Ppt1 gene therapy

Abstract

The present invention features PPT1 polypeptides and encoding nucleic acid constructs. Uses of the polypeptides and encoding nucleic acid constructs include producing a PPT1 polypeptide, increasing PPT1 activity in a subject; and treating a PPT1-related disorder, such as CLN1 disease, in the subject.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480031813.9 (22) Application Date 2024.03.19 (30) Priority Data 63 / 491,205 2023.03.20 US 63 / 584,000 2023.09.20 US 63 / 600,153 2023.11.17 US (85) PCT International Application Entering National Phase Date 2025.11.11 (86) PCT International Application Application Data PCT / US2024 / 020571 2024.03.19 (87) PCT International Application Publication Data WO2024 / 196947 EN 2024.09.26 (71) Applicant: Spark Medical Inc. Address: Pennsylvania, USA (72) Inventors: S. Allam, M.G. Bifferi, D. Cohen, J.P. Evans, A. Kativada, J. McBride, E. Ramsburg (74) Patent Agency: Beijing Kunrui Law Firm, 11494 Patent Attorney: Feng Xinqin (51) Int.Cl. A61K 38 / 46 (2006.01) A61K 31 / 711 (2006.01) (54) Invention Title: PPT1 Gene Therapy (57) Abstract: The present invention is characterized by a PPT1 polypeptide and a nucleic acid-encoding construct. Uses of the polypeptide and the nucleic acid-encoding construct include generating a PPT1 polypeptide, increasing PPT1 activity in a subject; and treating a subject with PPT1-related disorders, such as CLN1 disease. Claims 5 pages, Description 120 pages, Sequence Listing (Electronic Publication), Figures 22 pages, CN 121443306 A 2026.01.30 CN 1 21 44 33 06 A 1. A polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion or insertion; and / or (b) the PPT1 amino acid sequence comprises an aspartic acid (D) at its N-terminus substituted with glycine (G), valine (V) or leucine (L); and / or (c) the PPT1 sequence comprises an N-terminal amino acid sequence of leucine-glutamine-histidine-leucine; and / or (d) the nucleic acid sequence comprises a sequence of leucine-glutamine-histidine-leucine. NO: Any PPT1 encoded sequence from 61 to 94 that has at least 85% identity.2. The polynucleotide of claim 1, wherein the PPT1 polypeptide further comprises the signal sequence containing any one of the sequences of SEQ ID NO: 16-27. 3. The polynucleotide of claim 2, wherein the nucleic acid comprises the signal coding sequence of any one of SEQ ID NO: 43-58. 4. The polynucleotide of claim 2, wherein the polypeptide comprises the signal sequence of any one of SEQ ID NO: 16-21 and 24-27. 5. The polynucleotide of claim 4, wherein the signal sequence comprises the sequence of any one of SEQ ID NO: 16 or 19. 6. The polynucleotide of claim 5, wherein the signal sequence comprises the sequence of SEQ ID NO: 16, and the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 43; or the signal peptide comprises the sequence of SEQ ID NO: 19, and the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 50. 7. The polynucleotide of claim 2, wherein the polypeptide comprises the signal sequence of SEQ ID NO: 23. 8. The polynucleotide of claim 7, wherein the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 54. 9. The polynucleotide of any one of claims 1-6, wherein the PPT1 amino acid sequence comprises an N-terminal aspartic acid D substituted with G, V, or L, and the PPT1 amino acid sequence has at least 97% identity with the sequence of SEQ ID NO: 1. 10. The polynucleotide of claim 9, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G. 11. The polynucleotide of any one of claims 1-3, 7, or 8, wherein the PPT1 sequence comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine, and the PPT1 amino acid sequence has at least 97% identity with the sequence of SEQ ID NO: 1. 12. The polynucleotide of claim 11, wherein the PPT1 sequence comprises the sequence of SEQ ID NO: 4. 13. The polynucleotide of any one of claims 1-12, wherein the nucleic acid comprises a sequence having at least 85% identity with any one of SEQ ID NO: 61-94. 14. The polynucleotide of claim 1, wherein the PPT1 polypeptide comprises a sequence having at least 99% identity with any one of SEQ ID NO: 31-42. 15. The polynucleotide of claim 14, wherein the PPT1 polypeptide comprises SEQ ID NO: 31 or SEQ ID NO: 42. (Claims 1 / 5 page 2 CN)16. The polynucleotide of claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G, and the nucleic acid comprises a sequence having at least 85% identity with any one of SEQ ID NO: 107-125 and 168. 17. The polynucleotide of claim 16, wherein the nucleic acid comprises a sequence having at least 95% identity with any one of SEQ ID NO: 107-125 and 168. 18. The polynucleotide of claim 17, wherein the nucleic acid comprises a sequence of any one of SEQ ID NO: 107-125 and 168. 19. The polynucleotide of claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 34, wherein X is G, and the nucleic acid comprises a sequence having 85% identity with any one of SEQ ID NO: 126-140 and 161-167. 20. The polynucleotide of claim 19, wherein the nucleic acid comprises a sequence having 95% identity with any one of SEQ ID NO: 126-140 and 161-167. 21. The polynucleotide of claim 20, wherein the PPT1 coding sequence comprises any one of SEQ ID NO: 126-140 and 161-167. 22. The polynucleotide of claim 1, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 38. 23. A palmitoyl protein thioesterase-1 (PPT1) polypeptide, said PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, wherein: (a) said PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion or insertion; and / or (b) said PPT1 amino acid sequence comprises an N-terminal aspartic acid (D) substituted with glycine (G), valine (V) or leucine (L); and / or (c) said PPT1 sequence comprises an N-terminal amino acid sequence of leucine-glutamine-histidine-leucine. 24. The polypeptide of claim 23, wherein said PPT1 polypeptide further comprises a signal sequence containing the sequence of any one of SEQ ID NO: 16-27. 25. The polypeptide of claim 24, wherein said polypeptide comprises a signal sequence of any one of SEQ ID NO: 16-21 and 24-27. 26. The polypeptide of claim 25, wherein the signal sequence comprises the sequence of any one of SEQ ID NO: 16 or 19.27. The polypeptide of claim 24, wherein the polypeptide comprises the signal sequence of SEQ ID NO: 23. 28. The polypeptide of any one of claims 23-27, wherein the PPT1 amino acid sequence comprises an N-terminal aspartic acid D substituted with G, V, or L, and the PPT1 amino acid sequence has at least 97% identity with the sequence of SEQ ID NO: 1. 29. The polypeptide of claim 28, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G. 30. The polypeptide of claim 23, wherein the PPT1 sequence comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine and the PPT1 amino acid sequence has at least 97% identity with the sequence of SEQ ID NO: 1. 31. The polypeptide of claim 30, wherein the PPT1 sequence comprises SEQ ID NO: 4. Claims 2 / 5 Page 3 CN 121443306 A 32. The polypeptide of claim 23, wherein the PPT1 polypeptide comprises a sequence having at least 99% identity with any one of SEQ ID NO: 31-42. 33. The polypeptide of claim 32, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G; the sequence of SEQ ID NO: 34, wherein X is G; or the sequence of SEQ ID NO: 38. 34. A polynucleotide comprising a nucleic acid sequence encoding a PPT1 polypeptide, wherein the nucleic acid sequence encoding PPT1 encodes the PPT1 polypeptide according to any one of claims 23-33. 35. A polynucleotide comprising two or more exons and one or more introns that together encode the PPT1 polypeptide according to any one of claims 23-34. 36. The polynucleotide of any one of claims 1-22, 34, or 35, wherein the polynucleotide is an expression cassette comprising one or more expression control elements operatively linked to the nucleic acid encoding the PPT1 polypeptide. 37. The polynucleotide of claim 36, wherein the nucleic acid encoding the PPT1 polypeptide is operatively linked to an upstream promoter and a downstream polyadenylation signal. 38. The polynucleotide of claim 36, wherein the expression cassette from 5' to 3' comprises, operatively linked to, the nucleic acid encoding the PPT1 polypeptide: a promoter, a Kozak sequence, the nucleic acid sequence encoding the PPT1 polypeptide, and a polyadenylation signal. 39. The polynucleotide of claim 37 or 38, wherein the promoter comprises, with respect to SEQ ID NO: 5 or 17340. The polynucleotide of any one of claims 37-39, wherein the polyadenylation signal operatively linked to the nucleotide sequence encoding PPT1 comprises a sequence having at least 95% identity with the sequence of SEQ ID NO: 6. 41. The polynucleotide of any one of claims 36-40, wherein the expression cassette comprises a nucleotide sequence having at least 95% identity with the sequence of any one of SEQ ID NO: 141-143, 169, and 170. 42. The polynucleotide of any one of claims 1-22 and 34-41, wherein the polynucleotide is DNA. 43. A recombinant viral vector nucleic acid comprising the polynucleotide of any one of claims 1-22 and 34-42 and 5' and / or 3' viral elements providing viral packaging and replication. 44. The recombinant viral vector nucleic acid of claim 43, wherein the recombinant viral vector nucleic acid is DNA and comprises an adeno-associated virus (AAV) inverted repeat sequence (ITR) flanking the 5' end of the polynucleotide and an AAV ITR flanking the 3' end of the polynucleotide. 45. The recombinant viral vector nucleic acid of claim 44, wherein the recombinant viral vector nucleic acid comprises a 5' ITR and a 3' ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B. 46. The recombinant viral vector nucleic acid of claim 44, wherein the 5' ITR comprises a sequence having at least 95% identity with the sequence of SEQ ID NO: 8, and the 3' ITR comprises a sequence having at least 95% identity with the sequence of SEQ ID NO: 9. 47. The recombinant viral vector nucleic acid according to any one of claims 43-46, wherein the recombinant viral vector nucleic acid further comprises a polyadenylated sequence operatively linked to the 3' ITR. 48. The recombinant viral vector nucleic acid according to any one of claims 43-47, wherein the recombinant viral vector nucleic acid further comprises one or more filler sequences. Claims 3 / 5 pages 4 CN 121443306 A 49. The recombinant viral vector nucleic acid according to any one of claims 43-48, wherein the recombinant viral vector nucleic acid comprises a sequence having at least 95% identity with a sequence of any one of SEQ ID NO: 144-154, 171 and 172. 50. A gene delivery medium comprising a viral or non-viral vector and according to the claims.The polynucleotides described in any one of claims 1-22 and 34-42, or the recombinant viral vector nucleic acid described in any one of claims 43-49. 51. The gene delivery medium according to claim 50, wherein the gene delivery medium is a viral vector. 52. The gene delivery medium according to claim 51, wherein the viral vector is a recombinant AAV vector, a recombinant lentiviral vector, or a recombinant adenovirus vector. 53. The gene delivery medium of claim 52, wherein the viral vector is a recombinant AAV vector, and the recombinant AAV vector comprises a capsid having at least 90% identity with a VP1, VP2, or VP3 sequence having any one of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12, or SEQ ID NO: 15. 54. The gene delivery medium of claim 53, wherein the capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or the capsid comprises VP1 of SEQ ID NO: 12 or SEQ ID NO: 15. 55. The gene delivery medium of claim 54, wherein the capsid comprises VP1 containing the sequence of SEQ ID NO: 12, VP2 containing the sequence of SEQ ID NO: 13, and VP3 containing the sequence of SEQ ID NO: 14. 56. The gene delivery medium of claim 50, wherein the gene delivery medium is the non-viral vector. 57. The gene delivery medium of claim 56, wherein the non-viral vector is a nanoparticle selected from: lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein- or peptide-based nanoparticles, DNA dendritic polymers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. 58. The gene delivery medium of claim 56, wherein the non-viral vector is an LNP or LPNP.59. A pharmaceutical composition comprising a polynucleotide according to any one of claims 1-22 and 34-42, a PPT1 polypeptide according to any one of claims 23-33, a recombinant viral vector nucleic acid according to any one of claims 43-49, or a gene delivery medium according to any one of claims 50-58, and a pharmaceutically acceptable carrier. 60. A method of increasing PPT1 in a subject, the method comprising administering to the subject a polynucleotide according to any one of claims 1-22 and 34-42, a PPT1 polypeptide according to any one of claims 23-33, a recombinant viral vector nucleic acid according to any one of claims 43-49, a gene delivery medium according to any one of claims 50-58, or a pharmaceutical composition according to claim 59. 61. A method of treating neuronal ceroid lipofuscin deposition 1 in a subject, the method comprising administering to the subject a polynucleotide according to any one of claims 1-22 and 34-42, a PPT1 polypeptide according to any one of claims 23-33, a recombinant viral vector nucleic acid according to any one of claims 43-49, a gene delivery medium according to any one of claims 50-58, or a pharmaceutical composition according to claim 59. 62. The method of claim 60 or 61, wherein the administration comprises intraparenchymal, intracisional, or intraventricular administration. 63. The method of claim 62, wherein the administration is intraventricular and results in significant rAAV delivery to at least the frontal cortex, parietal cortex, temporal cortex, occipital cortex, thalamus, cerebellar cortex, hippocampus, corpus callosum, spinal cord, caudate nucleus, choroid plexus, optic chiasm, fornix, periaqueductal gray matter, olfactory bulb, and optic nerve. 64. The method of claim 60 or 61, wherein the administration comprises an initial administration outside the central nervous system (CNS). 65. The method of claim 63, wherein the subject is a sheep. 66. The method of any one of claims 60-62, wherein the administration is systemic. 67. The method of any one of claims 60-63, wherein the subject is a human. 68. An AAV vector genomic plasmid, said AAV vector genomic plasmid comprising recombinant viral vector nucleic acid according to any one of claims 43-49. 69. The AAV genomic plasmid according to claim 68, wherein said plasmid lacks the rep gene and cap gene. 70. A method for producing an rAAV vector, said method comprising culturing an rAAV production vector containing rAAV helper viral activity.The step of producing a cell line, wherein the genome of the producing cell comprises the recombinant viral vector nucleic acid, the rep gene, and the cap gene according to any one of claims 43-49, wherein the rAAV vector is produced. 71. A method of producing an rAAV vector, the method comprising the step of culturing rAAV-permitting cells comprising an AAV genome plasmid according to claim 68 or 69, wherein the rAAV-permitting cells further comprise (a) a rep gene and a cap gene provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper viral activity provided by the cell genome and / or provided by one or more separate plasmids. 72. The method of claim 71, wherein the rAAV-permitting cell is a packaging cell, wherein the packaged genome comprises a cap gene and a rep gene. 73. The method of claim 71, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the helper activity is provided by a helper plasmid. 74. A method for obtaining an rAAV vector, the method comprising the steps of: (a) generating the rAAV using the method of any one of claims 70-73 and (b) purifying the rAAV. Claims 5 / 5 Page 6 CN 121443306 A Cross-Reference to PPT1 Gene Therapy Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 491,205, filed March 20, 2023; U.S. Provisional Application No. 63 / 584,000, filed September 20, 2023; and U.S. Provisional Application No. 63 / 600,153, filed November 17, 2023, each of which is hereby incorporated herein by reference in its entirety. Reference to the electronically submitted sequence listing

[0002] The contents of the electronic sequence listing (065830.23WO1.xml; size: 315,901 bytes; creation date: March 8, 2024) are incorporated herein by reference in their entirety. Background Art

[0003] Palmitoyl protein thioesterase 1 (PPT1) is a glycoprotein involved in the removal of thioester-linked fatty acyl groups (such as palmitate esters) from proteins. Full-length PPT1 contains 306 amino acids and includes a 27-amino acid signal sequence. Removal of the signal sequence produces mature PPT1. (Bellizzi et al., (2000) PNAS 97:9 4573-4578.)

[0004] PPT1 is found in lysosomes, where it contributes to the catabolism of lipid-modified proteins. PPT1 is also found inIn other locations, such as synaptic bodies, synaptic vesicles, blood, and cerebrospinal fluid (CSF). PPT1 is encoded by the CLN1 gene.

[0005] Neuronal ceroid lipofuscin deposition disease 1 (NCL1) is a progressive neurodegenerative disease caused by reduced PPT1 activity. NCL1 can be caused by mutations that result in the elimination or reduction of PPT1 activity or expression, leading to a lack of lysosomal PPT1 activity. As the disease progresses, symptoms can include epilepsy, seizures, motor and cognitive decline, visual disturbances, behavioral disorders, sleep disturbances, and death. NCL1 is also known as CLN1, Batten disease, or infantile neuronal ceroid lipofuscin deposition disease (INCL), and symptoms typically begin around 12–18 months of age. In some cases, mutations in PPT1 can lead to late-onset symptoms, such as late infancy (around 2–4 years of age), adolescence, or adulthood. NCL1 symptoms can occur in various locations, including the brain and spinal cord. (Gorenberg et al., (2022) PLos Biol. 20(3): e3001590; Simonati and Williams (2022) Front. Neurol. Mar 11;13:811686; Shying et al., (2017) PNAS 114 (29) E5920-E5929; and Bellizzi et al., (2000) PNAS 97(9):4573-4578.)

[0006] References mentioning potential enzyme replacement therapy and gene therapy for NCL1 include Nelvagal et al., (2022) J. Clin. Invest. 132(20);e163107; Griffey et al., (2004) Neurobiology of Disease 16:360-369; Griffey et al., (2006) Molecular Therapy 13(3):538-547; Shyng et al., (2017) PNAS 114 (29) E5920-E5929; International Patent Publication No. WO 2017 / 219450; and International Patent Publication No. WO 2020 / 223322. Summary of the Invention

[0007] The present invention is characterized by a PPT1 polypeptide and a nucleic acid encoding construct. The PPT1 polypeptide described herein includes a polypeptide comprising a PPT1 amino acid sequence and, in various embodiments, further comprising a signal sequence, wherein either or both of the mature PPT1 or the signal sequence are modified relative to the sequence present in full-length naturally occurring human PPT1. The PPT1 encoding construct comprises a nucleic acid sequence encoding a PPT1 polypeptide, the PPT1 polypeptide being similar to that found in naturally occurring human PPT1.The PPT1 construct has one or more differences compared to a mature or full-length PPT1 sequence, and / or the construct contains a sequence with reduced CpG. Uses of the peptide and the encoding nucleic acid construct include generating a PPT1 peptide; increasing PPT1 activity in a subject; and treating PPT1-related disorders, such as CLN1 disease, in a subject.

[0008] Reference to the PPT1 peptide indicates the presence of a sequence associated with the mature sequence, and includes either a full-length sequence containing a signal sequence or a mature sequence not containing a signal sequence, or both. The size of the mature sequence may vary depending on the signal sequence and may be further processed. The PPT1 peptide sequence may be naturally occurring or a modification of the naturally occurring sequence. Reference to the full-length naturally occurring human PPT1 indicates SEQ ID NO: 29. The mature naturally occurring human PPT1 sequence is provided by SEQ ID NO: 1.

[0009] Therefore, a first aspect of the present invention describes a polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion or insertion; and / or (b) the PPT1 amino acid sequence comprises an aspartic acid (D) at its N-terminus substituted with glycine (G), valine (V) or leucine (L); and / or (c) the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) the nucleic acid sequence comprises a PPT1 coding sequence having at least 85% identity with any one of SEQ ID NO: 61-94.

[0010] The signal sequences of SEQ ID NO: 16-27 provide signal sequences not present in naturally occurring full-length human PPT1.

[0011] The substitution of the aspartic acid (D) at its amino terminus with glycine (G), valine (V), or leucine (L) indicates that glycine (G), valine (V), or leucine (L) is present at the position corresponding to the aspartic acid (D) in the naturally mature PPT1 sequence (SEQ ID NO: 1).

[0012] Another aspect of the invention describes a PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a signal sequence thereof.There is a variant with an amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence contains an amino-terminal aspartic acid (D) substituted with glycine (G), valine (V), or leucine (L); and / or (c) the PPT1 sequence contains an N-terminal amino acid sequence of leucine-glutamine-histidine-leucine.

[0013] Another aspect of the invention relates to an expression cassette comprising a nucleic acid sequence encoding a PPT1 polypeptide and one or more expression control elements operatively coupled to the encoding nucleic acid sequence.

[0014] The reference to one or more expression control elements “operatively linked” or “operatively coupled” to the nucleic acid encoding the PPT1 polypeptide indicates that the one or more expression control elements affect PPT1 polypeptide expression. PPT1 polypeptide expression can be affected in various ways, such as increased production of PPT1 polypeptide mRNA transcripts, increased nuclear transport and stability of mRNA transcripts, and increased mRNA translation.

[0015] Another aspect of the present invention relates to a recombinant viral vector nucleic acid comprising (a) an expression cassette comprising a nucleic acid sequence encoding a PPT1 polypeptide and one or more expression control elements operatively linked to the encoding nucleic acid sequence, and (b) 5' and / or 3' viral elements providing viral packaging and / or replication. Specification 2 / 120 pages 8 CN 121443306 A

[0016] Another aspect of the present invention relates to a delivery medium comprising a viral or non-viral vector and (a) a PPT1 polypeptide or (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid comprising a sequence encoding a PPT1 polypeptide.

[0017] Another aspect of the present invention relates to a pharmaceutical composition comprising (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid comprising a sequence encoding a PPT1 polypeptide; or (c) a delivery medium comprising (a) or (b); and a pharmaceutically acceptable carrier.

[0018] Another aspect of the invention relates to a method of increasing PPT1 activity in a subject, treating PPT1 disease or disorder, or treating CLN1, said method comprising administering: (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid comprising a sequence encoding the PPT1 polypeptide; (c) a delivery medium comprising (a) or (b); or (d) a pharmaceutical composition comprising (a), (b), or (c) and a pharmaceutically acceptable carrier.

[0019] A further aspect of the invention comprises: (a) a PPT1 polypeptide; (b) a polynucleotide, expression cassette, or recombinant viral vector nucleic acid comprising a sequence encoding the PPT1 polypeptide; (c) a delivery medium comprising (a) or (b); or (d)Pharmaceutical compositions comprising (a), (b), or (c) and a pharmaceutically acceptable carrier; (a), (b), (c), or (d) for use in a medicament to increase PPT1 activity in a subject, treat PPT1 disease or disorder, or treat CLN1; and (a), (b), (c), or (d) in the preparation of a medicament (e.g., for use in a medicament to increase PPT1 activity in a subject, treat PPT1-related disorder, or treat CLN1).

[0020] Further aspects of the invention include vector genomic plasmids containing recombinant viral vector nucleic acids encoding a PPT1 polypeptide, methods for producing recombinant viral vector nucleic acids encoding a PPT1 polypeptide, and methods for obtaining a PPT1 polypeptide.

[0021] Other features and advantages of the invention will be understood from the additional description provided herein, including different embodiments. The provided embodiments illustrate different components and methods useful in practicing the invention. Such embodiments do not limit the claimed invention. Based on this disclosure, those skilled in the art can identify and employ other components and methods useful in practicing the invention.

[0022] Figure 1 is a schematic diagram of the rAAV PPT1 expression construct (or expression cassette), which contains different nucleic acid regions: 5'-inverted terminal repeat (ITR), elongation factor-1α (EF-1α) promoter, Kozak sequence (Kozak), signal sequence (SS, also known as signal peptide), mature PPT1 sequence (human PPT1), bovine growth hormone polyadenylation sequence (bGH-pA), filler (filler sequence), synthetic polyadenylation sequence (synthetic pA), and 3'-ITR.

[0023] Figures 2A and 2B are bar graphs showing the PPT1 enzyme activity levels in PPT1 knockout HeLa cells transfected with rAAV plasmids carrying different engineered human PPT1 constructs. PPT1 activity is expressed as a percentage of PPT1 activity relative to PPT1 with its native signal peptide in total cell lysate (Figure 2A) and in cell culture supernatant (secreted PPT1) (Figure 2B). The term "inactivated" indicates a mutant PPT1 lacking enzyme activity.

[0024] Figures 3A-3C show the rAAV PPT1 nucleic acid / tdTomato reporter plasmid and the results of evaluating PPT1 secretion in transfected cells using said plasmid. Figure 3A shows the different rAAV nucleic acid regions and tdTomato regions. Figure 3B provides a bar graph showing the ratio of the number of cells with PPT1 to the number of cells transfected with the PPT1 / tdTomato reporter plasmid. Figure 3C provides a bar graph showing the results from Figure 3B normalized relative to the native construct. For both Figures 3B and 3C, each point (transparent circle; average of 30 fields of view) is an independent transfection (from 2-3 experiments), and the values ​​are averaged.Mean + SD. P-values ​​for one-way ANOVA, **** p < 0.0001, *** p < 0.001, ** p < 0.01.

[0025] Figure 4 shows the dose- and time-dependent increase in PPT1 activity in the culture medium of rat primary cortical neurons transduced with rAAV containing Sp7-F.PPT1. Neurons were transduced with three infection multiples (MOIs) (low, 1E+5; medium, 5E+5; and high, 1E+6), and PPT1 activity in the culture medium was analyzed at 3, 4, or 6 days after transduction. “Inactivated” PPT1 refers to PPT1 without catalytic activity. Untreated or diluted cells were used as negative controls. Purified recombinant PPT1 was used as a positive control for the assay. Each circle represents the value from cells independently transduced from a single well. Data are mean ± SD.

[0026] Figures 5A, 5B, and 5C show serum PPT1 expression and activity in mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid at different time points. Figure 5A shows the production of glycosylated PPT1 activity in the serum of mice administered intravenously (IV). Figure 5B shows serum PPT1 activity in IV-administered mice. Figure 5C shows serum activity in mice administered IPa (delivered to the hippocampus via stereotactic injection into the brain parenchyma). ND, not detected. Data are mean ± SD. P values ​​for ANOVA: *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0027] Figure 6 shows liver PPT1 activity in mice IV-administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Mice injected with the diluent were used as negative controls.

[0028] Figure 7 shows an immunohistochemical analysis demonstrating increased PPT1 staining in the mouse hippocampus (indicated by a black asterisk) after administration of rAAV containing Sp7-F.PPT1 viral vector nucleic acid to the hippocampus.

