Gene therapy for frontotemporal dementia

Abstract

The present disclosure describes improved vectors, such as adeno-associated virus (AAV) vectors, for expressing progranulin and its variants in transduced cells, and the use of such vectors to increase the amount of progranulin in subjects with progranulin deficiency, such as certain subjects with frontotemporal dementia (FTD) or frontotemporal lobar degeneration (FTLD).

Description

[Technical Field]

[0001] Frontotemporal dementia (FTD) refers to a clinical syndrome characterized by progressive deterioration of language, behavioral, and executive function due to selective neurodegeneration of the frontal and temporal cortical lobes (frontotemporal lobar degeneration or FTLD), in contrast to the global neurodegeneration commonly seen in Alzheimer's disease and certain other dementias. FTD often occurs during the prime of life, with onset of symptoms most commonly occurring between the ages of 45 and 64. Symptoms then rapidly progress, leading to functional decline and ultimately to death within an average of eight years. The only treatments for FTD focus on managing the effects of inevitable behavioral changes; none are effective in slowing or reversing the underlying neurodegeneration of the brain lobes that causes it. Given this overwhelming unmet medical need, there is a need in the art for effective methods to treat or prevent frontotemporal lobar degeneration and the resulting neurological damage and eventual death. Summary of the Invention

[0002] To address the 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 a subject characterized by a deficiency in the amount of human PGRN or variants 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 described below. These and related embodiments are described in further detail in the detailed description, including the examples and figures. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, equivalents to the specific embodiments described herein.

[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 of E1, wherein the PGRN polypeptide mutant is a carboxy-terminal truncation mutant lacking one or more amino acids that are originally present in the full-length wild-type human PGRN polypeptide, such that the mutant PGRN polypeptide has reduced binding to the human sortilin receptor (SORT1) protein compared to the full-length wild-type human PGRN polypeptide.

[0006] E3. An AAV vector of E2, in which the PGRN polypeptide mutant lacks the last three carboxy-terminal amino acids present in the full-length wild-type human PGRN polypeptide.

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

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

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

[0010] E7. An 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 a variant thereof.

[0011] E8. An AAV vector of E7, wherein the nucleotide sequence encoding the PGRN polypeptide or variant thereof 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 variant thereof.

[0012] E9. An AAV vector of E7 to E8, wherein the wild-type nucleotide sequence encoding the PGRN polypeptide or a variant thereof is contained in the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 15.

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

[0014] E11. An AAV vector of E1 to E10, wherein the nucleotide sequence encoding the PGRN polypeptide or 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. An AAV vector of 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. An AAV vector from E1 to E12 whose genome contains at least one AAV inverted terminal repeat (ITR).

[0017] E14. AAV vector at E13, in which the nucleotide sequence of the ITR is wild-type.

[0018] E15. An AAV vector at E13 in which the nucleotide sequence of the ITR has been modified.

[0019] E16. An AAV vector of E15, wherein the nucleotide sequence of the ITRs has been modified to reduce or eliminate the ability of the ITRs to undergo terminal degradation.

[0020] E17. An AAV vector at E15 in which the nucleotide sequence of the ITR has been modified to inactivate the terminal resolution site.

[0021] E18. An AAV vector at E15 in which the nucleotide sequences of the ITRs have been modified to reduce or eliminate the ability of the ITRs to support packaging into capsids.

[0022] E19. An AAV vector at E15 in which the nucleotide sequence of the ITR has been modified to inactivate the D region.

[0023] E20. E13-E14 AAV vector in which the ITRs are AAV2 ITRs.

[0024] E21. AAV vector of E20 with truncated AAV2 ITRs.

[0025] E22. The AAV vector of 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 complement or reverse complement of each of the above sequences.

[0026] E23. An AAV vector at E13-E14 in which the ITRs are other than the AAV2 ITRs.

[0027] E24. An AAV vector of 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 further comprising a third AAV ITR.

[0029] E26. An AAV vector of E25 in which the third ITR has been modified to inactivate the terminal degradation site.

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

[0031] E28. AAV vector of E27, in which the transcriptional control region is constitutive.

[0032] E29. AAV vector of E27, in which the transcriptional control region is inducible.

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

[0034] E31. The AAV vector of E30, wherein the transcriptional regulatory region is brain tissue-specific or neuronal cell-specific.

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

[0036] E33. An AAV vector of E27 to E32, in which the transcriptional regulatory region contains a promoter sequence.

[0037] E34. The AAV vector of E33, wherein the transcriptional control region further comprises an enhancer sequence.

[0038] E35. An AAV vector of E34 in which an enhancer sequence is located 5' to the promoter.

[0039] E36. The AAV vector of E34, in which the enhancer sequence is located 3' to the promoter.

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

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

[0042] E39. The AAV vector of E34, wherein each of the promoter and enhancer sequences is brain tissue-specific or neuronal cell-specific.

[0043] E40. The AAV vector of E33, wherein the promoter and / or enhancer sequences are derived from the synapsin gene.

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

[0045] E42. The AAV vector of E41, wherein the promoter sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6, or a promoter functional subsequence, variant, or mutant thereof.

[0046] E43. The AAV vector of E41, wherein the enhancer sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 6, respectively, or an enhancer functional subsequence, variant, or mutant thereof.

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

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

[0049] E46. Vectors E44 to E45, in which the 5'UTR sequence is derived from the synapsin gene.

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

[0051] E48. An AAV vector of 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, variant, or mutant thereof.

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

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

[0054] E51. An AAV vector of E50 in which the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene.

[0055] E52. The AAV vector of 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, variant, or mutant thereof.

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

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

[0058] E55. The AAV vector of E53, wherein no intron sequences interrupt the nucleotide sequence encoding the PGRN polypeptide or variant thereof.

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

[0060] E57. The AAV vector of E56, wherein an intron sequence is located 3' to the promoter and 5' to the nucleotide sequence encoding the PGRN polypeptide or 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. The AAV vector of E58, wherein the PRE sequence is located 3' to the nucleotide sequence encoding the PGRN polypeptide or variant thereof and 5' to the transcription termination signal sequence.

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

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

[0065] E62. An AAV vector of E61, wherein the miRNA binding site is located 3' to the nucleotide sequence encoding the PGRN polypeptide or a variant thereof and 5' to 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 total length of the AAV vector, including the ITRs, is about 3.5 to 5.0 kilobases.

[0067] E64. The AAV vector of E63, wherein the stuffer or filler nucleotide sequence is derived from the TATA binding protein (TBP) gene.

[0068] E65. The AAV vector of E64, wherein the stuffer or filler nucleotide sequence is a human TBP gene intron, or a subsequence, variant, or mutant thereof.

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

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

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

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

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

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

[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 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 of E73, wherein the vector further comprises a third AAV ITR located between the first and second AAV ITRs.

[0078] E75. An AAV vector of E74 in which the terminal degradation site in the third AAV ITR has been inactivated.

[0079] E76. An AAV vector according to any one of E68 to E75, wherein the transcriptional regulatory region is brain tissue-specific or neuronal cell-specific.

[0080] E77. The AAV vector of E76, wherein the transcriptional regulatory region comprises a promoter and / or enhancer sequence derived from a synapsin gene selected from the group consisting of a mammalian synapsin gene, a mouse synapsin gene, a rat synapsin gene, a primate synapsin gene, a monkey synapsin gene, a chimpanzee synapsin gene, and a human synapsin 1 gene (SYN1).

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

[0082] E79. An AAV vector of E67-E78, in which the transcription termination signal sequence is derived from the bovine growth hormone (bGH) gene.

[0083] E80. The AAV vector of 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, variant, or mutant thereof.

[0084] E81. The AAV vector of E67-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 vector is arranged in the order of 5' to 3': (a) a first AAV2 ITR; (b) a promoter sequence derived from the synapsin gene; (c) a 5'UTR sequence derived from the synapsin gene; (d) a nucleotide sequence encoding a human PGRN polypeptide or a variant thereof, operably linked to a promoter sequence; and (e) a transcription termination signal sequence derived from the bovine growth hormone (bGH) gene; and (f) a sequence derived from a TBP gene intron; (g) AAV vector E1 to E5 containing the second AAV2 ITR.

[0086] E83. The AAV vector of E82, wherein the promoter sequence derived from the synapsin gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:6; the 5'UTR sequence derived from the synapsin 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 derived 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 derived from the TBP gene intron comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:11.

[0087] E84. The AAV vector of E82-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 the foregoing sequences.

[0088] E85. The AAV vector of E82-E84, wherein the nucleotide sequence of the vector comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO: 19, or the reverse complement thereof.

[0089] E86. An AAV vector of E1 to E85, wherein the vector is 5 kilobases or less in length.

[0090] E87. An AAV vector of E1 to E86, wherein the vector is 4 kilobases or less in length.

[0091] An AAV vector comprising an E88.AAV capsid and an E1 to E87 AAV vector, wherein the vector is encapsidated by the capsid.

[0092] E89. An AAV vector of E88, wherein the AAV capsid is at least partially neuronotropic.

[0093] E90. An AAV vector of E89, wherein the AAV capsid is capable of crossing the blood-brain barrier (BBB) ​​in non-human primates or humans.

[0094] E91. An AAV vector of E90, wherein the AAV capsid crosses the BBB at least as efficiently as, or more efficiently than, the AAV9 capsid.

[0095] E92.AAV capsid is 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, AAV An AAV vector of E89 to E90 selected from the group consisting of SCH9, AAV4.18, AAV2-retro, AAV2 HBKO, AAV-TT and AAV-801.

[0096] E93. An AAV vector according to any one of 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.

[0097] E94. The AAV vector of 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.

[0098] E95. An AAV vector of E88-E94, wherein the vector is a single-stranded DNA genome.

[0099] E96. An AAV vector of E88 to E94, wherein the vector is a self-complementary DNA genome.

[0100] E97. AAV vector from E95 to E96, where the vector is positive polarity.

[0101] E98. AAV vectors from E95 to E96, where the vector is negative polarity.

[0102] 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 the reverse complement thereof.

[0103] A pharmaceutical composition comprising an AAV vector E100.E88-E100 and a pharmaceutically acceptable excipient.

[0104] E101. A method for preventing or treating a disease or disorder in a human subject caused by a deficiency in human PGRN polypeptide, comprising administering to the subject an AAV vector or composition of E1-E100 in an amount effective to increase the amount of PGRN polypeptide or a variant thereof in at least one biological fluid, tissue, or cell of the subject.

[0105] E102. The method of E101, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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 human, e.g., 6 ng / mL.

[0106] E103. The method of E101, 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 a healthy human brain.

[0107] E104. The method of E101, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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 human.

[0108] E105. The method of E101, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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 human.

[0109] E106. The method of E101, wherein the method is effective to increase the amount of bis(monoacylglycero)phosphate (BMP) 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 BMP in a healthy human brain, and the BMP can be any type of BMP, such as BMP 18:1 / 18:1 or BMP 22:6 / 22:6.

[0110] E107. The method of E101, wherein the method is effective to reduce beta-hexosaminidase (HexA) enzyme activity in the subject's brain to less than at or 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.

[0111] E108. The method of E101, wherein the method is effective to reduce β-galactosidase (β-Gal; β-galactosidase) enzyme activity in the subject's brain to less than at or about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of pre-treatment β-Gal enzyme activity.

[0112] E109. The method of E101, wherein the method is effective to reduce TDP43 fragmentation in the subject's brain to less than at or 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.

[0113] E110. The method of E101, wherein the method is effective to reduce lipofuscin levels in the subject's brain to less than at or about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of pre-treatment lipofuscin levels.

[0114] E111. A method for reducing the frequency or severity of at least one symptom or sign in a human subject caused by a deficiency in a human PGRN polypeptide, comprising administering to the subject an AAV vector or composition of E1-E100 in an amount effective to reduce the frequency or severity of such symptom or sign.

[0115] E112. The method of E111, wherein the symptom or sign is characteristic of frontotemporal lobar degeneration (FTLD), including, for example, FTLD-TDP type A.

[0116] E113. The method of E112, wherein the symptom or sign is atrophy in 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.

[0117] E114. The method of E112, wherein the symptom or sign is a behavioral change characteristic of behavioral-variant frontotemporal dementia (BV-FTD).

[0118] E115. The method of E112, wherein the symptom or sign is a behavioral change characteristic of non-fluent variant primary progressive aphasia (NFV-PPA).

[0119] E116. The method of E114 or E115, wherein the behavioral changes are selected from the group consisting of impaired word retrieval, apraxia of speech, agrammar, visual naming disorder, impaired word comprehension, phonological errors, impaired word repetition, impaired sentence repetition, impaired sentence comprehension, surface dyslexia, delusions, and hallucinations.

[0120] E117. The method of E112, wherein the symptom or sign is characteristic of Parkinsonism or corticobasal syndrome (CBS).

[0121] E118. The method of E101 to E117, wherein the subject has been diagnosed with frontotemporal lobar degeneration (FTLD) or frontotemporal dementia (FTD).

[0122] E119. The method of E101 to E118, wherein the deficiency of PGRN polypeptide in the subject is caused by a homozygous or heterozygous mutation in the GRN gene encoding PGRN polypeptide that reduces the amount or activity of PGRN polypeptide compared to a healthy human.

[0123] E120. A method for preventing or treating frontotemporal dementia in a human subject, comprising administering to the subject a prophylactically or therapeutically effective amount of an AAV vector or composition of E1-E100 effective to prevent or treat frontotemporal dementia in the subject.

[0124] E121. An effective dose of AAV vector is 1 × 10 per kilogram of subject body weight. 10 ~1×10 15 Methods E101 to E120, which are doses ranging from vector (vg / kg).

[0125] E122. The method of E101 to E121, wherein the AAV vector or composition is administered intracerebroventricularly to the subject.

[0126] E123. The method of E101 to E121, wherein the AAV vector or composition is administered intrathecally to the subject.

[0127] E124. The method of E101 to E121, wherein the AAV vector or composition is administered intravenously to the subject.

[0128] E125. Use of an AAV vector of E1 to E100 in the manufacture of a medicament for treating or preventing frontotemporal dementia in a human subject.

[0129] A DNA plasmid containing the nucleotide sequence of the AAV vector E126.E1 to E87.

[0130] Host cells for AAV vector production containing the DNA plasmids E127 and E126.

[0131] E128. The host cell of E127, wherein the host cell is a HEK293 cell.

[0132] E129. The host cell of E127-E128, wherein the host cell further contains genes encoding AAV Rep proteins, e.g., contained in a DNA plasmid.

[0133] E130. The host cell of E127-E129, wherein the host cell further comprises a gene encoding the AAV VP1 capsid protein, e.g., contained in a DNA plasmid.

[0134] E131. The host cell of E127-E130, wherein the host cell further comprises a gene encoding a viral helper factor, eg, contained in a DNA plasmid.

[0135] E132. A method of producing an AAV vector, comprising incubating the host cells of E131 under conditions sufficient to allow production of an AAV vector, and purifying the AAV vector produced thereby.

[0136] E133. An AAV vector produced by the method of E132. [Brief explanation of the drawings]

[0137] [Figure 1]Biacore 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 another source. [Figure 2A] Levels of mRNA encoding human progranulin in motor neurons differentiated from human iPSC cells transduced with an AAVDJ vector for expressing human full-length PGRN protein and an AAVDJ vector for expressing Δ3-truncated PGRN protein. [Figure 2B] Progranulin protein levels measured in conditioned medium for motor neurons differentiated from human iPSC cells transduced with an AAVDJ vector for expressing human full-length PGRN protein and an AAVDJ vector for expressing Δ3-truncated PGRN protein. [Figure 3A] Levels of mRNA encoding human progranulin in cortical glutaminergic neurons differentiated from human iPSC cells transduced with an AAV6 vector for expressing human full-length PGRN protein and an AAV6 vector for expressing Δ3-truncated PGRN protein. [Figure 3B] Figure 1 shows progranulin protein levels measured in the conditioned medium of cortical glutamatergic neurons differentiated from human iPSC cells transduced with an AAV6 vector expressing the full-length human PGRN protein and an AAV6 vector expressing the Δ3-truncated PGRN protein. Also shown are the results of a related experiment in which glutamatergic neurons were differentiated from iPSC cells derived from a human FTD patient and then transduced with an AAV6 vector expressing the Δ3-truncated human PGRN protein. [Figure 4]The human PGRNΔ3 protein concentration released into the culture medium by iPS cell-derived glutamatergic neurons after transduction with the AAV801-PGRNΔ3 vector was compared with that of untreated control cells and neurons transduced with the control vector AAV801-Luc. Statistical analysis was performed using one-way ANOVA followed by post-hoc analysis using Dunnett's multiple comparison test. P value****: <0.0001. [Figure 5] β-Hexosaminidase enzyme activity in lysates from glutamatergic neuron-differentiated iPSC cells. (Left) Results for control neurons derived from healthy iPSC cells: untreated neurons, neurons transduced with the control vector AAV801-Luc, and neurons transduced with the AAV801-PGRNΔ vector, a vector expressing the GRN transgene. (Right) Results for FTD iPSC cells: untreated neurons, neurons transduced with AAV801-Luc, and neurons transduced with the AAV801-PGRNΔ vector. Statistical analysis was performed using a two-way ANOVA test followed by post-hoc analysis using Sidak's multiple comparison test. P values ​​***: ≤0.0001; ***: =0.0004; ns: not significant. [Figure 6A] Transduction efficiency in brain tissue of AAV9 vectors expressing human full-length PGRN and AAV9 vectors expressing Δ3-truncated PGRN administered ICV to neonatal mice. [Figure 6B] Progranulin protein levels measured in CSF from neonatal mice 4 weeks after administration of AAV9 vectors for expressing human full-length PGRN and AAV9 vectors for expressing Δ3-truncated PGRN. [Figure 6C] Figure 1 shows progranulin protein levels measured in CSF from neonatal mice 4 weeks after administration of AAV9 vectors expressing human full-length PGRN and AAV9 vectors expressing Δ3-truncated PGRN. Data normalized to the amount of endogenous mouse PGRN in the samples. [Figure 6D]Progranulin protein levels measured in serum from neonatal mice 4 weeks after administration of AAV9 vectors expressing human full-length PGRN and AAV9 vectors expressing Δ3-truncated PGRN. [Figure 7A] Progranulin protein detected by immunohistochemistry in brain sections from neonatal mice 4 weeks after administration of a negative control AAV9 vector to express green fluorescent protein. [Figure 7B] Progranulin protein detected by immunohistochemistry in brain sections from neonatal mice 4 weeks after administration of an AAV9 vector expressing human full-length PGRN. [Figure 7C] Progranulin protein detected by immunohistochemistry in brain sections from neonatal mice 4 weeks after administration of an AAV9 vector to express Δ3-truncated PGRN. [Figure 8A] Transduction efficiency in brain tissue of AAV1 vectors, AAVDJ vectors, and AAV9 vectors for expressing human full-length PGRN, and AAV1 vectors, AAVDJ vectors, and AAV9 vectors for expressing Δ3-truncated PGRN, administered ICV to 6-month-old mice. [Figure 8B] Progranulin protein levels measured in CSF from mice 3 months after administration of AAV1, AAVDJ, and AAV9 vectors for expressing human full-length PGRN, and AAV1, AAVDJ, and AAV9 vectors for expressing Δ3-truncated PGRN, when the mice were 6 months old. [Figure 8C] Progranulin protein levels measured in CSF from mice 6 months after administration of AAV9 vectors expressing human full-length PGRN and AAV9 vectors expressing Δ3-truncated PGRN, when the mice were 6 months old. [Figure 9A] Transduction efficiency in brain tissue of AAVPHP.B vectors for expressing human full-length PGRN and AAVPHP.B vectors for expressing Δ3-truncated PGRN administered intravenously to 6-week-old mice. [Figure 9B]Progranulin protein levels measured in brain tissue from mice 4 weeks after administration of the AAVPHP.B vector for expressing human full-length PGRN and the AAVPHP.B vector for expressing Δ3-truncated PGRN, when the mice were 6 weeks old. [Figure 9C] Progranulin protein levels measured in CSF from mice 4 weeks after administration of AAVPHP.B vectors for expressing human full-length PGRN and AAVPHP.B vectors for expressing Δ3-truncated PGRN, when the mice were 6 weeks old. [Figure 10] Photomicrographs of the hippocampal region, including the CA3 region, from a mouse 3 months after administration of a negative control AAV9 vector to express green fluorescent protein, when the test animals were 6 months old. The top left photomicrograph shows negative staining for human progranulin protein. The bottom left photomicrograph shows negative staining for endogenous mouse Iba-1, a marker of microglial activation. The right photomicrograph shows intact tissue cytoarchitecture with H&E staining. [Figure 11] Photomicrographs of the hippocampal region, including CA3, of a mouse 3 months after administration of an AAV9 vector for human full-length PGRN, when the test animals were 6 months old. The top left photomicrograph shows positive and focal staining for human progranulin protein. The bottom left photomicrograph shows positive staining for endogenous mouse Iba-1. The right photomicrograph shows decreased cellularity by H&E staining. [Figure 12] Photomicrographs of the hippocampal region, including CA3, of a mouse 3 months after administration of an AAV9 vector for human Δ3-truncated PGRN, when the test animals were 6 months old. The top left photomicrograph shows positive and diffuse staining for human progranulin protein. The bottom left photomicrograph shows no or minimal staining for endogenous mouse Iba-1. The right photomicrograph shows normal cellularity by H&E staining. [Figure 13A]Vector copy number (VGC) counts in liver samples from Grn- / - KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector intracerebroventricularly (ICV) into one hemisphere. Age-matched wild-type and Grn- / - KO mice administered PBS by the same route served as controls. VGC counts are normalized to the amount of cellular genomic DNA (μg gDNA). [Figure 13B] Vector copy number (VGC) counts in brain samples from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn- / -KO mice administered PBS via the same route served as controls. VGC counts are normalized to the amount of cellular genomic DNA (μg gDNA). Pink dots represent results from brain samples from the injected hemisphere, and black dots represent data from the contralateral hemisphere. [Figure 13C] The amount of hGRNΔ3 mRNA in brain samples from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. RNA levels are normalized to the amount of RNA expressed from housekeeping genes. Pink dots represent results from brain samples from the injected hemisphere, and black dots represent data from the contralateral hemisphere. [Figure 13D] Figure 13 shows the concentration of PGRNΔ3 protein in fluid and tissue samples from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. Pink dots represent the results of fluid and tissue samples taken from the injected hemisphere, while black dots represent data from the contralateral hemisphere. Figure 13D shows the results of a cerebrospinal fluid (CSF) sample. Figure 13E shows the results of a serum sample. Figure 13F shows the results of a brain tissue sample. [Figure 13E]Figure 13 shows the concentration of PGRNΔ3 protein in fluid and tissue samples from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. Pink dots represent the results of fluid and tissue samples taken from the injected hemisphere, while black dots represent data from the contralateral hemisphere. Figure 13D shows the results of a cerebrospinal fluid (CSF) sample. Figure 13E shows the results of a serum sample. Figure 13F shows the results of a brain tissue sample. [Figure 13F] Figure 13 shows the concentration of PGRNΔ3 protein in fluid and tissue samples from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. Pink dots represent the results of fluid and tissue samples taken from the injected hemisphere, while black dots represent data from the contralateral hemisphere. Figure 13D shows the results of a cerebrospinal fluid (CSF) sample. Figure 13E shows the results of a serum sample. Figure 13F shows the results of a brain tissue sample. [Figure 14A] Concentrations of BMP 18:1 / 18:1 species (FIG. 14A) and BMP 22:6 / 22:6 species (FIG. 14B) in brain samples collected from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn- / -KO mice administered PBS by the same route served as controls. Pink dots represent results from brain samples collected 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. [Figure 14B]Concentrations of BMP 18:1 / 18:1 species (FIG. 14A) and BMP 22:6 / 22:6 species (FIG. 14B) in brain samples collected from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn- / -KO mice administered PBS by the same route served as controls. Pink dots represent results from brain samples collected 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. [Figure 15A] Enzyme activities of β-hexosaminidase (FIG. 15A) and β-galactosidase (FIG. 15B) in brain samples collected from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. Pink dots represent results from brain samples collected 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. [Figure 15B] Enzyme activities of β-hexosaminidase (FIG. 15A) and β-galactosidase (FIG. 15B) in brain samples collected from Grn− / − KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector into one hemisphere via ICV delivery. Age-matched wild-type and Grn− / − KO mice administered PBS via the same route served as controls. Pink dots represent results from brain samples collected 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. [Figure 16]TDP43 fragmentation in brain samples from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery to one hemisphere, and age-matched control wild-type and Grn- / -KO mice administered PBS. Fragmentation was estimated using semiquantitative Western blot analysis as the ratio of staining intensity of full-length TDP43 protein to its 20 kDa fragment, with lower ratios indicating greater fragmentation. Statistical analysis involved one-way ANOVA followed by post-hoc analysis using Dunnett's multiple comparison test. P value*: <0.05; ns: not significant. [Figure 17A] Figure 17 shows quantification of lipofuscin accumulation in different regions of brain samples taken from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery into the right hemisphere. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn- / -KO mice administered PBS via the same route served as controls. Figure 17A shows results from the CA2 / 3 hippocampal region. Figure 17B shows results from the whole hippocampus. Figure 17C shows results from the prefrontal cortex. Figure 17D shows results from the thalamus. Figure 17E shows results from the whole brain. The legend "Right" refers to data collected from the same hemisphere of the brain injected with the GRN vector, while "Left" refers to the contralateral hemisphere. [Figure 17B] Figure 17 shows quantification of lipofuscin accumulation in different regions of brain samples taken from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery into the right hemisphere. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn- / -KO mice administered PBS via the same route served as controls. Figure 17A shows results from the CA2 / 3 hippocampal region. Figure 17B shows results from the whole hippocampus. Figure 17C shows results from the prefrontal cortex. Figure 17D shows results from the thalamus. Figure 17E shows results from the whole brain. The legend "Right" refers to data collected from the same hemisphere of the brain injected with the GRN vector, while "Left" refers to the contralateral hemisphere. [Figure 17C]Figure 17 shows quantification of lipofuscin accumulation in different regions of brain samples taken from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery into the right hemisphere. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn- / -KO mice administered PBS via the same route served as controls. Figure 17A shows results from the CA2 / 3 hippocampal region. Figure 17B shows results from the whole hippocampus. Figure 17C shows results from the prefrontal cortex. Figure 17D shows results from the thalamus. Figure 17E shows results from the whole brain. The legend "Right" refers to data collected from the same hemisphere of the brain injected with the GRN vector, while "Left" refers to the contralateral hemisphere. [Figure 17D] Figure 17 shows quantification of lipofuscin accumulation in different regions of brain samples taken from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery into the right hemisphere. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn- / -KO mice administered PBS via the same route served as controls. Figure 17A shows results from the CA2 / 3 hippocampal region. Figure 17B shows results from the whole hippocampus. Figure 17C shows results from the prefrontal cortex. Figure 17D shows results from the thalamus. Figure 17E shows results from the whole brain. The legend "Right" refers to data collected from the same hemisphere of the brain injected with the GRN vector, while "Left" refers to the contralateral hemisphere. [Figure 17E] Figure 17 shows quantification of lipofuscin accumulation in different regions of brain samples taken from Grn- / -KO mice administered 1e11 vg of AAV9-PGRNΔ3 vector via ICV delivery into the right hemisphere. Age-matched wild-type mice administered the control vector AAV9-Luc and Grn- / -KO mice administered PBS via the same route served as controls. Figure 17A shows results from the CA2 / 3 hippocampal region. Figure 17B shows results from the whole hippocampus. Figure 17C shows results from the prefrontal cortex. Figure 17D shows results from the thalamus. Figure 17E shows results from the whole brain. The legend "Right" refers to data collected from the same hemisphere of the brain injected with the GRN vector, while "Left" refers to the contralateral hemisphere. [Figure 18A]Vector copy number (VGC) counts in liver samples from Grn-null knock-in (KI) mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched Grn-null KI mice administered PBS via the same route served as controls. VGC counts were normalized to the amount of cellular genomic DNA (μg gDNA). Statistical analysis involved one-way ANOVA followed by post-hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant. [Figure 18B] Vector copy number (VGC) counts in brain samples from Grn-null knock-in (KI) mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched Grn-null KI mice administered PBS via the same route served as controls. VGC counts were normalized to the amount of cellular genomic DNA (μg gDNA). Statistical analysis involved one-way ANOVA followed by post-hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant. [Figure 18C] The amount of hGRNΔ3 mRNA in brain samples from Grn-null KI mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched wild-type and Grn-null 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 involved one-way ANOVA followed by post-hoc analysis using Dunnett's multiple comparison test. P values ​​****: <0.0001; *: <0.05; ns: not significant. [Figure 18D]Figure 18D shows the results for cerebrospinal fluid (CSF) samples. Figure 18E shows the results for serum samples. Figure 18F shows the results for brain tissue samples. Statistical analysis was the same as that described for Figure 18C. [Figure 18E] Figure 18D shows the results for cerebrospinal fluid (CSF) samples. Figure 18E shows the results for serum samples. Figure 18F shows the results for brain tissue samples. Statistical analysis was the same as that described for Figure 18C. [Figure 18F] Figure 18D shows the results for cerebrospinal fluid (CSF) samples. Figure 18E shows the results for serum samples. Figure 18F shows the results for brain tissue samples. Statistical analysis was the same as that described for Figure 18C. [Figure 18G]Concentrations of BMP 18:1 / 18:1 species (FIG. 18G) and BMP 22:6 / 22:6 species (FIG. 15H) in brain samples collected from Grn-null KI mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery route. Age-matched wild-type and Grn-null KI mice administered PBS via the same route served as controls. Statistical analysis was the same as described for FIG. 18C. [Figure 18H] Concentrations of BMP 18:1 / 18:1 species (FIG. 18G) and BMP 22:6 / 22:6 species (FIG. 15H) in brain samples collected from Grn-null KI mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery route. Age-matched wild-type and Grn-null KI mice administered PBS via the same route served as controls. Statistical analysis was the same as described for FIG. 18C. [Figure 18I] β-hexosaminidase enzyme activity in brain samples from Grn-null KI mice administered the AAVPHP.B-PGRNΔ3 vector (5e12 vg / kg or 1e13 vg / kg) via intravenous (IV) delivery. Age-matched wild-type and Grn-null KI mice administered PBS via the same route served as controls. Statistical analysis was the same as that described for Figure 18C. [Figure 19A] Concentrations of PGRNΔ3 protein in CSF samples from cynomolgus monkeys 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 the intravenous (IV) delivery route. Two animals were administered each vector and dose. PGRNΔ3 protein was not detected in one test animal administered the low dose of AAV801-PGRNΔ3 and in both test animals administered 2e13 vg / kg of AAV9-PGRNΔ3. The dotted line labeled "Target" indicates the level of progranulin naturally occurring in humans. [Figure 19B]Concentration of PGRNΔ3 protein in serum samples from cynomolgus monkeys 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 were administered each vector and dose. [Figure 20] Vector copy number (VGC) counts in brain and other tissue samples collected 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). Samples were collected from multiple brain regions, spinal cord, and peripheral nervous and non-neuronal tissues. VGC counts are normalized to the amount of cellular genomic DNA (μg gDNA). [Figure 21] Quantity of hGRNΔ3 mRNA in brain and other tissue samples collected 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). Samples were collected from multiple brain regions, spinal cord, and peripheral nervous and non-neuronal tissues. RNA levels were normalized to the amount of RNA expressed from housekeeping genes. [Figure 22A] Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22B]Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22C] Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22D] Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22E]Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22F] Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 22G] Representative images of PGRNΔ3 transgene expression visualized by in situ hybridization performed on brain and spinal cord tissue samples from male cynomolgus monkeys 28 days after IV administration of 2e13 vg / kg of the AAV801-PGRNΔ3 vector. Figure 22A shows results from the motor cortex, Figure 22B shows results from the entorhinal cortex, Figure 22C shows results from hippocampal pyramidal cells, Figure 22D shows results from the thalamus, Figure 22E shows results from the dentate nucleus, Figure 22F shows results from the spinal cord, and Figure 22G shows results from the thalamus from a negative control animal. In the images, mRNA is labeled in red, and nuclei are labeled in blue. [Figure 23]Comparison of PGRNΔ3 protein concentrations with endogenous macaque progranulin levels in brain and other tissue samples collected 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). Samples were collected from multiple brain regions, spinal cord, and peripheral nervous and non-neuronal tissues. DETAILED DESCRIPTION OF THE INVENTION

