Gene therapy for frontotemporal dementia

Abstract

The present disclosure describes improved vectors, such as adeno-associated virus (AAV) vectors, for expressing pregranulin and variants thereof in transduced cells, and the use of such vectors for increasing the amount of pregranulin in subjects in which pregranulin deficiency occurs, such as certain subjects suffering from frontotemporal dementia (FTD) or frontotemporal degeneration (FTLD).

Description

Background Art

[0001] Frontotemporal dementia (FTD) refers to a clinical syndrome characterized by progressively worsening deficits in language, behavior, and executive function associated with selective neurodegeneration of the frontal and temporal cortical lobes (frontotemporal lobar degeneration or FTLD), rather than the more general neurodegeneration seen in Alzheimer's disease and certain other dementias. Patients with FTD typically develop FTD in their prime, with symptom onset most often occurring between the ages of 45 and 64. Symptoms then rapidly progress, leading to functional decline and ultimately death within an average of eight years. The only treatments for FTD focus on managing the effects of the inevitable behavioral changes, but these treatments are ineffective in slowing or reversing the underlying lobar neurodegeneration that leads to FTD. Given this significant unmet medical need, there is a need in the art for methods to effectively treat or prevent frontotemporal lobar degeneration and the resulting neurological deficits and eventual death. Summary of the Invention

[0002] To address this need in the art, the present disclosure provides improved adeno-associated virus (AAV) vectors for expressing human progranulin (PGRN) polypeptides or variants thereof, methods for producing such AAV vectors, and methods for using such AAV vectors to prevent or treat diseases or disorders in subjects characterized by a deficiency in the amount of human PGRN or a variant thereof, including but not limited to frontotemporal lobar degeneration (FTLD) and frontotemporal dementia (FTD).

[0003] Certain enumerated non-limiting embodiments (E) of the present disclosure are listed below. These and related embodiments are further described in detail in the detailed description (including the examples and figures). Those skilled in the art will recognize or will be able to ascertain equivalents to the specific embodiments described herein using only routine experimentation.

[0004] E1. A recombinant adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a human progranulin (PGRN) polypeptide or a variant thereof.

[0005] E2. An AAV vector according to E1, wherein the PGRN polypeptide variant is a carboxyl-terminal truncation variant lacking one or more amino acids originally present in the full-length wild-type human PGRN polypeptide, such that the binding of the variant PGRN polypeptide to the human sortilin receptor (SORT1) protein is reduced compared to the full-length wild-type human PGRN polypeptide.

[0006] E3. The AAV vector of E2, wherein the PGRN polypeptide variant lacks the last three carboxyl-terminal amino acids present in the full-length wild-type human PGRN polypeptide.

[0007] E4. The AAV vector according to E1 to E3, wherein the amino acid sequence of the PGRN polypeptide variant comprises, essentially consists of, or consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16.

[0008] E5. The AAV vector according to E1 to E4, wherein the nucleotide sequence encoding the PGRN polypeptide or a variant thereof is a wild-type nucleotide sequence.

[0009] E6. The AAV vector according to E1 to E4, wherein the nucleotide sequence encoding the PGRN polypeptide or a variant thereof is a codon-optimized nucleotide sequence.

[0010] E7. The AAV vector of E6, wherein the codon-optimized nucleotide sequence has a reduced number of CpG dinucleotides compared to the wild-type nucleotide sequence encoding the PGRN polypeptide or variant thereof.

[0011] E8. An AAV vector according to E7, wherein the nucleotide sequence encoding the PGRN polypeptide or its variant has 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 fewer CpG dinucleotides compared to the wild-type nucleotide sequence encoding the PGRN polypeptide or its variant.

[0012] E9. The AAV vector according to E7 to E8, wherein the wild-type nucleotide sequence encoding the PGRN polypeptide or its variant consists of the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 15.

[0013] E10. The AAV vector according to E7, wherein the nucleotide sequence encoding the PGRN polypeptide or variant thereof does not contain any CpG dinucleotides.

[0014] E11. An AAV vector according to E1 to E10, wherein the nucleotide sequence encoding the PGRN polypeptide or a variant thereof is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:15.

[0015] E12. The AAV vector according to E1 to E5, wherein the nucleotide sequence encoding the PGRN polypeptide or a variant thereof is identical to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 15.

[0016] E13. The AAV vector according to E1 to E12, wherein the genome comprises at least one AAV inverted terminal repeat (ITR).

[0017] E14. The AAV vector according to E13, wherein the nucleotide sequence of the ITR is wild type.

[0018] E15. The AAV vector according to E13, wherein the nucleotide sequence of the ITR is modified.

[0019] E16. The AAV vector according to E15, wherein the nucleotide sequence of the ITR is modified to reduce or eliminate the ability of the ITR to undergo terminal dissociation.

[0020] E17. The AAV vector according to E15, wherein the nucleotide sequence of the ITR is modified to inactivate the terminal dissociation site.

[0021] E18. The AAV vector according to E15, wherein the nucleotide sequence of the ITR is modified to reduce or eliminate the ability of the ITR to support packaging into capsids.

[0022] E19. The AAV vector according to E15, wherein the nucleotide sequence of the ITR is modified to inactivate the D region.

[0023] E20. The AAV vector according to E13 to E14, wherein the ITR is an AAV2 ITR.

[0024] E21. The AAV vector of E20, wherein the AAV2 ITR is truncated.

[0025] E22. An AAV vector according to E13, wherein the ITR comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, or SEQ ID NO:21, or the complementary sequence or reverse complementary sequence of each of said sequences.

[0026] E23. The AAV vector of E13 to E14, wherein the ITR is not an AAV2 ITR.

[0027] E24. The AAV vector according to E1 to E23, wherein the vector comprises a first AAV ITR located at its 5' end and a second AAV ITR located at its 3' end.

[0028] E25. The AAV vector of E24, wherein the vector further comprises a third AAV ITR.

[0029] E26. The AAV vector of E25, wherein the third ITR is modified to inactivate the terminal dissociation site.

[0030] E27. The AAV vector according to E1 to E26, wherein the vector further comprises a transcription control region operably linked to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0031] E28. The AAV vector of E27, wherein the transcription control region is constitutive.

[0032] E29. The AAV vector of E27, wherein the transcription control region is inducible.

[0033] E30. The AAV vector of E27, wherein the transcription control region is tissue or cell type specific.

[0034] E31. The AAV vector according to E30, wherein the transcription control region is brain tissue-specific or neuronal cell-specific.

[0035] E32. The AAV vector of E30, wherein the transcriptional control region is more transcriptionally active in CNS neurons than in hepatocytes.

[0036] E33. The AAV vector of E27 to E32, wherein the transcription control region comprises a promoter sequence.

[0037] E34. An AAV vector according to E33, wherein the transcription control region further comprises an enhancer sequence.

[0038] E35. An AAV vector according to E34, wherein the enhancer sequence is located 5' of the promoter.

[0039] E36. An AAV vector according to E34, wherein the enhancer sequence is located 3' to the promoter.

[0040] E37. The AAV vector according to E33, wherein the promoter sequence is brain tissue-specific or neuronal cell-specific.

[0041] E38. An AAV vector according to E34, wherein the enhancer sequence is brain tissue-specific or neuronal cell-specific.

[0042] E39. The AAV vector according to E34, wherein the promoter sequence and the enhancer sequence are each brain tissue-specific or neuronal cell-specific.

[0043] E40. The AAV vector according to E33, wherein the promoter sequence and / or the enhancer sequence are derived from the synaptophysin gene.

[0044] E41. The AAV vector according to E34, wherein the promoter sequence and / or the enhancer sequence are derived from a synaptophysin gene, the synaptophysin gene being selected from the group consisting of a mammalian synaptophysin gene, a mouse synaptophysin gene, a rat synaptophysin gene, a primate synaptophysin gene, a monkey synaptophysin gene, a chimpanzee synaptophysin gene, and a human synaptophysin 1 gene (SYN1).

[0045] E42. An AAV vector according to E41, wherein the promoter sequence comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO: 6 or a promoter function subsequence, modification or variant thereof.

[0046] E43. An AAV vector according to E41, wherein the enhancer sequence comprises, consists essentially of or consists of the nucleotide sequence of SEQ ID NO: 6 or an enhancer function subsequence, modification or variant thereof, respectively.

[0047] E44. The AAV vector according to E1 to E43, wherein the vector further comprises a 5' untranslated region (UTR) sequence.

[0048] E45. An AAV vector according to E44, wherein the 5'UTR sequence is located 3' to the promoter sequence and / or the enhancer sequence and 5' to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0049] E46. The vector according to E44 to E45, wherein the 5'UTR sequence is derived from the synaptophysin gene.

[0050] E47. The vector according to E46, wherein the 5'UTR sequence is derived from a synaptophysin gene, wherein the synaptophysin gene is selected from the group consisting of a mammalian synaptophysin gene, a mouse synaptophysin gene, a rat synaptophysin gene, a primate synaptophysin gene, a monkey synaptophysin gene, a chimpanzee synaptophysin gene and a human synaptophysin 1 gene (SYN1).

[0051] E48. The AAV vector according to E48, wherein the 5'UTR sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 7, or a 5'UTR functional subsequence, modification, or variant thereof.

[0052] E49. The AAV vector according to E1 to E48, wherein the vector further comprises a transcription termination signal sequence.

[0053] E50. The AAV vector according to E49, wherein the transcription termination signal sequence is a polyadenylation (poly(A)) signal sequence.

[0054] E51. The AAV vector according to E50, wherein the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene.

[0055] E52. An AAV vector according to E51, wherein the transcription termination signal sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a transcription termination signal functional subsequence, modification, or variant thereof.

[0056] E53. The AAV vector of E1 to E52, wherein the vector further comprises an intron sequence.

[0057] E54. An AAV vector according to E53, wherein the intron sequence is located within the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and interrupts the nucleotide sequence.

[0058] E55. The AAV vector according to E53, wherein the intron sequence does not interrupt the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0059] E56. An AAV vector according to E55, wherein the intron sequence is located 5' of the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0060] E57. An AAV vector according to E56, wherein the intron sequence is located 3' of the promoter and 5' of the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0061] E58. The AAV vector of E1 to E57, wherein the vector further comprises a post-transcriptional regulatory element (PRE) sequence.

[0062] E59. An AAV vector according to E58, wherein the PRE sequence is located 3' to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and 5' to the transcription termination signal sequence.

[0063] E60. The AAV vector according to E58 to E59, wherein the PRE sequence is a WPRE or HPRE sequence.

[0064] E61. The AAV vector of E1 to E60, wherein the vector further comprises a binding site for a microRNA (miRNA).

[0065] E62. An AAV vector according to E61, wherein the miRNA binding site is located 3' of the nucleotide sequence encoding the PGRN polypeptide or its variant and 5' of the transcription termination signal sequence.

[0066] E63. The AAV vector of E1 to E62, wherein the vector further comprises a stuffer or filler nucleotide sequence of sufficient length such that the entire length of the AAV vector including the ITRs is approximately 3.5 to 5.0 kilobases.

[0067] E64. An AAV vector according to E63, wherein the plug or stuffer nucleotide sequence is derived from a TATA binding protein (TBP) gene.

[0068] E65. An AAV vector according to E64, wherein the stuffing or filler nucleotide sequence is an intron of the human TBP gene or a subsequence, modification or variant thereof.

[0069] E66. The AAV vector according to E65, wherein the plug or filler nucleotide sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 11.

[0070] E67. The AAV vector according to E1 to E66, wherein the vector comprises a first AAV ITR, a transcription control region operably linked to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof, a transcription termination signal sequence and a second AAV ITR.

[0071] E68. The AAV vector according to E67, wherein the vector comprises, in 5' to 3' order, the first AAV ITR, the transcription control region operably linked to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof, the transcription termination signal sequence, and the second AAV ITR.

[0072] E69. An AAV vector according to E68, wherein the transcription control region comprises a promoter located 5' of the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and an enhancer located 5' of the promoter.

[0073] E70. The AAV vector according to E67 to E69, wherein the vector further comprises an intron located between the promoter and the nucleotide sequence encoding the PGRN polypeptide or a variant thereof.

[0074] E71. The AAV vector according to E68 to E70, wherein the vector further comprises an intron located within the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and interrupting the nucleotide sequence.

[0075] E72. The AAV vector of E68 to E71, wherein the vector further comprises a PRE located between the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and the poly(A) signal sequence.

[0076] E73. The AAV vector of E68 to E72, wherein the first AAV ITR is located at the 5' end of the vector and the second AAV ITR is located at the 3' end of the vector.

[0077] E74. The AAV vector according to E73, wherein the vector further comprises a third AAV ITR located between the first AAV ITR and the second AAV ITR.

[0078] E75. The AAV vector according to E74, wherein the terminal dissociation site of the third AAV ITR is inactivated.

[0079] E76. The AAV vector of E68 to E75, wherein the transcription control region is specific to brain tissue or neuronal cells.

[0080] E77. An AAV vector according to E76, wherein the transcription control region comprises a promoter sequence and / or an enhancer sequence derived from a synaptophysin gene, wherein the synaptophysin gene is selected from the group consisting of a mammalian synaptophysin gene, a mouse synaptophysin gene, a rat synaptophysin gene, a primate synaptophysin gene, a monkey synaptophysin gene, a chimpanzee synaptophysin gene and a human synaptophysin 1 gene (SYN1).

[0081] E78. The AAV vector according to E77, wherein the transcription control region comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6 or a transcription control region functional subsequence, modification, or variant thereof.

[0082] E79. The AAV vector according to E67 to E78, wherein the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene.

[0083] E80. An AAV vector according to E79, wherein the transcription termination signal sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a transcription termination signal functional subsequence, modification, or variant thereof.

[0084] E81. The AAV vector according to E67 to E80, further comprising a modified human TBP gene intron sequence located 3' to the transcription termination signal sequence and 5' to the second AAV ITR.

[0085] E82. The AAV vector of E1 to E5, wherein the vector comprises, in 5' to 3' order:

[0086] (a) First AAV2 ITR,

[0087] (b) the promoter sequence from the synaptophysin gene,

[0088] (c) 5'UTR sequence from the synaptophysin gene,

[0089] (d) a nucleotide sequence encoding a human PGRN polypeptide or a variant thereof operably linked to the promoter sequence,

[0090] (e) a transcription termination signal sequence from the bovine growth hormone (bGH) gene,

[0091] (f) sequences from an intron of the TBP gene, and

[0092] (g) Second AAV2 ITR.

[0093] E83. An AAV vector according to E82, wherein the promoter sequence from the synaptophysin gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:6; the 5'UTR sequence from the synaptophysin gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:7; the nucleotide sequence encoding the PGRN polypeptide or a variant thereof comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:15; the transcription termination signal sequence from the bovine growth hormone (bGH) gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10; and the sequence from the intron of the TBP gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:11.

[0094] E84. The AAV vector according to E82 to E83, wherein the first AAV2 ITR and the second AAV2 ITR each comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 21, or the complement or reverse complement of each of said sequences.

[0095] E85. The AAV vector according to E82 to E84, wherein the nucleotide sequence of the vector comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 19 or its reverse complement.

[0096] E86. The AAV vector of E1 to E85, wherein the vector is equal to or less than 5 kilobases in length.

[0097] E87. The AAV vector of E1 to E86, wherein the vector is equal to or less than 4 kilobases in length.

[0098] E88. An AAV vector comprising an AAV capsid and the AAV vector according to E1 to E87, wherein the vector is encapsidated by the capsid.

[0099] E89. An AAV vector according to E88, wherein the AAV capsid is at least partially neurotropic.

[0100] E90. The AAV vector according to E89, wherein the AAV capsid is capable of crossing the blood-brain barrier (BBB) ​​in non-human primates or humans.

[0101] E91. An AAV vector according to E90, wherein the AAV capsid crosses the BBB at least as efficiently or more efficiently than the AAV9 capsid.

[0102] E92. The AAV vector according to E89 to E90, wherein the AAV capsid is selected from the group consisting of: AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh.10, AAVv66, AAV-PHP.B, AAV-PHP.B / eB, AAV PHP.eB, AAV PHP.S, AAV-DJ, MNM008, MNM004, 9P31, 9P801, AAV-F, AAV-S, CAP-B10, CAP-B22, PHP.V1, AAV9-retro, T2 3Y+T+dH, AAV8 THR, AAV2.5, AAV-B1, AAV-AS, AAV-BR1, AAVSCH9, AAV4.18, AAV2-retro, AAV2 HBKO, AAV-TT and AAV-801.

[0103] E93. The AAV vector according to E89 to E92, wherein the AAV capsid is an AAV-801 capsid and comprises a VP3 protein consisting of the amino acid sequence of SEQ ID NO: 3.

[0104] E94. The AAV vector according to E93, wherein the AAV capsid further comprises a VP1 protein consisting of the amino acid sequence of SEQ ID NO: 1 or a VP2 protein consisting of the amino acid sequence of SEQ ID NO: 2.

[0105] E95. The AAV vector of E88 to E94, wherein the vector is a single-stranded DNA genome.

[0106] E96. The AAV vector of E88 to E94, wherein the vector is a self-complementary DNA genome.

[0107] E97. The AAV vector of E95 to E96, wherein the vector is of positive polarity.

[0108] E98. The AAV vector of E95 to E96, wherein the vector is of negative polarity.

[0109] E99. An AAV vector, comprising an AAV capsid encapsidating the AAV vector, wherein the AAV capsid is an AAV-801 capsid, and wherein the nucleotide sequence of the vector comprises or consists of the nucleotide sequence of SEQ ID NO: 17 or its reverse complement.

[0110] E100. A pharmaceutical composition comprising the AAV vector according to E88 to E100 and a pharmaceutically acceptable excipient.

[0111] E101. A method for preventing or treating a disease or disorder caused by a deficiency of human PGRN polypeptide in a human subject, the method comprising administering to the subject an amount of an AAV vector or composition according to E1 to E100 to effectively increase the amount of PGRN polypeptide or a variant thereof in at least one biological fluid, tissue or cell of the subject.

[0112] E102. A method according to E101, wherein the method effectively increases the amount of PGRN polypeptide or its variant in the cerebrospinal fluid (CSF) of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of endogenous PGRN polypeptide in the CSF of a healthy person, for example 6 ng / mL.

[0113] E103. A method according to E101, wherein the method effectively increases the amount of PGRN polypeptide or its variant in the brain of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of PGRN polypeptide in the brain of a healthy person.

[0114] E104. A method according to E101, wherein the method effectively increases the amount of PGRN polypeptide or its variant in the spinal cord of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of PGRN polypeptide in the spinal cord of a healthy person.

[0115] E105. A method according to E101, wherein the method effectively increases the amount of PGRN polypeptide or its variant in the serum of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of PGRN polypeptide in the serum of a healthy person.

[0116] E106. A method according to E101, wherein the method effectively increases the amount of bis(monoacylglycerol)phosphate (BMP) in the brain of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of BMP in the brain of a healthy person, wherein the BMP can be any kind of BMP, such as BMP 18:1 / 18:1 or BMP 22:6 / 22:6.

[0117] E107. The method according to E101, wherein the method effectively reduces the beta-hexosaminidase (HexA) enzyme activity in the brain of the subject to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the HexA enzyme activity before treatment.

[0118] E108. A method according to E101, wherein the method effectively reduces the β-galactosidase (β-Gal) enzyme activity in the brain of the subject to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the β-Gal enzyme activity before treatment.

[0119] E109. The method of E101, wherein the method effectively reduces TDP43 fragmentation in the brain of the subject to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of pre-treatment TDP43 fragmentation.

[0120] E110. A method according to E101, wherein the method is effective in reducing the level of lipofuscin in the subject's brain to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the level of lipofuscin before treatment.

[0121] E111. A method of reducing the frequency or severity of at least one symptom or sign caused by human PGRN polypeptide deficiency in a human subject, the method comprising administering to the subject an amount of an AAV vector or composition according to E1 to E100 effective to reduce the frequency or severity of such symptom or sign.

[0122] E112. The method according to E111, wherein the symptom or sign is characteristic of frontotemporal lobar degeneration (FTLD), including, for example, FTLD-TDP type A.

[0123] E113. The method according to E112, wherein the symptom or sign is atrophy of a brain region selected from the group consisting of: frontal lobe, anterior temporal lobe, medial temporal lobe, posterior temporal lobe, orbitofrontal cortex, anterior cingulate gyrus, inferior parietal lobe, striatum and thalamus.

[0124] E114. The method according to E112, wherein the symptom or sign is behavioral changes characteristic of behavioral variant frontotemporal dementia (BV-FTD).

[0125] E115. The method according to E112, wherein the symptom or sign is behavioral changes characteristic of nonfluent variant primary progressive aphasia (NFV-PPA).

[0126] E116. The method according to E114 or E115, wherein the behavioral changes are selected from the group consisting of word finding disorder, speech apraxia, agrammatism, verbal naming disorder, word comprehension disorder, phonological errors, word repetition errors, sentence repetition errors, sentence comprehension errors, surface alexia, delusions and hallucinations.

[0127] E117. The method according to E112, wherein the symptoms or signs are characteristic of parkinsonism or corticobasal syndrome (CBS).

[0128] E118. The method according to E101 to E117, wherein the subject is diagnosed with frontotemporal lobar degeneration (FTLD) or frontotemporal dementia (FTD).

[0129] E119. The method according to E101 to E118, wherein the PGRN polypeptide deficiency in the subject is caused by a homozygous or heterozygous mutation in the GRN gene encoding the PGRN polypeptide, which mutation reduces the amount or activity of the PGRN polypeptide relative to a healthy person.

[0130] E120. A method for preventing or treating frontotemporal dementia in a human subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of the AAV vector or composition according to E1 to E100, so as to effectively prevent or treat frontotemporal dementia in the subject.

[0131] E121. The method according to E101 to E120, wherein the effective amount of the AAV vector is in the range of 1×10 10 to 1×10 15 The dose was 100 mg of vehicle per kilogram (vg / kg) of subject body weight.

[0132] E122. The method according to E101 to E121, wherein the AAV vector or composition is administered intracerebroventricularly to the subject.

[0133] E123. The method according to E101 to E121, wherein the AAV vector or composition is administered intrathecally to the subject.

[0134] E124. The method according to E101 to E121, wherein the AAV vector or composition is administered intravenously to the subject.

[0135] E125. Use of the AAV vector according to E1 to E100 in the manufacture of a medicament for treating or preventing frontotemporal dementia in a human subject.

[0136] E126. A DNA plasmid comprising the nucleotide sequence of the AAV vector according to E1 to E87.

[0137] E127. A host cell for AAV vector production, the host cell comprising the DNA plasmid according to E126.

[0138] E128. The host cell according to E127, wherein the host cell is a HEK293 cell.

[0139] E129. The host cell according to E127 to E128, wherein the host cell further comprises a gene encoding an AAV Rep protein, such as a gene contained in a DNA plasmid.

[0140] E130. The host cell according to E127 to E129, wherein the host cell further comprises a gene encoding the AAV VP1 capsid protein, such as a gene contained in a DNA plasmid.

[0141] E131. The host cell of E127 to E130, wherein the host cell further comprises a gene encoding a viral accessory factor, such as a gene contained in a DNA plasmid.

[0142] E132. A method for preparing an AAV vector, the method comprising: incubating the host cell according to E131 under conditions sufficient to allow production of the AAV vector, and purifying the AAV vector thus produced.

[0143] E133. An AAV vector produced by the method according to E132. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1Biacore sensorgrams of human His-tagged full-length PGRN and PGRNΔ3 proteins binding to immobilized human sortilin receptor (SORT1) and human prosaposin (PSAP). Panel A shows binding of full-length PGRN to SORT1. Panel B shows binding of full-length PGRN and PGRNΔ3 to PSAP (top and bottom, respectively), while panel C shows similar binding to PSAP from an alternative source.

[0145] Figure 2A Levels of mRNA encoding human progranulin in motor neurons differentiated from human iPSC cells transduced with AAVDJ vectors expressing human full-length and Δ3-truncated PGRN proteins.

[0146] Figure 2B Progranulin levels were measured in the conditioned medium of motor neurons differentiated from human iPSC cells transduced with AAVDJ vectors expressing human full-length and Δ3-truncated PGRN proteins.

[0147] Figure 3A Levels of mRNA encoding human progranulin in cortical glutamatergic neurons differentiated from human iPSC cells transduced with AAV6 vectors expressing human full-length and Δ3-truncated PGRN proteins.

[0148] Figure 3B Progranulin levels measured in conditioned medium from cortical glutamatergic neurons differentiated from human iPSCs transduced with AAV6 vectors expressing full-length and Δ3-truncated human PGRN proteins. Also shown are results from related experiments in which glutamatergic neurons were differentiated from iPSCs from human FTD patients and then transduced with AAV6 vectors expressing Δ3-truncated human PGRN proteins.

[0149] Figure 4 Concentration of human PGRNΔ3 protein released into the culture medium by glutamatergic neurons derived from iPS cells following transduction with the AAV801-PGRNΔ3 vector, compared with untreated control cells and neurons transduced with the control vector AAV801-Luc. Statistical analysis was performed using one-way analysis of variance followed by post hoc analysis with Dunnett's multiple comparison test. P value ****: <0.0001.

[0150] Figure 5β-Hexosaminidase enzyme activity in lysates of iPSC cells differentiated from glutamatergic neurons. Left, results for control neurons from untreated healthy iPSC cells and those transduced with the control vector AAV801-Luc and the vector AAV801-PGRNΔ expressing the GRN transgene. Right, results for neurons from untreated FTD iPSC cells and those transduced with AAV801-Luc and AAV801-PGRNΔ vectors. Statistical analysis was performed using a two-way ANOVA followed by Post hoc analysis was performed using a multiple comparison test. P values: ***: = < 0.0001; ***: = 0.0004; ns: not significant.

[0151] Figure 6A . Transduction efficiency of AAV9 vectors expressing human full-length and Δ3-truncated PGRN in brain tissue administered ICV to neonatal mice.

[0152] Figure 6B Progranulin levels measured in CSF from neonatal mice 4 weeks after administration of AAV9 vectors expressing human full-length and Δ3-truncated PGRN.

[0153] Figure 6C Progranulin levels were measured in CSF from newborn mice 4 weeks after administration of AAV9 vectors expressing human full-length and Δ3-truncated PGRN. Data were normalized to the amount of endogenous mouse PGRN in the samples.

[0154] Figure 6D Progranulin levels measured in serum from newborn mice 4 weeks after administration of AAV9 vectors expressing human full-length and Δ3-truncated PGRN.

[0155] Figure 7A Progranulin detected by immunohistochemistry in brain sections from newborn mice 4 weeks after administration of a negative control AAV9 vector expressing green fluorescent protein.

[0156] Figure 7B Progranulin detected by immunohistochemistry in brain sections from newborn mice 4 weeks after administration of an AAV9 vector expressing human full-length PGRN.

[0157] Figure 7C Progranulin detected by immunohistochemistry in brain sections from newborn mice 4 weeks after administration of an AAV9 vector expressing human Δ3-truncated PGRN.

[0158] Figure 8ATransduction efficiency of AAV1, AAVDJ, and AAV9 vectors expressing human full-length and Δ3-truncated PGRN in brain tissue administered ICV to 6-month-old mice.

[0159] Figure 8B Progranulin levels were measured in CSF from mice 3 months after administration of AAV1, AAVDJ, and AAV9 vectors expressing human full-length and Δ3-truncated PGRN when the mice were 6 months old.

[0160] Figure 8C Progranulin levels were measured in CSF from mice 6 months after administration of AAV9 vectors expressing human full-length and Δ3-truncated PGRN when the mice were 6 months old.

[0161] Figure 9A . Transduction efficiency of AAVPHP.B vectors expressing human full-length and Δ3-truncated PGRN in brain tissue administered intravenously to 6-week-old mice.

[0162] Figure 9B Progranulin levels were measured in brain tissue from 6-week-old mice 4 weeks after administration of AAVPHP.B vectors expressing human full-length and Δ3-truncated PGRN.

[0163] Figure 9C Progranulin levels were measured in CSF from mice 4 weeks after administration of AAVPHP.B vectors expressing human full-length and Δ3-truncated PGRN when the mice were 6 weeks old.