[0029] Figure 8 provides an automated capillary-based immunoassay (JESS, ProteinSimple) analysis showing glycosylation of overexpressed PPT1 in the mouse brain. Recombinant AAV or a diluent containing Sp7-F.PPT1 viral vector nucleic acid was administered to the hippocampus (four mice per group).

[0030] Figure 9 provides an automated capillary-based immunoassay (JESS, ProteinSimple) of untreated (-) or deglycosylated (+) hippocampal protein extracts from two different mice after administration of rAAV containing Sp7-F.PPT1 viral vector to the hippocampus. The decrease in molecular weight after deglycosylation treatment (lanes marked with +) indicates glycosylation of PPT1.

[0031] Figure 10 shows the PPT1 activity assay in mouse hippocampal lysates, illustrating PPT1 expression in the mouse brain. Recombinant AAV containing Sp7-F.PPT1 viral vector nucleic acid was administered to the hippocampus.

[0032] Figure 11 is a bar graph showing the vector genome copy number (VGCN) as an indicator of viral transduction in different brain regions. Total DNA was isolated from frozen tissue, and VGCN was quantified by quantitative PCR (qPCR) using a standard curve. Each circle represents data from one mouse. The height of each bar indicates the mean. Hpc, hippocampus; Br. Stem, brainstem; crblm, cerebellum; Cerv, cervical spinal cord; Thor, thoracic spinal cord, lumb; lumbar spinal cord. Mice administered with the diluent were used as negative controls.

[0033] Figure 12 is a bar graph showing the fold change (FC) of PPT1 activity in different brain and spinal cord regions of animals injected with rAAV relative to mice injected with the diluent. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. PPT1 activity was determined using a standard curve (prepared using 4 MU at known concentrations). FC activity was calculated relative to the mean activity of animals injected with the diluent. Each circle represents the result for one mouse. Data are presented as mean ± SD. One-way ANOVA, Tukey test, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0034] Figure 13 is a bar graph showing the FC of PPT1 activity in cerebrospinal fluid (CSF) of mice that underwent rAAV injection compared to mice injected with the diluent. FC is the mean activity level relative to the diluent group. Each data point on the graph represents data from a single mouse. Data are presented as mean ± SD. Statistical analysis involved one-way ANOVA and subsequent Tukey test. *P < 0.05.

[0035] Figure 14 is a scatter plot showing the correlation between VGCN and PPT1 enzyme activity in the brain. Spearman correlation data 4 / 120 pages 10 CN 121443306 A Number r = 0.066, p < 0.0001. Each circle represents data from brain regions of mice administered rAAV.

[0036] Figure 15 shows the detection of glycosylated and deglycosylated PPT1 protein in brain lysates analyzed by JESS. Protein extracts from brain lysates of the cortex of two independent mice (mice 1: lanes 1 and 3; mice 2: lanes 2 and 4) administered rAAV containing Sp7-F. PPT1 were treated with deglycosylated enzymes and analyzed by JESS assay. In lanes 1-2The observed double bands indicate two glycosylation forms of PPT1. The decrease in molecular weight after deglycosylation enzyme treatment (lanes 3-4) indicates that PPT1 is glycosylated.

[0037] Figures 16A-16H are bar graphs depicting vector genome copy number (VGCN) as a measure of viral transduction in different regions: cortical regions (Figure 16A); thalamus (Figure 16B); cerebellar cortex (Figure 16C); hippocampus (Figure 16D); corpus callosum (Figure 16E); other indicated brain regions (Figure 16F); spinal cord (Figure 16G); and liver (Figure 16H). VGCN from frozen tissue DNA was quantified using quantitative PCR (qPCR) with a standard curve. Each data point represents a single sheep. Vertical arrows indicate samples from the contralateral brain region (compared to the injection side). Hollow circles correspond to sheep administered rAAV containing Sp7-F.PPT1, while solid circles depict results from sheep administered with a vector expressing GFP. Data are provided for the following regions: frontal cortex (FC), motor cortex (MC), somatosensory cortex (SSC), piriform cortex (PC), superior lateral gyrus (SSG), external lateral sulcus (EcG), internal lateral sulcus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaqueductal gray matter (Periaq G), olfactory bulb (Ol), optic nerve (Op nerve), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), cervical spinal cord (SC_Cer), thoracic spinal cord (SC_Tho), and lumbar spinal cord (SC_Lum).

[0038] Figures 17A-17D are bar graphs showing functional PPT1 expression and secretion in the brain and CSF of sheep injected with rAAV containing Sp7-F.PPT1 (n = 4 sheep) or GFP (n = 2 sheep). Figure 17A is a bar graph showing PPT1 activity in the cortex. Figure 17B is a bar graph showing PPT1 activity in the thalamus. Figure 17C is a bar graph showing PPT1 activity in the cerebellar cortex. Figure 17D is a bar graph showing PPT1 activity in the caudate nucleus. PPT1 activity in tissue lysates was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. Each circle represents the result of a tissue perforation sample from a brain region. N represents the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ± SEM. Statistical analysis was performed using the Mann-Whitney U test, *P < 0.05, ***P < 0.001.

[0039] Figure 18 provides 95% confidence intervals, showing the mean PPT1 activity of the overall treatment group. (Figure 17A-Figure 18 are provided.)Log-transformed activity results for all data in 17D and, after considering brain region differences in the number of perforated samples and mean values, hypothesis testing for differences in mean treatment type was performed at a 0.05 α level. The mean estimated fold change of PPT1 relative to GFP was 3.9, and the median of PPT1 was 73 nmol / mg / h, compared to a median of 19 nmol / mg / h for GFP. **** P < 0.0001, weighted two-way ANOVA.

[0040] Figure 19 is a bar chart showing the percentage change in PPT1 activity in the cerebrospinal fluid (CSF) of sheep administered rAAV carrying Sp7-F.PPT1 (n = 4 sheep) or GFP (n = 2 sheep). The percentage change is the mean activity relative to the control (GFP animals). Each circle represents one animal. N represents the number of animals. Data are mean ± SEM.

[0041] Figure 20 shows the results of the JESS assay, which detected recombinant PPT1 expression in tissue lysates of sheep spinal cord injected with rAAV vectors carrying PPT1 (sheep 1-4) or GFP (sheep 1-2). The first lane shows the position of the molecular weight standard (std). Although protein bands that may represent sheep PPT1 were observed in all subjects, bands of human PPT1 were identifiable only in animals treated with vectors encoding human PPT1. Abbreviations C, T, and L represent the cervical, thoracic, and lumbar segments of the spinal cord, respectively. KDa, kilodaltons.

[0042] Figure 21 shows the results, which show the mean increase in PPT1 activity levels in the thoracic and lumbar segments of sheep spinal cord administered with vectors carrying PPT1. Each circle represents the result from an animal spinal cord segment. Data are mean ± SEM.

[0043] Figure 22 is a bar graph showing rotarod evaluation of PPT1 knockout (KO) mice administered rAVV (1E+11 vg / animal) containing Sp7-F.PPT1(1) or SPARC.PPT1(2). KO mice were administered rAAV via bilateral ICV injection on day 1 after birth and evaluated at 7 months of age. Untreated (Un) or KO mice treated with the vector (Veh) served as negative controls. Natural refers to the rAAV vector encoding natural human PPT1. Each circle represents one mouse. These bars are mean + SEM. One-way ANOVA, Tukey post-hoc test, *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0044] Figure 23 shows the improvement in motor function of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 in Ppt1- / - mice (KO).The ability to coordinate and balance, as assessed by the latency of falling from the accelerator bar. These values ​​are mean + SEM. One-way ANOVA, Tukey post-hoc test. # # # # p < 0.0001, WT, 7 mo vs KO, Un, 7 mo; KO, Veh, 7 mo. **** p < 0.0001, KO, Veh, 7 mo vs all dosing groups except rAAV-Sp7-F .PPT1, hi, 9 mo. ** p < 0.005, KO, Veh, 7 mo vs rAAV-Sp7-F .PPT1, hi, 9 months. AAV = adeno-associated virus; ANOVA = analysis of variance; CNS - central nervous system; lo = low dose (1 x 10¹¹ vg / animal); hi = high dose (3.82 x 10¹¹ vg / animal); KO = Ppt1- / -; mo = month; PND = days after birth; sec = second; SEM = standard error of the mean; Un = untreated; Veh = vector; WT = wild type.

[0045] Figure 24 shows the effects of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 on grip strength in Ppt1- / - mice (KO). These values ​​are mean + SEM. One-way ANOVA, Tukey post-hoc test. # # # # p < 0.0001, WT, 7 mo vs KO, Un, 7 mo; KO, Veh, 7 mo. **** p < 0.0001, KO, Veh, 7 mo vs all treatment groups. AAV = Adeno-associated virus; ANOVA = Analysis of variance; CNS - Central nervous system; lo = Low dose (1 x 10¹¹ vg / animal); hi = High dose (3.82 x 10¹¹ vg / animal); KO = Ppt1 - / -; mo = Month; PND = Days after birth; sec = Second; SEM = Standard error of the mean; Un = Untreated; Veh = Vector; WT = Wild type.

[0046] Figures 25A and 25B show serum PPT1 activity in mice administered rAAV-Sp7-F .PPT1 or rAAV-SPARC .PPT1. Figure 25A shows the activity at different time points. Figure 25B shows the activity at 8 months. The number of mice in each group ranged from 11 to 18. The sex distribution within each group was roughly balanced. Each circle represents the result from one mouse. Each point in Figure 25A and each bar in Figure 25B shows the mean + SEM. One-way ANOVA and Tukey post-hoc test were performed on the transformed Log10 values. # # # # p < 0.0001, WT relative to KO, Un; WT relative to KO Veh; **p < 0.01, KO, Veh relative to natural, lo; ***p < 0.001, KO, Veh relative to rAAV-SPARC.PPT1, lo; ***p < 0.0001, KO, Veh relative to rAAV-Sp7-F .PPT1, lo; natural, hi; rAAV-Sp7-F .PPT1, hi; rAAV-SPARC.PPT1, hi. AAV = adeno-associated virus; ANOVA = analysis of variance; lo = low dose (1 x 10¹¹ vg / animal); hi = high dose (3.82 x 10¹¹ vg / animal); KO = Ppt1- / -; mo = month; LLOQ = lower limit of quantitation; PND = days after birth; sec = second; SD = standard deviation; Un = untreated; Veh = vector; WT = wild type.

[0047] Figures 26A-26C provide bar graphs showing PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C) of Ppt1- / - mice injected with rAAV-Sp7-F.PPT1 or rAAV-SPARC.PPT1. PND1 was administered to mice via bilateral intraventricular injection. “Natural” refers to an AAV vector containing the unmodified human PPT1 gene. These bars are mean ± SEM. Each circle represents a mouse.

[0048] Figure 27 is a schematic diagram showing the design of the rAAV nucleic acid present in the plasmid. Expression of the human PPT1 sequence is driven by a longer form of the EF1a promoter (SEQ ID NO: 173). SS = signal sequence.

[0049] Figures 28A and 28B are bar graphs depicting PPT1 expressed in HeLa cells transfected with different AAV plasmids carrying codon-optimized PPT1 sequences. Different codon-optimized PPT1 cDNA constructs (excluding the signal sequence, labeled CO and the numbers on the x-axis thereafter) are paired with the signal sequence. Figure 28A depicts Sp7F or SP7F (codon-optimized). Figure 28B depicts SpSPARC or SpSPARC (codon-optimized).

[0050] Figure 29 provides a survival curve demonstrating the ability of CNS-targeting AAV gene therapy delivering functional human PPT1 to prolong survival in Ppt1- / - mice.

[0051] Figure 30 is a bar graph showing the effect of rAAV containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 on brain weight in Ppt1- / - mice. Mean +SD. ***P < 0.0001, one-way ANOVA, Tukey post-hoc test. AAV = adeno-associated virus; CNS - central nervous system; lo = low dose (1 x 10¹¹ vg / animal); hi = high dose (3.82 x 10¹¹ vg / animal); KO = Ppt1 - / -; PND = days after birth; Un = untreated; Veh = vector; WT = wild type. Detailed Description

[0052] The present invention is characterized by a PPT1 polypeptide and a nucleic acid construct encoding the PPT1 polypeptide. The polypeptide and the nucleic acid construct encoding the polypeptide can be used, for example, to generate the PPT1 polypeptide, increase PPT1 activity in a subject, and / or treat PPT1-related diseases or disorders, such as CLN1.

[0053] The polynucleotide encoding the PPT1 polypeptide can be delivered to the subject by non-viral or viral delivery. Viral vectors that can be used include retroviral vectors, adenoviral vectors, AAV vectors, and herpes simplex virus vectors. Non-viral delivery includes naked DNA and the use of nanoparticles.

[0054] The term "subject" refers to mammals, such as humans; non-human primates, such as apes, gibbons, gorillas, chimpanzees, orangutans, and macaques; domesticated animals, such as dogs and cats; livestock, such as poultry, ducks, horses, cattle, goats, sheep, and pigs; and laboratory animals, such as mice, rats, rabbits, sheep, and guinea pigs. Humans are preferred subjects.

[0055] In some embodiments, sheep models are used to evaluate the expression and efficacy of the PPT1 peptide or nucleic acid constructs encoding the PPT1 peptide. (See, for example, Nelvagal et al., (2022) J. Clin. Invest. 132(20); e163107, which is hereby incorporated herein by reference in its entirety; and the Implementations section provided below.)

[0056] References to the indicated percentage of identity with one or more reference sequences and similar wording provided throughout the specification regarding the indicated percentage of identity with one or more reference sequences provide an indicated percentage of identity or a range of identity percentages independently of each of the reference sequences. In determining the percentage of identity of a polynucleotide, RNA and the corresponding DNA are considered to be identical unless the context in which the polynucleotide is used indicates otherwise, for example, that the polynucleotide is referred to as RNA or DNA. The corresponding RNA and DNA include uracil replaced by thymine and the ribose backbone replaced by the deoxyribose backbone.

[0057] References to “identical,” “percentage of identity,” and similar terms are in relation to two sequences having the maximum alignment in a particular region. The region provided is in relation to the indicated reference sequence. For example, the sequence “identical” or “identical” to the PPT1 polypeptide of SEQ ID NO: 1 can be calculated by determining the number of identical amino acids in the compared sequence and dividing by 1 / 2.The total number of amino acids in SEQ ID NO: 1 (279 amino acids) multiplied by 100. The percentage of “identity” or “identity” of a nucleic acid sequence can be determined in a similar manner, wherein the nucleotides are compared with a reference sequence, taking into account nucleotide differences and vacancies to achieve maximum alignment, divided by the total number of nucleotides in the reference sequence and multiplied by 100.

[0058] The percentage of identity or identification of a PPT1 coding sequence containing two or more exons is determined independently of any introns. For calculation purposes, one or more introns are removed before alignment.

[0059] When determining sequence identity with a reference sequence having one or more indicated variants, the specific variant selected for determining sequence identity is the variant that provides the maximum sequence identity. For example, SEQ ID NO: 2 provides that X is G, V, or L; and for the purpose of determining sequence identity with SEQ ID NO: 2, the X selected for determining sequence identity is the X that provides the maximum sequence identity.

[0060] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acids and oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). When discussing nucleic acids, the sequence or structure of a particular polynucleotide may be described herein according to the convention of providing sequences in the 5' to 3' orientation.

[0061] In some embodiments, nucleic acids include genomic DNA, cDNA, antisense DNA / RNA, plasmid DNA, linear DNA (polynucleotides and oligonucleotides), chromosomal DNA, spliced ​​or unspliced ​​mRNA, rRNA, tRNA, repressive DNA or RNA (RNAi, such as small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-spliced ​​RNA, or antisense RNA), locked nucleic acid analogs (LNA), single-stranded and double-stranded oligonucleotide DNA (ODN), immunostimulatory sequences (ISS), riboswitch, and ribozymes.

[0062] In some embodiments, nucleic acids include naturally occurring, synthetic, and intentionally modified or altered polynucleotides. Nucleic acids can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can have different lengths.

[0063] According to some embodiments, polynucleotides are single-stranded (ssDNA) or double-stranded DNA (dsDNA) molecules. According to some embodiments, dsDNA molecules are microcircles, nanoparticles, open linear double-stranded DNA, or closed-end linear double-stranded DNA (CELiD / ceDNA / doggybone DNA). According to some embodiments, ssDNA molecules are closed circular or open linear DNA.

[0064] “Transgenic” refers to nucleic acids that are intended or have been introduced into cells and are operatively linked to a promoter. Transgenic packagesThis includes heterologous polynucleotide sequences, such as nucleic acids encoding the PPT1 polypeptide and heterologous promoters.

[0065] Some embodiments involve “CpG reduced” or “CpG depleted”. “CpG reduced” or “CpG depleted” means (i) a nucleotide sequence in which one or more CpG dinucleotides (or motifs) have been removed from a reference nucleic acid sequence; and / or (ii) the percentage of CpG in the mentioned polynucleotide is 0% to 15%. In different implementations, the CpG percentage is 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% CpG; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpG.

[0066] CpG motifs may be reduced or eliminated in a suitable manner in the nucleotide sequence encoding the PPT1 protein and in other sequences that may be present in specific constructs (e.g., expression cassettes and viral vectors). Other possible sequences include non-coding sequences such as 5' and 3' untranslated regions (UTRs), filler sequences, promoters, enhancers, polyadenylation signals, ITRs, and introns.

[0067] Unless the context explicitly specifies otherwise, the singular forms “a”, “an”, and “the” include plural indicators.

[0068] The connecting term “and / or” between multiple statement elements covers both individual options and combined options. For example, in the case where two elements are combined by “and / or”, the first option refers to the applicability of the first option without the second option, the second option refers to the applicability of the second option without the first option, and the third option refers to the applicability of the first and second options together. Any one of the options is understood to fall within the meaning of the term “and / or” and thus satisfies the requirements of the term “and / or”. The coexistence of more than one of the options is also understood to fall within the meaning of the term “and / or”.

[0069] Unless the context clearly indicates otherwise, the terms “or” and “and” have the same meaning as “and / or”. Specification 8 / 120 pages 14 CN 121443306 A

[0070] References to terms such as “including,” “for example,” “eg,” “such as,” and subsequent different members or examples are open-ended descriptions, wherein the listed members or examples are illustrative and may be provided orUse other members or examples.

[0071] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably to refer to an amino acid sequence without regard to function. Polypeptides and peptides contain at least two amino acids, while proteins contain at least about 10 amino acids. Amino acids include naturally occurring amino acids and amino acids provided through cell modification.

[0072] References to “comprise” and variations such as “comprises” and “comprising” as used with respect to an element or group of elements are open-ended and do not exclude additional elements or method steps not listed. Terms such as “comprising,” “containing,” and “characterized in” are synonymous with “comprise.” In the various aspects and embodiments described herein, references to open-ended terms such as “comprise” may be replaced by “consisting of” or “substantially composed of.”

[0073] The reference to “consisting of” excludes any element, step, or component not specified in the listed claimed elements, wherein such element, step, or component relates to the claimed invention.

[0074] The reference to “consistent essentially of…” limits the scope of the claim to the specified materials or steps and those materials or steps that do not substantially affect one or more basic and novel features of the claimed invention.

[0075] The term “about” refers to a value within 10% of the base parameter (i.e., plus or minus 10%). For example, “about 1:10” includes 1.1:10.1 or 0.9:9.9, and “about 5 hours” includes 4.5 hours or 5.5 hours. The term “about” at the beginning of a series of values ​​modifies each value by 10%.

[0076] Unless the context clearly indicates otherwise, all numerical or numerical ranges include integers within such ranges as well as fractions of said values ​​or integers within such ranges. Therefore, for clarification, mentioning a reduction of 95% or more includes 95%, 96%, 97%, 98%, 99%, 100%, as well as 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, etc., 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, etc., and mentioning numerical ranges such as "1-4" includes 1, 2, 3, 4, as well as 1.1, 1.2, 1.3, 1.4, etc. As a further explanation, "1 to 4 weeks" includes 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0077] Furthermore, references to numerical ranges such as "0.01 to 10" include 0.011, 0.012, 0.013, etc., and 9.5, 9.6, 9.7, 9.8, 9.9, etc. For example, a dose of approximately "0.01 mg / kg to approximately 10 mg / kg" of the subject's body weight includes 0.011 mg / kg.mg / kg, 0.012 mg / kg, 0.013 mg / kg, 0.014 mg / kg, 0.015 mg / kg, etc., and 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, etc.

[0078] References to integers greater than or less than the reference number respectively include numbers greater than or less than the reference number. Thus, for example, references to more than 2 include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more; and application “twice or more times” includes 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times or more.

[0079] Various references, including articles and patent publications, are cited or described in the background art and throughout the specification. Each of these references is incorporated herein by reference in its entirety. No reference is acknowledged as prior art relating to any invention disclosed or claimed. In some cases, a particular reference is indicated to be incorporated herein by reference to emphasize the incorporation.

[0080] The definitions provided herein, including those in this section and other sections of this application, apply throughout this application.

[0081] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Specification 9 / 120 pages 15 CN 121443306 A

[0082] The specification has been divided into sections and paragraphs, and examples of various embodiments are provided. These divisions should not be considered to separate the substance of one paragraph or section or embodiment from the substance of another paragraph or section or embodiment. The description provided has a broad application and covers all combinations of various sections, paragraphs, and sentences that can be contemplated. The discussion of any embodiments is intended to be exemplary only and is not intended to imply that the scope of this disclosure (including the claims (unless otherwise stated in the claims)) is limited to these examples.

[0083] The present invention is disclosed herein using affirmative language in general to describe numerous embodiments thereof. The invention also explicitly includes embodiments in which specific subject matter, such as substances or materials, method steps and conditions, schemes or procedures, are excluded in whole or in part. For example, in some embodiments of the invention, materials and / or method steps are excluded. Therefore, embodiments not explicitly excluded in the invention are disclosed herein, even if the invention is not generally expressed in terms of what is not included herein. I. PPT1 polypeptide

[0084] The PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1. In some embodiments, the polypeptide comprises a sequence of amino acids having at least 95% identity with the sequence of SEQ ID NO: 1.The sequence of NO: 1 has at least 95% identity with the PPT1 amino acid sequence, wherein: (a) the PPT1 polypeptide further comprises the signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence comprises a D at its N-terminus substituted with G, V, or L; and / or (c) the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus. SEQ ID NO: 38 is an example of a full-length polypeptide wherein the region corresponding to the human mature sequence has leucine-glutamine-histidine-leucine added to the N-terminus of the naturally occurring sequence.

[0085] In some embodiments, the mature sequence contains a deletion at its N-terminus. SEQ ID NO: 37 is an example of a full-length polypeptide wherein the mature sequence has an aspartic-proline-proline-alanine deletion at the N-terminus of the naturally occurring sequence.

[0086] In some embodiments, the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1. In a further embodiment, the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is glycine; the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is valine; the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is leucine ... The sequence of SEQ ID NO: 4 has at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the PPT1 amino acid sequence; or the PPT1 polypeptide comprises PPT1 amino acids, said PPT1 amino acids comprising the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminal sequence and having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 4.

[0087] PPT1 is a well-characterized enzyme in which the different amino acids responsible for activity and the different mutations leading to decreased activity are well known. (See, for example, Kumar et al., Advances inProtein Chemistry and Structural Biology (2022) 132:89-109; Bellizzi et al., PNAS (2000) 97(9):4573-4578; and hyperlink: / / www.uniprot.org / uniprotkb / P50897 / entry (December 3, 2022), each of which is incorporated herein by reference in its entirety.)

[0088] The signal sequence provides the amino sequence of the signal peptide, wherein the signal peptide is a short N-terminal amino acid sequence that provides protein secretion. The terms signal sequence and sequence peptide are used interchangeably herein. The signal sequence directs the protein to or through the endoplasmic reticulum secretion pathway and is typically cleaved within the endoplasmic reticulum prior to secretion. Thus, the signal peptide enhances the secretion of the peptide from the cell compared to the secretion level of the corresponding peptide lacking the signal peptide.

[0089] The presence of a signal sequence in the PPT1 peptide promotes the extracellular secretion of the mature PPT1 peptide. The secreted peptide can be taken up by another cell, providing cross-correction.

[0090] In some embodiments, the signal peptide comprises the amino acid sequence of any one of SEQ ID NO: 16-27, or comprises an amino acid sequence that differs from any one of SEQ ID NO: 16-27 by one amino acid. In a further embodiment, the signal peptide comprises the amino acid sequence of SEQ ID NO: 16; comprises the amino acid sequence of SEQ ID NO: 19; or comprises the amino acid sequence of SEQ ID NO: 23.

[0091] In some embodiments, the PPT1 polypeptide comprises a signal sequence and a PPT1 sequence, wherein: a) the signal sequence comprises an amino acid sequence of any one of SEQ ID NO: 16-21 and 24-27, or differs from any one of SEQ ID NO: 16-21 and 24-27 by an addition, deletion, or substitution of one amino acid; and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; b) the signal sequence comprises an amino acid sequence of any one of SEQ ID NO: 16-21 and 24-27, the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2, and X is G, V, or L; c) the signal sequence comprises an amino acid sequence of SEQ ID NO: 16 or differs from SEQ ID NO: 16 by one amino acid.Acid addition, deletion, or substitution; and the PPT1 polypeptide sequence contains a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; d) the signal sequence contains the amino acid sequence of SEQ ID NO: 16, and the PPT1 polypeptide sequence contains a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2, and X is G, V, or L; and e) the signal sequence contains the amino acid sequence of SEQ ID NO: 16, and the PPT1 polypeptide sequence contains the sequence of SEQ ID NO: 2, wherein X is G. f) The signal sequence comprises the amino acid sequence of SEQ ID NO: 19 or differs from SEQ ID NO: 19 by one amino acid addition, deletion, or substitution; and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1; h) The signal sequence comprises the amino acid sequence of SEQ ID NO: 19, and the PPT1 polypeptide sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2, and X is G, V, or L; and i) The signal sequence comprises the amino acid sequence of SEQ ID NO: 19, and the PPT1 polypeptide sequence comprises the sequence of SEQ ID NO: 2, wherein X is G.