[0138] The following description is directed to various embodiments. The present disclosure is not intended to refer to any particular embodiment or otherwise limit the scope of the disclosure. While one or more of these embodiments may be preferred, the disclosed embodiments should not be construed or otherwise used as limiting the scope of the disclosure, including the claims. Furthermore, those skilled in the art will appreciate that the following description is broad in scope and that the description of any embodiment is intended only as an example of that embodiment and is not intended to imply that the scope of the disclosure, including the claims, is limited to that embodiment.

[0139] Specific Definitions As used herein, "adeno-associated viral vector" refers to an adeno-associated virus (AAV) that contains a naturally occurring or non-naturally occurring AAV capsid that encapsidates the vector. Adeno-associated viral vectors may be abbreviated as "AAV vectors" and, depending on the context, may be referred to by synonyms such as "recombinant AAV vectors," "rAAV vectors," "rAAV," or simply "vectors."

[0140] As used herein, "vector" refers to an AAV genome that contains a heterologous nucleotide sequence and that has been modified to render any AAV vector containing the vector replication-incompetent, for example, by inactivating or deleting the endogenous AAV rep and / or cap genes.

[0141] As used herein, "heterologous nucleotide sequence" means a nucleotide sequence that is introduced into one organism (including a virus) from a different organism (including an organism). The sequence of a heterologous nucleotide sequence can be the same as that occurring in nature, or it can be a modified version thereof, or it can even be partially or completely synthetic.

[0142] 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 to RNA.

[0143] As used herein, "transgene" means a nucleotide sequence that encodes at least one polypeptide and / or a nucleotide sequence that encodes at least one functional RNA molecule. A transgene may also be referred to by the synonym "gene of interest."

[0144] 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, whether unicellular or multicellular, can be derived from mammals or insects, or other organisms.

[0145] As used herein, the term "purify," and the related terms "purified" and "purification," when used in reference to an AAV vector, or a sample or preparation thereof, indicates a relative increase or improvement in purity compared to the starting material containing the vector and / or compared to a previous intermediate purification step in some schemes of sequential purification steps intended to purify a biological product, and does not require a particular qualitative or quantitative purity, unless otherwise specified.

[0146] As used herein, "transduction" refers to 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 a replication-competent adeno-associated virus genome into a cell.

[0147] As used herein, "target cell" means a cell that an AAV vector is designed or intended to transduce, or that is experimentally observed to be transduced by an AAV vector, whether in vitro or in vivo in a subject.

[0148] 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.

[0149] Frontotemporal dementia and frontotemporal lobar degeneration Frontotemporal dementia (FTD) refers to a clinical syndrome characterized by progressive deterioration of language, behavior, and executive function due to selective neurodegeneration of the frontal and temporal cortical lobes (frontotemporal lobar degeneration or FTLD), in contrast to the global neurodegeneration commonly seen in Alzheimer's disease and certain other dementias. Most patients with FTD present between their 40s and early 60s, with prevalence rates ranging from 10% in patients under 45 years of age to 60% in those aged 45 to 64 years, and 30% in those over 64 years of age. Following onset, symptoms progressively worsen, and survival times range from 6 to 11 years, with an average of 8 years, depending on the FTD subtype; however, some aggressive subtypes can be fatal in as little as 2 years.

[0150] FTD manifests with a variety of neurological symptoms depending on the underlying pattern of neurodegeneration, and three clinical variants have been defined. Behavioral variant frontotemporal dementia (BV-FTD) is characterized by early behavioral and executive impairments; nonfluent variant primary progressive aphasia (NFV-PPA) is characterized by progressive impairments in speech production, grammar, and word output; and semantic variant primary progressive aphasia (SV-PPA) is characterized by progressive impairments in semantic knowledge and naming. Diagnosis of FTD patients with one of the clinical variants depends on the predominance of behavioral and language deficits, especially early in the disease course. A diagnosis of BV-FTD variant requires the presence of at least three of the following behavioral changes: disinhibition; apathy or lethargy; loss of empathy or empathy; stereotypic, compulsive, or perseverative behaviors; changes in oral or eating habits; and executive dysfunction with relative preservation of visuospatial skills and memory. Both variants of primary progressive aphasia (PPA) require the presence of language impairment, of which aphasia is the first and most prominent. Early significant impairments in episodic memory, visual memory, or visual-perceptual abilities, or behavioral disturbances, exclude PPA. PPA is then further distinguished into semantic and non-fluent variants. In SV-PPA, patients exhibit impairments in visual naming and word comprehension, as well as at least three of the following language-related functional impairments or abilities: surface alexia or dysgraphia; preserved repetition; and preserved speech production. In contrast, NFV-PPA requires at least one agrammatism or apraxia of speech and at least two of the following: impairments in complex sentence comprehension, preserved word comprehension, and preserved object knowledge. Motor symptoms may also affect a small percentage of FTD patients. Motor neuron disease occurs in more than 12% of BV-FTD patients and less frequently in PPA-variant FTD patients. Approximately 20% of individuals with FTD also have parkinsonian symptoms and may show features of corticobasal syndrome or progressive supranuclear palsy syndrome.

[0151] Although many of the symptoms of FTD are also seen in patients with other types of dementia or psychiatric disorders, the brains of patients with FTD exhibit characteristic atrophy of the frontal or temporal lobes, with atrophy of the frotoinsular regions being particularly symptomatic of FTD. The 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.

[0152] Confirmation of FTD typically requires brain examination of suspected cases to detect neuropathological changes associated with the frontotemporal lobar degeneration (FTLD) disease process. FTLD is generally characterized by neuronal loss, gliosis, and microvacuolar changes in the frontal lobe, anterior temporal lobe, anterior cingulate cortex, and insular cortex. However, nearly all cases can be differentiated into three major subtypes: FTLD-tau, FTLD-TDP, and FTLD-FUS, based on the corresponding presence of abnormal deposits of specific proteins that also correlate with specific characteristic patterns of neurogenesis. FTLD-tau, which accounts for approximately 36–50% of all FTLD cases, is defined by the presence of microtubule-associated protein tau (MAPT) deposits on neuropathological examination and is associated with Pick's disease, corticobasal degeneration, and progressive supranuclear palsy FTLD subtypes. FTLD-FUS, which accounts for approximately 10% of FTLD cases, is defined by the presence of fused-in-sarcoma (FUS) protein deposits in the brain and is associated with early onset of FTD behavioral symptoms and the absence of motor and language disorders. FTLD-TDP, the most common, accounts for approximately 50% of FTLD cases and is defined by the presence of the 43-kDa TAR DNA-binding protein (TDP-43) in the brain.

[0153] FTLD-TDP is further distinguished into three subtypes, A, B, and C, based on both the pattern of abnormal protein deposition in the brain and the characteristic pattern of neurodegeneration. FTLD-TDP type A is associated with asymmetric dorsal atrophy affecting the frontal and temporal lobes (anterior, medial, and posterior regions), orbitofrontal cortex, anterior cingulate cortex, inferior parietal lobe, striatum, and thalamus. FTLD-TDP type B is associated with atrophy affecting the lateral and polar regions of the temporal lobe, anterior insular cortex, cingulate cortex, medial prefrontal cortex, and orbitofrontal cortex, with the frontal lobe being more severely affected in the posterior regions. FTLD-TDP type C is associated with right- or left-dominant anterior temporal lobe atrophy, further affecting the amygdala, hippocampus, orbitofrontal cortex, and insular cortex. Different FTLD-TDP subtypes have also been reported to correlate with specific FTD symptoms. Thus, TDP type A accounts for approximately 50% of NF-PPA cases and 25% of suspected corticobasal degeneration cases, but only a small proportion of BV-FTD (with or without motor neuron disease), type B accounts for approximately two-thirds of FTD cases with motor neuron disease and 25% of all BV-FTD cases, and type C accounts for approximately 90% of all SV-PPA or temporal lobe variant BV-FTD cases (Bang, J. et al., Lancet, 386:1672-82, 2015).

[0154] Human genetic studies have confirmed that perhaps 40% of FTLD cases are familial, and mutations in three genes—9ORF72, MAPT, and GRN—have been identified that are responsible for approximately 60% of all inherited cases. The GRN gene encodes progranulin (PGRN), a secreted protein involved in diverse functions, including cell cycle regulation, wound repair, axon growth, and inflammation. PGRN binds to tumor necrosis factor receptors (TNFRs), among other receptors, suggesting a mechanism for regulating inflammation triggered by TNFα or other inflammatory mediators. Heterozygous loss-of-function mutations in the GRN gene result in haploinsufficiency, with substantial loss of PGRN concentrations in cerebrospinal fluid (CSF) and serum. More than 70 pathogenic mutations in the GRN gene that result in FTD have been identified, the majority of which are nonsense mutations that induce degradation of the mRNA encoding progranulin. GRN mutations, which result in haploinsufficiency and reduced PGRN production and are found in approximately 5–20% of familial FTD cases, are associated with characteristic patterns of FTD clinical syndrome, brain atrophy, and neuropathology. As reported by Bang et al., loss-of-function mutations in GRN are associated with the neurological symptoms and signs of BV-FTD, NFV-PPA, parkinsonism, and corticobasal syndrome (CBS), as well as asymmetric patterns of neurodegeneration affecting primarily the anterior temporal, temporoparietal, and frontal lobes (the left side is more strongly associated with PPA syndrome, while the right side is more strongly associated with BV-FTD symptoms), anterior cingulate cortex, and insular cortex. Furthermore, neuropathological examinations of FTD patients with GRN mutations frequently reveal cellular changes and TDP protein deposition characteristic of FTLD-TDP type A.

[0155] Adeno-associated virus (AAV) The present disclosure provides vectors made from recombinantly modified adeno-associated viruses (AAV). AAV vectors can deliver genes, which may be under the control of transcriptional and other regulatory elements, to target cells via transduction. AAV vectors are useful in gene therapy for various diseases and disorders by providing functional copies of genes to target cells in which the endogenous version is defective or mutated.

[0156] AAV is a small, non-enveloped, apparently non-pathogenic parvovirus that depends on certain other viruses to provide gene products (known as helper factors) essential for its replication; this biological coincidence makes it well suited to function as a recombinant vector. For example, adenovirus (AdV) can function as a helper virus in cells co-infected with adenovirus and AAV by providing specific adenoviral factors, such as E1A, E1B55K, E2A, and E4ORF6 proteins, as well as VA RNA. Other helper viruses, such as herpes simplex virus, have also been identified. Because AAV replication depends on accessory factors provided by other viruses, AAV has been classified as a type of dependovirus. The AAV virion has two major structural features, termed the capsid and the genome. The capsid is an icosahedral protein shell that encloses and protects (encapsidates) the viral genome, which contains genes and other sequences necessary for viral replication within infected cells.

[0157] The AAV genome is a single strand of DNA containing two genes, rep and cap. In AAV2, a naturally occurring AAV that infects humans and is particularly well characterized biologically, the genome is approximately 4.7 kilobases in length. Alternative splicing of transcripts from two promoters allows the rep gene to produce four related multifunctional proteins called Reps (referred to in AAV2 as Rep 78, Rep 68, Rep 52, and Rep 40, named according to their apparent molecular weights), which are involved in viral gene expression and genome replication and packaging. Alternative splicing of transcripts from a single promoter controlling a single cap gene produces three related structural proteins, VP1, VP2, and VP3, which self-assemble into a total of 60 proteins that form the viral icosahedral capsid in a ratio of approximately 1:1:10, respectively. VP1 is the longest of the three VP proteins and contains amino acids in its amino-terminal region that are not present in VP2, which in turn is longer than VP3 and contains amino acids in its amino-terminal region that are not present in VP3. In addition to containing the genome, the capsid protein mediates interaction through specific binding with receptors on the surface of target cells. Based on this, AAVs can be limited in their infectivity to specific animal species and even tissues within the same type of animal, a phenomenon called tropism. For example, some types of AAVs can preferentially infect liver cells (e.g., hepatocytes) over muscle or nerve cells.

[0158] In addition to the rep and cap genes, the intact AAV genome contains relatively short (145 nucleotides in AAV2) sequence elements located at each of its 5' and 3' ends, called inverted terminal repeats (ITRs). The ITRs contain nested palindromic sequences that can self-anneal by Watson-Crick base pairing to form T-shaped or hairpin-shaped secondary structures. In AAV2, the ITRs have been shown to have important functions required for the viral life cycle, including converting the single-stranded DNA genome to the double-stranded form required for gene expression and packaging the single-stranded AAV genome into capsid assembly by the Rep protein.

[0159] Numerous naturally occurring types of AAV have been discovered in different species. Previously, only six types of primate AAVs were isolated from biological samples (AAV1, AAV2, AAV3, AAV4, AAV5, and AAV6), and the first five of these had sufficient structural differences to be classified as distinct serotypes based on antibody cross-reactivity experiments. Subsequently, two novel AAVs, designated AAV7 and AAV8, were discovered by PCR amplification of DNA from rhesus macaques using primers targeting highly conserved regions in the cap genes of previously discovered AAVs (Gao, G. et al., Proc. Natl. Acad. Sci. USA, 99:11854-9, 2002). Subsequently, using a similar approach, numerous novel AAVs were cloned from human and nonhuman primate tissues, significantly expanding the range of AAV capsid protein sequences (Gao, G. et al., J. Virol., 78:6381-8, 2004). Although many AAV capsid protein sequences are highly similar to each other and to previously identified AAVs and are often referred to as distinct AAV "serotypes," it is not necessarily expected that all such capsids will be immunologically distinguishable when tested by antibody cross-reactivity. AAVs or AAV capsids that are serologically indistinguishable from a defined serotype but contain capsid proteins with different amino acid sequences are better referred to as variants of known serotypes. Numerous capsids made from naturally occurring and non-naturally occurring capsid proteins have proven useful for generating AAV gene therapy vectors.

[0160] As confirmed by the study of AAV2, AAV viral particles enter cells via endocytosis after binding to one or more receptor molecules on the cell surface. 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 into the nucleus. Once inside the nucleus, the capsid disassembles, releasing the genome, which is then acted upon by cellular DNA polymerase to synthesize a second DNA strand starting from the 3'-end ITR, which acts as a primer after self-annealing. Expression of the rep and cap genes then begins, allowing the subsequent formation and release of new viral particles from the cell.

[0161] AAV vectors Motivated by the relative simplicity of AAV structure and life cycle, as well as the fact that AAV is known not to be pathogenic in humans, researchers investigated whether it could be engineered to convert AAV from a virus into a recombinant vector for gene therapy. Briefly, this was accomplished by cloning the entire AAV2 genome, including both ITRs, into a plasmid, moving the rep and cap genes to another plasmid, and replacing them with a heterologous gene expression cassette containing a promoter controlling the transgene-encoding protein. Thus, the only viral genomic sequences retained in the vector were the ITRs, because they perform essential functions in packaging and gene expression; without them, the AAV vector could neither be produced nor function to express a transgene after transduction of target cells. Finally, to avoid the need for coinfection with a helper virus, which is necessary for AAV virion replication, the genes for the so-called helper factors (e.g., for AdV, E1A helper factor, E1B55K helper factor, E2A helper factor, E4ORF6 helper factor, and VA RNA helper factor) were cloned into a third plasmid.

