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KR-20260062951-A - Method and composition for engineered DA neuron cells

KR20260062951AKR 20260062951 AKR20260062951 AKR 20260062951AKR-20260062951-A

Abstract

A novel strategy for the treatment of patients with Parkinson's disease and other secondary Parkinson's disorders is disclosed. In vitro, DA neuronal cells modified by the insertion of GDNF are disclosed. The GDNF coding sequence is inserted under the transcriptional control of a promoter, and after the administered engineered cells mature into a neuronal maturation cell type, the secreted protein is produced and absorbed by endogenous cells, thereby promoting the survival of endogenous neurons. In vitro, DA neuronal cells modified by the insertion of GBA and semi-zygous to SNCA are also disclosed. The GBA coding sequence is inserted under the transcriptional control of a universal promoter, and the secreted protein is produced and absorbed by the graft and endogenous cells immediately after transplantation, thereby promoting long-term graft integrity.

Inventors

  • 페더로프, 하워드

Assignees

  • 케나이 테라퓨틱스, 인크.

Dates

Publication Date
20260507
Application Date
20240829
Priority Date
20230831

Claims (20)

  1. A population of engineered cells, wherein the engineered cells comprise at least one exogenous GBA gene or a functional fragment thereof.
  2. In paragraph 1, a group of cells in which the manipulated cells are human DA neuron cells.
  3. In paragraph 1, a group of cells in which human DA neuron cells are derived from pluripotent cells.
  4. In paragraph 1, a group of cells that have been engineered so that the engineered cells become semi-zygous nulls to SNCA.
  5. A population of cells in which the exogenous GBA gene or a functional fragment thereof is located within or near the safe harbor locus in claim 1.
  6. In paragraph 5, a population of cells in which the safe harbor locus is a safe harbor locus selected from the list in Table 1.
  7. In paragraph 5, the safe harbor loci are chr1: 214186905-214187961; chr1: 91164906-91165855; chr1: 88180241-88181223; chr1: 72546731-72548018; chr1: 199684394-199685410; chr1: 104647871-104648861; chr10: 109456554-109457498; chr10: 84613037-84614143; chr10: 128540929-128541943; chr10: 128693340-128695044; chr10:36778899-36780002; chr10: 36725218-36726233; chr11: 121721801-121724194; chr11:42524286-42525334; chr11:81647375-81648903; chr11: 116192498-116194401; chr11: 114774331-114775325; chr11: 128021283-128022329; chr11: 116224105-116225916; chr11: 20309899-20311995; chr12:92649604-92650584; chr12:94763797-94764936; chr13: 103882813-103883879; chr13:52967785-52968920; chr13:52904228-52906942; chr13: 59317916-59319213; chr13:70309932-70311460; chr13:76473185-76474229; chr15:96947520-96948565; chr16:66127013-66128412; chr17: 56715590-56716621; chr18:61088416-61089493; chr18:40576326-40578671; chr18: 27749740-27750778; chr18:67199846-67201011; chr2: 133886374-133887591; chr2: 163347709-163348850; chr2: 147082542-147083491; chr2: 180125287-180126295; chr2: 121974721-121975718; chr2: 122660609-122661835; chr2:75920596-75921844; chr2: 103401408-103402401; chr2: 133843863-133844997; chr2: 180123973-180124967; chr2: 160582472-160583418; chr20:55667282-55669174; chr22:49103355-49104388; chr3:67113422-67114525; chr3:74614218-74615242; chr3:5426026-5426982; chr3: 117399281-117400399; chr3: 28915343-28917419; chr3: 117439560-117440548; chr3: 117237013-117239704; chr3: 137162914-137164165; chr3: 106609403-106610465; chr3:67109328-67110455; chr3: 16048355-16049420; chr3: 117397641-117399034; chr3: 104908816-104909868; chr3: 104596179-104597355; chr4: 180058397-180059507; chr4:180057145-180058232; chr4: 27684257-27685441; chr4: 116925637-116926672; chr4: 166165437-166166501; chr4: 156168620-156169693; chr4:18689105-18690087; chr4:85370123-85371213; chr4:64537927-64540013; chr4:154982074-154983202; chr4: 130563237-130564339; chr5:71937993-71940439; chr5: 113806007-113808129; chr5: 18393819-18394795; chr5: 18465687-18466781; chr5: 123874455-123875520; chr5: 123875580-123876580; chr5:71932489-71933717; chr5: 34474174-34475918; chr5: 18113286-18115121; chr5: 103808080-103809225; chr5: 144598204-144599350; chr5: 108521605-108522526; chr5: 113692453-113694380; chr5: 