[0092] In some embodiments, the PPT1 polypeptide comprises a signal sequence and a PPT1 sequence, wherein the signal sequence comprises the amino acid sequence of SEQ ID NO: 23 or differs from SEQ ID NO: 23 by one amino acid addition, deletion or substitution; and the PPT1 polypeptide sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus, and has a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 4.

[0093] In some embodiments of a PPT1 polypeptide containing a signal sequence and a PPT1 sequence, the polypeptide comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 31-42, or differing from any one of SEQ ID NO: 31-42 by 1-10 amino acid differences (1, ...).(Differences of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). Mentions of amino acid differences indicate any combination of additions, substitutions, and / or deletions.

[0094] In a further embodiment, the polypeptide comprises an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 31 or differing from SEQ ID NO: 31 by 1-5 amino acids; the polypeptide comprises the amino acid of SEQ ID NO: 31, wherein X is D or G; the polypeptide comprises an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 34 or differing from SEQ ID NO: 34 by 1-5 amino acids; the polypeptide comprises the amino acid of SEQ ID NO: 34, wherein X is D or G; the polypeptide comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine, and has at least 99% or 100% sequence identity with SEQ ID NO: 38 or differing from SEQ ID NO: 38 by 1-5 amino acids; or the polypeptide comprises SEQ ID NO: 30. II. Polynucleotides Containing PPT1 Encoding Sequences

[0095] Polynucleotides containing nucleic acid sequences encoding PPT1 polypeptides can be used to promote the production and intracellular delivery of PPT1 polypeptides. In some embodiments, the polynucleotide contains a nucleic acid sequence encoding a PPT1 polypeptide, wherein the PPT1 polypeptide contains a PPT1 amino acid sequence having at least 95% identity with the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further contains a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence contains an aspartic acid (D) at its N-terminus substituted with glycine (G), valine (V), or leucine (L); and / or (c) the PPT1 sequence contains the N-terminal amino acid sequence leucine-glutamine-histidine-leucine; and / or (d) the nucleic acid sequence contains a PPT1 encoding sequence having at least 85% identity with any one of SEQ ID NO: 61-94.

[0096] In some embodiments, (a) the polynucleotide encodes a PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 1; (b) the encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is glycine; (c)The encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is valine; or (d) the encoded PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 2, and X is leucine. In a further embodiment, the nucleotide sequence encoding any one of (a), (b), (c), or (d) comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 61-94 or a variant thereof, wherein the first three nucleotides encode G, D, V, or L, or encode G. Nucleotides encoding G, V, D, or L are provided in Table 1.

[0097] Table 1 Specification 12 / 120 pages 18 CN 121443306 A

[0098] In some embodiments, the nucleic acid encoding the mature PPT1 amino acid comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminal sequence and has at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the sequence of SEQ ID NO: 4.

[0099] In some embodiments, the nucleic acid encoding the mature PPT1 polypeptide further comprises a signal sequence comprising the amino acid sequence of any one of SEQ ID NO: 16-27, or comprising an amino acid sequence differing from any one of SEQ ID NO: 16-27 by one amino acid. In a further embodiment, the signal coding sequence is the sequence of any one of SEQ ID NO: 43-58. The reference to "signal coding sequence" indicates the nucleotide sequence encoding the signal sequence or signal peptide.

[0100] In some embodiments, the polynucleotide comprises the signal coding sequence of any one of SEQ ID NO: 16-21 and 24-27; SEQ ID NO: 16; or SEQ ID NO: 19.

[0101] In some embodiments, the signal coding sequence comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 43-47, and the signal sequence comprises the sequence of SEQ ID NO: 16, or differs from SEQ ID NO: 16 by one amino acid. In a further embodiment, the nucleic acid sequence comprises the signal coding sequence of any one of SEQ ID NO: 43, 44, 45, 46, or 47.

[0102] In some embodiments, the signal coding sequence comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 43, and the signal sequence comprises the sequence of SEQ ID NO: 16, or differs from SEQ ID NO: 16 by one amino acid. In a further embodiment, the signal coding sequence comprises the sequence of SEQ ID NO: 43.

[0103] In some embodiments, the signal coding sequence comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 50, and the signal coding sequence comprises the sequence of SEQ ID NO: 19, or differs from SEQ ID NO: 19 by one amino acid. In different embodiments, the nucleic acid sequence comprises the sequence of SEQ ID NO: 50.

[0104] In some embodiments, the signal coding sequence comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 54, and the signal coding sequence comprises the sequence of SEQ ID NO: 23, or differs from SEQ ID NO: 23 by one amino acid. In different embodiments, the nucleic acid sequence comprises the sequence of SEQ ID NO: 54.

[0105] In some embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence, wherein: a) the signal sequence comprises SEQ ID NO: 16, the signal coding sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 43, and the PPT1 sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2; b) the signal coding sequence comprises the sequence of SEQ ID NO: 43, and the PPT1 sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or 2; c) the signal coding sequence comprises SEQ ID NO: d) The signal-encoded sequence contains the sequence of SEQ ID NO: 43, and the PPT1 sequence contains the sequence of SEQ ID NO: 2 with at least 97% sequence identity; and X is G, V, or L;The signal coding sequence comprises the sequence of SEQ ID NO: 43, and the PPT1 sequence comprises a sequence having at least 99% sequence identity with SEQ ID NO: 2, and X is G; f) The signal coding sequence comprises the sequence of SEQ ID NO: 43; and the PPT1 sequence comprises the sequence of SEQ ID NO: 1; g) The signal coding sequence comprises the sequence of SEQ ID NO: 43; and the PPT1 sequence comprises the sequence SEQ ID NO: 2, wherein X is G; and h) In different embodiments, the sequence encoding the polypeptide sequence of any one of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 61-79 and 80-94, wherein the first three nucleotides are codons from Table 1. In a further embodiment, the sequence of the polypeptide sequence encoding any one of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 62-64, 71, 74, 78, 79, and 83, wherein the first three nucleotides are codons from Table 1; or the sequence of the polypeptide sequence encoding any one of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 64, wherein the first three nucleotides are codons from Table 1; or the sequence of the polypeptide sequence encoding any one of (a) to (g) comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 79, wherein the first three nucleotides are codons from Table 1.

[0106] In some embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence, wherein: a) the signal sequence comprises SEQ ID NO: 19, and the signal encoding sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 50; and the PPT1 sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 2; b) the signal encoding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1 or 2; c)The signal coding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises a sequence having at least 97% sequence identity with SEQ ID NO: 1; d) The signal coding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises a sequence having at least 97% sequence identity with SEQ ID NO: 2, and X is G, V, or L; e) The signal coding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises a sequence having at least 99% sequence identity with SEQ ID NO: 2, and X is G; f) The signal coding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises the sequence of SEQ ID NO: 1; g) The signal coding sequence comprises the sequence of SEQ ID NO: 50, and the PPT1 sequence comprises the sequence SEQ ID NO: 2, wherein X is G; and h) In different embodiments, the nucleic acid encoding the polypeptide sequence of any one of (a) to (g) comprises a sequence having at least 97% sequence identity with SEQ ID NO: 1. The sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 61-79 and 80-94, wherein the first three nucleotides are codons from Table 1.

[0107] In some embodiments, the polynucleotide encodes a PPT1 polypeptide comprising a signal sequence and a PPT1 sequence, wherein: a) the signal sequence comprises SEQ ID NO: 23, the signal encoding sequence comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 54, and the PPT1 sequence comprises a leucine-glutamine-histidine-leucine at its N-terminus and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4; b) the signal encoding sequence comprises the sequence of SEQ ID NO: 54, and the PPT1 sequence comprises a leucine-glutamine-histidine-leucine at its N-terminus and at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 4; c) the signal encoding sequence comprises SEQ ID NO: The sequence 54, and the PPT1 sequence contains leucine-glutamine-histidine-leucine at its N-terminus and has at least 97% sequence identity with SEQ ID NO: 1; d) The signal encoding sequence contains SEQ ID NO:e) The signal encoding sequence comprises the sequence of SEQ ID NO: 54, and the PPT1 sequence comprises leucine-glutamine-histidine-leucine at its N-terminus and has at least 98% sequence identity with SEQ ID NO: 4; and f) The signal encoding sequence comprises the sequence of SEQ ID NO: 54, and the PPT1 sequence comprises leucine-glutamine-histidine-leucine at its N-terminus and has at least 99% sequence identity with SEQ ID NO: 4; and f) The signal encoding sequence comprises the sequence of SEQ ID NO: 54, and the PPT1 sequence comprises SEQ ID NO: 4.

[0108] In some embodiments, the nucleic acid (a) encodes a PPT1 polypeptide comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 29-42, or differing from any one of SEQ ID NO: 29-42 by 1-10 amino acids (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); (b) encodes a PPT1 polypeptide comprising an amino acid sequence having at least 99% or 100% sequence identity with SEQ ID NO: 31, or differing from SEQ ID NO: 31 by 1-5 amino acids; (c) encodes a PPT1 polypeptide comprising the amino acid of SEQ ID NO: 31, wherein X is D or G; (d) encodes a PPT1 polypeptide comprising a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 29-42, or differing from any one of SEQ ID NO: 29-42 by 1-10 amino acids (1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids); 34. An amino acid sequence having at least 99% or 100% sequence identity or differing from SEQ ID NO: 34 by 1-5 amino acids; (e) Encoding a PPT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 34, wherein X is D or G; (f) Encoding a PPT1 polypeptide comprising the amino acid sequence of SEQ ID NO: 38; (g) Encoding (b), wherein the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 43; (h) Encoding (b), wherein the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 107-125 and 168; (i) Encoding (c), wherein the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 43; 99. A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity; (h) Encoding (c), wherein the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 126-140 and 161-167.A sequence with 100% sequence identity; or (i) encoding (f), wherein the nucleic acid comprises a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with SEQ ID NO: 103.

[0109] In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding the PPT1 polypeptide described in Section I of the previous article.

[0110] In some embodiments, the polynucleotide sequence encoding the PPT1 polypeptide comprises two or more exons and one or more introns encoding the PPT1 polypeptide.

[0111] In some embodiments, the polynucleotide comprises a PPT1 coding sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% sequence identity with any one of SEQ ID NO: 95-106.

[0112] The sequences containing stop codons provided in this application (as shown in Table 2 below) include embodiments in which a stop codon is absent, multiple stop codons are present, and different stop codons are present.

[0113] The sequences encoding proteins providing stop codons provided in this application (as shown in Table 2 below) include embodiments in which a stop codon is absent immediately following the provided sequence, a stop codon is present, multiple stop codons are present, and different stop codons are present.

[0114] In some embodiments, the nucleotide sequence encoding PPT1 contains 0-5, 0-10, or 0-15 CpGs; 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, or about 5.0% CpGs; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, or up to about 5.0% CpGs. III. Expression cassette

[0115] The polynucleotide expression cassette contains a nucleic acid encoding a PPT1 polypeptide operatively linked to one or more expression control elements. Expression control can be influenced, for example, at the levels of transcription, translation, splicing, and information stability. Expression control elements are typically located at the 5' (“upstream”) or 3' (“downstream”) of the transcribed nucleic acid. Expression control elements can also be located within the transcript (e.g., in introns), adjacent to the transcribed sequence, or at a distance from the transcribed sequence. One or more expression control elements of the same or different types may be present. Examples of expression control elements include promoters, enhancers, introns, polyadenylation signals, cozak sequences, post-transcriptional regulatory elements, and termination sequences.

[0116] A promoter is a DNA region in which transcription begins. Typically, the transcribed nucleic acid is located at the 3' end of the promoter sequence. In some embodiments, the promoter sequence is coupled to an enhancer. An enhancer is a DNA region that increases transcription of the promoter. An enhancer may be adjacent to or inside the promoter or may be distal to it. Typically, an enhancer is located upstream of the promoter, but may be located downstream of or within the promoter sequence.

[0117] Expression control elements such as promoters and enhancers may be selected to preferentially drive expression in a particular cell or tissue type. Expression control elements are typically active in a particular cell, tissue, or organ because they are recognized by transcription-activating proteins or other transcription regulators unique to that cell, tissue, or organ type. (See, for example, Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed., Vol. II, Cold Spring Harbor Laboratory Press, New York; and Ausubel et al. (2010) Current protocols in molecular biology, John Wiley & Sons, New York).

[0118] Incorporation of tissue-specific regulatory elements into the expression construct provides at least partial tissue tropism for the expression of the PPT1 protein. References to promoters or enhancers that are specific to a particular cell type of tissue indicate that the promoter or enhancer provides higher levels of expression and / or secretion in the indicated cell or tissue type. Examples of liver-specific promoters include the thyroxine transporter (TTR) gene promoter; the human α1-antitrypsin (hAAT) promoter; the apolipoprotein A-I promoter; albumin, Miyatake et al., J. Virol., 71:5124-32 (1997); the hepatitis B virus core promoter, Sandig et al., Gene Ther. 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot et al., Hum. Gene. Ther., 7:1503-14 (1996); the human factor IX promoter; the thyroxine-binding globulin (TBG) promoter; the TTR minimal enhancer / promoter; the α-antitrypsin promoter; LSP (845 nt) (requires intronless scAAV); and the LSP1 promoter. Examples of active enhancers in the liver are apolipoprotein E (apoE) HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272:29113-19 (1997)).

[0119] Expression control elements also include ubiquitous or promiscuous promoters and promoters / enhancers capable of driving polynucleotide expression in many different cell types. Such elements include the EF1-α promoter, cytomegalovirus (CMV) immediate early promoter / enhancer sequence, Rous sarcoma virus (RSV) promoter / enhancer sequence, phosphoglycerate kinase (PKG) promoter, CAG (a complex of CMV enhancer, chicken β-actin A protein promoter (CBA), and rabbit β-globin intron) (see, for example, Boshart et al., (1985) Cell, 41:521-530), SV40 promoter, dihydrofolate reductase promoter, and cytoplasmic β-actin promoter.

[0120] Examples of CNS-specific promoters include: neuron-specific promoters such as NSE (neuron-specific enolase), synaptic protein or NeuN, platelet-derived growth factor (PDGF), platelet-derived growth factor B chain (PDGF-β), methyl-CpG binding protein 2 (MeCP2), Ca2 / calmodulin-dependent protein kinase II (CaMKII), metabolite glutamate receptor 2 (mGluR2), neurofilament light chain (NFL) or neurofilament heavy chain (NFH), β-globin small gene nβ2, and proenkephalinogen (PPE). Promoters for enkephalin (Enk) and excitatory amino acid transporter 2 (EAAT2); promoters specific to astrocytes, such as glial fibrillary acidic protein (GFAP) and EAAT2 promoters; promoters specific to oligodendrocytes, such as myelin basic protein (MBP) / myelin-associated glycoprotein and oligodendrocyte transcription factor 2 promoters; promoters specific to neurons / hypothalamus, such as pro-opioid corticosteroid (POMC) promoters; and promoters specific to neurons / spinal cords, such as superoxide dismutase 1 (SOD1). (See, for example, U.S. Patent Publication No. 2021 / 214749 and Adeno-Associated Virus Vectors (2019), edited Castle., 1st edition, Springer New York, New York, NY.; both are incorporated herein by reference in their entirety.)

[0121] Other promoters include the SV40 early promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), the herpes simplex virus (HSV) promoter, the SFFV promoter, the rat insulin promoter, the TBG promoter, the desmin promoter, and similar muscle-specific promoters, synthetic promoters, hybrid promoters, and promoters with multi-tissue specificity.

[0122] Expression control elements can also influence expression in a manner that can be modulated by increasing or decreasing the expression of a signal or stimulus. A modifiable element that increases the expression of transcribed nucleic acid in response to a signal or stimulus is also called an "inducible element" (i.e., signal-induced). Typically, the amount of increase or decrease conferred by such an element is proportional to the amount of signal or stimulus present. Specific examples include the zinc-inducible sheep metallothionein (MT) promoter; the steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; the tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547–5551 (1992)); the tetracycline induction system (Gossen et al., Science 268: 1766–1769 (1995); see also Harvey et al., Curr. Opin. Chem. Biol. 2:512–518 (1998)); the RU486 induction system (Wang et al., Nat. Biotech. 15:239–243 (1997) and Wang et al., Gene Ther. 4:432–441 (1997); and the rapamycin induction system (Magari et al., J. Clin. Invest.). 100:2865-2872 (1997); and Rivera et al., Nat. Medicine. 2:1028-1032 (1996)). Other examples of adjustable control elements include those regulated by specific physiological states, such as temperature, acute phase, or development.

[0123] In some embodiments, the expression cassette further comprises one or more introns independent of the nucleotide encoding PPT1. A variety of different introns can be used to enhance gene expression. Examples of introns that can be used include rabbit β-globin introns with splice donors / acceptors, SV40 introns with splice donors / acceptors, human β-globin introns, intron 2 of the human hemoglobin β gene, hFIX int1 (intron 1 of the human coagulation factor IX gene), CBA-rHHB (a synthetic intron derived from a fusion of intron 1 of the chicken β-actin gene and intron 2 of rabbit hemoglobin β), CBA (intron 1 of the chicken β-actin gene), hGH (intron 1 of the human growth hormone gene), hFIX synth (synthetic introns derived from different parts of the human coagulation factor IX gene and present in the pLIVE vector, Mirus Bio, Madison, Wisconsin); synthetic introns of human hemoglobin subunit β (HBB2) and optimized HBB2; and chimeric introns, such as those derived from the followingThe introns consist of: a 5'-splicing donor from the first human β-globin intron, and a branch and 3'-receptor site from an intron located between the leader sequence and the body of the immunoglobulin gene heavy chain variable region. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197; Ronzitti et al., Mol. Ther. Methods Clin Dev. (2016) July 20, Specification 17 / 120 pages 23 CN 121443306 A day;3:16049; and the HBB-IGG intron provided by the pCMVNT™ vector.)

[0124] In some embodiments, the expression cassette contains post-transcriptional regulatory elements. Post-translational regulatory elements such as the marmot post-transcriptional regulatory element (WPRE) and the hepatitis B regulatory element can increase gene expression. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0125] Polyadenylation signaling sequences provide for the formation of poly-A tails, which promote nuclear export, translation, and / or mRNA stability, and may also participate in transcription termination. Examples of polyadenylation signaling sequences include SV40 late polyadenylation signal, bovine growth hormone poly-A (bGHpA) signal sequence, synthetic poly-A, mouse β-globin pA, rabbit β-globin pA, and H4-based pA. (Buck et al., Int. J. Mol. Sci. (2020), 21, 4197.)

[0126] In some embodiments, the expression cassette contains a Kozak concordant sequence or a variant thereof. The Kozak concordant sequence plays a role in translation initiation. Kozak co-sequences and variants are provided, for example, in McClements et al., (2021) Molecular Vision, 27, 233-242, which are hereby incorporated by reference.

[0127] In some embodiments, the expression cassette from 5' to 3' comprises: a promoter or promoter / enhancer, an intron, a Kozak sequence, a PPT1 coding sequence, and a polyadenylation signal operatively coupled to the PPT1 coding sequence. In some embodiments, the intron comprises the amino acid sequence of SEQ ID NO: 11.

[0128] In some embodiments, the expression cassette further comprises a miRNA target sequence, in a further embodiment, said miRNA target sequence being incorporated into the 3' UTR of the expression cassette. The miRNA target sequence is recognized by miRNAs present in a particular cell or tissue, resulting in degradation of the mRNA transcript. Based on the presence of certain miRNAs in a particular cell, incorporation of one or more miRNA target sequences can be used to reduce expression in certain cell or tissue types. Multiple tandem repeats of the miRNA target sequenceThe sequence can be used to increase degradation. (Geisle et al., (2016) World Journal of Experimental Medicine 6(2): 37-54.)

[0129] In some embodiments, the expression cassette encodes the PPT1 polypeptide provided in Section I above, the expression cassette contains a nucleic acid sequence encoding the PPT1 polynucleotide provided in Section II above, and / or the expression cassette contains a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any one of SEQ ID NO: 141-143, 169, and 170.

[0130] In some embodiments, the expression cassette nucleotide sequence contains any one of 0-5, 0-10, 0-15, 0-50, or 0-100 CpGs; 0, 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, or 30 CpGs; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%. CpG; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14%, or up to about 15% CpG. IV. Recombinant Viral Vector Nucleic Acid

[0131] The polynucleotide recombinant viral vector nucleic acid contains 5' and / or 3' viral elements that provide viral packaging and may provide additional activities such as self-initiation, DNA replication, promoter activity, genome integration, or episome multiplication. The 5' and 3' elements are typically located at or near the 5' and 3' ends of the recombinant viral vector nucleic acid and may be naturally occurring or modified forms of naturally occurring sequences. Examples of 5' and 3' elements include adenovirus ITRs, adeno-associated virus ITRs, and packaging sequences; as well as retrovirus 5' and 3' long terminal repeats (LTRs) and packaging sequences. (Naso et al., (2017) BioDrugs, 31(4), 317–334; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53 (2021); and Liu and Seol (2020) BMB Reports;53(11):565-575. ) Specification 18 / 120 pages 24 CN 121443306 A

[0132] The term “recombinant” as a modifier of nucleic acid or vector indicates a combination of elements that do not exist in nature. For example, a recombinant viral vector nucleic acid provides 5' and / or 3' viral elements, and an expression cassette containing one or more elements that are not naturally linked to the 5' and / or 3' elements. Similarly, a viral vector (such as an rAAV vector) may contain a naturally occurring or modified capsid that capsids the recombinant viral vector nucleic acid.

[0133] In some embodiments, the viral vector nucleic acid sequence comprises 5' UTR and 3' UTR, or 5' ITR and 3' ITR, and (1) comprises a sequence encoding a polypeptide of Section I above; (2) comprises a nucleic acid sequence encoding a PPT1 polynucleotide as provided in Section II above; and / or (3) comprises an expression cassette encoding a PPT1 polynucleotide as provided in Section III above.

[0134] In some embodiments, the viral vector includes a poly-A signal operatively linked to the 3'-ITR, wherein the poly-A signal antagonizes potential transcription initiated from the 3'-ITR. The operatively linked poly-A signal is located upstream of the 3'-ITR.

[0135] In some embodiments, the viral vector nucleic acid contains any one of 0-5, 0-10, 0-15, 0-50, 0-100, or 0 to 150 CpGs; 0, 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 1, 44, 44, 46, 47, 48, 49 or 50 CpG; 0%, about 0.5%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14% or about 15% CpG; and / or up to about 0.5%, up to about 1.0%, up to about 2.0%, up to about 3.0%, up to about 4.0%, up to about 5.0%, up to about 6%, up to about 7%, up to about 8%, up to about 9%, up to about 10%, up to about 11%, up to about 12%, up to about 13%, up to about 14% or up to 15% CpG.

[0136] In some embodiments, the recombinant viral vector nucleic acid comprises the same components as those in SEQ ID NO: 144-154, 171, and 172.The sequence of any one of them has at least 95% identity, at least 97% identity, at least 99% identity, or 100% identity. V. Viral Vectors

[0137] In some embodiments, the gene delivery medium is a viral vector containing a protein capsid that coats the nucleic acid of the recombinant viral vector. The viral vector can deliver the nucleic acid of the viral vector to cells or tissues. Depending on the specific vector, the viral vector may further contain a viral envelope. Examples of viral vectors that can be used for gene delivery include adenovirus vectors, rAAV, retrovirus vectors, and herpes simplex vectors.

[0138] Different serotypes exist in different types of viruses. Different serotypes can provide different activities, such as cell or tissue tropism and the likelihood of generating a host immune response. The term "serotype" broadly refers to both serologically different viruses and viruses that may not be serologically different within a subgroup or variant of a given serotype. Serological distinctiveness can be determined based on the lack of cross-reactivity between antibodies against one capsid compared to another. Such cross-reactivity differences are usually due to differences in capsid protein sequence / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0139] As more naturally occurring viral isolates are discovered or capsid mutants are generated, serological differences with any of the existing serotypes may or may not exist. Therefore, in the absence of serological differences in a new virus, the new virus will be a subgroup or variant of the corresponding serotype. VA Adenovirus Vector

[0140] Adenovirus is a non-enveloped double-stranded DNA virus. Recombinant adenovirus vectors contain recombinant adenovirus nucleic acid lacking one or more proteins involved in viral replication and further contain an adenovirus capsid. Recombinant adenovirus vectors containing varying amounts of adenovirus DNA can be generated. The adenovirus (Ad) genome has a hairpin-like inverted terminal repeat (ITR) sequence of varying length between 30 and 371 bp appended to its end. (See page 19 / 120 of CN 121443306 A) ITR serves as a self-initiating structure, which promotes non-initiator-dependent DNA replication. The packaging signal located on the left arm of the genome is required for viral genome packaging. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0141] In some embodiments, the recombinant adenovirus vector is a third-generation vector, also referred to as “gutless” or “helper-dependent”. Gutless vectors can be generated from recombinant adenovirus nucleic acid, in which, in addition to ITR and packaging signal,Apart from the number, all or substantially all viral sequences are absent. Empty-shell adenovirus vectors are high-capacity vectors capable of holding up to about 36 kb of DNA inserts. Preferred recombinant adenovirus nucleic acids are about 27 kb to about 37 kb. Filler sequences can be added to recombinant adenovirus nucleic acids to increase nucleic acid size and capsid incorporation. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences and immunogenic sequences. (Liu and Seol (2020) BMB Reports, 53(11):565-575; Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53; and Sandig et al., PNAS (2000) 97(3):1002-1007, each of which is hereby incorporated herein by reference in its entirety.)

[0142] In some embodiments, the recombinant adenovirus vector may be generated based on rare human serotypes or chimpanzee serotypes. The use of chimpanzee serotypes and rare human serotypes may help reduce the host immune response to recombinant adenovirus vectors due to pre-existing immunity. (Guo et al., (2018) Human vaccines & immunotherapeutics, 14(7):1679-1685 and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.)