[0162] When the three plasmids (sometimes referred to as the transgene, rep / cap, and helper plasmids) were transfected together into mammalian host cells, the Rep and capsid proteins, as well as the helper virus factors, were expressed from their respective plasmids. These gene products then functioned within the host cells to replicate the vector from the plasmid on which they resided into single-stranded DNA, assemble capsids, and package the single-stranded genome into the capsids to form the vector. The vector could then be purified from the host cells. Because the rep and cap genes were present in trans on a different plasmid, outside of their usual context adjacent to the ITRs, they were not packaged into the vector. As a result, the AAV vectors produced in this manner were able to bind to and deliver the expression cassette within their genome into target cells, but they were unable to replicate and generate new vector particles.

[0163] If the vector functions as intended, after transduction, the expression cassette becomes transcriptionally active in the target cell, producing the gene product encoded by the transgene. AAV vectors are extremely versatile because they can be designed to contain various transgenes in various configurations under the control of various functional sequences and regulatory elements, and can be combined with various naturally occurring capsids and engineered capsids with various tropisms and other properties. Thus, it is possible to produce a large number of types of gene products, with some control over the type of cell to be transduced and the amount of gene product produced.

[0164] An AAV vector comprises a vector encapsidated 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 a transduced target cell. In some embodiments, the heterologous nucleotide sequence is derived from a different type of virus or an entirely different type of organism, such as an animal, plant, protist, fungus, bacterium, archaea, or other type of organism. In some embodiments, the heterologous nucleotide sequence replaces part or all of the native AAV rep and / or cap genes such that the vector is unable to express functional Rep or VP proteins in the transduced target cell. In some embodiments, the entire sequence of the vector consists of the heterologous nucleotide sequence, except for the AAV inverted terminal repeats located at the termini of the genome.

[0165] The length of the genome of an AAV vector of the present disclosure, including the ITRs, can be any suitable length, which typically, but not necessarily, does not exceed the average genome size packaging capacity of a particular AAV capsid, which may be selected in the design and production of a particular AAV vector. Thus, in some non-limiting embodiments, the length of the genome of an AAV vector of the present disclosure, including the ITRs, is at least or about 1500 nucleotides, 1600 nucleotides, 1700 nucleotides, 1800 nucleotides, 1900 nucleotides, 2000 nucleotides, 2100 nucleotides, 2200 nucleotides, 2300 nucleotides, 2400 nucleotides, 2500 nucleotides, 2600 nucleotides, 2700 nucleotides, 2800 nucleotides, 2900 nucleotides, 3000 nucleotides, 3100 nucleotides, 3200 nucleotides, 3300 nucleotides, 3400 nucleotides, 3500 nucleotides, 3600 nucleotides, 3700 nucleotides, 3800 nucleotides, 3900 nucleotides, 4000 nucleotides, 4100 nucleotides, 4200 nucleotides, 4300 nucleotides, 4400 nucleotides, 4500 nucleotides, 4600 nucleotides, 4700 nucleotides, 4800 nucleotides, 4900 nucleotides, 5000 nucleotides, 5100 nucleotides, 5200 nucleotides, 5300 nucleotides, 5400 nucleotides, 5500 nucleotides, 5600 nucleotides, 5700 nucleotides, 5800 nucleotides, 5900 nucleotides, 6 The genomic sequence may be 0 nucleotides, 3600 nucleotides, 3700 nucleotides, 3800 nucleotides, 3900 nucleotides, 4000 nucleotides, 4100 nucleotides, 4200 nucleotides, 4300 nucleotides, 4400 nucleotides, 4500 nucleotides, 4600 nucleotides, 4700 nucleotides, 4800 nucleotides, 4900 nucleotides, 5000 nucleotides, 5100 nucleotides or 5200 nucleotides (or base pairs when the genomic sequence is embodied in a plasmid for vector production), or any integer value between or range inclusive of any of the specifically recited values ​​above.

[0166] Expression cassette 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, which act to control the initiation of transcription of the transgene from DNA to RNA, and a transcription termination element, such as a polyadenylation signal sequence, which acts to terminate transcription of the transgene into RNA. An AAV vector may contain two or more transgenes, either as part of a single transcription unit or each part of its own transcription unit. As described in later sections, the expression cassette may further contain additional sequence elements designed to affect transcription, transcript stability, translation, or other functions.

[0167] AAV vectors are typically designed so that the structure of the expression cassette and the entire genome are limited by the packaging capacity of the capsid, such that the length of the transgene, together with all other elements in the genome required for vector function, such as transcriptional control elements and ITRs, does not exceed approximately 5 kilobases for AAV2, although other types of capsids may have larger or smaller packaging limits. However, within size constraints, there is great flexibility in the selection of the transgene, ITRs, and other elements required for the vector to function for its intended purpose.

[0168] For purposes of gene therapy, it is understood that a transgene may be any gene whose product prevents or treats, but does not necessarily cure, any disease, disorder, or condition in a subject in need of such prevention or treatment. In some embodiments, gene therapy is intended to prevent or treat a disease, disorder, or condition characterized by an abnormally low or even absent amount of a product produced by a gene naturally present in the subject, such as may be caused by a loss-of-function mutation. For such embodiments, the transgene may be intended to compensate for a defective gene in the subject by providing the same or a similar gene product, when expressed, to at least a portion of the subject's cells. A non-limiting example would be a vector designed to express a functional form of coagulation factor IX for use in gene therapy of hemophilia B, which is caused by a loss-of-function mutation in the native factor IX gene. Meanwhile, in other embodiments, the transgene may be intended to counteract the deleterious effects of a functional mutation in target cells. In some embodiments, the transgene may encode a transcriptional activator that increases the activity of an endogenous gene that produces a desired gene product, or conversely, a transcriptional repressor that decreases the activity of an endogenous gene that produces a deleterious gene product.

[0169] In some embodiments, a transgene may encode a polypeptide or may encode an RNA molecule with a function different from the encoded 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.). Protein-coding sequences within a transgene may be codon-optimized, and translation start sites (e.g., Kozak sequences) may be modified to increase or decrease the propensity for translation to initiate. In some embodiments, a transgene encoding an amino acid sequence may contain one or more open reading frames and / or one or more splice donor and splice acceptor site pairs that allow alternative splicing of different messages and polypeptide sequences derived from such messages. A protein-encoding transgene further comprises one or more stop codons that terminate translation of the polypeptide chain.

[0170] In some embodiments, vectors can be designed to edit or otherwise modify the genome of a target cell. For example, the vector can include an expression cassette or transgene flanked by homology arms intended to promote homologous recombination between the vector and the target cell genome. In another example, vectors 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, which can bind to the gRNA and cleave the DNA sequence targeted by the gRNA.

[0171] PGRN transgene In some embodiments, the AAV vectors of the present disclosure include vectors comprising an expression cassette containing a coding sequence (transgene) for a Progranulin protein (abbreviated as "PGRN") or variants thereof, including human Progranulin protein or variants 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 contains 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 various approximately 6 kDa peptides with pleiotropic functions depending on the context of the cell or organism. In other embodiments, the expression cassette contains a coding sequence for a non-human Progranulin protein.

[0172] In yet other embodiments, the PGRN protein may include any naturally occurring variant of the human PGRN protein that does not contain a pathogenic mutation, such as a premature translation termination codon or an amino acid substitution, insertion, or deletion, that substantially impairs PGRN activity and / or protein stability. In still further embodiments, the PGRN protein may include engineered human PGRN protein variants, e.g., chimeric variants, that retain PGRN activity, and variants with amino acid substitutions, insertions, or deletions designed to modulate PGRN activity, add or remove glycosylation sites, add, remove, or alter internal cleavage sites in granulin that are normally cleaved from mature granulin, or sites 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 with a signal peptide sequence from a similar or completely different secreted protein.

[0173] In certain embodiments, the PGRN protein variant is a carboxy-terminal truncated variant in which one or more amino acids normally present in full-length wild-type human Progranulin are deleted from the Progranulin carboxy-terminus, e.g., 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, compared to the amino acid sequence of full-length wild-type human Progranulin protein (such as that provided by SEQ ID NO: 16). In some embodiments, the PGRN protein variant lacks the last three carboxy-terminal amino acids (QLL) naturally present in full-length wild-type human PGRN protein, and has the amino acid sequence of SEQ ID NO: 14. The latter PGRN protein variant may be referred to herein as "PGRNΔ3" or "PGRNDel3." In some embodiments, PGRN protein mutants, such as PGRNΔ3, exhibit reduced or no specific binding to the receptor protein known as sortilin 1 (SORT1) compared to full-length wild-type PGRN protein. In some embodiments, PGRNΔ3 mutants undergo further post-transcriptional and post-translational modifications after delivery by a viral vector. Further truncations are known to occur, including, for example, a single amino acid truncation at the carboxy-terminus that generates the PGRNΔ4 protein mutant. Proteins provided by PGRNΔ3 vectors can be further modified by other known post-transcriptional and post-translational processing events, 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 "tag(s)" or functional or signaling sequences, and the like.

[0174] In certain other embodiments, the PGRN protein variant comprises at least one amino acid substitution mutation, 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, compared to the full-length wild-type human Progranulin protein amino acid sequence (such as that provided by SEQ ID NO: 16), from the wild-type counterpart. In some of these embodiments, the substitution mutation may be a conservative amino acid substitution, in which a normally occurring amino acid is replaced with another amino acid that has similar physicochemical and / or size characteristics and an R group. Alternatively, in other embodiments, the substitution mutation may be a non-conservative amino acid substitution, in which a normally occurring amino acid is replaced with another amino acid that has dissimilar physicochemical and / or size characteristics and an R group.

[0175] 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 a cell type, such as a neuron, desired to be transduced by the vector. In some embodiments, the nucleotide sequence encoding the PGRN protein (i.e., the transgene) is a DNA sequence that is identical to that present in a naturally occurring gene encoding PGRN (i.e., the exons of such a gene) or corresponds to the mRNA sequence transcribed from such a gene. In some embodiments, the PGRN protein is a full-length wild-type human progranulin protein, the coding nucleotide sequence is provided by nucleotides 41-1822 of NCBI Reference Sequence: NM_002087.4, including the stop codon, or SEQ ID NO: 15. In some embodiments, the PGRN protein is a PGRNΔ3 protein variant, the coding nucleotide sequence is provided by SEQ ID NO: 8.

[0176] In other embodiments, the nucleotide sequence encoding the PGRN protein may differ at one or more nucleotide positions compared to a naturally occurring nucleotide sequence and, due to redundancy in the genetic code, may still encode a PGRN protein identical to the naturally occurring gene sequence or a PGRN protein variant encoded differently by the naturally occurring gene sequence, except for polypeptide differences relative to wild-type PGRN. In some embodiments, the nucleotide sequence encoding the PGRN protein may be intentionally modified to affect its function in transduced cells, for example, to eliminate sequence motifs that can stimulate an innate immune response, to eliminate potential splice junctions, to eliminate alternative start codons, to increase the stability of the corresponding mRNA, and / or to increase the rate of translation of the mRNA into protein. In other embodiments, the nucleotide sequence encoding the PGRN protein may be intron-free or may contain one or more introns that interrupt the coding sequence but are removed by the splicing machinery in the transduced cells to allow translation of the desired PGRN protein.

[0177] In some embodiments of the AAV vectors of the present disclosure, the transgene comprises a protein sequence that is highly similar or identical to the protein sequence encoded by a particular nucleotide reference sequence, although the nucleotide sequences of the transgene and the reference sequence are not identical; rather, they share a particular percent identity, with the differences corresponding to positions within the codons that do not change the corresponding amino acid (i.e., are silent changes). For example, in some embodiments, the transgene comprises or comprises a sequence that encodes 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 consisting of, comprising, or consisting of a sequence encoding the same PGRNΔ3 protein variant as SEQ ID NO:8 and having a nucleotide sequence 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:8.

[0178] The percentage of nucleotide sequence identity between a reference sequence and a transgene can be determined by any method known in the art. For example, in some embodiments, a computer algorithm can be used to align and compare the nucleotide sequences (or the amino acid sequences encoded by them) of a reference sequence and a transgene over their entire lengths, and calculate the percent nucleotide sequence identity. An exemplary algorithm for globally aligning and comparing nucleotide sequences is the Needleman-Wunsch algorithm. However, in other embodiments, local alignment algorithms such as the BLAST algorithm can be used (Needleman, S. & 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). In some embodiments, if one or the other of the reference sequence and the transgene sequence contains non-coding sequences, such as introns or stop codons, the non-coding sequence(s) are ignored, and only the protein-coding sequences in the reference sequence and the transgene sequence are aligned and compared. After an optimal global alignment between the reference sequence and the transgene is established, the percentage of identical nucleotides between the aligned sequences can be calculated.

[0179] As is known in the art, sequence comparison algorithms allow the user to define parameters, such as substitution scores and gap penalties, used to calculate alignment scores for the many possible alignments that can be created. The alignment with the highest score is then deemed optimal. Substitution scores involve assigning a numerical reward for a match and a penalty for a mismatch. An exemplary set of respective match and mismatch scores includes 1, -1; 1, -2; 1, -3; 1, -4; 2, -3; 4, -5, although others are possible. Gap costs involve assigning a numerical penalty for the existence of a gap (insertion or deletion of a nucleotide) and a penalty for extending the width of a gap once it has been formed. Increasing gap costs results in alignments that introduce fewer gaps. An exemplary set of respective costs for the existence and extension of a gap includes 0, -4; -2, -2; -2, -4; -3, -3; -4, -2; -4, -4; -5, -2; -6, -2, although others are possible. In some embodiments, alignment and comparison of the reference sequence and the transgene sequence is performed using default substitution scores and gap penalties, as well as any other default settings provided with the computer software or algorithm for performing the analysis.

[0180] signal peptide sequence In some embodiments, AAV vectors of the present disclosure include vectors that include an expression cassette containing a coding sequence (transgene) for a Progranulin protein comprising a mature granulin polypeptide sequence, where the signal peptide naturally present in the wild-type human Progranulin protein has been replaced with a signal peptide from a different secreted polypeptide from human or another species (i.e., a heterologous signal peptide sequence) for some reason, such as to improve the rate at which PGRN (or variants thereof) produced in transduced cells is secreted or to reduce immunogenicity. In some embodiments, the mature granulin polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 18 or a carboxy-terminal truncation thereof lacking the last three amino acids (QLL).

[0181] Any signal peptide sequence known in the art to be effective in secreting a protein from the cell in which it is synthesized can be used with the AAV vectors of the present disclosure. In some embodiments, such a signal peptide is removed from the protein by the cell during secretion. Numerous heterologous secretory signal peptides are known in the art and can be used to promote secretion of PGRN (or its variants) from cells, such as neurons or other brain cells, transduced with the AAV vectors of the present disclosure.

[0182] In some embodiments, the signal peptide sequence can originate from or be derived from any of a variety of proteins produced by and secreted from neurons or other cells of the central or peripheral nervous system. Non-limiting examples include signal peptide sequences from human proteins such as growth hormone, proenkephalin A, beta-neoendorphin-dynorphin, neuroendocrine protein 7B2, prolactin, gastrin-releasing peptide II, secretogranin I, growth hormone-releasing factor, axonin I, neuroendocrine convertase 2, and vasopressin copeptin, among many others known in the art. The amino acid and encoding nucleotide sequences of these and other secreted proteins are available in public sequence databases such as Genbank. The amino acid sequences of naturally occurring signal peptides can be modified to desirably alter their function, and similarly, nucleotide sequences encoding such wild-type or modified signal peptides can be modified to achieve desired types of sequence optimization, such as removal of CpG motifs. In yet other embodiments, entirely synthetic secretory signal peptide sequences can be used.

[0183] Transcriptional control region AAV vectors of the present disclosure intended to express PGRN protein in and / or from transduced cells may further include, as part of the vector, one or more transcriptional control regions operably linked to a transgene encoding a PGRN polypeptide sequence. As discussed further below, different types of transcriptional 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 terms "operably linked" and variants such as "operably linked," "operably linked," and "operably linked" refer to a functional relationship between a transcriptional control region and the transgene such that the transcriptional control region can affect (whether positively or negatively) the transcription of the transgene, without specifying any particular spatial or structural relationship between them. Thus, for example, a transcriptional control region can be operably linked to a transgene whether it is located 5' or 3' to the transgene and / or directly adjacent to the transgene or distal to the transgene. Transcriptional regulatory regions may be constitutively active, active in particular cells or tissues, inducibly active in response to some environmental stimulus, derived from a naturally occurring gene (of any appropriate species), modified to improve or alter its function, or even entirely synthetic.

[0184] In some embodiments, the transcriptional control region comprises a promoter region containing the minimal DNA sequence required to initiate transcription by the transcriptional machinery in the transduced cell (e.g., a TATA box or initiation sequence), and often one or more additional proximal elements that act alone or cooperatively to increase the rate of transcription from the basal promoter. Depending on the sequence, a promoter can initiate transcription by RNA polymerase I, RNA polymerase II, or RNA polymerase III; however, AAV vectors intended to express polypeptides such as PGRN or its variants, such as PGRNΔ3, in transduced cells often use promoters derived from protein-coding genes that are normally transcribed by RNA pol II.

[0185] In other embodiments, the transcriptional control region includes or further includes at least one enhancer region, which functions to increase the rate of gene transcription beyond that which can be sustained by the basal promoter alone. Enhancers, in their natural context, are often located distally from the promoter of the gene they act on, sometimes tens to hundreds or thousands of base pairs upstream (i.e., 5'), but can also be located elsewhere, such as within an intron or downstream (i.e., 3') of the gene they act on. Promoter regions may include proximal enhancer elements (subsequences that, when removed, can reduce transcription from the basal promoter), but enhancers typically do not include sequences that can function as basal promoters. In nature, enhancer regions are often located distal to the promoters of the genes they act on, but enhancer regions, or enhancer elements from within larger enhancer regions (such elements often correspond to DNA binding sites for transcription factors), when removed from their natural context and repositioned much closer to the promoter, whether from the same gene or even a different gene, may be able to retain at least some of their transcription-enhancing function.

[0186] The enhancer and promoter regions of genes described in the scientific literature, when combined with transgenes and other genomic elements required for vector function, can be too large to be accommodated by the packaging capacity of an AAV capsid. Therefore, in some embodiments, methods well known to those skilled in the art can be used to identify functional subsequences within longer enhancer or promoter regions, and then incorporate shorter functional subsequences into transcriptional control regions for use in the vectors of the present disclosure. In this way, the size of transcriptional control regions can be reduced while maintaining their desired function. Using this approach, functional elements from naturally occurring enhancers or promoters can be combined in novel ways, for example, by modifying their number, spacing, and / or positioning, to create hybrid or synthetic enhancers and / or promoters with improved properties. In some embodiments, the enhancer and promoter can each be derived from the same naturally occurring gene, while in other embodiments, the enhancer and promoter can be derived from completely different genes, including genes from different species.

[0187] In some embodiments, in terms of a coding strand (i.e., positive-sense) single-stranded DNA AAV vector, the promoter sequence is located 5' to a 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, an enhancer element or region, if present, may be located 5' to the promoter sequence, or alternatively, may be located elsewhere in the genome, such as within the 5' untranslated region (UTR) or 3' untranslated region adjacent to the transgene, within an intron, 3' to a transcription termination signal sequence, or elsewhere. In some embodiments, a vector may contain two or more enhancer regions (of the same or different types), which may be located adjacent to each other or spaced apart and / or separated by other functional elements in the genome. In some embodiments, the same enhancer element or region is provided as a tandem array of two, three, four, or more repeat units.

[0188] In some embodiments, transcriptional regulatory regions for use in the AAV vectors of the present disclosure are non-tissue-specific, meaning that they are constitutively active in many, but not necessarily all, different cell types. According to some embodiments, non-tissue-specific transcriptional regulatory regions include promoters derived from certain viruses, such as the human cytomegalovirus major immediate-early gene (CMV-IE), which may contain enhancer elements proximal to the basal promoter (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 derived from Rous sarcoma virus (RSV) and Moloney murine leukemia virus (MoMLV). In other embodiments, the non-tissue-specific transcriptional control region comprises a promoter (which may include a proximal enhancer element) derived from a different type of animal, such as the human polypeptide chain elongation factor (EF1α) gene; the phosphoglycerate kinase (PGK) gene; the ubiquitin C (UbiC) gene; the chicken beta-actin (CBA) gene; the U1a1 small nuclear RNA promoter or the 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); the histone H2 promoter or the histone H3 promoter (Hurt, M. et al., Mol. Cell. The genes may be derived from genes that are active in many different cell types (sometimes called "housekeeping" genes), including genes such as ribosomal proteins (e.g., ribosomal proteins), ribosomal proteins, and ribosomal proteins (e.g., ribosomal proteins).

[0189] Similarly, 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 from the same gene can be combined to create transcriptional control regions for use in the vectors of the present disclosure, although enhancers and promoters from different genes can be combined to create hybrid transcriptional control regions. A commonly used example is a 1.6 kilobase hybrid enh / pro region called CAG (or CAGGS) that contains 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 have been reduced in size, in which the CBA intron is replaced with a smaller simian virus 40 (SV40) intron (Wang, Z. et al., Gene Ther., 10:2105-11, 2003), and in which the SV40 intron is replaced with the CBA promoter (Wang, Z. et al., Gene Ther., 10:2105-11, 2003). This includes another one called the CBA hybrid intron (CBh) (Gray, S. et al., Hum. Gene Ther., 22:1143-53 2011), in which the 5' donor splice site from the 5' UTR is replaced with a hybrid intron consisting of the 3' acceptor splice site from the MVM intron.

[0190] In some embodiments, transcriptional regulatory regions for use 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 transgene expression in cell types within the CNS or brain compared to cells of other tissues or organs, such as muscle or liver. In some embodiments, CNS or brain cell types in which the transcriptional regulatory regions of the AAV vectors of the present disclosure are preferentially active include, but are not limited to, neurons, glial cells (e.g., microglial cells, astrocytes, and oligodendrocytes), and ependymal cells, although other cell types are possible. Without wishing to be bound by any particular theory of operation, one mechanism by which brain tissue or neuron (or any other cell type in the brain) gene transcription specificity can arise is the presence of one or more specific binding sites within the enhancer and / or promoter for a DNA-binding transcriptional activator protein that is preferentially expressed in brain cells, such as neurons or other cell types in the brain. The use of brain tissue or neuron (or other brain cell type) specific transcriptional control regions can be advantageous in some embodiments by reducing or even inhibiting transgene expression in cells other than brain or non-neuronal cells (or other brain cell types) that may be transduced by the vector, which can desirably reduce the risk of off-target effects.

[0191] Certain brain- or neuron- (or other brain cell type)-specific genes that are expressed at high levels have both enhancers and promoters that can be included within the transcriptional control region of the AAV vectors of the present disclosure. In some embodiments, enhancers and promoters from the same gene can be combined within a brain- or neuron- (or other brain cell type)-specific transcriptional control region, while in other embodiments, an enhancer from one gene can be combined with a promoter from a different gene within a hybrid brain- or neuron- (or other brain cell type)-specific transcriptional control region. If derived from the same gene, the transcriptional control region sequence, including one or more enhancers and promoters, can be copied as they exist in the context of the native gene from which they are derived, or can be engineered to reduce its length, such as by deleting non-transcriptionally active sequences that separate the one or more enhancers and promoters. In some embodiments, enhancers and promoters for use in brain- or neuron- (or other brain cell type)-specific transcriptional control regions can be derived from genes of different species. In yet other embodiments, the sequence of an enhancer and / or promoter within a transcriptional regulatory region may be modified relative to its original sequence by changing, adding, or removing nucleotides to improve its function, such as by increasing transcriptional activator binding, decreasing transcriptional repressor binding, or reducing the size of the transcriptional regulatory region.

[0192] In some embodiments, it is the enhancer that provides brain- or neuron- (or other brain cell type)-specific expression, and the promoter is not itself brain- or neuron- (or other brain cell type) specific, whereas in other embodiments, it is the promoter that provides brain- or neuron- (or other brain cell type)-specific expression, and the enhancer, when present, is not itself brain- or neuron- (or other brain cell type) specific, but can increase the rate of transcription from the 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 the synapsin 1 gene, can be paired with a strong viral promoter, such as the SV40 early promoter. In other embodiments, both 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 chimeric enhancer regions used in the transcription control regions in the AAV vectors of the present disclosure.

