Methods and compositions for tau reduction gene therapy

Abstract

The present disclosure provides methods and compositions for treating tauopathies, such as Alzheimer's disease or FTDP-17. The methods and compositions of the present disclosure include isolated nucleic acid molecules, rAAV vectors, and rAAV viral vectors that include polynucleotide sequences encoding artificial microRNAs (amiRNAs) that target MAPT.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 215,833, filed June 28, 2021, No. 63 / 267,440, filed February 2, 2022, and No. 63 / 342,240, filed May 16, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Incorporation by reference of sequence listing This application was submitted in ASCII format via EFS-Web and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The ASCII copy was created on Jun. 6, 2022, is named "TAYS-013_001WO_Seq_Listing_ST25.txt", and is approximately 148,953 bytes in size.

[0003] The present disclosure provides methods and compositions for treating tauopathies, including Alzheimer's disease (AD) and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).

[0004] The methods and compositions of the disclosure include isolated nucleic acid molecules comprising a polynucleotide sequence encoding an artificial microRNA (amiRNA) targeting MAPT, recombinant adeno-associated virus (rAAV) vectors, and rAAV viral vectors. [Background technology]

[0005] A common pathological feature of neurodegenerative diseases is the misfolding of certain proteins and their insoluble proteinaceous deposits in the CNS, accompanied by progressive loss of neurons in affected regions. Tauopathies are a group of neurodegenerative diseases broadly defined by the aggregation of highly phosphorylated fibrillar tau protein. The most common tauopathy is Alzheimer's disease (AD). AD is characterized by the aggregation of both b-amyloid as plaques and highly phosphorylated tau as neurofibrillary tangles (NFTs), but only NFT pathology has a close correlation with cognitive decline. Tau fibrils stably propagate tau pathology transcellularly from different brain regions, depending on the disease (Braak et al., Acta Neuropathol 82, 239-259, doi:10.1007 / bf00308809 (1991); Braak et al., Acta Neuropathol 121, 589-595, doi:10.1007 / s00401-011-0825-z (2011); Irwin et al., Parkinsonism Re / at Disord 22 Suppl 1, S29-33, doi:10.1016 / j.parkreldis.2015.09.020S1353-8020(15)00395-8[pii] (2016)).

[0006] Although the MAPT gene, which codes for tau, is not genetically associated with AD, mutations within MAPT cause other tauopathies, such as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (Hutton et al., Nature 393, pp. 702-705, doi:10.1038 / 31508 (1998); Spillantini et al., Proc Natl Acad Sci U S A 95, pp. 7737-7741, doi:10.1073 / pnas.95.13.7737 (1998)), and disruption of tau homeostasis has been shown to be sufficient to cause neurodegeneration.

[0007] Over the past decade, tremendous progress has been made in molecular therapies that target proteins that are misfolded and lead to degradation. Most approaches exploit the endogenous RNAi machinery by using one of two major RNAi platforms in development: oligonucleotide therapy and gene therapy. The former include antisense oligonucleotides (ASOs) targeting huntingtin for the treatment of Huntington's disease (HD) (Kordasiewicz et al., Neuron 74, pp. 1031-1044, doi:10.1016 / j.neuron.2012.05.009 (2012); NCT03761849), SOD1 for the treatment of amyotrophic lateral sclerosis (ALS) (Miller et al., Lancet Neural 12, pp. 435-442, doi:10.1016 / S1474-4422(13)70061-9 (2013); NCT02623699), and C90rf72 for the treatment of ALS (Mccampbell et al., J Clin Invest 128, pp. 3558-3567, doi:10.1172 / JCl99081 (2018)), and tau to treat AD and frontotemporal dementia (FTD) (DeVos et al., Sci Transl Med 9, doi:10.1126 / scitranslmed.aag0481 (2017); NCT03186989).

[0008] ASOs must be delivered into the cerebrospinal fluid by continuous infusion or repeated intrathecal injections, and their therapeutic effects are thought to be most potent in brain regions adjacent to the ventricular system (Kordasiewicz et al., Neuron 74, pp. 1031-1044, doi:10.1016 / j.Neuron.2012.05.009 (2012)). Although ASO therapy is promising, the caveats of these procedures include the ongoing expense of repeated drug administration throughout the patient's life. In humans, current ASO treatment methods require intrathecal injections every 3-4 months, may accumulate scar tissue over time, and may pose additional risk to infection in elderly populations. Alternatively, RNAi-based gene therapy approaches using small hairpin RNAs (shRNAs) and microRNAs (miRNAs) are also being developed for neurodegenerative diseases. RNAi-based gene therapy uses a single administration of a viral vector that drives the continuous expression of shRNA or artificial miRNA precursors and the subsequent long-lasting downregulation of target proteins. The artificial miRNA mimics the natural miRNA structure but can also be engineered to silence the expression of any gene of interest, presumably by base pairing with complementary sequences on the target mRNA. Gene therapy trials using vector-mediated delivery of miRNA shuttles have shown promise for the treatment of HD in preclinical models and have recently been brought to the clinical setting (Evers et al., Mol Ther 26, 2163-2177, doi:10.1016 / j.ymthe.2018.06.021(2018);NCT04120493).

[0009] For gene delivery, AAV has emerged as one of the safest and most commonly used vectors due to its ability to infect non-dividing cells, high transduction efficiency, long-term persistent expression from a single administration, and relatively low host immune response. Recombinant AAV (rAAV) vectors retain only the terminal inverted repeat sequences of the wild-type virus, which flank the transgene cassette consisting of a promoter together with the gene of interest to be delivered.

[0010] Advances in AAV vector design and delivery into the brain and spinal cord have made AAV ideal for treating neurological diseases, with the AAV9 serotype having the highest affinity for the CNS (Kantor, Adv Genet 87, pp. 125-197, doi:10.1016 / B978-0-12- 800149-3.00003-2 (2014)).

[0011] A giant axonal neuropathy IND application study was conducted to support a Phase I clinical trial in children using intrathecally administered AAV9 vectors (NCT02362438) and has been ongoing since 2015 (Bailey et al., Mol Ther Methods Clin Dev 9, pp. 160-171, doi:10.1016 / j.omtm.2018.02.005 (2018)). In addition to GAN, AAV9 gene therapy is being used in ongoing clinical trials for multiple disorders, and the FDA recently approved the use of AAV9 vector therapy for myeloarthritis.

[0012] Currently, there are no approved therapies for neurodegenerative diseases that significantly slow disease progression, and there remains a great need for disease-specific therapeutic agents. Summary of the Invention [Means for solving the problem]

[0013] Presented herein is an rAAV vector comprising at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, the amiRNA targeting MAPT comprising a nucleic acid sequence represented by any one of SEQ ID NOs: 139-186. In some embodiments, the polynucleotide sequence encoding at least one amiRNA targeting MAPT comprises a nucleic acid sequence represented by any one of SEQ ID NOs: 41-88. In some embodiments, the amiRNA targets human MAPT. In some embodiments, the amiRNA targeting human MAPT comprises a nucleic acid sequence represented by any one of SEQ ID NOs: 139-149. In some embodiments, the amiRNA encoded by the polynucleotide and targeting human MAPT comprises a nucleic acid sequence represented by any one of SEQ ID NOs: 41-49.

[0014] In some embodiments, the rAAV vector further comprises a first inverted terminal repeat (ITR) sequence, and the first ITR sequence is an AAV2 ITR sequence. In some embodiments, the first ITR sequence comprises the sequence represented by SEQ ID NO: 15. In some embodiments, the rAAV vector further comprises a second ITR sequence, and the second ITR sequence is an AAV2 ITR sequence. In some embodiments, the second ITR sequence comprises the sequence represented by SEQ ID NO: 16.

[0015] In some embodiments, the rAAV vector further comprises a first promoter sequence, and the first promoter sequence is a mouse U6 promoter sequence. In some embodiments, the mouse U6 promoter sequence comprises the nucleic acid sequence represented by SEQ ID NO:31.

[0016] In some embodiments, the rAAV vector further comprises a second promoter sequence, and the second promoter sequence is a CBh promoter sequence. In some embodiments, the CBh promoter sequence comprises the nucleic acid sequence represented by SEQ ID NO:29.

[0017] In some embodiments, the rAAV vector further comprises a termination signal. In some embodiments, the termination signal comprises the nucleic acid sequence represented by SEQ ID NO:40.

[0018] In another aspect, provided herein is an rAAV vector comprising, in a 5' to 3' direction, a first AAV2 ITR sequence, a mouse U6 promoter sequence, a polynucleotide sequence encoding at least one amiRNA targeting MAPT, a termination sequence, a CBh promoter sequence, a synthetic polyA sequence, and a second AAV2 ITR sequence, wherein the amiRNA targeting MAPT comprises a nucleic acid sequence represented by any one of SEQ ID NOs: 139 to 186. In some embodiments, the rAAV vector comprises a sequence represented by SEQ ID NO: 14.

[0019] In another aspect, the present invention provides a rAAV viral vector comprising the AAV capsid protein and the rAAV vector described herein. In some embodiments, the AAV capsid protein is an AAV9 capsid protein.

[0020] In another aspect, provided herein is a pharmaceutical composition comprising an rAAV vector described herein, or an rAAV viral vector described herein, and at least one pharma- ceutically acceptable excipient and / or additive.

[0021] In another aspect, the present specification provides a method for treating tauopathy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of rAAV vector, rAAV viral vector, or pharmaceutical composition as described herein.In some embodiments, the tauopathy is Alzheimer's disease or FTDP-17.In some embodiments, the subject has one or more mutations in MAPT gene.

[0022] In another aspect, the present specification provides rAAV vector, rAAV virus vector, or pharmaceutical composition as described herein for use in treating tauopathy.In some embodiments, tauopathy is Alzheimer's disease or FTDP-17.In some embodiments, tauopathy is associated with one or more mutations in MAPT gene.