[0143] Adenovirus vectors can be produced using, for example, appropriate helper viruses or plasmids and cell lines via trans-supply vectors to produce the desired viral proteins. (Liu and Seol (2020) BMB Reports; 53(11):565-575; and Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.) VB Recombinant AAV Vectors

[0144] Recombinant adeno-associated virus (referred to herein as “rAAV”) vectors are based on adeno-associated virus. Adeno-associated virus (AAV) is a single-stranded DNA virus containing a 4.7 kb genome with 145 nt ITRs flanking both ends of the genome. ITR activity is important for self-initiation and packaging, and can also provide additional activities such as promoter activity. The sizes of the AAV 5' and 3' ITRs can differ, and the 5' and 3' inverted repeat sequences do not need to be exact inverted repeat sequences.

[0145] The rAAV vector contains recombinant AAV nucleic acid and a viral capsid. The rAAV recombinant nucleic acid lacks one or more AAV proteins involved in viral replication. In some embodiments, the rAAV vector contains AAV 5' and / or 3' ITRs as well as a DNA insert. In some embodiments, the rAAV nucleic acid comprises 5' ITRs independently selected from the following...ITR and / or 3' ITR: 5' and 3' ITRs provided in AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, and AAV3B ITRs. In a further embodiment, 5' and 3' ITRs are present, and both ITRs originate from the same serotype genome.

[0146] In some embodiments, the 5' ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 8; and the 3' ITR independently comprises (a) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9; (b) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 158; (c) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 159; or (d) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 160.

[0147] In some embodiments, 3' ITR comprises a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 9; and 5' ITR independently comprises (a) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 156; (b) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 157; or (c) a sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 157.

[0148] Recombinant adeno-associated virus vectors typically accept DNA inserts ranging in size from about 4 kb to about 5.2 kb. If desired, a filler sequence can be used to increase the rAAV nucleic acid size and packaging efficiency. In different embodiments, the rAAV nucleic acid including the filler is 4–5.2 kb, 3.0–5.5 kb, 4.0–5.0 kb, 4.3–4.8 kb, or about 4.2 kb.kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, or about 4.7 kb. Preferred filler sequences avoid coding sequences, repetitive sequences, recombinant sequences, and immunogenic sequences.

[0149] In some embodiments, rAAV is a self-complementary adeno-associated virus vector (scAAV) or a short hairpin adeno-associated virus vector (shAAV). scAAV and shAAV provide double-stranded rAAV nucleic acid that can be incorporated into the AAV capsid. scAAV and shAAV contain an inverse dimer repeat sequence that provides intramolecular double-stranded DNA. scAAV can be generated by mutating the ITR terminal cleavage site so that rep cannot create a nick at the terminal cleavage site. shAAV can utilize a short hairpin to generate double-stranded AAV nucleic acid. scAAV and shAAV offer the advantage of double-stranded DNA by avoiding the DNA synthesis steps required for single-stranded rAAV nucleic acid after entry into the cell. A potential drawback of scAAV and shAAV is that the size of the DNA insert that can be incorporated is reduced by about half compared to single-stranded rAAV nucleic acid. (US Patent No. 10,457,940; Xie et al., Mol Ther. (2017) 25(6):1363-1374; and McCarty Mol. Ther. (2008) 16(10):1648-1656; each of which is incorporated herein by reference in its entirety.)

[0150] Naturally occurring AAV capsids contain viral proteins VP1, VP2, and VP3 in a ratio of about 1:1:10. AAV vectors can be generated in which all three viral proteins are based on a specific serotype, or one, two, or all three viral proteins are based on different serotypes.

[0151] Recombinant AAV capsids and nucleic acids can be based on the same serotype (or subgroup or variant), or they can be different from each other. In some embodiments, the rAAV nucleic acid has the same serotype genome (e.g., ITR) as the capsid protein.

[0152] In different embodiments, the rAAV capsid comprises proteins having sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.9%, or 100% identical to the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,VP1, VP2, or VP3 of any of AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10; or VP1 of SEQ ID NO: 12 or SEQ ID NO: 15.

[0153] Recombinant AAV capsids comprising VP1 of SEQ ID NO: 12 are described, for example, in U.S. Patent No. 9,840,719; and rAAV capsids comprising VP1 of SEQ ID NO: 15 are described, for example, in U.S. 9,169,299; both of these patents are incorporated herein by reference.

[0154] In some embodiments, the AAV capsid comprises VP1, VP2, and VP3, each having at least 80%, at least 90%, at least 95%, or 100% sequence identity with any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12, or SEQ ID NO: 15; and variants thereof (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions). (See, for example, U.S. Patent Nos. 9,909,142 and 9,840,719, which disclose RHM4-1, RHM15-1, RHM15-2, RHM15-3 / RHM15-5, RHM15-4, and RHM15-6; U.S. Patent Publication No. 21 / 120, page 27; CN 121443306 A; U.S. Patent Publication No. 2013 / 0059732 and U.S. Patent No. 9,169,299, which disclose LK01, LK02, and LK03; and U.S. Patent No. 11,110,153; the disclosures of these patents are incorporated herein by reference in their entirety.)

[0155] In some embodiments, the capsid comprises VP1 having the sequence of SEQ ID NO: 12; VP2 having the sequence of SEQ ID NO: 13; and VP3 having the sequence of SEQ ID NO: 14.

[0156] In some embodiments, the AAV capsid can cross the blood-brain barrier and provide CNS expression. Examples of such AAV capsids and designs of AAV capsids capable of providing CNS expression are provided in the following literature: Chen et al., (2021) J. Control. Release 333,References 129–138 (e.g., AAV9, AAV-PHP⋅B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, and Goertsen et al., (2022) Nat. Neurosci. 25, 106–115 (2022), are each incorporated herein by reference in their entirety.

[0157] The AAV genome contains two major genes: rep and cap. Transcription from the rep gene begins at two different promoters, resulting in the production of non-structural proteins named Rep78, Rep68, Rep52, and Rep40. The rep proteins play a role in genome replication and / or capsid formation. The cap gene encodes structural proteins (VP1, VP2, and Vp3) that make up the capsid; non-structural assembly activation proteins (APP) that perform functions related to capsid assembly; and membrane-associated accessory proteins that may be associated with the generation phase of the replication cycle. (Maurer and Weitzman (2020) Hum. Gene Ther. 31(9–10):499–511, which are incorporated herein by reference in their entirety.)

[0158] AAV requires helper viral functions to complete its replication cycle. Helper viral functions can be supplied by different viruses in permissive cell lines. Permissive cell lines are cell lines that are capable of supporting viral replication. Examples of helper viruses for AAV include adenoviruses, HSV-1, HPV-16, and HBoV1, which can be used in combination with, for example, permissive primate cells; and baculoviruses, which can be used in combination with, for example, permissive insect cells such as sf9. (Maurer and Weitzman (2020) Hum. Gene Ther. (2020) 31(9–10):499–511 and Meier et al., (2020) Viruses 19;12(6):662, both of which are incorporated herein by reference in their entirety.)

[0159] Recombinant AAV can be produced using, for example, a suitable helper virus or plasmid and cell line via a trans-supply vector to produce the desired viral proteins. In some embodiments, rAAV is produced using an rAAV vector genomic plasmid. The plasmid contains the portion of the rAAV nucleic acid that is ultimately packaged or capsidated to form a viral (e.g., rAAV) vector. The “plasmid backbone” contains elements important for replication and recombinant virus production. The plasmid backbone itself is not packaged or capsidated into the viral particle except for possible 3' ITR and / or 5' ITR clonal remnants.

[0160] The vector genomic plasmid may contain regions such as origin of replication and selectivity markers. Other possible sites include cloning sites.

[0161] Recombinant AAVs can be generated from different types of cell lines, including HeLa, A549, BHK, Vero, and HEK293 or derivatives thereof. In some embodiments, HEK293 cells (American Type Culture Collection accession number ATCC CRL1573) are used. Other host cell lines suitable for the generation of rAAV vectors are described, for example, in the following literature: Robert et al., (2017) Biotechnol. J. (2017) 12 (3), 1600193; and international application number PCT / US2017 / 024951, the disclosures of which are incorporated herein by reference in their entirety.

[0162] Recombinant AAVs can be cultured under a variety of different conditions suitable for providing cell growth and gene expression. References describing rAAV fabrication include Clément and Grieger (2016) Mol. Ther. Methods Clin. Dev. 16;3:16002; Robert et al. (2017) Biotechnol. J. 12(3), 1600193; and Adeno-Associated Virus Vectors (2019), edited Castle., 1st edition, Springer New York, New York, NY. 22 / 120 pages 28 CN 121443306 A York, NY.; each of which is incorporated herein by reference in its entirety.

[0163] In some embodiments, AAV helper functionality is introduced into host cells by transfecting host cells with an AAV helper construct prior to or concurrently with transfection of an AAV expression vector. Host cells with AAV helper functionality may be referred to as “helper cells” or “packaging helper cells”. Therefore, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to supplement the missing AAV function necessary for productive AAV transduction. AAV helper constructs typically lack the AAV ITR and are neither self-replicating nor self-packaging. These constructs can be in the form of, for example, plasmids, phages, transposons, colloids, viruses, or viral particles. Many AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 encoding both the rep and cap expression products. Many other vectors encoding the rep and / or cap expression products are known. For example, recombinant AAV can be generated, for example, as described in: U.S. Patent 9,408,904; and International Application No. PCT / The disclosures of US2017 / 025396 and PCT / US2016 / 064414 are incorporated herein by reference in their entirety.

[0164] In some embodiments, the rAAV vector is produced from rAAV-producing cells containing rAAV helper viral activity. The genome of the rAAV-producing cells contains rAAV nucleic acid, the rep gene, and the cap gene.

[0165] In some embodiments, the rAAV vector is produced by culturing rAAV-permitting cells containing an AAV genome plasmid, wherein the rAAV-permitting cells further contain the rep and cap genes provided as part of the cell genome and / or provided by one or more separate plasmids; and helper viral activity provided as part of the cell genome and / or provided by one or more separate plasmids. In a further embodiment, (a) the rAAV-permitting cell line is a packaging cell, wherein the genome of the packaging cell contains the cap gene and the rep gene; (b) the rep gene, the cap gene, and the helper activity are provided by the same plasmid; or (c) the rep gene and the cap gene are provided by a rep / cap plasmid, and the helper activity is provided by a helper plasmid.

[0166] In some embodiments involving the use of HSV helper functions, the helper functions are provided by genes encoding at least UL5, UL8, UL52, and ICP8.

[0167] In some embodiments involving the use of adenovirus helper functions, the helper functions are provided by genes encoding at least E1A, E1B19K, E1B55K, E2A, E4orf6, and VA RNA. In some embodiments, the E1, E2A, and VR RNA functions are provided by helper plasmids, wherein additional helper functions are provided by the host strain.

[0168] In some embodiments, the rAAV vector is obtained by generating rAAV and purifying rAAV using the methods described herein. Purification of rAAV can be performed using techniques such as gradient-based purification, column-based methods, and combination methods. (See, for example, Ayuso et al., (2010), Curr Gene Ther. (2010) 10(6):423-36, which is incorporated herein by reference in its entirety.) VC Retroviral Vectors

[0169] Retroviruses are enveloped single-stranded RNA viruses containing 5' and 3' LTRs and signal packaging sequences located just outside the LTRs. Different types of retroviral vectors may contain varying amounts of viral genome. In some embodiments, the retroviral vector is an HIV-based lentiviral vector that retains all cis-acting sequences required for viral RNA packaging, reverse transcription, and proviral DNA integration, while removing all HIV protein-coding genes. Lentiviral vectors have up to approximately 9Packaging capacity of kb. If desired, filler sequences can be used to increase rAAV nucleic acid size and packaging efficiency. Lentiviral vectors can be produced using appropriate plasmids and cell lines by trans-supplying the vector to produce the desired viral proteins. (Bulcha et al., (2021) Sig. Transduct. Target Ther. 6:53.) VI. Non-viral vectors

[0170] In some embodiments, the gene delivery medium is a non-viral vector. Non-viral vectors include nanoparticles and naked nucleic acids. Preferred non-viral vectors are nanoparticles. A variety of different nanoparticles can be used, including lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein and peptide-based nanoparticles, DNA tree dendritic polymers and DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles and exosomes. (See, for example, Riley and Vermerris, Nanomaterials (2017) 201, 7, 94; Thomas et al., Molecules (2019), 24, 3744; Bochicchio et al., (2021), 13, 198; Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., NanoImpact 20, 100261; Neshat et al., Current Opin. Biotechnol. 66:1-10; Ouranidis et al., Biomedicines, 10, 50; and Qin et al., Signal Transduct Target Ther. (2022) May 21;7(1):166; each of which is hereby incorporated herein by reference in its entirety.)

[0171] If desired, the nanoparticles can target cell types using, for example, targeting ligands that recognize target cell receptors. Examples of targeting ligands include carbohydrates (e.g., galactose, mannose, glucose, and galactomannan), endogenous ligands (e.g., folic acid and transferrin), antibodies and proteins / peptides (e.g., RGD, epidermal growth factor, and low-density lipoprotein) and peptides. (e.g., Teo et al., Advanced Drug Delivery Reviews (2016), 98, 41.)

[0172] The nanoparticles can be used to deliver polynucleotide constructs encoding PPT1 to cells. In different embodiments...In this context, nanoparticles can deliver additional therapeutic compounds; and one or more additional compounds are provided in different nanoparticles. The compounds mentioned include small molecules and macromolecules (e.g., therapeutic proteins and antibodies).

[0173] The generation of different nanoparticles and the incorporation of nucleic acids and other compounds are well known in the art. Examples of publications illustrating the incorporation of nucleic acids into specific nanoparticles (such as LPNP and LNP) include Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct;68:1-11 (each of which is hereby incorporated herein by reference in its entirety). Factors that may influence the incorporation of small molecules into nanoparticles include the presence of hydrophobic and ionizable moieties. (See, for example, Nii and Ishii, Int. J. Pharm. (2005) 298:198-205; and Chen et al., J. Control. Release (2018) 286:46-54.) VI.A. Lipid-Based Delivery Systems

[0174] Lipid-based delivery systems include those using lipids as components. Examples of lipid-based delivery systems include liposomes, LNPs, micelles, and extracellular vesicles.

[0175] “Lipid nanoparticles” or “LNPs” refer to lipid-based vesicles that can be used to deliver nucleic acid molecules and have a nanoscale size. In different embodiments, the nanoparticles are about 10 nm to about 1000 nm, about 50 nm to about 500 nm, or about 50 nm to about 200 nm.

[0176] DNA is negatively charged. Therefore, it may be advantageous for LNPs to contain cationic lipids (e.g., aminolipids). Exemplary aminolipids are described in the following documents: U.S. Patent Nos. 9,352,042, 9,220,683, 9,186,325, 9,139,554, 9,126,966, 9,018,187, 8,999,351, 8,722,082, 8,642,076, 8,569,256, 8,466,122, and 7,745,651, and U.S. Patent Publications Nos. 2016 / 0213785, 2016 / 0199485, 2015 / 0265708, and 2014 / 0288146.Documents 2013 / 0123338, 2013 / 0116307, 2013 / 0064894, 2012 / 0172411, and 2010 / 0117125 are incorporated herein in their entirety. In some embodiments, the LNP comprises the aminolipids described in U.S. Patent No. 9,512,073, which is hereby incorporated in its entirety.

[0177] The terms “cationic lipid” and “amino lipid” are used interchangeably herein to include lipids and their salts having one, two, three, or more fatty acid or aliphatic alkyl chains and pH-titrile amino groups (e.g., alkylamino or dialkylamino). Cationic lipids are generally protonated (i.e., positively charged) at pH below the cationic lipid pKa and substantially neutral at pH above the pKa. Cationic lipids may also be titrable cationic lipids. In some embodiments, page 24 / 120 of CN 121443306 A, the cationic lipid comprises a protonable tertiary amine (e.g., pH titratable) group; a C18 alkyl chain, wherein each alkyl chain may independently have one or more double bonds, one or more triple bonds; and an ether, ester, or ketal bond between the head group and the alkyl chain.

[0178] The cationic lipid includes 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenic acid-N,N-dimethylaminopropane (DLenDMA), 1,2-di-γ-linolenic acid-N,N-dimethylaminopropane (γ-DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K- C2-DMA, also known as DLin-C2K-DMA, XTC2 and C2K), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dilinoleoylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA, also known as MC2), (6Z,9Z,28Z,31Z)-heptahepta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (DLin-M-C3-DMA, also known as MC3), their salts and mixtures thereof. Other cationic lipids include 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DODMA), 2,2-dilinoleoyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleoyl-4-(3-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), DLen-C2K-DMA, γ-DLen-C2K-DMA and (DLin-MP-DMA) (also known as 1-B11).

[0179] Other cationic lipids include 2,2-dilinoleoyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleoyl-4-N-methylpiperazino-[1,3]-dioxolane (DLin-K-MPZ), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleoyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 2-Dilinoleoylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoyloxy-3-trimethylaminopropane chloride (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride (DLin-TAP.Cl), 1,2-dilinoleoyloxy-3-(N-methylpiperazino)propane (DLin-MPZ) 3-(N,N-dilinoleoylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleoyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N- Distearate-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleoyloxy-N-[ 2-(spermine-formamido)ethyl]-N,N-dimethyl-1-propanetrifluoroacetate ammonium (DOSPA), bis(octadecylamidoglycyl)spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecyldienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-β-oxy)-3'-oxaproxy)-3-dimethyl1-(cis,cis-9′,1-2′-octadecyldienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleoyloxybenzylamine (DMOBA), 1,2-N,N′-dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N′-dilinoleoylcarbamoyl-3-dimethylaminopropane (DLincarbDAP), dexamethasone-spermine (DS) and disubstituted spermine (D2S) or mixtures thereof.

[0180] Many commercial formulations of cationic lipids can be used, such as LIPOFECTIN® (containing DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE® (containing DOSPA and DOPE, available from GIBCO / BRL).

[0181] Other ionizable lipids that may be used include C12-200, 306Oi10, MC3, cKK-E12, bCKK-E12, lipid 5, lipid 9, ATX-002, ATX-003, and Merck-32. U.S. Patent Application Publication No. 2017 / 0367988 describes Merck-32 on pages 25 / 120 of CN 121443306 A.

[0182] In a further embodiment, the cationic lipid may be present in an amount from about 10% to about 85% of the molar ratio of the LNP, or from about 50% to about 75% of the molar ratio of the LNP.

[0183] The LNP may comprise neutral lipids. Neutral lipids may comprise lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramides, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids is typically guided by considerations including particle size and stability. In some embodiments, the neutral lipid component may be a lipid having two acyl groups (e.g., diacylphosphatidylcholine and diacylphosphatidylethanolamine).

[0184] Lipids with various acyl chain groups having different chain lengths and degrees of saturation are available or may be isolated or synthesized. In some embodiments, lipids containing saturated fatty acids with carbon chain lengths in the C14 to C22 range may be used. In some embodiments, lipids having monounsaturated or diunsaturated fatty acids with carbon chain lengths in the C14 to C22 range are used. Alternatively, lipids having a mixture of saturated and unsaturated fatty acid chains may be used. Exemplary neutral lipids include 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-Phosphocholine (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), or phosphatidylcholine. Neutral lipids can also be composed of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups such as serine and inositol.

[0185] In a further embodiment, a neutral lipid is provided, which may be present in amounts of about 0.1% to about 99% by weight of LNP, or about 5% to about 15% by weight of LNP, for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0186] The LNP may contain additional components, such as sterols and polyethylene glycol. Sterols can impart fluidity to the LNP. As used herein, “sterol” refers to naturally occurring sterols of plant (phytosterol) or animal (animal sterol) origin, as well as synthetic sterols not naturally occurring, all characterized by the presence of a hydroxyl group at the 3-position of the steroid A ring. Suitable sterols include those conventionally used in the field of liposome, lipovesicle, or lipogranule formulations, most commonly cholesterol. Phytosterols include campesterol, sitosterol, and stigmasterol. Sterols also include sterol-modified lipids, such as those described in U.S. Patent Application Publication No. 2011 / 0177156. In various embodiments providing sterols, the sterols are present in an amount from about 1% to about 80% by weight of LNP, or from about 10% to about 25% by weight of LNP.

[0187] Polyethylene glycol (PEG) is a water-soluble polymer of ethylene PEG repeating units having terminal hydroxyl groups. PEGs are classified according to their molecular weight; for example, PEG 2000 has an average molecular weight of about 2,000 Daltons, and PEG 5000 has an average molecular weight of about 5,000 Daltons. PEGs commercially available from Sigma Chemical Co. and other companies include monomethoxy polyethylene glycol (MePEG-OH), monomethoxy polyethylene glycol-succinate (MePEG-S), monomethoxy polyethylene glycol-succinimide succinate (MePEG-S-NHS), monomethoxy polyethylene glycol-amine (MePEG-NH2), monomethoxy polyethylene glycol-toluenesulfonate (MePEG-TRES), and monomethoxy polyethylene glycol-imidazolyl-carbonyl (MePEG-IM).

[0188] In some embodiments involving PEG, the PEG has an average molecular weight of about 550 to about 10,000 Daltons, andAnd optionally substituted with alkyl, alkoxy, acyl or aryl groups. In a further embodiment, the PEG is substituted with a methyl group at the terminal hydroxyl position. In a further embodiment, the PEG has an average molecular weight of about 750 to about 5,000 Daltons, or about 1,000 to about 5,000 Daltons, or about 1,500 to about 3,000 Daltons, or from about 2,000 Daltons or from about 750 Daltons.

[0189] PEG-modified lipids include PEG-diane conjugates (PEG-DAA) as described in U.S. Patent Nos. 8,936,942 and 7,803,397. PEG-modified lipids (or lipid-polyoxyethylene conjugates) may have a variety of "anchoring" lipid moieties to fix the PEG moieties to the surface of lipid vesicles. Examples of suitable PEG-modified lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) described in U.S. Patent No. 5,820,873, PEG-modified dialkylamines, and PEG-modified 1,2-diacyloxypropyl-3-amine. In some embodiments, the PEG-modified lipid may be PEG-modified diacylglycerols and dialkylglycerols. In some embodiments, the PEG may have an amount from about 0.1% to about 50% by weight of the LNP, or from about 5% to about 15% by weight of the LNP.

[0190] In a further embodiment relating to the size of the LNP, the LNP has a size range of about 10 nm to 500 nm, or about 50 nm to about 200 nm, or 75 nm to about 125 nm prior to encapsulation of the nucleic acid.

[0191] In some embodiments involving LNPs, the LNPs are described by the following literature: Billingsley et al., Nano Lett. 2020, 20, 1578 or Billingsley et al., International Patent Publication No. WO 2021 / 077066 (both literatures are incorporated herein by reference in their entirety). Billingsley et al. and WO 2021 / 077066 describe LNPs containing lipid-anchored PEG, cholesterol, phospholipids, and ionizable lipids. In some embodiments, the LNP contains a C14-4 polyamine core and / or has a particle size of about 70 nm. C14-4 has the following structure.

[0192] In some embodiments, the LNPs are composed of cationic lipids or lipopeptides as described in the following literature: U.S. Patent No. 10,493,031, U.S. Patent No. 10,682,374 or International Patent Publication No. WO 2021 / 077066 (each of which is incorporated herein by reference in its entirety).(Hereinafter incorporated herein by reference in its entirety). In some embodiments, the LNP contains cationic lipids, cholesterol-based lipids, and / or one or more PEG-modified lipids. In some embodiments, the LNP contains cKK-E12 (Dong et al., PNAS (2014) 111(11), 3955):

[0193] In some embodiments, the LNP contains a modified form of cKK-E12, referred herein as “bCKK-E12”, having the following structure: Specification 27 / 120 pages 33 CN 121443306 A

[0194] In some embodiments, the LNP contains lipids 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 as described in the following literature: Sabnis et al., Molecular Therapy 2018, 26:6, 1509-1519 (hereinafter incorporated herein by reference in its entirety). In some embodiments, LNPs include lipids 5, 8, 9, 10, or 11 as described by Sabnis et al.

[0195] Lipid 5 of Sabnis et al. has the following structure:

[0196] Lipid 9 of Sabnis et al. has the following structure:

[0197] Other lipids that can be utilized include those described in the following literature: Rocees et al., Pharmaceutics, 2020, 12, 1095; Jayaraman et al., Angew. Chem. Int. Ed., 2012, 51, 8529-8533; Maier et al., www.moleculartherapy.org, 2013, Vol. 21, No. 8, 1570-1578; Liu et al., Adv. Mater. 2019, 31, 1902575, such as BAMEA-O16B; Cheng et al., Adv. Mater., 2018, 30, 1805308, e.g. 5A2-SC8; Hajj et al., Small, 2019 15, 1805097, e.g. 306Oi10; Du et al., U.S. Patent Application Publication No. 20160376224; and Tanaka et al., Adv. Funct. Mater., 2020, 30, 1910575; each of which is incorporated herein by reference in its entirety.

[0198] In a further embodiment, the nanoparticles are LNPs. In a further embodiment, the LNPs comprise, consist substantially of, or consist of the following components in an elliptical percentage: (1) about 20% to 65% of one or more of the following components.The phospholipids comprise, or (2) about 1% to about 50% of one or more phospholipids, about 0.1% to 10% of one or more PEG-conjugated lipids, and about 0% to about 70% of cholesterol; or (3) about 20% to 50% of one or more cationic lipids, about 5% to about 20% of one or more phospholipids, about 0.1% to 5% of one or more PEG-conjugated lipids, and about 20% to about 60% of cholesterol. In a further embodiment, the phospholipids are neutral lipids; and the phospholipids are DOPE or DSPC.