[0193] Brain tissue or neuron (or other brain cell type) specific transcriptional control regions (either enhancers, promoters, or both) for use 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 particular regions of the brain or neuron subtypes.For example, but not limited to, the synapsin 1 gene (SYN1), neuron-specific enolase (NSA) gene, and tubulin α1 gene each contain a neuron-specific promoter; the glial fibrillary acidic protein (GFAP) gene promoter is at least partially astrocyte-specific; the L7-6 gene promoter is at least partially cerebellar Purkinje cell-specific; the Ca2+ / calmodulin-dependent protein kinase II (CaMKII) gene promoter is at least partially forebrain excitatory neuron-specific; the distalless homeobox (DLX) gene enhancer is at least partially forebrain inhibitory neuron-specific; and the glutamic acid decarboxylase (GAD) gene promoter is at least partially forebrain inhibitory neuron-specific. The promoter of the tyrosine hydroxylase gene is also at least partially specific to inhibitory neurons, and the promoter of the tyrosine hydroxylase gene is at least partially specific to catecholaminergic neurons, and is involved in the expression of the following genes: ADORA2A, ATP6V1C2, AVP, C8ORF46, CARTPT, CCKBR, CCL27, CD68, CLDN5, CRH, CX3CR1, DBH, DCX, DDC, DRD1, FEV, FEZF2, GABRA6, GAL, GCHFR, GFAP, GPR88, GPX3, GR The transcriptional regulatory regions (promoters and / or enhancers) of P, HAP1, HBEGF, HCRT, HSPA12B, HTR1A, ICMT, LCT, MKI67, NR2E1, NTSR1, OLIG1, OXT, PCP2, PITX3, PKP2, POGZ, RAMP3, RGS16, RLBP1L2, S100B, SLC6A2, SLC6A3, SLC6A4, SLC6A5, SLC7A5, SLITRK6, TAC1, TAC3, TBR1, THY1, TNNT1, TRH, UGT8, VIM, and VIP are at least partially neuron-specific.

[0194] In some embodiments, the transcriptional control region of the AAV vector of the present disclosure for expressing PGRN protein or a variant thereof, such as PGRNΔ3, can comprise or consist of the promoter region of the human synapsin 1 gene (SYN1), which has been shown to confer neuronal cell-specific transcriptional regulation in transduced neurons, or a subsequence thereof that retains such neuron-specific transcriptional regulation. The promoter region of the human synapsin 1 gene is described in further detail, for example, in Thiel, G, et al., Characterization of tissue-specific transcription by the human synapsin I gene promoter, PNAS 88:3431-3435 (1991) and Schoch, S, et al., Neuron-specific Gene Expression of Synapsin I, JBC 271(6):3317-3323 (1996). In some embodiments, the transcriptional control region can 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 transcriptionally functional subsequence thereof.

[0195] A variety of brain- or neuron- (or other brain cell type)-specific transcriptional control regions for use in gene therapy vectors (e.g., AAV, adenoviral, or lentiviral vectors) have been created by adapting, e.g., reducing the length of, enhancers and / or promoters from brain- or neuron- (or other brain cell type)-specific genes, any of which can be used in the AAV vectors of the present disclosure to express PGRN protein or variants thereof, such as PGRNΔ3, in transduced cells, such as neurons or other brain cell types in the brain. Non-limiting examples of such brain- or neuron- (or other brain cell type)-specific transcriptional regulatory regions include the transcriptionally functional portion of the human synapsin 1 gene promoter (Kugler, S. et al., Mol. Cell Neurosci., 17:78-96, 2001; Kugler, 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. 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;US Patent No. 7,341,847).

[0196] transcription termination signal sequence In some aspects, the AAV vectors of the present disclosure include vectors that include a transcription terminator sequence located 3' of the transgene in terms of the coding (plus) strand single-stranded DNA vector. In some embodiments, additional sequences, such as a 3' untranslated region (UTR) sequence, can be located between the transgene sequence and the transcription 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. & Libri, D., Nat. Rev. Mol. Cell Biol., 16:190-202, 2015).

[0197] In some embodiments, particularly when the transgene comprises a protein-coding sequence (as opposed to a sequence of RNA that has some function other than encoding a protein), the transcription terminator sequence may be a polyadenylation signal sequence (variously abbreviated as "polyA," "pA," "poly(A)," or "p(A)"). In some embodiments, pA signal sequences are derived from naturally occurring genes and can be used in vectors, while in other embodiments, pA signals can be modified, for example, by shortening them compared to their natural counterparts or by altering their sequence to increase their efficiency in terminating transcription. In other embodiments, pA signals can be hybrid sequences combining pA sequences from different genes, or synthetic sequences.

[0198] 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 beta-globin genes; SV40 late genes; sNRP1; spA; herpes simplex virus thymidine kinase gene (HSV TK); or the adenovirus type 5 L3 polyadenylation site, among others. In other embodiments, transcription terminators for use 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)).

[0199] In some embodiments, AAV vectors of the present disclosure include vectors containing a transgene, the transcription of which is terminated by the inclusion of a poly(A) site derived from the bovine growth hormone gene (bGH), which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10; a poly(A) site derived from the SV40 virus, which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO:26 or SEQ ID NO:27; or a poly(A) site derived from the rabbit beta-globin gene, which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO:28.

[0200] Other vector genome elements In addition to transcriptional control regions and transcription termination signals, other sequences, including cis-regulatory elements, can be included in the genome of the AAV vectors of the present disclosure to improve, control, or regulate transgene expression and / or translation in 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 regulatory 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 experimentally shown to improve transgene expression, even though the mechanism may be uncertain. In other embodiments, the vector may contain so-called stuffer or filler sequences, which are intended only to increase the overall length of the vector to a desired size, e.g., to achieve a length that approaches, but is still lower than, the packaging capacity of a particular capsid, thereby reducing the likelihood of accidental packaging of truncated vector or non-vector DNA within the capsid.

[0201] In some embodiments, vectors can include introns to increase transgene expression and / or transcript stability. In some embodiments, protein-coding transgenes are provided in which the exon and intron(s) are identical in sequence to the naturally occurring gene. However, in genes with multiple exons and introns, one or more introns can be removed to minimize overall length while still facilitating the inclusion of other elements, while still maintaining the packaging capacity of the capsid. In other embodiments, however, the intron can be provided from an entirely different gene than the one providing the coding sequence for the vector transgene. Whether the intron is from the same gene as the transgene or a different gene, the intron can be modified from its original sequence, for example, by changing specific nucleotides or by removing internal sequences to shorten its overall length while maintaining splice donor and acceptor sequence motifs required for efficient splicing to occur, or other intron cis elements important for function (e.g., enhancers that may be present in the original, unmodified intron sequence). Introns may also be hybrid, in which a splice donor portion of an intron from one gene is paired with a splice acceptor portion of an intron from a different gene, or synthetic, with a sequence that does not correspond to an intron of any known gene. In some embodiments, the intron is located within the coding sequence of the transgene and may therefore interrupt it (and may be provided with the donor and acceptor sites necessary for efficient splicing to occur), while in other embodiments, the intron is present but does not interrupt the protein-coding sequence; instead, it is located either 5' or 3' to the coding sequence. If the intron does not interrupt the coding sequence, it may be provided with some exon sequence carried over from its original genetic context, as long as the exon sequence does not contain a potential translation initiation signal. 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 distally in the vector, either upstream or downstream from the coding sequence.

[0202] 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. & Pintel, D., J. Virol., 72:1834-43, 1998; Haut, D. & Pintel, D., Virology, 258:84-94, 1999); an internal deletion of intron 1 from human coagulation factor IX (FIXm1 and FIXm2) (Kurachi, S. et al., J. Biol. Chem., 270:5276-81, 1995); chimeric beta globin splice donor and immunoglobulin heavy chain splice acceptor introns (GenBank U47120.2 nucleotides 890-1022); intron 1 from the mouse alpha globin gene; and the SV40 small t antigen intron (Nathwani, A. et al., Blood, 107:2653-61, 2006), which may comprise or consist of base pairs 4644 to 4552 of GenBank entry J02400.1 and may be modified at positions 4582(g-c), 4580(g-c), 4578(a-c), and 4561(a-t).

[0203] In some embodiments, post-transcriptional regulatory elements (PREs) can be included in the vector to increase transgene expression. Examples of PREs include woodchuck hepatitis virus post-transcriptional regulatory elements (WPREs), hepatitis B virus post-transcriptional regulatory elements (HPREs), and variants 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. Patent Application Publication No. 2018-0353620(A1)). In some embodiments, in terms of a coding (positive) strand single-stranded DNA vector, at least one WPRE sequence may be located downstream of the transgene (and 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 (e.g., two, three, or more PREs) of the same or different types may be included, which may be arranged in tandem. In some embodiments, AAV vectors of the present disclosure include vectors that include a WPRE element, which in some embodiments comprises or consists of the nucleotide sequence of SEQ ID NO:29.

[0204] AAV inverted terminal repeat (ITR) The ends of the adeno-associated virus genome contain unique nucleotide sequences called inverted terminal repeats (ITRs), which function as origins of viral DNA replication in infected cells and as priming sites that aid in the conversion of the single-stranded (ssDNA) genome into a double-stranded (dsDNA) form suitable for supporting the transcription of the rep and cap protein-encoding genes. The ITRs also function in packaging the replicated ssDNA genome into AAV capsids. AAV ITRs contain multi-palindromic sequences that can fold back on themselves by intrastrand complementary base pairing to form dsDNA T-shaped hairpin secondary structures.

[0205] As described further below, the vectors of the AAV vectors of the present disclosure can contain one or more AAV ITRs that function similarly to how they function in the unmodified virus. Unless otherwise specified, the use of the term "inverted terminal repeat" or "ITR" herein includes intact, full-length ITRs and ITRs that have been modified (e.g., truncations, internal deletions, modified sequences (such as trs or D sequences), additions, and substitutions of one or more nucleotides) to retain one or more of the functions attributed to an ITR, including vector rescue from recombinant DNA (such as a plasmid), vector replication, and / or packaging of the vector into assembled capsids, even if less efficient than an intact ITR of the same type.

[0206] As they exist in packaged viruses and vectors, ITRs located at the 3' end of the ssDNA genome have a free 3' hydroxyl group, while ITRs located at the opposite 5' end of the ssDNA genome have a free 5' end. The 5' ITR is sometimes referred to as the "left" ITR, and the 3' ITR is sometimes referred to as the "right" ITR. However, in plasmids, such as those that can be used for vector production, the vector sequence exists in double-stranded form, resulting in two sets of 5' and 3' ITRs. Therefore, to avoid ambiguity, the ITRs should be distinguished by specifying on which strand they are located. Unless otherwise specified, reference to ITRs in a double-stranded form of a vector, such as a plasmid, refers to the plus or sense strand, i.e., the DNA strand in which the transgene sequence is identical to the coding sequence for the transgene's polypeptide product or, if the transgene does not encode a protein, to the coding sequence for a functional RNA.

[0207] In wild-type AAV2, the ITRs are 145 bases long, with the terminal 125 bases containing a palindromic subsequence. When annealed, the AAV2 ITRs contain two double-stranded palindromes, B-B' and C-C', that form the arms of a hairpin, which are linked to a larger double-stranded palindrome, A-A', that forms the stem of the hairpin. Toward the non-end of the ITR, the ITRs further contain a D sequence, which has no complementary sequence within the ITRs and therefore remains single-stranded, but has a complementary sequence to the D region of the ITR at the opposite end of the genome (hence D and D'). The A-A' stem structure contains a Rep-binding element (RBE), which contains a tetranucleotide repeat motif to which the AAV Rep protein binds in order to introduce a sequence-specific and strand-specific nick at the terminal resolution 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.

[0208] When they are restored, ITRs can fold into two structures called flip and flop, in which the sequence between the A and A' inverted repeats is present as the reverse complement of the other structure. For the 5' ITR (left ITR), the order of the terminal palindrome sequences in the flip structure is 5'-ABB'CC'A'D-3', and the order of the flop structure is 5'-ACC'BB'A'D-3'. For the 3' ITR (right ITR), the order of the terminal palindrome sequences in the flip structure is 3'-A'B'BC'CAD'-5', and the order of the flop structure is 3'-A'C'CB'BAD'-5'. As a result, flip structures have a B'B palindrome closest to the free 3' end, whereas flop structures have 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).

[0209] The secondary structure of the ITRs is hypothesized to support viral DNA replication by a self-priming single-strand displacement elongation mechanism initiated by endogenous cellular DNA polymerases at ITRs with free 3' hydroxyl groups. Strand elongation results in the formation of a monomeric dsDNA genome replication intermediate with one covalently closed end. The open-end double-stranded ITR refolds (isomerizes) into a double hairpin structure, displacing the complementary strand and forming a new, elongating 3' ITR. The large AAV Rep protein binds to the ITR at the closed end (downstream), nicks the DNA at its terminal resolution site, and initiates a second DNA replication complex that copies the downstream ITR before the DNA replication complex initiated at the open end can reach it. The original replication complex then displaces the opposite strand (its newly synthesized ITR) and completes replication to what was previously the closed end of the genome, opening up the double-stranded ITR, making it available for isomerization into a double hairpin. Thus, a monomeric dsDNA genome replication intermediate is reassembled to restart the replication cycle, while the displaced ssDNA genome (with its newly generated 3' ITR) can be packaged into viral particles.

[0210] The replicated ssDNA genome contains both positive (plus or sense) and negative (minus or antisense) strand polarities, and evidence suggests that they are individually packaged into capsids with similar efficiency. Consequently, a preparation of AAV vector particles, like the virus to which it is adapted, may, in some embodiments, contain roughly equal proportions of sense or antisense ssDNA genomes. Alternatively, the ITRs used to generate AAV vectors can be modified by selectively removing the D sequence from one of the ITRs, thereby restricting packaging to either the minus or plus strand of the vector (Wang, XS, et al., J Virol, 70(3):1668-77 (1996)). Thus, in some other embodiments, an AAV vector preparation may contain vector particles in which most or substantially all of the vector is either the plus or minus strand.

[0211] After infection, AAV virions are transported to the nucleus, where the ssDNA genome is released from the capsid. Before the viral rep and cap genes can be expressed, the ssDNA genome must first be converted to dsDNA through complementary strand synthesis by cellular DNA polymerases that initiate chain elongation at the 3' ITR; this process is thought to be slow and inefficient. It has also been hypothesized that a faster mechanism may exist to form intracellular dsDNA genomes, in which complementary positive and negative ssDNA genomes from different virions infecting the same cell encounter each other in the nucleus and hybridize through intermolecular base pairing. Such double-stranded genomes can then support transcription without first requiring elongation by cellular DNA polymerases.

[0212] In AAV vector design, the only AAV viral DNA sequences retained in the vector are the ITRs, as they play an important role in DNA replication and packaging during production and in converting the ssDNA genome to dsDNA after transduction. Sequences encoding the Rep and Cap proteins, as well as viral helper functions, are also required for vector production and can be provided in trans by a variety of methods known in the art. When vector sequences, such as those contained in plasmids used for AAV vector production, contain two intact ITRs and are not longer than the AAV capsid packaging capacity of approximately 5 kb, the ssDNA genome can be packaged as described above, but the conditions required for dsDNA conversion may result in less than desirable transduction efficiencies due to the inefficiency of this step before gene expression can occur. A potential strategy to overcome the requirement for dsDNA conversion by endogenous cellular DNA polymerases and improve transduction efficiency and expression of heterologous sequences, such as therapeutic transgenes, relies on the replication and packaging of "self-complementary" AAV vectors (scAAV), which contain plus- and minus-strand sequences in the same DNA molecule and can rapidly revert by intramolecular base pairing (i.e., intramolecular hybridization) to form a dsDNA transcription template after encapsidation in the target cell nucleus.

[0213] The production of self-complementary genomes can be promoted in at least two ways, both of which depend on the inability of Rep to nick the ITRs at the terminal resolution site during replication of the DNA molecule that will become the genome. In the natural AAV replication cycle, a dimeric dsDNA genome replication intermediate molecule can arise when Rep is unable to nick the downstream ITR at the terminal resolution site of a monomeric genome replication intermediate with a double hairpin at its open end and a single downstream ITR at its closed end. When this occurs, replication by the initial DNA replication complex (initiated from the open-ended 3' ITR) continues through 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 open ends with double-stranded ITRs, it can isomerize and initiate a cycle of DNA replication just like the monomeric form. Alternatively, the closed-end hairpin can undergo terminal resolution to form a double-stranded ITR that can isomerize to initiate DNA synthesis from the degraded end. In both cases, replication of the dimeric template generates a new dimeric dsDNA genome replication intermediate, replacing the ssDNA dimeric inverted repeat genome containing the 5' ITR, viral genomic sequences of one polarity, central ITR, viral genomic sequences of the opposite polarity, and 3' ITR.

[0214] Normally, ssDNA dimer inverted repeat viral genomes exceed the packaging capacity of normal capsids and are therefore not packaged. However, by designing a short vector so that the packaging capacity of AAV is not exceeded when the ssDNA dimer inverted repeat genome is formed, it is possible to produce vector particles containing self-complementary genomes. In practice, the vector preparation produced in this way may contain a mixture of particles packaged with one scDNA genome or one or two monomeric ssDNA genomes, and the proportions of all of these may vary between preparations.

[0215] After packaging, scDNA genomes exist within capsids in a single-stranded form (as do ssDNA non-self-complementary genomes), but they likely rapidly anneal after encapsidation to form dsDNA molecules with a covalently closed ITR at one end and two open ITRs at the other, similar to the structure of conventional viral genomes after self-priming dsDNA conversion. Thus, without wishing to be bound by theory, the primary difference between so-called ssDNA and scDNA genomes is not their topology during encapsidation, but rather the topology each type of genome likely acquires after encapsidation and genome release in transduced cells.

[0216] However, further modifications can provide increased control over the production of vector particles containing scDNA genomes. Specifically, by mutating or deleting the terminal resolution site from one ITR, such as in a plasmid containing the vector sequence used for vector production, it is possible to inhibit or eliminate single-strand nicking at that ITR during the vector replication cycle. As a result, a replication complex initiated at the non-mutated ITR proceeds back to the initiation end via the mutant hairpin, resulting in a dimeric dsDNA genome replication intermediate, just as would occur if terminal resolution of the wild-type ITR did not occur by chance. However, this intermediate contains a closed wild-type ITR at one end, a mutated double-stranded ITR in the center of the molecule, and an open double-stranded ITR capable of isomerization at the opposite end. This molecule can then undergo normal replication and strand displacement from the wild-type ITR at each end to generate a displaced daughter genome copy containing the 5' wild-type ITR, vector sequence of one polarity, the central mutant ITR, vector sequence of the opposite polarity, and the wild-type ITR at the 3' end. When the heterologous sequence includes a transgene encoding a protein, the scDNA genome contains both the coding sequence and its complement in the same DNA molecule. Such genomes are thought to be packaged in capsids in a single-stranded form, but due to the presence of a significant amount of self-complementary sequences outside the ITRs, they may be able to rapidly self-anneal into a double-stranded form in the nucleus of transduced cells, in which form they may support the transcription of the heterologous sequence. By producing scAAV from constructs containing mutated ITRs, more than 90% of the scDNA genome can be obtained.

[0217] For AAV2 capsids with a packaging capacity of about 4.7 kb, excluding ITR sequences, the ssDNA genome can accommodate about 4.4 kb of heterologous sequence, while the scDNA vector can accommodate about 2.2 kb. According to certain non-limiting embodiments, the size of the genome construct for producing scAAV vectors (for example, it can be contained in a plasmid for AAV vector production) is about 2,500 nucleotides long, including about 2,200 nucleotides of heterologous sequence and two ITRs (one wild-type and one mutated). This results in a scAAV genome of about 4,700 nucleotides long, which is less than the typical AAV capsid packaging capacity.

[0218] ITR terminal resolution sites can be disrupted in various ways to facilitate scAAV vector production. For example, exogenous sequences can be inserted into the terminal resolution site (trs) sequence itself or into adjacent sequences of the ITR, such as between the Rep binding element and the trs. Alternatively, the trs sequence can be deleted in part or in its entirety. In other embodiments, the adjacent D region can be deleted in part or in its entirety. In still other embodiments, nucleotides within the trs can be replaced with different nucleotides that reduce the frequency of trs nicking by Rep. Other methods of rendering ITRs non-degradable are within the ordinary skill in the art.

[0219] Further information regarding AAV virus and AAV 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. Patent No. 7,790,154.

[0220] In some embodiments, the AAV vectors of the present disclosure comprise one or more AAV ITRs from different AAV serotypes and variants, which may have different sequences and lengths and be located at different positions within the genome. The AAV ITR sequences for use in the vectors of the present disclosure may be wild-type or modified. In other embodiments, AAV ITR sequences may also be included as part of the vector sequence in other types of vectors, such as plasmids and baculoviruses, which are used to introduce vector sequences into host cells for vector production purposes.

[0221] While ITRs from AAV2 are often used in the production of AAV vectors, 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, so long as such ITRs function in vector replication and packaging and transgene expression. ITRs may also contain modifications to the sequence of wild-type ITR sequences or may be entirely synthetic. Modified ITRs may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the wild-type ITR sequence, such as the ITR sequence from AAV2. In some embodiments, the ITRs are modified by adding, deleting, and / or changing nucleotides to disrupt the terminal resolution site (trs) and / or D sequence of the ITR, or to alter some other subsequence comprising the ITR. Vectors, in some embodiments, may contain ITRs from different AAV serotypes or variants.

[0222] In some embodiments, the ITRs selected for use in vector production are from the same serotype or variant as the capsid. For example, AAV2 ITRs can be used in combination with an AAV2 capsid. In other embodiments, however, the vectors can be pseudotyped or hybrid, meaning that a vector with ITRs from one serotype or variant can be packaged within a capsid from a different serotype or variant. For example, a vector with ITRs from AAV2 can be packaged within a capsid from AAV8 (or any other serotype or variant). This pseudotype is often abbreviated as AAV2 / 8, where the number before the slash indicates the origin of the ITRs and the number after the slash indicates the origin of the capsid.

[0223] According to some embodiments, AAV vectors of the present disclosure may include vectors with different numbers of ITRs. For example, intact AAV viral genomes typically have two ITRs, each located at the 5' and 3' ends, respectively, and AAV vectors produced with intact ITRs and packaged with ssDNA genomes may similarly have two ITRs located there. However, as described above, vectors may be designed such that their genomes contain three ITRs, with two ITRs at the ends of the genome as in viruses or conventional vectors, and one ITR added (intact or mutated) in or near the center as in self-complementary vectors. However, it has also been observed that in some vectors, the 5' ITR may be shortened or completely lost, particularly when the genome length exceeds the average packaging capacity of the capsid, and this is hypothesized to be due to premature or variable packaging termination. It is also hypothesized that although defective interfering particles may occur, such particles may nevertheless support transgene expression as a result of inherently incomplete genome complementation (Kapranov, P. et al., Hum. Gene Ther., 23:46-55, 2012). Thus, in some embodiments, AAV vector particles of the present disclosure may comprise a genome containing a single functional ITR, such as at the 3' or 5' end, with a non-functional truncated ITR or no ITR sequence at the opposite end. Thus, in some embodiments of the AAV vectors of the present disclosure, the AAV ITRs 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 other locations.

[0224] Another source of heterogeneity in AAV vectors may arise from differences in the presence of flipped and flopped ITR structures within any particular genome. Thus, for example, in some embodiments, any particular vector particle in a sample may contain a genome with flipped ITRs at both ends of the genome, or with flopped ITRs at both ends of the genome, or with a flipped ITR at the 5' end and a flopped ITR at the 3' end, or with a flopped ITR at the 5' end and a flipped ITR at the 3' end. Combined with the observation that single-stranded DNA genomes can occur as either plus or minus strands, a sample of AAV vectors may contain similar or potentially different proportions of the eight possible structures. Unless otherwise specified, the genomes of the AAV vectors of the present disclosure are not limited to any of these structures, and any or all genomes may be packaged by such vectors.