[0164] Figure 10 . Micrographs of the hippocampus (including CA3) from mice 3 months after administration of a negative control AAV9 vector expressing green fluorescent protein when the test animals were 6 months old. The upper left micrograph shows negative staining for human progranulin. The lower left micrograph shows negative staining for endogenous mouse Iba-1 (a marker of microglial activation). The right micrograph shows intact tissue cell structure by H&E staining.

[0165] Figure 11 . Micrographs of the hippocampus (including CA3) from mice 3 months after administration of an AAV9 vector of human full-length PGRN when the test animals were 6 months old. The upper left micrograph shows positive and focal staining for human progranulin. The lower left micrograph shows positive staining for endogenous mouse Iba-1. The right micrograph shows reduced cellularity by H&E staining.

[0166] Figure 12Photomicrographs of the hippocampus (including CA3) from mice 3 months after administration of an AAV9 vector of human Δ3-truncated PGRN when the test animals were 6 months old. The upper left photomicrograph shows positive and diffuse staining for human progranulin. The lower left photomicrograph shows no or minimal staining for endogenous mouse Iba-1. The right photomicrograph shows normal cellularity by H&E staining.

[0167] Figure 13A . From Grn administered with 1e11 vg AAV9-PGRNΔ3 vector via intracerebroventricular (ICV) delivery to one hemisphere - / - The number of vector copies (VGC) in liver samples harvested from KO mice. Age-matched wild-type mice and Grn mice were administered PBS by the same route. - / - KO mice were used as controls. VGC numbers were normalized to the amount of cellular genomic DNA (μg gDNA).

[0168] Figure 13B . From Grn administered 1e11 vg AAV9-PGRNΔ3 vector to one hemisphere via ICV delivery route - / - The number of vector copies (VGC) in brain samples harvested from KO mice. Age-matched wild-type mice and Grn mice were administered PBS by the same route. - / - KO mice were used as controls. VGC numbers were normalized to the amount of genomic DNA in the cells (μg gDNA). Pink dots represent results from brain samples harvested from the injected hemisphere, and black dots represent data from the contralateral hemisphere.

[0169] Figure 13C . From Grn administered 1e11 vg AAV9-PGRNΔ3 vector to one hemisphere via ICV delivery route - / - hGRNΔ3 mRNA levels in brain samples harvested from KO mice. - / - KO mice were used as controls. RNA levels were normalized to the amount of RNA expressed from housekeeping genes. Pink dots represent results from brain samples harvested from the injected hemisphere, and black dots represent data from the contralateral hemisphere.

[0170] Figures 13D to 13F .Grn from 1e11 vg AAV9-PGRNΔ3 vector administered via ICV delivery into one hemisphere - / - PGRNΔ3 protein concentrations in fluid and tissue samples of KO mice. Age-matched wild-type mice and PGRNΔ3 mice were administered PBS by the same route. - / -KO mice served as controls. Pink dots represent results from fluid and tissue samples taken from the injected hemisphere, and black dots represent data from the contralateral hemisphere. Figure 13D Results for cerebrospinal fluid (CSF) samples are shown. Figure 13E Results for serum samples are shown. Figure 13F Results for brain tissue samples are shown.

[0171] FIG. 14A to FIG. 14B . From Grn administered 1e11 vg AAV9-PGRNΔ3 vector to one hemisphere via ICV delivery route - / - BMP 18:1 / 18:1 species in brain samples harvested from KO mice ( Figure 14A ) and BMP 22:6 / 22:6 types ( Figure 14B ) concentration. Age-matched wild-type mice and Grn mice were administered PBS by the same route. - / - KO mice served as controls. Pink dots represent results from brain samples harvested from the injected hemisphere, and black dots represent data from the contralateral hemisphere. Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P values: ****: <0.0001; **: <0.01, *: <0.05; ns: not significant.

[0172] FIG. 15A to FIG. 15B . From Grn administered 1e11 vg AAV9-PGRNΔ3 vector to one hemisphere via ICV delivery route - / - β-Hexosaminidase ( Figure 15A ) and β-galactosidase ( Figure 15B ) enzyme activity. Age-matched wild-type mice and Grn mice were administered PBS by the same route. - / - KO mice served as controls. Pink dots represent results from brain samples harvested from the injected hemisphere, and black dots represent data from the contralateral hemisphere. Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P values: ****: <0.0001; **: <0.01; ns: not significant.

[0173] Figure 16 .Grn from 1e11 vg AAV9-PGRNΔ3 vector administered via ICV delivery into one hemisphere - / - KO mice and age-matched wild-type control mice and Grn - / -TDP43 fragmentation in brain samples from KO mice. Fragmentation was estimated using semiquantitative Western blot analysis as the ratio of the staining intensity of the full-length TDP43 protein to its 20 kDa fragment, with a lower ratio indicating greater fragmentation. Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P value*: <0.05; ns: not significant.

[0174] 17A to 17E . From Grn administered 1e11 vg AAV9-PGRNΔ3 vector via ICV delivery to one right hemisphere - / - Quantification of lipofuscin accumulation in different regions of brain samples harvested from KO mice. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn mice administered PBS by the same route. - / - KO mice were used as controls. Figure 17A Results for the CA2 / 3 hippocampal region are shown. Figure 17B Results for the entire hippocampus are shown. Figure 17C Results for the prefrontal cortex are shown. Figure 17D Results for the thalamus are shown. Figure 17E Results for the whole brain are shown. Legends indicating "right" refer to data collected from the same hemisphere injected with the GRN vector, while "left" refers to the contralateral hemisphere.

[0175] Figure 18A .The number of vector copies (VGCs) in liver samples harvested from Grn-deficient knock-in (KI) mice administered AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched Grn-deficient KI mice administered PBS via the same route were used as controls. The number of VGCs was normalized to the amount of cellular genomic DNA (μg gDNA). Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant.

[0176] Figure 18B.The number of vector copies (VGCs) in brain samples harvested from Grn-deficient knock-in (KI) mice administered AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched Grn-deficient KI mice administered PBS via the same route served as controls. The number of VGCs was normalized to the amount of cellular genomic DNA (μg gDNA). Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant.

[0177] Figure 18C .Amount of hGRNΔ3 mRNA in brain samples harvested from Grn-deficient KI mice administered AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched wild-type mice and Grn-deficient KI mice administered PBS via the same route served as controls. RNA levels were normalized to the amount of RNA expressed from housekeeping genes. Statistical analysis was performed using one-way ANOVA followed by post hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant.

[0178] Figures 18D to 18F PGRNΔ3 protein concentrations in fluid and tissue samples from Grn-deficient KI mice administered AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched wild-type mice and Grn-deficient KI mice administered PBS by the same route served as controls. Figure 18D Results for cerebrospinal fluid (CSF) samples are shown. Figure 18E Results for serum samples are shown. Figure 18F Results for brain tissue samples are shown. Figure 18C Same as described.

[0179] Figures 18G to 18H BMP 18:1 / 18:1 species in brain samples harvested from Grn-deficient KI mice administered AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery ( Figure 18G ) and BMP 22:6 / 22:6 species (Figure 15H). Age-matched wild-type mice and Grn-deficient KI mice administered PBS by the same route were used as controls. Figure 18C Same as described.

[0180] Figure 18I β-hexosaminidase enzyme activity in brain samples harvested from Grn-deficient KI mice administered with AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched wild-type mice and Grn-deficient KI mice administered with PBS via the same route served as controls. Figure 18C Same as described.

[0181] Figure 19A PGRNΔ3 protein concentrations in CSF samples from cynomolgus macaques 14 and 30 days after administration of AAV801-PGRNΔ3 vector (5e12 vg / kg or 2e13 vg / kg) or AAV9-PGRNΔ3 vector (2e13 vg / kg) via intravenous (IV) delivery. Two animals received each vector and dose. PGRNΔ3 protein was undetectable in one test animal that received the low dose of AAV801-PGRNΔ3 and in two test animals that received 2e13 vg / kg of AAV9-PGRNΔ3. The dashed line labeled "target" indicates the level of progranulin naturally occurring in humans.

[0182] Figure 19B PGRNΔ3 protein concentrations in serum samples from cynomolgus macaques before IV administration of AAV801-PGRNΔ3 vector (5e12 vg / kg or 2e13 vg / kg) or AAV9-PGRNΔ3 vector (2e13 vg / kg), and on days 3, 7, 14, 21, and 28 after treatment. Two animals received each vector and dose.

[0183] Figure 20 Vector copy (VGC) counts in brain and other tissue samples harvested from cynomolgus monkeys 28 days after IV administration of AAV801-PGRNΔ3 vector (5e12 vg / kg or 2e13 vg / kg) or AAV9-PGRNΔ3 vector (2e13 vg / kg). Multiple brain regions, spinal cord, and peripheral nerve and non-neural tissues were sampled. VGC counts were normalized to the amount of cellular genomic DNA (μg gDNA).

[0184] Figure 21 hGRNΔ3 mRNA levels in brain and other tissue samples harvested from cynomolgus monkeys 28 days after IV administration of AAV801-PGRNΔ3 vector (5e12 vg / kg or 2e13 vg / kg) or AAV9-PGRNΔ3 vector (2e13 vg / kg). Multiple brain regions, spinal cord, and peripheral nerve and non-neural tissues were sampled. RNA levels were normalized to the amount of RNA expressed from housekeeping genes.

[0185] Figure 22A to Figure 22G Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization of brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg AAV801-PGRNΔ3 vector. Figure 22A shows results for the motor cortex. Figure 22B shows results for the entorhinal cortex. Figure 22C shows results for hippocampal pyramidal cells. Figure 22D shows results for the thalamus. Figure 22E shows results for the dentate nucleus. Figure 22F shows results for the spinal cord. Figure 22G Results from the thalamus of negative control animals are shown. In these images, mRNA is labeled in red and cell nuclei are labeled in blue.

[0186] Figure 23 PGRNΔ3 protein concentrations in brain and other tissue samples harvested from cynomolgus macaques 28 days after IV administration of AAV801-PGRNΔ3 vector (5e12 vg / kg or 2e13 vg / kg) or AAV9-PGRNΔ3 vector (2e13 vg / kg) compared to levels of endogenous macaque progranulin. Multiple brain regions, spinal cord, and peripheral nerve and non-neural tissues were sampled. DETAILED DESCRIPTION

[0187] The following discussion relates to various embodiments. The present disclosure is not intended to refer to any particular embodiment or otherwise limit the scope of the present disclosure. Although one or more of these embodiments may be preferred, the disclosed embodiments should not be interpreted or otherwise used to limit the scope of the present disclosure (including the claims). In addition, it should be understood by those skilled in the art that the following description has a wide range of applications, and the discussion of any embodiment is intended only as an example of that embodiment, and is not intended to imply that the scope of the present disclosure (including the claims) is limited to that embodiment.

[0188] Certain definitions

[0189] As used herein, "adeno-associated viral vector" means an adeno-associated virus (AAV) comprising a naturally occurring or non-naturally occurring AAV capsid encapsidating the vector. An adeno-associated viral vector may be abbreviated as an "AAV vector" and, depending on the context, may be represented by synonymous terms such as "recombinant AAV vector," "rAAV vector," "rAAV," or simply "vector."

[0190] As used herein, "vector" refers to an AAV genome that has been modified to both comprise a heterologous nucleotide sequence and to render any AAV vector containing the vector replication-incompetent, such as by inactivating or deleting the endogenous AAV rep and / or cap genes.

[0191] As used herein, "heterologous nucleotide sequence" means a nucleotide sequence introduced into an organism (including a virus) from a different organism (including an organism). The sequence of a heterologous nucleotide sequence may be identical to a naturally occurring sequence, or may be a modified form thereof, or even partially or completely synthetic.

[0192] As used herein, "expression cassette" means a nucleotide sequence comprising a transgene operably linked to regulatory regions or elements that control the initiation and termination of transcription of the transgene from DNA into RNA.

[0193] As used herein, "transgene" means a nucleotide sequence encoding at least one polypeptide, and / or a nucleotide sequence encoding at least one functional RNA molecule. Transgene can be represented by the synonymous term "gene of interest".

[0194] As used herein, "host cell" refers to a cell in which an AAV vector is produced. Producer cells and packaging cells are examples of host cells. Host cells can be mammalian or insect, or from other organisms, whether unicellular or multicellular.

[0195] As used herein, the term "purify" and the related terms "purified" and "purification" when used in connection with an AAV vector or a sample or preparation thereof, means a relative increase or improvement in purity compared to the starting material containing the vector and / or a previous intermediate purification step in a scheme of sequential purification steps aimed at purifying a biological product, and does not require a specific qualitative or quantitative purity unless otherwise indicated.

[0196] As used herein, "transduction" means the introduction of the genome of an AAV vector into a target cell. Transduction is distinguished from infection, the latter term being used to refer to the introduction of the genome of a replicative adeno-associated virus into a cell.

[0197] As used herein, "target cell" means a cell that an AAV vector is designed or intended to transduce, or a cell that is experimentally observed to be transduced by an AAV vector, whether in vitro or in a subject.

[0198] As used herein, "subject" means an organism to which an AAV vector is administered for the purpose of preventing or treating a disease, disorder, or condition.

[0199] Frontotemporal dementia and frontotemporal lobar degeneration

[0200] Frontotemporal dementia (FTD) refers to a clinical syndrome characterized by progressively worsening deficits in language, behavior, and executive function associated with selective neurodegeneration of the frontal and temporal cortical lobes (frontotemporal lobar degeneration or FTLD), rather than the more generalized neurodegeneration seen in Alzheimer's disease and certain other dementias. Onset of disease in many patients with FTD occurs in their fourth to early sixth decade, with a prevalence of 10% for those younger than 45 years, 60% for those aged 45 to 64 years, and 30% for those older than 64 years. Following onset, symptoms progressively worsen, and survival ranges from 6 to 11 years, with an average of 8 years, depending on the subtype of FTD, although some aggressive subtypes can lead to death in as few as 2 years.

[0201] FTD presents with distinct neurological symptoms, depending on the underlying pattern of neurodegeneration, and three clinical variants have been defined. Behavioral variant frontotemporal dementia (BV-FTD) is associated with early behavioral and executive function deficits, nonfluent variant primary progressive aphasia (NFV-PPA) is typically characterized by progressive impairments in speech, grammar, and word production, and semantic variant primary progressive aphasia (SV-PPA) is characterized by progressive impairments in semantic knowledge and naming. The diagnosis of one of these clinical variants in patients with FTD depends on the predominant behavioral and language deficits, particularly early in the disease process. The diagnosis of the BV-FTD variant requires the presence of at least three of the following behavioral changes: disinhibition; flattened or dulled affect; loss of sympathy or empathy; stereotyped, compulsive, or persuasive behavior; hyperorality or dietary changes; and deficits in executive function, with relatively preserved visuospatial skills and memory. All PPA variants require the presence of language deficits, with aphasia being the most prominent initially. PPA is ruled out if there are significant early deficits in episodic memory, visual memory, or visual perceptual skills, or if behavioral disturbances are present. PPA is then further differentiated into semantic and nonfluent variants. For SV-PPA, patients demonstrate impairments in verbal naming and single-word comprehension, as well as at least three of the following language-related deficits or abilities: object knowledge impairment; surface alexia or dysgraphia; preserved repetition; and preserved verbal expression. NFV-PPA, on the other hand, requires at least one of agrammatism or apraxia in verbal expression, and at least two of the following: impairment in complex sentence comprehension, preserved single-word comprehension, and preserved object knowledge. Motor symptoms can also affect a minority of FTD patients. Slightly over 12% of patients with BV-FTD also develop motor neuron disease, which is less common in patients with the PPA variant of FTD. Approximately 20% of patients with FTD also exhibit parkinsonian symptoms and may also present with features of corticobasal syndrome or progressive supranuclear palsy.

[0202] Although many symptoms of FTD can be seen in patients with other types of dementia or psychiatric disorders, the brains of patients with FTD will show characteristic atrophy of the frontal or temporal lobes, with atrophy of the fronto-insular region being particularly indicative of FTD. Patterns of cortical atrophy associated with FTD can be detected using structural neuroimaging methods such as MRI or CT or functional methods such as fluorodeoxyglucose PET, functional MRI, and SPECT.

[0203] Confirmation of FTD typically requires examination of the brain of a suspected case to detect neuropathological changes associated with the frontotemporal lobar degeneration (FTLD) disease process. While FTLD is typically characterized by neuronal loss, gliosis, and microvesicular changes in the frontal, anterior temporal, anterior cingulate, and insular cortices, nearly all cases can be distinguished into three main types: FTLD-tau, FTLD-TDP, and FTLD-FUS, based on the presence of abnormal deposits of certain proteins that are also associated with certain characteristic patterns of neurogeneration. FTLD-tau, which accounts for approximately 36% to 50% of all FTLD cases, is defined by the presence of deposits of the microtubule-associated protein tau (MAPT) on neuropathological examination and is associated with the FTLD subtypes of Pick's disease, corticobasal degeneration, and progressive supranuclear palsy. FTLD-FUS, which accounts for approximately 10% of FTLD cases, is defined by the presence of brain deposits of the fusion protein sarcoma (FUS) and is associated with the early onset of FTD behavioral symptoms, the absence of movement, and language deficits. FTLD-TDP is the most prevalent, accounting for approximately 50% of FTLD cases, and is defined by brain deposits of the 43 kDa TAR DNA-binding protein (TDP-43).

[0204] Based on the pattern of abnormal protein deposition in the brain and characteristic patterns of neurodegeneration, FTLD-TDP is further divided into three subtypes: A, B, and C. FTLD-TDP type A is associated with asymmetric dorsal atrophy, including the frontal and temporal lobes (anterior, medial, and posterior regions), the orbitofrontal cortex, the anterior cingulate gyrus, the inferior parietal lobe, the striatum, and the thalamus. FTLD-TDP type B is associated with atrophy involving the medial and polar temporal lobes, the anterior insula, the cingulate and medial prefrontal cortex, and the orbitofrontal cortex, with the frontal lobe being more severely affected in the posterior regions. FTLD-TDP type C is associated with atrophy of the right or left dominant anterior temporal lobe, with additional involvement of the amygdala, hippocampus, orbitofrontal cortex, and insular cortex. Different FTLD-TDP subtypes have also been reported to be associated with certain FTD symptoms. Thus, TDP type A accounts for approximately 50% of NF-PPA cases, 25% of suspected corticobasal degeneration cases, but only a small fraction of BV-FTD (with or without motor neuron disease); type B accounts for approximately two-thirds of FTD cases manifesting with motor neuron disease and 25% of BV-FTD overall; and type C accounts for approximately 90% of all cases of SV-PPA or temporal variant BV-FTD. Bang, J. et al., Lancet, 386:1672-82, 2015.

[0205] Human genetic studies have determined that up to 40% of FTLD cases may be familial, with mutations identified in three genes—9ORF72, MAPT, and GRN—responsible for approximately 60% of inherited cases. The GRN gene encodes progranulin (PGRN), a secreted protein involved in multiple functions, including cell cycle regulation, wound repair, axonal growth, and inflammation. PGRN binds to the tumor necrosis factor receptor (TNFR), among other receptors, suggesting a mechanism for regulating inflammation induced by TNFα or other inflammatory mediators. Heterozygous loss-of-function mutations in the GRN gene result in haploinsufficiency, accompanied by a substantial decrease in PGRN concentrations in cerebrospinal fluid (CSF) and serum. Over 70 pathogenic mutations in the GRN gene have been identified that cause FTD, the majority of which are nonsense mutations that trigger degradation of the mRNA encoding progranulin. In approximately 5% to 20% of familial FTD cases, GRN mutations that result in haploinsufficiency and reduced PGRN production are associated with the characteristic pattern of FTD clinical syndromes, brain atrophy, and neuropathology. As reported by Bang et al., GRN loss-of-function mutations are associated with neurological symptoms and signs of BV-FTD, NFV-PPA, parkinsonism, and corticobasal syndrome (CBS), as well as an asymmetric pattern of neurodegeneration, primarily affecting the anterior temporal lobe, temporoparietal lobe, frontal lobe (the left side is more associated with PPA syndrome and the right side is more associated with BV-FTD symptoms), anterior cingulate cortex, and insular cortex. In addition, on neuropathological examination, the brains of FTD patients with GRN mutations often show cellular changes and TDP protein deposits characteristic of FTLD-TDP A type.

[0206] Adeno-associated virus (AAV)

[0207] The present disclosure provides vectors produced by recombinantly modified adeno-associated viruses (AAVs). AAV vectors are capable of delivering genes that may be controlled by transcriptional elements and other regulatory elements into target cells by transduction. By providing functional copies of genes to target cells that have lost or mutated endogenous forms, AAV vectors can be used for gene therapy of various diseases and disorders.

[0208] AAV is a small, non-enveloped, apparently non-pathogenic parvovirus that relies on certain other viruses to provide the gene products (called accessory factors) necessary for its own replication, a biological characteristic that makes AAV very suitable for use as a recombinant vector. For example, adenovirus (AdV) can be used as a helper virus by providing certain adenoviral factors (such as E1A, E1B55K, E2A and E4ORF6 proteins and VA RNA) in cells co-infected by adenovirus and AAV. Other helper viruses, such as herpes simplex virus, have also been identified. AAV replication depends on accessory factors provided by other viruses, which has led to AAV being characterized as a dependent virus. The AAV virion has two main structural features, respectively referred to as the capsid and the genome. The capsid is an icosahedral protein shell that surrounds and protects (encapsulates) the viral genome, which contains genes and other sequences required for viral replication in infected cells.

[0209] The AAV genome is a single-stranded DNA containing two genes called rep and cap. In AAV2 (a naturally occurring AAV that infects humans and is particularly well characterized biologically), the genome length is about 4.7 kilobases. Due to the selective splicing of transcripts from two promoters, the rep gene can produce four related multifunctional proteins, known as Rep (referred to as Rep 78, Rep 68, Rep 52 and Rep 40 in AAV2, named according to their apparent molecular weight), which are involved in viral gene expression and genome replication and packaging. The selective splicing of transcripts from a single promoter that controls a single cap gene produces three related structural proteins VP1, VP2 and VP3, wherein a total of 60 structural proteins self-assemble to form the icosahedral capsid of the virus at a ratio of approximately 1:1:10. VP1 is the longest of the three VP proteins and contains amino acids that do not exist in VP2 in its amino terminal region, while VP2 is longer than VP3 and contains amino acids that do not exist in VP3 in its amino terminal region. In addition to housing the genome, the capsid protein mediates specific binding interactions with receptors on the surface of target cells. Based on this, AAV can be restricted in its ability to infect certain animal species, or even tissues within the same type of animal, a phenomenon known as tropism. For example, one type of AAV may preferentially infect liver cells (e.g., hepatocytes) compared to muscle or neuronal cells.

[0210] In addition to the rep and cap genes, the complete AAV genome also has relatively short (145 nucleotides in AAV2) sequence elements located at each of its 5' and 3' ends, known as inverted terminal repeats (ITRs). ITRs contain nested palindromic sequences that can self-anneal to form T-shaped or hairpin secondary structures through Watson-Crick base pairing. In AAV2, ITRs have been shown to have important functions required for the viral life cycle, including converting the single-stranded DNA genome into the double-stranded form required for gene expression, and packaging the single-stranded AAV genome into capsid assemblies by the Rep proteins.

[0211] Many naturally occurring AAV types have been found in different species. At one time, only six types of primate AAV (AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6) were isolated from biological samples, of which the first five were structurally different enough to be classified as different serotypes based on antibody cross-reactivity experiments. Later, two new AAVs were discovered by PCR amplification of DNA from rhesus monkeys using primers targeting a highly conserved region in the cap gene of previously discovered AAV, known as AAV7 and AAV8 (Gao, G. et al., Proc. Natl. Acad. Sci. USA, 99: 11854-9, 2002). Subsequently, similar methods were used to clone many new AAVs from human and non-human primate tissues, greatly expanding the range of AAV capsid protein sequences (Gao, G. et al., J. Virol., 78: 6381-8, 2004). Many AAV capsid protein sequences are highly similar to each other or to previously identified AAVs, and while often referred to as distinct AAV "serotypes," not all such capsids would necessarily be expected to be immunologically distinguishable if tested for antibody cross-reactivity. AAV or AAV capsids that are serologically indistinguishable from a defined serotype but contain capsid proteins with different amino acid sequences are best referred to as variants of a known serotype. A wide variety of capsids made from both naturally occurring and non-naturally occurring capsid proteins have been found to be useful in generating AAV gene therapy vectors.

[0212] Studies of AAV2 have demonstrated that, after binding to one or more receptor molecules on the cell surface, AAV viral particles enter cells via endocytosis. Upon reaching the low pH of the lysosome, the capsid protein undergoes a conformational change, allowing the capsid to escape into the cytosol and then be transported to the nucleus. Once there, the capsid disassembles, releasing the genome. Cellular DNA polymerases initiate the synthesis of the second DNA strand, starting with the 3'-terminal ITR, which acts as a primer after self-annealing. Expression of the rep and cap genes then begins, followed by the formation of new viral particles and their release from the cell.

[0213] AAV vectors

[0214] The relative simplicity of AAV's structure and life cycle, and the fact that it is not known to be pathogenic to humans, inspired researchers to engineer AAV and investigate whether it could be converted from a virus into a recombinant vector for gene therapy. Briefly, this is accomplished by cloning the entire genome of AAV2 (including the two ITRs) into a plasmid, removing the rep and cap genes into a separate plasmid, and replacing them with a heterologous gene expression cassette containing a promoter controlling a protein-encoding transgene. Thus, the only viral genomic sequences retained in the vector are the ITRs, as they have critical functions in packaging and gene expression, and without them, AAV vectors cannot be produced and transgenes cannot be expressed after transduction of target cells. Finally, to avoid the need for co-infection with a helper virus, which is required for AAV virion replication, the genes for so-called auxiliary factors (such as, in the case of AdV, E1A, E1B55K, E2A, E4ORF6, and the VA RNA auxiliary factor) are cloned into a third plasmid.

[0215] When these three plasmids (sometimes referred to as transgene, rep / cap, and helper plasmid) are transfected together into a mammalian host cell, the Rep and capsid proteins, as well as the helper viral factors, are expressed from their respective plasmids. These gene products then act in the host cell to replicate the vector from the plasmid where its sequence resides into single-stranded DNA, assemble the capsid, and package the single-stranded genome into the capsid, thereby forming a vector. The vector can then be purified from the host cell. Because the rep and cap genes are present on different plasmids in a trans form, outside their usual range of ITRs on both sides, they are not packaged into the vector. Therefore, although the AAV vectors produced in this way are able to bind to target cells and deliver the expression cassette within their genome into target cells, they are unable to replicate and produce new vector particles.

[0216] If the vector functions as intended, upon transduction, the expression cassette will be transcriptionally active and produce the gene product encoded by the transgene in the target cell. AAV vectors are highly versatile because a variety of vector configurations can be designed containing a variety of transgenes controlled by different functional sequences and regulatory elements, and these vectors can be paired with a variety of naturally occurring and engineered capsids with varying tropism and other properties. Consequently, a wide range of gene products can be produced, with a degree of control over the type of cell transduced and the amount of gene product produced.

[0217] AAV vectors comprise vectors wrapped by an AAV capsid. In some embodiments, the AAV vector comprises at least one AAV inverted terminal repeat (ITR) and a heterologous nucleotide sequence that has a desired function when present or expressed in the transduced target cell. In some embodiments, the heterologous nucleotide sequence is derived from different types of viruses, or completely different types of organisms, such as animals, plants, protozoa, fungi, bacteria, archaea or other types of organisms. In some embodiments, the heterologous nucleotide sequence replaces some or all of the natural AAV rep and / or cap genes so that the vector cannot express functional Rep or VP proteins in the transduced target cells. In some embodiments, the entire sequence of the vector consists of a heterologous nucleotide sequence except the AAV inverted terminal repeats located at the ends of the genome.