[0199] In some embodiments, LNP, by molar percentage, comprises, is substantially composed of, or is composed of the following components: (1) cKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (2) bCKK-E12, about 35%; C14-PEG2000, about 2.5%; cholesterol, about 46.5%; and DOPE, about 16%; (3) lipid 9, about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; (4) lipid 5, about 50%; C14-PEG2000, about 1.5%; cholesterol, about 38.5%; and DSPC, about 10%; (5) Ionizable lipids, approximately 50%; DSPC, approximately 10%; cholesterol, approximately 37.5%; and stabilizers (PEG-lipids), approximately 2.5%; or (6) GenVoy-ILM™ LNP (Precision NanoSystems). VI.B. Polymer-based Nanoparticles

[0200] Polymer-based delivery systems can be made from a variety of different natural and synthetic materials. DNA and other compounds can be trapped in the polymer matrix of polymer nanoparticles, or can be adsorbed or conjugated onto the surface of the nanoparticles. Examples of commonly used polymers for nucleic acid delivery include poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly(ethyleneimine) (PEI) and PEI derivatives, chitosan, dendritic polymers, polyanhydrides, polycaprolactone, polymethacrylates, poly-L-lysine, pachymannan, dextran and hyaluronic acid, and poly-β-amino esters. (Thomas et al., (2019) Molecules 24, 3744.)

[0201] Polymer-based nanoparticles can have different sizes, ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, and about 150 nm or smaller. VI.C. Lipid Polymer Nanoparticles

[0202] Lipid polymer nanoparticles are hybrid nanoparticles that provide both lipid and polymer components, and can therefore be considered as LNPs or LPNPs. LPNP configurations can provide an external polymer and an internal lipid or an external lipid and an internal polymer. The presence of two different types of materials helps in the design of nanoparticles to provide delayed release of the components. Different lipid and polymer components can be selected by considering the materials to be delivered. (See, for example, Teo et al., Advanced Drug Delivery Reviews (2016) 98, 41; Bochicchio et al., Pharmaceutics (2021) 13, 198; Mahzabin and Das, IJPSR (2021) 12(1), 65; and Teixeira et al., (2017) Prog. Lipid Res. Oct; 68:1-11.) IV.D. Protein and Peptide-Based Nanoparticles

[0203] Protein and peptide-based systems can employ a variety of different proteins and peptides. Examples of proteins that can be employed include gelatin and elastin. Peptide-based systems may employ, for example, cell-penetrating peptides (CPPs).

[0204] CPPs are short peptides (6–30 amino acid residues) that are potentially capable of intracellular penetration to deliver therapeutic molecules. Most CPPs are composed primarily of arginine and lysine residues, making them cationic and hydrophilic, but CPPs can also be amphiphilic, anionic, or hydrophobic. CPPs can be derived from natural biomolecules (e.g., HIV-1 Tat protein) or obtained through synthetic methods (e.g., poly-L-lysine, polyarginine) (Singh et al., Drug Deliv. 2018;25(1):1996–2006). Examples of CPPs include cationic CPPs (highly positively charged) (such as Tat peptide, penetratin, protamine, poly-L-lysine, and polyarginine); amphiphilic CPPs (chimeric or fusion peptides constructed from different sources, containing both positively and negatively charged amino acid sequences) (such as transportan, VT5, bovine antimicrobial peptide 7 (Bac7), proline-rich peptide (PPR), SAP (VRLPPP) 3, TP10, pep-1, and MPG); membrane-loving CPPs (exhibiting both hydrophobic and amphiphilic properties, and containing both large aromatic residues and small residues) (such as H625, SPION-PEG-CPP, and NP); and hydrophobic CPPs (containing only nonpolar motifs or residues) (such as SG3, PFVYLI, pep-7, and fibroblast growth factor).

[0205] Protein and peptide nanoparticles can be provided in different sizes, for example, ranging from about 1nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm or smaller.

[0206] VI.E. Specification for Peptide Cage Nanoparticles 29 / 120 pages 35 CN 121443306 A

[0207] Peptide cage-based delivery systems can be generated from protein materials capable of assembling into cage-like structures to form a confined internal environment. Peptide cages may comprise protein shells (e.g., structures with internal cavities that are naturally accessible to the solvent, or can be made so by changing the solvent concentration, pH, or equilibrium ratio). The monomers of the protein cages can be in naturally occurring forms or variant forms, including amino acid substitutions, insertions, and deletions (e.g., fragments).

[0208] Different types of protein “shells” can be assembled and loaded using different types of materials. Protein cages can be generated using one or more viral capsid proteins (e.g., protein capsids from cowpea chlorotic mottle virus) and non-viral proteins (e.g., U.S. Patent Nos. 6,180,389 and 6,984,386, U.S. Patent Publication No. 20040028694, and U.S. Patent Publication No. 20090035389, each of which is incorporated herein by reference in its entirety).

[0209] Examples of protein cages derived from non-viral proteins include: ferritin and deferroferritin of eukaryotic or prokaryotic origin, such as 12- and 24-subunit ferritin; and heat shock proteins (HSPs), such as the class of 24-subunit heat shock proteins that form the internal core space, small HSPs of Methanococcus jannaschii, dodecimal Dsp HSPs of Escherichia coli; and MrgA proteins.

[0210] Protein cages can have different core sizes, for example, ranging from about 1 nm to about 1000 nm, about 10 nm to about 500 nm, about 50 nm to about 200 nm, about 100 nm to about 150 nm, or about 150 nm or smaller. VI.F. Exosomes

[0211] Exosomes are small biomembrane vesicles and have been used to deliver a variety of cargoes, including small molecules, peptides, proteins, and nucleic acids. Exosomes are typically in the size range of about 30 nm to 100 nm and can be absorbed by cells to deliver their cargoes. The cargoes can associate with exosome surface structures or can be encapsulated within an exosome bilayer.

[0212] Various modifications can be made to exosomes to facilitate cargo delivery and cell targeting. Modifications for facilitating cargo delivery include structures for association with the cargo, such as protein scaffolds and polymers. Modifications for cell targeting include targetTo ligands and modify surface charge. Publications describing the generation, modification and use of exosomes for delivering various cargoes include Munagala et al., Cancer Letters (2021), 505, 58; Fu et al., NanoImpact 20, 100261 (2020); and Dooley et al., Molecular Therapy 29(5), 1729 (each of which is hereby incorporated by reference). VII. Pharmaceutical Compositions

[0213] Pharmaceutical compositions comprise pharmaceutically acceptable carriers that facilitate the administration and / or storage of PPT1 peptides, encoding polynucleotides, viral vectors or non-viral vectors. The reference to “pharmaceutically acceptable” indicates that the component will not cause serious undesirable biological effects at the amount used. Pharmaceutically acceptable carriers may comprise different components, such as one or more pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include salts, sugars, buffers, solvents, preservatives, proteins and surfactants. A particular excipient may have more than one function. Examples of pharmaceutically acceptable excipients and carriers that can be used with viral vectors are provided, for example, in International Patent Publication No. WO 2021 / 071835.

[0214] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery. Compositions suitable for parenteral administration include aqueous and non-aqueous solutions, suspensions, or emulsions, which are generally sterile and isotonic with the blood of the intended recipient. Illustrative examples include water, buffered saline, Hanks' solution, Ringer's solution, glucose, fructose, ethanol, animal oils, vegetable oils, and synthetic oils. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0215] In one embodiment, the pharmaceutical composition contains a formulation capable of being injected into a subject. Examples of injectable formulation components include isotonic sterile saline solutions, salts (e.g., sodium dihydrogen phosphate or disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and mixtures of such salts), buffered saline solutions, sugars (e.g., glucose), and water for injection. Pharmaceutical compositions include dried (e.g., freeze-dried) compositions that, upon addition of sterile water or physiological saline, allow for the formation of a solution suitable for administration.

[0216] Alternatively, suspensions can be prepared as suitable oil-injection suspensions. Suitable lipophilic solvents or mediators include fatty oils (e.g., sesame oil), or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, thereby facilitating the preparation of a concentrated solution.

[0217] "Effective amount" or "sufficient amount" refers to an amount that provides an indicated or desired effect. An effective amount may be administered alone or in combination with one or more other compositions (e.g., additional therapeutic agents or immunosuppressants), treatments, regimens, or treatment regimens; and provides a long-term or short-term response.

[0218] A pharmaceutical composition comprising a transgenic protein encoding a PPT1 polypeptide may be delivered to a subject to allow for the production of the encoded protein. Delivery may be in vivo or ex vivo. In some embodiments, the pharmaceutical composition contains sufficient genetic material to enable the recipient to produce a therapeutically effective amount of the protein in the subject.

[0219] "Therapeutically effective amount" refers to an amount of active ingredient or component that elicits a desired or indicated biological or medical response in a subject. A therapeutically effective amount may be determined based on observed symptoms and / or by using biomarkers associated with a specific disease or disorder. The selection of a specific effective amount may be optimized considering various factors, including the disease or disorder to be treated or prevented, the symptoms involved, safety and efficacy in animal models, the patient's weight, and the patient's immune status. The optimal dosage used in the formulation will also depend on the route of administration and the severity of the disease or disorder, and can be evaluated based on the patient's condition. The effective dosage can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0220] In some embodiments, the pharmaceutical composition comprising the rAAV carrier comprises an empty AAV capsid. In some embodiments, in the pharmaceutical composition comprising the rAAV carrier and the empty AAV capsid, the ratio of the empty AAV capsid to the rAAV carrier is within or between about 100:1-50:1, about 50:1-25:1, about 25:1-10:1, about 10:1-1:1, about 1:1-1:10, about 1:10-1:25, about 1:25-1:50, or about 1:50-1:100. In some embodiments, the ratio of empty AAV capsid to rAAV carrier is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0221] Further guidance and examples of pharmaceutical compositions and delivery systems are provided, for example, in the following literature: Remington: The Science and Practice of Pharmacy (2020) 23rd edition, University of the Sciences in Philadelphia, published by Elsevier; The Merck Index (2013) 15th edition, Whitehouse, NJ; Pharmaceutical Principles ofSolid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; and Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th Edition, Lippincott Williams & Wilkins, Baltimore, MD. VIII. Administration and Treatment

[0222] The PPT1 peptide and its encoding polynucleotide construct, viral vector, and nonviral vector may be administered to a subject, preferably a human subject, to provide preventative treatment and / or treatment of a diagnosed disease or disorder that reduces the likelihood or severity of the disease or disorder. In some embodiments, a particular therapeutic agent, route of administration, and / or pharmaceutical composition is selected in consideration of the specific disease or disorder to be treated.

[0223] Subjects with a particular disease or disorder or an increased risk of having a particular disease or disorder may be identified, for example, based on symptoms, PPT1 activity, biomarkers, and genetic markers. For example, subjects with increased risk of developing NCL1 symptoms and subjects diagnosed with NCL1 can be treated.

[0224] NCL1 is an autosomal recessive progressive neurodegenerative disease whose symptoms vary depending on the onset. The main symptoms include: onset at birth (congenital), microcephaly, malformation features, seizures and hypermotor activity; onset at 6–18 months (infants), decreased head growth, neurodevelopmental regression and seizures; onset at 2–4 years (variant), seizures, neurodevelopmental regression and behavioral disorders; and onset at 5–7 years (adolescents), vision loss and cognitive decline. (Simonati and Williams (2022) Front. Neurol. 11;13:811686.)

[0225] Different mutations are associated with NCL1. References providing examples of NCL1-related mutations include Sheth et al., (2018) BMC Neurol. 12;18(1):203; Kumar et al., Advances in Protein Chemistry and Structural Biology (2022) 132:89-109; Kousi et al., (2012) Hum. Mutat. 33(1): 42-63; and hyperlink: / / www.uniprot.org / uniprotkb / P50897 / entry#disease_variants(January 17, 2023); each of these are incorporated herein by reference in their entirety.

[0226] Due to the progressive nature of NCL1, early treatment is very important. In some embodiments, treatment is administered before the primary symptoms are identified. Such patients can be identified, for example, based on PPT1 enzyme activity levels and / or the presence of PPT1 mutations.

[0227] In some embodiments, patients diagnosed with NCL1 are treated. Diagnosis may be based on symptoms, genetic testing, and / or measurement of enzyme activity.

[0228] Potential routes of administration include subcutaneous, epidermal, intradermal, intrathecal, orbital, mucosal, nasal, intraperitoneal, intravenous, intrapleural, intraarterial, intracavitary, oral, intrahepatic, via portal vein, intramuscular, intracranial, intraparenchymal, intracisional, intracranial, intracerebellomedullary cistern, intraventricular, or intraventricular administration. In some embodiments, a viral or nonviral vector is administered to the patient via infusion in a drug carrier.

[0229] A suitable route of administration should provide therapeutic delivery to the CNS. Delivery to the CNS can be achieved using various initial routes of administration, including extra-CNS administration, such as intravenous administration in the brain and spinal cord, and delivery to the eye (e.g., intravitreal and subretinal); and direct administration to the brain (e.g., intraparenchymal, intravenous, and intracisional) and / or the spine (e.g., intrathecal). (Zhu et al., (2021) Trends Mol. Med. 27(6):524-537, which is incorporated herein by reference in its entirety.)

[0230] When the initial site of administration is extra-brain, techniques that provide transport across the blood-brain barrier can be used to facilitate administration to the brain. Examples of such techniques include disrupting the blood-brain barrier and utilizing blood-brain barrier carriers (Chen et al., (2021) J. Control. Release 333:129-138; Bellettato and Scrapa, Italian Journal of Pediatrics (2018) 44(Supplement 2):131; Haumann et al., (2020) CNS Drugs 34, 1121-1131; and Cammilleri et al., (2020) J. Clin. Neurophysiol. 37(2):104-117; each of which is hereby incorporated herein by reference in its entirety). Techniques that facilitate crossing the blood-brain barrier can be used to deliver mediators and / or PPT1 protein.

[0231] In some embodiments, treatment is performed using an expression system that provides peptide expression outside the CNS (e.g., high hepatic expression) in combination with techniques that facilitate PPT1 transport across the blood-brain barrier.

[0232] In some embodiments, treatment is performed using techniques that facilitate the crossing of the delivery medium across the blood-brain barrier. In other embodiments, focused ultrasound in combination with microbubbles is used to facilitate crossing the blood-brain barrier (see, for example, Cammilleri et al., (2020) J Clin Neurophysiol. 37(2):104-117, which is incorporated herein by reference in its entirety).

[0233] In some embodiments, an AAV capsid that provides CNS or crosses the blood-brain barrier is used. Examples of such capsids are provided in the specification, page 32 / 120, CN 121443306 A, and are cited in: Chen et al., (2021) J. Control. Release 333:129-138 (e.g., AAV9, AAV-PHP⋅B, AAV-PHP.eB, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, and AAV1 / rh.10), U.S. Patent No. 9,585,971, and U.S. Patent Publication No. US 202 / 1214749, each of which is incorporated herein by reference in its entirety.

[0234] CNS administration can also be performed, for example, by using a needle or catheter to administer directly to the brain. (e.g., International Publication No. WO 2021 / 108809; Cohen-Pferrer et al., Pediatric Neurology 67 (2017) 23-35; and U.S. Patent No. 10,369,329; each of which is incorporated herein by reference in its entirety.)

[0235] Another example of a technique for CNS administration is convection-enhanced delivery. Convection-enhanced delivery involves surgical exposure of the brain followed by placement of a catheter directly into the target region, followed by infusion of a therapeutic agent (e.g., U.S. Patent Publication No. 2022 / 010001; and Debinski et al., (2009) Expert Rev Neurother. 9(10):1519-27; both of which are incorporated herein by reference in their entirety).

[0236] CNS delivery devices, systems, and technologies also include, for example, those described in the following documents: U.S. Patent No. 8,128,600, U.S. Patent Publication No. 2020 / 0324089, U.S. Patent No. 1,112,9643, U.S. Patent No. 1,115,4377, U.S. Patent Publication No. 2021 / 0343,397, U.S. Patent Publication No. 2021 / 0282,866, U.S. Patent No. 9,572,928, U.S. Patent No. 8,337,458, U.S. Patent No. 10,722,265, and U.S. Patent Publication No. 2021 / 214,749, each of which is incorporated herein by reference in its entirety.

[0237] Delivery of the PPT1 peptide and encoding nucleic acid can also provide benefits in treating PPT1 deficiency or defective lysosomal storage outside the CNS. In some embodiments, administration provides systemic delivery. In further embodiments, treatment includes the use of an expression cassette or viral vector containing a pervasive or hybrid promoter. In further embodiments, techniques and / or vectors that facilitate transport across the blood-brain barrier are used to facilitate CNS entry.

[0238] In some embodiments, the expression cassette contains a PGK promoter, a CBh promoter, or an E1F α promoter.

[0239] The optimal dose can vary depending on various factors such as the specific therapeutic agent, the desired endpoint, and the route of administration. The amount, quantity, frequency, or duration of the dose may be increased or decreased proportionally, taking into account adverse side effects, complications, or other risk factors of the treatment or therapy, as well as the condition of the subject.

[0240] A “unit dosage form” refers to a physically discrete unit containing a predetermined effective amount of the active ingredient combined with a pharmaceutically acceptable carrier. Unit dosage forms may be provided, for example, in ampoules and vials that may include pharmaceutically acceptable carriers, or in compositions in a lyophilized or freeze-dried state. In the case of a lyophilized or freeze-dried state, a sterile liquid carrier may be added prior to administration. Individual unit dosage forms may be included in multi-dose kits or containers.

[0241] An “effective amount” achieves the desired or indicated effect. For example, an effective amount for treatment reduces one or more adverse symptoms, reduces the likelihood of one or more symptoms associated with a disease or disorder, or reduces the progression of a disease or disorder. A preferred effective amount for treatment can effectively reduce multiple or all adverse symptoms.

[0242] In some embodiments, the pharmaceutical composition is administered to the subject at a dose suitable for increasing PPT1 activity. In some embodiments, the dose is sufficient to increase PPT1 activity to the following levels: at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of normal activity; about 10% to 200% average activity, 20% to 150% average activity, or 30% to 100% average activity; or about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% average activity. In some embodiments, PPT1 activity is increased to 10X, 100X, or 1000X of average activity. Average activity refers to the average activity occurring in the general population.

[0243] In different embodiments, a suitable dose is about 0.01 mg / kg to about 10 mg / kg of the subject's body weight. (Instructions for Use, Page 33 / 120, 39 CN 121443306 A)The vector, approximately 0.01 mg / kg to approximately 0.1 mg / kg of the vector per kg of subject body weight, approximately 0.1 mg / kg to approximately 1.0 mg / kg of the vector per kg of subject body weight, or approximately 1.0 mg / kg to approximately 10 mg / kg of the vector per kg of subject body weight.

[0244] Typically, the rAAV dose range is at least 1 x 10⁸ vector genomes / kg (vg / kg) of subject body weight, or more, such as 1 x 10⁹, 1 x 10¹⁰, 1 x 10¹¹, 1 x 10¹², 1 x 10¹³, or 1 x 10¹⁴ or more vector genomes / kg (vg / kg) of subject body weight, to achieve a therapeutic effect. In different implementations, the rAAV dose is approximately 5 x 10¹¹ rAAV vg / kg or greater than approximately 5 x 10¹¹ rAAV vg / kg; approximately 1 x 10¹² rAAV vg / kg or greater than approximately 1 x 10¹² rAAV vg / kg; approximately 2 x 10¹² rAAV vg / kg or greater than approximately 2 x 10¹² rAAV vg / kg; approximately 3 x 10¹² rAAV vg / kg or greater than approximately 3 x 10¹² rAAV vg / kg; approximately 4 x 10¹² rAAV vg / kg or greater than approximately 4 x 10¹² rAAV vg / kg; approximately 5 x 10¹² rAAV vg / kg or greater than approximately 5 x 10¹² rAAV vg / kg; approximately 1 x 10¹³ rAAV vg / kg or greater than approximately 1 x 10¹³ rAAV vg / kg; approximately 2 x 10¹³ rAAV vg / kg or greater than about 2 x 10¹³ rAAV vg / kg; about 3 x 10¹³ rAAV vg / kg or greater than about 3 x 10¹³ rAAV vg / kg; about 4 x 10¹³ rAAV vg / kg or greater than about 4 x 10¹³ rAAV vg / kg; about 5 x 10¹³ rAAV vg / kg or greater than about 5 x 10¹³ rAAV vg / kg; about 6 x 10¹³ rAAV vg / kg or greater than about 6 x 10¹³ rAAV vg / kg.

[0245] Examples of dose ranges for rAAV vg / kg include a dose range of about 5 x 10¹¹ to about 6 x 10¹³ rAAV vg / kg; a dose range of about 5 x 10¹¹ to about 5.5 x 10¹¹ rAAV vg / kg; a dose range of about 5.5 x 10¹¹ to about 6 x 10¹¹ rAAV vg / kg; a dose range of about 6 x 10¹¹ to about 6.5 x 10¹¹ rAAV vg / kg; and a dose range of about 6.5 x 10¹¹ to about 7 x 10¹¹ rAAV vg / kg.Dosage range of approximately 7 x 10¹¹ to approximately 7.5 x 10¹¹ rAAV vg / kg; dosage range of approximately 7.5 x 10¹¹ to approximately 8 x 10¹¹ rAAV vg / kg; dosage range of approximately 8 x 10¹¹ to approximately 8.5 x 10¹¹ rAAV vg / kg; dosage range of approximately 8.5 x 10¹¹ to approximately 9 x 10¹¹ rAAV vg / kg; dosage range of approximately 9 x 10¹¹ to approximately 9.5 x 10¹¹ rAAV vg / kg; dosage range of approximately 9.5 x 10¹¹ to approximately 1 x 10¹² rAAV vg / kg; dosage range of approximately 1 x 10¹² to approximately 1.5 x 10¹² rAAV vg / kg; dosage range of approximately 1.5 x 10¹² to approximately 2 x 10¹² rAAV vg / kg; dosage range of approximately 2 x Dosage range from approximately 10¹² to approximately 2.5 x 10¹² rAAV vg / kg; dosage range from approximately 2.5 x 10¹² to approximately 3 x 10¹² rAAV vg / kg; dosage range from approximately 3 x 10¹² to approximately 3.5 x 10¹² rAAV vg / kg; dosage range from approximately 3.5 x 10¹² to approximately 4 x 10¹² rAAV vg / kg; dosage range from approximately 4 x 10¹² to approximately 4.5 x 10¹² rAAV vg / kg; dosage range from approximately 4.5 x 10¹² to approximately 5 x 10¹² rAAV vg / kg; dosage range from approximately 5 x 10¹² to approximately 5.5 x 10¹² rAAV vg / kg; dosage range from approximately 5.5 x 10¹² to approximately 6 x 10¹² rAAV vg / kg; dosage range from approximately 6 x 10¹² to approximately 6.5 x 10¹² rAAV vg / kg. Dosage range of 10¹² rAAV vg / kg; dosage range of approximately 6.5 x 10¹² to approximately 7 x 10¹² rAAV vg / kg; dosage range of approximately 7 x 10¹² to approximately 7.5 x 10¹² rAAV vg / kg; dosage range of approximately 7.5 x 10¹² to approximately 8 x 10¹² rAAV vg / kg; dosage range of approximately 8 x 10¹² to approximately 8.5 x 10¹² rAAV vg / kg; dosage range of approximately 8.5 x 10¹² to approximately 9 x 10¹² rAAV vg / kg; dosage range of approximately 9 x 10¹² to approximately 9.5 x 10¹² rAAV vg / kg; dosage range of approximately 9.5 x 10¹² to approximately 1 x 10¹³ rAAV vg / kg; dosage range of approximately 1 x 10¹³ to approximately 1.5 x 10¹³ rAAV vg / kg. Dosage range (vg / kg): approximately 1.5 x 10¹³ to approximately 2 x 10¹³ rAAVDosage range of approximately 2 x 10¹³ to approximately 2.5 x 10¹³ rAAV vg / kg; dosage range of approximately 2.5 x 10¹³ to approximately 3 x 10¹³ rAAV vg / kg; dosage range of approximately 3 x 10¹³ to approximately 3.5 x 10¹³ rAAV vg / kg; dosage range of approximately 3.5 x 10¹³ to approximately 4 x 10¹³ rAAV vg / kg; dosage range of approximately 4 x 10¹³ to approximately 4.5 x 10¹³ rAAV vg / kg; dosage range of approximately 4.5 x 10¹³ to approximately 5 x 10¹³ rAAV vg / kg; dosage range of approximately 5 x 10¹³ to approximately 5.5 x 10¹³ rAAV vg / kg; dosage range of approximately 5.5 x 10¹³ to approximately 6 x 10¹³ rAAV vg / kg; dosage range of approximately 6 x The dosage range is from 10¹³ to approximately 1 x 10¹⁴ rAAV vg / kg.

[0246] In some embodiments, rAAV vg / kg is administered at the following doses: about 5 x 10¹¹ vg / kg, about 6 x 10¹¹ vg / kg, about 7 x 10¹¹ vg / kg, about 8 x 10¹¹ vg / kg, about 9 x 10¹¹ vg / kg, about 1 x 10¹² vg / kg, about 2 x 10¹² vg / kg, about 3 x 10¹² vg / kg, about 4 x 10¹² vg / kg, about 5 x 10¹² vg / kg, about 6 x 10¹² vg / kg, about 7 x 10¹² vg / kg, about 8 x 10¹² vg / kg, about 9 x 10¹² vg / kg, about 1 x 10¹³ vg / kg, about 2 x 10¹³ vg / kg, about 3 x 10¹³ vg / kg, about 4 x 10¹³ vg / kg, about 5 x 10¹³ vg / kg, or about 6 x 10¹³ vg / kg.

[0247] In some embodiments, the dosages and dosage ranges of other viral vectors are as provided herein with respect to rAAV. For example, in some embodiments, the dosages and dosage ranges of recombinant adenovirus vectors, recombinant retroviral vectors (e.g., lentiviruses), and recombinant herpes simplex virus vectors are the same as those described above with respect to rAAV.

[0248] In different embodiments, a suitable dosage for PPT1 administration is about 0.01 mg / kg to about 25 mg / kg protein per kg of subject body weight or about 0.1 mg / kg to about 1.0 mg / kg protein per kg of subject body weight.