[0023] The above and further features will be more clearly understood from the following detailed description when taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows a schematic of a self-complementary AAV9 vector containing an anti-tau miRNA driven by the U6 promoter. [Diagram 2] FIG. 1 shows knockdown of human MAPT using various miRNAs targeting human tau as measured by dual reporter assay; scr=scrambled tau miRNA; hTau=human specific tau miRNA. [Figure 3A] FIG. 1 shows knockdown of human tau mRNA and protein using various miRNAs targeting human tau, respectively. [Figure 3B] FIG. 1 shows knockdown of human tau mRNA and protein using various miRNAs targeting human tau, respectively. [Figure 3C] FIG. 1 shows tau expression in HEK293 cells following treatment with a scrambled control miRNA and a miRNA specific for human tau, respectively, as determined by immunofluorescence. [Figure 3D] FIG. 1 shows tau expression in HEK293 cells following treatment with a scrambled control miRNA and a miRNA specific for human tau, respectively, as determined by immunofluorescence. [Figure 4A]FIG. 1 shows vector distribution by glial and neuronal GFP staining and corresponding reduction in tau protein after delivery into mice overexpressing human P301S mutant tau with vehicle control or an AAV9 vector expressing an miRNA targeting human tau and also encoding a GFP reporter protein. [Figure 4B] FIG. Quantification of MAPT mRNA in treated mouse brains. [Diagram 5] FIG. 1 shows knockdown of mouse Mapt using various miRNAs targeting mouse tau as measured by dual reporter assay; scr=scrambled tau miRNA; mTau=mouse specific tau miRNA. [Figure 6A] FIG. 1 shows knockdown of mouse Mapt mRNA using various miRNAs targeting mouse tau. [Figure 6B] FIG. 1 shows knockdown of mouse tau protein, but not human tau protein, using various miRNAs targeting mouse tau. [Figure 7A] FIG. 1 shows knockdown of mouse Mapt and human MAPT using various miRNAs targeting both human and mouse tau, respectively. [Figure 7B] FIG. 1 shows knockdown of mouse Mapt and human MAPT using various miRNAs targeting both human and mouse tau, respectively. [Figure 8] FIG. 1 shows survival rates at 3 months of age for wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP. [Figure 9A] FIG. 9 shows changes in body weight for wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP (data for male and female mice are shown in FIG. 9A and FIG. 9B, respectively). [Figure 9B]FIG. 9 shows changes in body weight for wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP (data for male and female mice are shown in FIG. 9A and FIG. 9B, respectively). [Figure 10] FIG. 13 shows representative images of immunohistochemical staining using GFP antibody in mouse brain following ICM injection of AAV9 / hTau5i-GFP or vehicle. [Figure 11] FIG. 13 shows MAPT expression as determined by qPCR in brainstem tissue from wild-type and human P301S mutant tau-overexpressing mice treated with vehicle control or AAV9 / hTau5i-GFP. [Figure 12A] FIG. 1 shows tau expression as determined by ELISA in brainstem, cerebellum, and cortex tissues from wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP. [Figure 12B] FIG. 1 shows tau expression as determined by ELISA in brainstem, cerebellum, and cortex tissues from wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP. [Figure 12C] FIG. 1 shows tau expression as determined by ELISA in brainstem, cerebellum, and cortex tissues from wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control or AAV9 / hTau5i-GFP. [Figure 13A]FIG. 13. Seeding assay results: transfection of brain homogenates from wild-type mice treated with vehicle or AAV9 / hTau5i-GFP did not result in FRET-positive inclusions, whereas addition of brain lysates from the P301S-treated cohort induced intracellular FRET-positive aggregates, while P301S mice treated with AAV9 / hTau5i-GFP induced fewer FRET-positive aggregates compared to vehicle-treated P301S mice (one-way ANOVA, Dunnett's multiple comparisons test compared to P301S+vehicle, ****p<0.0001). [Figure 13B] FIG. 13. Seeding assay results: transfection of brain homogenates from wild-type mice treated with vehicle or AAV9 / hTau5i-GFP did not result in FRET-positive inclusions, whereas addition of brain lysates from the P301S-treated cohort induced intracellular FRET-positive aggregates, while P301S mice treated with AAV9 / hTau5i-GFP induced fewer FRET-positive aggregates compared to vehicle-treated P301S mice (one-way ANOVA, Dunnett's multiple comparisons test compared to P301S+vehicle, ****p<0.0001). [Figure 13C] FIG. 13. Seeding assay results: transfection of brain homogenates from wild-type mice treated with vehicle or AAV9 / hTau5i-GFP did not result in FRET-positive inclusions, whereas addition of brain lysates from the P301S-treated cohort induced intracellular FRET-positive aggregates, while P301S mice treated with AAV9 / hTau5i-GFP induced fewer FRET-positive aggregates compared to vehicle-treated P301S mice (one-way ANOVA, Dunnett's multiple comparisons test compared to P301S+vehicle, ****p<0.0001). [Figure 14]FIG. 1 shows survival rates at 6 months of age for wild-type and human P301S mutant tau-overexpressing mice treated with vehicle control, AAV9 / scrambled (Scr) control, AAV9 / hTau5i-GFP, or AAV9 / hTau5i. [Figure 15A] FIG. 13 shows the change in body weight at 6 months of age for wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control, AAV9 / Scr control, AAV9 / hTau5i-GFP, or AAV9 / hTau5i. [Figure 15B] FIG. 13 shows the change in body weight at 6 months of age for wild-type mice and mice overexpressing human P301S mutant tau treated with vehicle control, AAV9 / Scr control, AAV9 / hTau5i-GFP, or AAV9 / hTau5i. [Figure 16A] Figure 16 shows the results of seeding assay. Figure 16A and Figure 16B show that AAV9 / Scr did not change seeding activity in WT or P301S mice compared to vehicle treatment. Figure 16C shows that mice treated with AAV9 / hTau5i-GFP or AAV9 / hTau5i had similar seeding activity in the brainstem. Figure 16D shows that treatment with AAV9 / hTau5i at 6 months of age significantly reduced tau seeding activity in the brainstem. [Figure 16B] Figure 16 shows the results of seeding assay. Figure 16A and Figure 16B show that AAV9 / Scr did not change seeding activity in WT or P301S mice compared to vehicle treatment. Figure 16C shows that mice treated with AAV9 / hTau5i-GFP or AAV9 / hTau5i had similar seeding activity in the brainstem. Figure 16D shows that treatment with AAV9 / hTau5i at 6 months of age significantly reduced tau seeding activity in the brainstem. [Figure 16C]Figure 16 shows the results of seeding assay. Figure 16A and Figure 16B show that AAV9 / Scr did not change seeding activity in WT or P301S mice compared to vehicle treatment. Figure 16C shows that mice treated with AAV9 / hTau5i-GFP or AAV9 / hTau5i had similar seeding activity in the brainstem. Figure 16D shows that treatment with AAV9 / hTau5i at 6 months of age significantly reduced tau seeding activity in the brainstem. [Figure 16D] Figure 16 shows the results of seeding assay. Figure 16A and Figure 16B show that AAV9 / Scr did not change seeding activity in WT or P301S mice compared to vehicle treatment. Figure 16C shows that mice treated with AAV9 / hTau5i-GFP or AAV9 / hTau5i had similar seeding activity in the brainstem. Figure 16D shows that treatment with AAV9 / hTau5i at 6 months of age significantly reduced tau seeding activity in the brainstem. [Figure 17] FIG. 1 shows survival rates at 9 months of age for wild-type and human P301S mutant tau-overexpressing mice treated with AAV9 / Scr control or AAV9 / hTau5i. [Figure 18A] FIG. 1 shows body weight changes at 9 months of age in wild-type mice and mice overexpressing human P301S mutant tau treated with AAV9 / Scr control or AAV9 / hTau5i. [Figure 18B] FIG. 1 shows body weight changes at 9 months of age in wild-type mice and mice overexpressing human P301S mutant tau treated with AAV9 / Scr control or AAV9 / hTau5i. [Figure 19A] FIG. 1 shows no changes in clinical blood chemistry one month post-injection from wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i. [Figure 19B]FIG. 1 shows no changes in clinical blood chemistry one month post-injection from wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i. [Figure 19C] FIG. 1 shows no changes in clinical blood chemistry one month post-injection from wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i. [Figure 19D] FIG. 1 shows no changes in clinical blood chemistry one month post-injection from wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i. [Figure 19E] FIG. 1 shows no changes in clinical blood chemistry one month post-injection from wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i. [Figure 20A] FIG. 1 shows that wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i show no changes in behavior one month post-injection. [Figure 20B] FIG. 1 shows that wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i show no changes in behavior one month post-injection. [Figure 20C] FIG. 1 shows that wild-type mice treated at 3 months of age with vehicle, AAV9 / Scr control, or AAV9 / mTau2i show no changes in behavior one month post-injection. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In particular, the present disclosure provides an isolated nucleic acid molecule, a recombinant adeno-associated virus (rAAV) vector, and a rAAV virus vector, which comprises at least one polynucleotide sequence encoding at least one amiRNA molecule that targets MAPT.The present disclosure also provides the method for producing these isolated polynucleotides, rAAV vectors, and rAAV virus vectors, and their use for delivering shRNA molecules to treat or prevent tauopathy.

[0026] The term "adeno-associated virus" or "AAV" as used herein refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus of the family Parvoviridae. Adeno-associated viruses are single-stranded DNA viruses that grow in cells, in which certain functions are provided by a coinfecting helper virus. General information and reviews on AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228 and Berns, 1990, Virology, pp. 1743-1764, Raven Press (New York). Since it is well known that the various serotypes are extremely closely related, both structurally and functionally, even at the genetic level, it is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized since the publication date of the reviews (see, for example, Blacklowe, 1988, Parvoviruses and Human Disease, edited by J.R. Puttson, pp. 165-174, and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit highly similar replication properties mediated by the homologous rep genes, and all have three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by extensive cross-hybridization between serotypes along the length of the genome and heteroduplex analysis revealing the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery, and all known serotypes are capable of infecting cells of a variety of tissue types. At least eleven AAV serotypes, numbered consecutively, are known in the art.Non-limiting exemplary serotypes useful in the methods disclosed herein include any of eleven serotypes, such as AAV2, AAV8, AAV9, or variant serotypes, such as AAV-DJ and AAV PHP.B. AAV particles comprise, consist essentially of, or consist of three major viral proteins, namely VP1, VP2, and VP3. In some embodiments, AAV refers to serotypes AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh.10.

[0027] Exemplary adeno-associated and recombinant adeno-associated viruses include, but are not limited to, all serotypes (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh.10). Exemplary adeno-associated and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids that contain the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ, and AAV-DJ8). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019;4(22):e131610) and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018;26(1):289-303).

[0028] AAV structure and function AAV is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length and contains two 145-nucleotide inverted terminal repeats (ITRs). There are numerous serotypes of AAV. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided under GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided under GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided under GenBank Accession No. NC_001829, the complete genome of AAV-5 is provided under GenBank Accession No. AF085716, and the complete genome of AAV-6 is provided under GenBank Accession No. AF085716. The genome is provided under GenBank Accession No. NC_001862, at least a portion of the genomes of AAV-7 and AAV-8 are provided under GenBank Accession Nos. AX753246 and AX753249, respectively, the genome of AAV-9 is provided in Gao et al., J. Virol., 78:6381-6388 (2004), the genome of AAV-10 is provided in Mol. Ther., 13(1):67-76 (2006), and the genome of AAV-11 is provided in Virology, 330(2):375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928. U.S. Patent No. 9,434,928 also provides the sequences of the capsid proteins and the self-complementary genome. In one embodiment, the AAV genome is a self-complementary genome. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and integration into host cell chromosomes are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 according to their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19) coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately involved in the replication of the viral genome.

[0029] The cap gene is expressed from the p40 promoter and encodes three capsid proteins, namely VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are involved in the production of the three related capsid proteins. More specifically, after a single mRNA is transcribed from which each of the VP1, VP2, and VP3 proteins is translated, this mRNA can be spliced ​​in two different ways. A long or short intron is excised, resulting in the formation of two pools of mRNA, namely 2.3 kb and 2.6 kb long mRNA pools. The long intron is often preferred, and thus the 2.3 kb long mRNA can be referred to as the major splice variant. This form lacks the first AUG codon from which synthesis of the VP1 protein begins, resulting in a reduction in the overall level of VP1 protein synthesis. The first AUG codon remaining in the major splice variant is the start codon for the VP3 protein. However, upstream of that codon in the same open reading frame is an ACG sequence (encoding threonine) surrounded by an optimal Kozak (translation initiation) context, which contributes to the low level of synthesis of the VP2 protein.The VP2 protein is actually a member of the VP2 protein family, as described in Becerra SP et al., (December 1985). "Direct mapping of adeno-associated viral capsid proteins B and C: a possible ACG initiation codon". Proceedings of the National Academy of Sciences of the United States of America. 82(23):7919-23; Cassinotti P et al., (November 1988). "Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced ​​mRNA coding for viral capsid protein 1". Virology. 167(1):176-84; Muralidhar S et al., (January 1994). "Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity". Journal of Virology. 68(1):170-6; and Trempe JP, Carter BJ (September 1988). "Alternate mRNA splicing is required for synthesis of adeno-associated viral VP1 capsid protein". Journal of Virology. 62(9):3356-63. Like VP1, it is the addition of N-terminal residues to the VP3 protein. A single consensus polyA site is located at map position 95 of the AAV genome. The life cycle and genetic features of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0030] Each VP1 protein contains a VP1 portion, a VP2 portion, and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is an amino acid sequence present in the VP1 protein that is also found in the N-terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared by the VP1 protein and the VP2 protein.

[0031] The VP3 protein can be further divided into discontinuous variable surface regions I to IX (VR-I to IX). Each of the variable surface regions (VRs) can contain specific amino acid sequences that, alone or in combination with specific amino acid sequences of each of the other VRs, can confer a unique infection phenotype (e.g., reduced antigenicity, improved transduction, and / or tissue-specific tropism compared to other AAV serotypes) to a particular serotype, as described in DiMatta et al., "Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9," J. Virol., Vol. 86(12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0032] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture does not cause cytopathic changes, and natural infection of humans and other animals is symptomless and asymptomatic. Moreover, AAV infects many mammalian cells, allowing it to target many different tissues in vivo. Moreover, AAV can slowly transduce dividing and non-dividing cells and persist as transcriptionally active nuclear episomes (extrachromosomal elements) for virtually the lifetime of these cells. The proviral genome of AAV is inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal ∼4.3 kb of the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA to generate rAAV vectors. The rep and cap proteins can be provided in trans. Another striking feature of AAV is that it is an extremely stable and viable virus. It easily survives the conditions used to inactivate adenovirus (56-65°C for several hours), making the problem of cryopreservation of AAV minor. AAV can even be lyophilized. Finally, cells infected with AAV do not resist superinfection.

[0033] Many studies have demonstrated long-term (>1.5 years) recombinant AAV-mediated protein expression in muscle. See Clark et al., Hum Gene Ther, 8:659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93:14082-14087 (1996); and Xiao et al., J Virol, 70:8098-8108 (1996). See also Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has led to the appearance of the transgene product in the systemic circulation after intramuscular injection, as described by Herzog et al., Proc Natl Acad Sci USA, 94:5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94:13921-13926 (1997). Furthermore, Lewis et al., J Virol, 76:8769-8775 (2002) demonstrated that skeletal muscle myofibers possess the cellular factors necessary for the correct glycosylation, folding, and secretion of antibodies, showing that muscle can stably express secreted protein therapeutics.

[0034] The recombinant AAV (rAAV) genome of the present invention comprises, consists essentially of, or consists of a nucleic acid molecule comprising a polynucleotide sequence encoding at least one amiRNA targeting MAPT, the nucleic acid molecule comprising one or more AAV ITRs adjacent to the nucleic acid molecule. The production of pseudotyped rAAV is disclosed, for example, in WO2001083692. Other types of rAAV variants are also contemplated, such as rAAVs with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0035] Isolated Nucleic Acid Molecules The present disclosure provides an isolated nucleic acid molecule comprising a polynucleotide sequence encoding at least one amiRNA that targets MAPT.