[0218] The length of the genome (including ITRs) of the AAV vectors of the present disclosure can be any suitable length, which generally (but not necessarily) does not exceed the average genome size packaging capacity of a particular AAV capsid, which can be selected in the design and production of a particular AAV vector. Thus, in some non-limiting embodiments, the length of the genome (including ITRs) of the AAV vectors of the present disclosure can be at least or about 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100 or 5200 nucleotides (or base pairs when the genomic sequence is embodied in a plasmid for vector production), or an integer value between or within a range encompassing any of the foregoing specific recited values.

[0219] Expression cassette

[0220] In some embodiments, the heterologous nucleotide sequence comprises or consists of an expression cassette containing a transgene operably linked to a promoter and optionally one or more enhancers for controlling the initiation of transcription of the transgene from DNA to RNA, and operably linked to a transcription termination element (such as a polyadenylation signal sequence) for terminating transcription of the transgene into RNA. An AAV vector may contain more than one transgene, either as part of a single transcription unit or each as part of its own transcription unit. As described in subsequent sections, the expression cassette may also contain additional sequence elements designed to affect transcription, transcript stability, translation, or other functions.

[0221] As is generally the case with AAV vectors, the structure of the expression cassette and the genome as a whole is limited by the packaging capacity of the capsid, so the length of the transgene, when combined with all other elements of the genome required for vector function (such as transcriptional control elements and ITRs), does not exceed approximately 5 kilobases in the case of AAV2, although other types of capsids may have larger or smaller packaging limits. However, within the size limit, there is considerable flexibility in the selection of the transgene, ITRs, and other elements required for the vector to perform its intended function.

[0222] For the purposes of gene therapy, a transgene may be any gene, the product of which will be understood to prevent or treat (although not necessarily cure) any disease, disorder or condition of a subject in need of prevention or treatment. In some embodiments, gene therapy is intended to prevent or treat a disease, disorder or condition characterized in that the amount of product produced by a naturally occurring gene in the subject is abnormally low or even absent, such as may occur due to a loss-of-function mutation. With respect to such embodiments, a transgene may be a transgene that is intended to compensate for a subject's defective gene by providing the same or similar gene product to at least some cells of the subject when expressed. A non-limiting example would be a vector designed to express a functional form of coagulation factor IX for gene therapy of hemophilia B, which is caused by a loss-of-function mutation in a natural factor IX gene. However, in other embodiments, a transgene may be a transgene that is intended to counteract the effects of a deleterious gain-of-function mutation in a target cell. In some embodiments, a transgene may encode a transcriptional activator to increase the activity of an endogenous gene that produces a desired gene product, or conversely, a transcriptional repressor may be encoded to reduce the activity of an endogenous gene that produces a deleterious gene product.

[0223] In some embodiments, the transgene may encode a polypeptide, or encode an RNA molecule having a function different from that of encoding a protein, such as a regulatory non-coding RNA molecule (e.g., microRNA, small interfering RNA, piwi-acting RNA, enhancer RNA, long non-coding RNA, etc.). The protein coding sequence in the transgene may be codon optimized, and the translation initiation site (e.g., Kozak sequence) may be modified to increase or decrease its tendency to initiate translation. In some embodiments, the transgene encoding the amino acid sequence may contain one or more open reading frames, and / or contain one or more splice donor and acceptor site pairs to allow selective splicing of different messengers and polypeptide sequences from such messengers. The protein-encoding transgene also includes one or more stop codons to terminate the translation of the polypeptide chain.

[0224] In some embodiments, the vector can be designed for the purpose of editing or otherwise modifying the genome of the target cell. For example, the vector can contain an expression cassette or transgene flanked by homology arms designed to promote homologous recombination between the vector and the target cell genome. In another example, the vector can be designed to perform CRISPR gene editing by expressing a guide RNA (gRNA) and / or an endonuclease (such as Cas9) or a related endonuclease (such as SaCas9) that can bind to the gRNA and cleave the DNA sequence targeted by the gRNA.

[0225] PGRN transgene

[0226] In some embodiments, the AAV vector of the present disclosure comprises a vector containing an expression cassette comprising a coding sequence (transgene) of a progranulin protein (abbreviated as "PGRN") or a variant thereof (including human progranulin or a variant thereof). In some embodiments, the progranulin protein is identical to the 593 amino acid long 88 kDa human progranulin precursor protein (NCBI Reference Sequence: NP_002078.1 or SEQ ID NO: 16), which comprises a 17 amino acid long signal peptide (SEQ ID NO: 18) and a 576 amino acid long mature granulin polypeptide (SEQ ID NO: 18). In some embodiments, after cleavage of the signal peptide, the mature granulin polypeptide is further cleaved into a variety of approximately 6 kDa peptides that have pleiotropic functions, depending on the cell or organism context. In other embodiments, the expression cassette comprises a coding sequence for a non-human progranulin protein.

[0227] In yet other embodiments, the PGRN protein may comprise any naturally occurring variant of the human PGRN protein that does not contain a pathogenic mutation, such as a premature translation stop codon, or an amino acid substitution, insertion, or deletion that substantially impairs PGRN activity and / or protein stability. In yet further embodiments, the PGRN protein may comprise an engineered variant of the human PGRN protein that retains PGRN activity, such as a chimeric variant and a variant with an amino acid substitution, insertion, or deletion that is designed to modulate PGRN activity, add or remove a glycosylation site, add or remove or alter an internal cleavage site of a granule protein that is normally cleaved from the mature granule protein, or a site for other post-translational modifications, or alter other aspects of PGRN structure or function. In some embodiments, the native signal peptide sequence is modified or completely replaced by a signal peptide sequence from a similar or completely different secretory protein.

[0228] In certain embodiments, the PGRN protein variant is a carboxyl terminal truncation variant in which one or more amino acids normally present in the full-length wild-type human progranulin are deleted, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids are deleted from the carboxyl terminus of the progranulin, compared to the full-length wild-type human progranulin amino acid sequence (such as the amino acid sequence provided by SEQ ID NO: 16). In some embodiments, the PGRN protein variant has deleted the last three carboxyl terminal amino acids (QLL) originally present in the full-length wild-type human PGRN protein and has the amino acid sequence of SEQ ID NO: 14. The latter PGRN protein variant is sometimes referred to herein as "PGRNΔ3" or "PGRNDel3". In some embodiments, the PGRN protein variant (such as PGRNΔ3) has reduced or no specific binding to the receptor protein called sortilin 1 (SORT1) compared to the full-length wild-type PGRN protein. In some embodiments, the PGRNΔ3 variant undergoes further post-transcriptional and post-translational modifications after delivery via a viral vector. Additional terminal cleavages are known to occur, including, for example, single amino acid cleavage at the carboxyl terminus to produce PGRNΔ4 protein variants. Other known post-transcriptional and post-translational processing events may further modify the protein provided by the PGRNΔ3 vector, including, but not limited to, glycosylation, isomerization, complete or partial degradation, cleavage of amino acid sequences (e.g., cleavage of one or more signal sequences), addition of molecules (e.g., peptide "tags" or functional or signaling sequences), and the like.

[0229] In certain other embodiments, the PGRN protein variant comprises at least one amino acid substitution mutation compared to the full-length wild-type human progranulin amino acid sequence (such as provided by SEQ ID NO: 16), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids are changed compared to its wild-type counterpart. In some of these embodiments, the substitution mutation can be a conservative amino acid substitution, wherein the normally occurring amino acid is replaced by another amino acid with an R group having similar physicochemical and / or size characteristics. Alternatively, in other embodiments, the substitution mutation can be a non-conservative amino acid substitution, wherein the normally occurring amino acid is replaced by another amino acid with an R group having non-similar physicochemical and / or size characteristics.

[0230] For use in the AAV vectors of the present disclosure, the nucleotide sequence encoding the PGRN protein can be any nucleotide sequence capable of encoding the desired PGRN protein in the cell type desired to be transduced by the vector (such as neurons). In some embodiments, the nucleotide sequence encoding the PGRN protein (i.e., transgene) is identical to the nucleotide sequence present in the naturally occurring gene encoding PGRN (i.e., the exons of such a gene), or is a DNA sequence corresponding to the mRNA sequence transcribed from such a gene. In some embodiments, wherein the PGRN protein is full-length wild-type human progranulin, the encoding nucleotide sequence is provided by nucleotides 41 to 1822 (including the stop codon) of the NCBI reference sequence: NM_002087.4 or is provided by SEQ ID NO: 15. In some embodiments, wherein the PGRN protein is a PGRNΔ3 protein variant, the encoding nucleotide sequence is provided by SEQ ID NO: 8.

[0231] In other embodiments, the nucleotide sequence encoding the PGRN protein may differ from the naturally occurring nucleotide sequence at one or more nucleotide positions and, due to the redundancy of the genetic code, still encode a PGRN protein identical to the naturally occurring gene sequence, or encode a PGRN protein variant that is encoded by the naturally occurring gene sequence except for the differences in the polypeptide relative to wild-type PGRN. In some embodiments, the nucleotide sequence encoding the PGRN protein may be intentionally modified to affect its function in the transduced cell, such as eliminating sequence motifs that can stimulate an innate immune response, eliminating cryptic splice junctions, eliminating alternative start codons, increasing the stability of the corresponding mRNA, and / or increasing the rate at which the mRNA is translated into protein. In other embodiments, the nucleotide sequence encoding the PGRN protein may be intronless, or may contain one or more introns that interrupt the coding sequence, but these introns will be removed by the splicing machinery in the transduced cell to allow translation of the desired PGRN protein.

[0232] In some embodiments of the AAV vectors of the present disclosure, the transgene comprises a protein sequence that is highly similar or identical to a protein sequence encoded by a particular nucleotide reference sequence, but wherein the nucleotide sequences of the transgene and the reference sequence are not identical, but rather share a particular percent identity, the differences corresponding to positions within the codon that do not change the corresponding amino acid (i.e., are silent changes). For example, in some embodiments, the transgene comprises or consists of a sequence encoding a full-length PGRN protein identical to SEQ ID NO: 15 and has a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 15; or comprises or consists of a sequence encoding a PGRNΔ3 protein variant identical to SEQ ID NO: 8 and has a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: NO:8 is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the nucleotide sequence.

[0233] The percentage of nucleotide sequence identity between the reference sequence and the transgene can be determined by any method known in the art. For example, in some embodiments, the nucleotide sequence of the reference sequence and the transgene (or the amino acid sequence encoded by them) can be aligned and compared over their entire length, and the percentage of nucleotide sequence identity can be calculated using a computer algorithm. An exemplary algorithm for global alignment and comparison of nucleotide sequences is the Needleman-Wunsch algorithm. However, in other embodiments, a local alignment algorithm such as the BLAST algorithm (Needleman, S. and Wunsch, C., J. Mol. Biol., 48:443-53, 1970; States D. et al., Methods, 3:66-70, 1991; Pearson, W., Curr. Protoc. Bioinformatics, 43:3.5.1–3.5.9, 2013) can be used. In some embodiments, where one or the other of the reference and transgenic sequences contains non-coding sequences (such as introns or stop codons), the non-coding sequences are ignored and only the protein-coding sequences within the reference and transgenic sequences are aligned and compared. Once an optimal global alignment between the reference sequence and the transgenic sequence is established, the percentage of identical nucleotides between the aligned sequences can be calculated.

[0234] As known in the art, sequence comparison algorithms can allow users to define permutation scores and gap penalties, which are parameters used to calculate the alignment scores of many possible alignments that can be performed. The alignment with the highest score is then considered the best. The permutation score involves assigning a numerical reward for matching and a penalty for mismatching. Exemplary sets of corresponding match and mismatch scores include 1, -1; 1, -2; 1, -3; 1, -4; 2, -3; 4, -5, but other sets are also possible. The gap cost involves assigning a numerical penalty for the presence of a gap (insertion or deletion of a nucleotide) and a penalty for the gap width expansion after formation. Increasing the gap cost will produce an alignment with a reduced number of gaps introduced. Exemplary sets of corresponding costs for gap existence and expansion include 0, -4; -2, -2; -2, -4; -3, -3; -4, -2; -4, -4; -5, -2; -6, -2, but other sets are also possible. In some embodiments, alignment and comparison of a reference sequence and a transgene sequence are performed using default substitution scores and gap penalties, and any other default settings, provided with the computer software or algorithm performing the analysis.

[0235] Signal peptide sequence

[0236] In some embodiments, the AAV vectors of the present disclosure comprise a vector comprising an expression cassette comprising a coding sequence for a progranulin protein having a mature progranulin polypeptide sequence (a transgene), but wherein the signal peptide naturally present in wild-type human progranulin is replaced with a signal peptide of a different secreted polypeptide from human or another species (i.e., a heterologous signal peptide sequence), for example, to increase the secretion rate of PGRN (or its variant) produced in the transduced cells, or for some other reason, such as reduced immunogenicity. In some embodiments, the mature progranulin polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a carboxyl-terminal truncation thereof lacking the last three amino acids (QLL).

[0237] Any signal peptide sequence known in the art that effectively promotes secretion of a protein from the cell in which it is synthesized can be used in conjunction with the AAV vectors of the present disclosure. In some embodiments, cells remove these signal peptides from the protein during the secretion process. Many heterologous secretion signal peptides are known in the art and can be used to promote the secretion of PGRN (or its variants) by cells (such as neurons or other brain cells) transduced with the AAV vectors of the present disclosure.

[0238] In some embodiments, the signal peptide sequence may be derived from or derived from any of a variety of proteins produced and secreted by neurons or other cells in the central or peripheral nervous system. Non-limiting examples include signal peptide sequences from human proteins, such as growth hormone, proenkephalin A, β-neoendorphin-dynorphin, neuroendocrine protein 7B2, prolactin, gastrin-releasing peptide II, secretagogue protein I, growth hormone-releasing factor, axonal protein I, neuroendocrine converting enzyme 2, vasopressin and copeptin, as well as many other proteins known in the art. The amino acid sequences and encoding nucleotide sequences of these and other secretory proteins can be obtained in public sequence databases (such as Genbank). The amino acid sequence of the naturally occurring signal peptide can be modified to ideally change its function, and the nucleotide sequence encoding such a wild-type or modified signal peptide can also be modified to achieve the desired type of sequence optimization, such as removing the CpG motif. In yet other embodiments, a completely synthetic secretory signal peptide sequence can be used.

[0239] transcription control region

[0240] The AAV vectors of the present disclosure intended to express PGRN protein in and / or from transduced cells may also include one or more transcription control regions operably connected to a transgene encoding a PGRN polypeptide sequence as part of the vector. As discussed further below, different types of transcription control regions are known in the art that can be used to control the initiation of transcription of a transgene into RNA. As used herein, the term "operably connected" and variations such as "operably connected," "operably connected," and "operably connected" refer to the functional relationship between a transcription control region and a transgene such that the control region can affect the transcription of the transgene (whether positive or negative) without specifying any specific spatial or structural relationship between them. Thus, for example, a transcription control region can be operably connected to a transgene even if it is located 5' or 3' of the transgene and / or is adjacent to or away from the transgene. A transcription control region may have constitutive activity, be active in specific cells or tissues, have inducible activity in response to some environmental stimuli, be derived from naturally occurring genes (of any suitable species), and be modified to improve or alter their function, or even be fully synthetic.

[0241] In some embodiments, the transcription control region comprises a promoter region comprising the minimal DNA sequence required for the transcription machinery to initiate transcription in the transduced cell (e.g., a TATA box or initiator sequence), and typically also comprises one or more additional proximal elements that act alone or synergistically to increase the rate of transcription from the basic promoter. Depending on its sequence, a promoter can initiate transcription by RNA polymerase I, II, or III, but promoters from protein-coding genes that are typically transcribed by RNA pol II are commonly used in AAV vectors intended to express polypeptides (such as PGRN or variants thereof, such as PGRNΔ3) in transduced cells.

[0242] In other embodiments, the transcription control region comprises or further comprises at least one enhancer region, the function of which is to further increase the gene transcription rate to exceed the rate that the basic promoter can maintain alone. In its natural context, enhancers are usually located distal to the promoter of the gene on which they act, sometimes tens to hundreds or thousands of base pairs upstream (i.e., 5'), but enhancers can also appear elsewhere, such as in introns or downstream (i.e., 3') of the gene on which they act. Although the promoter region may contain proximal enhancer elements (which, if removed, will reduce the subsequence transcribed from the basic promoter), enhancers usually do not contain sequences that can act as basic promoters. Although enhancer regions are usually located distal to the promoter of the gene on which they act in nature, when removed from their natural context and relocated closer to the promoter (whether from the same or even different genes), enhancer regions or enhancer elements within larger enhancer regions (such elements usually correspond to DNA binding sites for transcription factors) can sometimes retain at least some of their transcription enhancing functions.

[0243] When combined with other genomic elements required for transgenic and vector function, the enhancer and promoter regions of genes described in the scientific literature may be too large to be accommodated by the packaging capacity of the AAV capsid. Therefore, in some embodiments, methods familiar to those of ordinary skill in the art can be used to identify functional subsequences within longer enhancer or promoter regions, and shorter functional subsequences can be integrated into the transcription control region of the vector for the present disclosure. In this way, the size of the transcription control region can be reduced while maintaining its desired function. Using this approach, functional elements from naturally occurring enhancers or promoters can be combined in novel ways, such as by modifying their number, spacing, and / or arrangement, to produce hybrid or synthetic enhancers and / or promoters with improved properties. In some embodiments, enhancers and promoters can each be derived from the same naturally occurring gene, while in other embodiments, enhancers and promoters can be derived from completely different genes, including genes from different species.

[0244] In some embodiments, from the perspective of the coding strand (i.e., positive strand) single-stranded DNA AAV vector, the promoter sequence is located 5' of the downstream sequence to be transcribed into RNA, such as a transgene encoding a protein (such as PGRN or a variant thereof, such as PGRNΔ3). In some embodiments, the enhancer element or enhancer region (if present) may be located 5' of the promoter sequence, or elsewhere in the genome, such as in the 5' or 3' untranslated region (UTR) adjacent to the transgene, in an intron, 3' of the transcription termination signal sequence, or elsewhere. In some embodiments, the vector may contain more than one enhancer region (same or different types), which may be positioned adjacent to each other, or spaced apart, and / or separated by other functional elements within the genome. In some embodiments, the same enhancer element or enhancer region is provided in a tandem array of repeat units (such as 2, 3, 4 or more).

[0245] In some embodiments, the transcription control regions used in the AAV vectors of the present disclosure are non-tissue specific, meaning that they are constitutively active in many different cell types, although not necessarily all cell types. According to some embodiments, non-tissue specific transcription control regions include promoters derived from certain viruses (which may contain enhancer elements near the basal promoter), such as the human cytomegalovirus major immediate early gene (CMV-IE) (Boshart, M. et al., Cell, 41:521-30, 1985; Yew, N. et al., Hum. Gene Ther., 8:575-84, 1997); Simian virus 40 (SV40); and retroviral long terminal repeat (LTR) promoters from Rous sarcoma virus (RSV) and Moloney murine leukemia virus (MoMLV). In other embodiments, the non-tissue-specific transcriptional control region includes a promoter (which may include a proximal enhancer element) that can be derived from a gene (sometimes referred to as a "housekeeping" gene) that is active in many different cell types, including from different types of animals, such as the human polypeptide chain elongation factor (EF1α) gene; the phosphoglycerate kinase (PGK) gene; the ubiquitin C (UbiC) gene; the chicken β-actin (CBA) gene; the U1a1 or U1b2 small nuclear RNA promoter (Bartlett, J. et al., Proc. Natl. Acad. Sci. USA, 93:8852-7, 1996; Wu, Z. et al., Mol. Ther., 16:280-9, 2008); or the histone H2 or histone H3 promoter (Hurt, M. et al., Mol. Cell Biol., 11:2929-36, 1991).

[0246] Likewise, enhancer regions can be derived from viruses and genes that are active in different cell types from different types of animals. As noted, in some embodiments, promoters and enhancers derived from the same gene can be combined to create a transcriptional control region for use in the vectors of the present disclosure, but enhancers and promoters from different genes can be combined to create a hybrid transcriptional control region. A commonly used example is the 1.6 kilobase hybrid enh / pro region known as CAG (or CAGGS), which encompasses the CMV immediate early enhancer, the chicken beta actin (CBA) gene promoter, and the CBA intron / exon 1 (Niwa, H. et al., Gene, 108:193-9, 1991; Ikawa, M. et al., Dev. Growth Differ., 37:455-9, 1995), and subsequent modifications that reduced its size, including one in which the CBA intron was replaced with a smaller simian virus 40 (SV40) intron (Wang, Z. et al., Gene Ther., 10:2105-11, 2003), and another, known as the CBA hybrid intron (CBh), which replaces the SV40 intron with a CBA intron consisting of a CBA intron. The 5' donor splice site of the 5' UTR and the 3' acceptor splice site from the MVM intron are combined into a hybrid intron (Gray, S. et al., Hum. Gene Ther., 22: 1143-53 2011).

[0247] In some embodiments, the transcription control regions used in the AAV vectors of the present disclosure can be central nervous system (CNS) or brain tissue specific, meaning that they are more active or most active in directing the expression of transgenes in cell types within the CNS or brain compared to cells of other tissues or organs (such as muscle or liver). In some embodiments, the CNS or brain cell types in which the transcription control regions of the AAV vectors of the present disclosure are preferentially active include, but are not limited to, neurons, glial cells (such as microglia, astrocytes, and oligodendrocytes), and ependymal cells, with other cell types also possible. Without wishing to be bound by any particular theory of operation, one mechanism by which brain tissue or neuronal (or any other cell type in the brain) gene transcription specificity may occur is the presence of one or more specific binding sites in enhancers and / or promoters for DNA binding transcription activator proteins that are preferentially expressed in brain cells (such as neurons or other cell types in the brain). In some embodiments, the use of brain tissue or neuron (or other brain cell type) specific transcriptional control regions can be advantageous in that they reduce or even prevent transgene expression in cells outside the brain or non-neuronal cells (or other brain cell types) that can be transduced by the vector, thereby ideally reducing the risk of off-target effects.

[0248] Certain brain or neuron (or other brain cell type) specific genes expressed at high levels have enhancers and promoters that can be included in the transcription control region of the AAV vectors disclosed herein. In some embodiments, enhancers and promoters derived from the same gene can be combined in a brain or neuron (or other brain cell type) specific transcription control region, while in other embodiments, enhancers from one gene and promoters from different genes can be combined in a hybrid brain or neuron (or other brain cell type) specific transcription control region. When derived from the same gene, the transcription control region sequence comprising one or more enhancers and promoters can be copied when it is present in the natural gene background from which it is derived, or engineered to reduce its length, such as by deleting non-transcriptionally active sequences that separate one or more enhancers and promoters. In some embodiments, enhancers and promoters for brain or neuron (or other brain cell type) specific transcription control regions can be derived from genes of different species. In yet other embodiments, the sequence of the enhancer and / or promoter in the transcriptional control region can be modified relative to its original sequence by changing, adding or removing nucleotides to improve its function, such as increasing the binding of transcriptional activators, reducing the binding of transcriptional repressors, or reducing the size of the transcriptional control region.

[0249] In some embodiments, it is the enhancer that provides brain or neuron (or other brain cell type) specific expression, and the promoter itself is not brain or neuron (or other brain cell type) specific, while in other embodiments, it is the promoter that provides brain or neuron (or other brain cell type) specific expression, and the enhancer (if present) itself is not brain or neuron (or other brain cell type) specific, but is capable of increasing the rate of transcription from a brain or neuron (or other brain cell type) specific promoter. For example, a strong viral enhancer (such as the human CMV major immediate early gene enhancer) can be paired with a brain or neuron (or other brain cell type) specific promoter, or a strong brain or neuron (or other brain cell type) specific enhancer (such as from the synapsin 1 gene) can be paired with a strong viral promoter (such as the SV40 early promoter). In other embodiments, the enhancer and promoter are each brain or neuron (or other brain cell type) specific. In some embodiments, different enhancer regions can be combined to form a chimeric enhancer region for use in the transcription control region of the AAV vectors of the present disclosure.

[0250] Brain tissue or neuron (or other brain cell type) specific transcriptional control regions (whether enhancers, promoters, or both) used in the AAV vectors of the present disclosure can be derived from genes that are naturally expressed at high levels in the brain or neurons (or other brain cell types), or even in specific regions of the brain or neuronal subtypes. For example, but not limited to, the synaptophysin 1 gene (SYN1), the neuron-specific enolase (NSA) gene, and the tubulin alpha 1 gene each contain a neuron-specific promoter, the glial fibrillary acidic protein (GFAP) gene promoter is at least partially specific for astrocytes, the L7-6 gene promoter is at least specific for cerebellar Purkinje cells, the Ca2+ / calmodulin-dependent protein kinase II (CaMKII) gene promoter is at least partially specific for forebrain excitatory neurons, the distalless homeobox II (DLX) gene enhancer is at least partially specific for forebrain inhibitory neurons, the glutamate decarboxylase (GAD) 65 gene promoter is also at least partially specific for inhibitory neurons, the tyrosine hydroxylase gene promoter is at least partially specific for catecholaminergic neurons, and the transcriptional control regions (promoters and / or enhancers) of the following genes are at least partially specific for neurons: ADORA2A, A TP6V1C2, AVP, C8ORF46, CARTPT, CCKBR, CCL27, CD68, CLDN5, CRH, CX3CR1, DBH, DCX, DDC, DRD1, FEV , FEZF2, GABRA6, GAL, GCHFR, GFAP, GPR88, GPX3, GRP, HAP1, HBEGF, HCRT, HSPA12B, HTR1A, ICMT, LC T, MKI67, NR2E1, NTSR1, OLIG1, OXT, PCP2, PITX3, PKP2, POGZ, RAMP3, RGS16, RLBP1L2, S100B, SLC6 A2, SLC6A3, SLC6A4, SLC6A5, SLC7A5, SLITRK6, TAC1, TAC3, TBR1, THY1, TNNT1, TRH, UGT8, VIM and VIP.

[0251] In some embodiments, the transcriptional control region of the AAV vector of the present disclosure that expresses PGRN protein or a variant thereof (such as PGRNΔ3) in transduced neurons may comprise or consist of the following: the promoter region of the human synapsin 1 gene (SYN1), which has been shown to confer neuronal cell-specific transcriptional regulation; or a subsequence thereof that retains such neuron-specific transcriptional regulation. The promoter region of the human synapsin 1 gene is further described in detail in the following literature: for example, Thiel, G et al., Characterization of tissue-specific transcription by the human synapsin I gene promoter, PNAS 88: 3431-3435 (1991), Schoch, S et al., Neuron-specific Gene Expression of Synapsin I, JBC 271 (6): 3317–3323 (1996). In some embodiments, the transcriptional control region may comprise, consist of, or further comprise the human synapsin 1 gene (SYN1) promoter, which in some embodiments comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6 or a neuron-specific transcriptional function subsequence thereof.

[0252] A variety of brain or neuron (or other brain cell type) specific transcriptional control regions for gene therapy vectors (e.g., AAV, adenovirus, or lentivirus vectors) have been generated by modifying enhancers and / or promoters from brain or neuron (or other brain cell type) specific genes, for example by reducing their length, any of which can be used in the AAV vectors of the present disclosure to express PGRN protein or a variant thereof, such as PGRNΔ3, in transduced cells in the brain (such as neurons or other brain cell types). Non-limiting examples of such brain or neuron (or other brain cell type) specific transcriptional control regions include the transcriptionally functional portion of the human synapsin 1 gene promoter (Kügler, S. et al., Mol. Cell Neurosci., 17:78-96, 2001; Kügler, S. et al., Gene Ther., 10:337-47, 2003; Hioki, H. et al., Gene Ther., 14:872-82, 2007; Portales-Casamar, E. et al., Proc. Natl. Acad. Sci. USA, 107:16589-94, 2010; Jackson, K. et al., Front. Mol. Neuro., 9:1-11, 2016; Massaro, G. et al., Hum. Mol. Genet., 29:1933-49, 2020; Finneran, D. et al., Front. Neurol., 12:1-12, 2021; Radhiyanti, P. et al., Neurosci. Lett., 756:1-6, 2021; U.S. Patent No. 7,341,847).