[0249] In some embodiments, the polypeptide constructs, polynucleotide constructs, viral vectors, and nonviral vectors described herein are administered in combination with additional compounds or treatments for a specific disease or disorder; and / or in combination with compounds that reduce the immune response to the provided or generated polypeptide, polynucleotide, and / or delivery medium. The additional compounds or treatments may be provided in different ways, such as by administration alone; and may be administered or performed before, substantially simultaneously with, or after the administration of the polypeptide constructs, encoding polynucleotide constructs, viral vectors, and nonviral vectors described herein.

[0250] In some embodiments, the administration of the polypeptide constructs, encoding polynucleotide constructs, viral vectors, and nonviral vectors described herein is performed in combination with immunosuppressants or regimens. Such agents and regimens can be used as needed to achieve immune tolerance to the generated PPT1 protein, the provided polynucleotide, or the provided delivery medium, or to reduce the immune response to the generated PPT1 protein, the provided polynucleotide, or the provided delivery medium. Examples of immunosuppressants and regimens include methotrexate, rituximab, intravenous gamma globulin (IVIG), omalizumab, ImmTOR® (synthetic vaccine particle (SVP)-rapamycin (rapamycin encapsulated in biodegradable nanoparticles)), ImmTOR-ILTM (ImmTOR with a Treg-selective IL-2 agonist), B cell depletion, immunoadsorption, and plasma removal.

[0251] In some embodiments, a viral or non-viral vector is administered in combination with one or more immunosuppressants, wherein one or more immunosuppressants are administered before, substantially simultaneously with, or after the administration of the vector or non-viral vector. In some embodiments, one or more immunosuppressants are administered simultaneously with the vector or non-viral vector. In some embodiments, one or more immunosuppressants are administered 1–12 hours, 12–24 hours, or 24–48 hours before administration of the viral or non-viral vector; or 2–4 days, 4–6 days, 6–8 days, 8–10 days, 10–14 days, 14–20 days, 20–25 days, 25–30 days, 30–50 days, or more than 50 days after administration of the viral or non-viral vector. Immunosuppressants can be administered some time after the administration of the vector or non-viral vector, for example, some time after the administration of the vector or non-viral vector (e.g., 20–25 days, 25–30 days, 30–50 days, 50–75 days, 75–100 days, 100–150 days, 150–200 days, or more than 200 days) when the protein encoded by the vector is initially...In cases where expression levels subsequently decrease.

[0252] In some embodiments, the immunosuppressant is an anti-inflammatory agent. In some embodiments, the immunosuppressant is a steroid, such as a corticosteroid. In some implementations, the immunosuppressants are prednisone, prednisolone, calcineurin inhibitors (e.g., cyclosporine, tacrolimus), MMF (mycophenolate mofetil, e.g., CellCept®, Myfortic®), CD52 inhibitors (e.g., alemtuzumab), CTLA4-Ig (e.g., abatacept, beraccept), anti-CD3 mAb, anti-LFA-1 mAb (e.g., efazolin), anti-CD40 mAb (e.g., ASKP1240), anti-CD22 mAb (e.g., epazolizumab), anti-CD20 mAb (e.g., rituximab, olizumab, olfamumab, vetozumab), proteasome inhibitors (e.g., bortezomib), TACI-Ig (e.g., acecicept), anti-C5 mAb (e.g., eculizumab), mycophenolate mofetil, azathioprine, sirolimus everolimus, TNFR-Ig, and anti-TNF. mAb, tofacitinib, anti-IL-2R (e.g., baribizumab, page 35 / 120, CN 121443306 A), anti-IL-17 mAb (e.g., secukinumab), anti-IL-6 mAb (e.g., sirukumab), anti-IL-6 receptor antibody tocilizumab (Actemra®), IL-10 inhibitors, TGF-β inhibitors, B-cell targeting antibodies (e.g., rituximab), mammalian target of rapamycin (mTOR) inhibitors (e.g., rapamycin), synthetic vaccine particles (SVP™) - rapamycin (rapamycin encapsulated in biodegradable nanoparticles), intravenous gamma globulin (IVIG), omalizumab, methotrexate, tyrosine kinase inhibitors (e.g., ibrutinib), cyclophosphamide, fingolimod, B-cell activating factor (BAFF) inhibitors (e.g., anti-BAFF). mAbs, such as belimumab), proliferation-inducing ligand (APRIL) inhibitors, anti-IL-1b mAbs (such as canakinumab (Haris®)), C3a inhibitors, tregitope (see, for example, U.S. Patent No. 10,213,496), or combinations and / or derivatives thereof.

[0253] Immunosuppressive regimens (including the use of rapamycin alone or in combination with IL-10) can be used to reduce, decrease, suppress, prevent, or block humoral and cellular immune responses to the PPT1 protein. Viral vectors (e.g., rAAV) and non-viral vectors are used.Liver gene transfer can be used to induce immune tolerance to the PPT1 protein by inducing regulatory T cells (Tregs).

[0254] Strategies to reduce (overcome) or avoid humoral immunity to viral vectors (such as rAAV) in systemic gene transfer include: administering high vector doses; using empty AAV capsids as bait to adsorb anti-AAV antibodies; administering immunosuppressive drugs to reduce, decrease, suppress, prevent, or eradicate humoral immune responses to rAAV; altering the rAAV capsid serotype or engineering the rAAV capsid to make it less sensitive to neutralizing antibodies; using plasma exchange circulation to adsorb anti-AAV immunoglobulins, thereby reducing anti-AAV antibody titers; and using delivery techniques, such as balloon catheters followed by saline flushing. Such strategies are described in Mingozzi et al., (2013) Blood, 122:23-36. Other strategies include using AAV-specific plasma depletion columns to selectively deplete anti-AAV antibodies from plasma without depleting the total immunoglobulin library, as described in Bertin et al., 2020, Sci. Rep. 10:864. Similar techniques and strategies can be used for other types of viral vectors.

[0255] Empty capsids used as decoy probes can be provided at different ratios to the viral vector. The amount of empty capsids applied can be calibrated based on the amount (titer) of antibodies produced in a particular subject. In some embodiments, the ratio of empty AAV capsid to rAAV vector is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. In certain aspects, the applied ratio of empty AAV capsid to rAAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Preferably, the serotype of the empty capsid is the same as the rAAV serotype.

[0256] Strategies to reduce humoral immunity to rAAV (which can be applied to other viral vectors) include methods for removing, depleting, capturing, and / or inactivating AAV antibodies, commonly referred to as apheresis, and more specifically, involving plasma removal from blood products. Apheresis, or plasma removal, is a process of circulating plasma from a human subject outside the body (in vitro) via a device that alters the plasma by adding, removing, and / or replacing components. Plasma removal can be used to remove human immunoglobulins (e.g., IgG, IgE, IgA, IgD) from blood products (e.g., plasma). This procedure can be used to deplete, capture, and / or inactivate AAV antibodies.Activating, reducing, or removing immunoglobulins (antibodies) that bind to AAV, thereby lowering the titer of AAV antibodies in the treated subject, may contribute to rAAV neutralization. One example is the use of a device consisting of an AAV capsid affinity matrix column, and the passage of blood products (e.g., plasma) through the AAV capsid affinity matrix, resulting in the binding of different isotypes of AAV antibodies. (See, for example, Bertin et al., (2020) Sci. Rep. 10, 864, which is incorporated herein by reference in its entirety.)

[0257] In some embodiments, peptide constructs, polynucleotide constructs, viral vectors, and nonviral vectors may be used in combination with agents that block, inhibit, or reduce the interaction of IgG with neonatal Fc receptors (FcRn), such as anti-FcRn antibodies, to reduce IgG recycling and enhance IgG clearance in vivo; and / or may be used in combination with agents that reduce circulating antibodies binding to PPT1 peptides, encoding nucleic acids, or delivery mediators. In some embodiments, antibody binding is reduced or inhibited by an agent that reduces the interaction of IgG with FcRn, protease, or glycosidase. Specification 36 / 120 pages 42 CN 121443306 A

[0258] In some embodiments, the polypeptide constructs, polynucleotide constructs, viral vectors, and nonviral vectors described herein are used in combination with endopeptidases (e.g., IdeS from Streptococcus pyogenes) or modified variants thereof, or endoglucosidases (e.g., EndoS from Streptococcus pyogenes) or modified variants thereof. For example, such treatment can be performed to reduce or eliminate neutralizing antibodies and make it possible to treat patients previously considered ineligible for treatment. Such strategies are described, for example, in Leborgne et al., (2020) Nat. Med., 26:1096-1101.

[0259] In some embodiments, the treatment of the subject is combined with a compound that reduces the expression of naturally occurring mutant PPT1 (which provides mutant PPT1 with reduced activity). Mutant PPT1 expression can be inhibited, for example, using a repressive nucleic acid that selectively targets the coding sequence of mutant PPT1. The reference to “selectively targeting” mutant PPT1 activity indicates that the expression of the polynucleotide encoding the PPT1 protein, which provides increased activity, is not significantly affected. The repressive nucleic acid can be provided on the same polynucleotide and / or vector encoding the PPT1 protein, or using a separate viral or non-viral vector. Examples of repressive nucleic acids include short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), ribozymes, and antisense RNA. IX. Kits

[0260] The present invention includes kits having packaging materials and one or more components therein. Kits typically includeA label or packaging insert may include a description of the components or instructions for use of the components in vitro, in vivo, or ex vivo. The kit may contain a collection of such components, such as a PPT1 peptide, a viral or non-viral vector, and optionally a second active substance, such as another compound, agent, drug, or composition.

[0261] A kit refers to a physical structure that contains one or more components. Packaging materials may sterilely hold the components and may be made of materials commonly used for such purposes, such as paper, corrugated fiber, glass, plastic, foil, ampoules, vials, and tubes.

[0262] The label or insert may include identification information for one or more of the components, dosage amounts, and clinical pharmacology (including mechanism of action), pharmacokinetics, and pharmacodynamics of one or more active ingredients. The label or insert may include information identifying the manufacturer, batch number, place and date of manufacture, and expiration date. The label or insert may include information about the diseases that the kit components may target. Labels or inserts may include instructions for use by clinicians or subjects in the use of one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions may include dosage, frequency, or duration, and instructions for carrying out any of the methods, uses, treatment protocols, or prophylactic or therapeutic regimens described herein.

[0263] Labels or inserts may include information about one or more benefits that the components may provide, such as prophylactic or therapeutic benefits. Labels or inserts may include information about potential adverse side effects, complications, or reactions, such as warnings to subjects or clinicians about situations where the use of a particular composition may be unsuitable. Adverse side effects or complications may also occur when subjects are taking, will take, or are currently taking one or more other drugs that may be incompatible with the composition, or when subjects are experiencing, will take, or are currently undergoing another treatment protocol (treatment protocol or therapeutic regimen) that will be incompatible with the composition; therefore, instructions may include information about such incompatibilities.

[0264] Labels or inserts include “printed matter,” such as paper or cardboard, either alone or attached to components, kits, or packaging materials (e.g., boxes), or affixed to ampoules, tubes, or vials containing kit components. Labels or inserts may additionally include computer-readable media such as barcode-printed labels, disks, optical discs (e.g., CD-ROM / RAM or DVD-ROM / RAM, DVD), MP3s, magnetic tapes, or electrical storage media (e.g., RAM and ROM), or hybrids of these media (e.g., magnetic / optical storage media, flash memory media, or memory cards). X. mRNA Therapeutic Agent Instructions for Use 37 / 120 pages 43 CN 121443306 A

[0265] In some embodiments, the RNA form of the nucleic acid encoding the PPT1 polypeptide described herein is provided as an mRNA construct capable of expressing the encoded polypeptide within cells. The mRNA construct comprises a 5' cap, a 5' UTR, the encoding RNA, a 3' UTR, and a poly(a) tail. The UTR and poly(a) tail can provide different functions, such as participating in subcellular localization of mRNA, regulating translation efficiency, and mRNA stability. The design and generation of mRNA constructs (including various modifications) have been described in various publications, such as Ouranidis et al., (2022) Biomedicines, 10, 50; Qin et al., Signal Transduct Target Ther. (2022) 21;7(1):166; and U.S. Patent Publication No. US 2013 / 0259924, each of which is incorporated herein by reference in its entirety.

[0266] In some embodiments, nanoparticles are used to deliver the mRNA construct to cells or a subject. Examples of nanoparticles include those described in Section VI (including VI.A. to VI.E) of the previous article; Ouranidis et al., (2022) Biomedicines, 10, 50; and those provided in U.S. Patent Publication No. US 2013 / 0259924. XI. Other Aspects and Embodiments

[0267] Examples of other aspects, embodiments, and combinations thereof include the following: 1) a polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase-1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence having at least 95%, at least 97%, or at least 99% identity with the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence comprises a substitution of aspartic acid (D) at its N-terminus with glycine (G), valine (V), or leucine (L); and / or (c) the PPT1 sequence comprises an N-terminal amino acid sequence of leucine-glutamine-histidine-leucine; and / or (d) the nucleic acid sequence comprises a sequence of leucine-glutamine-histidine-leucine. 1) Any one of SEQ ID NO: 16-94 having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or 100% identity. 2) The polynucleotide according to claim 1, wherein the PPT1 polypeptide further comprises the signal sequence containing the sequence of any one of SEQ ID NO: 16-27.3) The polynucleotide of claim 2, wherein the nucleic acid comprises the signal coding sequence of any one of SEQ ID NO: 43-58. 4) The polynucleotide of claim 2, wherein the polypeptide comprises the signal sequence of any one of SEQ ID NO: 16-21 and 24-27. 5) The polynucleotide of claim 4, wherein the signal sequence comprises the sequence of SEQ ID NO: 16 or 19. 6) The polynucleotide of claim 5, wherein the signal sequence comprises the sequence of SEQ ID NO: 16, and the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 43, or the signal peptide comprises the sequence of SEQ ID NO: 19, and the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 50. 7) The polynucleotide of claim 2, wherein the polypeptide comprises the signal coding sequence of SEQ ID NO: 23. 8) The polynucleotide of claim 7, wherein the nucleic acid sequence comprises the signal coding sequence of SEQ ID NO: 54. 9) The polynucleotide according to any one of 1-6, wherein the PPT1 amino acid sequence comprises an N-terminal aspartic acid D substituted with G, V or L, and the PPT1 amino acid sequence has at least 95%, at least 97%, or at least 99% identity with SEQ ID NO: 1; or comprises SEQ ID NO: 1. (Specification 38 / 120 pages 44 CN 121443306 A) 10) The polynucleotide according to 9, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G, X is V, or X is L. 11) The polynucleotide according to any one of 1-3, 7 or 8, wherein the PPT1 sequence comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine and the PPT1 amino acid sequence has at least 95%, at least 97%, or at least 99% identity with the sequence of SEQ ID NO: 1. 12) The polynucleotide according to 11, wherein the PPT1 sequence comprises SEQ ID NO: 4. 13) The polynucleotide according to any one of 1-12, wherein the nucleic acid comprising the PPT1 coding sequence comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO: 61-94, or comprises any one of SEQ ID NO: 61-94; in a further embodiment, the PPT1 coding sequence comprises a sequence having at least 85% identity with any one of SEQ ID NO: 62-64, 71, 74, 78, 79, or 83.The PPT1 encoded sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity with any of the sequences SEQ ID NO: 62-64, 71, 74, 78, 79, or 83; in a further embodiment, the PPT1 encoded sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity with any of the sequences SEQ ID NO: 62-64, 71, 74, 78, 79, or 83; in a further embodiment, the PPT1 encoded sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity with any of the sequences SEQ ID NO: 64; in a further embodiment, the PPT1 encoded sequence comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity with any of the sequences SEQ ID NO: 64; The sequence of SEQ ID NO: 79 has at least 85% identity, at least 90% identity, at least 95% identity, at least 99% identity, or contains the sequence of SEQ ID NO: 79. 14) The polynucleotide according to claim 1, wherein the PPT1 polypeptide contains a sequence having at least 99% identity with any one of SEQ ID NO: 31-42 or contains any one of SEQ ID NO: 31-42. 15) The polynucleotide according to claim 14, wherein the PPT1 polypeptide contains the sequence of SEQ ID NO: 31 or SEQ ID NO: 34. 16) The polynucleotide according to claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO: 107-125 and 168; or the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO: 107-125, or comprises any one of SEQ NO: 107-125 and 168. In a further embodiment, the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO: 64, or a sequence comprising SEQ ID NO: 64; in a further embodimentIn the scheme, the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, where X is G, and the nucleic acid comprises a sequence having at least 85% identity, at least 90% identity, at least 95% identity, or at least 99% identity with SEQ ID NO: 79, or a sequence comprising SEQ ID NO: 79. 17) The polynucleotide of claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 34, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO: 126-140 and 161-167; or the PPT1 polypeptide comprises the sequence of SEQ ID NO: 34, wherein X is G, and the nucleic acid comprises a sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity with any one of SEQ ID NO: 126-140 and 161-167, or comprises any one of SEQ NO: 126-140 and 161-167. 18) The polynucleotide of claim 1, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 38. 19) A palmitoyl protein thioesterase-1 (PPT1) polypeptide, said PPT1 polypeptide comprising a PPT1 amino acid sequence having at least 95%, at least 97%, and at least 99% identity with the sequence of SEQ ID NO: 1, wherein: (a) said PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NO: 16-27 or a variant thereof having an amino acid substitution, deletion, or insertion; and / or (b) said PPT1 amino acid sequence comprising a substitution of its N-terminal aspartic acid (D) with glycine (G), valine (V), or leucine (L); and / or (c) said PPT1 sequence comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine. 20) The polypeptide according to claim 19, wherein said PPT1 polypeptide further comprises a signal sequence containing the sequence of any one of SEQ ID NO: 16-27. 21) The polypeptide of claim 20, wherein the polypeptide comprises the signal sequence of any one of SEQ ID NO: 16-21 and 24-27. 22) The polypeptide of claim 21, wherein the signal sequence comprises the sequence of any one of SEQ ID NO: 16 or 19. 23) The polypeptide of claim 20, wherein the polypeptide comprises the signal sequence of SEQ ID NO: 23.24) The polypeptide according to any one of 19-23, wherein the PPT1 amino acid sequence comprises an N-terminal aspartic acid D substituted with G, V, or L, and the PPT1 amino acid sequence has at least 97% or at least 99% identity with the sequence of SEQ ID NO: 1. 25) The polypeptide according to 24, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G. 26) The polypeptide according to 19, wherein the PPT1 sequence comprises the N-terminal amino acid sequence leucine-glutamine-histidine-leucine, and the PPT1 amino acid sequence has at least 97% or at least 99% identity with the sequence of SEQ ID NO: 1. 27) The polypeptide according to 26, wherein the PPT1 sequence comprises SEQ ID NO: 4. 28) The polypeptide according to 19, wherein the PPT1 polypeptide comprises a sequence having at least 99% or 100% identity with any one of SEQ ID NO: 31-42. 29) The polypeptide of claim 28, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G; the sequence of SEQ ID NO: 34, wherein X is G; or the sequence of SEQ ID NO: 38. 30) A polynucleotide comprising a nucleic acid sequence encoding a PPT1 polypeptide, wherein the nucleic acid sequence encoding PPT1 encodes the PPT1 polypeptide according to any one of 19-29. 31) A polynucleotide comprising two or more exons and one or more introns together encoding the PPT1 polypeptide according to any one of 19-29. 32) A polynucleotide according to any one of 1-18, 30, or 31, wherein the polynucleotide is an expression cassette comprising one or more expression control elements operatively linked to the nucleic acid encoding the PPT1 polypeptide. 33) A polynucleotide according to claim 32, wherein the nucleic acid encoding the PPT1 polypeptide is operatively linked to an upstream promoter and a downstream polyadenylation signal. Specification 40 / 120 pages 46 CN 121443306 A 34) The polynucleotide according to claim 32, wherein the expression cassette from 5' to 3' comprises, operatively linked to, the nucleic acid encoding the PPT1 polypeptide: a promoter, a Kozak sequence, the nucleic acid sequence encoding the PPT1 polypeptide, and a polyadenylation signal. 35) The polynucleotide according to claim 33 or 34, wherein the promoter comprises a sequence having at least 95%, at least 97%, or at least 99% identity with the sequence of SEQ ID NO: 5, or comprises SEQ ID NO: 5, or the promoter comprises a sequence having at least 95%, at least 97%, or at least 99% identity with the sequence of SEQ ID NO: 5.The sequence of SEQ ID NO: 173 has at least 95%, at least 97%, or at least 99% identity, or contains SEQ ID NO: 173. 36) The polynucleotide according to any one of 33-35, wherein the polyadenylation signal operatively linked to the PPT1-encoding nucleotide sequence comprises a sequence having at least 95%, at least 97%, or at least 99% identity with the sequence of SEQ ID NO: 6, or contains SEQ ID NO: 6. 37) A polynucleotide according to any one of 32-36, wherein the expression cassette comprises a nucleotide sequence having at least 95% and at least 97% identity with any one of SEQ ID NO: 141-143, or comprises any one of SEQ ID NO: 141-143; or the expression cassette comprises a nucleotide sequence having at least 95% and at least 97% identity with SEQ ID NO: 169, or comprises SEQ ID NO: 169; or the expression cassette comprises a nucleotide sequence having at least 95% and at least 97% identity with SEQ ID NO: 170, or comprises SEQ ID NO: 170. 38) A polynucleotide according to any one of 1-18 and 30-37, wherein the polynucleotide is DNA. 39) A recombinant viral vector nucleic acid comprising a polynucleotide according to any one of 1-18 and 30-38 and 5' and / or 3' viral elements providing viral packaging and replication. 40) The recombinant viral vector nucleic acid according to claim 39, wherein the recombinant viral vector nucleic acid is DNA and comprises an adeno-associated virus (AAV) inverted repeat sequence (ITR) located on the 5' end flanking of the polynucleotide and an AAV ITR located on the 3' end flanking of the polynucleotide. 41) The recombinant viral vector nucleic acid according to claim 40, wherein the recombinant viral vector nucleic acid comprises a 5' ITR and a 3' ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B. 42) The recombinant viral vector nucleic acid according to claim 40, wherein the 5' ITR comprises a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with the sequence of SEQ ID NO: 8, or comprises SEQ ID NO: 8; and the 3' ITR comprises a sequence having at least 95% identity, at least 97% identity, or at least 99% identity with the sequence of SEQ ID NO: 9, or comprises SEQ ID NO: 9. 43) The recombinant viral vector nucleic acid according to any one of claims 39-42, wherein the recombinant viral vector nucleic acid further...44) A recombinant viral vector nucleic acid according to any one of 39-43, wherein the recombinant viral vector nucleic acid further comprises one or more filling sequences. 45) A recombinant viral vector nucleic acid according to any one of 39-44, wherein the recombinant viral vector nucleic acid comprises a sequence having at least 95%, at least 97%, at least 99%, or 100% identity with any one of SEQ ID NO: 144-154; or the recombinant viral vector nucleic acid comprises a sequence having at least 95%, at least 97%, at least 99%, or 100% identity with sequence SEQ ID NO: 171; or the recombinant viral vector nucleic acid comprises a sequence having at least 95%, at least 97%, at least 99%, or 100% identity with sequence SEQ ID NO: 172. 46) A gene delivery medium comprising a viral or non-viral vector and a polynucleotide according to any one of 18, 30-38, or a recombinant viral vector nucleic acid according to any one of 39-45. 47) The gene delivery medium according to claim 46, wherein the gene delivery medium is a viral vector. 48) The gene delivery medium according to claim 47, wherein the viral vector is a recombinant AAV vector, a recombinant lentiviral vector, or a recombinant adenovirus vector. 49) The gene delivery medium according to claim 48, wherein the viral vector is a recombinant AAV vector, and the recombinant AAV vector comprises a capsid having at least 90%, at least 95%, or 100% identity with any of the following VP1, VP2, or VP3 sequences: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12, or SEQ ID NO: 15. 50) The gene delivery medium according to claim 49, wherein the capsid is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10,AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or the capsid comprises VP1 of SEQ ID NO: 12 or SEQ ID NO: 15; in a further embodiment, the capsid comprises VP1 containing the sequence of SEQ ID NO: 12, VP2 containing the sequence of SEQ ID NO: 13, and VP3 containing the sequence of SEQ ID NO: 14. 51) The gene delivery medium according to claim 46, wherein the gene delivery medium is a non-viral vector. 52) The gene delivery medium according to claim 51, wherein the non-viral vector is a nanoparticle selected from: lipid nanoparticles (LNP), polymer nanoparticles, lipid polymer nanoparticles (LPNP), protein- or peptide-based nanoparticles, DNA dendritic polymers or DNA-based nanocarriers, carbon nanotubes, microparticles, microcapsules, inorganic nanoparticles, peptide cage nanoparticles, and exosomes. 53) The gene delivery medium according to claim 52, wherein the non-viral vector is an LNP or LPNP. 54) A pharmaceutical composition comprising a polynucleotide according to any one of 1-18 or 30-38, a polypeptide according to any one of 19-29, a recombinant viral vector nucleic acid according to any one of 39-45, or a gene delivery medium according to any one of 46-53, and a pharmaceutically acceptable carrier. 55) A method of increasing PPT1 in a subject, the method comprising administering to the subject a polynucleotide according to any one of 1-18 or 30-38, a polypeptide according to any one of 19-29, a recombinant viral vector nucleic acid according to any one of 39-45, or a gene delivery medium according to any one of 46-53, or the pharmaceutical composition according to 54. 56) A method of treating neuronal ceroid lipofuscin deposition 1 in a subject, the method comprising administering to the subject a polynucleotide according to any one of 1-18 or 30-38, a polypeptide according to any one of 19-29, a recombinant viral vector nucleic acid according to any one of 39-45, or a gene delivery medium according to any one of 46-53, or the pharmaceutical composition according to 54. 57) The method of claim 55 or 56, wherein the administration comprises intraparenchymal, intracisional, or intraventricular administration. In a further embodiment, the administration is intraventricular; and the administration is intraventricular and results in significant rAAV delivery to at least the frontal cortex, parietal cortex, temporal cortex, occipital cortex, thalamus, cerebellar cortex, hippocampus, corpus callosum, spinal cord, caudate nucleus, choroid plexus, optic chiasm, fornix, periaqueductal gray matter, olfactory bulb, and optic nerve. 58) The method of claim 55 or 56, wherein the administration comprises initial administration outside the central nervous system (CNS).59) The method according to any one of 55-58, wherein the administration is systemic. 60) The method according to any one of 55-59, wherein the subject is a human. Specification 42 / 120 pages 48 CN 121443306 A 61) An AAV vector genomic plasmid comprising recombinant viral vector nucleic acid according to any one of 39-45. 62) The AAV genomic plasmid according to 61, wherein the plasmid lacks the rep and cap genes. 63) A method for producing an rAAV vector, the method comprising the step of culturing an rAAV production cell line containing rAAV helper viral activity, wherein the genome of the production cell comprises recombinant viral vector nucleic acid, the rep gene, and the cap gene according to any one of 39-45, wherein the rAAV vector is produced. 64) A method for producing an rAAV vector, the method comprising culturing rAAV-permitting cells containing an AAV genomic plasmid according to 61 or 62, wherein the rAAV-permitting cells further contain (a) a rep gene and a cap gene provided as part of a cell genome and / or provided by one or more separate plasmids, and (b) helper viral activity provided by the cell genome and / or provided by one or more separate plasmids. 65) The method according to 64, wherein the rAAV-permitting cell is a packaging cell, wherein the packaged genome contains a cap gene and a rep gene. 66) The method according to 64, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the helper activity is provided by a helper plasmid. 67) A method for obtaining an rAAV vector, the method comprising the steps of: (a) producing the rAAV using the method according to any one of 63-67 and (b) purifying the rAAV. XII. Sequence

[0268] Table 2 provides different nucleic acid and amino acid sequences. In some cases, variable sequences are indicated in the description. When a signal sequence is present in the full-length PPT1 sequence, the signal sequence is underlined. Some nucleic acid sequences indicated in bold provide codons. The mention of "derived" regarding the signal amino acid sequence indicates that the natural sequence has been modified.