[0036] Exemplary polynucleotide sequences encoding amiRNA sequences targeting human MAPT ("shuttle DNA"), as well as the corresponding amiRNA sequences targeting human MAPT, are set forth in Table 1. Exemplary polynucleotide sequences encoding amiRNA sequences targeting mouse MAPT are set forth in SEQ ID NOs:52-70, and the corresponding amiRNA sequences targeting mouse MAPT are set forth in SEQ ID NOs:150-168. Exemplary polynucleotide sequences encoding amiRNA sequences targeting mouse and human MAPT are set forth in SEQ ID NOs:71-88, and the corresponding amiRNA sequences targeting mouse and human MAPT are set forth in SEQ ID NOs:169-168.

[0037] [Table 1]

[0038] In some embodiments, an amiRNA targeting MAPT comprises, consists essentially of, or consists of a nucleic acid sequence set forth in any one of SEQ ID NOs: 139-186. In some embodiments, an amiRNA targeting MAPT comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to a nucleic acid sequence set forth in any one of SEQ ID NOs: 139-186.

[0039] In some embodiments, the polynucleotide sequence encoding the amiRNA targeting MAPT comprises, consists essentially of, or consists of a nucleic acid sequence represented by any one of SEQ ID NOs: 41-88. In some embodiments, the polynucleotide sequence encoding the amiRNA targeting MAPT comprises, consists essentially of, or consists of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent therebetween) identical to a nucleic acid sequence represented by any one of SEQ ID NOs: 41-88. SEQ ID NOs: 41-88 are DNA sequences, and the corresponding RNA sequences are represented by SEQ ID NOs: 90-137, respectively.

[0040] Generally, the polynucleotide sequences presented herein encoding an amiRNA targeting MAPT include an amiRNA targeting MAPT (mature antisense sequence), a loop sequence, and passenger sequences adjacent to the 5' and 3' overhangs.

[0041] The loop sequence and the overhang together constitute the miRNA "backbone". An exemplary loop sequence is CTGTAAAGCCACAGATGGG (DNA, SEQ ID NO: 188) or CUGUAAAGCCACAGAUGGG (RNA, SEQ ID NO: 189). An exemplary 5' overhang sequence is GAGTGAGCG (DNA, SEQ ID NO: 190) or GAGUGAGCG (RNA, SEQ ID NO: 191). An exemplary 3' overhang sequence is TGCCTACT (DNA, SEQ ID NO: 192) or UGCCUACU (RNA, SEQ ID NO: 193). Without wishing to be bound by theory, it is believed that the passenger sequence and the amiRNA targeting MAPT are tightly packed together to form a hairpin loop structure. It is believed that the hairpin loop structure is then further processed by Dicer, an endoribonuclease that removes the hairpin loop and leaves the miRNA duplex intact. The miRNA duplex, an amiRNA targeting MAPT, is then incorporated into the RNA-induced silencing complex (RISC) and interacts with its target mRNA (e.g., MAPT mRNA), thus blocking translation. See Myburgh et al., Molecular Therapy-Nucleic Acids (2014) 3, p. e207.

[0042] The loop and overhang sequence form a miRNA scaffold. It is clear to those skilled in the art that any suitable scaffold can be used to deliver the amiRNA targeting MAPT presented herein. Exemplary scaffolds include the miR-30 scaffold (Chang et al., Cold Spring Harb Protoc; 2013; doi:10.1101 / pdb.prot075853) and the miR-33 scaffold (Xie et al., 2020 Mor Ther. 28(2):422). As will be recognized by those skilled in the art, the amiRNA targeting MAPT can be cloned into any suitable miR scaffold.

[0043] In some embodiments, the MAPT transcript for the amiRNA described herein is a human MAPT transcript. Illustrative human MAPT transcripts that can be targeted by the amiRNAs presented herein include those represented by NCBI reference sequences NM_016835.5, NM_005910.6, NM_016834.5, NM_016841.5, NM_001123067.4, NM_001123066.4, NM_001203251.2, NM_001203252.2, NM_001377265.1, NM_001377266.1, NM_001377267.1, and NM_001377268.1 (SEQ ID NOs: 2-13, respectively). In some embodiments, the amiRNAs described herein target 1, 2, 3, 4, 5, or 6 MAPT transcripts. In some embodiments, the amiRNAs described herein target human MAPT transcripts. In some embodiments, the amiRNAs described herein target 1, 2, 3, 4, 5, or 6 human MAPT transcripts.

[0044] As will be appreciated by those skilled in the art, the phrase "amiRNA targeting MAPT" refers to an RNA molecule that, once produced in a cell, directs an endogenous RNAi pathway (e.g., the Dicer pathway, the RNA-induced silencing complex (RISC) pathway) to initiate degradation and / or downregulation of MAPT mRNA. The term "AAV-amiRNA targeting MAPT" refers to a rAAV vector that includes at least one polynucleotide sequence encoding an amiRNA targeting MAPT.

[0045] In some aspects, isolated nucleic acid molecule can comprise two or more polynucleotide sequences encoding amiRNA targeting MAPT.Thus, in some embodiments, isolated nucleic acid molecule comprises at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20 polynucleotide sequences encoding amiRNA targeting MAPT, respectively.

[0046] In some embodiments, where the isolated nucleic acid molecule comprises two or more polynucleotide sequences that code for amiRNA that targets MAPT, any number of the polynucleotide sequences can be identical, and any number can be different.In a non-limiting example, where the isolated nucleic acid molecule comprises three polynucleotide sequences (i.e., a first polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence), each of which codes for amiRNA that targets MAPT, all three polynucleotide sequences can have the same sequence.Alternatively, all three polynucleotide sequences can have different sequences.Also alternatively, two of the three polynucleotide sequences can have the same sequence, and the third polynucleotide sequence can have a different sequence.

[0047] rAAV vectors In some aspects, the isolated polynucleotide comprising at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT described herein may be a recombinant AAV (rAAV) vector.

[0048] As used herein, the term "vector" refers to a nucleic acid that comprises, consists essentially of, or consists of an intact replicon, such that the vector is replicated when placed in a cell, for example, by a process of transfection, infection, or transformation. Once in the cell, it is understood in the art that the vector may replicate as an extrachromosomal (episomal) element or may integrate into the host cell's chromosome. Vectors may include nucleic acids derived from retroviruses, adenoviruses, herpes viruses, baculoviruses, modified baculoviruses, papovaviruses, or other modified naturally occurring viruses. Exemplary non-viral vectors for delivering nucleic acids include the use of naked DNA, complexes of DNA with cationic lipids alone or in combination with cationic polymers, anionic and cationic liposomes, DNA-protein complexes, and particles comprising, consisting essentially of, or consisting of DNA condensed with cationic polymers such as heterogeneous polylysine, oligopeptides of defined length, and polyethyleneimine, in some cases contained in liposomes, as well as ternary complexes comprising, consisting essentially of, or consisting of viruses and polylysine-DNA.

[0049] With regard to general recombinant techniques, vectors containing both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Agilent Technologies (Santa Clara, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or change the 5' and / or 3' untranslated portions of the cloned transgene to eliminate redundant, potentially inappropriate alternative translation initiation codons or other sequences that may interfere with or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' of the initiation codon to enhance expression. The nucleic acid sequence of the AAV vector can be codon optimized.

[0050] "rAAV vector" as used herein refers to a vector that comprises, consists essentially of, or consists of one or more transgenes and / or exogenous polynucleotide sequences and one or more AAV inverted terminal repeats (ITRs). Such rAAV vectors, when present in a host cell, can replicate and be packaged into infectious viral particles that provide the function of the rep and cap gene products, for example, upon transfection of the host cell. In some embodiments, the rAAV vector comprises a promoter, at least one nucleic acid that can encode at least one protein or RNA, and / or an enhancer and / or terminator in the flanking ITRs that are packaged into the infectious AAV particle. The encapsidated nucleic acid portion may be referred to as the rAAV vector genome. The plasmids that contain the rAAV vectors may contain elements for manufacturing purposes, such as antibiotic resistance genes, sequences for origins of replication, etc., but these are not encapsidated and therefore do not form part of the AAV particle.

[0051] In some embodiments, rAAV vectors may comprise at least one polynucleotide sequence encoding at least one amiRNA that targets MAPT.In some embodiments, rAAV vectors may comprise at least one AAV inverted terminal (ITR) sequence.In some embodiments, rAAV vectors may comprise at least one promoter sequence.In some embodiments, rAAV vectors may comprise at least one enhancer sequence.In some embodiments, rAAV vectors may comprise at least one polyA sequence.

[0052] In some embodiments, the rAAV vector may include a first AAV ITR sequence, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, and a second AAV ITR sequence. In some embodiments, the rAAV vector may include, in the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, and a second AAV ITR sequence.

[0053] In some embodiments, the rAAV vector may include a first AAV ITR sequence, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, a polyA sequence, and a second AAV ITR sequence. In some embodiments, the rAAV vector may include, in the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, a polyA sequence, and a second AAV ITR sequence.

[0054] In some embodiments, a rAAV vector may contain two or more polynucleotide sequences encoding at least one amiRNA that targets MAPT.

[0055] In some aspects, the disclosure provides rAAV vectors that contain at least about 2, or at least about 3, or at least about 4, or at least about 5, or at least about 6, or at least about 7, or at least about 8, or at least about 9, or at least about 10, or at least about 11, or at least about 12, or at least about 13, or at least about 14, or at least about 15, or at least about 16, or at least about 17, or at least about 18, or at least about 19, or at least about 20 polynucleotide sequences, each encoding at least one amiRNA that targets MAPT.

[0056] In some aspects, the disclosure provides rAAV vectors that include about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20 polynucleotide sequences, each encoding at least one amiRNA that targets MAPT.

[0057] In the embodiment in which the rAAV vector comprises two or more polynucleotide sequences encoding at least one amiRNA targeting MAPT, any number of the polynucleotide sequences can be the same sequence, and any number can be different sequences.In a non-limiting example in which the rAAV vector comprises three polynucleotide sequences encoding at least one amiRNA targeting MAPT (i.e., a first polynucleotide sequence, a second polynucleotide sequence, and a third polynucleotide sequence), all three polynucleotide sequences can have the same sequence.Alternatively, all three polynucleotide sequences can have different sequences.Also alternatively, two of the three polynucleotide sequences can have the same sequence, and the third polynucleotide sequence can have a different sequence.

[0058] In some embodiments, the rAAV vector may include two or more promoter sequences. In some embodiments, the rAAV vector may include at least two promoter sequences, whereby the rAAV vector includes a first promoter sequence and at least a second promoter sequence. In some embodiments, the first and at least a second promoter sequence may include the same sequence. In some embodiments, the first and at least a second promoter sequence may include different sequences. In some embodiments, the first and at least a second promoter sequence may be adjacent to each other. In some embodiments, where the rAAV vector also includes a first polynucleotide sequence encoding at least one amiRNA that targets MAPT and at least a second polynucleotide sequence encoding at least one amiRNA that targets MAPT, the first promoter may be located upstream (5') of the first polynucleotide sequence, and the at least a second promoter may be located between the first polynucleotide sequence and the at least a second polynucleotide sequence, whereby the at least a second promoter is located downstream (3') of the first polynucleotide sequence and upstream (5') of the at least a second polynucleotide sequence.

[0059] Any of the preceding rAAV vectors can further comprise at least one enhancer. The at least one enhancer can be located anywhere in the rAAV vector.

[0060] In some embodiments, at least one enhancer may be located immediately upstream (5') of the promoter. That is, the rAAV vector may include, in the 5' to 3' direction, a first AAV ITR sequence, an enhancer, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, and a second AAV ITR sequence. In some embodiments, at least one enhancer may be located immediately downstream (3') of the promoter. That is, the rAAV vector may include, in the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, an enhancer, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, and a second AAV ITR sequence. In some embodiments, at least one enhancer may be located immediately downstream of the at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT. That is, the rAAV vector can include, from the 5' to 3' direction, a first AAV ITR sequence, a promoter sequence, at least one polynucleotide sequence encoding at least one amiRNA targeting MAPT, an enhancer, a polyA sequence, and a second AAV ITR sequence.

[0061] In some embodiments, the rAAV vector comprises a mutant AAV2 ITR with a deleted D element, a U6 promoter, a polynucleotide sequence encoding an amiRNA targeting MAPT, a T6 termination signal, a CBh promoter, a polyadenylation signal, and a wild-type AAV2 ITR.

[0062] An exemplary sequence of a rAAV vector containing a polynucleotide encoding an amiRNA targeting MAPT is shown in SEQ ID NO: 14. The ITR sequences are underlined in SEQ ID NO: 14; the promoter region is in italics, and the sequence encoding hTau5i is in bold italics.

[0063] [ka] JPEG2024527313000004.jpg218154JPEG2024527313000005.jpg214151JPEG2024527313000006.jpg76156

[0064] AAV ITR sequences In some embodiments, the AAV ITR sequence can include any AAV ITR sequence known in the art. In some embodiments, the AAV ITR sequence can be an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh.10 ITR sequence.