[0253] Transcription termination signal sequence

[0254] In some embodiments, the AAV vectors of the present disclosure include vectors containing a transcriptional terminator sequence that is located 3' to the transgene from the perspective of the encoding (plus) strand single-stranded DNA vector. In some embodiments, another sequence (such as a 3' untranslated region (UTR) sequence) may be located between the transgene sequence and the transcriptional terminator sequence (Proudfoot, N., Genes Dev., 25: 1770-82, 2011; Kuehner, J. et al., Nat. Rev. Mol. Cell Biol., 12: 283-94, 2011; Porrua, O and Libri, D., Nat. Rev. Mol. Cell Biol., 16: 190-202, 2015).

[0255] In some embodiments, particularly when the transgene contains a protein-coding sequence (rather than a sequence of RNA having some function other than encoding a protein), the transcription terminator sequence can be a polyadenylation signal sequence (variously abbreviated as "poly A," "pA," "poly(A)," or "p(A)"). In some embodiments, the pA signal sequence can be derived from a naturally occurring gene and used in the vector, while in other embodiments, the pA signal can be modified, such as by reducing its length or altering its sequence compared to its natural counterpart to make it more efficient at transcription termination. In other embodiments, the pA signal can be a hybrid sequence, combining pA sequences from different genes, or can be synthetic.

[0256] Non-limiting examples of pA signals that can be used in the vectors of the present disclosure include the pA signal from the bovine growth hormone gene (bGH pA); human, mouse or rabbit β-globin genes; SV40 late genes; sNRP1; spA; herpes simplex virus thymidine kinase gene (HSV TK); or adenovirus type 5 L3 polyadenylation site, among which others are also possible. In other embodiments, transcription terminators used in the vectors of the present disclosure include those that terminate RNA transcripts without directing polyadenylation, such as the histone H4 gene mRNA 3' end processing signal (Whitelaw, E et al., Nucleic Acids Res, 14:7059-70 (1986)).

[0257] In some embodiments, the AAV vectors of the present disclosure comprise a vector comprising a transgene, the transcription of which is terminated by comprising a poly(A) site: a poly(A) site derived from the bovine growth hormone gene (bGH), in some embodiments, the poly(A) site comprising or consisting of the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10; a poly(A) site from the SV40 virus, in some embodiments, the poly(A) site comprising or consisting of the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:27; or a poly(A) site from the rabbit β-globin gene, in some embodiments, the poly(A) site comprising or consisting of the nucleotide sequence of SEQ ID NO:28.

[0258] Other vector genome elements

[0259] In addition to the transcription control region and the transcription termination signal, the genome of the AAV vector of the present disclosure may also contain other sequences (including cis-regulatory elements) to improve, control or regulate transgene expression and / or translation in the transduced cells, or to confer other functions to the vector. Such elements include, but are not limited to, untranslated regions from the 5' and / or 3' ends of the gene, non-coding exons, introns, splice donor and acceptor sites, lox sites, internal ribosome entry sites (IRES), sequences encoding 2A peptides, elements that stabilize RNA transcripts, binding sites for regulating miRNAs, microRNA (miRNA) sequences, elements that enhance mRNA nuclear export, including viral post-transcriptional regulatory elements, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and any other elements that have been empirically shown to improve transgene expression, even if the mechanism may not be certain. In other embodiments, the vector may contain so-called stuffing or filler sequences, which are intended only to increase the total length of the vector to the desired size, for example, to a length that is close to but still below the packaging capacity of a particular capsid, thereby reducing the possibility of accidentally packaging truncated vector or non-vector DNA into the capsid.

[0260] In some embodiments, introns may be included in the vector to increase transgenic expression and / or transcript stability. In some embodiments, a transgenic protein is provided, wherein the sequence of exons and introns is identical to that of naturally occurring genes. However, in genes with multiple exons and introns, one or more introns in these introns can be removed to minimize the total length, thereby being convenient to include other elements without exceeding capsid packaging capacity. However, in other embodiments, introns can be provided by genes completely different from the genes providing the coding sequence of the vector transgenic. Regardless of whether introns and transgenic are from identical or different genes, introns can be modified from their original sequence, such as by changing certain nucleotides, or removing internal sequences to reduce their total length, while maintaining the splicing donor and acceptor sequence motifs required for effective splicing, or other intron cis elements important to function (such as, enhancers that may be present in the original unmodified intron sequence). Introns can also be hybrid, where the splice donor portion of an intron from one gene is paired with the splice acceptor portion of an intron from a different gene; or synthetic, where the sequence does not correspond to an intron from any known gene. In some embodiments, the intron may be located within the coding sequence of the transgene and thus interrupt the coding sequence of the transgene (and have the donor and acceptor sites required for efficient splicing to occur), while in other embodiments, the intron is present but does not interrupt the protein coding sequence, but is located 5' or 3' to the coding sequence. In cases where the intron does not interrupt the coding sequence, it may have some exonic sequence carried over from its original genetic background, as long as the exonic sequence does not contain cryptic translation initiation signals. In some embodiments, the intron may be located 3' to the promoter (from the perspective of the positive-strand ssDNA vector) and 5' to the coding sequence. In other embodiments, the intron may be located distal to the coding sequence in the vector, either upstream or downstream.

[0261] Non-limiting examples of introns that can be used in the AAV vectors of the present disclosure include the small intron from minute virus of mice (MVM) (Haut, D. and Pintel, D., J. Virol., 72:1834-43, 1998; Haut, D. and Pintel, D., Virology, 258:84-94, 1999); the internal deletion intron 1 from human coagulation factor IX (FIXm1 and FIXm2) (Kurachi, S. et al., J. Biol. Chem., 270:5276-81, 1995); the chimeric beta globin splice donor and immunoglobulin heavy chain splice acceptor introns (GenBank Accession No. 200100103); U47120.2 nucleotides 890-1022); intron 1 from the mouse alpha globin gene; and the SV40 small t antigen intron, which may comprise or consist of base pairs 4644 to 4552 of GenBank record J02400.1 and may be modified at positions 4582 (g to c), 4580 (g to c), 4578 (a to c), and 4561 (a to t) (Nathwani, A. et al., Blood, 107:2653-61, 2006).

[0262] In some embodiments, a post-transcriptional regulatory element (PRE) can be included in the vector to increase transgene expression. Examples of PRE include woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), hepatitis B virus post-transcriptional regulatory element (HPRE), and modifications thereof (Donello, J. et al., J. Virol., 72:5085-92, 1998; Loeb, J. et al., Hum. Gene Ther., 10:2295-305, 1999; Zanta-Boussif, M. et al., Gene Ther., 16:605-19, 2009; Patricio, M. et al., Mol. Ther. Nucleic Acids, 6:198-208, 2017; U.S. Published Patent Application 2018-0353620A1). In some embodiments, from the perspective of a single-stranded DNA vector encoding a (plus) strand, at least one WPRE sequence can be located downstream of the transgene (thus, 3' to the stop codon of the transgene encoding the polypeptide) and upstream of the poly(A) signal sequence. In some embodiments, multiple PREs can be included, such as two, three, or more PREs of the same or different types, which can be arranged in tandem. In some embodiments, the AAV vectors of the present disclosure comprise a vector containing a WPRE element, and in some embodiments, the WPRE element comprises or consists of the nucleotide sequence of SEQ ID NO: 29.

[0263] AAV inverted terminal repeats (ITRs)

[0264] Located at the end of the adeno-associated virus genome is a unique nucleotide sequence known as an inverted terminal repeat (abbreviated as "ITR"), which serves as both the origin of viral DNA replication and as a priming site to support the conversion of the single-stranded (ssDNA) genome into a double-stranded form (dsDNA) in infected cells. The double-stranded form (dsDNA) has the ability to support the transcription of the rep and cap protein-encoding genes also contained in the viral genome. ITR also serves to package the replicated ssDNA genome into the AAV capsid. AAV ITR contains a polypalindromic sequence that can fold back on itself by intrachain complementary base pairing, thereby forming a dsDNA T-shaped hairpin secondary structure.

[0265] As further explained below, the vectors in the AAV vectors of the present disclosure may comprise one or more AAV ITRs that function similarly to their function in the unmodified virus. Unless otherwise indicated, the term "inverted terminal repeat sequence" or "ITR" as used herein includes intact, full-length ITRs, as well as ITRs with modified sequences (such as truncations of one or more nucleotides, internal deletions (such as internal deletions of trs or D sequences), additions, and substitutions) that retain one or more of the functions attributable to ITRs (even if less efficiently than intact ITRs of the same type), including but not limited to rescue of the vector from recombinant DNA (such as a plasmid), vector replication, and / or packaging of the vector into assembled capsids.

[0266] When present in packaged viruses and vectors, the ITR located at the 3' end of the ssDNA genome will have a free 3' hydroxyl group, while the ITR located at the opposite 5' end of the ssDNA genome will have a free 5' end. The 5' ITR may also be referred to as the "left" ITR, and the 3' ITR may also be referred to as the "right" ITR. However, in the context of a plasmid (such as that used for vector production), the vector sequence will be present in a double-stranded form, so that there will be two sets of 5' and 3' ITRs each. Therefore, to avoid ambiguity, the strand on which the ITRs are located should be specified to distinguish them. In the absence of such a specification, reference to the ITRs of a double-stranded vector (such as in a plasmid) is with respect to the plus or sense strand, i.e., the DNA strand on which the sequence of the transgene is identical to the coding sequence of the polypeptide product of the transgene, or, if the transgene does not encode a protein, the DNA strand on which the sequence of the functional RNA is identical.

[0267] In wild-type AAV2, the ITR is 145 bases long, of which the terminal 125 bases contain a palindrome sequence. When annealed, the AAV2 ITR contains two double-stranded palindromes, B-B' and C-C' (forming the arms of the hairpin), which are connected to a larger double-stranded palindrome, A-A' (forming the stem of the hairpin). The ITR also contains a D sequence towards the non-terminus of the ITR, which has no complementary sequence within the ITR and therefore remains single-stranded, but has a complementary sequence in the D region of the ITR at the opposite end of the genome (thus, having D and D'). The A-A' stem structure contains a Rep binding element (RBE) containing a tetranucleotide repeat motif, and the large AAV Rep protein binds to these tetranucleotide repeat motifs to introduce sequence-specific and strand-specific nicks at the terminal dissociation site (trs) in the ITR sequence (between nucleotides 124 and 125 in the AAV2 ITR, counting from the 3' end), a step required for DNA replication of the viral genome to occur.

[0268] When they renature, ITRs can fold into two configurations, called flip and flop, in which the sequence between the A and A' inverted repeats exists as the reverse complement sequence relative to the other configuration. For the 5' ITR (left ITR), the order of the terminal palindrome sequence of the flip configuration is 5'-ABB'CC'A'D-3', and the order of the flop configuration is 5'-ACC'BB'A'D-3'. For the 3' ITR (right ITR), the order of the terminal palindrome sequence of the flip configuration is 3'-A'B'BC'CAD'-5', and the order of the flop configuration is 3'-A'C'CB'BAD'-5'. Thus, the flip configuration has a B'B palindrome closest to the free 3' end, while the flop configuration has a C'C palindrome closest to the free 3' end (Lusby, E. et al., J. Virol., 34:402-9, 1980; Srivastava, A. et al., J. Virol., 45:555-64, 1983; Samulski, R. et al., Cell, 33:135-43, 1983).

[0269] It is speculated that the ITR secondary structure supports viral DNA replication through a self-priming single-strand displacement extension mechanism initiated by endogenous cellular DNA polymerase at the ITR with a free 3' hydroxyl group. Chain extension leads to the formation of a monomeric dsDNA genome replication intermediate with a covalently closed end. The duplex ITR refolds (isomerizes) into a double hairpin structure at the open end, forming a new 3' ITR, which extends when the complementary chain is displaced. The large AAV Rep protein binds to the ITR at the closed end (downstream) and cuts the DNA at its terminal dissociation site, thereby initiating a second DNA replication complex that copies the downstream ITR before the DNA replication complex initiated at the open end reaches the downstream ITR. The original replication complex displaces the opposite strand (whose ITR has just been newly synthesized) and completes replication to the once closed end of the genome, now the open end, where the duplex ITR can be used to isomerize into a double hairpin. Therefore, the monomeric dsDNA genome replication intermediate is regenerated to restart the replication cycle, and the displaced ssDNA genome (whose 3' ITR is newly generated) can be packaged into viral particles.

[0270] The replicated ssDNA genome will include both positive (positive or sense) and negative (negative or antisense) strand polarity, and evidence suggests that they are individually packaged into capsids with equal efficiency. Thus, preparations of AAV vector particles, like the viruses from which they are derived, may in some embodiments contain approximately equal proportions of sense or antisense ssDNA genomes. However, the ITRs may also be modified by selectively removing the D sequence from one of the two ITRs used to generate the AAV vector, thereby restricting packaging to either the negative or positive strand of the vector (Wang, XS et al., J Virol, 70(3):1668-77 (1996)). Thus, in some other embodiments, preparations of AAV vectors may contain vector particles in which most or substantially all of the vector is positive or negative strand.

[0271] Following infection, AAV virions are transported to the cell nucleus, where the ssDNA genome is released from the capsid. It has been hypothesized that before the viral rep and cap genes can be expressed, the ssDNA genome must first be converted to dsDNA by complementary strand synthesis initiated by cellular DNA polymerases at the 3' ITR, a process considered slow and inefficient. It has also been hypothesized that a faster mechanism for the formation of intracellular dsDNA genomes may exist, in which complementary positive and negative ssDNA genomes originating from different virions infecting the same cell encounter each other in the nucleus and hybridize through intermolecular base pairing. Such duplex genomes can then support transcription without the need for initial extension by cellular DNA polymerases.

[0272] When designing an AAV vector, the only AAV viral DNA sequences that are retained in the vector are the ITRs, as they play a key role in DNA replication and packaging during production and in the conversion of the ssDNA genome to dsDNA after transduction. Sequences encoding the Rep and Cap proteins, as well as viral helper functions (which are also required for vector production), can be provided in trans in a variety of ways known in the art. When a vector sequence (such as might be contained in a plasmid for AAV vector production) contains two intact ITRs and does not exceed approximately 5 kb of AAV capsid packaging capacity in length, as described above, the ssDNA genome can be packaged, but the need for this dsDNA conversion step may result in lower transduction efficiencies than would be expected due to the low efficiency of dsDNA conversion before gene expression can occur. One strategy to overcome the need for dsDNA conversion by endogenous cellular DNA polymerases and potentially improve the transduction efficiency and expression of heterologous sequences, such as therapeutic transgenes, has been to rely on replicating and packaging “self-complementary” AAV vectors (scAAVs), which contain both positive- and negative-strand sequences in the same DNA and can therefore rapidly anneal to form dsDNA transcription templates via intramolecular base pairing (i.e., intramolecular hybridization) after capsid uncoating in the target cell nucleus.

[0273] There are at least two ways to promote the production of self-complementary genomes, both of which rely on the inability of Rep to cleave the ITR at the terminal dissociation site during the replication of the DNA molecule to become the genome. In the natural replication cycle of AAV, a dimeric dsDNA genome replication intermediate molecule appears when Rep is unable to cleave at the terminal dissociation site of the downstream ITR of a monomeric genome replication intermediate with a double hairpin at the open end and a single downstream ITR at the closed end. When this occurs, replication by the initial DNA replication complex (starting from the 3' ITR at the open end) continues until the downstream (closed end) ITR and the displaced strand, forming a dimeric dsDNA genome. This molecule is similar to the monomeric dsDNA genome replication intermediate described above, but contains two genomes. Because it has an open end with a duplex ITR, it can isomerize to initiate the DNA replication cycle as a monomer. Alternatively, the closed-end hairpin can undergo terminal dissociation, forming a duplex ITR, which can isomerize, allowing DNA synthesis to initiate from the dissociated end. In either case, replication of the dimeric template generates a new dimeric dsDNA genome replication intermediate and displaces the ssDNA dimeric inverted repeat genome containing the 5' ITR (viral genomic sequence of one polarity), the central ITR (viral genomic sequence of the opposite polarity), and the 3' ITR.

[0274] Normally, a ssDNA dimer inverted repeat viral genome will not be packaged because it exceeds the normal capsid packaging capacity. However, by designing the vector to reduce the length so that the ssDNA dimer inverted repeat genome does not exceed the AAV packaging capacity when formed, vector particles containing the self-complementary genome can be generated. In practice, vector preparations generated in this way can contain a mixture of particles in which a single scDNA genome or one or two monomeric ssDNA genomes are packaged, and the proportions of all these genomes can vary between preparations.

[0275] After packaging, the scDNA genome likely exists in a single-stranded form within the capsid (similar to ssDNA non-self-complementary genomes), but then rapidly anneals after capsid uncoating to form a dsDNA molecule with a covalently closed ITR at one end and two open ITRs at the other end, similar to the structure of conventional viral genomes after self-priming dsDNA transformation. Thus, while not wishing to be bound by theory, the main difference between so-called ssDNA and scDNA genomes is not the topological structure when encapsidated, but rather the topological structure that each type of genome may acquire after capsid uncoating and genome release within the transduced cell.

[0276] However, further modifications allow for greater control over the production of vector particles containing scDNA genomes. Specifically, by mutating or deleting the end-dissociation site from one ITR, such as in a plasmid containing the vector sequence used for vector production, single-stranded cleavage at that ITR can be inhibited or eliminated during the vector replication cycle. Thus, a replication complex initiating at the unmutated ITR would step through the mutated hairpin and return to the origin, generating a dimeric dsDNA genome replication intermediate, much like one in which end-dissociation of the wild-type ITR would not occur inadvertently. However, this intermediate would contain a closed wild-type ITR at one end, a mutated duplex ITR in the middle of the molecule, and a duplex open ITR capable of isomerization at the opposite end. This molecule could then undergo normal cycles of replication and strand displacement from the wild-type ITR at each end to produce a displaced daughter genome copy containing a 5' wild-type ITR, vector sequence of one polarity, a mutated ITR in the middle, vector sequence of the opposite polarity, and a wild-type ITR at the 3' end. If the heterologous sequence contains a protein-coding transgene, the scDNA genome will contain the coding sequence and its complement within the same DNA molecule. Although such genomes are thought to be packaged within the capsid as single-stranded sequences, the presence of numerous self-complementary sequences outside the ITRs suggests that they are capable of rapidly self-annealing to a double-stranded form within the nucleus of transduced cells, where they can support transcription of the heterologous sequence. scAAVs generated from constructs containing mutant ITRs have yielded over 90% scDNA genomes.

[0277] In an AAV2 capsid, which has a packaging capacity of approximately 4.7 kb, the ssDNA genome can accommodate approximately 4.4 kb of heterologous sequence, excluding ITR sequences, while the scDNA vector can accommodate approximately 2.2 kb. According to a specific non-limiting embodiment, the size of the genome construct used to generate the scAAV vector (such as may be contained in a plasmid for AAV vector production) is approximately 2,500 nucleotides long, including a heterologous sequence of approximately 2,200 nucleotides long plus two ITRs (one wild-type and one mutant). This will result in an scAAV genome of approximately 4,700 nucleotides long, which is less than the typical AAV capsid packaging capacity.

[0278] The ITR terminal dissociation site can be destroyed in various ways to promote the production of scAAV vectors. For example, the exogenous sequence can be inserted into the terminal dissociation site (trs) sequence itself, or into the adjacent sequence of the ITR, such as between the Rep binding element and the trs. Alternatively, the trs sequence can be partially or completely deleted. In other embodiments, the adjacent D region can be partially or completely deleted. In yet other embodiments, the nucleotides in the trs can be replaced with different nucleotides, thereby reducing the cutting frequency of Rep to trs. Other ways of making the ITR non-dissociable belong to the common skills in the art.

[0279] Further information on AAV virus and vector replication and scAAV design can be found in McCarty, DM et al., Gene Therapy, 10:2112-18 (2003); McCarty, DM et al., Gene Therapy, 8:1248-54 (2001); McCarty, DM, Molecular Therapy, 16(10):1648-56 (2008); U.S. Pat. No. 7,790,154.

[0280] In some embodiments, the AAV vectors of the present disclosure may contain one or more AAV ITRs that are derived from different AAV serotypes and variants, have different sequences and lengths, and are located at different positions within the genome. The sequences of the AAV ITRs used in the vectors of the present disclosure may be wild-type or modified. In other embodiments, the AAV ITR sequences may also be included as part of the vector sequence in plasmids and other types of vectors (such as baculovirus) for the purpose of introducing the vector sequence into a host cell to produce the vector.

[0281] ITRs from AAV2 are often used to generate AAV vectors, but alternative embodiments may include the use of ITRs from any AAV serotype or variant, including, for example, ITRs from AAV1, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or any other AAV serotype or variant known or yet to be discovered, as long as such ITRs are functional in terms of vector replication and packaging and transgene expression. The ITRs may also comprise modifications to the sequence of a wild-type ITR sequence or be completely synthetic. The modified ITRs may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a wild-type ITR sequence (such as an ITR sequence from AAV2). In some embodiments, the ITRs are modified by adding, deleting, and / or changing nucleotides to disrupt the terminal dissociation site (trs) and / or D sequence of the ITR, or to alter some other subsequence of the ITR. In some embodiments, the vector may comprise ITRs from different AAV serotypes or variants.

[0282] In some embodiments, the ITRs selected for vector production will be from the same serotype or variant as the capsid. For example, AAV2 ITRs can be used in conjunction with AAV2 capsids. However, in other embodiments, the vector can be pseudotyped or hybrid, meaning that a vector with ITRs from one serotype or variant can be packaged into a capsid from a different serotype or variant. For example, a vector with ITRs from AAV2 can be packaged into a capsid from AAV8 (or any other serotype or variant capsid). This pseudotype is often abbreviated as AAV2 / 8, where the number before the slash indicates the origin of the ITR and the number after the slash indicates the origin of the capsid.

[0283] According to some embodiments, the AAV vectors of the present disclosure may contain vectors with different numbers of ITRs. For example, the complete AAV viral genome typically has two ITRs, one at each of the 5' and 3' ends, and AAV vectors produced using complete ITRs and packaged with ssDNA genomes may also have two similarly positioned ITRs. However, as discussed above, vectors can be designed in such a way that their genomes contain three ITRs, two of which are located at the ends of the genome, as in viruses or conventional vectors, but the other is located in the middle or near the middle (complete or mutated), as in self-complementary vectors. However, it has also been observed that for some vectors, particularly when the genome length exceeds the average packaging capacity of the capsid, the 5' ITR may be truncated or completely deleted, presumably due to premature or variable packaging termination. Although defective interfering particles can be produced, it is also presumed that such particles can still support transgene expression due to the complementation of the originally incomplete genome (Kapranov, P. et al., Hum. Gene Ther., 23:46-55, 2012). Thus, in some embodiments, the AAV vector particles of the present disclosure may contain a genome comprising a single functional ITR, such as at the 3' or 5' end, and wherein at the opposite end is a non-functional truncated ITR, or no ITR sequence. Thus, in some embodiments of the AAV vectors of the present disclosure, the AAV ITR may be located at the 5' end of the genome, or at the 3' end of the genome, or at both the 5' and 3' ends of the genome, as well as at other locations.

[0284] Another source of heterogeneity that may occur in AAV vectors may be due to the presence of differences in flip and flop ITR configurations in any particular genome. Thus, for example, in some embodiments, any particular vector particle in a sample may contain a genome having ITRs in a flip configuration at both ends of the genome, or having ITRs in a flop configuration at both ends of the genome, or having a flip ITR at the 5' end and a flop ITR at the 3' end, or having a flop ITR at the 5' end and a flip ITR at the 3' end. When combined with the observation that single-stranded DNA genomes can exist as either a plus or minus strand, a sample of AAV vectors may contain eight possible configurations in similar or potentially different proportions. Unless otherwise indicated, the genomes of the AAV vectors of the present disclosure are not limited to any one of these configurations; any or all configurations may be packaged by such vectors.

[0285] In some embodiments, the AAV vector comprises complete, full-length ITRs at each of its 5' and 3' ends. Thus, for example, if both ITRs are from AAV2, such full-length ITRs will be 145 nucleotides long. However, in other embodiments, one or more of these ITRs may be truncated, deleting one or more terminal nucleotides relative to the typical full-length sequence of that type of ITR. Thus, for example, relative to the typical full-length sequence of that type of ITR, the ITR may lack at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more from its termini. Such truncated ITRs may be present in the genome packaged by the AAV vectors of the present disclosure, but may also be present in the vector sequences used to generate the AAV vectors, such as in plasmids. For example, it has been observed that if the sequence deleted in one ITR is retained in another ITR, the truncated ITR can still function to produce AAV due to its ability to self-repair (Wang, XS. et al., J. Mol. Biol., 250:573-80, 1995; Samulski, R. et al., Cell, 33:135-43, 1983).

[0286] In some non-limiting and purely exemplary embodiments, the genome of the AAV vectors of the present disclosure may contain one or more AAV ITRs comprising, consisting essentially of, or consisting of the nucleotide sequence of any one or more of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 21 (other ITR sequences are also possible), or the complement or reverse complement of any of the foregoing specifically enumerated sequences.

[0287] Sequence optimization

[0288] In some embodiments, one or more sequences within the AAV vector can be optimized to improve its functional characteristics relative to the starting reference sequence. For example, but not limited to, based on the genetic code degeneracy and codon usage preferences known to exist between different species and between proteins expressed at high or low levels in the same species, any protein coding sequence in the vector can be codon optimized relative to the wild-type sequence. For example, the codon adaptation index of a particular species can be used to identify such codon preferences. The codon adaptation index (CAI) is explained in more detail in Sharp, PM and Li, WH, Nucleic Acids Res, 15: 1281-95 (1987). In some embodiments, the coding sequence is human codon optimized, which means that the coding sequence is optimized based on human codon preferences. Various algorithms known in the art can be used to promote codon optimization. As is known in the art, different CAIs can be constructed based on analyzing which highly expressed genes (such as human genes). Exemplary human CAIs are reported in Haas, J et al., Current Biology, 6 (3): 315-24 (1996). If desired, protein coding sequences can also be codon-optimized for species other than human.

[0289] In order to improve protein expression levels, different codon optimization strategies have been proposed and implemented. For example, the most commonly used synonymous codons (i.e., codons encoding the same amino acid) can be replaced at every position where the codon does not occur. Alternatively, codon usage can be adjusted across the entire coding sequence to be proportional to the natural codon preference distribution of the host organism. In some embodiments, codon replacement is limited to codons that occur relatively rarely in highly expressed proteins in a species, e.g., as reflected in the CAI, with a frequency of 10% or less.

[0290] In some embodiments, the protein coding sequence to be expressed by the AAV vector of the present disclosure can be codon-optimized by replacing at least one rare codon with a more common synonymous codon. In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%, and in some embodiments 100%, of the rare codons in the protein coding sequence are replaced with more commonly used synonymous codons as reflected in the CAI (such as the human CAI). In some embodiments, rare codons are codons that occur at a frequency of less than or equal to 10%, 9%, 8%, 7%, 6% or 5%, as reflected in the CAI (such as the human CAI).

[0291] In some embodiments, a protein coding sequence to be expressed by an AAV vector of the present disclosure can be codon-optimized by replacing one or more codons with more frequently used synonymous codons as reflected in a CAI (such as a human CAI) such that the CAI value calculated for the entire coding sequence is increased relative to the starting non-codon-optimized sequence (which in some embodiments is the wild-type coding sequence for the protein). Thus, in some embodiments, the CAI value of the starting reference sequence is calculated with reference to a specific CAI reference table, and one or more codons are replaced with more frequently used synonymous codons such that the overall CAI value of the now codon-optimized coding sequence is increased by at least or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.55, 0.60, or 0.70.