[0269] In different embodiments, the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any nucleic acid sequence provided in Table 2; the polynucleotide comprises a nucleic acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any nucleic acid sequence provided in Table 2.The nucleic acid sequence wherein the stop codon shown in bold is absent and / or is replaced by a different stop codon; or the polypeptide contains an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any amino acid sequence provided in Table 2.

[0270] Table 2 Specification 43 / 120 pages 49 CN 121443306 A Specification 44 / 120 pages 50 CN 121443306 A Specification 45 / 120 pages 51 CN 121443306 A Specification 46 / 120 pages 52 CN 121443306 A Specification 47 / 120 pages 53 CN 121443306 A Specification 48 / 120 pages 54 CN 121443306 A Specification 49 / 120 pages 55 CN 121443306 A Specification 50 / 120 pages 56 CN 121443306 A Specification 51 / 120 pages 57 CN 121443306 A Specification 52 / 120 pages 58 CN 121443306 A Specification 53 / 120 Page 59 CN 121443306 A Instruction Manual 54 / 120 Page 60 CN 121443306 A Instruction Manual 55 / 120 Page 61 CN 121443306 A Instruction Manual 56 / 120 Page 62 CN 121443306 A Instruction Manual 57 / 120 Page 63 CN 121443306 A Instruction Manual 58 / 120 Page 64 CN 121443306 A Instruction Manual 59 / 120 Page 65 CN 121443306 A Instruction Manual 60 / 120 Page 66 CN 121443306 A Instruction Manual 61 / 120 Page 67 CN 121443306 A Instruction Manual 62 / 120 Page 68 CN 121443306 A Instruction Manual 63 / 120 Page 69 CN 121443306 A Instruction manual 64 / 120 pages 70 CN 121443306 A Instruction manual 65 / 120 pages 71 CN 121443306 A Instruction manual 66 / 120 pages 72 CN 121443306 A Instruction manual 67 / 120 pages 73 CN 121443306 A Instruction manual 68 / 120 pages 74 CN 121443306 A Instruction manual 69 / 120 pages 75 CN121443306 A Instruction Manual 70 / 120 pages 76 CN 121443306 A Instruction Manual 71 / 120 pages 77 CN 121443306 A Instruction Manual 72 / 120 pages 78 CN 121443306 A Instruction Manual 73 / 120 pages 79 CN 121443306 A Instruction Manual 74 / 120 pages 80 CN 121443306 A Instruction Manual 75 / 120 pages 81 CN 121443306 A Instruction Manual 76 / 120 pages 82 CN 121443306 A Instruction Manual 77 / 120 pages 83 CN 121443306 A Instruction Manual 78 / 120 pages 84 CN 121443306 A Instruction Manual 79 / 120 pages 85 CN 121443306 A Instruction manual, pages 80 / 120, 86 CN 121443306 A; Instruction manual, pages 81 / 120, 87 CN 121443306 A; Instruction manual, pages 82 / 120, 88 CN 121443306 A; Instruction manual, pages 83 / 120, 89 CN 121443306 A; Instruction manual, pages 84 / 120, 90 CN 121443306 A; Instruction manual, pages 85 / 120, 91 CN 121443306 A; Instruction manual, pages 86 / 120, 92 CN 121443306 A; Instruction manual, pages 87 / 120, 93 CN 121443306 A; Instruction manual, pages 88 / 120, 94 CN 121443306 A; Instruction manual, pages 89 / 120, 95 CN 121443306 A; Instruction manual, pages 90 / 120, 96 CN. 121443306 A Instruction Manual 91 / 120 pages 97 CN 121443306 A Instruction Manual 92 / 120 pages 98 CN 121443306 A Instruction Manual 93 / 120 pages 99 CN 121443306 A Instruction Manual 94 / 120 pages 100 CN 121443306 A Instruction Manual 95 / 120 pages 101 CN 121443306 A Instruction Manual 96 / 120 pages 102 CN 121443306 A Instruction Manual 97 / 120 pages 103 CN 121443306 A Instruction Manual 98 / 120 pages 104 CN 121443306 A Instruction Manual 99 / 120 pages 105 CN121443306 A Instruction Manual 100 / 120 pages 106 CN 121443306 A Instruction Manual 101 / 120 pages 107 CN 121443306 A Instruction Manual 102 / 120 pages 108 CN 121443306 A Instruction Manual 103 / 120 pages 109 CN 121443306 A Instruction Manual 104 / 120 pages 110 CN 121443306 A Instruction Manual 105 / 120 pages 111 CN 121443306 A Instruction Manual 106 / 120 pages 112 CN 121443306 A Instruction Manual 107 / 120 pages 113 CN 121443306 A Instruction Manual 108 / 120 pages 114 CN 121443306 A Instruction Manual 109 / 120 Page 115 CN 121443306 A Specification 110 / 120 Page 116 CN 121443306 A Examples

[0271] Examples are provided below to further illustrate different features of the invention and methods of carrying out the invention. The examples provided do not limit the claimed invention.

[0272] Example 1: PPT1 expression construct with exogenous signal peptide

[0273] The activity of endocrine PPT1 was measured in cells transfected with plasmids containing different PPT1 rAAV expression constructs. To evaluate the depalmitoylation activity of palmitoyl-protein thioesterase (PPT1), 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) was used as a substrate. The release of MU from MU-6SPalm-βGal cannot be accomplished by the action of PPT1 alone, because the enzyme only hydrolyzes the palmitoyl thioester bond, producing a non-fluorescent reaction intermediate. Therefore, the intermediate is hydrolyzed using exogenous β-galactosidase or β-glucosidase, resulting in the release of fluorescent 4 MU that can be detected by a fluorescence plate reader. A known amount of free 4 MU is run in parallel and used to plot a standard curve. The level of PPT1 in the sample is quantified using the standard curve, or simply expressed as relative fluorescence use (RFU).

[0274] Figure 1 is a basic schematic diagram of the PPT1 rAAV expression construct, in which the following nucleic acid regions are indicated: 5'-ITR, EF-1α promoter (EF1a), Kozak sequence (Kozak), signal sequence (SS), mature PPT1 sequence (human PPT1), bovine polyadenylation sequence (bGH-pA), filler (filler sequence), synthetic polyadenylation sequence (synthetic pA), and 3'-ITR.

[0275] Table 3 provides the construct names of the AAV nucleic acids and mentions the following SEQ ID NO: the encoded signal peptide sequence.The sequence includes the encoded mature PPT1 amino acid sequence, the encoded polynucleotide signal sequence, the encoded mature PPT1 polynucleotide sequence, and the complete rAAV sequence.

[0276] Table 3 Specification 111 / 120 pages 117 CN 121443306 A

[0277] Table 3 characterizes the different constructs as Group 1, Group 2, or Group 3. Group 1 provides a polypeptide in which (a) the N-terminus of mature PPT1 is substituted and (b) a heterologous signal peptide. Group 2 provides a polypeptide in which (a) the N-terminus of mature PPT1 is substituted and (b) the heterologous signal peptide is modified (derived). Group 3 provides a polypeptide comprising (a) mature PPT1 with N-terminal addition (tPA) or deletion (OSM) and (b) a heterologous signal peptide.

[0278] Figure 2A provides a bar graph showing the PPT1 enzyme activity levels detected in total cell lysates (intracellular) from PPT1 knockout HeLa cells transfected with different AAV plasmid constructs. The x-axis indicates the signal sequence present in full-length PPT1. The data demonstrate the ability of different constructs to express functional PPT1 protein. These values ​​are expressed relative to native (unmodified PPT1, set as 100%). Data are normalized based on transfection efficiency. The number of experiments n = 3–4, except for tPA, where n = 2. These values ​​are mean + SEM.

[0279] Figure 2B provides a bar graph showing the PPT1 enzyme activity levels detected in secretory medium (secreted) collected from PPT1 knockout HeLa cells transfected with AAV plasmids carrying different engineered human PPT1 candidates. The x-axis indicates the secretory signal present in full-length PPT1. The data demonstrate the ability of different constructs to express functional PPT1 protein. These values ​​are expressed relative to native (unmodified PPT1, set as 100%). Data were normalized based on transfection efficiency. Number of experiments n = 3–4. These values ​​are mean + SEM. *p < 0.05, native vs. others, Mann-Whitney test.

[0280] Example 2: PPT1 uptake in untransfected cells

[0281] Uptake of secreted PPT1 expressed from different constructs was measured using plasmids containing different rAAV nucleic acids and TdTomato (red fluorescent protein) reporter. Figure 3A presents a schematic diagram of the plasmid, with the following rAAV nucleic acid regions noted: (Instructions 112 / 120, Page 118, CN 121443306, A 5'-ITR, EF-1α promoter (EF1a), Kozak sequence (Kozak), signal sequence (SS), mature PPT1 sequence (human PPT1), bovine growth hormone polyadenylation sequence (bGH-pA), filler (filler sequence), synthetic polyadenylation sequence (synthetic...)The rAAV nucleic acid constructs are named in Table 4 and the following SEQ ID NOs are noted: the encoded signal peptide sequence, the encoded mature PPT1 amino acid sequence, the encoded polynucleotide signal sequence, the encoded mature PPT1 polynucleotide sequence, and the complete rAAV sequence.

[0283] Table 4

[0284] PPT1 KO HeLa cells were transfected with different plasmids and fixed with 4% paraformaldehyde for 48 hours after transfection. Immunostaining was performed with mouse anti-PPT1 (Novus Biologicals (OTI1F10)) and Alexa 488 conjugated anti-mouse secondary antibody (green fluorescence). The cell nuclei were labeled with Hoechst and were considered blue.

[0285] Images were captured in the Opera Phenix Plus HCS system (Perkin Elmer) (image data not shown). Cells highlighted in green indicate the presence of PPT1, and cells highlighted in yellow indicate the presence of both PPT1 and TdTomato (plasmid-transfected cells). Cells highlighted only in green indicate the uptake of PPT1 secreted by transfected (red) cells present in the culture medium. Figure 3B is a bar graph reflecting the image analysis of immunostaining and showing the ratio of cells with PPT1 (green) to transfected cells (red). The increased ratios of Sp7-F, SPARC, and tPA compared to PPT1 with the native signal peptide indicate an increased number of PPT1-positive cells due to increased uptake of engineered PPT1 carrying the exogenous signal sequence. Each circle (mean of 30 fields) represents independent transfection (from 2–3 experiments). These values ​​are mean + SD. P-values ​​for one-way ANOVA: **** p < 0.0001, *** p < 0.001, ** p < 0.01.

[0286] Figure 3C provides a bar chart showing the results from Figure 3B normalized relative to the native construct. White circles indicate independent transfections. Each point (mean of 30 fields) is an independent transfection (from 2–3 experiments). These values ​​are mean + SD. P-values ​​for one-way ANOVA: **** p < 0.0001, *** p < 0.001, ** p < 0.01.

[0287] Example 3: In vitro expression of PPT1 in cortical neurons transduced by recombinant AAV

[0288] The dose-dependent expression and secretion of PPT1 from primary rat cortical neurons transduced by AAV were evaluated. Recombinant AAVThe overall design of the vector is shown in Figure 1. The recombinant rAAV vector was named “Sp7-F.PPT1” or “inactivated PPT1”. Sp7-F.PPT1 corresponds to Sp7-F as described in Table 3. Inactivated PPT1 encodes a mutant full-length PPT1, wherein the mutation renders the protein non-catalytically active.

[0289] The recombinant rAAV vector was generated by triple transfection of human embryonic kidney cells with a capsid containing VP1 of SEQ ID NO: 12, VP2 of SEQ ID NO: 13, and VP3 of SEQ ID NO: 14, and purified by CsCl purification.

[0290] Cultured neurons were transduced with rAAV at three MOIs (low, 1E+5; medium, 5E+5; and high, 1E+6), and the PPT1 activity in the culture medium was analyzed 3, 4, or 6 days after transduction instructions (pages 113 / 120, CN 121443306 A). The results are shown in Figure 4. Cells transduced with rAAV encoding non-catalytically active PPT1 showed no activity. Untreated or diluted cells were used as negative controls. Purified recombinant PPT1 was used as a positive control for the assay. Each circle represents the value from cells independently transduced from a single well. Data are mean ± SD.

[0291] Glycosylation of secretory PPT1 bands from rat primary cortical neurons transduced at medium and high doses was tested on day 6. PPT1 protein was not observed in the mediator or untransduced cells. Glycosylated PPT1 was detected in cells transduced with the rAAV vector (data not shown).

[0292] Example 4: In vivo expression of PPT1 via recombinant AAV

[0293] PPT1 expression and secretion were measured in C57BL6 / J mice administered rAAV containing Sp7-1.PPT1 viral vector nucleic acid (SEQ ID NO: 143, see Table 4) or viral vector nucleic acid encoding inactivated PPT1 (non-enzymatically active PPT1). Recombinant rAAV vectors were generated using capsids containing VP1 (SEQ ID NO: 12), VP2 (SEQ ID NO: 13), and VP3 (SEQ ID NO: 14), and purified by CsCl purification. The vectors were administered to the hippocampus via stereotactic injection or direct IV administration. Table 5 summarizes the experimental protocols.

[0294] Table 5 N = Number of animals

[0295] Brains were analyzed 6 weeks post-injection. Animals administered only with the diluent served as negative controls.

[0296] Figures 5A, 5B, and 5C show serum PPT1 expression and activity in mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid at different time points. Figure 5A shows the expression and activity of PPT1 in the serum of mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid or diluent.Production of PPT1 in the serum of mice IV-administered with an AAV containing live PPT1 viral vector nucleic acid. Serum samples were collected at 2, 4, or 6 weeks post-administration and analyzed by a capillary-based automated immunoassay (WES; Protein Simple) to detect PPT1. Mice administered with a diluent were used as negative controls. The presence of PPT1 was detected in mice administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid at 2 weeks (indicated by arrows in Figure 5A), and levels increased further at 4 weeks. The signal saturated at 6 weeks. Purified recombinant PPT1 was used as a positive control. Figure 5B shows serum activity in IV-administered mice. Figure 5C shows serum activity in IPa-administered mice (administered via stereotactic injection into the hippocampus). ND, not detected. Data are mean ± SD. P values ​​for ANOVA: *P < 0.05, ***P < 0.001, ****P < 0.0001.

[0297] Figure 6 shows liver PPT1 activity from mice (n = 4, indicated by circles) that were IV-administered with rAAV containing Sp7-F.PPT1 viral vector nucleic acid. Mice injected with the diluent were used as negative controls.

[0298] Figures 7, 8, 9, and 10 show the localization, expression, glycosylation, and activity of PPT1 expressed in the brain after administration of rAAV containing Sp7-F.PPT1 viral vector nucleic acid to the hippocampus in mice. Figure 7 shows an immunohistochemical analysis, according to the specification 114 / 120 pages 120 CN 121443306 A, showing increased PPT1 staining in the hippocampus (indicated by asterisks). Figure 8 shows the detection of PPT1 protein in hippocampal protein lysates analyzed by JESS. The double bands seen in low exposure (indicated as low) indicate two glycosylated forms of PPT1. Figure 9 confirms that PPT1 expressed in the hippocampus is glycosylated. Hippocampal protein extracts were treated with deglycosylation enzymes and analyzed by JESS assays. The decrease in molecular weight after deglycosylation enzyme treatment (using +-labeled lanes) indicated that PPT1 was glycosylated. Figure 10 provides an assay of PPT1 activity in hippocampal lysates, demonstrating that PPT1 expressed in the brain possesses biological activity as described in Example 1.

[0299] Example 5: CNS Distribution of rAAV Encoding PPT1

[0300] Wild-type C57BL / 6J male mice were administered rAAV containing Sp7-F.PPT1 viral vector nucleic acid via bilateral intraventricular (ICV) injections on day 1 after birth. Recombinant AAV containing Sp7-F.PPT1 viral vector nucleic acid is described in Example 3. The injections involved administration of 1E+10 vector genomes (vg) in the low-dose group and 1E+11 vg in the high-dose group, 3 µL per side, for a total of 6µL. Six weeks after injection, the brain was extracted and the left hemisphere was dissected into specific regions: cortex, hippocampus, thalamus, brainstem, and cerebellum. Each of these regions was further divided into two equal sections. One section was processed for vector genome analysis, while the other was used to test transgenic (PPT1 protein) expression. The right hemisphere of the brain was fixed and subjected to histological evaluation. The spinal cord was coronally dissected into cervical, thoracic, and lumbar segments, with half of each segment used for vector genome analysis. Cerebrospinal fluid (CSF) was collected by cerebellomedullary cistern puncture and subsequently analyzed for PPT1 levels.

[0301] ICV delivery of the viral vector was shown as a dose-dependent distribution in the brain and spinal cord of mice. Figure 11 shows the vector genome copy number (VGCN) in different brain and spinal cord regions, which is an indicator of viral transduction. Figure 12 shows the fold change (FC) of PPT1 activity in different brain regions of animals injected with rAAV relative to mice injected with the diluent. PPT1 activity in tissue lysates was quantified using MUTG as described in Example 1. Figure 13 shows the FC of PPT1 activity in the cerebrospinal fluid (CSF) of mice that underwent rAAV injection compared to mice injected with the diluent. FC is the mean activity level relative to the diluent group. Figure 14 is a scatter plot showing the correlation between VGCN and PPT1 enzyme activity in the brain. Spearman correlation coefficient r = 0.66, p < 0.0001.

[0302] Figure 15 shows the detection of glycosylated and deglycosylated PPT1 protein in brain lysates analyzed by JESS. Protein extracts were treated with deglycosylation enzymes and analyzed by JESS assay. The double bands seen in lanes 1–2 indicate two glycosylated forms of PPT1. The decrease in molecular weight after deglycosylation enzyme treatment (lanes 3–4) indicates that PPT1 is glycosylated.

[0303] Histological evaluation was performed using an antibody against PPT1, followed by detection using a fluorophore-conjugated secondary antibody. Images were captured using fluorescence microscopy. Animals receiving the diluent showed baseline PPT1 signaling. Animals given a low dose of rAAV showed a slight increase in PPT1 expression compared to the diluent, while animals receiving a high dose showed significantly higher expression. PPT1 expression was evident throughout the cortex (from the rostral region to the caudal region) and was also significant in the hippocampus, striatum, and olfactory bulb. PPT1 signaling surrounded NeuN signaling, which means that PPT1 is primarily expressed within neurons. (Data not shown.)

[0304] Example 6: Biodistribution of rAAV and encoded PPT1 in sheep

[0305] The CNS distribution of rAAV containing Sp7-F.PPT1 (Example 3) and encoded PPT1 was evaluated in sheep. Animals were injected with either Sp7-F.PPT1 (n = 4 sheep) or GFP-encoded viral vector nucleic acid (n = 2 sheep) via unilateral intraventricular (ICV) injection.rAAV (in sheep) was administered to PPT1+ / - male sheep aged nine to ten months. A dose of 1E+14 vg was infused in 2 mL. Eight weeks post-injection, the brain was extracted, coronally sectioned, and the vector genome and PPT1 protein were analyzed from brain perforation samples (3 mm wide) from the target region. Contralateral hemisphere brain slabs were fixed and used for histological evaluation. The spinal cord was coronally dissected into cervical, thoracic, and lumbar segments and underwent vector genome and PPT1 protein analysis. PPT1 levels in cerebrospinal fluid (CSF) were analyzed. The liver was collected and the vector genome was analyzed.

[0306] ICV administration in sheep resulted in vector delivery throughout different brain regions (beak-to-tail) and the spinal cord. In sheep, the vector genome copy number (VGCN) as a measure of viral transduction was measured in different regions as per instructions (pages 115 / 120, CN 121443306 A): cortical regions (Fig. 16A), (Fig. 16B) thalamus, (Fig. 16C) cerebellar cortex, (Fig. 16D) hippocampus, (Fig. 16E) corpus callosum, (Fig. 16F) other indicated brain regions, (Fig. 16G) spinal cord, and (Fig. 16H) liver. Vector genomes from frozen tissue DNA were quantified using quantitative PCR (qPCR) with a standard curve. Vertical arrows indicate samples from contralateral brain regions. Hollow circles correspond to sheep administered the Sp7-F.PPT1 vector, while solid circles depict results from sheep administered the GFP-expressing vector. Data were provided for the following regions: frontal cortex (FC), motor cortex (CM), somatosensory cortex (SSC), piriform cortex (PC), superior lateral fissure gyrus (SSG), external lateral sulcus (EcG), internal lateral sulcus (EnG), caudate nucleus (Cau), choroid plexus (Ch Ple), optic chiasm (Opt chi), fornix (For), periaqueductal gray matter (Periaq G), olfactory bulb (Ol), optic nerve (Op), hippocampus (HPC), thalamus (Tha), corpus callosum (Cca), cerebellar cortex (Cer ctx), cervical spinal cord (SC Cer), thoracic spinal cord (SC Tho), and lumbar spinal cord (SC Lum).

[0307] PPT1 activity assays showed the expression and secretion of functional PPT1 in the brain and CSF. PPT1 activity was measured using MUTG as described in Example 1. Figures 17A-17D show PPT1 activity in the cortex (Figure 17A), thalamus (Figure 17B), cerebellar cortex (Figure 17C), and caudate nucleus (Figure 17D) of sheep injected with Sp7-F.PPT1 (n = 4 sheep) or GFP (n = 2 sheep) rAAV. Each circle represents the result of a tissue perforation sample from one brain region. N indicates the number of regions from 2 GFP-treated animals and 4 PPT1-treated animals. Data are mean ±SEM. Statistical analysis was performed using the Mann-Whitney U test, *P < 0.05, ***P < 0.001.

[0308] Figure 18 provides 95% confidence intervals, showing the mean PPT1 activity of the overall treatment groups. After obtaining the logarithmically transformed activity results for all data in Figures 17A-17D and considering brain region differences in the number of perforated samples and mean, a hypothesis test for differences in mean treatment type was performed at the 0.05 α level. The test resulted in a significant difference in the mean activity level of PPT1 relative to GFP. The mean estimated fold change of PPT1 relative to GFP was 3.9, and the median of PPT1 was 73 nmol / mg / h, compared to a median of 19 nmol / mg / h for GFP. ****P < 0.0001, weighted two-way ANOVA.

[0309] Figure 19 is a bar graph showing the percentage change in PPT1 activity in the cerebrospinal fluid (CSF) of sheep administered rAAV carrying Sp7-F.PPT1 (n = 4 sheep) or GFP (n = 2 sheep). The percentage change is the mean activity relative to the control (GFP animals). Each circle represents one animal. N represents the number of animals. Data are mean ± SEM.

[0310] Figure 20 provides the results of a JESS assay that detected PPT1 expression in tissue lysates of the spinal cord of sheep injected with rAAV vectors encoding nucleic acids containing PPT1 (sheep 1–4) or GFP (sheep 1–2).

[0311] Figure 21 shows the results, which demonstrate the increased mean PPT1 activity levels in the thoracic and lumbar segments of the spinal cord of sheep administered rAAV containing nucleic acids encoding PPT1.

[0312] Immunofluorescence micrographs were taken from sheep cerebellar lobes for histological evaluation to assess transduction and transgene expression. Contralateral hemisphere brain sections were fixed and frozen sections were prepared for immunohistochemical analysis. A blue fluorescent DNA staining agent indicating dsDNA was generated using DAPI (4′,6-diamidinyl-2-phenylindole). PPT1 was immunostained with an antibody against purified PPT1 protein, followed by subsequent staining with a fluorophore-conjugated secondary antibody. Images showing the expression and localization of GFP in PPT1 were visualized and captured using fluorescence microscopy. Extensive expression of transgenes (GFP reporter gene and PPT1) was observed in the cerebellar lobes of sheep brains.