[0065] That is, in some embodiments, the AAV ITR sequence may comprise, consist essentially of, or consist of an AAV1 ITR sequence, an AAV2 ITR sequence, an AAV4 ITR sequence, an AAV5 ITR sequence, an AAV6 ITR sequence, an AAV7 ITR sequence, an AAV8 ITR sequence, an AAV9 ITR sequence, an AAV10 ITR sequence, an AAV11 ITR sequence, an AAV12 ITR sequence, an AAV13 ITR sequence, an AAVrh74 ITR sequence, or an AAVrh.10 ITR sequence.

[0066] In some embodiments, the rAAV vector of the present disclosure can comprise, consist essentially of, or consist of AAV2 ITR sequence. In some embodiments, the rAAV vector of the present disclosure can comprise, consist essentially of, or consist of AAV2 ITR sequence or modified AAV2 ITR sequence. The AAV2 ITR can be modified, for example, by deleting the D region.

[0067] In some embodiments, the AAV ITR sequence comprises, consists essentially of, or can consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percent in between) to any one of the nucleic acid sequences represented by any one of SEQ ID NOs: 15-24, or a complement thereof.

[0068] In some embodiments, the first AAV ITR sequence comprises, consists essentially of, or may consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to a nucleic acid sequence represented by SEQ ID NO:15, or its complement.

[0069] In some embodiments, the second AAVITR sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the nucleic acid sequence represented by SEQ ID NO:16, or its complement.

[0070] Promoter sequences and enhancers As used herein, the terms "promoter" and "promoter sequence" refer to a regulatory sequence that is a region of a polynucleotide sequence at which the initiation and rate of transcription of a coding sequence, such as a gene or transgene or shRNA sequence, is controlled. Promoters can be, for example, constitutive, inducible, repressible, or tissue-specific. Promoters can include genetic elements to which regulatory proteins and molecules, such as RNA polymerase and transcription factors, can bind. Non-limiting exemplary promoters include the Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, the U6 promoter, the H1 promoter, the ubiquitous chicken β-actin hybrid (CBh) promoter, the small nuclear RNA (U1a or U1b) promoter, the MeCP2 promoter, the MeP418 promoter, the MeP426 promoter, the minimal MeCP2 promoter, the VMD2 promoter, the mRho promoter, or the EF1 promoter. The promoter can be, for example, a human promoter sequence or a mouse promoter sequence.

[0071] Further non-limiting exemplary promoters provided herein include, but are not limited to, EFla, Ubc, human β-actin, CAG, TRE, Ac5, polyhedrin, CaMKIIa, Gal1, TEF1, GDS, ADH1, Ubi, and alpha-1-antitrypsin (hAAT). It is known in the art that the nucleotide sequences of such promoters can be modified to increase or decrease the efficiency of mRNA transcription. See, for example, Gao et al. (2018) Mol. Ther.: Nucleic Acids 12:135-145 (modifying the TATA box of 7SK, U6, and H1 promoters to disable RNA polymerase III transcription and stimulate RNA polymerase II-dependent mRNA transcription). Synthetic derived promoters may be used for ubiquitous or tissue-specific expression. In addition, promoters derived from viruses, some of which are mentioned above, such as CMV, HIV, adenovirus and AAV promoters, can be used in the methods described herein.In some embodiments, promoters are used with at least one enhancer to increase transcription efficiency.Non-limiting examples of enhancers include interstitial retinoid binding protein (IRBP) enhancer, RSV enhancer, or CMV enhancer.

[0072] In some embodiments, the promoter sequence is a Rous sarcoma virus (RSV) LTR promoter sequence (optionally with the RSV enhancer), a cytomegalovirus (CMV) promoter sequence, an SV40 promoter sequence, a dihydrofolate reductase promoter sequence, a β-actin promoter sequence, a phosphoglycerol kinase (PGK) promoter sequence, a human U6 promoter sequence, a mouse U6 promoter sequence, an H1 promoter sequence, a ubiquitous chicken β-actin hybrid (CBh) promoter sequence, a small nuclear RNA (U1a or U1b) promoter sequence, a MeCP2 promoter sequence, a MeP418 promoter sequence, a MeP426 promoter sequence, a MeP428 promoter sequence, a MeP429 promoter sequence, a MeP430 promoter sequence, a MeP432 promoter sequence, a MeP434 promoter sequence, a MeP436 promoter sequence, a MeP438 promoter sequence, a MeP438 promoter sequence, a MeP439 ... The promoter sequence may comprise, consist essentially of, or consist of a sequence, a meP229 promoter sequence, a minimal MeCP2 promoter sequence, a VMD2 promoter sequence, a mRho promoter sequence, an EFI promoter sequence, an EFla promoter sequence, a Ubc promoter sequence, a human β-actin promoter sequence, a CAG promoter sequence, a TRE promoter sequence, an Ac5 promoter sequence, a polyhedrin promoter sequence, a CaMKIIa promoter sequence, a Gal1 promoter sequence, a TEF1 promoter sequence, a GDS promoter sequence, an ADH1 promoter sequence, a Ubi promoter sequence, or an alpha-1 antitrypsin (hAAT) promoter sequence.

[0073] Enhancers are control elements that increase the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains a sequence that can provide both promoter and enhancer functions. For example, retroviral long terminal repeats contain both promoter and enhancer functions. Enhancers / promoters can be "endogenous" or "exogenous" or "heterologous". An "endogenous" enhancer / promoter is an enhancer / promoter that is naturally linked to a given gene in a genome. An "exogenous" or "heterologous" enhancer / promoter is an enhancer / promoter that is juxtaposed to a gene by means of genetic engineering (i.e., molecular biological techniques) or synthetic techniques, whereby transcription of that gene is directed by the linked enhancer / promoter. Non-limiting examples of linked enhancers / promoters for use in the methods, compositions, and constructs provided herein include the PDE promoter plus IRBP enhancer, or the CMV enhancer plus U1a promoter. It is understood in the art that enhancers can act from a distance and regardless of their orientation relative to the location of the endogenous or heterologous promoter, i.e., an enhancer that acts at a distance from a promoter is thus further understood to be "operably linked" to that promoter regardless of its location in the vector or its orientation relative to the location of the promoter.

[0074] As used throughout this disclosure, the term "operably linked" refers to the expression of a polynucleotide sequence (i.e., a polynucleotide sequence encoding at least one amiRNA targeting MAPT) that is under the control of a promoter and spatially linked thereto. A promoter can be located 5' (upstream) or 3' (downstream) of the polynucleotide sequence that is under its control. A promoter can be located 5' (upstream) of the polynucleotide sequence that is under its control. The distance between a promoter and its polynucleotide sequence under its control can be approximately the same as the distance between the promoter and the polynucleotide sequence that the promoter controls in the gene from which it is derived. Variation in the distance between the promoter and the polynucleotide sequence can be accommodated without loss of promoter function.

[0075] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of a JeT promoter sequence. The JeT promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO:25 or its complement.

[0076] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of a MeP229 promoter sequence, which may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO:26 or its complement.

[0077] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of the MeP426 promoter sequence, which may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO:27 or its complement.

[0078] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of a CBh promoter sequence. The CBh promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO: 28 or its complement. The CBh promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO: 29 or its complement.

[0079] In some embodiments, the promoter sequence may comprise, consist essentially of, or consist of a U6 promoter sequence. The U6 promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO: 30 or its complement. The mouse U6 promoter sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to SEQ ID NO: 31 or its complement.

[0080] In some embodiments, the bacterial plasmids of the present disclosure may comprise a prokaryotic promoter that comprises, consists essentially of, or can consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical (or any percentage in between) to SEQ ID NO:32 or its complement.

[0081] In some embodiments, the rAAV vectors provided herein comprise two promoters, such as the mouse U6 promoter and the CBh promoter.

[0082] PolyA sequence In some aspects, the polyadenylation (polyA) sequence may comprise any polyA sequence known in the art. Non-limiting examples of polyA sequences include, but are not limited to, MeCP2 polyA sequence, retinol dehydrogenase 1 (RDH1) polyA sequence, bovine growth hormone (BGH) polyA sequence, SV40 polyA sequence, SPA49 polyA sequence, sNRP-TK65 polyA sequence, sNRP polyA sequence, or TK65 polyA sequence. In some embodiments, the polyA sequence is a synthetic polyA sequence.

[0083] That is, the polyA sequence may comprise, consist essentially of, or consist of a MeCP2 polyA sequence, a retinol dehydrogenase 1 (RDH1) polyA sequence, a bovine growth hormone (BGH) polyA sequence, an SV40 polyA sequence, an SPA49 polyA sequence, a sNRP-TK65 polyA sequence, a sNRP polyA sequence, or a TK65 polyA sequence.

[0084] In some embodiments, the polyA sequence can comprise, consist essentially of, or consist of the SV40pA sequence, hi some embodiments, the SV40pA sequence can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage therebetween) identical to the sequence set forth in SEQ ID NO:33, or its complement.

[0085] In some embodiments, the polyA sequence may comprise, consist essentially of, or consist of the BGHpA sequence. In some embodiments, the BGHpA sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to the sequence set forth in SEQ ID NO: 34 or its complement. In some embodiments, the BGHpA sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percent in between) identical to the sequence set forth in SEQ ID NO: 35 or its complement.

[0086] In some embodiments, the synthetic polyA sequence may comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to the sequence set forth in SEQ ID NO:36 or its complement.

[0087] Termination sequence In some embodiments, the rAAV vectors presented herein may contain a termination sequence. The termination sequence defines the end of the transcription unit and initiates the process of releasing newly synthesized RNA from the transcription mechanism. The termination sequence may contain several thymine residues. For example, the T6 termination contains the sequence TTTTTT (SEQ ID NO: 40). The T7 terminator contains the sequence TTTTTTT (SEQ ID NO: 41).

[0088] Bacterial plasmids In some aspects, the rAAV vectors of the disclosure may be included in a bacterial plasmid to allow for propagation of the rAAV vector in vitro, i.e., the disclosure provides a bacterial plasmid that includes any of the rAAV vectors described herein.

[0089] The bacterial plasmid, rAAV vector, or rAAV viral vector may further comprise an origin of replication sequence. In some aspects, the origin of replication sequence may comprise, consist essentially of, or consist of any origin of replication sequence known in the art. The origin of replication sequence may be a bacterial origin of replication sequence, allowing the rAAV vector comprising said bacterial origin of replication sequence to be produced, propagated, and maintained in bacteria by using standard methods in the art. In some embodiments, the origin of replication comprises, consists essentially of, or consists of the sequence represented by SEQ ID NO:36.

[0090] The bacterial plasmid, rAAV vector, or rAAV viral vector may further comprise an antibiotic resistance gene. In some aspects, the antibiotic resistance gene may comprise, consist essentially of, or consist of any antibiotic resistance gene known in the art. Examples of antibiotic resistance genes known in the art include, but are not limited to, kanamycin resistance gene, spectinomycin resistance gene, streptomycin resistance gene, ampicillin resistance gene, carbenicillin resistance gene, bleomycin resistance gene, erythromycin resistance gene, polymyxin B resistance gene, tetracycline resistance gene, and chloramphenicol resistance gene. In some embodiments, the kanamycin resistance gene comprises, consists essentially of, or consists of the sequence represented by SEQ ID NO:38. In some embodiments, the kanamycin resistance gene is operably linked to a promoter. In some embodiments, the kanamycin resistance gene is operably linked to an ampicillin resistance gene promoter (AmpR promoter). In some embodiments, the AmpR promoter comprises, consists essentially of, or consists of the sequence represented by SEQ ID NO:37.

[0091] The bacterial plasmid, rAAV vector, or rAAV viral vector may further comprise a prokaryotic promoter.

[0092] AAV viral vectors A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide that is delivered into a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, AAV vectors, lentiviral vectors, adenoviral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, for example, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying et al., (1999) Nat. Med. 5(7):823-827.

[0093] "AAV virion" or "AAV viral particle" or "AAV viral vector" or "rAAV viral vector" or "AAV vector particle" or "AAV particle" refers to a viral particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. That is, the production of a rAAV viral vector necessarily includes the production of a rAAV vector, and thus the vector is contained within the rAAV vector.

[0094] As used herein, the term "viral capsid" or "capsid" refers to the proteinaceous shell or coat of a virus particle. Capsids function to encapsidate, protect, transport, and release the viral genome into the host cell. Capsids are generally composed of oligomeric structural subunits of proteins ("capsid proteins"). As used herein, the term "encapsidation" means being enclosed in a viral capsid. The viral capsid of AAV is composed of a mixture of three viral capsid proteins, namely VP1, VP2, and VP3. The mixture of VP1, VP2, and VP3 contains 60 monomers arranged in a T=1 icosahedral symmetry in a ratio of 1:1:10 (VP1:VP2:VP3) or 1:1:20 (VP1:VP2:VP3), as described in Sonntag F et al., (June 2010) "A viral assembly factor promotes AAV2 capsid formation in the nucleolus". Proceedings of the National Academy of Sciences of the United States of America. 107(22):10220-5, and Rabinowitz JE, Samulski RJ (December 2000). "Building a better vector: the manipulation of AAV virions". Virology. 278(2):301-8, each of which is incorporated herein by reference in its entirety.