[0292] As is known in the art, the presence of hypomethylated CpG dinucleotides in nucleic acids can stimulate an immune response, thereby eliminating transduced cells. Therefore, depleting CpG dinucleotides in a vector can increase the likelihood that vector transduction will result in long-term gene expression. Wright, JF, Mol Ther, 28(3): 701-3(2020). In view of the potential harmful effects of CpG dinucleotides, in some embodiments, any sequence in the genome, including, for example, enhancers, promoters, introns, open reading frames encoding proteins or functional RNAs, transcription terminators, 5' and / or 3' untranslated region (UTR) sequences, ITRs, or any other sequences, can be modified to remove one or more CpG dinucleotides, as long as doing so does not unacceptably interfere with or destroy some desired functions of the modified elements. Since the functions of certain elements (such as ITRs, promoters, and enhancers) in a vector may be highly dependent on the identity of specific nucleotides in certain positions, the chances of significantly depleting such elements of CpG dinucleotides may be more limited. Because AAV vectors of both polarities (e.g., sense and antisense relative to the coding sequence of the transgene within the genome) are packaged into capsids in approximately equal proportions, in some embodiments, CpG depletion strategies can involve reducing or eliminating CpG motifs from the nucleotide sequences of vectors of both polarities, rather than just from the nucleotide sequences of vectors containing protein-coding sequences in the sense orientation.

[0293] In some embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the CpG dinucleotides in the coding sequence or the entire vector sequence (relative to the sense and / or antisense strand) are deleted or replaced relative to the reference starting sequence. In other embodiments, at least 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more CpG dinucleotides, or ranges between any of the foregoing values, are deleted or replaced relative to the starting reference sequence. In other embodiments, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, 45 to 50, 50 to 55, 55 to 60, 60 to 65, 65 to 70, 70 to 75, 75 to 80, 80 to 85, 85 to 90, 90 to 95, or 95 to 100 CpG dinucleotides are deleted or substituted relative to the reference starting sequence.

[0294] In other embodiments, sequence optimization can increase or decrease the total GC content relative to the starting reference sequence. Thus, in some embodiments, the total percentage of G or C nucleotides in the transgene or the entire genome can be increased by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, or 40% or more relative to the starting reference sequence (such as the wild-type protein coding sequence). In other embodiments, the total percentage of G or C nucleotides in the transgene or the entire genome can be reduced by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 35%, or 40% or more relative to a starting reference sequence (such as a wild-type protein coding sequence).

[0295] As will be appreciated by those of ordinary skill in the art, when optimizing coding sequences, the goal of replacing any particular amino acid in a species (such as humans) with a more common codon may be incompatible with other optimization strategies because the introduction of more commonly used codons may introduce CpG motifs, or elimination of CpGs may require the use of rarely occurring codons, or codon optimization may increase or decrease GC content in an undesirable manner. In these cases, it may be necessary to design and test different optimized coding sequences (encoding the same polypeptide) to identify a form that achieves an acceptable balance between the different optimization strategies, thereby achieving improved protein expression.

[0296] In addition to codon bias and CpG content, transgene and vector sequences can also be optimized by altering features. For example, any of the following features that sometimes occur in sequences and negatively impact transgene expression can be identified (conceptually (such as by using an algorithm) or empirically) and altered or eliminated to reduce their impact: cryptic splice sites; premature transcription termination signal sequences (e.g., poly A sequences); translation initiation sites other than the expected initiator methionine (e.g., IRES); sequence regions with high GC content; sequences at the 5' end of the mRNA that can form a hairpin; and AU-rich elements (AREs) in the 3' untranslated region of the mRNA that can be bound by destabilizing RNA-binding proteins. Other sequence features that can be present in transgenes and vectors and that, when altered, can enhance transgene expression will be familiar to those of ordinary skill in the art.

[0297] In other embodiments, the transgene or vector sequence can be modified to enhance functionality. For example, the first expected start codon in a protein coding sequence may only weakly support translation initiation from that site. In this case, the surrounding sequence can be altered to match the so-called Kozak consensus sequence for translation initiation in eukaryotes. Kozak, M., Gene, 234(2):187-208 (1999).

[0298] In some embodiments, CpG depletion (partial or complete) or other types of sequence optimization (such as codon optimization) of a transgenic coding sequence can improve protein expression from the transgene compared to the same vector comprising a non-optimized reference starting sequence (such as the wild-type coding sequence from which the optimized sequence was derived). Thus, for example, the optimized coding sequence of a transgene can be expressed with an efficiency of at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more compared to a non-optimized reference starting sequence (such as the wild-type form of the coding sequence).

[0299] AAV capsid

[0300] The AAV vectors of the present disclosure can utilize any AAV capsid, whether naturally occurring, modified, or engineered, including those currently known or yet to be discovered or developed, that is suitable for transducing cells in a subject to transgeneically express PGRN protein or a variant thereof from the vector.

[0301] The choice of which capsid to use when designing and producing AAV vectors (and the corresponding cap gene sequences used in their production) can be guided by many considerations and factors. As described above, different AAV capsids can have different cell or tissue tropisms due to specific interactions with certain cell surface receptors, which can be an advantage when it is desired to preferentially transduce certain tissues rather than other tissues. For example, in order to preferentially express a transgenic product in the brain (such as in neurons), a capsid with a greater tropism for neurons than, for example, muscle or liver can be used to design and produce a vector. Conversely, in order to express a transgenic product in hepatocytes, a capsid with a greater tropism for hepatocytes than for neurons, muscle or other tissues can be used to design and produce a vector.

[0302] Other factors may also be important. For example, some individuals have been reported to have high neutralizing antibody titers against certain capsids due to exposure to naturally occurring AAV, which may interfere with the ability of AAV vectors with the same or similar capsids to transduce target cells. Therefore, when designing vectors for gene therapy, the choice of capsid may in some cases be guided by the immunogenicity of the capsid and / or the seroprevalence of the patients to be treated. Other considerations that may influence capsid selection include manufacturability and stability during storage, as well as other relevant guiding factors known in the art.

[0303] The AAV vectors of the present disclosure can use capsids made of capsid proteins from naturally occurring AAVs as well as modified or engineered capsid proteins. For example, naturally occurring capsid proteins can be modified by inserting or deleting amino acids or peptides or introducing amino acid substitutions in the VP1, VP2 and / or VP3 protein sequences to improve capsid function in some way, such as tissue tropism, immunogenicity, stability or manufacturability. Other examples include new capsids with improved properties, which are generated by exchanging amino acids or domains from one known capsid to another (e.g., mosaic or chimeric capsids), or using DNA shuffling and directed evolution methods to discover capsid protein sequences with desired properties.

[0304] In some embodiments, the AAV vectors of the present disclosure may comprise capsids from known AAV serotypes and variants, as well as non-naturally occurring capsids, including but not limited to AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5, and AAV2i8, among many other capsids. In some embodiments, the capsids of the AAV vectors of the present disclosure comprise VP1, VP2, and / or VP3 AAV capsid proteins that are variants or derivatives of known VP1, VP2, or VP3 AAV capsid proteins. In some embodiments, the amino acid sequence of such variant or derivative AAV capsid protein may be at least or about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.10%, 99.11%, 99.12%, 99.13%, 99.14%, 99.15%, 99.16%, 99.17%, 99.18%, 99.19 ... 7%, 99.8% or 99.9% identical to the known AAV capsid VP1, VP2 or VP3 protein sequences, including but not limited to the following AAV capsid VP1, VP2 or VP3 proteins: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5 and AAV2i8, or any other suitable AAV capsid, including, for example, the neurotropic capsids discussed below.In some other embodiments, the amino acid sequence of such variant or derivative AAV capsid protein differs (whether by deletion, insertion, or substitution of amino acids) from the known AAV capsid VP1, VP2, or VP3 protein amino acid sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids, the known AAV capsid VP1, VP2 or VP3 protein amino acid sequence includes but is not limited to the following AAV capsid VP1, VP2 or VP3 proteins: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh10, AAVrh74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5 and AAV2i8, or any other suitable AAV capsid, including, for example, the neurotropic capsids discussed below.

[0305] Neurotropic AAV capsid

[0306] In some embodiments, the AAV vectors of the present disclosure include neurotropic capsids. Neurotropic capsids are AAV capsids that have tropism for neurons. In some embodiments, such neurons may be neurons in the brain, including neurons in certain areas of the brain, such as but not limited to the cerebral cortex, including the cerebral cortex of the frontal lobe, temporal lobe, parietal lobe or occipital lobe of the brain, or other areas such as the basal ganglia, thalamus, hippocampus, limbic system, olfactory bulb, retina, midbrain or brainstem, and other brain regions are also possible. In other embodiments, such neurons may be neurons in the spinal cord, peripheral nervous system or enteric nervous system. In other embodiments, the AAV vectors of the present disclosure include capsids that have tropism for cells (except neurons) in or outside the CNS, non-limiting examples of these cells include astrocytes, microglia, oligodendrocytes or ependymal cells, and other cell types are also possible. In some non-limiting embodiments, the AAV vectors of the present disclosure include capsids that have tropism for neurons (such as those present in the human brain). As will be understood by one of ordinary skill in the art, neurotropism does not necessarily mean that the capsid is capable of transducing only neurons to the exclusion of non-neuronal cells. Rather, a neurotropic capsid is one that exhibits some greater propensity to transduce neurons rather than some other cell type, even if that propensity is not absolute, or even if the capsid in question exhibits a greater propensity to transduce non-neuronal cell types rather than neurons. In the latter case, one might call the capsid neurotropic, even if that is not predominantly so.

[0307] Examples of neurotropic capsids include, but are not limited to: AAV1; AAV2; AAV4; AAV5; AAV7; AAV8; AAV9; AAV-rh.10; AAVv66 (Hsu, HL. et al., Nat. Commun., 11:3279, 2020); AAV-DJ (Grimm, D. et al., J. Virol., 82:5887-911, 2008); MNM008, MNM004, 9P31, 9P801, AAV-F, AAV-S, CAP-B10, CAP-B22, PHP.V1, AAV9-retro, T2 3Y+T+dH, AAV8 THR, AAV2.5, AAV-B1, AAV-AS (Bjorklund, T. and Davidsson, M., J. Parkinson's Dis., 11:S209-17, 2021); AAV2 HBKO (Sullivan, J. et al., Gene Ther., 25:205-19, 2018); AAV4.18 (Murlidharan, G. et al., J. Virol., 89:3976-87, 2015; Ojala, D. et al., Mol. Ther., 26:304-19, 2017); AAV2-retro (Tervo, D. et al., Neuron, 92:372-82, 2016); AAV-TT (Tordo, J. et al., Brain, 141:2014-31, 2018); AAV-PHP.B (Deverman, B. et al., Nat. Biotechnol., 34:204-9, 2016); AAV-PHP.S and AAV-PHP.eB (Chan, K. et al., Nat. Neurosci., 20:1172-9, 2017); AAV-CAP-B10 and AAV-CAP-B22 (Goertsen, D. et al., Nat. Neurosci., 25:106-15, 2022); AAV-SCH9 (Ojala, D. et al., Mol. Ther., 26:304-19, 2018); neurotropic capsid (US2022-0042044 and WO 2021 / 230987); and many other capsids, including an AAV capsid comprising a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1, and / or a VP2 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and / or a VP3 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3, which AAV capsid may be referred to herein as an AAV-801 capsid.

[0308] AAV vectors for expressing progranulin

[0309] According to certain embodiments, the AAV vector of the present disclosure comprises an AAV vector containing a transgene encoding a wild-type progranulin (PGRN), such as a human PGRN protein, or a naturally occurring or engineered variant thereof. In some embodiments, the transgene comprises a coding sequence for a signal peptide sequence, and a coding sequence for a mature form of the PGRN protein. In some embodiments, the signal peptide sequence is identical to the signal peptide sequence in the naturally occurring PGRN protein, but a signal peptide from a heterologous protein can also be used. In some embodiments, the mature human PGRN polypeptide comprises the amino acid sequence of SEQ ID NO: 18, and the amino acid sequence of the human PGRN signal peptide includes SEQ ID NO: 17. In some embodiments, the amino acid sequence of the wild-type human PGRN protein (including the natural signal peptide) includes the amino acid sequence of SEQ ID NO: 16.

[0310] In some embodiments, the AAV vectors of the present disclosure include AAV vectors comprising a transgene encoding a variant of a human PGRN protein, such as a variant comprising an insertion, deletion, and / or substitution of at least one amino acid compared to the amino acid sequence of a wild-type human PGRN protein (e.g., such as the amino acid sequence provided by SEQ ID NO: 16). In some embodiments, the transgene encodes a human variant PGRN protein having at least one (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more) amino acids deleted from the PGRN carboxyl terminus (C-terminus) of the human variant PGRN protein compared to the amino acid sequence of a wild-type human PGRN amino acid sequence (e.g., such as the amino acid sequence provided by SEQ ID NO: 16). In some embodiments, the transgene encodes a human PGRN variant protein having the last three C-terminal amino acids (QLL) present in the wild-type human PGRN protein deleted (PGRNΔ3). In some embodiments, the amino acid sequence of PGRNΔ3 is identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, C-terminal deletion variants of human PGRN, including PGRNΔ3 variants, have reduced binding to Sortilin (Zheng, Y. et al., PLOS One, 6:1-7, 2011).

[0311] In some embodiments, the nucleotide sequence of the transgene encoding the human PGRN protein is the wild-type coding sequence, which is the same as the coding sequence when present in the exons of a naturally occurring gene encoding the human PGRN protein (i.e., GRN) or in a cDNA sequence corresponding to an mRNA transcribed from a GRN gene and encoding the human PGRN protein. In some embodiments, the wild-type coding sequence for the wild-type human PGRN protein is provided by SEQ ID NO: 15, and the wild-type coding sequence for human PGRNΔ3 (but with the codons for the last three amino acids present at the C-terminus of the wild-type human PGRN deleted) is provided by SEQ ID NO: 8. However, in other embodiments, the coding sequence can be optimized, such as by deleting some or all CpG dinucleotides, while still encoding the same PGRN or PGRNΔ3 protein. 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, or more CpG dinucleotides (or any range encompassing any of the foregoing specifically recited values) are removed from SEQ ID NO: 15 or SEQ ID NO: 8 by replacing the codons in which the CpG dinucleotides occur with synonymous codons lacking the CpG dinucleotides. In some embodiments, the nucleotide sequence encoding the PGRN or PGRNΔ3 protein does not contain CpG dinucleotides. In some embodiments, the nucleotide sequence encoding the PGRN or PGRNΔ3 protein comprises 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 CpG dinucleotides (or any range encompassing any of the foregoing specific recited values) relative to SEQ ID NO: 15 or SEQ ID NO: 8, respectively. In some embodiments, CpG dinucleotides are identified relative to the order of nucleotides in the sense strand, while in other embodiments, CpG dinucleotides are identified relative to the order of nucleotides in the antisense strand.

[0312] In other embodiments, the nucleotide sequence of the transgene encoding the wild-type human PGRN protein is identical to the nucleotide sequence provided in SEQ NO: 15 (excluding the stop codon), and in other embodiments, the transgene is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the nucleotide sequence of SEQ NO: 15 and encodes an amino acid sequence identical to the amino acid sequence encoded by SEQ NO: 15 (i.e., SEQ ID NO: 16). In other embodiments, the nucleotide sequence of the transgene encoding the human PGRNΔ3 protein is identical to the nucleotide sequence provided in SEQ NO:8 (excluding the stop codon), and in other embodiments, the transgene is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the nucleotide sequence of SEQ NO:8 and encodes an amino acid sequence identical to the amino acid sequence encoded by SEQ NO:8 (i.e., SEQ ID NO:14).

[0313] In some embodiments, the vector of the AAV vector of the present disclosure further comprises at least one AAV inverted terminal repeat (ITR) located at the 5' and / or 3' end of the genome. In some embodiments, the vector may further comprise at least a second AAV ITR located at the end of the genome opposite to the first AAV ITR. In some embodiments, the vector comprises an AAV ITR located at its 5' end. In some embodiments, the vector comprises an AAV ITR located at its 3' end. In some embodiments, the vector comprises a first AAV ITR located at its 5' end and a second AAV ITR located at its 3' end. In some embodiments, the vector comprises a first AAV ITR located at its 5' end and a second AAV ITR located at its 3' end and a third AAV ITR located between the first AAV ITR and the second AAV ITR.

[0314] The AAV ITRs used in the vectors of the present disclosure can be of any type, such as AAV2 ITRs or non-AAV2 ITRs, and can be full-length or truncated, and can have the same sequence as any known AAV viral ITR as it exists in nature (wild-type sequence), or can be modified. Exemplary non-limiting types of modifications include reducing the number of CpG dinucleotides present in the ITR sequence, reducing or eliminating the ability of the ITR sequence to undergo terminal dissociation by the AAV Rep protein, such as by mutating, deleting, or otherwise inactivating the terminal dissociation site (trs), and reducing or eliminating the ability of the ITR to support packaging into the capsid, such as by mutating, deleting, or otherwise inactivating the D sequence of the ITR sequence. In some embodiments, the vector may further comprise at least a third AAV ITR, such as an AAV ITR located between the two ends of the genome (such as near or at the center of the vector sequence). In some of these embodiments, the third ITR may be modified, such as by inactivating its terminal dissociation site, so that the vector comprising the transgene is self-complementary. In some embodiments, the vector comprises an AAV ITR comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 21, or the complement or reverse complement of each of these sequences.

[0315] In some embodiments, the vector of the AAV vector of the present disclosure further comprises a transcriptional control region operably linked to a transgene encoding a PGRN or PGRNΔ3 protein. In some embodiments, the transcriptional control region can be inducible, constitutively active, or cell type or tissue type specific, such as being active primarily or only in neurons or the brain (thus being neuron-specific or brain tissue-specific). In some embodiments, the transcriptional control region comprises or consists of a promoter, and may also comprise at least one enhancer region or element. Any region or element in the transcriptional control region may be derived from a human gene or a non-human gene, such as a rat, mouse, cow, non-human primate, chicken, or viral gene, or other species or type of organism. The regions or elements of the transcriptional control region may be adjacent to each other or separated by other functional sequences of the vector. Thus, for example, the promoter region may be proximal and 5' (upstream) of the transgene, while the enhancer region or element may be anywhere else in the vector, such as distal upstream, or elsewhere, such as distal 3' (downstream) of the transgene. Any region or element of the transcriptional control region can be cell type or tissue specific, such as brain tissue or neuron (or other brain cell type) specific. Thus, the promoter can be brain tissue or neuron (or other brain cell type) specific, the enhancer region or element can be brain tissue or neuron (or other brain cell type) specific, or both the promoter and enhancer, acting alone or in concert, can be brain tissue or neuron (or other brain cell type) specific.

[0316] In some embodiments, the transcription control region of the vectors of the present disclosure may contain a promoter sequence derived from the human synapsin 1 (SYN1) gene, wherein the promoter is the entire human SYN1 gene promoter, or a promoter functional subsequence of the human SYN1 gene promoter. Thus, for example, the promoter may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 6, or a promoter functional subsequence, modification, or variant thereof.

[0317] In some embodiments, the vector of the AAV vector of the present disclosure further comprises a 5' untranslated region (UTR) from a gene located 3' of the promoter and 5' of the transgene encoding the PGRN or PGRNΔ3 protein. In some embodiments, the 5'UTR sequence is the entire 5'UTR sequence from the gene or a subsequence thereof. In some embodiments, the 5'UTR sequence is from the human synapsin 1 gene (SYN1), while in other embodiments, the 5'UTR sequence is from other human synapsin genes, or synapsin genes from other species, or from genes other than synapsin 1. In some embodiments, the 5'UTR sequence comprises, consists essentially of, or consists of SEQ ID NO: 7.

[0318] In some embodiments, the vector of the AAV vector of the present disclosure further comprises a transcription termination signal sequence, such as a polyadenylation (poly(A)) signal sequence, such as a poly(A) signal sequence derived from the bovine growth hormone (bGH) gene, or a transcription termination functional subsequence of the poly(A) signal sequence of such a bGH gene. Thus, for example, the transcription termination signal sequence may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a transcription termination functional subsequence, modification, or variant thereof.

[0319] In some embodiments, the vectors of the AAV vectors of the present disclosure further comprise additional functional sequences, such as introns, viral post-transcriptional regulatory element (PRE) sequences, such as woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or hepatitis B virus post-transcriptional regulatory element (HPRE), any of which may be located 3' to the transgene and 5' to the transcription termination signal sequence, or elsewhere in the genome. In some embodiments, the PRE may be a WPRE comprising, consisting essentially of, or consisting of the nucleotide sequence of SEQ ID NO: 29, or a functional subsequence, modification, or variant thereof. Other sequences that may be used in the vectors of the present disclosure include, but are not limited to, microRNA (miRNA) binding sites, which may be located 3' to the transgene and 5' to the transcription termination signal sequence, or elsewhere in the genome; and stuffing or filler nucleotide sequences, which, while not necessarily intended to directly affect the expression of the transgene (although such properties may exist), are added to bring the overall length of the vector to a specific size, for example, long enough to approach the packaging capacity of a particular AAV capsid, thereby reducing the amount of contaminating non-full-length vector genomic DNA packaged in the capsid.

[0320] In some embodiments, the vector of the AAV vector of the present disclosure further comprises a stuffing or filler sequence located 3' of the poly (A) signal sequence and 5' of the ITR. In some embodiments, the stuffing or filler sequence is derived from an intron of a gene, such as a gene encoding a TATA box binding protein (TBP) of a human or another species. In some embodiments, the stuffing or filler sequence is an intron of the TBP gene or a modification or variant thereof, such as the sequence provided by the nucleotide sequence of SEQ ID NO: 11.

[0321] In some embodiments, the vector of the AAV vector of the present disclosure comprises a first AAV ITR, a transcriptional control region, a 5'UTR sequence, a transgene encoding a human PGRN or PGRNΔ3 protein operably linked to the transcriptional control region, a transcription termination signal sequence, a stuffer sequence, and a second AAV ITR. In related embodiments, these elements may be arranged sequentially in a 5' to 3' order, as would appear in a single-stranded vector in the sense direction, or in the sense strand of a double-stranded DNA molecule comprising the vector sequence, such as in a plasmid for producing the vector in a host cell. Conversely, these elements may be arranged sequentially in a 3' to 5' order in an antisense direction in a single-stranded vector or in the antisense strand of a double-stranded DNA molecule comprising the vector sequence. Thus, for example, but not limited to, if a sense-oriented vector comprises a promoter, a transgene, and a poly(A) signal sequence in a 5' to 3' order, the complementary antisense vector sequence will comprise those same elements in reverse order starting from its 5' end, but it will be understood that the nucleotide sequence of the antisense strand genome read in the 5' to 3' direction will be the reverse complement of the nucleotide sequence of the sense strand genome. In the case of a self-complementary vector (scAAV), the arrangement of the elements will occur in 5' to 3' order in approximately half of its sequence, and then in 3' to 5' order in the complementary half.

[0322] In some embodiments, the vector of the AAV vector of the present disclosure comprises, in 5' to 3' order, a first AAV ITR from AAV2 at the 5' end of the genome, a transcriptional control region, a 5'UTR sequence, a transgene encoding a PGRN or PGRNΔ3 protein operably linked to the transcriptional control region, a transcriptional termination signal sequence, a stuffer sequence, and a second AAV ITR from AAV2 at the 3' end of the genome. In some embodiments, the transcriptional control region comprises a promoter, which may be neuron-specific, such as a promoter derived from the human synapsin 1 gene, which in some embodiments may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 6. In some embodiments, the 5'UTR is also derived from the human synapsin 1 gene, which in some embodiments may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 7. In some embodiments, the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene, and in some embodiments, it may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the stuffer sequence is derived from an intron from the TBP gene, and in some embodiments, it may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 11.

[0323] In any of the preceding embodiments, the nucleotide sequence of the transgene encoding the human PGRN protein can comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 15, or a nucleotide sequence that is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the nucleotide sequence of SEQ ID NO: 15 and encodes the same amino acid sequence as SEQ ID NO: 16. Alternatively, in any of the preceding embodiments, the nucleotide sequence of the transgene encoding the human PGRNΔ3 protein may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:8, or a nucleotide sequence that is at least or about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the nucleotide sequence of SEQ ID NO:8 and encodes the same amino acid sequence as SEQ ID NO:14.

[0324] In any of the foregoing embodiments, either or both of the first AAV2 ITR and the second AAV2 ITR can be full-length or truncated and can be in a flip or flop configuration. In any of the foregoing embodiments, either or both of the first AAV2 ITR and the second AAV2 ITR can comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 21, or the complement or reverse complement of each of such sequences.

[0325] In some embodiments, the vector of the AAV vectors of the present disclosure further comprises a third AAV ITR located between the first and second AAV ITRs, such as in the middle of the vector (even if not exactly in the middle), and in some embodiments, the third AAV ITR may have a mutated or altered terminal dissociation site that does not undergo terminal dissociation. In some of these embodiments, the vector may be self-complementary and may have a length ranging from about 3000 to 5000 or 4000 to 5000 nucleotides when packaged in a capsid, or may have a length ranging from about 1500 to 2500 or 2000 to 2500 nucleotides when its sequence is contained in a plasmid suitable for producing a scAAV vector in a host cell.

[0326] In any of the foregoing embodiments, the vector may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 19 or its reverse complement. In any of the foregoing embodiments, the vector may be single-stranded, meaning that it is not self-complementary (outside the ITRs), and may have a length ranging from about 3500 to 5000 nucleotides, about 3500 to 4700 nucleotides, about 3800 to 4500 nucleotides, about 3800 to 4300 nucleotides, about 3800 to 4100 nucleotides, about 3800 to 4000 nucleotides, about 3900 to 4000 nucleotides, about 3950 nucleotides, or about 3942 nucleotides. In any of the foregoing embodiments, the vector may be in a sense orientation or in an antisense orientation.

[0327] In any of the foregoing embodiments, the vector can be encapsidated by an AAV capsid, such as a neurotropic AAV capsid, non-limiting examples of which include capsids AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAV-rh.10, AAVv66, AAV-DJ, MNM008, MNM004, 9P31, 9P801, AAV-F, AAV-S, CAP-B10, CAP-B22, PHP.V1, AAV9-retro, T2 3Y+T+dH, AAV8 THR, AAV2.5, AAV-B1, AAV-AS, AAV2 HBKO, AAV4.18, AAV2-retro, AAV-TT, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, AAV-CAP-B10, AAV-CAP-B22 or AAV-SCH9 or AAV-801 capsid, which comprises the following proteins: a VP1 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 1, a VP2 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 2, and a VP3 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 3.

[0328] In some embodiments, the AAV vector of the present disclosure comprises an AAV-801 capsid encapsidating (packaging) an AAV vector in a sense or antisense orientation, wherein the genome in the sense orientation comprises, in 5' to 3' order, a first AAV ITR located at the 5' end of the genome, a neuron-specific transcriptional control region, a 5'UTR sequence, a transgene encoding human PGRNΔ3 protein operably linked to the transcriptional control region, a transcription termination signal sequence, a stuffer sequence, and a second AAV ITR located at the 3' end of the genome, wherein either or both of the AAV ITRs are AAV2 ITRs, wherein the promoter is derived from the human synapsin 1 gene, wherein the 5'UTR is also derived from the human synapsin 1 gene, wherein the transcription termination signal sequence is a poly(A) signal sequence derived from the bovine growth hormone (bGH) gene, wherein the stuffer sequence is a modified intron from the human TBP gene, wherein the nucleotide sequence of the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6, wherein the nucleotide sequence of the 5'UTR comprises SEQ ID NO: In certain related embodiments, the nucleotide sequence of the transgene encoding the human PGRNΔ3 protein comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 8, or a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO: 8 and encodes a polypeptide identical to the polypeptide encoded by SEQ ID NO: 8, wherein the nucleotide sequence of the poly(A) signal sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, wherein the nucleotide sequence of the stuffer sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 11, and wherein the genome in the antisense orientation comprises a nucleotide sequence that is the reverse complement of the nucleotide sequence of the vector in the sense orientation. In certain related embodiments, the nucleotide sequence of the vector in the sense orientation comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 19.