[0225] In some embodiments, an AAV vector comprises intact, full-length ITRs at each of its 5' and 3' ends. Thus, for example, if both ITRs are derived from AAV2, such full-length ITRs would be 145 nucleotides in length. Meanwhile, in other embodiments, one or more of the ITRs may be truncated, lacking one or more terminal nucleotides compared to the canonical full-length sequence of that type of ITR. Thus, for example, an 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, or more nucleotides from its terminus compared to the canonical full-length sequence of that type of ITR. Such truncated ITRs may be present in the genome packaged by the AAV vectors of the present disclosure, but may also be present in vector sequences used for AAV vector production, such as in plasmids. For example, it has been observed that if the sequence lost from one ITR is retained in the other ITR, the truncated ITR can still function to produce AAV due to its ability to self-repair (Wang, X.S. et al., J. Mol. Biol., 250:573-80, 1995; Samulski, R. et al., Cell, 33:135-43, 1983).

[0226] In some non-limiting, merely exemplary embodiments, the genome of an AAV vector of the present disclosure may comprise one or more AAV ITRs comprising, consisting essentially of, or consisting of any one or more of the nucleotide sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:20, or SEQ ID NO:21, although other ITR sequences are also possible, or may comprise the complement or reverse complement of any of the sequences specifically listed above.

[0227] Array Optimization In some embodiments, one or more sequences within an AAV vector can be optimized to improve its functional characteristics compared to the starting reference sequence. For example, without limitation, any protein-coding sequence in the vector can be codon-optimized compared to the wild-type sequence based on the degeneracy of the genetic code and codon usage bias known to exist between different species and between proteins expressed at high or low levels within the same species. Such codon bias can be identified, for example, using a codon adaptation index (CAI) for a particular species. The codon adaptation index (CAI) is described 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, meaning that the coding sequence is optimized based on human codon bias. Codon optimization can be facilitated using various algorithms known in the art. As known in the art, different CAIs can be constructed based on the analysis of highly expressed genes, such as human genes. An exemplary human CAI is reported in Haas, J, et al., Current Biology, 6(3):315-24 (1996). If desired, protein coding sequences can be codon-optimized for species other than human.

[0228] To increase protein expression levels, different codon optimization strategies have been proposed and implemented. For example, the most frequently used synonymous codons (i.e., those that code for the same amino acid) can be substituted at each position where they do not occur. Alternatively, codon usage can be adjusted throughout the coding sequence to be proportional to the natural codon bias distribution of the host organism. In some embodiments, codon substitution is limited to codons that occur relatively rarely, for example, at a frequency of 10% or less, in highly expressed proteins in a species, as reflected by CAI.

[0229] In some embodiments, protein-coding sequences expressed by AAV vectors 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 a more frequently used synonymous codon, as reflected in a CAI, such as human CAI. In some embodiments, the rare codon occurs at a frequency of 10%, 9%, 8%, 7%, 6%, or 5% or less, as reflected in a CAI, such as human CAI.

[0230] In some embodiments, a protein-coding sequence 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 the human CAI, such that the calculated CAI value for the overall coding sequence is increased compared 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 by reference to a particular CAI lookup 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 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, 0.19, 10.20, 10.21, 10.22, 10.23, 10.24, 10.25, 10.26, 10.27, 10.28, 10.29, 10.30, 10.31, 10.32, 10.33, 10.34, 10.35, 10.36, 10.37, 10.38, 10.39, 11.40, 11.41, 11.42, 11.43, 11.44, 11.45, 11.46, 11.47, 11.48, 11.49, 12.50, 12.51, 0.16, 0.17, 0.18, 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 increase.

[0231] As is known in the art, the presence of hypomethylated CpG dinucleotides in nucleic acids can stimulate an immune response that eliminates transduced cells. Therefore, depleting CpG dinucleotides in vectors can increase the likelihood that vector transduction will result in long-term gene expression. Wright, JF, Mol Ther, 28(3):701-3 (2020). Taking into account the potentially harmful effects of CpG dinucleotides, in some embodiments, any sequence within a genome, including, for example, enhancers, promoters, introns, open reading frames encoding proteins or functional RNAs, transcription terminators, 5' untranslated region (UTR) and / or 3' untranslated region sequences, ITRs, or any other sequence, can be modified to remove one or more CpG dinucleotides, as long as it does not unacceptably interfere with or destroy some desired function of the modified element. Because the function of certain elements within vectors, such as ITRs, promoters, and enhancers, may be highly dependent on the identity of specific nucleotides at specific positions, the opportunities for significantly depleting such elements of CpG dinucleotides may be more limited. Because AAV vectors of both polarities (e.g., sense and antisense with respect to the coding sequence of the transgene within the genome) are packaged into capsids in approximately equal proportions, CpG depletion strategies may, in some embodiments, involve reducing or eliminating CpG motifs from the nucleotide sequences of vectors of both polarities, as well as vectors containing protein coding sequences in the sense orientation.

[0232] 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 (with respect to the sense and / or antisense strand) are deleted or substituted compared to a reference starting sequence, while 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 a range between any of the above values, are deleted or substituted compared to the starting reference sequence. In other embodiments, between 1 and 5, 5 and 10, 10 and 15, 15 and 20, 20 and 25, 25 and 30, 30 and 35, 35 and 40, 40 and 45, 45 and 50, 50 and 55, 55 and 60, 60 and 65, 65 and 70, 70 and 75, 75 and 80, 80 and 85, 85 and 90, 90 and 95 or 95 and 100 CpG dinucleotides are deleted or substituted relative to the reference starting sequence.

[0233] In other embodiments, sequence optimization can increase or decrease the overall GC content compared to a starting reference sequence. Thus, in some embodiments, the overall percentage of G or C nucleotides in a transgene or the entire genome can be increased by at least or about 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, 6 percent, 7 percent, 8 percent, 9 percent, 10 percent, 11 percent, 12 percent, 13 percent, 14 percent, 15 percent, 16 percent, 17 percent, 18 percent, 19 percent, 20 percent, 21 percent, 22 percent, 23 percent, 24 percent, 25 percent, 26 percent, 27 percent, 28 percent, 29 percent, 30 percent, 35 percent, or 40 percent or more compared to a starting reference sequence, such as a wild-type protein-coding sequence. In other embodiments, the overall percentage of G or C nucleotides in the transgene or the entire genome may be reduced by at least or about 1 percent, 2 percent, 3 percent, 4 percent, 5 percent, 6 percent, 7 percent, 8 percent, 9 percent, 10 percent, 11 percent, 12 percent, 13 percent, 14 percent, 15 percent, 16 percent, 17 percent, 18 percent, 19 percent, 20 percent, 21 percent, 22 percent, 23 percent, 24 percent, 25 percent, 26 percent, 27 percent, 28 percent, 29 percent, 30 percent, 35 percent, or 40 percent or more as compared to a starting reference sequence, such as a wild-type protein-coding sequence.

[0234] As will be appreciated by those skilled in the art, when optimizing coding sequences, the goal of substituting a more common codon for any particular amino acid in a species (such as human) may be incompatible with other optimization strategies because the introduction of a more frequently used codon may introduce CpG motifs, or the 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, to achieve improved protein expression, it may be necessary to design and test different optimized coding sequences (encoding the same polypeptide) to identify an acceptable balance between the different optimization strategies.

[0235] In addition to codon bias and CpG content, transgene and vector sequences can be optimized by varying various features. For example, any of the following features that may be found in the sequence and negatively affect transgene expression can be identified (conceptually, such as by using an algorithm, or experimentally) and altered to reduce or eliminate their effects: potential splice sites; premature transcription termination signal sequences (e.g., polyA sequences); translation initiation sites other than the intended initiating methionine (e.g., IRES); GC-rich sequence regions; mRNA 5'-terminal sequences that can form hairpins; and AU-rich elements (AREs) in the mRNA 3'-untranslated region to which destabilizing RNA-binding proteins may bind. Other sequence features that may appear in transgenes and vectors that, when altered, can enhance transgene expression are well known to those skilled in the art.

[0236] In other embodiments, the transgene or vector sequence can be modified to enhance functionality. For example, the original intended start codon in a protein-coding sequence may only slightly support translation initiation from that site, in which case the surrounding sequence can be changed to conform to the so-called Kozak consensus sequence for translation initiation in eukaryotes. Kozak, M., Gene, 234(2):187-208 (1999).

[0237] In some embodiments, other types of sequence optimization of a transgene coding sequence, such as (partial or complete) CpG depletion or codon optimization, can improve protein expression from the transgene compared to the same vector containing a non-optimized reference starting sequence, such as the wild-type coding sequence from which the optimized sequence is derived. Thus, for example, an optimized coding sequence of a transgene may be expressed at least 25%, 50%, 75%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more efficiently compared to a non-optimized reference starting sequence, such as the wild-type coding sequence.

[0238] AAV capsid 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 of a subject to express a PGRN protein or variant thereof from a vector transgene.

[0239] The choice of capsid (and the corresponding cap gene sequence used in its production) for designing and producing an AAV vector can be guided by numerous considerations and factors. As noted above, by specifically interacting with particular cell surface receptors, different AAV capsids can have different cell or tissue tropisms, which can be advantageous when it is desired to preferentially transduce certain tissues relative to other tissues. For example, to express a transgene product preferentially in the brain, such as neurons, a vector can be designed and produced with a capsid that has a stronger affinity for neurons than for muscle or liver. Conversely, to express a transgene product in hepatocytes, a vector can be designed and produced with a capsid that has a stronger affinity for hepatocytes than for neurons, muscle, or other tissues.

[0240] Other factors may also be important. For example, it has been reported that some humans have high neutralizing antibody titers against certain capsids as a result of 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 selection of a capsid may in some cases be guided by the capsid's immunogenicity and / or seroprevalence in the patient to be treated. Other considerations that may influence the selection of a capsid include manufacturability and stability during storage, and other relevant guiding factors are known in the art.

[0241] The AAV vectors of the present disclosure can use capsids made from capsid proteins derived 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 by introducing amino acid substitutions, in the VP1, VP2, and / or VP3 protein sequences, with the intention of improving capsid function in some respect, such as tissue tropism, immunogenicity, stability, or manufacturability. Other examples include novel capsids with improved properties created by exchanging amino acids or domains from one known capsid to another (e.g., a mosaic or chimeric capsid), or by using DNA shuffling and directed evolution methods to discover capsid protein sequences with desired properties.

[0242] In some embodiments, AAV vectors of the disclosure may comprise capsids from known AAV serotypes and variants, including but not limited to AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrhl74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5, and AAV2i8, as well as non-native capsids, and many others are possible. In some embodiments, the capsid of an AAV vector of the disclosure comprises VP1, VP2, and / or VP3 AAV capsid proteins that are variants or derivatives of the known VP1, VP2, or VP3 AAV capsid proteins. In some embodiments, the amino acid sequence of such a mutant or derivative AAV capsid protein is selected from the group consisting of, but not limited to, the AAV capsid VP1 protein, VP2 protein, or VP3 protein of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrhl74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5, and AAV2i8, or any other suitable AAV capsid, including, for example, the neurotropic capsids described below. The amino acid sequence of the VP1 protein sequence, VP2 protein sequence, or VP3 protein sequence of a known AAV capsid, including a known AAV capsid, 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% or 99.9% identical to the amino acid sequence of a known AAV capsid VP1 protein sequence, VP2 protein sequence, or VP3 protein sequence, including a known AAV capsid.In some other embodiments, the amino acid sequence of such mutant or derivative AAV capsid proteins is selected from known AAV capsid VP1, VP2, or VP3 proteins, including, but not limited to, the AAV capsid VP1, VP2, or VP3 proteins of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, AAVrhl74, AAV-DJ, AAV-PHP.B, Anc80, AAV2.5, and AAV2i8, or any other suitable AAV capsid, including, for example, the neurotropic capsids described below. a protein amino acid sequence and 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 (whether resulting from amino acid deletion, insertion, or substitution) , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids differ.

[0243] Neurotropic AAV capsid In some embodiments, the AAV vectors of the present disclosure comprise a neurotropic capsid. A neurotropic capsid is an AAV capsid that has tropism for neurons. In some embodiments, such neurons may be neurons in specific regions of the brain, such as, but not limited to, the cerebral cortex, including the frontal, temporal, parietal, or occipital lobes of the brain, or neurons in other regions, such as neurons in 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 comprise capsids that have tropism for cells within or outside the CNS other than neurons, non-limiting examples of which include astrocytes, microglia, oligodendrocytes, or ependymal cells, although other cell types are also possible. In some non-limiting embodiments, the AAV vectors of the present disclosure comprise capsids that have tropism for neurons, such as neurons present in the human brain. As will be understood by those skilled in the art, neurotropism does not necessarily mean that the capsid can transduce only neuronal cells to the exclusion of non-neuronal cells. Rather, a neurotropic capsid is one that has a greater propensity to transduce neurons than some other cell types, even if that propensity is not absolute, or even if the capsid in question has a greater propensity to transduce non-neuronal cell types than neurons. In the latter case, such capsids may be referred to as such even if they are not primarily neurotropic.

[0244] Examples of neurotropic capsids include, but are not particularly limited to, AAV1; AAV2; AAV4; AAV5; AAV7; AAV8; AAV9; AAV-rh.10; AAVv66 (Hsu, H-L. 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. & 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); AAV 2-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 capsids (U.S. Patent Application Publication No. 2022-0042044 and International Patent Application Publication No. 2021 / 230987); and numerous others, 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 may be referred to herein as an AAV-801 capsid.

[0245] AAV vector for expressing progranulin protein According to certain embodiments, the AAV vectors of the present disclosure include AAV vectors comprising a transgene encoding a wild-type progranulin (PGRN) protein, such as the 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 the same as that of the naturally occurring PGRN protein, although signal peptides from heterologous proteins can be used as well. 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 comprises SEQ ID NO: 17. In some embodiments, the amino acid sequence of the wild-type human PGRN protein (including the native signal peptide) comprises the amino acid sequence of SEQ ID NO: 16.

[0246] In some embodiments, AAV vectors of the present disclosure include AAV vectors comprising a transgene encoding a variant of human PGRN protein, such as a variant comprising at least one amino acid insertion, deletion, and / or substitution compared to the amino acid sequence of a wild-type human PGRN protein, such as that provided by SEQ ID NO: 16. In some embodiments, the transgene encodes a human variant PGRN protein in which at least one amino acid, 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, is deleted from the PGRN carboxy-terminus (C-terminus) compared to the amino acid sequence of a wild-type human PGRN amino acid sequence, such as that provided by SEQ ID NO: 16. In some embodiments, the transgene encodes a human PGRN variant protein (PGRNΔ3) in which the last three C-terminal amino acids (QLL) present in the wild-type human PGRN protein are deleted. 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 mutants of human PGRN, including the PGRNΔ3 mutant, have reduced binding to sortilin (Zheng, Y. et al., PLOS One, 6:1-7, 2011).

[0247] In some embodiments, the nucleotide sequence of the transgene encoding the human PGRN protein is a wild-type coding sequence that is identical to the coding sequence present within the exons of a naturally occurring gene encoding the human PGRN protein (i.e., GRN) or within the cDNA sequence corresponding to the mRNA transcribed from the GRN gene and encoding the human PGRN protein. In some embodiments, the wild-type coding sequence for wild-type human PGRN protein is provided by SEQ ID NO: 15, and the wild-type coding sequence for human PGRNΔ3, excluding the deletion of the codons for the last three amino acids present at the C-terminus of wild-type human PGRN, is provided by SEQ ID NO: 8. While in other embodiments, the coding sequence can be optimized, such as by deletion of some or all CpG dinucleotides, while still encoding the same PGRN or PGRNΔ3 protein. In some embodiments, at least 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 12 4, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 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 specifically recited values ​​above) are removed from SEQ ID NO: 15 or SEQ ID NO: 8. In some embodiments, the nucleotide sequence encoding the PGRN protein or PGRNΔ3 protein does not contain any CpG dinucleotides.In some embodiments, the nucleotide sequence encoding the PGRN protein or the PGRNΔ3 protein includes 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 above specifically recited values) compared to SEQ ID NO: 15 or SEQ ID NO: 8, respectively. In some embodiments, the CpG dinucleotides are identified with respect to the order of nucleotides in the sense strand, while in other embodiments, the CpG dinucleotides are identified with respect to the order of nucleotides in the antisense strand.

[0248] In other embodiments, the nucleotide sequence of the transgene encoding the wild-type human PGRN protein is the same as that provided in SEQ ID NO: 15 (regardless of the stop codon), while 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 ID NO: 15, but encodes an amino acid sequence identical to that encoded by SEQ ID NO: 15 (i.e., SEQ ID NO: 16). In other embodiments, the nucleotide sequence of the transgene encoding the human PGRNΔ3 protein is the same as that provided in SEQ ID NO:8 (regardless of the stop codon), while 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 ID NO:8, but encodes an amino acid sequence identical to that encoded by SEQ ID NO:8 (i.e., SEQ ID NO:14).

[0249] In some embodiments, 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 opposite end of the genome from 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, a second AAV ITR located at its 3' end, and a third AAV ITR located between the first and second AAV ITRs.

[0250] AAV ITRs for use in the vectors of the present disclosure can be of any type, such as AAV2 ITRs or non-AAV2 ITRs, can be full-length or truncated, and can have the same sequence as any known naturally occurring AAV viral ITR (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 degradation by AAV Rep proteins, such as by mutating, deleting, or otherwise inactivating terminal resolution sites (trs), and reducing or eliminating the ability of the ITR to support packaging into capsids, such as by mutating, deleting, or otherwise inactivating D sequences in the ITR sequence. In some embodiments, the vector may further comprise at least a third AAV ITR, such as an ITR located between the ends of the genome, such as near or in the middle of the vector sequence. In some of these embodiments, the third ITR may be modified, such as by inactivating its terminal resolution site, so that the vector containing the transgene is self-complementary. In some embodiments, the vector comprises an AAV ITR that 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 complement or reverse complement of each such sequence.

[0251] In some embodiments, the AAV vector of the present disclosure further comprises a transcriptional control region operably linked to a transgene encoding a PGRN protein or a PGRNΔ3 protein. In some embodiments, the transcriptional control region may be inducible, constitutively active, or cell-type- or tissue-type-specific, such as being primarily or exclusively active in neurons or the brain (thus neuron- or brain tissue-specific). In some embodiments, the transcriptional control region comprises or consists of a promoter and may further comprise at least one enhancer region or element. Any region or element within the transcriptional control region may be derived from a human gene or a non-human gene, such as a rat, mouse, bovine, non-human primate, chicken, or viral gene, or from another species or type of organism. The regions or elements of the transcriptional control region may be contiguous with 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 located anywhere else within the vector, such as distal upstream or distal 3' (downstream) of the transgene. Any region or element of the transcriptional control region may be cell type- or tissue-specific, such as brain tissue-specific or neuron (or other brain cell type)-specific. Thus, the promoter may be brain tissue-specific or neuron (or other brain cell type)-specific, the enhancer region or element may be brain tissue-specific or neuron (or other brain cell type)-specific, or both the promoter and enhancer(s), acting alone or in concert, may be brain tissue-specific or neuron (or other brain cell type)-specific.

[0252] In some embodiments, a transcriptional control region for use in a vector of the present disclosure may comprise a promoter sequence derived from the human synapsin 1 (SYN1) gene, where such a promoter is the entire human SYN1 gene promoter or a functional promoter subsequence of such a human SYN1 gene promoter. Thus, for example, a promoter can comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:6, or a promoter functional subsequence, variant, or mutant thereof.

[0253] In some embodiments, the AAV vector of the present disclosure further comprises a 5' untranslated region (UTR) from a gene located 3' to the promoter and 5' to the transgene encoding the PGRN protein or PGRNΔ3 protein. In some embodiments, the 5' UTR sequence is the entire 5' UTR sequence from the gene, or a partial sequence 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 of other species, or from a gene other than synapsin 1. In some embodiments, the 5' UTR sequence comprises, consists essentially of, or consists of SEQ ID NO:7.

[0254] In some embodiments, the AAV vectors of the present disclosure further comprise a transcription termination signal sequence, such as a polyadenylation (poly(A)) signal sequence, such as the poly(A) signal sequence 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 can 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, variant, or mutant thereof.

[0255] In some embodiments, the AAV vectors of the present disclosure further comprise additional functional sequences, such as introns, viral post-transcriptional regulatory element (PRE) sequences such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) or the 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 that comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:29, or a functional subsequence, variant, or mutant thereof. Other sequences that may be useful 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 stuffer or filler nucleotide sequences that are not necessarily intended to directly affect transgene expression (although such properties may be present), but are included to bring the total length of the vector to a particular size, e.g., long enough to approximate the packaging capacity of a particular AAV capsid so as to reduce the amount of contaminating non-full-length vector genomic DNA packaged into the capsid.

[0256] In some embodiments, the AAV vectors of the present disclosure further comprise a stuffer or filler sequence located 3' to the poly(A) signal sequence and 5' to the ITR. In some embodiments, the stuffer or filler sequence is derived from an intron of a gene, such as the gene encoding the human or other species TATA box binding protein (TBP). In some embodiments, the stuffer or filler sequence is a TBP gene intron, or a variant or mutant thereof, such as that provided by the nucleotide sequence of SEQ ID NO:11.

[0257] In some embodiments, an AAV vector of the present disclosure comprises a first AAV ITR, a transcriptional regulatory region, a 5' UTR sequence, a transgene encoding a human PGRN protein or a PGRNΔ3 protein operably linked to the transcriptional regulatory region, a transcription termination signal sequence, a filler sequence, and a second AAV ITR. In related embodiments, these elements may be arranged contiguously in 5' to 3' order in a single-stranded vector in a sense orientation or in the sense strand of a double-stranded DNA molecule containing the vector sequence, such as in a plasmid used to produce the vector in a host cell. Conversely, these elements may be arranged contiguously in 3' to 5' order in a single-stranded vector in an antisense orientation or in the antisense strand of a double-stranded DNA molecule containing the vector sequence. Thus, for example, and not by way of limitation, if a sense-oriented vector contains, in 5' to 3' order, a promoter, a transgene, and a poly(A) signal sequence, a complementary antisense vector sequence will contain those same elements in the opposite order, starting from its 5' end, with the understanding that the nucleotide sequence of the antisense strand genome read in the 5' to 3' direction is the reverse complement of the nucleotide sequence of the sense strand genome. In the case of a self-complementary vector (scAAV), the arrangement of elements occurs in both 5' to 3' order across approximately half of the sequence, and then 3' to 5' order across the complementary half.

[0258] In some embodiments, an 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 regulatory region, a 5' UTR sequence, a transgene encoding a PGRN protein or a PGRNΔ3 protein operably linked to the transcriptional regulatory region, a transcription termination signal sequence, a filler sequence, and a second AAV ITR from AAV2 at the 3' end of the genome. In some embodiments, the transcriptional regulatory region comprises a promoter that may be neuron-specific, such as a promoter 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 may also be 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, which in some embodiments 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 filler sequence is derived from an intron from the TBP gene, which in some embodiments may comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO:11.

[0259] In any of the above embodiments, the nucleotide sequence of the transgene encoding the human PGRN protein may 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 an amino acid sequence identical to SEQ ID NO:16. Alternatively, in any of the above 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 an amino acid sequence identical to SEQ ID NO:14.

[0260] In any of the above embodiments, either or both of the first and second AAV2 ITRs may be full-length or truncated, and may have a flip or flop structure. In any of the above embodiments, either or both of the first and second AAV2 ITRs may 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 such sequence.