[0095] The present disclosure provides an rAAV viral vector comprising: a) any of the rAAV vectors described herein, or a complement thereof; and b) an AAV capsid protein.

[0096] The AAV capsid protein may be any AAV capsid protein known in the art. The AAV capsid protein may be an AAV1 capsid protein, an AAV2 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVPHP.B capsid protein, an AAVrh74 capsid protein, or an AAVrh.10 capsid protein.

[0097] Compositions and pharmaceutical compositions The present disclosure provides a composition comprising any of the isolated polynucleotides, rAAV vectors, and / or rAAV viral vectors described herein.In some aspects, the composition can be a pharmaceutical composition.Therefore, the present disclosure provides a pharmaceutical composition comprising any of the isolated polynucleotides, rAAV vectors, and / or rAAV viral vectors described herein, and optionally a pharma- ceutical acceptable carrier.

[0098] Pharmaceutical compositions, as described herein, can be formulated by any method known or developed in the art of pharmacology, including, but not limited to, contacting the active ingredient (e.g., viral particle or recombinant vector) with excipients and / or additives and / or other auxiliary ingredients and dividing or packaging the product into dosage units. Viral particles of the present disclosure can be formulated with desirable characteristics, such as increased stability, increased cell transfection, sustained or delayed release, biodistribution or tropism, regulated or enhanced translation of the encoded protein in vivo, and in vivo release profile of the encoded protein.

[0099] Thus, the pharmaceutical composition may further comprise saline, lipids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for implantation into a subject), nanoparticle mimics, or combinations thereof. In some embodiments, the pharmaceutical composition is formulated as nanoparticles. In some embodiments, the nanoparticles are self-assembled nucleic acid nanoparticles.

[0100] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. The amount of active ingredient is generally equal to the dosage of active ingredient administered to a subject and / or a convenient fraction of such a dosage, such as, for example, half or a third of such a dosage. The formulations of the present invention may include one or more excipients and / or additives in amounts that together increase the stability of the viral vector, increase the transfection or transduction of cells by the viral vector, increase the expression of a protein encoded by the viral vector, and / or modify the release profile of a protein encoded by the viral vector. In some embodiments, the pharmaceutical composition includes an excipient and / or additive. Non-limiting examples of excipients and / or additives include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, or combinations thereof.

[0101] In some embodiments, the pharmaceutical composition comprises a cryoprotectant. The term "cryoprotectant" refers to an agent that can reduce or eliminate damage to a substance during freezing. Non-limiting examples of cryoprotectants include sucrose, trehalose, lactose, glycerol, dextrose, raffinose, and / or mannitol.

[0102] As used herein, the term "pharmaceutical acceptable carrier" includes any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, water, and emulsions, such as oil-in-water or water-in-oil emulsions, as well as various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see Martin (1975) Remington's Pharm.Sci., 15th Edition (Mack Publ.Co., Easton).

[0103] In some embodiments, pharmaceutical compositions of the present disclosure may include phosphate buffered saline, D-sorbitol, sodium chloride, Pluronic® F-68, or any combination thereof.

[0104] In some embodiments, the pharmaceutical composition may include sodium chloride, and the sodium chloride is present at a concentration of about 100 mM to about 500 mM, or about 200 mM to about 400 mM, or about 300 mM to about 400 mM. In some embodiments, the sodium chloride may be present at a concentration of about 350 mM.

[0105] In some embodiments, the pharmaceutical composition may include D-sorbitol, where D-sorbitol is present at a concentration of about 1% to about 10%, or about 2.5% to about 7.5%. In some embodiments, D-sorbitol may be present at a concentration of about 5%.

[0106] In some embodiments, the pharmaceutical composition may include Pluronic® F-68, where Pluronic® F-68 is present at a concentration of about 0.00001% to about 0.01%, or about 0.0005% to about 0.005%. In some embodiments, Pluronic® F-68 may be present at a concentration of about 0.001%.

[0107] The disclosure then provides a pharmaceutical composition comprising an rAAV vector and / or rAAV viral vector of the disclosure in a phosphate-buffered saline solution, where the pharmaceutical composition further comprises sodium chloride at a concentration of 350 mM, D-sorbitol at a concentration of 5%, and Pluronic® F-68 at a concentration of 0.001%.

[0108] The disclosure then provides pharmaceutical compositions comprising the rAAV vectors and / or rAAV viral vectors of the disclosure, where the pharmaceutical compositions further comprise sodium chloride at a concentration of 350 mM, D-sorbitol at a concentration of 5%, and Pluronic® F-68 at a concentration of 0.001%.

[0109] The disclosure then provides a pharmaceutical composition comprising an rAAV vector and / or rAAV viral vector of the disclosure in a phosphate-buffered saline solution, wherein the pharmaceutical composition further comprises sodium chloride at a concentration of 350 mM and D-sorbitol at a concentration of 5%.

[0110] The disclosure then provides pharmaceutical compositions comprising the rAAV vectors and / or rAAV viral vectors of the disclosure, where the pharmaceutical compositions further comprise sodium chloride at a concentration of 350 mM and D-sorbitol at a concentration of 5%.

[0111] Methods of Using the Disclosed Compositions The present disclosure provides for the use of the disclosed compositions or pharmaceutical compositions for the treatment of a disease or disorder in a cell, tissue, organ, animal, or subject known in the art or described herein, for example, by administering or contacting a therapeutically effective amount of the composition or pharmaceutical composition to the cell, tissue, organ, animal, or subject. In one embodiment, the subject is a mammal. Preferably, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0112] The present disclosure provides a method for preventing or treating a disorder comprising, consisting essentially of, or consisting of administering to a subject a therapeutically effective amount of any one of the isolated nucleic acid molecules, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein.

[0113] In one aspect, provided herein is a method of treating a tauopathy in a subject, the method comprising administering to the subject an effective amount of an isolated nucleic acid, rAAV vector, rAAV viral vector, or composition described herein.

[0114] In some embodiments, the subject suffers from a disorder associated with a mutation in the MAPT gene.In some embodiments, the subject treated according to the methods described herein has one or more mutations in the MAPT gene.Mutations in MAPT that have an effect on tau function are known in the art, see, for example, Strang et al., Lab Invest. July 2019; 99(7):912-928. In some embodiments, the mutation is selected from the group consisting of R5H, R5L, G55R, K257T, I260V, L266V, G272V, N279K, Δ280K, S285R, K298E, P301L, P301S, P301T, G303V, S305I, S305N, K317M, K317N, S320F, P332S, G335V, Q336H, Q336R, V337M, S352L, S356T, P364S, K369I, E372G, G389R(G→A), G389R(G→C), and N410H.

[0115] In some embodiments, the disease is AD. In some embodiments, the disease is FNTD-17.

[0116] Thus, the present disclosure provides a method for preventing or treating tauopathy in a subject, comprising, consisting essentially of, or consisting of administering to a subject a therapeutically effective amount of any one of the isolated nucleic acid molecules, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein. The present disclosure provides the use of any one of the isolated nucleic acid molecules, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein for the manufacture of a medicament for treating or preventing tauopathy. The present disclosure provides any one of the isolated nucleic acid molecules, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions disclosed herein for use in treating or preventing tauopathy. In some embodiments, the tauopathy is AD. In some embodiments, the disease is FNTD-17.

[0117] The methods of treatment and prevention disclosed herein may be combined with appropriate diagnostic procedures to identify and select patients for treatment or prevention.

[0118] The present disclosure provides a method for reducing the level of one or more proteins in a host cell, comprising contacting the host cell with any one of the rAAV viral vectors disclosed herein, wherein the rAAV viral vector comprises any one of the rAAV vectors disclosed herein.In some embodiments, the host cell is in vitro, in vivo, or ex vivo.In some embodiments, the host cell is derived from a subject, for example, a subject suffering from tauopathy.

[0119] In some aspects, administration of an isolated nucleic acid, rAAV vector, or rAAV viral vector herein results in a decrease in tau protein expression in host cells. Tau levels are reduced to about 1×10 -7 ng, about 3x10 -7 ng, about 5x10 -7 ng, about 7x10-7 ng, about 9x10 -7 ng, about 1x10 -6 ng, about 2x10 -6 ng, about 3x10 -6 ng, about 4x10 -6 ng, about 6x10 -6 ng, about 7x10 -6 ng, about 8x10 -6 ng, about 9x10 -6 ng, about 10x10 -6 ng, about 12x10 -6 ng, approx. 14x10 -6 ng, about 16x10 -6 ng, approx. 18x10 -6 ng, about 20x10 -6 ng, about 25x10 -6 ng, about 30x10 -6 ng, approx. 35x10 -6 ng, about 40x10 -6 ng, about 45x10 -6 ng, about 50x10 -6 ng, about 55x10 -6 ng, about 60x10 -6 ng, about 65x10 -6 ng, about 70x10 -6 ng, about 75x10 -6 ng, about 80x10 -6 ng, about 85x10 -6 ng, about 90x10 -6 ng, about 95x10 -6 ng, about 10x10 -5 ng, about 20x10 -5 ng, about 30x10 -5 ng, about 40x10 -5 ng, about 50x10 -5 ng, about 60x10 -5 ng, about 70x10 -5 ng, approximately 80 × 10 -5 ng, or approximately 90 × 10 -5 It can be as low as ng levels.

[0120] In some embodiments, levels of tau protein may be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or about 100%.

[0121] In some embodiments, levels of tau protein may be reduced by at least about 1.25-fold, at least about 1.5-fold, at least about 1.25-fold, at least about 2-fold, at least about 2.25-fold, at least about 2.5-fold, at least about 2.75-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0122] The present disclosure provides a method for reducing the level of an RNA molecule in a host cell, comprising contacting the host cell with any one of the rAAV viral vectors disclosed herein, wherein the rAAV viral vector comprises any one of the rAAV vectors disclosed herein. In some aspects, the RNA molecule is an mRNA molecule or a non-coding RNA molecule. In some aspects, the host cell is in vitro, in vivo, or ex vivo. In some aspects, the host cell is derived from a subject, for example, a subject suffering from tauopathy. In some embodiments, the host cell is a cell derived from the brainstem, cerebellum, or cortex of a subject suffering from tauopathy.

[0123] In some embodiments, administration of an isolated nucleic acid, rAAV vector, or rAAV viral vector herein results in a decrease in MAPT mRNA in a host cell. The level of mRNA molecules is reduced to about 1×10 -7 ng, approximately 3 × 10 -7 ng, about 5x10 -7ng, about 7x10 -7 ng, about 9x10 -7 ng, about 1x10 -6 ng, about 2x10 -6 ng, about 3x10 -6 ng, about 4x10 -6 ng, about 6x10 -6 ng, about 7x10 -6 ng, about 8x10 -6 ng, about 9x10 -6 ng, about 10x10 -6 ng, about 12x10 -6 ng, approx. 14x10 -6 ng, about 16x10 -6 ng, approx. 18x10 -6 ng, about 20x10 -6 ng, about 25x10 -6 ng, about 30x10 -6 ng, approx. 35x10 -6 ng, about 40x10 -6 ng, about 45x10 -6 ng, about 50x10 -6 ng, about 55x10 -6 ng, about 60x10 -6 ng, about 65x10 -6 ng, about 70x10 -6 ng, about 75x10 -6 ng, about 80x10 -6 ng, about 85x10 -6 ng, about 90x10 -6 ng, about 95x10 -6 ng, about 10x10 -5 ng, about 20x10 -5 ng, about 30x10 -5 ng, about 40x10 -5 ng, about 50x10 -5 ng, about 60x10 -5 ng, about 70x10 -5 ng, approximately 80 × 10 -5 ng, or approximately 90 × 10 -5 It can be as low as ng levels.

[0124] In some embodiments, the level of MAPT mRNA is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, or about 100%.

[0125] In some embodiments, the level of MAPT mRNA is reduced by at least about 1.25-fold, at least about 1.5-fold, at least about 1.25-fold, at least about 2-fold, at least about 2.25-fold, at least about 2.5-fold, at least about 2.75-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold.

[0126] The present disclosure provides a method for introducing a polynucleotide sequence of interest (e.g., a polynucleotide sequence encoding at least one amiRNA that targets MAPT) into a cell in a subject, the method comprising contacting the cell with an effective amount of any one of the rAAV viral vectors disclosed herein, wherein the rAAV viral vector contains any one of the rAAV vectors disclosed herein that includes the polynucleotide sequence of interest.