[0313] Example 7: Improvement of motor function

[0314] The effects of rAAV provided with (1) Sp7-PPT1 or (2) SPARC.PPT1 on PPT1 knockout (KO) mice were evaluated using a rotarod apparatus. The evaluation of the rotator is described, for example, in Deacon J. Vis. Exp. May 29, 2013;(75):e2609 (which is hereby incorporated herein by reference in its entirety). Sp7-PPT1, SPARC.PPT1, and rAVV are described in Table 3, page 116 / 120, CN 121443306 A. KO mice were administered rAAV carrying the PPT1 gene via bilateral ICV injection on day 1 after birth and evaluated at 7 months of age. rAAV was produced as described in Example 3 and administered at a dose of 1E+11 vg / animal.

[0315] The results are shown in Figure 22. Untreated (Un) or KO mice treated with a medium (Veh) were used as negative controls. Natural refers to the rAAV vector encoding natural human PPT1 (SEQ ID NO: 29). Sp7-PPT1 is indicated by “1” and SPARC.PPT1 by “2”. Each circle represents one mouse. These bars are mean + SEM. One-way ANOVA, Tukey post-hoc test, *P < 0.05, ***P < 0.001, ****P < 0.0001. CNS-targeted rAAV therapy successfully rescued coordination and balance in PPT1 KO mice by delivering functional human PPT1.

[0316] Example 8: Improvement of motor function and balance

[0317] The ability of CNS-targeted rAAV vectors containing nucleic acids encoding Sp7-PPT1 (rAAV-Sp7-PPT1) and SPARC.PPT1 (rAAV-SPARC.PPT1) to rescue motor coordination and balance in Ppt1- / - mice was evaluated on an accelerator rotarod apparatus. rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 were generated as described in Example 3. The nucleic acids of the rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 vectors are described in Table 4.

[0318] Ppt1- / - mice were administered rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1 via bilateral intraventricular injection of PND 1 (low dose, 1 x 10¹¹ vg / animal) or PND 1 and 3 (high dose, 3.82 x 10¹¹ vg / animal) and evaluated at different time points. Mice underwent four trials at each time point. Each phase included a 5-minute training trial at 4 RPM on a rotarod apparatus (Rotamex, OH). One hour after the training trial, animals were tested with three consecutive accelerated trials, each lasting 5 minutes, during which the speed was varied over 300 seconds. The interval between trials was at least 30 minutes. The latency of falling from the accelerator was recorded and quantitatively analyzed. Untreated or vector-treated Ppt1- / - mice were used as negative controls. “Natural” refers to an AAV vector containing the unmodified human PPT1 gene. In each group, the number of mice ranged from...The number of sexes ranged from 10 to 18, except for the WT and 11 mo groups, where n = 7. The sex distribution within each group was roughly balanced.

[0319] Figure 23 shows the ability of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 to improve motor coordination and balance, as assessed by the latency of falling from the accelerator bar. (See, for example, Kovács and Pearce, Dis Model Mech. April 2015;8(4):351-61): PMC4381334. Both rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 shortened the fall latency.

[0320] Example 9: Muscle Strength

[0321] Based on grip strength assessment, the ability of CNS-targeting rAAV vectors containing nucleic acids encoding Sp7-PPT1 (rAAV-Sp7-PPT1) or SPARC.PPT1 (rAAV-SPARC.PPT1) to improve muscle strength in Ppt1- / - mice was evaluated. rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 were generated as described in Example 3. The nucleic acids of rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 vectors are described in Table 4.

[0322] Ppt1- / - mice were administered rAAV-Sp7-PPT1 and rAAV-SPARC.PPT1 via bilateral intraventricular injection at PND 1 (low dose, 1.00 x 10¹¹ vg / animal) or PND 1 and 3 (high dose, 3.82 x 10¹¹ vg / animal) and evaluated at indicated time points. Forelimb muscle strength was assessed using grip strength in five consecutive trials (San Diego Instruments, San Diego, California). A plot was created using the average of all five trials. The animal was lowered toward the platform and gently pulled back by the experimenter with a consistent force until it released its grip.

[0323] Figure 24 illustrates the effects of rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 on grip strength at different time points. Untreated or vector-treated Ppt1- / - mice were used as negative controls. The term "natural" refers to AAV vectors carrying the unmodified human PPT1 gene. In each group, the number of mice at each time point ranged from 7 to 18, with roughly balanced sexes. Both rAAV-Sp7-F.PPT1 and rAAV-SPARC.PPT1 improved grip strength.

[0324] Example 10: PPT1 activity specification 117 / 120 pages 123 CN 121443306 A

[0325] In PND 1 (low dose, 1.00 x 10¹¹ vg / animal) or PND 1 and 3 (high dose, 3.82 x 10¹¹ vg / animal)Ppt1- / - mice were administered rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1 via bilateral intraventricular injection. As described in Example 1, PPT1 activity in serum at specified time points was quantified using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate. A standard curve (prepared using known concentrations of 4 MU) was used to determine PPT1 activity.

[0326] The results of the PPT1 assay are shown in Figures 25A and 25B. Figure 25A shows the activity at different time points. Figure 25B shows the activity at 8 months. An increase in serum PPT1 levels was observed in mice administered with AAV, indicating sustained long-term expression of PPT1. Untreated or vector-treated Ppt1- / - mice were used as negative controls. “Natural” refers to an AAV vector containing the unmodified human PPT1 gene.

[0327] Example 11: PPT1 activity in the cortex, brainstem, and cerebellum

[0328] Ppt1- / - mice were administered PND 1 (low dose, 1.00 x 10¹¹ vg / animal) via bilateral intraventricular injection of rAAV-Sp7-PPT1 or rAAV-SPARC.PPT1. PPT1 activity was measured in the cortex, brainstem, and cerebellum using 4-methylumbelliferyl-6-thiopalmitoyl-β-D-glucopyranoside (MUTG) as a substrate, as described in Example 1. PPT1 activity was determined using a standard curve (prepared by using 4 MU at a known concentration). Tissues were analyzed at 10 months of age, except for untreated Ppt1- / - mice, which were tested at 8 months of age.

[0329] Figures 26A-26C provide bar graphs illustrating PPT1 activity in the cortex (Figure 26A), brainstem (Figure 26B), and cerebellum (Figure 26C). Increased PPT1 activity in mice administered rAAV at 10 months of age indicates persistent expression of PPT1 in the brains of Ppt1- / - mice. “Natural” refers to an AAV vector containing the unmodified human PPT1 gene. These bars are mean ± SEM. Each circle represents one mouse.

[0330] Example 12: Codon-Optimized PPT1 Coding Sequence

[0331] Expression of different codon-optimized constructs was evaluated in vitro. Plasmids containing rAAV nucleic acids are shown in Figure 27. Constructs are summarized in Tables 6 and 7.

[0332] Table 6

[0333] Table 7 Specification 118 / 120 pages 124 CN 121443306 A

[0334] Examples of full-length rAAV nucleic acid constructs are provided by SEQ ID NO: 171 and SEQ ID NO: 172.Sequence 171 provides the full-length rAAV nucleic acid sequence of a construct named “SpF7-co4”, and Sequence 172 provides the full-length rAAV nucleic acid sequence of a construct named SpF7-co19. Other rAAV sequences were generated by substituting the signal and PPT coding sequence.

[0335] PPT1 knockout HeLa cells were transfected with different AAV plasmids carrying codon-optimized PPT1 coding sequences. PPT1 expression was assessed by measuring enzyme activity in the culture medium 48 hours after transfection.

[0336] Figures 28A and 28B are bar graphs depicting the expression levels of PPT1 in the culture medium of in vitro cultured cells transfected with multiple codon-optimized variants of human PPT1. Different codon-optimized PPT1 cDNA constructs (excluding the signal sequence, labeled CO and the numbers on the x-axis thereafter) were paired with signal sequences (B) Sp7F, SP7F (codon-optimized), (C) SpSPARC, or SpSPARC (codon-optimized) and cloned downstream of the long EF1a promoter, as shown in Figure 27. Each circle represents a well of an independently transfected 96-well plate. These values ​​are presented as mean ± SEM.

[0337] Example 13: Survival Data

[0338] Ppt1- / - mice were administered rAAV containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 via bilateral intraventricular injection at PND 1 (low dose, 1 x 10¹¹ vg / animal) or PND 1 and 3 (high dose, 3.82 x 10¹¹ vg / animal). The rAAV Sp7F.PPT1 and SPARC.PPT1 constructs are summarized in Table 4. The number of mice enrolled in the study was: WT, n = 20; KO, Un = 16; KO, Veh and natural Hi, n = 17. The remaining treatment groups each consisted of 18 mice. Untreated or vector-treated Ppt1- / - mice were used as negative controls. “Natural” refers to an AAV vector containing the unmodified human PPT1 gene.

[0339] Survival data are shown in Figure 29. Mouse data were fitted with a Cox proportional hazards model to determine whether the survival probabilities differed between treatment groups. The survival rate of the KO, Veh group was significantly lower than that of the WT and all AAV treatment groups (all p < 0.01). There was no difference between the KO, Veh and KO, Un groups (p = 0.22). AAV = Adeno-associated virus; CNS - Central nervous system; lo = Low dose (1 x 10¹¹ vg / animal); hi = High dose (3.82 x 10¹¹ vg / animal); KO = Ppt1- / -; PND = Days after birth; Un = Untreated; Veh = Vector; WT = Wild type.

[0340] Example 14: Brain weight reduction

[0341] Ppt1- / - mice were administered rAAV containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 via bilateral intraventricular injection in PND 1 (low dose, 1 x 10¹¹ vg / animal) or PND 1 and 3 (high dose, 3.82 x 10¹¹). The rAAV Sp7F.PPT1 and SPARC.PPT1 constructs are summarized in Table 4. Untreated or mediator-treated Ppt1- / - mice were used as negative controls. “Natural” means AAV vector containing the unmodified human PPT1 gene. Mice were euthanized at 8–10 months of age, and brains were collected and weights recorded. Number of mice, n = 6 / group, except for KO, Veh, n = 4 and Lead 1, hi, n = 9.

[0342] Figure 30 is a bar graph showing the effect of rAAV containing nucleic acids encoding Sp7F.PPT1 or SPARC.PPT1 on brain weight in Ppt1- / - mice. Sp7F.PPT1 and SPARC.PPT1 inhibited brain weight loss in Ppt1- / - mice at both low and high doses.

[0343] Although the invention has been described and illustrated with reference to certain specific embodiments thereof, those skilled in the art will understand that various adjustments, changes, modifications, substitutions, deletions or additions can be made to the procedures and schemes without departing from the spirit and scope of the invention. Instruction manual 120 / 120 pages 126 CN 121443306 A; Instruction manual illustration 1 / 22 pages 127 CN 121443306 A; Instruction manual illustration 2 / 22 pages 128 CN 121443306 A; Instruction manual illustration 3 / 22 pages 129 CN 121443306 A; Instruction manual illustration 4 / 22 pages 130 CN 121443306 A; Instruction manual illustration 5 / 22 pages 131 CN 121443306 A; Instruction manual illustration 6 / 22 pages 132 CN 121443306 A; Instruction manual illustration 7 / 22 pages 133 CN 121443306 A; Instruction manual illustration 8 / 22 pages 134 CN 121443306 A; Instruction manual illustration 9 / 22 pages 135 CN 121443306 A; Instruction manual illustration 10 / 22 pages 136 CN 121443306 A Instruction Manual, Figures 11 / 22, Page 137 CN 121443306 A Instruction Manual, Figures 12 / 22, Page 138 CN121443306 A Instruction Manual Illustrations, Page 13 / 22, 139 CN; 121443306 A Instruction Manual Illustrations, Page 14 / 22, 140 CN; 121443306 A Instruction Manual Illustrations, Page 15 / 22, 141 CN; 121443306 A Instruction Manual Illustrations, Page 16 / 22, 142 CN; 121443306 A Instruction Manual Illustrations, Page 17 / 22, 143 CN; 121443306 A Instruction Manual Illustrations, Page 18 / 22, 144 CN; 121443306 A Instruction Manual Illustrations, Page 19 / 22, 145 CN; 121443306 A Instruction Manual Illustrations, Page 20 / 22, 146 CN; 121443306 A Instruction Manual Illustrations, Page 21 / 22, 147 CN; 121443306 A Instruction Manual Illustrations, Page 22 / 22, 148 CN 121443306 A

Claims

1. A polynucleotide comprising a nucleic acid sequence encoding a palmitoyl protein thioesterase- 1 (PPT1) polypeptide, wherein the PPT1 polypeptide comprises a PPT1 amino acid sequence that is at least 95% identical to the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NOs: 16-27, or a variant thereof having one amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence comprises substitution of an aspartate (D) at its amino terminus with a glycine (G), valine (V), or leucine (L); and / or (c) the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus; and / or (d) the nucleic acid sequence comprises a PPT1 coding sequence that is at least 85% identical to any one of SEQ ID NOs: 61-94.

2. The polynucleotide of claim 1, wherein the PPT1 polypeptide further comprises the signal sequence comprising the sequence of any one of SEQ ID NOs: 16-27.

3. The polynucleotide of claim 2, wherein the nucleic acid comprises a signal-encoding sequence of any one of SEQ ID NOs: 43-58.

4. The polynucleotide of claim 2, wherein the polypeptide comprises a signal sequence of any one of SEQ ID NOs: 16-21 and 24-27.

5. The polynucleotide of claim 4, wherein the signal sequence comprises the sequence of any one of SEQ ID NOs: 16 or 19.

6. The polynucleotide of claim 5, wherein the signal sequence comprises the sequence of SEQ ID NO: 16 and the nucleic acid sequence comprises the signal-encoding sequence of SEQ ID NO: 43; or the signal peptide comprises the sequence of SEQ ID NO: 19 and the nucleic acid sequence comprises the signal-encoding sequence of SEQ ID NO:

50.

7. The polynucleotide of claim 2, wherein the polypeptide comprises the signal sequence of SEQ ID NO:

23.

8. The polynucleotide of claim 7, wherein the nucleic acid sequence comprises the signal-encoding sequence of SEQ ID NO:

54.

9. The polynucleotide of any one of claims 1-6, wherein the PPT1 amino acid sequence comprises substitution of an aspartate D at its amino terminus with a G, V, or L, and the PPT1 amino acid sequence is at least 97% identical to the sequence of SEQ ID NO:

1.

10. The polynucleotide of claim 9, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G. ​ ​ ​ ​ 11. The polynucleotide of any one of claims 1-3, 7, or 8, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus and the PPT1 amino acid sequence has at least 97% identity to the sequence of SEQ ID NO:

1.

12. The polynucleotide of claim 11, wherein the PPT1 sequence comprises the sequence of SEQ ID NO:

4.

13. The polynucleotide of any one of claims 1-12, wherein the nucleic acid comprises a sequence having at least 85% identity to any one of SEQ ID NOs: 61-94.

14. The polynucleotide of claim 1, wherein the PPT1 polypeptide comprises a sequence having at least 99% identity to any one of SEQ ID NOs: 31-42.

15. The polynucleotide of claim 14, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31 or SEQ ID NO:

34.

16. The polynucleotide of claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G, and the nucleic acid comprises a sequence having at least 85% identity to the sequence of any one of SEQ ID NOs: 107-125 and 168.

17. The polynucleotide of claim 16, wherein the nucleic acid comprises a sequence having at least 95% identity to the sequence of any one of SEQ ID NOs: 107-125 and 168.

18. The polynucleotide of claim 17, wherein the nucleic acid comprises the sequence of any one of SEQ ID NOs: 107-125 and 168.

19. The polynucleotide of claim 15, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 34, wherein X is G, and the nucleic acid comprises a sequence having 85% identity to the sequence of any one of SEQ ID NOs: 126-140 and 161-167.

20. The polynucleotide of claim 19, wherein the nucleic acid comprises a sequence having 95% identity to the sequence of any one of SEQ ID NOs: 126-140 and 161-167.

21. The polynucleotide of claim 20, wherein the PPT1 coding sequence comprises any one of SEQ ID NOs: 126-140 and 161-167.

22. The polynucleotide of claim 1, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO:

38.

23. A palmitoyl protein thioesterase-1 (PPT1) polypeptide comprising a PPT1 amino acid sequence having at least 95% identity to the sequence of SEQ ID NO: 1, wherein: (a) the PPT1 polypeptide further comprises a signal sequence of any one of SEQ ID NOs: 16-27 or a variant thereof having one amino acid substitution, deletion, or insertion; and / or (b) the PPT1 amino acid sequence comprises a substitution of aspartate (D) at the amino terminus with glycine (G), valine (V), or leucine (L); and / or (c) the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus.

24. The polypeptide of claim 23, wherein the PPT1 polypeptide further comprises a signal sequence comprising the sequence of any one of SEQ ID NOs: 16-27.

25. The polypeptide of claim 24, wherein the polypeptide comprises the signal sequence of any one of SEQ ID NOs: 16-21 and 24-27.

26. The polypeptide of claim 25, wherein the signal sequence comprises the sequence of any one of SEQ ID NOs: 16 or 19.

27. The polypeptide of claim 24, wherein the polypeptide comprises the signal sequence of SEQ ID NO:

23.

28. The polypeptide of any one of claims 23-27, wherein the PPT1 amino acid sequence comprises a substitution of aspartate (D) at the amino terminus with G, V, or L, and the PPT1 amino acid sequence has at least 97% identity to the sequence of SEQ ID NO:

1.

29. The polypeptide of claim 28, wherein the PPT1 amino acid sequence comprises the sequence of SEQ ID NO: 2, wherein X is G.

30. The polypeptide of claim 23, wherein the PPT1 sequence comprises the amino acid sequence leucine-glutamine-histidine-leucine at its N-terminus and the PPT1 amino acid sequence has at least 97% identity to the sequence of SEQ ID NO:

1.

31. The polypeptide of claim 30, wherein the PPT1 sequence comprises SEQ ID NO:

4.

32. The polypeptide of claim 23, wherein the PPT1 polypeptide comprises a sequence having at least 99% identity to any one of SEQ ID NOs: 31-42.

33. The polypeptide of claim 32, wherein the PPT1 polypeptide comprises the sequence of SEQ ID NO: 31, wherein X is G; the sequence of SEQ ID NO: 34, wherein X is G; or the sequence of SEQ ID NO:

38.

34. A polynucleotide comprising a nucleic acid sequence encoding a PPT1 polypeptide, wherein the PPT1-encoding nucleic acid sequence encodes a PPT1 polypeptide of any one of claims 23-33.

35. A polynucleotide comprising two or more exons and one or more introns that together encode a PPT1 polypeptide of any one of claims 23-34.

36. The polynucleotide of any one of claims 1-22, 34, or 35, wherein the polynucleotide is an expression cassette comprising one or more expression control elements operably linked to the nucleic acid encoding the PPT1 polypeptide.

37. The polynucleotide of claim 36, wherein the nucleic acid encoding the PPT1 polypeptide is operably linked to an upstream promoter and a downstream polyadenylation signal.

38. The polynucleotide of claim 36, wherein the expression cassette comprises, from 5’ to 3’, operably linked to the nucleic acid encoding the PPT1 polypeptide: a promoter, a Kozak sequence, the nucleic acid sequence encoding PPT1 polypeptide, and a polyadenylation signal.

39. The polynucleotide of claim 37 or 38, wherein the promoter comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 5 or 173.

40. The polynucleotide of any one of claims 37-39, wherein the polyadenylation signal operably linked to the nucleotide sequence encoding PPT1 comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:

6.

41. The polynucleotide of any one of claims 36-40, wherein the expression cassette comprises a nucleotide sequence having at least 95% identity to the sequence of any one of SEQ ID NOs: 141-143, 169, and 170.

42. The polynucleotide of any one of claims 1-22 and 34-41, wherein the polynucleotide is DNA.

43. A recombinant viral vector nucleic acid comprising the polynucleotide of any one of claims 1-22 and 34-42 and 5’ and / or 3’ viral elements that provide for viral packaging and replication.

44. The recombinant viral vector nucleic acid of claim 43, wherein the recombinant viral vector nucleic acid is DNA and comprises an adeno-associated virus (AAV) inverted terminal repeat (ITR) flanking the 5’ end of the polynucleotide and an AAV ITR flanking the 3’ end of the polynucleotide.

45. The recombinant viral vector nucleic acid of claim 44, wherein the recombinant viral vector nucleic acid comprises a 5’ ITR and a 3’ ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.10, AAVrh.74, or AAV3B.

46. The recombinant viral vector nucleic acid of claim 44, wherein the 5’ ITR comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 8 and the 3’ ITR comprises a sequence having at least 95% identity to the sequence of SEQ ID NO:

9.

47. The recombinant viral vector nucleic acid of any one of claims 43-46, further comprising a polyadenylation sequence operably linked to the 3’ ITR.

48. The recombinant viral vector nucleic acid of any one of claims 43-47, further comprising one or more stuffer sequences.

49. The recombinant viral vector nucleic acid of any one of claims 43-48, wherein the recombinant viral vector nucleic acid comprises a sequence that is at least 95% identical to the sequence of any one of SEQ ID NOs: 144-154, 171, and 172.

50. A gene delivery vehicle comprising a viral or non-viral vector and the polynucleotide of any one of claims 1-22 and 34-42, or the recombinant viral vector nucleic acid of any one of claims 43-49.

51. The gene delivery vehicle of claim 50, wherein the gene delivery vehicle is a viral vector.

52. The gene delivery vehicle of claim 51, wherein the viral vector is a recombinant AAV vector, a recombinant lentivirus vector, or a recombinant adenovirus vector.

53. The gene delivery vehicle of claim 52, wherein the viral vector is a recombinant AAV vector, and the recombinant AAV vector comprises a capsid comprising a VP1, VP2, or VP3 that is at least 90% identical to the VP1, VP2, or VP3 sequence of any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, AAV1 / rh.10, SEQ ID NO: 12, or SEQ ID NO:

15.

54. The gene delivery vehicle of claim 53, wherein the capsid is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.74, AAV3B, AAV-2i8, AAVrh.10, AAVrh.8, AAVHSC, AAV-B1, AAV-AS, or AAV1 / rh.10 capsid; or the capsid comprises a VP1 of SEQ ID NO: 12 or SEQ ID NO:

15.

55. The gene delivery vehicle of claim 54, wherein the capsid comprises a VP1 comprising the sequence of SEQ ID NO: 12, a VP2 comprising the sequence of SEQ ID NO: 13, and a VP3 comprising the sequence of SEQ ID NO:

14.

56. The gene delivery vehicle of claim 50, wherein the gene delivery vehicle is the non-viral vector.

57. The gene delivery vehicle of claim 56, wherein the non-viral vector is a nanoparticle selected from the group consisting of a lipid nanoparticle (LNP), a polymeric nanoparticle, a lipopolymeric nanoparticle (LPNP), a protein or peptide-based nanoparticle, a DNA dendrimer or DNA-based nanocarrier, a carbon nanotube, a microparticle, a microcapsule, an inorganic nanoparticle, a peptide cage nanoparticle, and an exosome.

58. The gene delivery vehicle of claim 56, wherein the non-viral vector is an LNP or an LPNP.

59. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-22 and 34-42, the PPT1 polypeptide of any one of claims 23-33, the recombinant viral vector nucleic acid of any one of claims 43-49, or the gene delivery vehicle of any one of claims 50-58, and a pharmaceutically acceptable carrier.

60. A method of increasing PPT1 in a subject, the method comprising administering to the subject the polynucleotide of any one of claims 1-22 and 34-42, the PPT1 polypeptide of any one of claims 23-33, the recombinant viral vector nucleic acid of any one of claims 43-49, the gene delivery vehicle of any one of claims 50-58, or the pharmaceutical composition of claim 59.

61. A method of treating neuronal ceroid lipofuscinosis 1 in a subject, the method comprising administering to the subject the polynucleotide of any one of claims 1-22 and 34-42, the PPT1 polypeptide of any one of claims 23-33, the recombinant viral vector nucleic acid of any one of claims 43-49, the gene delivery vehicle of any one of claims 50-58, or the pharmaceutical composition of claim 59.

62. The method of claim 60 or 61, wherein the administering comprises intraparenchymal, intracisternal, or intracerebroventricular administration.

63. The method of claim 62, wherein the administering is intracerebroventricular and results in substantial rAAV delivery to at least frontal cortex, parietal cortex, temporal cortex, occipital cortex, thalamus, cerebellar cortex, hippocampus, corpus callosum, spinal cord, caudate nucleus, choroid plexus, optic chiasm, fornix, periaqueductal gray, olfactory bulb, and optic nerve.

64. The method of claim 60 or 61, wherein the administering comprises an initial administration outside the central nervous system (CNS).

65. The method of claim 63, wherein the subject is a sheep.

66. The method of any one of claims 60-62, wherein the administering is systemic.

67. The method of any one of claims 60-63, wherein the subject is a human.

68. An AAV vector genome plasmid comprising the recombinant viral vector nucleic acid of any one of claims 43-49.

69. The AAV genome plasmid of claim 68, wherein the plasmid lacks a rep gene and a cap gene.

70. A method of producing a rAAV vector, the method comprising the step of culturing a rAAV producer cell line comprising rAAV helper virus activity, wherein the genome of the producer cell comprises the recombinant viral vector nucleic acid of any one of claims 43-49, a rep gene, and a cap gene, wherein the rAAV vector is produced.

71. A method of producing a rAAV vector, the method comprising the step of culturing a rAAV permissive cell comprising the AAV genome plasmid of claim 68 or 69, wherein the rAAV permissive cell further comprises (a) a rep gene and a cap gene provided as part of the cell genome and / or provided by one or more separate plasmids, and (b) helper virus activity provided by the cell genome and / or provided by one or more separate plasmids.

72. The method of claim 71, wherein the rAAV permissive cell is a packaging cell, wherein the packaged genome comprises a cap gene and a rep gene.

73. The method of claim 71, wherein (a) the rep gene, the cap gene, and the helper activity are provided in a single plasmid, or (b) the rep gene and the cap gene are provided by a rep / cap plasmid and the helper activity is provided by a helper plasmid.

74. A method of obtaining an rAAV vector, the method comprising the steps of: (a) producing the rAAV using the method of any one of claims 70-73 and (b) purifying the rAAV.

74. The method of claim 73, wherein the rAAV is purified by affinity chromatography.

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