, comprising administering a composition described in any one of claims 1 to 13 to a human subject in need of treatment, wherein the subject has one or more mutations in at least one allele of PSEN1, preferably a mutation encoding a dominant-negative PSEN1 protein isoform.

15. 15. The method of claim 14, wherein the neurodegenerative disease, disorder or condition is Alzheimer's disease.

16. 16. The method of claim 15, wherein the Alzheimer's disease is familial Alzheimer's disease.

17. 17. The method of claim 15 or 16, wherein the subject has an E280A, Y115H, L166P, C410Y, Δex9, G548, D257A, R278I, L435F, G384A or L392V mutation in the PSEN1 gene, or an N141I, G206A, H163R, A79V, S290C, A260P, A426P, A431E, R269H, L271V, C1410Y, E280G, P264L, E185D, L235V or M146V mutation in the PSEN1 gene.

18. 16. The method of claim 15, wherein the Alzheimer's disease is sporadic Alzheimer's disease.

19. 16. The method of claim 15, wherein the Alzheimer's disease is late-onset or early-onset Alzheimer's disease.

20. 15. The method of claim 14, wherein the neurodegenerative disease, disorder or condition is frontotemporal dementia, memory loss, cognitive decline or impairment.

21. 21. The method of claim 20, wherein the cognitive impairment is mild cognitive impairment (MCI).

22. 21. The method of any one of claims 14 to 20, wherein the composition is administered to the CNS of the subject in need of treatment.

23. 23. The method of claim 22, wherein the polynucleotide encoding the PSEN1 and / or PSEN2 gene or mRNA is administered to the CNS by intravenous delivery, intrathecal delivery, intracisternal delivery, intraventricular delivery, or stereotactic intraparenchymal injection into specific brain regions, optionally the cisterna magna, ventricles, lumbar spinal cavity, or direct injection into the hippocampus or cortical area.

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