[0127] In some embodiments of the method of the present disclosure, the subject can also be administered a preventive immunosuppressive treatment regimen in addition to administering the rAAV vector or rAAV viral vector of the present disclosure.In some embodiments, the immunosuppressive treatment regimen includes administering at least one immunosuppressive therapeutic agent.Non-limiting examples of immunosuppressive therapeutic agents include, but are not limited to, sirolimus (rapamycin), acetaminophen, diphenhydramine, IV methylprednisolone, prednisone, or any combination thereof.The immunosuppressive therapeutic agent can be administered prior to the day of administration of the rAAV vector and / or rAAV viral vector, on the same day as administration of the rAAV vector and / or rAAV viral vector, or on any day after administration of the rAAV vector and / or rAAV viral vector.

[0128] A "subject" of diagnosis or treatment is a cell or an animal, such as a mammal, or a human. A subject is not limited to a particular species, and includes non-human animals that are the subject of diagnosis or treatment, including, without limitation, ape, murine, rat, dog, or rabbit species, and those that are the subject of infection or animal models, as well as other farm animals, sport animals, or pets. In some embodiments, a subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0129] As used herein, "treating" a disease in a subject or "treatment" thereof refers to (1) delaying the onset of a symptom or disease in a subject susceptible to or not yet exhibiting symptoms of the disease; (2) inhibiting or preventing the onset of the disease; or (3) ameliorating or causing regression of the disease or symptoms of the disease. Delaying, inhibiting, and ameliorating may be compared to another subject (e.g., a subject suffering from the same tauopathy (e.g., AD or FTDP-17)) to which a composition, isolated nucleic acid molecule, rAAV vector, or rAAV viral vector described herein is not administered. 。The inhibition or amelioration can be compared to another subject (e.g., a subject suffering from a tauopathy (e.g., AD or FTDP-17) to which a composition, isolated nucleic acid molecule, rAAV vector, or rAAV viral vector described herein is not administered), or to the subject being treated prior to the first administration of a composition, isolated nucleic acid molecule, rAAV vector, or rAAV viral vector described herein.

[0130] As understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results may include, but are not limited to, one or more, alleviation or amelioration of one or more symptoms, whether detectable or not, diminishment of the severity of a condition (including a disease), stabilization of the condition (i.e., not worsening), delay or slowing of the condition (including a disease), progression, improvement, or relief, condition, and remission (whether partial or complete) of a condition (including a disease).

[0131] In some embodiments, the condition that is ameliorated by administration of the compositions, isolated nucleic acid molecules, rAAV vectors, or rAAV viral vectors described herein is the accumulation of tau in the brain, particularly highly phosphorylated fibrillar tau.Tau protein can be detected using any suitable method known in the art or described herein, including, for example, immunohistochemistry.

[0132] In some embodiments, the condition that will be ameliorated by administration of a composition, isolated nucleic acid molecule, rAAV vector, or rAAV viral vector described herein is dementia.

[0133] As used herein, the term "effective amount" is intended to mean an amount sufficient to achieve a desired effect. For therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition in question, as well as the characteristics of the individual subject, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In the context of gene therapy, the effective amount may be an amount sufficient to cause the normalization or improvement of the function of a gene that is deficient in a subject. In some embodiments, an effective amount of an isolated polynucleotide, rAAV vector, rAAV viral vector, or composition provided herein is an amount sufficient to cause the expression of an amiRNA targeting MAPT in a subject, for example, in the brainstem, cortex, and / or cerebellum of a subject. In some embodiments, an effective amount of an isolated polynucleotide, rAAV vector, rAAV viral vector, or composition provided herein is an amount sufficient to cause the expression of an amiRNA targeting MAPT in a subject, such as, for example, the expression of tau is upregulated. In some embodiments, the expression of tau is upregulated in the brainstem, cortex, and / or cerebellum. One of skill in the art would be able to determine appropriate amounts depending on these and other factors.

[0134] In some aspects, effective amount will depend on the size and nature of the application in question.It will also depend on the nature and sensitivity of the target subject and the method used.Those skilled in the art will be able to determine effective amount based on these and other considerations.Effective amount can include, consist essentially of, or consist of one or more doses of the composition according to the embodiment.

[0135] As used herein, the term "administer" or "administration" is intended to mean the delivery of a substance to a subject, such as a human or animal. Administration can be accomplished in one dose, continuously, or intermittently throughout the course of treatment. Methods for determining the most effective means and dosages of administration are known to those skilled in the art and will vary with the composition used for treatment, the purpose of the treatment, and the age, health, or sex of the subject being treated. Single or multiple administrations may be performed with the dose level and pattern selected by the treating clinician or, in the case of pets and other animals, the treating veterinarian.

[0136] Methods for determining the most effective means of administration and dosage are known to those skilled in the art and will vary depending on the composition used for treatment, the purpose of treatment, and the subject being treated. Single or multiple administrations may be performed with the dosage level and pattern selected by the treating clinician. It is noted that dosage may be influenced by the route of administration. Suitable dosage formulations and methods of administering drugs are known in the art. A non-limiting example of such a suitable dosage is 10 mg / kg per administration. 9 Low to 10% vector genome 17 There may be up to high amounts of vector genomes (or "viral particles").

[0137] In some aspects, the amount of viral particles in a composition, pharmaceutical composition, or administered to a patient can be calculated based on the percentage of viral particles predicted to contain the viral genome.

[0138] In some embodiments, the rAAV viral vector of the present disclosure may be introduced into a subject intravenously, intrathecally, intracerebrally, intracisternomaduo (ICM), intraventricularly, intranasally, intratracheally, intraaurally, intraocularly or periocularly, orally, intrarectally, transmucosally, by inhalation, transdermal, parenterally, subcutaneously, intradermal, intramuscularly, intracisternomaduo, intraneuronally, intrapleurally, topically, intralymph node, intracisternomaduo; such introduction may also be intraarterially, intracardiacly, subventricularly, epidurally, intracerebrally, intraventricularly, subretinally, intravitreally, intraarticularly, intraperitoneally, intrauterinely, intraneuronally, or any combination thereof. In some embodiments, the viral particles are delivered to a desired target tissue, for example, as non-limiting examples, to the lung, eye, or CNS. In some embodiments, the delivery of the viral particles is systemic. Intracisternomaduo routes of administration include the direct administration of the drug into the cerebrospinal fluid of the ventricles. This may be performed by direct injection into the cisterna magna or by a permanently placed tube. In some embodiments, the rAAV viral vectors of the present disclosure are administered intrathecally. In some embodiments, the rAAV viral vectors of the present disclosure are administered intracisternally.

[0139] Administration of the disclosed rAAV vectors, rAAV viral vectors, compositions, or pharmaceutical compositions can be accomplished in one dose, continuously, or intermittently throughout the course of treatment. In some embodiments, the disclosed rAAV vectors, rAAV viral vectors, compositions, or pharmaceutical compositions are administered parenterally by injection, infusion, or implantation.

[0140] In some embodiments, the rAAV viral vectors of the present disclosure exhibit enhanced tropism to the brain and cervical spine. In some embodiments, the rAAV viral vectors of the present disclosure are capable of crossing the blood-brain barrier (BBB).

[0141] Manufacturing method Various approaches can be used to produce the rAAV viral vector of the present disclosure.In some embodiments, packaging is achieved by using helper virus or helper plasmid and cell line.Helper virus or helper plasmid contains elements and sequences that facilitate the production of viral vector.In another embodiment, helper plasmid is stably integrated into the genome of packaging cell line, so that packaging cell line does not need further transfection with helper plasmid.

[0142] In some embodiments, the cell is a packaging cell line or a helper cell line. In some embodiments, the helper cell line is a eukaryotic cell, such as a HEK293 cell or a 293T cell. In some embodiments, the helper cell is a yeast cell or an insect cell.

[0143] In some embodiments, the cell comprises a nucleic acid encoding a tetracycline activator protein and a promoter that controls the expression of the tetracycline activator protein.In some embodiments, the promoter that controls the expression of the tetracycline activator protein is a constitutive promoter.In some embodiments, the promoter is a phosphoglycerate kinase promoter (PGK) or a CMV promoter.

[0144] The helper plasmid can, for example, contain at least one viral helper DNA sequence derived from a replication-incompetent viral genome that encodes the trans-all virion proteins necessary for packaging replication-incompetent AAV and for producing high titers of virion proteins capable of packaging replication-incompetent AAV without producing replication-competent AAV.

[0145] Helper plasmids for packaging AAV are known in the art, see, for example, US Patent Application Publication No. 2004 / 0235174A1, which is incorporated herein by reference. As described therein, AAV helper plasmids can contain DNA sequences as helper viruses, including, but not limited to, Ad5 genes E2A, E4, and VA, controlled by their native promoters or heterologous promoters. AAV helper plasmids can further contain expression cassettes for the expression of marker proteins, such as fluorescent proteins, to allow simple detection of transfection of desired target cells.

[0146] The present disclosure provides a method for producing a rAAV viral vector, comprising transfecting a packaging cell line with any one of the AAV helper plasmids disclosed herein and any one of the rAAV vectors disclosed herein. In some embodiments, the AAV helper plasmid and the rAAV vector are co-transfected into the packaging cell line. In some embodiments, the cell line is a mammalian cell line, for example, a human embryonic kidney (HEK) 293 cell line. The present disclosure provides a cell comprising any one of the rAAV vectors and / or rAAV viral vectors disclosed herein.

[0147] As used herein, the term "helper" in relation to a virus or plasmid refers to a virus or plasmid that is used to provide additional components required for the replication and packaging of any one of the rAAV vectors described herein. The components encoded by the helper virus can include any genes required for virion assembly, encapsidation, genome replication, and / or packaging. For example, the helper virus or plasmid can encode the necessary enzymes for viral genome replication. Non-limiting examples of helper viruses and plasmids suitable for use with AAV constructs include pHELP (plasmid), adenovirus (virus), or herpesvirus (virus). In some embodiments, the pHELP plasmid can be a pHELPK plasmid, in which the ampicillin expression cassette is replaced with a kanamycin expression cassette.

[0148] As used herein, packaging cells (or helper cells) are cells used to produce viral vectors. The production of recombinant AAV viral vectors (rAAV viral vectors) requires Rep and Cap proteins provided in trans, as well as gene sequences from adenovirus that help AAV replication. In some embodiments, packaging / helper cells contain plasmids and are stably integrated into the genome of the cells. In other embodiments, packaging cells can be transiently transfected. Typically, packaging cells are eukaryotic cells, such as mammalian cells or insect cells.

[0149] kit The isolated polynucleotides, rAAV vectors, rAAV viral vectors, compositions, and / or pharmaceutical compositions described herein may be assembled into pharmaceutical or diagnostic or research kits to facilitate their use in therapeutic, diagnostic, or research applications. In some embodiments, the kits of the present disclosure include any one of the isolated polynucleotides, rAAV vectors, rAAV viral vectors, compositions, pharmaceutical compositions, host cells, and isolated tissues as described herein.

[0150] In some embodiments, the kit further comprises instructions for use. Specifically, such kits may comprise one or more agents as described herein, together with instructions that describe the intended use and correct use of these agents. In some embodiments, the kit may comprise instructions for mixing one or more components of the kit, and / or isolating and mixing samples and applying them to a subject. In some embodiments, the agents in the kit are in pharmaceutical formulations, and in dosages appropriate for the particular use and method of administration of the agents. Kits for research purposes may comprise components in appropriate concentrations or amounts to carry out various experiments.

[0151] The kits are designed to facilitate the use of the methods described herein and can take many forms. Each of the components of the kit can be provided in liquid form (e.g., in solution) or solid form (e.g., dry powder), where applicable. In certain instances, some of the compositions can be reconstituted or otherwise processed (e.g., to an active form) with, for example, a suitable solvent or other species (e.g., water or cell culture medium) that may or may not be provided with the kit. In some embodiments, the compositions can be provided in a storage solution (e.g., a cryopreservation solution). Non-limiting examples of storage solutions include DMSO, paraformaldehyde, and CryoStor® (Stem Cell Technologies, Vancouver, Canada). In some embodiments, the storage solution includes an amount of a metalloprotease inhibitor.

[0152] In some embodiments, the kit includes any one or more of the components described herein in one or more containers. That is, in some embodiments, the kit may include a container that contains the agent described herein. The agent may be in liquid, gel, or solid (powder) form. The agent may be aseptically prepared, packaged in a syringe, and shipped refrigerated. Alternatively, the agent may be contained in a vial or other container for storage. A second container may have another agent that is aseptically prepared. Alternatively, the kit may include an active agent that is premixed and shipped in a syringe, vial, tube, or other container. The kit may have one or more or all of the parts necessary to administer the agent to a subject, such as a syringe, a topical application device, or an IV needle tube and bag.

[0153] Further definitions It is specifically contemplated that the various features of the invention described herein may be used in any combination unless the context dictates otherwise.Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein may be excluded or omitted.For purposes of illustration, if the specification states that a composite comprises components A, B, and C, it is specifically contemplated that any or any combination of A, B, or C may be omitted or discarded, either alone or in any combination.