[0261] In some embodiments, the AAV vector of the present disclosure further comprises a third AAV ITR located between the first and second AAV ITRs, e.g., in the middle (even if not exactly in the middle) of the vector, which in some embodiments may have a mutated or modified terminal resolution site that is not subject to terminal resolution. In some of these embodiments, the vector may be self-complementary and may have a length in the range of about 3,000-5,000 nucleotides or 4,000-5,000 nucleotides when packaged into a capsid, or in the range of about 1,500-2,500 nucleotides or 2,000-2,500 nucleotides when the sequence is contained in a plasmid suitable for use in producing an scAAV vector in a host cell.

[0262] In any of the above embodiments, the vector can comprise, consist essentially of, or consist of the nucleotide sequence of SEQ ID NO: 19 or its reverse complement. In any of the above embodiments, the vector can be single-stranded, meaning that the vector is not self-complementary (other than the ITRs) and can have a length in the range of about 3500-5000 nucleotides, about 3500-4700 nucleotides, about 3800-4500 nucleotides, about 3800-4300 nucleotides, about 3800-4100 nucleotides, about 3800-4000 nucleotides, about 3900-4000 nucleotides, about 3950 nucleotides, or about 3942 nucleotides. In any of the above embodiments, the vector can be in the sense or antisense orientation.

[0263] In any of the above embodiments, the vector may be encapsidated with an AAV capsid, such as a neurotropic AAV capsid, non-limiting examples of such capsids 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 Examples of the capsid include 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 an AAV-801 capsid comprising 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.

[0264] In some embodiments, the AAV vectors of the present disclosure comprise an AAV-801 capsid that encapsidates (packages) the AAV vector in a sense or antisense orientation, and 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 a human PGRNΔ3 protein operably linked to the transcriptional control region, a transcription termination signal sequence, a filler sequence, and a second AAV ITR located at the 3' end of the genome, wherein either or both of the AAV ITRs are AAV2 the promoter is derived from the human synapsin 1 gene, the 5'UTR is also derived from the human synapsin 1 gene, the transcription termination signal sequence is a poly(A) signal sequence derived from the bovine growth hormone (bGH) gene, the filler sequence is a modified intron derived from the human TBP gene, the nucleotide sequence of the promoter comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:6, the nucleotide sequence of the 5'UTR comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:7, and the nucleotide sequence of the transgene encoding the human PGRNΔ3 protein is the nucleotide sequence of SEQ ID NO:8. The vector in the antisense orientation comprises a nucleotide sequence that is at least 70% identical to the nucleotide sequence of SEQ ID NO:8 and encodes the same polypeptide as that encoded by SEQ ID NO:8; 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; the nucleotide sequence of the filler sequence comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:11; and the antisense orientation genome 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.

[0265] AAV vector production As is known in the art, AAV vectors can be produced in a variety of ways, including on a large scale. AAV vectors can be produced in mammalian or insect cells, for example, and then purified. A traditional approach that does not rely on coinfection with a helper virus involves the use of three plasmids: one plasmid contains genes for helper virus factors, a second plasmid contains the AAV genome sequence in double-stranded form, and a third plasmid contains the AAV rep and cap genes. While the rep / cap plasmid often contains the rep gene from AAV2, this is not a requirement; the cap gene sequence is selected based on which AAV capsid proteins are desired to comprise the capsid. In practice, the three plasmids are often replicated separately in bacteria, purified, mixed together in solution in 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), which are incubated for a sufficient time (e.g., 48-72 hours) and under sufficient conditions for the host cells to express the helper factors and the rep and cap genes and for the AAV vector to replicate from its plasmid template and be packaged into capsids. In some embodiments, the host cells are HEK293 cells, which constitutively express AdV helper factors E1A and E1B; thus, the helper plasmid need only contain the AdV E2A, E4ORF6, and VA RNA genes. Use of other mammalian host cells that do not themselves produce AdV or other viral helper factors requires the use of helper plasmids containing the missing or otherwise required helper factors. While the so-called triple transfection method described above is commonly used, it is not necessary for the genes for the helper factors and the rep and cap genes to be provided on separate plasmids. In principle, all these genes can be contained in one plasmid, for example, and in this case two plasmids can be used for transfection.

[0266] In search of a more efficient method for large-scale production of AAV vectors, stable cell lines have been generated that contain some, but not all, of the components that would normally be required for transient transfection. Packaging cell lines contain stably integrated AAV rep and cap genes. AAV production in packaging cells requires transient transfection of the cells with a plasmid containing an AAV vector and infection with a helper virus. Alternatively, AAV vectors can be produced in packaging cells without transfection by first infecting the cells with AdV (either wild-type or E2b gene-deleted AdV) that provides the AdV E1 gene products that drive rep and cap expression in the cells, as well as helper factors required for AAV replication, and then infecting them with a replication-deficient hybrid AdV in which the AAV vector replaces the E1 gene in the hybrid virus genome.

[0267] Alternatively, the producer cell line also contains stably integrated AAV rep and cap genes and an AAV vector. AAV production in the producer cells requires infection of these cells with a helper virus. Packaging and producer 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; Clement, N. & Grieger, J., Mol. Ther. Methods Clin. Dev., 3:16002, 2016). Other cell lines are contemplated for producing AAV vectors in mammalian cells, including on a commercial scale.

[0268] The baculovirus system has also been used to produce AAV vectors, in which Sf9 insect cells are infected with recombinant baculovirus vectors that variously contain the AAV rep and cap genes and the AAV genome. The exogenous 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. Subsequent modifications included combining rep and cap into a single baculovirus, thereby requiring only two types of baculovirus, and creating the Sf9 cell line, which contains stably integrated AAV rep and cap genes and requires only infection with a single type of recombinant baculovirus containing an 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 lines are contemplated for producing AAV vectors in insect cells, including on a commercial scale.

[0269] host cell As used herein, "host cells" refer to cells suitable or adapted for in vitro production of AAV vectors. Host cells are often clonal cell lines capable of dividing for multiple generations before aging causes growth arrest, or they may even be immortal. To produce vectors, host cells can be transiently or non-transiently modified by the introduction of exogenous genetic information designed to direct the biosynthesis in the host cell of various components required for AAV vector assembly, particularly AAV capsid proteins, Rep proteins, helper virus factors, and vectors. For example, host cells can be transfected with exogenously supplied nucleic acid, such as in the form of one or more DNA plasmids, containing nucleotide sequences encoding the necessary vector components.

[0270] Various methods for transfecting host cells with nucleic acids are known in the art. These methods include, but are not limited to, mixing the nucleic acid with certain compounds that can form complexes with the nucleic acid and then be taken up by the cell, such as calcium phosphate or cationic organic compounds (e.g., DEAE-dextran, polyethylenimine (PEI), polylysine, polyornithine, polybrene, cyclodextrins, cationic lipids, and others known in the art. Transfection can also be performed non-chemically by electroporation and more exotic techniques, such as biolistic particle delivery. As known in the art, transfection can be transient or stable. In transient transfection, the transfected nucleic acid is present in the cell for a limited period of time and, in the case of DNA, does not integrate into the genome. In stable transfection, the DNA introduced into the cell is distributed within an episomal plasmid. Stably transfected cells can persist for long periods of time, either as a vector or integrated into a chromosome. Stably transfected cells are typically generated by transfecting cells with a plasmid containing a nucleotide sequence encoding a selectable marker gene and one or more necessary vector components, and then growing and maintaining the cells under selection, i.e., conditions under which untransfected cells or transfected cells that have lost the exogenous DNA, including the selectable marker, for some reason, are killed. For example, the plasmid may contain an antibiotic resistance gene, and transfected cells can be selected by adding an antibiotic to the medium in which the cells are grown. In some embodiments, the nucleotide sequence introduced into a stably transfected host cell and encoding one or more of the necessary vector components is under the control of an inducible promoter, such that it is not expressed, or is expressed only at low levels, unless an environmental factor, such as a drug, metal ion, or elevated temperature, is introduced that induces the promoter as the cells grow.

[0271] In other embodiments, genetic engineering methods, such as knock-in or gene editing, can be used to non-transiently and targetedly modify the host cell genome to direct the host cell to produce one or more of the required vector components. In other embodiments, nucleotide sequences encoding one or more of the required vector components can be introduced into host cells for the purpose of directing the production of AAV vectors by transduction, where the host cells are infected with a modified virus containing such nucleotide sequences. Examples of viral vectors useful for this purpose include adenoviruses, retroviruses (including lentiviruses), baculoviruses, vaccinia viruses, and herpes simplex viruses, among others.

[0272] Host cells can be any type of cell known in the art to be useful for producing AAV vectors. Host cells are often animal cells and can be of various types or species, such as insect cells or mammalian cells, including rat, mouse, or human cells, among others. In some embodiments, host cells useful for producing the AAV vectors of the disclosure are mammalian host cells, such as HeLa cells, Cos cells, HEK293 cells (and variants of HEK293 cells such as HEK293E cells, HEK293F cells, HEK293H cells, HEK293T cells, 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, among many others. In some embodiments, host cells useful for producing the AAV vectors of the invention are insect host cells, such as Sf9, ExpiSf9, ​​Sf21, S2, D.Mel2, Tn-368, or BTI-Tn-5B1-4 cells, among many others. In some embodiments, host cells, including but not limited to HEK293 cells and variants thereof, can be adapted for growth in suspension culture.

[0273] For the purpose of producing AAV vectors, host cells are grown or maintained in culture under controlled conditions conducive to host cell growth and vector biosynthesis. For example, host cells can be grown in a chemically defined liquid medium that provides all nutrients necessary for cell growth and biosynthesis. Exemplary media include DMEM, DMEM / F12, MEM, and RPMI 1640 for mammalian host cells, and Express Five SFM, Sf-900 II SFM, Sf-900 III, or ExpiSf CD for certain insect cells. Such media may be supplemented with antibiotics, growth factors, or cytokines (recombinantly produced or present in animal serum, such as FBS) known to stimulate growth of the particular type of cell being used, as well as other components that may be required for optimal biosynthesis of AAV vectors but are naturally in low supply. Exemplary supplements include essential amino acids, glutamine, vitamin K, insulin, BSA, or transferrin. In addition to the growth medium, other culture conditions, such as pH, temperature, and CO2 and oxygen concentrations, may be controlled to optimize cell growth and / or productivity.

[0274] Host cells in culture can be grown or maintained in a number of vessels 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 or multiple use. Depending on the characteristics of the host cells in question, host cells can be grown in adherent cell culture, in which cells attach to and grow in contact with a physical substrate, or in suspension cell culture, in which single cells either float freely in the medium that sustains them or attach to bead microcarriers suspended in the medium. As is known in the art, various techniques have been developed, such as perfusion culture, that can increase the total amount of AAV vector produced in a single production run and can be used to grow host cells to high cell densities.

[0275] As is known in the art, samples of host cells are often maintained in frozen cell banks, such as master cell banks and working cell banks, facilitating the production of biological products in multiple batches over time while ensuring consistent performance by the host cells. Prior to AAV vector production activities, frozen samples of host cells from the cell bank are typically thawed, seeded into small culture volumes, and grown to further increase in density or number, with increasing culture volumes. 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, to cause the host cells to begin producing AAV vectors. Alternatively, when using host cells that have been non-transiently modified so that a nucleotide sequence encoding one or more required vector components is under inducible control, environmental factors necessary to induce expression can be introduced. The host cells can then be grown or maintained in culture for a time and under conditions sufficient to produce the AAV vector.

[0276] AAV vector purification Following biosynthesis in the host cell, the AAV vector can be purified by a variety of methods known in the art. For example, in some embodiments, the host cells can be lysed mechanically or chemically, such as with detergents, followed by removal of host cell DNA and other components, followed by a step such as density gradient centrifugation or the use of one or more chromatographic separation methods to yield a highly purified preparation of the AAV vector for use in research or therapeutic methods.

[0277] Chromatographic methods useful for purifying AAV vectors include, but are not limited to, size exclusion chromatography (SEC); affinity chromatography, which uses any affinity ligand, such as an antibody, lectin, or glycan, that can specifically bind to the capsid and is 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).

[0278] In some embodiments, AAV vectors can be purified using antibody-based affinity chromatography, in which an antibody or antibody fragment thereof is bound to a stationary phase (matrix or resin) packed in a chromatography column, host cell lysate is pumped through the column, and the antibody-bound vector is then washed and eluted. The antibody bound to the solid phase can be an IgG or fragment thereof, or a single-chain camelid antibody (such as a heavy chain variable region camelid antibody), although 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).

[0279] In other embodiments, AAV vectors can be purified using ligand chromatography, in which the stationary phase is conjugated to the same type of ligand that a particular AAV is known to use to bind to cells, such as a glycan, sialic acid (e.g., O-linked sialic acid or N-linked sialic acid), galactose, heparin, heparan sulfate, or a proteoglycan, such as heparan sulfate proteoglycan or heparin 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).

[0280] Depending on the physicochemical characteristics of the vector, such as the charge on the capsid, the AAV vector can be further purified by anion exchange chromatography, cation exchange chromatography, 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. Two or more downstream processing steps can be used, and multiple downstream processing steps can be performed in any order according to the knowledge of those skilled in the art.

[0281] Treatment methods In addition to AAV vectors for expressing PGRN protein or its variants, such as PGRNΔ3, and compositions comprising such AAV vectors, the present disclosure provides methods for treating a subject, such as a human subject, in need of treatment for frontotemporal dementia (FTD), frontotemporal lobar degeneration (FTLD), or PGRN deficiency by administering a therapeutically effective amount of an AAV vector of the present disclosure or a composition comprising such an AAV vector to the subject. Also provided is the use of an AAV vector for expressing PGRN protein or its variants, such as PGRNΔ3, in the manufacture of a medicament for use in the therapeutic methods disclosed herein. Further provided is an AAV vector for expressing PGRN protein or its variants, such as PGRNΔ3, or a pharmaceutical composition comprising such an AAV vector, for use in the therapeutic methods disclosed herein. In some embodiments, the AAV vector used in the therapeutic method or included in the medicament or pharmaceutical composition is the vector described herein as AAV801-PGRNΔ3, including the vector described herein as clone 249.

[0282] In some embodiments, the subject has been diagnosed with FTD or FTLD (or suspected FTD or FTLD) by the time of treatment based on standard diagnostic criteria, including, but not limited to, evaluation 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 the pattern of brain atrophy characteristic of FTD or FTLD, in each case. In some embodiments, the subject has been diagnosed with behavioral variant frontotemporal dementia (BV-FTD) or non-fluent variant primary progressive aphasia (NFV-) by the time of treatment. In some embodiments, the subject has been diagnosed with atrophy in one or more brain regions, including, but not limited to, the frontal lobe (e.g., the orbitofrontal cortex or anterior cingulate gyrus), the temporal lobe (e.g., the anterior temporal lobe, the medial temporal lobe, or the posterior temporal lobe), or other brain regions such as the inferior parietal lobe, the striatum, or the thalamus, or other brain regions, by the time of treatment.

[0283] In some embodiments, the diagnosis is confirmed using a biochemical test, for example, by detecting lower-than-normal levels of progranulin protein (PGRN) in a serum or cerebrospinal fluid (CSF) sample taken from the subject, and / or in some other embodiments, by identifying a heterozygous or homozygous deleterious (complete or partial loss of function) mutation in the GRN gene, which encodes PGRN, in the subject. In some embodiments, the subject is haploinsufficient with respect to the amount of PGRN produced by the GRN gene and either or both GRN alleles. In some embodiments, the subject is diagnosed with FTLD-TDP type A, FTLD-TDP type B, or FTLD-TDP type C, for example, based on neuropathological analysis of brain tissue.

[0284] Treatment of a subject with FTD, FTLD, or PGRN deficiency need not result in a cure to be considered effective, where "cure" is defined as either halting the progression of the disease or partially or completely restoring the subject's health status to that before the onset or worsening of symptoms, or compared to a healthy person without FTD, FTLD, or PGRN deficiency. Rather, a therapeutically effective amount of an AAV vector of the present disclosure (including but not limited to the vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector may function to at least partially reverse, alleviate, or ameliorate the degree or severity of at least one symptom or sign associated with FTD, FTLD, or PGRN deficiency in a subject; or to at least partially reverse, alleviate, or ameliorate the degree 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 to slow the progression of other adverse effects of FTD, FTLD, or PGRN deficiency in a subject; or to improve the quality of life of a subject with FTD, FTLD, or experiencing adverse effects of PGRN deficiency. Examples of symptoms, signs, disorders, or dysfunctions 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, inferior parietal lobe, or anterior, medial, and posterior regions of the temporal lobe), subcortical brain atrophy (e.g., affecting the striatum, thalamus, amygdala, or hippocampus), and behavioral consequences of brain atrophy, such as Parkinsonism, corticobasal syndrome (CBS), impaired word retrieval, apraxia of speech, agrammar, visual naming disorder, impaired word comprehension, phonological errors, impaired word repetition, impaired sentence repetition, impaired sentence comprehension, surface dyslexia, delusions, hallucinations, and reduced quality of life (QoL).

[0285] In other embodiments, a therapeutically effective amount of an AAV vector of the present disclosure (including, but not limited to, the vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector at least partially corrects or alters the level of a biomarker associated with FTD, FTLD, or PGRN deficiency to a level more reflective of normal function. Examples of biomarkers associated with FTD, FTLD, or PGRN deficiency include, but are not limited to, lower than normal levels of PGRN protein in CSF, serum, or plasma, or in brain tissue, lower than normal levels of bis(monoacylglycero)phosphate (BMP) in brain tissue, higher than normal levels of β-hexosaminidase (HexA) and β-galactosidase (β-Gal) enzyme activity in brain tissue, higher than normal levels of TDP43 fragmentation in brain tissue, and higher than normal levels of lipofuscin in brain tissue.

[0286] In some merely illustrative and non-limiting embodiments, the mean or median PGRN protein levels in plasma of humans with deleterious GRN mutations are about 28% of those in healthy humans, and the mean or median PGRN protein levels in CSF of humans with deleterious GRN mutations are about 39% of those in healthy humans (Meeter, L. et al., Dement. Geriatr. Cogn. Dis. Extra, 6:330-40, 2016). Those skilled in the art will understand that mean or median PGRN protein concentrations in biological fluid 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.

[0287] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency disclosed herein 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, recombinations, splice site variants, missense or nonsense mutations in one or both alleles of the GRN gene, mutations affecting the transcriptional regulatory regions (e.g., enhancers or promoters) of one or both alleles of the GRN gene, and / or mutations that reduce the stability of mRNA expressed from one or both alleles of the GRN gene or reduce the amount of protein translated from such mRNA transcripts, so long as the mutation(s) result in a reduction or loss of the amount of PGRN protein produced. Methods for determining a subject's genotype as having a deleterious mutation in one or both alleles of the GRN gene, such as by RFLP analysis or genetic or genomic sequencing, are well known to those of 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.

[0288] The therapeutic efficacy of the treatment methods disclosed herein can be assessed in individual subjects with FTD, FTLD, or PGRN deficiency by observing or measuring and comparing the severity or degree of any symptom, sign, disorder, dysfunction, or biomarker value characteristic of FTD, FTLD, or PGRN deficiency before (baseline) and after treatment. Such comparisons can be performed at one or more time points 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 after treatment, or at other time points. Data from individual subjects used for comparison can be a single data point or, if available, an average of multiple data points. In some other embodiments, the efficacy of treatment can be assessed in a population (i.e., two or more) of subjects with FTD, FTLD, or PGRN deficiency that serve as their own control by observing or measuring the severity or degree of any symptom, sign, disorder, impairment, or biomarker value characteristic of FTD, FTLD, or PGRN deficiency among individuals in the population before (baseline) and after treatment, and comparing the mean pre-treatment data with the mean post-treatment data. Such a comparison can be performed at one or more time points after treatment, for example, 0, 1, 2, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 60, 72, or 84 months after treatment, or at other time points. In some other embodiments, studies intended to establish and quantify therapeutic efficacy can be designed to compare the therapeutic effect in a population of subjects treated with an AAV vector of the present disclosure (treatment group) with the therapeutic effect in a population of subjects administered a placebo (control group). Typically, but not necessarily, subjects within the treatment and control groups in a study are matched for relevant subject characteristics, such as age, sex, and disease severity at the time of intervention.In other embodiments, the control population is instead statistically defined or determined from a natural history study that tracks disease progression in patients with FTD, FTLD, or PGRN deficiency in the absence of intervention (other than what is considered the standard of care).

[0289] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to treat subjects with FTD, FTLD, or PGRN deficiency of any age, including but not limited to, 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 above specifically recited ages.

[0290] In some embodiments, upon treatment with an AAV vector of the disclosure (including but not limited to, the vector described herein as AAV801-PGRNΔ3) or a pharmaceutical composition containing such an AAV vector, the subject does not exhibit any overt signs or symptoms of FTD, FTLD, or PGRN deficiency, but has been diagnosed as likely to develop such signs or symptoms in the absence of treatment based on a genetic test that indicates the subject has at least one deleterious mutation in one or both alleles of the GRN gene.

[0291] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to treat a subject with FTD, FTLD, or PGRN deficiency for a period of time following administration of an AAV vector of the disclosure (including, but not limited to, the vector described herein as AAV801-PGRNΔ3), or a pharmaceutical composition containing such an AAV vector, during which such subject experiences no symptoms or signs of FTD, FTLD, or PGRN deficiency, or experiences no worsening of symptoms or signs of FTD, FTLD, or PGRN deficiency that may be present at the time of treatment, or at most, minimal 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, functioning, quality of life, and / or lifespan is not substantially or materially affected. In some embodiments, this period, referred to herein as the period of treatment duration, can be any suitable or desired period, including, for example, but not limited to, at least or about 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21 months, 24 months or more, or at least or about 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years or more, or at least or about 10 years, 20 years, 30 years, 40 years, 50 years, 60 years, 70 years or more, or any integer value between or encompassing any of the above specifically recited times, or even, in some embodiments, the remainder of the subject's lifespan after receiving gene therapy as described herein.

[0292] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein involve increasing the concentration of PGRN protein or a variant 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 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 CSF of a healthy human, for example, about 2.9 ng / mL, 3.0 ng / mL, 3.1 ng / mL, 3.2 ng / mL, 3.3 ng / mL, 3.4 ng / mL, 3.5 ng / mL, 3.6 ng / mL, 3.7 ng / mL, 3.8 ng / mL, 3.9 ng / mL, 3.10 ng / mL, 3.11 ng / mL, 3.12 ng / mL, 3.13 ng / mL, 3.14 ng / mL, 3.15 ng / mL, 3.16 ng / mL, 3.17 ng / mL, 3.18 ng / mL, 3.19 ng / mL, 3.20 ng / mL, 3.21 ng / mL, 3.22 ng / mL, 3.23 ng / mL, 3.24 ng / mL, 3.25 ng / mL, 3.26 ng / mL, 3.27 ng / mL, 3.28 ng / mL, 3.29 ng / mL, 3.30 ng / mL, 3.31 ng / mL, 3.32 ng / mL, 3.3 0.7ng / mL, 3.8ng / mL, 3.9ng / mL, 4.0ng / mL, 4.1ng / mL, 4.2ng / mL, 4.4ng / mL, 4.5ng / mL, 4.6ng / mL, 4.7ng / mL, 4.8ng / mL, 4.9ng / mL, 5.0ng / mL, 5.1ng / mL, 5.2ng / mL, 5.3ng / mL, 5.4ng / mL, 5.5ng / mL, 5.6ng / mL, 5.7ng / mL, 5.8ng / mL, 5.9ng / mL, 6.0ng / mL or more, or a range encompassing any of the specifically recited values ​​above, with other values ​​possible depending on the type of assay used to detect and quantify PGRN protein levels.

[0293] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to 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 than the average concentration of endogenous PGRN protein in the CSF of such subject before treatment, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months before treatment.

[0294] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein comprise increasing the concentration of PGRN protein or a variant 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%, for example, about 100 ng, of the average concentration of endogenous PGRN protein in the serum or plasma of a healthy human. ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL, 150 ng / mL, 155 ng / mL, 160 ng / mL, 165 ng / mL, 170 ng / mL, 175 ng / mL or more, or a range encompassing any of the specifically recited values ​​above, with other values ​​possible depending on the type of assay used to detect and quantify PGRN protein levels.