101678253-101679223; chr5:87944237-87945265; chr5: 101389859-101390987; chr5: 113678518-113679693; chr5:88089287-88091255; chr5: 121797421-121798482; chr5:87949701-87952291; chr5: 103894512-103895553; chr5:63401153-63402160; chr6:85922657-85923735; chr6: 104472033-104473578; chr6: 104525429-104526360; chr6:47097062-47097998; chr6:87968250-87969278; chr6: 16964556-16965560; chr6: 137415153-137416177; chr6:91081186-91082896; chr6:99746005-99747236; chr6:99822427-99823526; chr6: 140439792-140441984; chr6: 137313918-137314874; chr6:48765024-48766063; chr6:90890535-90891762; chr7:152925055-152926297; chr7: 12111174-12112347; chr7:42493270-42494306; chr7: 31192574-31193687; chr7:22000950-22002150; chr7:96785947-96787328; chr8: 141715497-141716580; chr8:26189940-26191013; chr8: 131803831-131805003; chr8: 106856111-106857217; chr8:75975412-75977242; chr8: 115076228-115077322; chr8: 131815871-131817817; chr8: 137492454-137493716; chr9:85258346-85259505; chr9:17904998-17907161; chr9:78439098-78440151; chr9: 16132791-16134459; chr9:29513212-29515120; chr9:75265233-75266237; chr9:7542343-7543943; chr9: 118169738-118170714; chr9:71517517-71519983; chr9:7401713-7402906; chr9: 26325995-26327156; chr9: 1453442-1454897; chr9: 105156511-105157922; chr9: 12425914-12426947; chrX:68894085-68895495; A population of cells selected from chrX: 20530099-20531285; chrX: 40996550-40997770; chrX: 20527023-20528155; chrX: 94058277-94059471; or chrX: 138127607-138128791.
  8. A population of engineered cells, wherein the engineered cells comprise at least one exogenous GDNF gene or a functional fragment thereof.
  9. In paragraph 8, a population of cells in which the manipulated cells are human DA neuron cells.
  10. In paragraph 8, a group of cells in which human DA neuron cells are derived from pluripotent cells.
  11. A method for manufacturing engineered pluripotent cells, the method - Provides a population of cells including pluripotent cells; - Includes introducing the following into a part of the pluripotent cell: i) at least one nucleic acid comprising an exogenous polynucleotide sequence for encoding at least one GDNF gene, which is incorporated into a selected endogenous locus; ii) at least one sequence-specific reagent that specifically targets a selected endogenous locus, Here, the method in which an exogenous polynucleotide sequence is inserted into an endogenous locus by targeted gene integration.
  12. In paragraph 11, a method in which the sequence-specific reagent is a nuclease.
  13. A method according to claim 11 or 12 in which the targeted gene integration is operated by homologous recombination into pluripotent cells or NHEJ.
  14. A method according to any one of claims 11 to 13, wherein an exogenous polynucleotide sequence is incorporated under the transcriptional control of an endogenous promoter present at a locus.
  15. A method according to any one of claims 11 to 14, wherein the engineered pluripotent cells differentiate into DA neuron cells to form engineered DA neuron cells.
  16. In paragraph 15, a method in which a DA neuron cell engineered to form a DA neuron cell is transplanted into a human.
  17. In paragraph 16, a method in which the transplanted engineered DA neuron cells do not initially express GDNF.
  18. In paragraph 16, a method in which the transplanted engineered DA neuron cells do not express GDNF until the transplanted engineered DA neuron cells differentiate into a mature neuron cell type.
  19. A method for manufacturing engineered pluripotent cells, the method - Provides a population of cells including pluripotent cells; - Includes introducing the following into a part of the pluripotent cell: i) at least one nucleic acid comprising an exogenous polynucleotide sequence for encoding at least one GBA gene and at least one hemizygous null SCNA gene, which is integrated into a selected endogenous locus; ii) at least one sequence-specific reagent that specifically targets a selected endogenous locus, Here, the method in which an exogenous polynucleotide sequence is inserted into an endogenous locus by targeted gene integration.
  20. In paragraph 19, a method in which the engineered pluripotent cells differentiate into DA neuron cells to form engineered DA neuron cells.