[0329] AAV vector production

[0330] As is known in the art, AAV vectors can be produced in a variety of ways, including large-scale production. For example, AAV vectors can be prepared in mammalian or insect cells and then purified. Traditional methods that do not rely on co-infection with a helper virus involve the use of three plasmids as discussed above. One plasmid contains genes for helper virus factors, a second plasmid contains the AAV genomic sequence in double-stranded form, and a third plasmid contains the AAV rep and cap genes. The rep / cap plasmid typically contains the rep gene from AAV2, but this is not necessary, and the cap gene sequence is selected based on which AAV capsid protein is desired to constitute the capsid. In practice, these three plasmids are usually replicated separately in bacteria, purified, mixed together in a solution at a predetermined ratio, and then mixed with a transfection agent. The transfection mixture is then used to transfect suitable mammalian host cells (in adherent or suspension cell culture) and the host cells are incubated for a sufficient time (e.g., 48 to 72 hours, etc.) under conditions sufficient to allow the host cells to express the helper factors and the rep and cap genes, and sufficient to allow the AAV vector to replicate and package into the capsid from its plasmid template. In some embodiments, the host cell is a HEK293 cell, which constitutively expresses the AdV helper factors E1A and E1B, so that the helper plasmid only needs to contain the AdV E2A, E4ORF6 and VA RNA genes. When using other mammalian host cells that do not produce AdV or other viral helper factors themselves, it is necessary to use a helper plasmid containing any helper factors that are missing or otherwise required. Although the so-called triple transfection method described above is often used, it is not required to provide the genes for the helper factors and the rep and cap genes on separate plasmids. In principle, all of these genes can be accommodated on a single plasmid, for example, in which case two plasmids can be used for transfection.

[0331] In order to find more efficient methods for large-scale production of AAV vectors, stable cell lines have been established that contain some, but not all, of the components that would otherwise need to be introduced into the cells by transient transfection. The packaging cell lines contain stably integrated AAV rep and cap genes. Producing AAV in packaging cells requires transient transfection of the cells with a plasmid containing the AAV vector and infection of the cells with a helper virus. AAV vectors can also be produced in packaging cells without transfection by first infecting the packaging cells with AdV (wild-type or E2b gene-deficient AdV), which provides the AdV E1 gene product (which can induce rep and cap expression in the cell) and the auxiliary factors required for AAV replication; and then infecting with a replication-deficient hybrid AdV, in which the AAV vector replaces the E1 gene in the genome of the hybrid virus.

[0332] In another option, the production cell line contains stably integrated AAV rep and cap genes and also contains an AAV vector. Producing AAV in production cells requires infecting them with a helper virus. Packaging cells and production cells have been described (Martin, J. et al., Hum. Gene Methods, 24: 253-69, 2013; Gao, G. et al., Hum. Gene Ther., 9: 2353-62, 1998; Clément, N. and Grieger, J., Mol. Ther. Methods Clin. Dev., 3: 16002, 2016). Other cell systems for producing AAV vectors (including production on a commercial scale) in mammalian cells are also possible.

[0333] A baculovirus system has also been used to produce AAV vectors, in which Sf9 insect cells are infected with recombinant baculovirus vectors containing various amounts of the AAV rep and cap genes, along with the AAV genome. The foreign gene is expressed, and the genome is then packaged into vector particles within the cell. In early versions of this system, each component, rep, cap, and genome, was carried by three separate baculoviruses. Later modifications were made, such as combining rep and cap into a single baculovirus, so that only two types of baculovirus were needed, and generating an Sf9 cell line containing stably integrated AAV rep and cap genes, which only needed to be infected with a single type of recombinant baculovirus containing the AAV vector (Urabe, M. et al., Hum. Gene Ther., 13: 1935-43, 2002; Virag, T. et al., Hum. Gene Ther., 20: 807-17, 2009; Smith, R. et al., Mol. Ther., 17: 1888-96, 2009; Mietzsch, M. et al., Hum. Gene Ther., 25: 212-22, 2014). Other cell systems for producing AAV vectors in insect cells (including production on a commercial scale) are also possible.

[0334] host cells

[0335] As used herein, "host cell" means a cell that is suitable or adapted for in vitro production of AAV vectors. Host cells are typically clonal cell lines that are able to divide for multiple generations before aging prevents growth or can even be immortal. In order to produce vectors, host cells can be modified transiently or non-transiently by introducing exogenous genetic information designed to direct the biosynthesis of various components required for AAV vector assembly (particularly AAV capsid proteins, Rep proteins, helper viral factors and vectors) in the host cell. For example, host cells can be transfected with exogenously provided nucleic acids (such as in the form of one or more DNA plasmids) containing nucleotide sequences encoding the desired vector components.

[0336] Various methods of transfecting host cells with nucleic acids are known in the art. These methods include, but are not limited to, mixing nucleic acids with certain compounds that can be complexed with nucleic acids and then taken into cells. These compounds include calcium phosphate or cationic organic compounds, such as DEAE-dextran, polyethyleneimine (PEI), polylysine, polyornithine, polybrene, cyclodextrin, cationic lipids, and other compounds known in the art. Non-chemical transfection can also be performed by electroporation and more specific techniques (such as gene gun particle delivery). As known in the art, transfection can be transient or stable. During transient transfection, the transfected nucleic acid exists in the cell for a limited period of time, and in the case of DNA, is not integrated into the genome. During stable transfection, the DNA introduced into the cell can exist as an episome plasmid or be integrated into the chromosome for a long time. Typically, in order to produce stably transfected cells, a plasmid containing a selection marker gene and a nucleotide sequence encoding one or more desired vector components is transfected into the cell, and the cell is then grown and maintained under selective pressure, i.e., under conditions where non-transfected cells are killed or transfected cells lose exogenous DNA (including its selection marker) for some reason. For example, the plasmid may contain an antibiotic resistance gene, and transfected cells can be selected by adding the antibiotic to the culture medium in which the cells are grown. In some embodiments, the nucleotide sequence encoding one or more desired vector components introduced into stably transfected host cells is under the control of an inducible promoter and is not expressed or is expressed only at a low level unless an environmental factor that induces the promoter, such as a drug, metal ion, or increased temperature, is introduced during cell growth.

[0337] In other embodiments, genetic engineering methods (such as knock-in or gene editing methods) can be used to modify the host cell genome in a non-transient and targeted manner to instruct the host cell to produce one or more desired vector components. In other embodiments, nucleotide sequences encoding one or more desired vector components can be introduced into the host cell by transduction to instruct the production of AAV vectors, in which the host cell is infected with a modified virus containing such nucleotide sequences. Examples of viral vectors that can be used for such purposes include adenovirus, retrovirus (including lentivirus), baculovirus, vaccinia virus, and herpes simplex virus, among which other viruses are also possible.

[0338] The host cell can be any type of cell known in the art that can be used to produce AAV vectors. The host cell is typically an animal cell, of which different types or species are also possible, such as insect cells or mammalian cells, including rat, mouse or human cells, of which other cells are also possible. In some embodiments, the host cell that can be used to produce the AAV vectors of the present disclosure is a mammalian host cell, examples of which include HeLa cells, Cos cells, HEK293 cells (and variants of HEK293 cells, such as HEK293E, HEK293F, HEK293H, HEK293T or HEK293FT cells), A549 cells, BHK cells, Vero cells, NIH 3T3 cells, HT-1080 cells, Sp2 / 0 cells, NS0 cells, C127 cells, AGE1.HN cells, CAP cells, HKB-11 cells, WI-38 cells, MRC-5 cells or PER.C6 cells, of which many other cells are also possible. In some embodiments, host cells useful for producing the AAV vectors of the present disclosure are insect host cells, examples of which include Sf9 cells, ExpiSf9, ​​Sf21 cells, S2 cells, D.Mel2 cells, Tn-368 cells, or BTI-Tn-5B1-4 cells, among which many other cells are also possible. In some embodiments, host cells (including but not limited to HEK293 cells and variants thereof) can be adapted to grow in suspension culture.

[0339] In order to produce AAV vectors, host cells are usually grown or maintained in culture under controlled conditions that are conducive to their growth and vector biosynthesis. For example, host cells can be grown in a liquid culture medium with a determined chemical composition that provides all nutrients required for cell growth and biosynthesis. Exemplary culture medium includes DMEM, DMEM / F12, MEM, RPMI 1640 for mammalian host cells and Express Five SFM, Sf-900 II SFM, Sf-900 III or ExpiSf CD for some insect cells. Such culture medium may be supplemented with antibiotics, growth factors or cytokines (recombinantly produced or present in animal serum such as FBS) that are known to stimulate the growth of specific cell types in use, as well as other ingredients that may be needed for the optimal biosynthesis of AAV vectors but that their supply may be limited. Exemplary supplements include essential amino acids, glutamine, vitamin K, insulin, BSA or transferrin. In addition to growth medium, other culture conditions may also be controlled to optimize the growth and / or productivity of cells, such as pH, temperature and CO and oxygen concentration.

[0340] Host cells in culture can be grown or maintained in a number of containers known in the art, such as stirred tank bioreactors, wave bags, spinner flasks, hollow fiber bioreactors, or roller bottles, some of which can be designed and configured for single use or multiple use. Depending on the characteristics of the host cell in question, the host cells can be grown in adherent cell culture, in which the cells are attached to and grow in contact with a physical matrix; or in suspension cell culture, in which single cells float freely in a culture medium that supports them, or are attached to bead microcarriers suspended in the culture medium. As is known in the art, various techniques have been developed and can be used to grow host cells to high cell densities, such as perfusion cultures, thereby increasing the total amount of AAV vectors generated per production run.

[0341] As is known in the art, host cell samples are typically maintained in frozen cell banks, such as master cell banks and working cell banks, which facilitate the production of biological products in multiple batches over time while ensuring consistent performance of the host cells. Before the creation of an AAV vector, a frozen sample of host cells from a cell bank is typically thawed, inoculated into a small culture volume, and grown to a higher density or quantity in a culture of increasing volume. When the host cells reach a desired cell density and / or volume in culture, exogenous genetic material can be introduced, such as by transfection with plasmid DNA or infection or transduction with a viral vector, so that they begin to produce an AAV vector. Alternatively, if a non-transiently modified host cell is used, in which the nucleotide sequence encoding one or more desired vector components is under inducible control, the environmental factors necessary for inducing expression can be introduced. The host cells can then be grown or maintained in culture for a certain period of time under conditions sufficient to allow the host cells to produce an AAV vector.

[0342] AAV vector purification

[0343] After biosynthesis in host cells, AAV vectors can be purified in a variety of ways known in the art. For example, in some embodiments, host cells can be lysed mechanically or chemically (such as with detergents), followed by removal of host cell DNA and other components, followed by steps such as density gradient centrifugation or use of one or more chromatographic separation methods to obtain highly purified preparations of AAV vectors for use in research or therapeutic methods.

[0344] Chromatographic methods for purifying AAV vectors include, but are not limited to, size exclusion chromatography (SEC); affinity chromatography, which uses any affinity ligand capable of specifically binding to the capsid, such as an antibody, lectin, or glycan, attached to a chromatography resin or matrix; immobilized metal chelate chromatography (IMAC); thiophilic adsorption chromatography; hydrophobic interaction chromatography (HIC); multimodal chromatography (MMC); pseudo-affinity chromatography; and ion exchange chromatography (IEX or IEC), such as anion exchange chromatography (AEX) or cation exchange chromatography (CEX).

[0345] In some embodiments, an antibody-based affinity chromatography can be used to purify an AAV vector, wherein the antibody or its antibody fragment is attached to a stationary phase (matrix or resin) loaded into a chromatographic column, and the host cell lysate is pumped through the chromatographic column, and then washed and eluted with the carrier bound to the antibody. The antibody bound to the solid phase can be IgG or its fragment, or a single-chain camel antibody (such as a heavy chain variable region camel antibody), and other types of antibodies are also possible. Non-limiting examples of ligand affinity resins include Sepharose AVB, POROS CaptureSelect AAVX, POROS CaptureSelect AAV8, and POROS CaptureSelect AAV9 (Terova, O. et al., BioPharm Intl. eBook pp. 27-35, 2017; Mietzsch, M. et al., Mol. Ther. Methods Clin. Dev., 19: 362-73, 2020; Rieser, R. et al., Pharmaceutics, 13: 748, 2021).

[0346] In other embodiments, AAV vectors can be purified using ligand chromatography, wherein the stationary phase has attached thereto the same type of ligand that is known to be used by certain AAVs when binding to cells, such as a glycan, sialic acid (e.g., O-linked or N-linked sialic acid), galactose, heparan, heparan sulfate, or a proteoglycan, such as a heparan sulfate proteoglycan or a heparan sulfate proteoglycan (HSPG). For example, an affinity matrix containing sialic acid residues can be used to purify AAV vectors having capsids that specifically bind to sialic acid (e.g., AAV1, AAV4, AAV5, or AAV6); an affinity matrix containing galactose can be used to purify AAV vectors having capsids that specifically bind to galactose (e.g., AAV9); and an affinity matrix containing heparin, heparan, or HSPG can be used to purify AAV vectors having capsids that specifically bind to HSPG (e.g., AAV2, AAV3A, AAV3B, AAV6, or AAV13).

[0347] Depending on the physicochemical characteristics of the vector, such as the charge on the capsid, the AAV vector can be further purified by performing anion exchange, cation exchange, or hydrophobic interaction chromatography. Other downstream processing steps useful for purifying AAV vectors, such as, but not limited to, desalting and buffer exchange, ultrafiltration, nanofiltration, diafiltration, and tangential flow filtration (TFF), can also be used. More than one downstream processing step can be used, and multiple downstream processing steps can be performed in any order, according to the knowledge of those skilled in the art.

[0348] Treatment

[0349] In addition to AAV vectors for expressing PGRN protein or variants thereof (such as PGRNΔ3) and compositions comprising such AAV vectors, the present disclosure also provides methods for treating subjects (such as human subjects) in need of treatment for frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD) or PGRN deficiency by administering to the subject a therapeutically effective amount of an AAV vector of the present disclosure or a composition comprising such an AAV vector. Also provided is the use of an AAV vector for expressing PGRN protein or variants thereof (such as PGRNΔ3) in the manufacture of a medicament for the treatment methods disclosed herein. Additionally provided are AAV vectors for expressing PGRN protein or variants thereof (such as PGRNΔ3) or pharmaceutical compositions containing such AAV vectors for use in the treatment methods disclosed herein. In some embodiments, the AAV vector employed in the treatment method or contained in the medicament or pharmaceutical composition is a vector described herein as AAV801-PGRNΔ3, which contains a vector described herein as clone 249.

[0350] In some embodiments, the subject has been diagnosed with FTD or FTLD (or suspected FTD or FTLD) at the time of treatment based on standard diagnostic criteria, such as, but not limited to, assessment of neurological or psychiatric symptoms or signs, and / or structural or functional brain imaging using MRI, CT, PET, or other brain imaging methods to identify patterns of brain atrophy, in each case characteristic of FTD or FTLD. In some embodiments, the subject has been diagnosed with behavioral variant frontotemporal dementia (BV-FTD) or nonfluent variant primary progressive aphasia (NFV-PPA) at the time of treatment. In some embodiments, the subject has been diagnosed with atrophy of one or more brain regions, such as, but not limited to, atrophy of the frontal lobe (e.g., orbitofrontal cortex or anterior cingulate gyrus), temporal lobe (e.g., anterior temporal lobe, medial temporal lobe, or posterior temporal lobe), or other brain regions (such as the inferior parietal lobe, striatum, or thalamus, or other brain regions) at the time of treatment.

[0351] In some embodiments, a biochemical test is used to confirm the diagnosis, for example, by detecting lower than normal levels of progranulin (PGRN) in a serum or cerebrospinal fluid (CSF) sample obtained from the subject, and / or in some other embodiments, identifying one or more heterozygous or homozygous deleterious (complete or partial loss of function) mutations in the GRN gene encoding PGRN in the subject. In some embodiments, the subject has a haploinsufficient amount of PGRN for the GRN gene and for the amount of PGRN produced from either or both GRN alleles. In some embodiments, for example, based on neuropathological analysis of brain tissue, the subject is diagnosed with FTLD-TDP type A, FTLD-TDP type B, or FTLD-TDP type C.

[0352] Treatment of a subject with FTD, FTLD, or PGRN deficiency does not necessarily require a cure to be considered effective, where "cure" is defined as halting disease progression, or partially or completely restoring the subject's health status prior to the onset or exacerbation of symptoms, or the health status relative to a healthy person without FTD, FTLD, or PGRN deficiency. Instead, a therapeutically effective amount of an AAV vector of the present disclosure (including but not limited to a vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector can be an amount that is used to at least partially reverse, alleviate, or improve the extent or severity of at least one symptom or sign associated with FTD, FTLD, or PGRN deficiency in a subject; or at least partially reverse, alleviate, or improve the extent or severity of at least one disorder or dysfunction of the body, organ, tissue, or cell caused by FTD, FTLD, or PGRN deficiency in a subject; or slow the progression of other deleterious effects of FTD, FTLD, or PGRN deficiency in a subject; or improve the quality of life of a subject suffering from FTD, FTLD, or experiencing deleterious effects of PGRN deficiency. Examples of symptoms, signs, disorders, or functional disturbances associated with FTD, FTLD, or PGRN deficiency include, but are not limited to, cortical or lobar brain atrophy (e.g., affecting the orbitofrontal cortex, medial prefrontal cortex, anterior cingulate gyrus, anterior insular cortex, cingulate cortex, insular cortex, anterior, medial, and posterior regions of the inferior parietal or temporal lobes), subcortical brain atrophy (e.g., affecting the striatum, thalamus, amygdala, or hippocampus), and behavioral consequences of brain atrophy, including, for example, parkinsonism, corticobasal syndrome (CBS), word-finding impairment, apraxia of speech, agrammatism, verbal naming impairment, word comprehension impairment, phonological errors, word repetition errors, sentence repetition errors, sentence comprehension errors, surface alexia, delusions, hallucinations, and decreased quality of life (QoL).

[0353] In other embodiments, a therapeutically effective amount of an AAV vector of the present disclosure (including but not limited to vectors described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector is an amount that at least partially corrects or alters the value of a biomarker associated with FTD, FTLD, or PGRN deficiency to a value that is more reflective of normal function. Examples of biomarkers associated with FTD, FTLD, or PGRN deficiency include, but are not limited to, lower than normal amounts of PGRN protein in CSF, serum or plasma, or brain tissue, lower than normal amounts of bis(monoacylglycerol)phosphate (BMP) in brain tissue, higher than normal amounts of β-hexosaminidase (HexA) and β-galactosidase (β-Gal) enzyme activity in brain tissue, higher than normal amounts of TDP43 fragmentation in brain tissue, and higher than normal amounts of lipofuscin in brain tissue.

[0354] In some purely exemplary and non-limiting embodiments, the mean or median amount of PGRN protein in the plasma of humans with deleterious GRN mutations is about 28% of that in healthy humans, and the mean or median amount of PGRN protein in the CSF of humans with deleterious GRN mutations is about 39% of that in healthy humans (Meeter, L. et al., Dement. Geriatr. Cogn. Dis. Extra, 6:330-40, 2016). One of ordinary skill in the art will appreciate that the mean or median value of PGRN protein concentration in biological fluids and tissue samples from humans with GRN mutations or healthy humans may vary depending on the assay and sample population, as well as other variables.

[0355] In some embodiments, the methods disclosed herein for treating FTD, FTLD or PGRN deficiency can be used to treat FTD, FTLD or PGRN deficiency in human subjects with any type of homozygous or heterozygous deleterious mutation in or affecting the GRN gene. Non-limiting examples of deleterious mutations include deletions, insertions, recombination, splice site variations, missense or nonsense mutations in either or both alleles of the GRN gene, mutations in the transcriptional control region (e.g., enhancer or promoter) affecting either or both alleles of the GRN gene, and / or mutations that reduce the stability of mRNA expressed from either or both alleles of the GRN gene or the amount of protein translated from such mRNA transcripts, as long as the mutation results in a reduction or loss of the amount of PGRN protein produced. Methods for genotyping a subject as having a deleterious mutation in either or both alleles of the GRN gene (such as by RFLP analysis or gene or genomic sequencing) are familiar to those of ordinary skill in the art, as are methods for detecting and quantifying the amount of PGRN protein in a biological fluid or tissue sample from a subject.

[0356] The therapeutic efficacy of the methods disclosed herein can be assessed in individual subjects suffering from FTD, FTLD or PGRN deficiency by observing or measuring and comparing the severity or magnitude of any symptom, sign, obstacle, dysfunction or biomarker value specific to FTD, FTLD or PGRN deficiency before (baseline) and after treatment. This comparison can be performed at one or more times after treatment, such as 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 60, 72 or 84 months or some other time after treatment. The data for comparison from an individual subject can be a single data point, or an average of multiple data points (if available). In some other embodiments, the efficacy of the treatment can be assessed in a population of subjects (i.e., two or more) with FTD, FTLD, or PGRN deficiency that serves as its own control by observing or measuring the severity or magnitude of any symptom, sign, disorder, functional disorder, or biomarker value specific to FTD, FTLD, or PGRN deficiency before (baseline) and after treatment among individuals within the population and comparing the average data before treatment with the average data after treatment. This comparison can be made at one or more times after treatment, such as at 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 60, 72, or 84 months or some other time after treatment. In some other embodiments, studies designed to establish and quantify the efficacy of the treatment can be designed to compare the therapeutic effect of a population of subjects treated with the AAV vectors of the present invention (treatment group) with the therapeutic effect of a population of subjects receiving a placebo (control group). Typically, although not necessarily, subjects within the treatment and control groups of a study are matched with respect to relevant subject characteristics, such as age, sex, and disease severity at the time of intervention. In other embodiments, the control population is statistically defined or determined through a natural history study in which disease progression is followed in patients with FTD, FTLD, or PGRN deficiency without any intervention (except perhaps standard of care).

[0357] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency are effective for treating subjects with FTD, FTLD, or PGRN deficiency of any age, including but not limited to, subjects who are at least or about 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 years of age or older, or an age range encompassing any of the foregoing specifically recited ages.

[0358] In some embodiments, the subject has not yet exhibited any overt signs or symptoms of FTD, FTLD, or PGRN deficiency when treated with an AAV vector of the present disclosure (including but not limited to vectors described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector, but has been diagnosed as being at risk of developing such signs or symptoms in the absence of treatment based on genetic testing demonstrating the presence of at least one deleterious mutation in either or both alleles of the subject's GRN gene.

[0359] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively treat a subject suffering from FTD, FTLD, or PGRN deficiency for a period of time following administration of an AAV vector of the present disclosure (including but not limited to a vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector, during which the subject does not develop any symptoms or signs of FTD, FTLD, or PGRN deficiency, or does not develop any worsening of symptoms or signs of FTD, FTLD, or PGRN deficiency that may be present at the time of treatment, or develops at most a slight worsening of symptoms or signs of FTD, FTLD, or PGRN deficiency that may be present at the time of treatment, such that the subject's overall health, function, quality of life, and / or lifespan is not substantially or substantially affected. In some embodiments, the time period (referred to herein as the treatment persistence time period) can be any suitable or desired time period, including, for example, but not limited to, at least or about 3, 6, 9, 12, 15, 18, 21, 24 months, or more, or at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 years, or more, or at least or about 1, 2, 3, 4, 5, 6, 7 decades, or more, or any integer value therebetween, or a timespan encompassing any of the foregoing specifically recited times, or even, in some embodiments, the remaining lifespan of the subject after receiving a gene therapy as described herein.

[0360] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively increase the concentration of PGRN protein or variants thereof, including PGRNΔ3, in the cerebrospinal fluid (CSF) of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or 100% of the average concentration of endogenous PGRN protein in the CSF of healthy subjects. 110%, for example, about 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 ng / mL or more, or a range encompassing any of the foregoing specifically recited values, and other values ​​are possible depending on the type of assay used to detect and quantify PGRN protein levels.

[0361] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively increase the concentration of PGRN protein or variants thereof, including PGRNΔ3, in the cerebrospinal fluid (CSF) of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency by at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more above the average concentration of endogenous PGRN protein in the subject's CSF before treatment, such as 1, 2, 3, 4, 5, or 6 months before treatment.

[0362] In some embodiments, the methods described herein for treating FTD, FTLD or PGRN deficiency effectively increase the concentration of PGRN protein or variants thereof, including PGRNΔ3, in the serum or plasma of a human subject in need of treatment for FTD, FTLD or PGRN deficiency to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100% or 110% of the average concentration of endogenous PGRN protein in the serum or plasma of a healthy person, for example, about 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175 ng / mL or more, or a range encompassing any of the foregoing specifically recited values, although other values ​​are possible depending on the type of assay used to detect and quantify PGRN protein levels.

[0363] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively increase the concentration of PGRN protein or variants thereof, including PGRNΔ3, in the serum or plasma of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency by at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% or more above the average concentration of endogenous PGRN protein in the serum or plasma of the subject before treatment, such as 1, 2, 3, 4, 5, or 6 months before treatment.

[0364] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively increase the concentration of PGRN protein or variants thereof (including PGRNΔ3) in the brain or spinal cord of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the average concentration of endogenous PGRN protein in the brain or spinal cord of a healthy person.

[0365] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively increase the concentration of bis(monoacylglycerol)phosphate (BMP) in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, or 110% of the average concentration of endogenous BMP in the brain of a healthy person. In some embodiments, the species of BMP that is elevated is BMP 18:1 / 18:1, BMP 22:6 / 22:6, or some other species of BMP.

[0366] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively reduce the level of beta-hexosaminidase (HexA) enzyme activity in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the HexA enzyme activity in the brain prior to treatment.

[0367] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively reduce the level of β-galactosidase (β-Gal) enzyme activity in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the β-Gal enzyme activity in the brain prior to treatment.

[0368] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively reduce the extent or degree of TDP43 fragmentation in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the extent or degree of TDP43 fragmentation in the brain prior to treatment.

[0369] In some embodiments, the methods described herein for treating FTD, FTLD, or PGRN deficiency effectively reduce the amount of lipofuscin in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of the amount of lipofuscin in the brain prior to treatment.

[0370] The concentration of PGRN protein or its variants (including PGRNΔ3) and the concentration of BMP or lipofuscin in biological fluids or tissue samples from subjects treated with the AAV vectors of the present invention or from healthy or other controls can be detected and quantified using any method known in the art, such as, but not limited to, ELISA, RIA, or LCMS-MS, or any other method known in the art. The enzyme activity of HexA or β-Gal in biological fluids or tissue samples from subjects treated with the AAV vectors of the present invention or from healthy or other controls can be detected and quantified using any method known in the art, such as, but not limited to, fluorogenic substrate enzymatic assays, or other methods known in the art. TDP43 fragmentation can be detected and quantified in tissue samples from subjects treated with the AAV vectors of the present invention or from healthy or other controls using any method known in the art, such as, but not limited to, semi-quantitative Western blot analysis, or any other method known in the art.

[0371] Prevention methods

[0372] In other embodiments, the present disclosure provides methods for preventing FTD, FTLD or PGRN deficiency by administering a prophylactically effective amount of an AAV vector of the present disclosure (including but not limited to a vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector to a subject (such as a human subject) in need of prevention of FTD, FTLD or PGRN deficiency. Also provided is the use of the AAV vector of the present disclosure in the manufacture of a medicament for the prevention method disclosed herein. In addition, the AAV vector of the present disclosure or a pharmaceutical composition containing such an AAV vector is provided for use in the prevention method disclosed herein.

[0373] In some embodiments, administration of a prophylactically effective amount of an AAV vector of the present disclosure (including but not limited to a vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector to a subject in need of prevention of FTD, FTLD, or PGRN deficiency is effective to prevent the onset or onset of FTD or FTLD in the subject; effectively prevent the onset or onset of any deleterious effect of PGRN deficiency in the subject; effectively prevent the onset or onset of a decrease in the amount of PGRN in the subject; effectively prevent the onset or onset of at least one symptom or sign associated with FTD, FTLD, or PGRN deficiency in the subject; effectively prevent the onset or onset of at least one disorder or dysfunction of the body, organ, tissue, or cell caused by FTD, FTLD, or PGRN deficiency in the subject; and effectively prevent the onset or onset of any decrease in quality of life in the subject caused by FTD, FTLD, or PGRN deficiency, in each case which would have occurred if prophylactic measures were not taken.

[0374] According to some embodiments of the prevention methods described herein, the subject is a human subject with a homozygous or heterozygous deleterious mutation in the GRN gene, and the presence of this mutation is determined by genotyping before the onset of any detectable symptoms or signs of FTD or FTLD or other symptoms or signs associated with PGRN deficiency. Methods for genotyping (such as by RFLP analysis or gene or genome sequencing) are familiar to those of ordinary skill in the art.