[0154] Unless expressly stated otherwise, all specified aspects, embodiments, features, and terms are intended to include both the recited aspect, embodiment, feature, or term and its biological equivalents.

[0155] The practice of the present technique will employ, unless otherwise indicated, conventional techniques of organic chemistry, pharmacology, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); Current Protocols In Molecular Biology (FMAusubel et al., eds., (1987)); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)), Antibodies, a Laboratory Manual, Harlow and Lane, eds. (1988), and Animal Cell Culture (RI Freshney, ed., (1987)).

[0156] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude others. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted to include the recited materials or steps, and materials or steps that do not substantially affect the basic and novel characteristics of the recited embodiment. That is, the term "consisting essentially of" as used herein should not be interpreted as equivalent to "comprising." "Consisting of" means excluding more than trace elements of other ingredients and substantial method steps for administering the compositions disclosed herein. The aspects defined by each of these transitional phrases are within the scope of the present disclosure. In each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by the other two phrases while retaining their original meaning.

[0157] All numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that vary (+) or (-) by increments of 1.0 or 0.1, or by ±15%, 10%, 5%, 2% variation, as appropriate. Although not always explicitly stated, it is understood that all numerical designations are preceded by the term "about". Although not always explicitly stated, it is also understood that the reagents described herein are merely exemplary, and equivalents thereof are known in the art. The term "about" as used herein when referring to amounts or concentrations or other measurable values ​​is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0158] The terms "acceptable," "effective," or "sufficient," when used to describe any component, range, dosage form, or other selection disclosed herein, are intended to mean that said component, range, dosage form, etc. is suitable for the disclosed purpose.

[0159] Also, as used herein, "and / or" refers to and includes every possible combination of one or more of the associated listed items, as well as the lack of combinations when stated in the alternative ("or").

[0160] Unless specifically recited, the term "host cell" includes eukaryotic host cells, including, for example, fungal cells, yeast cells, higher plant cells, insect cells, and mammalian cells. Non-limiting examples of eukaryotic host cells include ape, bovine, porcine, murine, rat, avian, reptilian, and human, e.g., HEK293 and 293T cells.

[0161] As used herein, the term "isolated" refers to a molecule or biological product or cellular material that is substantially free of other materials.

[0162] As used herein, the terms "nucleic acid sequence" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, i.e., the term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers comprising, consisting essentially of, or consisting of purine and pyrimidine bases or other naturally occurring, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0163] "Gene" refers to a polynucleotide containing at least one open reading frame (ORF) capable of encoding a particular polypeptide or protein. "Gene product" or "gene expression product" refers to the amino acid sequence (e.g., a peptide or polypeptide) produced when a gene is transcribed and translated.

[0164] As used herein, "expression" refers to the two-step process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.

[0165] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, is dependent on being operably linked to elements that contribute to or facilitate the initiation of transcription. "Operably linked" refers to being positioned in a manner that allows the polynucleotide to function in a cell. In one embodiment, a promoter may be operably linked to a downstream sequence.

[0166] The term "encoding" when applied to a polynucleotide and / or nucleic acid sequence refers to a polynucleotide and / or nucleic acid sequence that can be said to "encode" an RNA molecule (e.g., an shRNA molecule) if, in its natural state, the polynucleotide and / or nucleic acid sequence corresponds to the sequence of a biologically active shRNA molecule. The antisense strand is the complement of such a polynucleotide and / or nucleic acid sequence, and a coding sequence can be deduced therefrom.

[0167] The terms "equivalent" or "biologically equivalent" when referring to a particular molecule, biological material, or cellular material are used interchangeably and contemplate a particular molecule, biological material, or cellular material that has minimal homology while still maintaining a desired structure or functionality. Non-limiting examples of equivalent polypeptides include polypeptides having at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, or at least about 99% identity to a reference polypeptide (e.g., a wild-type polypeptide), or polypeptides encoded by a polynucleotide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% identity, at least about 97% sequence identity, or at least about 99% sequence identity to a reference polynucleotide (e.g., a wild-type polynucleotide).

[0168] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing positions in each sequence aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are identical at that position. The degree of identity between sequences is a function of the number of matching positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, less than 25% identity with one of the sequences of the present disclosure. Alignment and percent sequence identity can be determined for the nucleic acid or amino acid sequences provided herein by importing the nucleic acid or amino acid sequence into and using ClustalW (available at https: / / genome.jp / tools-bin / clustalw / ). For example, the ClustalW parameters used to perform the protein sequence alignments found herein were generated using the Gonnet (for proteins) weight matrix. In some embodiments, the ClustalW parameters used to perform nucleic acid sequence alignments using the nucleic acid sequences found herein are generated using the ClustalW (for DNA) weight matrix.

[0169] The polynucleotides disclosed herein can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes referred to as a "transgene") into a host cell, regardless of the method used for the introduction. Such methods include a variety of known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or a variety of other protein- or lipid-based gene delivery complexes), as well as techniques that facilitate the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and a variety of other techniques used for the introduction of polynucleotides). The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell or is integrated into a host cell replicon, such as an extrachromosomal replicon (e.g., a plasmid), or a nuclear or mitochondrial chromosome. As known in the art and described herein, it is known that several vectors can mediate the transfer of genes into mammalian cells.

[0170] A "plasmid" is a DNA molecule that is typically capable of replicating separately and independently from chromosomal DNA. In many instances, it is circular and double stranded. Plasmids provide a mechanism for horizontal gene transfer within a population of microorganisms, typically providing a selective advantage under a given environmental situation. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances. While plasmid vectors often exist as extrachromosomal circular DNA molecules, they can also be designed to stably integrate into host chromosomes in a random or targeted manner, and it is known in the art that such integration can be achieved using circular plasmids or plasmids that are linearized prior to introduction into the host cell.

[0171] "Plasmids" used in genetic engineering are called "plasmid vectors". Many plasmids are commercially available for such use. The gene to be replicated is inserted into a copy of the plasmid, including a gene that makes the cell resistant to a particular antibiotic, and into the multiple cloning site (MCS or polylinker), a short region that contains several commonly used restriction sites and facilitates the insertion of DNA fragments at this location. Another major use of plasmids is to make large amounts of proteins. In this case, bacteria or eukaryotic cells containing the plasmid with the gene of interest are grown by the researcher, and these can be induced to produce large amounts of protein from the inserted gene.

[0172] In embodiments in which gene transfer is mediated by a DNA viral vector, such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that comprises, consists essentially of, or consists of the viral genome or a portion thereof and a transgene / exogenous polynucleotide sequence.

[0173] The term "tissue" is used herein to refer to tissue of a living or dead organism or any tissue derived from or designed to mimic a living or dead organism. Tissues may be healthy, diseased, and / or genetically mutated. Biological tissues may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues that make up an organ or part or region of the body of an organism. Tissues may comprise, consist essentially of, or consist of homogenous cellular material, or may be composite structures such as those found in regions of the body including the chest, which may include, for example, lung tissue, skeletal tissue, and / or muscle tissue. Exemplary tissues include, but are not limited to, tissues derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary system, duodenum, abdominal aorta, iliac vein, heart, and intestine, and may include any combination thereof.

[0174] All references cited herein are incorporated by reference in their entirety. EXAMPLES

[0175] The examples described in this section are provided for illustrative purposes only and are not intended to limit the invention in any way.

[0176] Example 1: Screening for miRNAs targeting human or mouse MAPT As a novel therapeutic approach for tauopathy, AAV delivery of an artificial miRNA shuttle was used to target tau mRNA, encoded by the MAPT gene. The antisense guide strand of the miRNA has perfect complementarity with tau mRNA over a 22-nt stretch of sequence, thereby directing the transcript towards the RNAi degradation pathway. Figure 1 shows a schematic of tau targeting using miRNA. Evidence supports that miRNA shuttles are more predictably processed, more effective and safer than shRNAs (Boudreau et al., Mol Ther 17, pp. 169-175, doi:10.1038 / mt.2008.231 (2009); Grimm et al., Nature 441, pp. 537-541, doi:10.1038 / nature04791 (2006)), and can be expressed in vivo from DNA-based delivery systems, such as viral vectors.

[0177] A panel of artificial miRNA shuttles based on mir-30 was designed to be specific for sequences common to all tau brain isoforms. Reference sequences for tau or mouse-specific miRNA shuttles were generated using an AppleScript program (using published constraints to predict miRNA shuttle sequences based on target genes input by the user). For resolution, mature guide strands were designed to specifically target only human tau (hTaui), mouse tau (mTaui), or both human and mouse tau (hmTaui) mRNAs. From the list generated by the program, promising candidates were selected based on species specificity and screening of potential off-target genes. Boudreau et al. (RNA Interference Techniques Neuromethods (ed. SQHarper) Ch. 2, 173 (Humana Press, 2011) ) The miRNA shuttle sequence was synthesized and cloned into the U6T6 plasmid using published protocols from.

[0178] To screen for target association, 10-20 candidates from each species-specific library were cloned into the PsiCheck2 plasmid, and dual luciferase reporter assays (DLRA) were performed according to standard practices. As shown in Table 2, the shuttle DNA sequences encoding the tested constructs are represented by SEQ ID NOs: 41-89, the corresponding RNA sequences are represented by SEQ ID NOs: 90-138, and the amiRNA sequences targeting MAPT are represented by SEQ ID NOs: 139-187.

[0179] [Table 2] JPEG2024527313000008.jpg133164

[0180] The results of the primary screen for miRNAs targeting human tau are shown in Figure 2, and the results of the primary screen for miRNAs targeting mouse tau are shown in Figure 5. The results of the primary screen for miRNAs targeting both human and mouse tau are shown in Figures 7A and 7B.

[0181] From this primary screen, the top three candidates were then tested in a secondary screen (in HEK293T cells, where the miRNA shuttle plasmid was co-transfected with plasmids expressing human or mouse cDNA). The results of the secondary screen for miRNAs targeting human tau are shown in Figure 3A-D, and the results of the secondary screen for miRNAs targeting mouse tau are shown in Figure 6A and Figure 6B.

[0182] Top candidates from the secondary screen were then cloned into AAV vectors for viral delivery. The first generation tau miRNA shuttle vectors have an hTaui or mTaui shuttle upstream of a GFP reporter transgene in the same cassette. This design allows expression of GFP protein in transduced cells, allowing assessment of viral biodistribution. Figure 4A and Figure 4B show vector expression and target association of AAV9 / hTau-i-GFP in P301S tau mice. In the second generation tau miRNA shuttle, the GFP coding sequence was removed to allow for use in humans.

[0183] Example 2: Study to evaluate efficacy of AAV9 / hTau5i when administered intracisternally in 3-month-old P301S tau mice This study was designed to evaluate the therapeutic benefit of AAV9 / hTau5i in the PS19 mouse model of tauopathy early in the disease course. The sequence of AAV9 / hTau5i is depicted in SEQ ID NO: 14. PS19 mice overexpress human P301S mutant tau at levels 7-fold higher than endogenous mouse tau. By 8 months, this model develops neuronal loss and brain atrophy, primarily in the hippocampus, and spreading to other brain regions, such as the neocortex and entorhinal cortex. These mice develop widespread neurofibrillary tangle-like inclusions in the neocortex, hippocampus, brainstem, and spinal cord (Yoshiyama et al., Neuron. 2007 Feb. 1;53(3):337-51). Before histological methods reveal overt tau pathology, the brains of such mice show seeding activity (tau may propagate from one cell to another and along synaptically connected brain networks) (Holmes et al., Proc Natl Acad Sci USA. 2013 Aug 13;110(33):E3138-47). It has been shown that tau aggregates present in brain homogenates can induce further aggregation of tau, which is first detected at 1.5 months of age (Holmes et al., Proc Natl Acad Sci USA. 2014 Oct 14;111(41):E4376-85). Behaviorally, such mice develop motor disorders (associated with defects in nesting behavior and feeding difficulties) that progress to paralysis within 9-10 months. In mixed genetic background strains, more than 50% of PS19 mice die prematurely by 12 months of age. In this study, non-transgenic and P301S tau transgenic littermates were administered 6E+11vg AAV9 / hTau5i-GFP or vehicle intracisternally (ICM) per mouse at 3 months of age.AAV9 / hTau5i-GFP contains an AAV9 capsid packaged with a self-complementary AAV genome containing a mutant AAV2 inverted terminal repeat (ITR) with a deleted D element, a U6 promoter, an hTau5i microRNA shuttle sequence, a T6 termination signal, a CBh promoter, a GFP DNA coding sequence, a polyadenylation signal, and the wild-type AAV2 ITR.

[0184] method At 3 months of age, non-transgenic (wild type; WT) and P301S tau transgenic male and female littermates were ICM injected with 10 uL of either vehicle (WT: n=7, 4 females, 3 males; P301S: n=6, 3 females, 3 males) or 6E+11vg AAV9 / hTau5i-GFP (WT: n=8, 4 females, 4 males; P301S: n=7, 3 females, 4 males). Animals were observed and weighed three times per week for 4 weeks after dosing, then once per week. Mice were monitored weekly for clinical signs, adverse events, and death after treatment. At 3 months after dosing, animals were postmortem dissected for brain histology and biochemistry analysis. Biochemical analyses included qPCR analysis of human tau (MAPT) mRNA, enzyme-linked immunoassay (ELISA) analysis of total tau protein using tau5 antibody, and a seeding assay to assess the presence of pathological tau species.