[0295] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to 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 than the average concentration of endogenous PGRN protein in the serum or plasma of such subject before treatment, e.g., 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months before treatment.

[0296] In some embodiments, the methods for treating FTD, FTLD or PGRN deficiency described herein are effective to increase the concentration of PGRN protein or a variant 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 human.

[0297] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to increase the concentration of bis(monoacylglycero)phosphate (BMP) in the brain 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%, or 110% of the average concentration of endogenous BMP in the brain of a healthy human. In some embodiments, the elevated BMP species is BMP 18:1 / 18:1, BMP 22:6 / 22:6, or some other BMP species.

[0298] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to reduce the level of β-hexosaminidase (HexA) enzyme activity in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to up to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the HexA enzyme activity in the brain before treatment.

[0299] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to 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 up to about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the β-Gal enzyme activity in the brain before treatment.

[0300] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to 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 about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the extent or degree of TDP43 fragmentation in the brain before treatment.

[0301] In some embodiments, the methods for treating FTD, FTLD, or PGRN deficiency described herein are effective to reduce the amount of lipofusin in the brain of a human subject in need of treatment for FTD, FTLD, or PGRN deficiency to at most about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% or less of the amount of lipofuscin in the brain before treatment.

[0302] The concentrations of PGRN protein or its variants, including PGRNΔ3, and BMP or lipofuscin in biological fluid or tissue samples from subjects receiving treatment with the AAV vectors of the present disclosure, or from healthy subjects or other controls, can be detected and quantified using any method known in the art, including, but not limited to, ELISA, RIA, or LCMS-MS, or any other method known in the art. The enzymatic activity of HexA or β-Gal in biological fluid or tissue samples from subjects receiving treatment with the AAV vectors of the present disclosure, or from healthy subjects or other controls, can be detected and quantified using any method known in the art, including, but not limited to, fluorogenic substrate enzyme assays or other methods known in the art. TDP43 fragmentation in tissue samples from subjects receiving treatment with the AAV vectors of the present disclosure, or from healthy subjects or other controls, can be detected and quantified using any method known in the art, including, but not limited to, semi-quantitative Western blot analysis or any other method known in the art.

[0303] Prevention methods Among 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, the 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 such prevention. Also provided is the use of an AAV vector of the present disclosure in the manufacture of a medicament for use in the preventive methods disclosed herein. In addition, provided is an AAV vector of the present disclosure or a pharmaceutical composition containing such an AAV vector for use in the preventive methods disclosed herein.

[0304] In some embodiments, administering to a subject in need of prevention of FTD, FTLD, or PGRN deficiency a prophylactically effective amount of an AAV vector of the present disclosure (including, but not limited to, the vector described herein as AAV801-PGRNΔ3), or a pharmaceutical composition containing such an AAV vector, is effective for preventing the onset or development of FTD or FTLD in the subject; effective for preventing the onset or development of any adverse effects due to PGRN deficiency in the subject; effective for preventing the onset or development of a decrease in PGRN amount in the subject; effective for preventing the onset or development of at least one symptom or sign associated with FTD, FTLD, or PGRN deficiency in the subject; effective for preventing the onset or development 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 effective for preventing the onset or development of a decrease in quality of life caused by FTD, FTLD, or PGRN deficiency in the subject, in each case, that would have occurred in the absence of the prevention.

[0305] 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, the presence of which is determined by genotyping prior to 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 well known to those skilled in the art.

[0306] AAV vector compositions, formulations, administration methods, and dosages In addition to AAV vectors, the present disclosure provides compositions comprising such vectors and at least one pharmaceutically acceptable excipient, diluent, or carrier. Such vectors can be used, inter alia, in methods for preventing and treating FTD, FTLD, or PGRN deficiency, as also described herein.

[0307] Compositions comprising the AAV vectors of the present disclosure can be provided as aqueous solutions or suspensions, emulsions, and other forms such as lyophilized cakes. The vector compositions can be formulated using any appropriate diluents and excipients necessary to achieve desired properties, such as pH, ionic strength, tonicity, stability, shelf life, resistance to freeze-thaw cycles, and lyophilization potential, as well as taking into account 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 numerous others known in the art. Compositions comprising the AAV vectors of the present disclosure for use in preventing or treating a disease or disorder in a subject, such as FTD, FTLD, or PGRN deficiency, can be packaged in any suitable form, such as vials or prefilled syringes. In some embodiments, kits are provided that include multiple vials containing a total amount of vector sufficient to achieve the desired total dose to be delivered to a particular subject based on relevant variables, such as the subject's disease severity, weight, sex, etc.

[0308] Compositions comprising the AAV vectors of the present disclosure 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, intra-arterial administration, intralymphatic administration, intraperitoneal administration, intramuscular administration, intraparenchymal administration, intrathecal administration, intraventricular administration, or intracisternal administration, among others.

[0309] Compositions comprising the AAV vectors of the present disclosure may be administered alone without any other type of therapy, or may be administered simultaneously, contemporaneously, or at any suitable dosing interval with standard of care treatment or any other agent, compound, drug, treatment, or therapeutic regimen. In some embodiments, compositions comprising the AAV vectors of the present disclosure may be administered following prophylaxis with immunosuppressants such as steroids or tacrolimus, or other immunosuppressants, or immunosuppressants may be administered subsequently to control any humoral and / or cellular immune response to the gene therapy.

[0310] The vector composition may contain any suitable amount of 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 a subject and / or is expected not to cause any discomfort or undesirable side effects to the subject.

[0311] In connection with the prophylactic and therapeutic methods provided by the present disclosure, AAV vectors and compositions comprising such vectors can be administered at any appropriate dose predicted or determined to be effective in achieving the desired degree of prophylaxis or treatment. In some embodiments, the dose of an AAV vector of the present disclosure for preventing or treating FTD, FTLD, or PGRN deficiency can be quantified and expressed as vectors per kilogram of subject body weight (vg), abbreviated as "vg / kg." In some embodiments, exemplary effective doses of an AAV vector of the present disclosure, including, for example, an AAV vector comprising an AAV801 capsid and a genome comprising the nucleotide sequence of SEQ ID NO: 19 or its reverse complement, include, but are not limited to, at least or about 1 x 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 15vg / kg, or a range of doses between and including any of the doses specifically recited above, and other doses are also possible.

[0312] Other objects, features, and advantages of the present disclosure will be apparent from the foregoing detailed description. However, since various changes, modifications, and equivalents within the concept and scope of the present disclosure will become apparent to those skilled in the art from the detailed description and examples, and fall within the scope of the appended claims, it should be understood that the detailed description and specific examples which follow indicate particular embodiments of the disclosure and are given by way of illustration only.

[0313] Unless otherwise specified, the use of the term "or" with respect to one or more elements of a set of embodiments is equivalent in meaning to "and / or" and does not require them to be mutually exclusive. Unless otherwise specified, explicitly recited numerical ranges also describe ranges whose lower limit is derived from any one lower or upper limit of the explicitly recited ranges, and whose upper limit is derived from any other lower or upper limit of the explicitly recited ranges. Thus, for example, the set of explicitly recited ranges "10 to 20, 20 to 30, 30 to 40, 40 to 50, 100 to 150, 200 to 250, 275 to 300" also describes the ranges 10 to 50, 50 to 100, 100 to 200, and 150 to 250, among many other ranges. Unless otherwise stated, 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 immediately appears thereafter, but also any subsequent values ​​or ranges that appear in the same series or range. Thus, for example, the phrase "about 1, 2, or 3" is equivalent to "about 1, about 2, or about 3."

[0314] All publications and references cited herein, including but not limited to, articles, abstracts, patents, patent applications (published and unpublished), and biological sequences (including but not limited to those 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 claims priority, directly or indirectly, is also hereby incorporated by reference in its entirety.

[0315] Unless otherwise stated, the following examples describe experiments that have been or will be performed using standard techniques that are well known and routine to those skilled in the art. The examples are illustrative. [Example]

[0316] The following examples and figures are included to demonstrate preferred embodiments. Those of skill in the art will recognize that many changes can be made to the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope described herein.

[0317] Example 1 Design and production of AAV vectors expressing progranulin (PGRN) An AAV vector (PGRNDel3 or PGRNΔ3) containing a transgene for expressing human Progranulin lacking the last three carboxy-terminal amino acids was designed, constructed, and produced for in vitro and in vivo testing. In optimization experiments, the expression of various transgenes encoding truncated human PGRN proteins (originally the wild-type amino acid sequence) was compared in transfected Neuro-2A cells. The codon-optimized and CpG-depleted transgenes produced less Progranulin protein compared to the wild-type coding sequence (data not shown); therefore, the latter was used as the transgene.

[0318] The AAV genome, designated Syn-PGRNΔ3 clone 249, contains, from 5' to 3', the 5' ITR from AAV2, a promoter from the human synapsin gene, a 5' untranslated region (UTR) from a primate synapsin I gene transcript (NCBI Reference Sequence XM_034950363.1), the coding sequence for the human progranulin polypeptide (hGRNΔ3) lacking the three carboxy-terminal amino acids, a stop codon, the bovine growth hormone (bGH) gene polyadenylation (polyA) signal sequence (i.e., the transcription terminator), an intron from the human TATA-box binding protein (TBP) gene (NCBI Reference Sequence NG_008165.1), and the 3' ITR from AAV2. The complete sequence of the clone 249 vector is reported as SEQ ID NO: 17 (including both ITRs), and the nominal start and stop nucleotide numbers for each genomic component are listed in Table 1 below. Additional polypeptide and nucleotide sequences related to the vectors described herein are set forth in the sequence listing.

[0319] [Table 1]

[0320] To produce vector particles, HEK293 cells in suspension culture were transfected using a classical triple transfection method. HEK293 cells were expanded from an aliquot of a working cell bank through multiple passages, starting in shake flasks, then through wave bags, and finally into a 250 L-scale single-use bioreactor. Cells were transfected by adding a transfection cocktail containing PEI and three different plasmids: pHelper for expressing AdV helper factors; pRepCap for expressing AAV capsid proteins (variously AAV6, AAV9, AAVDJ, AAVPHP.B, or AAV801, depending on the experiment); and a transgene plasmid. After 3.5 hours of transfection, transfection was stopped by adding CDM4 medium, followed by a 72-hour incubation period to allow the cells to produce AAV vectors. The vector was recovered by lysing the cells with Triton X-100, adding domiphen bromide to aggregate host cell DNA, filtering the supernatant, and then purifying the vector in three steps, including affinity chromatography, anion exchange chromatography, and tangential flow filtration. After recovery and purification, the vector was titered 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 protein (PGRNΔ3) can improve biomarkers associated with GRN haploinsufficiency, which leads to FTD in humans.

[0321] Example 2 In vitro testing of AAV GRN vectors Several reports have shown that carboxy-terminal truncations of the 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). Progranulin binding to sortilin requires the terminal three amino acids of WT PGRN, but not to prosaposin. WT PGRN binds to both human sortilin and prosaposin recombinant proteins. PGRNΔ3 does not bind to sortilin but maintains binding to prosaposin protein, as observed with two different prosaposin sources (Abcam and Mybiosource).

[0322] Experiments were designed to determine whether human PGRNΔ3 could be produced by different types of neurons in vitro after transduction with an AAV vector expressing a GRN transgene under the control of a neuron-specific promoter, compared with full-length human PGRN expressed from an otherwise identical vector. Using standard techniques, spinal motor neurons and cortical glutamatergic neurons were differentiated from human induced pluripotent stem cells (iPSCs) and then transduced with an AAV vector expressing a truncated form of human PGRN under the control of the SYN promoter and a vector expressing full-length human PGRN. Two weeks after differentiation, motor neurons were transduced with the vector at a multiplicity of infection (MOI) of 1E5 using AAVDJ capsids. Because AAVDJ demonstrated poor transduction of glutamatergic neurons, AAV vectors with AAV6 capsids were used instead for experiments testing two doses: an MOI of 1E5 and an MOI of 3E5. As shown in Figure 2A, transduction of motor neurons with AAVDJ vectors resulted in comparable levels of mRNA encoding truncated and full-length PGRN. However, at the protein level, as shown in Figure 2B, motor neurons transduced with the AAVDJ vector expressing PGRNΔ3 had higher levels of PGRN protein in the conditioned medium in which the cells were grown compared with neurons transduced with the vector expressing the full-length protein. A similar pattern was observed when glutamatergic neurons were transduced with the two types of AAV6 vectors. As shown in Figure 3A, these neurons produced comparable levels of mRNA encoding PGRNΔ3 and full-length PGRN, which were dose-responsive and increased with a 3-fold increase in MOI.Similar to what was observed in motor neurons, cells transduced with an AAV6 vector expressing PGRNΔ3 produced significantly higher levels of progranulin measured in the conditioned medium than cells transduced with a similar vector expressing the full-length protein, again in a dose-responsive manner (Figure 3B). While the amounts of truncated progranulin mRNA in the conditioned medium of cultured neurons were comparable to those of the full-length form, these results suggest that the truncated form is more abundant than the full-length form, possibly due to the inability of the truncated protein to bind to sortilin, thereby preventing its reuptake into cells after secretion. In a separate in vitro experiment, transduction of cortical glutamatergic neurons differentiated from iPSCs obtained from a human FTD patient with an AAV6 vector expressing PGRNΔ3 also produced detectable levels of progranulin in the conditioned medium, exceeding those produced by similar neurons from healthy individuals (Figure 3B).

[0323] Collectively, the results from in vitro experiments testing AAVDJ and AAV6 vectors suggest that transducing neurons in vivo by administering an AAV vector expressing PGRNΔ3 to the brain may be more effective than previous attempts relying on a vector expressing full-length Progranulin. We extended these studies by testing whether transducing neurons in vitro with a vector containing a capsid capable of crossing the blood-brain barrier in NHPs could be as effective as AAVDJ and AAV6 vectors. Late-stage neuronal progenitor cells derived from control (WC-30) iPSCs and FTD (ND50017) iPSCs were differentiated into glutamatergic neurons in culture for 14 days, after which the AAV801 vector expressing human PGRNΔ3 from the clone 249 vector or a control AAV801 vector designed to produce luciferase (AAV801-Luc) was added. As shown in Figure 4, the mean levels of endogenous human progranulin in the culture medium of untreated neurons and neurons treated with the control vector AAV801-Luc (MOI 1E6 cells) were 0.69 (SD 0.06) ng / mL and 0.61 (SD 0.09) ng / mL, respectively, whereas the detectable total hPGRN level in neurons transduced with the AAV801-PGRNΔ3 vector (MOI 1E6 cells) was substantially increased to 2.39 (SD 0.23) ng / mL, reflecting the production of endogenous full-length and mutant PGRN by the transduced cells.

[0324] Levels of the lysosomal enzyme β-hexosaminidase (HexA) have been reported to be reduced in neurons derived from FTD patients with GRN haploinsufficiency. We designed an experiment to determine whether expressing PGRN in such neurons via vector transduction could increase the levels of this enzyme. Basal β-hexosaminidase activity in control iPSC-derived neurons averaged 1641.8 (SD 249.7) relative fluorescent units (RFU). As expected, it was lower in neurons differentiated from FTD iPSCs, averaging 790.4 (SD 88.8) RFU (Figure 5). When control and FTD neurons were transduced with AAV801-Luc (MOI 1E6 cells), HexA activity was comparable to that in untreated control cells of the same type, averaging 1606.5 (SD 225.4) RFU and 956.5 (SD 79.2) RFU in control and FTD neurons, respectively. In contrast, transduction of control and FTD neurons with AAV801-PGRNΔ3 (MOI 1E6 cells) increased HexA activity in both neurons by approximately 1.9-fold compared with the control AAV801-Luc vector, to 3055.1 (SD 344.8) RFU in control neurons and 1825 (SD 160.05) in FTD neurons. HexA activity in FTD neurons after transduction with AAV801-PGRNΔ3 was comparable to that in untreated control neurons (1825 RFU and 1642 RFU, respectively), suggesting that the AAV801-PGRNΔ3 vector can increase progranulin levels in FTD neurons sufficiently to restore HexA activity to levels comparable to those typically seen in normal neurons.

[0325] Example 3 In vivo testing of AAV vectors expressing full-length and truncated PGRN Since we demonstrated that Progranulin was present at higher levels than the full-length protein in the conditioned medium of neurons transduced in vitro with an AAV vector expressing PGRNΔ3 and that PGRNΔ3 is bioactive, we designed an experiment to test whether similar results could be demonstrated in vivo. We produced AAV vectors expressing truncated and full-length human PGRN and administered them to mice by different routes of administration, and then measured the levels of human Progranulin expressed by the transduced cells.

[0326] In the first set of experiments, on day P0, AAV9 vectors expressing PGRNΔ3 under the control of the neuron-specific synapsin (SYN) promoter and AAV9 vectors expressing full-length PGRN were administered bilaterally into the brains of neonatal mice via the intracerebroventricular route (ICV) (1.5e10 vg / ventricle). Four weeks later, the animals were sacrificed, and brain tissue, cerebrospinal fluid (CSF), and serum samples were collected and analyzed to measure vector transduction and the level and pattern of PGRN expression. As shown in Figure 6A, 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 the constitutive CMV promoter. Despite comparable levels of transduction, and similar to the in vitro experiments described above, substantially more human PGRN protein was detected in the CSF of mice transduced with the vector expressing truncated PGRNΔ3 compared to full-length (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 synapsin promoter, much lower levels of expressed protein were detected in CSF from test animals administered another similar vector in which the transgene was controlled by the CMV promoter (Figure 6D). In serum from test animals, the pattern was reversed, with the CMV promoter-containing vector producing higher amounts of full-length human PGRN protein. Only background levels of PGRN signal were detected in samples from test animals transduced with a negative control vector expressing a GFP reporter transgene. Brain sections from test animals were also analyzed by immunohistochemistry (IHC) using an antibody specific for human progranulin. No human PGRN signal was detected by IHC in brain sections from animals treated with an AAV9 vector expressing GFP (Figure 7A).In contrast, PGRN staining was detected in brain sections from animals administered AAV9 vectors expressing full-length and truncated human PGRN (Figures 7B and 7C, respectively), although expression of the truncated PGRNΔ3 protein appeared to be stronger and more diffusely distributed throughout the brain compared with full-length PGRN.

[0327] In a second set of experiments, AAV1, AAVDJ, and AAV9 vectors expressing PGRNΔ3, and AAV1, AAVDJ, and AAV9 vectors expressing full-length PGRN, controlled by different promoters, were unilaterally administered via ICV into the brains of 6-month-old mice (5e10 vg / animal). After 3 or 6 months, the test animals were sacrificed and the levels of progranulin protein secreted into the CSF were analyzed. As shown in Figure 8A, both vectors transduced brain tissue in the hemisphere contralateral to the injection site, but vectors using the AAVDJ capsid were approximately 10-fold more efficient at transduction than AAV1 and AAV9. Both vectors expressing human progranulin, whether truncated or full-length, produced detectable levels of protein in the CSF of test animals 3 months after treatment. Although the data were variable, there was a trend toward higher expression of PGRNΔ3 compared with full-length PGRN, a consistent pattern across different capsids used to package the expression cassette and whether the transgene was driven by the neuron-specific synapsin or nonspecific EF1a promoter (Figure 8B). In contrast, another nonspecific promoter, CMV, appeared to be inactive in the brain, even though AAV9 vectors containing it successfully transduced cells. A subset of test animals administered AAV9 vectors demonstrated longer duration of expression, where both truncated and full-length progranulin were detected in the CSF 6 months after treatment at levels comparable to those measured 3 months prior (Figure 8C).

[0328] The above experiments demonstrated that ICV administration of different AAV vectors resulted in detectable expression of truncated and full-length human progranulin in the CSF of treated animals. In a third series of experiments, 6-week-old mice were intravenously (retroorbitally) administered the AAV PHP.B vector (2e13 vg / kg) to express PGRNΔ3 and full-length PGRN under the control of the synapsin promoter. After 4 weeks of treatment, the test animals were sacrificed, and tissues were collected and analyzed to determine whether systemically administered vectors could express proteins in the nervous system. As shown in Figure 9A, both vectors equally transduced brain tissue, and truncated progranulin was present at higher levels in both brain tissue (Figure 9B) and CSF (Figure 9C) compared with full-length PGRN, similar to the results obtained when the vectors were administered via the ICV route.

[0329] The results of the in vivo experiments described above showed that PGRNΔ3 was present at higher levels than full-length PGRN in the brains of mice of different ages that received AAV vectors with different capsids and two different routes. Because vectors expressing the two different forms of PGRN protein transduced brain tissue with similar efficiency, the most likely explanation for this difference is that the truncated protein, unlike full-length progranulin, does not bind to sortilin and therefore remains in the extracellular space after secretion rather than being internalized into cells.

[0330] Example 4 Testing AAV vector-expressed PGRN neurotoxicity in wild-type mice As reported by others, expression of full-length human PGRN protein from an AAV9 vector administered intravenously into the brains of Grn-null mice resulted 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 undesirable side effects may be manageable in humans, for example, by administering immunosuppressants, we designed an experiment to test whether expression of a truncated form of PGRN could reduce or eliminate the neurotoxicity seen with the full-length protein.

[0331] AAV9 vectors expressing human full-length PGRN under the control of the SYN gene promoter and AAV9 vectors expressing PGRNΔ3 (5e10 vg each) were intracerebrally administered intravenously to 6-month-old mice. After 3 months of treatment, the test animals were sacrificed, and brain tissue was collected and analyzed. The histology and cellularity of the hippocampal region in coronal brain slices were assessed by H&E staining, and the levels of human progranulin protein and the mouse microglial marker Iba-1 were detected by immunohistochemistry (IHC). Before sacrifice, no significant differences in body weight or survival rate were observed between animals treated with the PGRN expression vector and negative control animals administered PBS or an AAV9 vector expressing green fluorescent protein (GFP) (data not shown). In the hippocampal region of test animals administered an AAV9 vector expressing GFP, ICH detected normal levels of mouse Iba-1, but not human PGRN protein (Figure 10, bottom left and top left micrographs, respectively). Similarly, normal neuronal cellularity was observed in the hippocampus (Figure 10, right micrograph). In test animals administered an AAV9 vector expressing human full-length PGRN, progranulin protein was readily detectable in the CA3 region of the hippocampus (Figure 11, top left micrograph). However, unlike negative control animals, the same region showed decreased cellularity (Figure 11, right micrograph) and increased signal intensity of the microglial marker Iba-1, which colocalized with PGRN expression (Figure 11, bottom left micrograph). Collectively, these results suggest that expression of full-length human PGRN protein is toxic to hippocampal neurons and stimulates a strong microglial response, consistent with previous reports. Similar results were observed when the full-length human PGRN protein was expressed from a vector using the AAVDJ capsid (results shown here), confirming that the toxicity was most likely due to the expressed protein and not the vector capsid.

[0332] Test animals administered an AAV9 vector expressing human PGRNΔ3 also had detectable levels of protein in the hippocampus, but the expression pattern was more diffuse than that of the full-length form of the same protein (Figure 12, top left micrograph). Surprisingly, and in stark contrast, the hippocampal CA3 region from these same test animals appeared to have normal neuronal cellularity (Figure 12, right micrograph), and normal to slightly increased levels of Iba-1 protein (Figure 12, bottom left micrograph), suggesting the absence of significant microglial activation. Importantly, these results demonstrate that AAV vector-mediated expression of the truncated PGRNΔ3 protein in wild-type mouse brain did not cause the toxicity to hippocampal neurons seen with full-length PGRN, suggesting that expression of the truncated protein via gene therapy may be safer.

[0333] Example 5 Testing AAV GRN vectors in a mouse GRN knockout model A potential advantage of the AAV801 capsid over other capsids is that it has been shown to cross the blood-brain barrier (BBB) ​​in nonhuman primates. However, AAV801 does not do so in mice, which limits the feasibility of testing vectors in its capsid in mouse models of human disease. To circumvent this limitation, we designed experiments to package 249 clone vectors into surrogate AAV9 (AAV9-PGRNΔ3) and AAVPHP.B (AAVPHP.B-PGRNΔ3) capsids for testing in a mouse model of FTD in which endogenous mouse Grn was knocked out (KO). As described further below, Grn null 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.