[0375] AAV vector compositions, formulations, administration methods, and dosages

[0376] In addition to AAV vectors, the present disclosure also provides compositions comprising such vectors and at least one pharmaceutically acceptable excipient, diluent, or carrier. Such vectors can also be used in, among other things, the methods of preventing and treating FTD, FTLD, or PGRN deficiency, also described herein.

[0377] Compositions comprising the AAV vectors of the present invention can be provided as aqueous solutions or suspensions, emulsions, and in other forms (such as lyophilized cakes). The vector compositions can be formulated using any suitable diluents and excipients, which may be necessary to achieve the desired properties (such as pH, ionic strength, tension, stability, shelf life, resistance to freeze-thaw cycles, and ability to freeze-dry) and to consider the mode of administration. Exemplary diluents and carriers include, but are not limited to, sterile water for injection, ethanol, and glycerol. Exemplary excipients include, but are not limited to, salts, buffers, acids, bases, surfactants, sugars, sugar alcohols, and many other excipients known in the art. Compositions comprising the AAV vectors of the present invention for preventing or treating a disease or disorder (such as FTD, FTLD, or PGRN deficiency) in a subject can be packaged in any suitable form, such as vials or prefilled syringes. In some embodiments, the kit is provided with a plurality of vials containing sufficient total amount of vector to deliver the desired total dose to a particular subject based on relevant variables (such as the severity of the subject's disease, weight, sex, or other variables).

[0378] Compositions comprising the disclosed AAV vectors can be administered by any suitable route of administration, non-limiting examples of which include systemic administration, direct administration into a tissue or organ, intravenous administration, intraarterial administration, intralymphatic administration, intraperitoneal administration, intramuscular administration, intraparenchymal administration, intrathecal administration, intracerebroventricular administration, or intracisterna magna administration, where other routes of administration are also possible.

[0379] The compositions comprising the AAV vectors of the present disclosure can be administered alone without any other type of therapy, or can be administered simultaneously, concurrently, or at any suitable dosing interval with standard of care treatment or some other agent, compound, drug, treatment, or therapeutic regimen. In some embodiments, the compositions comprising the AAV vectors of the present disclosure can be administered after prophylaxis with an immunosuppressant (such as a steroid or tacrolimus) or other immunosuppressant drug, or the immunosuppressant drug can be administered afterwards to control any humoral and / or cellular immune response to the gene therapy.

[0380] The vector composition can contain any suitable amount of an AAV vector calculated to deliver a prophylactically or therapeutically effective amount of such vector to a subject in a volume that is easy to handle or administer to the subject and / or is not expected to cause any discomfort or undesirable side effects to the subject.

[0381] In conjunction with the prevention and treatment methods provided herein, AAV vectors and compositions comprising such vectors can be administered at any suitable dose predicted or determined to be effective to achieve the desired degree of prevention or treatment. In some embodiments, the dose of the AAV vector of the present disclosure for preventing or treating FTD, FTLD, or PGRN deficiency can be quantified and expressed as vector per kilogram of subject body weight (vg), abbreviated as "vg / kg". In some embodiments, exemplary effective doses of the AAV vectors of the present disclosure (including, for example, AAV vectors comprising an AAV801 capsid and a genome having a nucleotide sequence of SEQ ID NO: 19 or its reverse complement) include, but are not limited to, at least or about 1×10 9 vg / kg, 1×10 10 vg / kg, 1×10 11 vg / kg, 1×10 12 vg / kg, 1×10 13 vg / kg, 1×10 14 vg / kg or 1×10 15 vg / kg, or a dosage range between and including any of the aforementioned specifically recited dosages; other dosages are also possible.

[0382] Based on the above detailed description, other purposes, features and advantages of the present disclosure will be apparent. However, it should be understood that the following detailed description and specific examples, although indicating specific embodiments of the present disclosure, are given by way of illustration only, because various changes, modifications and equivalents within the spirit and scope of the present disclosure will be apparent to those skilled in the art based on the detailed description and examples, and fall within the scope of the appended claims.

[0383] Unless otherwise indicated, the use of the term "or" when referring to one or more members of a group of embodiments is equivalent to "and / or" in meaning and does not require that they are mutually exclusive. Unless otherwise indicated, multiple explicitly enumerated numerical ranges also describe such ranges, whose lower limit is derived from the lower limit or upper limit of any one explicitly enumerated range, and whose upper limit is derived from the lower limit or upper limit of any other explicitly enumerated range. Thus, for example, a series of explicitly enumerated ranges "10-20, 20-30, 30-40, 40-50, 100-150, 200-250, 275-300" also describes ranges 10-50, 50-100, 100-200, and 150-250, etc. Unless otherwise indicated, the use of the term "about" before a series of numerical values ​​or ranges is intended to modify not only the value or range that appears immediately thereafter, but also each value or range that appears thereafter in the same series. Thus, for example, the phrase "about 1, 2, or 3" is equivalent to "about 1, about 2, or about 3."

[0384] All publications and references cited herein, including but not limited to articles, abstracts, patents, patent applications (whether published or unpublished), and biological sequences (including but not limited to biological sequences identified by specific database reference numbers), are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or reference was specifically and individually indicated to be incorporated by reference. Any patent application to which this application directly or indirectly claims priority is also incorporated by reference in its entirety.

[0385] Unless otherwise indicated, the following examples describe experiments that were or are currently performed using standard techniques that are well known and routine to those of ordinary skill in the art. These examples are illustrative.

[0386] Example

[0387] The following examples and figures are included to demonstrate preferred embodiments. Those of skill in the art will appreciate that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of what is described herein.

[0388] Example 1

[0389] Design and generation of AAV vectors expressing progranulin (PGRN)

[0390] AAV vectors containing a transgene for expressing human progranulin (PGRNDel3 or PGRNΔ3) lacking the last three carboxyl-terminal amino acids were designed, constructed, and generated for in vitro and in vivo testing. In optimization experiments, expression of various transgenes encoding truncated human PGRN protein (otherwise wild-type amino acid sequence) was compared in transfected Neuro-2A cells. Codon-optimized and CpG-depleted transgenes produced less progranulin than the wild-type coding sequence (data not shown), so the latter was used for transgenesis.

[0391] The AAV genome (designated Syn-PGRNΔ3 clone 249) comprises, in 5' to 3' order, a 5' ITR from AAV2, a promoter from the human synaptophysin gene, a 5' untranslated region (UTR) from the primate synaptophysin I gene transcript (NCBI reference sequence XM_034950363.1), the coding sequence for the human progranulin polypeptide (hGRNΔ3) lacking the three carboxyl-terminal amino acids, a stop codon, a bovine growth hormone (bGH) gene polyadenylation (poly A) signal sequence (i.e., a transcription terminator), an intron from the human TATA box binding protein (TBP) gene (NCBI reference sequence NG_008165.1), and a 3' ITR from AAV2. For the vector of clone 249, its complete sequence is reported as SEQ ID NO: 17 (including both ITRs), and the nominal start and end nucleotide numbers for each genome component are listed in Table 1 below. Accessory polypeptide and nucleotide sequences associated with the vectors described herein are listed in the Sequence Listing.

[0392] Table 1

[0393]

[0394] To produce vector particles, HEK 293 cells in suspension culture were transfected using a classic triple transfection method. HEK 293 cells were amplified from a working cell bank aliquot through multiple passages, starting from a shake flask, through a wave bag, to a 250L scale disposable bioreactor (SUB). Cells were transfected by adding a transfection mixture containing PEI and the following three different plasmids: pHelper, for expressing AdV auxiliary factors; pRepCap, for expressing AAV capsid proteins (variously AAV6, AAV9, AAVDJ, AAVPHP.B or AAV801, depending on the experiment) and AAV Rep proteins; and a transgenic plasmid. After 3.5 hours of transfection, the transfection was quenched by adding CDM4 medium, followed by a 72-hour incubation period to allow the cells to produce AAV vectors. The vector was harvested by lysing cells with Triton X-100, adding domiphene bromide to flocculate host cell DNA, filtering the supernatant, and then purifying the vector in three stages, including affinity chromatography, anion exchange chromatography, and tangential flow filtration. After harvesting and purification, the vector was titrated by quantitative PCR and then tested for efficacy and toxicity in vitro and in vivo. As described below, the vector was then tested in vitro and in vivo to determine whether expression of a modified progranulin (PGRNΔ3) could improve biomarkers associated with GRN haploinsufficiency, which causes FTD in humans.

[0395] Example 2

[0396] In vitro testing of AAV GRN vectors

[0397] It has been reported that carboxyl-terminal truncations of human PGRN protein are unable to bind to sortilin while retaining the ability to bind to other receptors such as prosaposin (Zheng, Y. et al., PLoS ONE, 6:e21023, 2011). To confirm this observation, recombinant WT progranulin (PGRNwt) and progranulin Δ3 (PGRNΔ3) were expressed with a HIS tag fused to the N-terminal portion of the protein and binding to sortilin and prosaposin was assessed in vitro ( Figure 1 The terminal three amino acids of WT PGRN are required for progranulin binding to Sortilin but not to prosaposin. WT PGRN binds to both human Sortilin and recombinant prosaposin. PGRNΔ3 does not bind to Sortilin but retains binding to prosaposin, as shown by two different sources of prosaposin (Abcam and Mybiosource).

[0398] Experiments were designed to determine whether different types of neurons could produce human PGRNΔ3 in vitro after transduction with AAV vectors expressing the GRN transgene under the control of a neuron-restricted promoter, compared to full-length human PGRN expressed from otherwise identical vectors. Using standard techniques, spinal cord motor neurons and cortical glutamatergic neurons were differentiated from human induced pluripotent stem cells (iPSCs) and then transduced with AAV vectors expressing truncated and full-length human PGRN controlled by the SYN promoter. After two weeks of differentiation, motor neurons were transduced with vectors using AAVDJ capsids at a multiplicity of infection (MOI) of 1E5. Because AAVDJ did not transduce glutamatergic neurons well, AAV vectors using AAV6 capsids were used in those experiments testing two doses of MOI 1E5 and 3E5. Figure 2A As shown, AAVDJ vectors transduced motor neurons produced comparable levels of mRNA encoding truncated and full-length PGRN. However, at the protein level, motor neurons transduced with AAVDJ vectors expressing PGRNΔ3 produced higher levels of PGRN protein in the conditioned medium of cell growth compared to neurons transduced with vectors expressing the full-length protein. Figure 2B Similar patterns were observed when glutamatergic neurons were transduced with these two types of AAV6 vectors. Figure 3AAs shown, those neurons produced comparable levels of mRNA encoding PGRNΔ3 and full-length PGRN, which was dose-responsive, increasing with tripling of the MOI. Furthermore, similar to the observations in motor neurons, significantly higher levels of progranulin were measured in conditioned medium from cells transduced with AAV6 vectors expressing PGRNΔ3 than with similar vectors expressing the full-length protein, which was also dose-responsive ( Figure 3B The results of these experiments showed that the amount of mRNA in the conditioned medium from cultured neuronal cells was similar but higher for the truncated progranulin compared to the full-length protein, which was attributed to the inability of the truncated protein to bind sortilin, thereby preventing its uptake back into the cell after secretion. In another in vitro experiment, it was shown that transduction of cortical glutamatergic neurons differentiated from iPSCs obtained from human FTD patients was also transduced with an AAV6 vector expressing PGRNΔ3 and produced detectable levels of progranulin in the conditioned medium, which exceeded the levels produced by similar neurons from healthy humans ( Figure 3B ).

[0399] Collectively, the results from in vitro experiments testing AAVDJ and AAV6 vectors suggest that transduction of neurons in vivo by administering AAV vectors expressing PGRNΔ3 to the brain may be more effective than previous attempts that relied on vectors expressing full-length progranulin. These studies were extended by testing whether transduction of neurons in vitro with vectors using capsids capable of crossing the blood-brain barrier in NHPs could be similarly effective as vectors using AAVDJ and AAV6. Late neuronal progenitor cells from control (WC-30) and FTD (ND50017) iPSCs were differentiated into glutamatergic neurons over 14 days in culture, followed by the addition of either an AAV801 vector expressing human PGRNΔ3 from a clone 249 vector or a control AAV801 vector engineered to produce luciferase (AAV801-Luc). Figure 4 As shown, the mean endogenous human progranulin levels in the culture medium from untreated neurons and neurons treated with the control vector AAV801-Luc (MOI 1E6 cells) were 0.69 (SD 0.06) and 0.61 (SD 0.09) ng / mL, respectively, whereas the amount of total detectable hPGRN from neurons transduced with the AAV801-PGRNΔ3 vector (MOI 1E6 cells) was greatly increased to 2.39 (SD 0.23) ng / mL (reflecting endogenous full-length PGRN and variant PGRN produced by the transduced cells).

[0400] Low levels of the lysosomal enzyme β-hexosaminidase (HexA) have been reported in neurons from FTD patients with GRN haploinsufficiency, and experiments were designed to determine whether expression of PGRN in such neurons via vector transduction could increase levels of this enzyme. Basal β-hexosaminidase activity in neurons derived from control iPSCs averaged 1641.8 (SD 249.7) relative fluorescence units (RFUs) and, as expected, was lower in differentiated neurons derived from FTD iPSCs, averaging 790.4 (SD 88.8) RFUs. Figure 5 When control and FTD neurons were transduced with AAV801-Luc (MOI 1E6 cells), HexA activity was comparable to that of untreated control cells of the same type, with an average of 1606.5 (SD 225.4) RFU in control and 956.5 (SD 79.2) RFU in FTD neurons. However, transduction of both control and FTD neurons with AAV801-PGRNΔ3 (MOI 1E6 cells) increased HexA activity by approximately 1.9-fold compared to the control AAV801-Luc vector, increasing it to 3055.1 (SD 344.8) RFU in control neurons and 1825 (SD 160.05) RFU in FTD neurons. HexA activity in FTD neurons transduced with AAV801-PGRNΔ3 was comparable to that in untreated control neurons (1825 RFU vs. 1642 RFU, respectively), indicating that the AAV801-PGRNΔ3 vector can sufficiently elevate progranulin levels in FTD neurons to restore HexA activity to levels comparable to those prevalent in normal neurons.

[0401] Example 3

[0402] In vivo testing of AAV vectors expressing full-length and truncated PGRN

[0403] Since we have shown that progranulin is present at higher levels in the conditioned medium of neurons transduced in vitro with AAV vectors expressing PGRNΔ3 compared to the full-length protein and that PGRNΔ3 is biologically active, we designed experiments to test whether similar results could be demonstrated in vivo. We generated AAV vectors expressing truncated and full-length human PGRN and administered these AAV vectors to mice via different routes of administration, after which we measured the levels of human progranulin expressed in the transduced cells.

[0404] In the first set of experiments, AAV9 vectors expressing PGRNΔ3 and full-length PGRN under the control of the neuron-specific synaptophysin (SYN) promoter were bilaterally administered into the brains of newborn mice (1.5e10 vg / ventricle) via the intracerebroventricular route (ICV) on day P0. The test animals were sacrificed 4 weeks later, and brain tissue, cerebrospinal fluid (CSF), and serum samples were removed and analyzed to measure the level and pattern of vector transduction and PGRN expression. Figure 6A As shown, both vectors transduced mouse brain tissue at comparable levels, as did a third AAV9 vector designed to express full-length human PGRN under the control of a constitutive CMV promoter. Despite similar transduction levels, and consistent with the in vitro experiments described above, substantially more human PGRN protein was detected in the CSF of mice transduced with a vector expressing the truncated PGRNΔ3 compared to the full-length PGRN. Figure 6B Similar results were obtained when the expressed protein levels were normalized to the amount of endogenous mouse PGRN present in CSF samples from test animals ( Figure 6C In contrast to test animals administered a vector containing a transgene encoding full-length human PGRN controlled by the synaptophysin promoter, greatly reduced levels of expressed protein were detected in CSF from test animals administered an otherwise similar vector in which the transgene was controlled by the CMV promoter ( Figure 6D ). However, this pattern was reversed in serum from test animals in which vectors containing the CMV promoter produced greater amounts of full-length human PGRN protein. In samples from test animals transduced with a negative control vector expressing a GFP reporter transgene, only background levels of PGRN signal were detected. Brain sections from test animals were also analyzed by immunohistochemistry (IHC) using an antibody specific for human progranulin. In brain sections from animals treated with AAV9 vectors expressing GFP, no human PGRN signal was detected by IHC ( Figure 7A In contrast, PGRN staining was detected in brain sections from animals administered AAV9 vectors expressing full-length and truncated human PGRN ( Figure 7B and Figure 7C ), but compared with full-length PGRN, the expression of the truncated PGRNΔ3 protein appeared to be more robust and diffusely distributed throughout the brain.

[0405] In the second set of experiments, AAV1, AAVDJ, and AAV9 vectors expressing PGRNΔ3 and full-length PGRN controlled by different promoters were unilaterally administered into the brains of 6-month-old mice (5e10 vg / animal) via the ICV route. The test animals were sacrificed 3 or 6 months later, and the levels of progranulin secreted into the CSF were analyzed. Figure 8AAs shown, all vectors transduced brain tissue in the hemisphere contralateral to the injection site, but the transduction efficiency of vectors using the AAVDJ capsid was approximately 10-fold higher compared to AAV1 and AAV9. All vectors used to express human progranulin, whether truncated or full-length, produced detectable levels of protein in the CSF of test animals after 3 months of treatment. Although the data were variable, there was a trend toward increased expression of PGRNΔ3 compared to full-length PGRN, a pattern that was consistent among the different capsids used to package the expression cassette and regardless of whether the transgene was driven by the neuron-specific synaptophysin promoter or the nonspecific EF1a promoter ( Figure 8B In contrast, another nonspecific promoter, CMV, appeared to be inactive in the brain, even when AAV9 vectors containing it successfully transduced cells. A longer duration of expression was demonstrated in a subset of test animals administered with AAV9 vectors, where both truncated and full-length progranulin were detected in the CSF six months after treatment at levels similar to those measured three months earlier ( Figure 8C ).

[0406] The above experiments demonstrated that ICV administration of different AAV vectors resulted in the expression of truncated and full-length human progranulin detectable in the CSF of treated animals. In a third series of experiments, AAVPHP.B vectors (2e13 vg / kg) were administered intravenously (retroorbitally) to 6-week-old mice to express PGRNΔ3 and full-length PGRN under the control of the synaptophysin promoter. After four weeks of treatment, the test animals were sacrificed and tissues were collected for analysis to test whether systemic administration of the vectors could result in protein expression in the nervous system. Figure 9A As shown, both vectors transduced brain tissue equally, and consistent with the results of ICV administration of the vectors, truncated progranulin was expressed in brain tissue ( Figure 9B ) and CSF( Figure 9C ) are present at higher levels.

[0407] The results from the in vivo experiments described above demonstrate that PGRNΔ3 is present at higher levels than full-length PGRN in the brains of mice of different ages to which AAV vectors using different capsids were administered by two different routes. Since vectors expressing the two different forms of the PGRN protein transduced brain tissue with equal efficiency, the most likely explanation for the discrepancy is that, unlike the full-length progranulin, the truncated protein does not bind to sortilin and, therefore, after secretion, remains in the extracellular space rather than being taken up into cells.

[0408] Example 4

[0409] Testing the neurotoxicity of AAV-expressed PGRN in wild-type mice

[0410] As reported by others, expression of full-length human PGRN protein by AAV9 vectors administered ICV into the brain of Grn-deficient mice results in selective and significant hippocampal toxicity and degeneration, affecting neurons and glia, reportedly mediated by T cell responses (Amado D. et al., Mol. Ther., 27:465-78, 2019). Although such unwanted side effects may be manageable in humans, such as by administering immunosuppressants, experiments were designed to test whether expression of truncated PGRN could reduce or eliminate the neurotoxicity seen with the full-length protein.

[0411] AAV9 vectors (5e10 vg each) expressing human full-length PGRN and PGRNΔ3 under the control of the SYN gene promoter were administered ICV to the brains of 6-month-old mice. After three months of treatment, the test animals were sacrificed and brain tissue was removed and analyzed. The histological structure and cell composition of the hippocampus in coronal brain sections were assessed by H&E staining, and the levels of human progranulin and mouse microglial marker Iba-1 were detected by immunohistochemistry (IHC). Before being sacrificed, no significant differences in body weight or survival were observed between animals treated with vectors expressing PGRN and negative control animals administered PBS or AAV9 vectors expressing green fluorescent protein (GFP) (data not shown). In the hippocampus of test animals administered with AAV9 vectors expressing GFP, normal levels of mouse Iba-1 were detected by ICH, but no human PGRN protein was detected ( Figure 10 , lower left and upper left micrographs, respectively). Similarly, the hippocampus exhibited normal neuronal cellularity ( Figure 10 , right micrograph). In test animals that received an AAV9 vector expressing full-length human PGRN, progranulin was readily detected in the CA3 region of the hippocampus ( Figure 11 , upper left micrograph). However, unlike negative control animals, the same region showed reduced cellularity ( Figure 11 , right micrograph), and the signal intensity of the microglial marker Iba-1 increased ( Figure 11 , lower left micrograph), which colocalizes with PGRN expression. In summary, these results show that expression of full-length human PGRN protein is toxic to neurons in the hippocampus and stimulates a strong microglial response, which is similar to previous reports. When full-length human PGRN protein was expressed from a vector using an AAVDJ capsid, similar results were observed (now shown), confirming that toxicity is most likely due to the expressed protein rather than the vector capsid.

[0412] Test animals that received an AAV9 vector expressing human PGRNΔ3 also had detectable levels of the protein in the hippocampus, but the expression pattern was more diffuse compared to the full-length form of the same protein ( Figure 12 , upper left micrograph). Surprisingly, in stark contrast, the hippocampal CA3 region from these same test animals had a neuronal cellularity that appeared normal ( Figure 12 , right micrograph), and normal to slightly increased Iba-1 protein levels ( Figure 12 , lower left micrograph), showing no significant microglial activation. Importantly, these results demonstrate that AAV-mediated expression of the truncated PGRNΔ3 protein in the brains of wild-type mice does not cause hippocampal neuronal toxicity, as seen with full-length PGRN, suggesting that gene therapy may be safer for expressing truncated forms of the protein.

[0413] Example 5

[0414] Testing AAV GRN vectors in a mouse GRN knockout model

[0415] A potential advantage of the AAV801 capsid over other capsids is that it has been shown to cross the blood-brain barrier (BBB) ​​in non-human primates. However, AAV801 does not possess this capability in mice, which has limited the ability to test vectors encapsidated in this capsid in mouse models of human disease. To circumvent this limitation, experiments were designed in which the 249 clone vector was packaged in alternative AAV9 (AAV9-PGRNΔ3) and AAVPHP.B (AAVPHP.B-PGRNΔ3) capsids for testing in a FTD mouse model in which the endogenous murine Grn was knocked out (KO). As described further below, Grn-deficient mice exhibited decreased expression of two different BMP species (18:1 / 18:1, 22:6 / 22:6), increased HexA, increased β-gal activity and increased TDP43 fragmentation, as well as accumulation of lipofuscin in multiple brain regions, all of which were reversed after treatment with a vector expressing PGRNΔ3.

[0416] Through intraventricular route (ICV) with 1e11 vg to Grn - / - KO mice were injected unilaterally with AAV9-PGRNΔ3, and age-matched Grn - / -KO and WT mice were injected with PBS as a control. All test animals survived to autopsy and no significant differences in body weight were noted after treatment. Tissues (brain and liver) and biofluids (CSF and serum) were harvested approximately 2 months after injection for analysis. One hemisphere of the brain was removed for biochemical processing and the other half was immunostained. Eight right hemispheres and six left hemispheres were removed from mice injected with ICV in the right lateral ventricle to compare the distribution of injection sites and non-injection sites from the brain (the right hemisphere was in the left hemisphere). Figure 13B 、 Figure 13C 、 Figure 13E The vector genome copies (VGC) in the liver and brain (left or right hemisphere) of mice injected with AAV9-PGRNΔ3 averaged 6.64e5 and 6.23e5 vg / μg gDNA, respectively ( Figure 13A and Figure 13B The average VGC in the right hemisphere was 1.02e6 vg / μg gDNA and in the left hemisphere was 9.38e4 vg / μg gDNA, indicating a higher distribution at the injection site relative to the non-injection site ( Figure 13B VGC was also high in the liver, likely due to leakage into the blood when the test animals received ICV injection.

[0417] PGRNΔ3 transgenic mRNA was also detectable in the brain, averaging 57-fold higher than endogenous mouse progranulin mRNA transcripts overall, but relatively higher in the right hemisphere (i.e., injected side) at 77-fold and relatively lower in the left hemisphere at 31-fold ( Figure 13C The average level of human progranulin (PGRNΔ3) protein in CSF was 156 ng / mL, while the concentration of endogenous mouse progranulin was approximately 2 ng / mL ( Figure 13D PGRNΔ3 protein levels in serum were low (3.8 ng / mL), likely due to the use of the synaptophysin promoter, which is expected to restrict transgene expression to neurons ( Figure 13E PGRNΔ3 was not detected in the liver (data not shown). In the brain, PGRNΔ3 protein levels were on average 34-fold higher than endogenous mouse progranulin and, similar to mRNA, were 51-fold higher in the right hemisphere and 10-fold higher in the left hemisphere ( Figure 13F ).

[0418] Compared with age-matched WT mice, Grn – / – The KO mouse brain showed a significant deficiency of 18:1 / 18:1 and 22:6 / 22:6 bis(monoacylglycerol) phosphate (BMP; also known as lysobisphosphatidic acid) ( Figure 14A and Figure 14B). BMP is an endolysosomal phospholipid that has been identified as interacting with progranulin in a pH-dependent manner and is a redox-sensitive enhancer of lysosomal proteolysis and lipolysis (Logan, T. et al., Cell, 184:4651-68, 2021). BMP deficiency reflects the lysosomal defect in progranulin deficiency. Tissues from test animals were tested to determine whether vehicle treatment affects BMP levels. In brain tissue from control WT mice, BMP 18:1 / 18:1 levels averaged 433 ng / g, and as expected, in brain tissue from Grn treated with vehicle, BMP 18:1 / 18:1 levels averaged 433 ng / g. - / - The brain tissue of KO mice was lower, with an average of 215ng / g ( Figure 14A However, when Grn was treated with AAV9-PGRNΔ3 vector - / - In KO mice, the BMP level in the brain increased to an average of 583 ng / g ( Figure 11 A). Similarly, in WT mice, the concentration of BMP 22:6 / 22:6 in the brain averaged 3947 ng / g and in Grn treated with vehicle. - / - KO mice was 2074 ng / g, which was increased to 5074 ng / g in brain tissue from KO mice treated with AAV9-PGRNΔ3 ( Figure 14B ).

[0419] Compared with age-matched WT control mice, Grn - / - The KO mouse brains showed elevated levels of two lysosomal enzymes, β-hexosaminidase (HexA) and β-galactosidase (β-Gal). Figure 15A and Figure 15B Tissues from the test animals were tested to determine whether vehicle treatment affected BMP levels. In brain tissue from control WT mice, HexA activity averaged 40,309 RFU (SD 5,296) and β-Gal activity averaged 38,700 RFU (SD 5,624), whereas in brain tissue from vehicle-treated Grn mice, HexA activity averaged 40,309 RFU (SD 5,296) and β-Gal activity averaged 38,700 RFU (SD 5,624). - / - In the brains of KO mice, the average HexA activity was 55430 RFU (7681 SD), and the average β-Gal activity was 50078 RFU (12403 SD). - / -In KO mice, both HexA and β-Gal enzyme activities decreased to control levels. In vehicle-treated test animals, HexA activity decreased to an average of 35,300 RFU (SD 7,371), which is similar to the WT control level of 40,309 RFU (SD 5,296), and β-Gal activity decreased to an average of 41,299 RFU (SD 7,095), which is similar to the WT control level of 38,700 RFU (SD 5,624). Figure 15A and Figure 15B ).