[0185] result The safety of ICM-delivered 6E+11vg AAV9 / hTau5i-GFP is supported by survival data showing that there were no early deaths in any of the treatment groups prior to the planned 3-month post-dose time point (Figure 8). No outward signs of toxicity were noted throughout the study period. To assess the overall health of the animals, weight change was monitored after injection. Figures 9A and 9B show that there was no significant difference in weight change in AAV9 / hTau5i-GFP-treated animals compared to vehicle-treated animals of the same genotype after injection. Additionally, there was no significant difference in weight gain between vehicle-treated WT and P301S mice at this age (Figures 9A and 9B). Overall, the survival and weight data support that the ICM-delivered 6E+11vg AAV9 / hTau5i-GFP dose was well tolerated in WT and P301S tau littermates up to 3 months after treatment.

[0186] Tissues were evaluated histologically and biochemically 3 months after injection. In FIG. 10, representative images of immunohistochemical staining using GFP antibody show that ICM injection of AAV9 / hTau5i-GFP results in vector distribution throughout the brain, while no positivity is observed in vehicle-treated mice. As shown in FIG. 11, a dose of 6E+11vg AAV9 / hTau5i-GFP delivered by ICM significantly reduced human tau mRNA levels in P301S tau mice compared to vehicle-treated P301S tau mice, although quantitative PCR analysis of brainstem tissue from treated mice confirmed target engagement (one-way ANOVA with Dunnett's multiple comparison test, **** p<0.0001). As expected, human tau mRNA detected in WT littermates was not significant (Figure 11, one-way ANOVA with Dunnett's multiple comparison test, ****p<0.0001). Figure 12 shows ELISA analysis of total tau levels in the brainstem was performed using a tau5 antibody that detects both mouse and human tau. WT littermates had significantly less total tau compared to P301S tau mice, which was unchanged when treated with AAV9 / hTau5i-GFP, confirming that hTau5i does not reduce mouse tau levels (Figure 12; one-way ANOVA with Dunnett's multiple comparison test). In contrast, treatment with AAV9 / hTau5i-GFP significantly reduced total tau levels in P301S tau mice in the brain regions closest to the delivery site (Figure 12; one-way ANOVA with Dunnett's multiple comparison test compared to P301S+vehicle), confirming that reduced human tau mRNA results in reduced tau protein.

[0187] To assess the potential benefit of reducing tau protein levels, we used a tau "seeding" assay, an in vitro method that measures the ability of pathological tau aggregates to induce misfolding of naive monomeric tau. In the seeding assay, HEK293 cells stably express the repeat domain of tau fused to cyan or yellow fluorescent protein, which creates Förster resonance energy transfer (FRET) when the cells are treated with pathological tau to induce aggregate formation. Figure 13 shows the results of a seeding assay in which transfection of brain homogenates from WT mice treated with vehicle or AAV9 / hTau5i-GFP resulted in the absence of FRET-positive inclusions, and addition of brain lysates from the P301S-treated cohort induced intracellular FRET-positive aggregates (one-way ANOVA, Dunnett's multiple comparison test compared to P301S+vehicle). Importantly, treatment with 6E+11vg AAV9 / hTau5i-GFP at 3 months of age prevented tau aggregate formation when assessed in the brainstem, cerebellum, and cortex at 6 months of age (Figure 13, one-way ANOVA, Dunnett's multiple comparison test compared to P301S+vehicle). Pathological tau was reduced in the cortex, a brain region not significantly targeted by viral vectors via this delivery route (Figure 10 and Figure 12C), suggesting that distal brain regions may benefit from reduced tau levels within connected neural networks. Collectively, this data supports that AAV9 delivery of the hTau5i microRNA shuttle early in the disease process can selectively reduce human tau mRNA, reduce tau protein levels, and inhibit pathogenic tau formation in the brain.

[0188] Example 3: Study to evaluate the efficacy of AAV9 / hTau5i when administered intracisternally in 6-month-old P301S tau mice This study was designed to evaluate the therapeutic benefit of AAV9 / hTau5i in the PS19 mouse model of tauopathy when administered at intermediate disease progression. In this study, non-transgenic and P301S tau transgenic littermates were injected ICM with vehicle or AAV9 / hTau5i-GFP, AAV9 / hTau5i or AAV9 / Scr at 6E+11vg per mouse at 6 months of age. AAV9 / hTau5i contains AAV9 capsids packaged with a self-complementary AAV genome containing mutant AAV2 ITRs with the D element deleted, U6 promoter, hTau5i microRNA shuttle sequence, T6 termination signal, CBh promoter, polyadenylation signal, and wild-type AAV2 ITRs. AAV9 / Scr contains an AAV9 capsid packaged with a self-complementary AAV genome containing a mutant AAV2 ITR with the D element deleted, a U6 promoter, a scrambled / non-specific microRNA shuttle sequence, a T6 termination signal, a CBh promoter, a polyadenylation signal, and a wild-type AAV2 ITR. An additional purpose of this design was to perform bridging studies between the AAV9 / hTau5i-GFP vector, designed for preclinical trials, and the AAV9 / hTau5i vector, which can be directly transferred to clinical use without modifying its design. The AAV9 / Scr vector is an additional control for viral vector delivery and microRNA expression.

[0189] method At 6 months of age, WT and P301S tau transgenic male and female littermates were ICM injected with 10 uL of vehicle (WT: n=12, 6 females, 6 males; P301S: n=11, 6 females, 5 males), 6E+11vg AAV9 / Scr (WT: n=12, 6 females, 6 males; P301S: n=10, 5 females, 5 males), 6E+11vg AAV9 / hTau5i-GFP (P301S: n=11, 6 females, 5 males), or 6E+11vg AAV9 / hTau5i (P301S: n=11, 6 females, 5 males). Animals were observed and weighed 3 times per week for 4 weeks after injection, then once per week. Mice were monitored weekly for clinical signs, adverse events, and deaths after treatment. Three months after treatment, animals were post-mortem examined.

[0190] result As shown in FIG. 14, no early deaths were observed in WT mice injected with vehicle or AAV9 / Scr vectors prior to the scheduled 3-month post-dosing time point. In P301S mice, one early death was observed in each of the four P301S treatment groups (FIG. 14). Overall, the survival data confirm that ICM delivery of AAV9 / Scr, AAV9 / hTau5i-GFP, or AAV9 / hTau5i is not detrimental to the survival of WT or P301S mice when treated at 6 months of age. No external signs of toxicity were noted throughout the study period. To assess the overall health of the animals, weight changes after injection were monitored. As shown in FIG. 15, there was no significant difference in weight changes between WT and P301S mice at this age. Additionally, no significant differences were observed in the body weight of mice treated with AAV9 / Scr, AAV9 / hTau5i, or AAV9 / hTau5i-GFP, demonstrating that all vectors were well tolerated in 6-month-old P301S tau mice up to 3 months after treatment (Figure 15). Seeding assays were performed using brainstem lysates from treated mice. Treatment with AAV9 / Scr did not alter seeding activity in WT mice (Figure 16A) or P301S mice (Figure 16B) compared to vehicle-treated animals. Figure 16C shows that P301S mice treated with AAV9 / hTau5i-GFP or AAV9 / hTau5i had comparable seeding levels, confirming that the two vectors have similar activity. Treatment with 6E+11vg AAV9 / hTau5 at 6 months of age prevented tau aggregate formation in the brainstem compared to P301S mice treated with AAV9 / Scr (FIG. 16D), unpaired Student's t-test.

[0191] Example 4: Study to evaluate the efficacy of AAV9 / hTau5i when administered intracisternally to 9-month-old P301S tau mice This study was designed to evaluate the therapeutic benefit of AAV9 / hTau5i administered late during disease progression in the PS19 mouse model of tauopathy, in which non-transgenic and P301S tau transgenic littermates received ICM administration of 6E+11vg AAV9 / hTau5i or AAV9 / Scr at 9 months of age.

[0192] method At 9 months of age, WT and P301S tau transgenic male and female littermates were ICM injected with 10 μL of 6E+11vg AAV9 / Scr (WT: n=20, 10 females, 10 males; P301S: n=23, 15 females, 8 males) or 6E+11vg AAV9 / hTau5i (P301S: n=18, 11 females, 7 males). Animals were observed and weighed three times per week for 4 weeks after dosing, then once per week. Mice were monitored weekly for clinical signs, adverse events, and deaths after treatment. Three months after dosing, animals were postmortem dissected.

[0193] result As shown in FIG. 17, all animals survived to the study endpoint compared to AAV9 / Scr injected WT littermates, and the survival rate of AAV9 / Scr injected P301S mice was significantly lower, with only 39% surviving to the study endpoint (Mantel-Cox, **** p<0.0001). Despite treatment at a later age, ICM injection of 6E+11vg AAV9 / hTau5i significantly extended survival of P301S mice by 72% at the study endpoint (Figure 17, Mantel-Cox, * p<0.05). To assess the overall health of the animals, body weight change after injection was monitored. As shown in FIG. 18, at this late stage of disease progression, body weight change in AAV9 / Scr-treated P301S male and female mice was significantly reduced compared to AAV9 / ScrWT mice (two-way ANOVA, Sidak's multiple comparison test, ***p<0.001). Treatment with AAV9 / hTau5i significantly slowed weight loss in P301S males and had no effect on weight loss in P301S females compared with AAV9 / Scr-treated P301S sex-matched mice (Figure 18; two-way ANOVA, Sidak's multiple comparison test, * p<0.05). Overall, this in-life data supports the potential treatment benefit of AAV9 / hTau5i for tauopathies, even when provided late in the disease course.

[0194] Example 5: Study to evaluate the safety of AAV9 / hTau5i when administered intracisternally in 3-month-old WT C57BL / 6J mice This study was designed to evaluate the safety of lowering endogenous tau levels in mice, in which WT C57BL / 6J mice were administered ICM vehicle, 4.7E+11vg AAV9 / mTau2i, or AAV9 / Scr at 3 months of age.

[0195] method At 3 months of age, male and female littermates of WT C57BL / 6J mice were ICM injected with 10 μL of vehicle (n=30, 15 females, 5 males), 4.7E+11vg AAV9 / Scr (n=30, 15 females, 15 males), or 4.7E+11vg AAV9 / mTau2i (n=30, 15 females, 15 males). After treatment, mice were monitored weekly for clinical signs, adverse events, and death. An intermediate group of animals (n=10, 5 females, 5 males per treatment group) was harvested 1 month post-injection for clinical blood chemistry and histopathology analysis. The remaining animals will undergo behavioral testing 1 month post-treatment and will be aged to the study endpoint (12 months post-treatment).

[0196] result To date, no differences in clinical signs or mortality have been found between treatment groups. Tissues from the intermediate group were histologically normal and are shown in FIG. 19. Groups aged to the study endpoint underwent behavioral assessments for motor learning and coordination (rotarod), general activity (open field), and anxiety (elevated plus maze) one month after injection. FIG. 20 shows that knockdown of endogenous tau in the AAV9 / mTau2i-treated group did not result in significant changes in behavior in these three tasks compared to vehicle and AAV9 / Scr-treated mice. Overall, these data support that knockdown of endogenous tau was well tolerated in the short term.

Claims

1. In the 5' to 3' direction, a. a first AAV2 ITR sequence; b. a mouse U6 promoter sequence; c. a polynucleotide sequence encoding at least one amiRNA targeting MAPT; d. a termination sequence; e. a CBh promoter sequence; f. a synthetic polyA sequence; g. a second AAV2 ITR sequence and comprising a recombinant adeno-associated virus (rAAV) vector comprising the sequence represented by SEQ ID NO:

14.

2. An rAAV viral vector comprising (a) an AAV capsid protein and (b) the rAAV vector according to claim 1.

3. The rAAV viral vector according to claim 2, wherein the AAV capsid protein is an AAV9 capsid protein.

4. A pharmaceutical composition comprising the rAAV vector according to claim 1, or the rAAV viral vector according to claim 2 or 3, and at least one pharmaceutically acceptable excipient and / or additive.

5. The rAAV vector according to claim 1, the rAAV viral vector according to claim 2 or 3, or the pharmaceutical composition according to claim 4, for use in the treatment of tauopathy.

6. The rAAV vector for use according to claim 5, the rAAV viral vector for use according to claim 5, or the pharmaceutical composition for use according to claim 5, wherein the tauopathy is Alzheimer's disease or frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17).

7. The rAAV vector for use according to claim 5 or 6, the rAAV viral vector for use according to claim 5 or 6, or the pharmaceutical composition for use according to claim 5 or 6, wherein the tauopathy is associated with one or more mutations within the MAPT gene. ​ ​ ​

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