[0334] Grn - / - KO mice were unilaterally injected with AAV9-PGRNΔ3 at 1e11 vg via the intracerebroventricular route (ICV), and age-matched Grn mice were - / - KO and WT mice were injected with control PBS. All test animals survived until necropsy, and no significant differences in body weight were observed after treatment. Approximately 2 months after injection, tissues (brain and liver) and biological fluids (CSF and serum) were collected for analysis. One hemisphere of the brain was collected for biochemistry, and the other half for immunostaining. Eight right hemispheres and six left hemispheres were collected from mice injected ICV into the right lateral ventricle to compare the distribution of vector genome copies from injected and non-injected brain sites (right hemispheres are indicated by pink dots in Figures 13B, 13C, 13E, and 13G). The vector genome copy numbers (VGCs) 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 (Figures 13A and 13B). The mean VGC in the right hemisphere was 1.02e6 vg / μg gDNA and 9.38e4 vg / μg gDNA, indicating a greater distribution in the injected than non-injected sites (FIG. 13B). VGC in the liver was also high, likely due to leakage into the blood during ICV injection in the test animals.

[0335] PGRNΔ3 transgene mRNA was also detectable in the brain, where it was 57-fold higher overall than endogenous mouse progranulin mRNA transcripts on average, but was 77-fold higher in the right hemisphere (i.e., the injected side) and 31-fold lower in the left hemisphere (Figure 13C). In CSF, human progranulin (PGRNΔ3) protein levels averaged 156 ng / mL, whereas endogenous mouse progranulin concentrations were approximately 2 ng / mL (Figure 13D). In serum, PGRNΔ3 protein levels were low (3.8 ng / mL), likely due to the use of the synapsin promoter, which was 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 similarly, mRNA was 51-fold higher in the right hemisphere compared with 10-fold in the left hemisphere (Figure 13F).

[0336] Grn - / - The brains of KO mice showed a significant deficiency of 18:1 / 18:1 and 22:6 / 22:6 bis(monoacylglycero)phosphate (BMP; also known as lysobisphosphatidic acid) compared to age-matched WT mice (Figures 14A and 14B). BMP is an endolysosomal phospholipid that has been shown to interact 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). In progranulin-deficient conditions, BMP deficiency reflects lysosomal abnormalities. Tissues from test animals were examined to determine whether vector treatment affected BMP levels. In brain tissue from control WT mice, BMP 18:1 / 18:1 levels averaged 433 ng / g, and as expected, vehicle-treated Grn mice exhibited significantly higher BMP levels. - / - The levels were lower in brain tissue from KO mice, averaging 215 ng / g (Fig. 14A). - / -Treatment of KO mice with the AAV9-PGRNΔ3 vector increased brain BMP levels to an average of 583 ng / g (Fig. 11A). Similarly, in WT mice, brain concentrations of BMP 22:6 / 22:6 averaged 3947 ng / g, significantly higher than those of vehicle-treated Grn mice. - / - The mean was 2074 ng / g in the brains of KO mice, and increased to 5074 ng / g in brain tissue from KO mice treated with AAV9-PGRNΔ3 (FIG. 14B).

[0337] Grn - / - The brains of KO mice showed increased levels of two lysosomal enzymes, β-hexosaminidase (HexA) and β-galactosidase (β-Gal; β-galactosidase), compared with age-matched WT control mice (Figures 15A and 15B). Tissues from test animals were examined to determine whether vector 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 vehicle-treated Grn mice showed significantly increased levels of HexA activity. - / - In the brains of KO mice, HexA activity was an average of 55,430 RFU (7,681 SD), and β-Gal activity was an average of 50,078 RFU (12,403 SD). - / - Treatment of KO mice with the AAV9-PGRNΔ3 vector reduced both HexA and β-Gal enzyme activity to control levels. In vector-treated animals, HexA activity was reduced to an average of 35,300 RFU (SD 7,371), comparable to the WT control level of 40,309 RFU (SD 5,296), and β-Gal activity was reduced to an average of 41,299 RFU (SD 7,095), comparable to the WT control level of 38,700 RFU (SD 5,624) (Figures 15A and 15B).

[0338] Aggregation of TDP43 fragments is a postmortem brain marker of FTD caused by GRN haploinsufficiency, and Grn - / -Experiments were designed to test the presence or absence of such fragments in KO mice and whether vector treatment could affect TDP43 fragmentation. Control WT mice, vehicle-treated control Grn mice, and WT mice were also tested. - / - KO mice and Grn treated with AAV9-PGRNΔ3 vector - / - Brain lysates from KO mice were prepared and analyzed by Western blot analysis using an antibody that binds to both full-length TDP43 and the TDP43 fragment, migrating at 43 kDa and 20 kDa, respectively. The staining intensity of both bands was quantified, and the ratio of the staining intensity of the 43 kDa band to the 20 kDa band (a smaller ratio indicates relatively more fragmentation) was calculated. As shown in Figure 16, Western blot analysis showed that PBS-treated Grn - / - The KO mice showed a lower staining ratio of the 43 kDa band to the 20 kDa band compared with age-matched WT controls, indicating that Grn - / - KO mice showed a greater degree of TDP43 fragmentation in the absence of progranulin expression. - / - When KO mice were treated with the AAV9-PGRNΔ3 vector, the staining ratio increased and was comparable to that observed in WT controls, suggesting that vector treatment and restoration of PGRN expression reduced TDP43 fragmentation.

[0339] Grn - / - Another biomarker of FTD in KO mice is the rapid progression of lipofuscinosis, characterized by excessive accumulation of lipofuscin throughout brain regions, which is visualized by autofluorescence staining compared to WT mice. WT mice, as well as treated Grn- / - KO mice and control Grn- / - mice, were also significantly affected. - / - Lipofuscin staining intensity was quantified in KO mice to determine whether this biomarker also responded to treatment with the AAV9-PGRNΔ3 vector. - / -Representative images of lipofuscin staining (shown in red) in the hippocampus and thalamus from KO and vehicle-treated Grn- / - KO mice are shown in Figure 17A. Results from this analysis are shown in Figures 17C-17F, which report lipofuscin levels in different brain regions, moving from lateral to distal across one hemisphere. Lipofuscin levels were low in WT mice and significantly increased in Grn- / - mice treated with PBS vehicle or AAV9-Luc negative control vector. - / - KO mice had higher levels. - / - In contrast to KO mice, when mice were treated with AAV9-PGRNΔ3 by intravenous administration of the vector into the right ventricle, reduced lipofuscin accumulation was observed in all treated animals, tending to approach levels in WT mice, as measured in the whole brain (Fig. 17B), hippocampal region CA2 / 3 (Fig. 17C), whole hippocampus (Fig. 17D), prefrontal cortex (Fig. 17E), and thalamus (Fig. 17F). These data also indicate that vector treatment was more effective in reducing lipofuscin levels in the right hemisphere of the brain, the side where the vector was administered, than in the contralateral left hemisphere.

[0340] These results suggest that intravenous administration of a vector engineered to express PGRNΔ3 is effective in correcting biomarkers associated with FTD in humans. Experiments were designed to test whether similar results could be achieved in mice by intravenous administration of a vector capable of crossing the BBB. The same vector packaged and tested in AAV801 capsids as described above was packaged in AAVPHP.B capsids, which, unlike AAV801, can cross the BBB in mice (but not in NHPs). Grn-null knock-in (KI) mice were intravenously administered the AAVPHP.B-PGRNΔ3 vector at doses of 5e12 and 1e13 vg / kg. Both the liver and brain showed high levels of transduction, as detected by quantifying the number of vectors per microgram of genomic DNA from those tissues (Figures 18A and 18B, respectively) (6.9e5 vg / μg gDNA and 8.2e5 vg / μg gDNA in the brain, 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 PGRNΔ3 protein levels were approximately 57-fold higher than that of endogenous mouse PGRN protein (Figure 18F). PGRNΔ3 protein levels were also elevated in cerebrospinal fluid (CSF) (Figure 18D). Mice receiving the higher vector dose had approximately two-fold higher RNA and protein levels, indicating a dose-responsive therapeutic effect. In serum, PGRNΔ3 protein levels were low (FIG. 18E), likely due to the use of the synapsin promoter, restricting transgene expression to neurons.

[0341] At both doses, brain tissue from Grn-null KI mice treated with the AAVPHP.B-PGRNΔ3 vector showed increased levels of BMP 18:1 / 18:1 (mean 384.1 ng / g at a dose of 5e12 vg / kg) and BMP 22:6 / 22:6 (mean 427.8 ng / g at a dose of 5e12 vg / kg) compared with WT controls (Figures 18G and 18H, respectively), whereas HexA enzyme activity was decreased (Figure 18I). Collectively, these results suggest that intravenous administration of a transgene expressing PGRNΔ3 protein via an AAV capsid capable of crossing the blood-brain barrier can transduce brain cells to express PGRNΔ3 protein at levels sufficient to correct biomarkers associated with FTD in humans.

[0342] Example 6 Testing of AAV GRN vectors in non-human primates A study was conducted to compare the biodistribution of AAV vectors containing 249 clone vectors packaged in AAV801 and AAV9 capsids in cynomolgus monkeys.

[0343] Cynomolgus monkeys were intravenously administered two doses of AAV801-PGRNΔ3 and AAV9-PGRNΔ3, containing the 249 clone vector expressing hPGRNΔ3, at 5e12 vg / kg and 2e13 vg / kg, respectively. Progranulin protein concentrations were measured in serum and CSF samples collected immediately before and up to 28 days after administration, as were brain and other tissue samples tested to quantify vector copy number (VGC) and transgene RNA and protein expression. PGRNΔ3 levels in CSF and serum were detected using a human ligand binding assay (LBA), which does not detect endogenous macaque progranulin.

[0344] PGRNΔ3 protein was detected in the CSF of test animals treated with AAV801-PGRNΔ3 on days 14 and 28 (Figure 19A). At the high dose (2e13 vg / kg), PGRNΔ3 protein levels in two test animals were approximately 50 ng / mL and 20 ng / mL, both of which were higher than the mean value of 6 ng / mL for naturally occurring progranulin in humans. Lower, but still detectable, levels of PGRNΔ3 protein were detected in one of two test animals administered the lower dose of 5e12 vg / kg. In contrast, two test animals administered AAV9-PGRNΔ3 at a dose of 2e13 vg / kg did not express detectable PGRNΔ3 protein. These results suggest that the AAV801 capsid is much more efficient than AAV9 at crossing the BBB to reach the brain and transducing neurons there. Serum concentrations of PGRNΔ3 protein in test animals treated with both the AAV801-PGRNΔ3 and AAV9-PGRNΔ3 vectors were similar (Figure 19B) and substantially lower than the approximately 200 ng / mL naturally present in human serum. In test animals administered a high vector dose (2e13 vg / kg) delivered by either the 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 less than 2 ng / mL. The low serum PGRNΔ3 protein levels are likely due to the use of a neuron-restricted promoter to drive transgene expression in transduced cells.

[0345] Brain and other tissue samples were collected to examine transduction efficiency. Quantification of vector presence confirmed transduction by the AAV801-PGRNΔ3 vector in all brain regions tested and was at least 100-fold higher than transduction by the AAV9-PGRNΔ3 vector at the same dose (2e13 vg / kg) (Figure 20), again demonstrating the superiority of AAV801 over AAV9 in crossing the BBB and transducing the brain, including deep brain structures. The spinal cord was also transduced approximately 10-fold more efficiently with AAV801-PGRNΔ3 than with 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, particularly non-neuronal tissues, the VGC of AAV801-PGRNΔ3 was lower or comparable to that of AAV9-PGRNΔ3. For example, in the liver, which tends to be highly transduced by intravenous administration of multiple capsids, AAV801-PGRNΔ3 produced approximately half the number of VGCs as AAV9-PGRNΔ3. Transduction of the dorsal root ganglion (DRG) and trigeminal ganglia was relatively low, even at the highest dose tested (2e13 vg / kg).

[0346] Consistent with the VGC data, PGRNΔ3 RNA levels produced in test animals treated with AAV801-PGRNΔ3 were 100- to 1000-fold higher than the RNA levels in brain tissue samples collected from test animals administered the AAV9-PGRNΔ3 vector (Figure 21). PGRNΔ3 RNA levels were particularly high in the frontal and temporal cortices (at least 5-fold higher than the levels of the MfHPRT housekeeping gene) and thalamus, compared with other brain regions such as the amygdala or hippocampus, and were also high in the spinal cord. In contrast, PGRNΔ3 RNA levels were low in the liver, despite vector quantification revealing relatively high transduction levels (approximately 0.1 copies of vector per cellular MfHPRT gene copy), again suggesting the effectiveness of using the synapsin promoter in restricting transgene expression to neurons. In the DRG and trigeminal ganglion, PGRNΔ3 RNA levels obtained from AAV801-PGRNΔ3 transduction were comparable to those obtained from AAV9-PGRNΔ3 and were lower than in most brain regions. PGRNΔ3 RNA expression was dose-responsive, but both doses resulted in similarly widespread transgene expression throughout the brain.

[0347] RNA expression from the PGRNΔ3 transgene was also analyzed by in situ hybridization (ISH). As shown in Figures 22A–22G, widespread and strong RNA expression was detected in multiple brain regions affected by FTD, including the motor cortex, entorhinal cortex, hippocampal pyramidal cells, thalamus, and dentate nucleus, from animals treated with AAV801-PGRNΔ3 at a dose of 2e13 vg / kg. A similar pattern was observed in animals treated with the lower dose of 5e12 vg / kg, although the frequency and intensity of neuronal staining was reduced. Substantial RNA expression from the AAV801-PGRNΔ3 vector was also observed in the spinal cord. In animals treated with the AAV9-PGRNΔ3 vector at a dose of 2e13 vg / kg, RNA expression was observed by ISH in the DRG, spinal cord, and trigeminal ganglion, but no positively stained neurons were detected in the brain. Furthermore, no RNA expression was detected by ISH in peripheral sympathetic ganglia, such as paravertebral ganglia or GI tract neurons.

[0348] PGRNΔ3 protein was highly expressed in multiple brain regions from test animals administered 2e13 vg / kg of the AAV801-PGRNΔ3 vector, exceeding the levels of progranulin naturally present in the brains of the test animals (Figure 23). For example, in the frontal cortex, PGRNΔ3 protein levels were approximately 500 ng per gram of brain tissue, which was approximately 27-fold higher than the concentration of endogenous macaque progranulin. In the temporal cortex, PGRNΔ3 protein levels were approximately 30 ng per gram of brain tissue, i.e., approximately two-fold higher than endogenous progranulin and approximately 40-fold higher than macaque progranulin levels in the spinal cord. In the periphery, PGRNΔ3 levels were approximately 10 ng / g DRG tissue, which was lower than endogenous macaque progranulin (approximately 50 ng / g tissue). In several brain regions, including the frontal cortex, thalamus, and spinal cord, PGRNΔ3 protein was detectable 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, in the liver, PGRNΔ3 protein from AAV801-PGRNΔ3 was not detected despite the presence of endogenous progranulin at 100 ng / g tissue, again confirming restricted tissue expression driven by the synapsin promoter. PGRNΔ3 protein expression was also analyzed by immunohistochemistry (ICH) in several brain sections. The staining pattern was consistent with that of transgene mRNA determined using ISH, suggesting that there was no significant uptake of PGRNΔ3 protein in cells that did not express vector mRNA.

[0349] Example 7 Quantification of vector-derived PGRN in NHP samples An immunoaffinity liquid chromatography mass spectrometry (LCMS) assay was developed to quantify the amount of expressed human PGRN protein in tissue or biofluid samples from cynomolgus monkeys treated with an AAV vector to express PGRNΔ3. In this method, tissue or biofluid samples from NHP test animals treated with the PGRNΔ3 vector are mixed with a lysis buffer to release proteins from the cells. A polyclonal antibody specific for PGRN is then used to immunoprecipitate endogenous full-length monkey PGRN and truncated human PGRN forms (e.g., Δ3 and post-translationally modified Δ4) produced as a result of vector treatment. After removal of non-immunoprecipitated proteins, the immunoprecipitated PGRN protein is digested with Staphylococcus aureus V8 GluC protease, which cleaves proteins at the C-terminus of aspartic acid or glutamic acid residues. In both full-length human and full-length cynomolgus monkey PGRN, GluC cleaves between amino acid residues 576 (E) and 577 (A) to release a 17-amino acid C-terminal peptide, whereas GluC cleavage of the Δ3-truncated PGRN releases a 14-amino acid 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.

[0350] Using the immunoaffinity LCMS assay described above, we quantified the amount of truncated PGRN protein in pooled CSF samples from NHP test animals treated with the AAV801-PGRNΔ3 vector as described in Example 6. Unexpectedly, the predominant truncated C-terminal peptide in the samples did not correspond to the 14-amino acid peptide (577-590) generated by digestion of human PGRNΔ3 with GluC, but instead to the 13-amino acid peptide (577-589) thought to be released by digestion of human PGRN with a four-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 expressed human PGRNΔ3 protein, which was then modified, presumably intracellularly before secretion, extracellularly after secretion, or both, to remove the C-terminal arginine (corresponding to position 590 of full-length human PGRN), producing PGRNΔ4 protein as the predominant human truncated PGRN species in the samples.

[0351] [Table 2-1]

[0352] [Table 2-2]

[0353] [Table 2-3]

[0354] [Table 2-4]

[0355] [Table 2-5]

[0356] [Table 2-6]

[0357] Table 2-7

[0358] Table 2-8

Claims

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

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

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

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

5. 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 carboxy-terminal arginine removed and, optionally, 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 variant thereof is a codon-optimized nucleotide sequence.

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

8. 8. The AAV vector of claim 7, wherein the nucleotide sequence encoding the PGRN polypeptide or variant thereof 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 a wild-type nucleotide sequence encoding the PGRN polypeptide or variant thereof.

9. 9. The AAV vector of claim 7 or 8, wherein the wild-type nucleotide sequence encoding the PGRN polypeptide or variant thereof is contained in the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:

15.

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

11. 11. The AAV vector of claims 1-10, wherein the nucleotide sequence encoding the PGRN polypeptide or 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.

12. The AAV vector of claims 1 to 5, wherein the nucleotide sequence encoding the PGRN polypeptide or variant thereof is identical to the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:

15.

13. 13. The AAV vector according to claim 1, comprising at least one AAV inverted terminal repeat (ITR).

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

15. 15. The AAV vector of claim 14, wherein the nucleotide sequence of the ITR has been modified to reduce or eliminate the ability of the ITR to undergo terminal degradation.

16. 15. The AAV vector of claim 14, wherein the nucleotide sequence of the ITR has been modified to reduce or eliminate the ability of the ITR to assist packaging into a capsid.

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

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

19. 19. The AAV vector of claim 18, wherein the AAV2 ITRs are truncated.

20. 14. The AAV vector of claim 13, wherein the ITR comprises 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 or reverse complementary sequence of each of said sequences.

21. 14. The AAV vector of claim 13, further comprising a third AAV ITR.

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

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

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

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

26. 25. 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 of claim 23, wherein the transcriptional regulatory region comprises a promoter sequence and / or an enhancer sequence that is brain tissue-specific or neuronal cell-specific.

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

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

30. The AAV vector of claims 1 to 29, wherein the vector further comprises a 5' untranslated region (UTR) sequence.

31. The vector of claim 30, wherein the 5'UTR sequence is derived from a synapsin gene.

32. 31. 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, variant, or mutant thereof.

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

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

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

36. 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, variant, or mutant 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. 39. The AAV vector of claim 38, wherein the PRE sequence is a woodchuck hepatitis virus PRE sequence 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. 41. The AAV vector of claims 1 to 40, wherein the vector further comprises a stuffer or filler nucleotide sequence of sufficient length so that the total length of the AAV vector, including the ITRs, is about 3.5 to 5.0 kilobases.

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

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

11.

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

45. The vector comprises, in 5' to 3' order: (a) a first AAV2 ITR; (b) a promoter sequence derived from the synapsin gene; and (c) a 5'UTR sequence derived from the synapsin gene; and (d) a nucleotide sequence encoding a human PGRN polypeptide or a variant thereof, operably linked to the promoter sequence; and (e) a transcription termination signal sequence derived from the bovine growth hormone (bGH) gene; and (f) a sequence derived from a TBP gene intron; and (g) a second AAV2 ITR; and The AAV vector according to claims 1 to 5, comprising:

46. 46. ​​The AAV vector of claim 45, wherein the promoter sequence derived from the synapsin gene comprises, consists essentially of, or consists of the nucleotide sequence of SEQ ID NO:6, the 5'UTR sequence derived from the synapsin 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 derived from the bovine growth hormone (bGH) gene comprises the nucleotide sequence of SEQ ID NO:9 or SEQ ID NO:10, and the sequence derived from the TBP gene intron comprises the nucleotide sequence of SEQ ID NO:

11.

47. 47. The AAV vector of claim 45 or 46, wherein each of the first AAV2 ITR and the second AAV2 ITR comprises 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 a complementary or reverse complementary sequence of each of said sequences.

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

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

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

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

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

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

3.

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

2.

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

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

57. 57. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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%, e.g., 6 ng / mL, of the amount of endogenous PGRN polypeptide in CSF of a healthy human.

58. 57. 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 a healthy human brain.

59. 57. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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 human.

60. 57. The method of claim 56, wherein the method is effective to increase the amount of PGRN polypeptide or a variant thereof 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 human.

61. 57. The method of claim 56, wherein the method is effective to increase the amount of bis(monoacylglycero)phosphate (BMP) 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 BMP in a healthy human brain, and the BMP can be any type of BMP, such as BMP 18:1 / 18:1 or BMP 22:6 / 22:

6.

62. 57. The method of claim 56, wherein the method is effective to reduce beta-hexosaminidase (HexA) enzyme activity in the subject's brain to less than at or 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.

63. 57. The method of claim 56, wherein the method is effective to reduce β-galactosidase (β-Gal) enzyme activity in the subject's brain to less than at or 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.

64. 57. The method of claim 56, wherein the method is effective to reduce TDP43 fragmentation in the subject's brain to less than at or 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.

65. 57. The method of claim 56, wherein the method is effective to reduce lipofuscin levels in the subject's brain to less than at or about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% of pre-treatment lipofuscin levels.

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

67. 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. 68. The method of claim 67, wherein the symptom or sign is atrophy in 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. 68. The method of claim 67, wherein the symptom or sign is behavioral changes characteristic of behavioral variant frontotemporal dementia (BV-FTD).

70. 68. The method of claim 67, wherein the symptom or sign is a behavioral change characteristic of non-fluent variant primary progressive aphasia (NFV-PPA).

71. 68. The method of claim 67, wherein the symptoms or signs are characteristic of Parkinsonism or Corticobasal Syndrome (CBS).

72. 72. The method of claims 56-71, wherein the subject has been diagnosed with frontotemporal lobar degeneration (FTLD) or frontotemporal dementia (FTD).

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

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

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

76. 56. Use of an AAV vector according to 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 an AAV vector according to claims 1 to 49.

78. A host cell for producing an AAV vector, comprising the DNA plasmid of claim 77.

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

80. 80. The host cell of claim 78 or 79, wherein the host cell further comprises genes encoding AAV Rep proteins, e.g., contained in a DNA plasmid.

81. 81. The host cell of claims 78-80, wherein the host cell further comprises a gene encoding the AAV VP1 capsid protein, for example contained in a DNA plasmid.

82. 82. The host cell of claims 78 to 81, wherein the host cell further comprises a gene encoding a viral helper factor, for example contained in a DNA plasmid.

83. 83. A method of producing an AAV vector, comprising incubating the host cell of claim 82 under conditions sufficient to allow production of an AAV vector, and purifying the AAV vector produced thereby.

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

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