[0420] A hallmark of FTD caused by GRN haploinsufficiency in postmortem brain is the accumulation of TDP43 fragments, and experiments were designed to test Grn - / - The presence of such fragments in KO mice and whether vehicle treatment affects TDP43 fragmentation. Control Grn from control WT mice treated with vehicle were prepared. - / - Grn KO mice and treated with AAV9-PGRNΔ3 vector - / - Brain lysates of KO mice were analyzed by Western blot analysis using antibodies that bind to full-length TDP43 and TDP43 fragments (migrating at 43 kDa and 20 kDa, respectively). The staining intensity of these two bands was quantified, and the ratio of the staining intensity of the 43 kDa band to the 20 kDa band was calculated, with lower ratios indicating relatively more fragmentation. Figure 16 As shown, Western blot analysis demonstrated that Grn treated with PBS had increased expression of 5-6 N-terminal sera compared with age-matched WT controls. - / - KO mice had a lower staining ratio of the 43 kDa band to the 20 kDa band, indicating that Grn - / - KO mice showed greater TDP43 fragmentation in the absence of progranulin expression. However, when Grn was treated with AAV9-PGRNΔ3 vector - / - In KO mice, the staining ratio increased and was comparable to that observed in WT controls, indicating that vehicle treatment and restoration of PGRN expression reduced TDP43 fragmentation.

[0421] Grn - / - Another biomarker of FTD in KO mice is accelerated lipofuscin deposition, characterized by excessive accumulation of lipofuscin in all brain regions compared with WT mice, as visualized by autofluorescence staining. - / - Lipofuscin staining intensity was quantified in KO mice to determine whether this biomarker would also respond to AAV9-PGRNΔ3 vector treatment. Figure 17A Grn treated with carrier and vehicle are provided in - / -Representative images of lipofuscin staining in the hippocampus and thalamus of KO mice (shown in red). Figure 17C to Figure 1 Results from this analysis are presented in Figure 7F, which reports lipofuscin levels in different brain regions moving laterally and distally across one hemisphere. Lipofuscin levels were low in WT mice and elevated in Grn mice administered PBS vehicle or AAV9-Luc negative control vector. - / - KO mice were higher than control Grn - / - In contrast, when mice were treated with AAV9-PGRNΔ3 by ICV administration of the vector into the right ventricle, reduced lipofuscin accumulation was observed in all treated animals tested, with a trend toward levels in WT mice, as observed throughout the brain ( Figure 17B ), hippocampus CA2 / 3 ( Figure 17C ), the entire hippocampus ( Figure 17D ), prefrontal cortex ( Figure 17E ) and thalamus ( FIG17F ). The data also reflect that vehicle treatment reduces lipofuscin levels more in the right hemisphere (the side of the brain to which vehicle was administered) than in the contralateral left hemisphere.

[0422] The above results indicate that administration of vectors designed to express PGRNΔ3 by the ICV route is effective in correcting biomarkers associated with FTD in humans. Experiments were designed to test whether similar results could be achieved by intravenous administration of vectors capable of crossing the BBB in mice. The same vectors packaged into AAV801 capsids and tested as described above were packaged into AAVPHP.B capsids, which, unlike AAV801, are capable of crossing the BBB in mice (but not in NHPs). The AAVPHP.B-PGRNΔ3 vectors were administered intravenously (IV) to Grn-deficient knock-in (KI) mice at doses of 5e12 and 1e13 vg / kg. Both tissues showed high levels of transduction (100% and 100% respectively) as detected by quantifying the amount of vector per microgram of genomic DNA from the liver and brain. Figure 18A and Figure 18B (In the brain, 6.9e5 vg / μg and 8.2e5 vg / μg gDNA at the low and high doses, respectively). In the brain, transduction resulted in high levels of transgene mRNA and PGRNΔ3 protein expression. At the lower vector dose, mRNA expressed from the GRN transgene was approximately 32-fold higher than that produced by the endogenous mouse Grn gene ( Figure 18C ), and the PGRNΔ3 protein level was approximately 57-fold higher than the endogenous mouse PGRN protein level ( Figure 18F PGRNΔ3 protein levels were also elevated in cerebrospinal fluid (CSF). Figure 18DIn mice receiving the higher vector dose, RNA and protein levels were approximately 2-fold higher, indicating a dose-responsive effect of treatment. PGRNΔ3 protein levels were lower in serum ( Figure 18E ), likely due to the use of the synaptophysin promoter, which provides restricted expression of the transgene in neurons.

[0423] At both doses, brain tissue from all Grn-deficient KI mice treated with the AAVPHP.B-PGRNΔ3 vector exhibited elevated levels of BMP 18:1 / 18:1 (mean of 384.1 ng / g at 5e12 vg / kg) and BMP 22:6 / 22:6 (mean of 427.8 ng / g at 5e12 vg / kg) compared to WT controls (P < 0.05). Figure 18G and Figure 18H ), while HexA enzyme activity decreased ( Figure 18I Collectively, these results demonstrate that IV administration of a transgene expressing PGRNΔ3 protein via an AAV capsid capable of crossing the blood-brain barrier can transduce brain cells and express PGRNΔ3 protein at sufficient levels to correct biomarkers associated with FTD in humans.

[0424] Example 6

[0425] Testing AAV GRN vectors in nonhuman primates

[0426] A study was conducted to compare the biodistribution of AAV vectors containing 249 clone vectors packaged in AAV801 and AAV9 capsids in cynomolgus monkeys.

[0427] Cynomolgus monkeys were intravenously administered AAV801-PGRNΔ3 and AAV9-PGRNΔ3 vectors, each containing a 249-clone vector expressing hPGRNΔ3, at two doses of 5e12 vg / kg and 2e13 vg / kg. Progranulin concentrations were measured in serum and CSF samples collected immediately before and up to 28 days after dosing, while samples from brain and other tissues were also tested to quantify vector copy numbers (VGCs) and transgenic RNA and protein expression. PGRNΔ3 levels in CSF and serum were measured using a human ligand binding assay (LBA), which does not detect endogenous macaque progranulin.

[0428] PGRNΔ3 protein was detected in the CSF of test animals treated with AAV801-PGRNΔ3 on days 14 and 28 ( Figure 19A). The high dose (2e13 vg / kg) produced PGRNΔ3 protein levels of approximately 50 ng / mL and 20 ng / mL in two test animals, both of which are higher than the average 6 ng / mL progranulin naturally occurring in humans. Lower but still detectable levels of PGRNΔ3 protein were detected in one of the two test animals that received the lower 5e12 vg / kg dose. In contrast, there was no detectable PGRNΔ3 protein expression in the two test animals that received the 2e13 vg / kg dose of AAV9-PGRNΔ3. These results suggest that the AAV801 capsid can more efficiently cross the BBB to reach the brain to transduce neurons there than AAV9. Serum concentrations of PGRNΔ3 protein were comparable in test animals treated with both AAV801-PGRNΔ3 and AAV9-PGRNΔ3 vectors ( Figure 19B ), and is significantly lower than the approximately 200 ng / mL naturally present in human serum. In test animals receiving a high vector dose (2e13 vg / kg) delivered by AAV801 or AAV9 capsid, protein was first detected on day 7 and rose to a peak of approximately 5 ng / mL on day 14. In test animals administered a low dose (5e12 vg / kg) of the AAV801-PGRNΔ3 vector, PGRNΔ3 protein levels were below 2 ng / mL. The low levels of PGRNΔ3 protein in serum can be attributed to the use of a neuron-restricted promoter to drive expression of the transgene in transduced cells.

[0429] Brain and other tissue samples were harvested to investigate transduction efficiency. Quantification of vector presence confirmed transduction with the AAV801-PGRNΔ3 vector in all brain regions tested and was significantly higher, at least 100-fold, than transduction with the same dose of AAV9-PGRNΔ3 vector (2e13 vg / kg). Figure 20 ), again demonstrating the superiority of AAV801 in transducing the brain (including deep brain structures) across the BBB compared to AAV9. AAV801-PGRNΔ3 also transduced the spinal cord approximately 10-fold more than AAV9-PGRNΔ3. Transduction by the AAV801-PGRNΔ3 vector was dose-responsive, but both doses resulted in widespread distribution of the vector throughout the brain. In other tissues, especially non-neural tissues, the VGC of AAV801-PGRNΔ3 was lower than or equal to that of AAV9-PGRNΔ3. For example, in the liver (which tends to be highly transduced by many intravenously administered capsids), the VGC of AAV801-PGRNΔ3 was approximately half that of AAV9-PGRNΔ3. Transduction of the dorsal root ganglion (DRG) and trigeminal ganglion was relatively low, even at the highest dose tested (2e13 vg / kg).

[0430] Consistent with the VGC data, PGRNΔ3 RNA levels were 100- to 1000-fold higher in test animals treated with AAV801-PGRNΔ3 compared to RNA levels in brain tissue samples taken from test animals that received the AAV9-PGRNΔ3 vector ( Figure 21 ). PGRNΔ3 RNA levels were particularly high in the frontal and temporal cortex (at least 5-fold higher than levels from the MfHPRT housekeeping gene) and thalamus, and were also high in the spinal cord, compared to other brain regions (such as the amygdala or hippocampus). In contrast, in the liver, PGRNΔ3 RNA levels were low despite relatively high transduction as determined by quantification of the vector (approximately 0.1 copies of vector per copy of the MfHPRT gene per cell), again suggesting an effect of restricting transgene expression to neuronal cells by using the synaptophysin promoter. In the DRG and trigeminal ganglia, PGRNΔ3 RNA levels produced by AAV801-PGRNΔ3 transduction were equivalent to those from AAV9-PGRNΔ3 and were lower than in most brain regions. PGRNΔ3 RNA expression was dose-responsive, but both doses resulted in similar widespread transgene expression throughout the brain.

[0431] RNA expression from the PGRNΔ3 transgene was also analyzed by in situ hybridization (ISH). Figure 22A to Figure 22G As shown, extensive and robust RNA expression was detected in multiple brain regions affected by FTD from test animals administered AAV801-PGRNΔ3 at a dose of 2e13vg / kg, including the motor cortex, entorhinal cortex, hippocampal pyramidal cells, thalamus, and dentate nucleus. A similar pattern was observed in test animals receiving a lower dose of 5e12vg / kg, although the frequency and intensity of neuronal staining were reduced. Extensive RNA expression from the AAV801-PGRNΔ3 vector was also observed in the spinal cord. In test animals receiving a dose of 2e13vg / kg of the AAV9-PGRNΔ3 vector, RNA expression was observed in the DRG, spinal cord, and trigeminal ganglia by ISH, but no positively stained neurons were detected in the brain. In addition, no RNA expression was detected in peripheral sympathetic ganglia (e.g., paravertebral ganglia or gastrointestinal neurons) by ISH.

[0432] PGRNΔ3 protein was highly expressed in multiple brain regions from test animals that received 2e13 vg / kg of AAV801-PGRNΔ3 vector, exceeding the levels of naturally occurring progranulin in the brains of test animals ( Figure 23For example, in the frontal cortex, PGRNΔ3 protein levels were approximately 500 ng / g brain tissue, which is approximately 27-fold higher than the concentration of endogenous macaque progranulin. In the temporal cortex, PGRNΔ3 protein levels were approximately 30 ng / g brain tissue, or approximately 2-fold higher than endogenous progranulin, and approximately 40-fold higher than the level of macaque progranulin in the spinal cord. Peripherally, PGRNΔ3 levels were approximately 10 ng / g DRG tissue, which is lower than endogenous macaque progranulin (approximately 50 ng / g tissue). PGRNΔ3 protein was detectable in some brain regions, including the frontal cortex, thalamus, and spinal cord, from the AAV801-PGRNΔ3 vector administered at a lower dose (5e12 vg / kg), whereas no protein was detected from the AAV9-PGRNΔ3 vector even at a higher dose (2e13 vg / kg). Finally, despite the presence of 100 ng / g of endogenous progranulin, PGRNΔ3 protein from AAV801-PGRNΔ3 was not detected in the liver, again confirming tissue-restricted expression under the synaptophysin promoter. PGRNΔ3 protein expression was also analyzed in numerous brain sections by immunohistochemistry (ISH). The staining pattern matched that of transgenic mRNA determined using ISH, indicating that PGRNΔ3 protein was not significantly taken up into cells that do not express the vector mRNA.

[0433] Example 7

[0434] Quantification of vector-derived PGRN in NHP samples

[0435] An immunoaffinity liquid chromatography mass spectrometry (LCMS) assay was developed to quantify the amount of human PGRN protein expressed in tissue or biofluid samples from cynomolgus macaques treated with an AAV vector expressing PGRNΔ3. In this method, tissue or biofluid samples from NHP test animals treated with a PGRNΔ3 vector were mixed with a lysis buffer to release the protein from the cells. Polyclonal antibodies specific for PGRN were then used to immunoprecipitate endogenous full-length monkey PGRN and truncated human PGRN (including, for example, Δ3 and post-translational modification Δ4) produced as a result of vector treatment. After removing non-immunoprecipitated proteins, the immunoprecipitated PGRN protein was digested with Staphylococcus aureus (S. aureus) V8GluC protease, which cleaves the protein to the C-terminus at aspartic acid or glutamic acid residues. In full-length human and cynomolgus macaque PGRN, GluC is cleaved between amino acid residue numbers 576 (E) and 577 (A) to release a 17-amino acid-long C-terminal peptide, while GluC cleavage of the Δ3-truncated PGRN releases a 14-amino acid-long peptide. Mass spectrometry was then used to distinguish the mass of the longer endogenous full-length monkey PGRN peptide from the shorter human PGRN Δ3 peptide.

[0436] The amount of truncated PGRN protein in pooled CSF samples from NHP test animals treated with the AAV801-PGRNΔ3 vector was quantified using the above-described immunoaffinity LCMS assay, as described in Example 6. Unexpectedly, the major truncated C-terminal peptide in the sample did not correspond to the 14 amino acid long peptide (577-590) produced by digestion of human PGRNΔ3 with GluC, but rather to the 13 amino acid long peptide (577-589) released by digestion of human PGRN with a 4 amino acid C-terminal truncation (i.e., PGRNΔ4). This observation suggests that after administration of the AAV801-PGRNΔ3 vector to NHP test animals, transduced brain cells express human PGRNΔ3 protein, which is then modified intracellularly, presumably before secretion, or extracellularly after secretion, or both, to remove the C-terminal arginine (corresponding to position 590 in full-length human PGRN), resulting in PGRNΔ4 protein, which becomes the major human truncated PGRN species in the sample.

[0437] Sequence Listing

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

Claims

1. A recombinant adeno-associated virus (AAV) vector comprising a nucleotide sequence encoding a human progranulin (PGRN) polypeptide or a variant thereof.

2. The AAV vector of claim 1 , wherein the PGRN polypeptide variant is a carboxyl-terminal truncation variant lacking one or more amino acids originally present in the full-length wild-type human PGRN polypeptide, such that the variant PGRN polypeptide has reduced binding to the human Sortilin receptor (SORT1) protein compared to the full-length wild-type human PGRN polypeptide.

3. The AAV vector of claim 2, wherein the PGRN polypeptide variant lacks the last three or four carboxyl-terminal amino acids present in the full-length wild-type human PGRN polypeptide.

4. The AAV vector according to any one of claims 1 to 3, wherein the amino acid sequence of the PGRN polypeptide variant comprises SEQ ID NO: 14, SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 16 with the amino terminal signal sequence of SEQ ID NO: 17 removed.

5. The AAV vector of claim 4, wherein the amino acid sequence of the PGRN polypeptide variant comprises the amino acid sequence of SEQ ID NO: 14 with the carboxyl-terminal arginine removed and optionally with the amino-terminal signal sequence of SEQ ID NO: 17 removed.

6. The AAV vector of any one of claims 1 to 5, wherein the nucleotide sequence encoding the PGRN polypeptide or a variant thereof is a codon-optimized nucleotide sequence.

7. The AAV vector of claim 6, wherein the codon-optimized nucleotide sequence has a reduced number of CpG dinucleotides compared to the wild-type nucleotide sequence encoding the PGRN polypeptide or variant thereof.

8. The AAV vector of claim 7, wherein the nucleotide sequence encoding the PGRN polypeptide or its variant has 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10 or 1 to 5 fewer CpG dinucleotides than the wild-type nucleotide sequence encoding the PGRN polypeptide or its variant.

9. The AAV vector according to claim 7 or claim 8, wherein the wild-type nucleotide sequence encoding the PGRN polypeptide or its variant consists of the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

15.

10. The AAV vector of claim 7, wherein the nucleotide sequence encoding the PGRN polypeptide or variant thereof does not contain any CpG dinucleotides.

11. The AAV vector according to claim 1 to claim 10, wherein the nucleotide sequence encoding the PGRN polypeptide or its variant is the same as SEQ ID NO: 8 or SEQ ID NO: The nucleotide sequence of NO:15 is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% the same.

12. The AAV vector according to claim 1 to claim 5, wherein the PGRN is encoded The nucleotide sequence of the polypeptide or its variant is identical to the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO:

15.

13. The AAV vector of claim 1 to claim 12, wherein the vector comprises at least one AAV inverted terminal repeat (ITR).

14. The AAV vector of claim 13, wherein the nucleotide sequence comprises a wild-type or modified AAV inverted terminal repeat (ITR).

15. The AAV vector of claim 14, wherein the nucleotide sequence of the ITR is modified to reduce or eliminate the ability of the ITR to undergo end dissociation.

16. The AAV vector of claim 14, wherein the nucleotide sequence of the ITR is modified to reduce or eliminate the ability of the ITR to support packaging into capsids.

17. The AAV vector of claim 14, wherein the nucleotide sequence of the ITR is modified to alter the activity of the D region.

18. The AAV vector of claim 13, wherein the ITR is an AAV2 ITR.

19. The AAV vector of claim 18, wherein the AAV2 ITR is truncated.

20. The AAV vector of claim 13, wherein the ITR comprises SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20 or SEQ ID The nucleotide sequence of NO:21 or the complementary sequence or reverse complementary sequence of each of said sequences.

21. The AAV vector of claim 13, wherein the vector further comprises a third AAV ITR.

22. The AAV vector of claim 21, wherein the third ITR is modified to inactivate the terminal dissociation site.

23. The AAV vector of claim 1 to claim 22, wherein the vector further comprises a transcription control region operably linked to the nucleotide sequence encoding the PGRN polypeptide or variant thereof.

24. The AAV vector of claim 23, wherein the transcriptional control region is tissue or cell type specific.

25. The AAV vector of claim 24, wherein the transcriptional control region is brain tissue-specific or neuron-specific.

26. The AAV vector of claim 24, wherein the transcriptional control region is more transcriptionally active in CNS neurons than in hepatocytes.

27. The AAV vector according to claim 23, wherein the transcription control region comprises a brain tissue-specific or neuronal cell-specific promoter sequence and / or enhancer sequence.

28. The AAV vector of claim 23, wherein the promoter sequence and / or the enhancer sequence are derived from a synaptophysin gene.

29. The AAV vector of claim 28, wherein the promoter sequence and / or the enhancer sequence comprises the nucleotide sequence of SEQ ID NO: 6 or a functional subsequence, modification or variant thereof.

30. The AAV vector of claims 1 to 29, wherein the vector further comprises a 5' untranslated region (UTR) sequence. The vector according to claim 30 , wherein the 5′UTR sequence is derived from the synaptophysin gene.

32. The AAV vector of claim 30, wherein the 5'UTR sequence comprises, consists essentially of, or consists of: The nucleotide sequence of SEQ ID NO: 7 or a functional subsequence, modification or variant thereof.

33. The AAV vector of claims 1 to 32, wherein the vector further comprises a transcription termination signal sequence.

34. The AAV vector of claim 33, wherein the transcription termination signal sequence is a polyadenylation (poly(A)) signal sequence.

35. The AAV vector of claim 34, wherein the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene.

36. The AAV vector of claim 35, wherein the transcription termination signal sequence comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10, or a functional subsequence, modification or variant thereof.

37. The AAV vector of claims 1 to 36, wherein the vector further comprises an intron sequence.

38. The AAV vector of claims 1 to 37, wherein the vector further comprises a post-transcriptional regulatory element (PRE) sequence.

39. The AAV vector of claim 38, wherein the PRE sequence is a woodchuck hepatitis virus PRE or a hepatitis B virus PRE sequence.

40. The AAV vector of claims 1 to 60, wherein the vector further comprises a binding site for a microRNA (miRNA).

41. The AAV vector of claims 1 to 40, wherein the vector further comprises a stuffer or filler nucleotide sequence of sufficient length such that the entire length of the AAV vector, including the ITRs, is approximately 3.5 to 5.0 kilobases.

42. The AAV vector of claim 41, wherein the plug or stuffer nucleotide sequence is derived from a TATA binding protein (TBP) gene.

43. The AAV vector of claim 42, wherein the plug or stuffer nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:

11.

44. The AAV vector of claims 1 to 43, wherein the vector comprises a first AAV ITR, a transcription control region operably linked to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof, a transcription termination signal sequence, and a second AAV ITR.

45. The AAV vector of claims 1 to 5, wherein the vector comprises, in 5' to 3' order: (a) First AAV2 ITR, (b) the promoter sequence from the synaptophysin gene, (c) 5'UTR sequence from the synaptophysin gene, (d) a nucleotide sequence encoding a human PGRN polypeptide or a variant thereof operably linked to the promoter sequence, (e) a transcription termination signal sequence from the bovine growth hormone (bGH) gene, (f) sequences from an intron of the TBP gene, and (g) Second AAV2 ITR.

46. ​​The AAV vector of claim 45, wherein the promoter sequence from the synaptophysin gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6; the 5'UTR sequence from the synaptophysin gene comprises the nucleotide sequence of SEQ ID NO: 7; the nucleotide sequence encoding the PGRN polypeptide or variant thereof comprises the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 15; the transcription termination signal sequence from the bovine growth hormone (bGH) gene comprises the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 10; and the sequence from an intron of the TBP gene comprises the nucleotide sequence of SEQ ID NO:

11.

47. The AAV vector of claim 45 or claim 46, wherein the first AAV2 ITR and the second AAV2 ITR each comprise the nucleotide sequence of SEQ ID NO: 5, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, or SEQ ID NO: 21, or the complement or reverse complement of each of said sequences.

48. The AAV vector of claim 45 to claim 47, wherein the nucleotide sequence of the vector comprises the nucleotide sequence of SEQ ID NO: 19 or its reverse complement.

49. The AAV vector of claims 1 to 48, wherein the vector is 5 kilobases in length or less or 4 kilobases in length.

50. An AAV vector comprising an AAV capsid and the AAV vector of claim 1 to claim 49, wherein the vector is encapsidated by the capsid.

51. The AAV vector of claim 50, wherein the AAV capsid is at least partially neurotropic.

52. The AAV vector of claim 51, wherein the AAV capsid crosses the BBB at least as efficiently or more efficiently than the AAV9 capsid.

53. The AAV vector of claim 51 or claim 52, wherein the AAV capsid is an AAV-801 capsid comprising a VP3 protein having the amino acid sequence of SEQ ID NO:

3.

54. The AAV vector of claim 53, wherein the AAV capsid further comprises a VP1 protein having the amino acid sequence of SEQ ID NO: 1 and / or a VP2 protein having the amino acid sequence of SEQ ID NO:

2.

55. An AAV vector comprising an AAV capsid encapsidating the AAV vector, wherein the AAV capsid is an AAV-801 capsid, and wherein the nucleotide sequence of the vector comprises the nucleotide sequence of AAV-801 or its reverse complement.

56. A method for preventing or treating a disease or disorder caused by a deficiency of human PGRN polypeptide in a human subject, the method comprising administering to the subject an amount of an AAV vector or composition according to claim 1 to claim 55 to effectively increase the amount of PGRN polypeptide or a variant thereof in at least one biological fluid, tissue or cell of the subject.

57. The method of claim 56, wherein the method effectively increases the amount of PGRN polypeptide or a variant thereof in the subject's cerebrospinal fluid (CSF) to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of endogenous PGRN polypeptide in the CSF of a healthy person, e.g., 6 ng / mL.

58. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof in the subject's brain to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% of the amount of PGRN polypeptide in the brain of a healthy person.

59. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or its variant in the spinal cord of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 4 45%, 50%, 60%, 70%, 80%, 90% or 100%.

60. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or its variant in the serum of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 510%, 520 45%, 50%, 60%, 70%, 80%, 90% or 100%.

61. The method of claim 56, wherein the method is effective to increase the amount of bis(monoacylglycerol)phosphate (BMP) in the brain of the subject to at least or about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 45%, 50%, 60%, 70%, 80%, 90% or 100%, wherein the BMP can be any kind of BMP, such as BMP 18:1 / 18:1 or BMP 22:6 / 22:

6.

62. The method of claim 56, wherein the method is effective to reduce β-hexosaminidase (HexA) enzyme activity in the subject's brain to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 80%, 8 ... 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

63. The method of claim 56, wherein the method is effective in reducing beta-galactosidase (β-Gal) enzyme activity in the subject's brain to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% of the beta-Gal enzyme activity before treatment.

64. The method of claim 56, wherein the method is effective to reduce TDP43 fragmentation in the subject's brain to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 600%, 700%, 8 ... 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

65. The method of claim 56, wherein the method is effective to reduce lipofuscin levels in the subject's brain to at most or less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 500%, 600%, 600%, 700%, 600%, 700%, 8 ... 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%.

66. A method of reducing the frequency or severity of at least one symptom or sign caused by human PGRN polypeptide deficiency in a human subject, the method comprising administering to the subject an amount of the AAV vector or composition of claim 1 to claim 55 effective to reduce the frequency or severity of such symptom or sign.

67. The method of claim 66, wherein the symptoms or signs are characteristic of frontotemporal lobar degeneration (FTLD), including, for example, FTLD-TDP Type A.

68. The method of claim 67, wherein the symptom or sign is atrophy of a brain region selected from the group consisting of the frontal lobe, anterior temporal lobe, medial temporal lobe, posterior temporal lobe, orbitofrontal cortex, anterior cingulate gyrus, inferior parietal lobe, striatum, and thalamus.

69. The method of claim 67, wherein the symptom or sign is behavioral changes characteristic of behavioral variant frontotemporal dementia (BV-FTD).

70. The method of claim 67, wherein the symptom or sign is behavioral changes characteristic of nonfluent variant primary progressive aphasia (NFV-PPA).

71. The method of claim 67, wherein the symptoms or signs are characteristic of parkinsonism or corticobasal syndrome (CBS).

72. The method of claim 56 to claim 71, wherein the subject is diagnosed with frontotemporal lobar degeneration (FTLD) or frontotemporal dementia (FTD).

73. The method of claim 56 to claim 71, wherein the PGRN polypeptide deficiency in the subject is caused by a homozygous or heterozygous mutation in the GRN gene encoding the PGRN polypeptide, which mutation reduces the amount or activity of the PGRN polypeptide relative to a healthy person.

74. A method for preventing or treating frontotemporal dementia in a human subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of the AAV vector or composition of claim 1 to claim 55 to effectively prevent or treat frontotemporal dementia in the subject.

75. The method of claim 56 to claim 74, wherein the effective amount of the AAV vector is in the range of 1×10 10 to 1×10 15 The dose is vector genomes per kilogram (vg / kg) of subject body weight.

76. Use of the AAV vector of claims 1 to 55 in the manufacture of a medicament for treating or preventing frontotemporal dementia in a human subject.

77. A DNA plasmid comprising the nucleotide sequence of the AAV vector according to claim 1 to claim 49.

78. A host cell for AAV vector production, said host cell comprising the DNA plasmid according to claim 77.

79. The host cell of claim 78, wherein the host cell is a HEK293 cell.

80. The host cell of claim 78 or claim 79, wherein the host cell further comprises a gene encoding an AAVRep protein, such as a gene contained in a DNA plasmid.

81. The host cell of claim 78 to claim 80, wherein the host cell further comprises a gene encoding the AAV VP1 capsid protein, such as a gene contained in a DNA plasmid.

82. The host cell of claim 78 to claim 81 , wherein the host cell further comprises a gene encoding a viral accessory factor, such as a gene contained in a DNA plasmid.

83. A method for preparing an AAV vector, the method comprising: The host cell of claim 82 is incubated under conditions sufficient to allow production of the AAV vector, and the AAV vector produced thereby is purified.

84. An AAV vector produced by the method of claim 83.

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