Description

Method and composition for engineered DA neuron cells Cross-reference of related applications This application was filed on August 31, 2023, and claims priority to U.S. provisional patent application No. 63/579,861, titled “Method and composition for engineered DA neuron cells,” the entire contents of which are incorporated herein by reference. Sequence list The present application comprises a sequence list XML submitted electronically in XML format, the full text of which is incorporated herein by reference. The attached sequence list has a size of 108 KB, a filename of "87874_00116.xml", and a creation date of August 13, 2024. Technology field The present disclosure generally relates to gene therapy and/or gene editing for the treatment of disorders associated with central nervous system degeneration, such as Parkinson's disease. Gene editing technology Gene editing technology has proven to be a useful tool in the development of in vitro disease models since its inception. The replacement of endogenous genomic sequences with exogenous donor DNA/RNA through homologous recombination (HR) and precise insertion of exogenous DNA/RNA at defined mammalian chromosomal locations was first developed in the 1980s (Smithies et al., 1984) and subsequently applied to genome modification in mouse embryonic stem cells (PSCs) (Hasty et al., 1991). With the discovery of the yeast meganuclease I-SceI (Jacquier and Dujon, 1985), which promotes the HR endogenous cellular mechanism for repairing DNA/RNA double-strand breaks (DSBs) in the presence of donor DNA/RNA, genome-editing strategies were established in murine cells (Choulika et al., 1995) and PSCs (Cohen-Tannoudji et al., 1998) based on proteins derived from unicellular organisms. The emergence of zinc-finger nuclease (ZFN) technology improved efficiency in genome editing in mammalian cells (Bibikova et al., 2001), leading to the creation of the first knockout rat (Geurts et al., 2009). Since its use in animal and cell models (Petersen and Niemann, 2015), ZFN-based genome editing has been utilized for the correction of gene mutations in patient-derived iPSCs (Soldner et al., 2011; Reinhardt et al., 2013; Kiskinis et al., 2018; Wang et al., 2018; Korecka et al., 2019) or for the insertion of known disease-associated mutations in iPSCs derived from healthy individuals (Verheyen et al., 2018), enabling specific genome alterations and the direct investigation of disease phenotypes. In addition, ZFNs have been applied to the generation of engineered lineages to study cell fate determination and improve iPSC differentiation protocols (Hockemeyer et al., 2009), as well as to generate cell type-specific reporter systems for investigating disease pathogenesis (Zhang et al., 2016). Genome editing technology has been further advanced with the emergence of transcriptional activator-like effector nucleases (TALENs), which have proven to be an efficient technique for generating animal models (Tesson et al., 2011). TALENs have been further utilized in the study of neurological disorders through the introduction of disease-causing mutations into control iPSCs (Wen et al., 2014; Lenzi et al., 2015; Akiyama et al., 2019) and/or the correction of gene mutations in patient-derived iPSCs (Maetzel et al., 2014; Wen et al., 2014; Li H. L. et al., 2015; Tanaka et al., 2018; Akiyama et al., 2019), thereby increasing confidence in the underlying mechanisms of the disease and leading to the development of therapeutic approaches. Furthermore, a reporter family for stem cell-based research was developed using TALEN technology (Cerbini et al., 2015; Pei et al., 2015). Rapidly following the development of TALEN technology, regularly spaced short palindromic repeat sequences (CRISPR) by the CRISPR-associated protein (Cas13) system (Gasiunas et al., 2012; Jinek et al., 2012) demonstrated revolutionary potential for manipulating the genomes of mammalian cells in cultures (Cong et al., 2013; Mali et al., 2013) and animal models (Wang H. et al., 2013). As with ZFN or TALEN, CRISPR-Cas13 utilizes distinct RNA cleavage and binding modules. However, the CRISPR-Cas13 system uses its own natural endonuclease and relies on CRISPR RNA (crRNA) and trans-activating RNA (transRNA) to bind to target RNA sequences and activate Cas13. Therefore, the long and complex process of generating engineered nucleases was rapidly overcome by the flexibility and simplicity of generating different CRISPR-based approaches that require only the design of RNA matching specific targets. The exceptional efficacy of CRISPR-Cas13, along with its superior versatility for generating various complex alterations such as extensive substitutions, duplications, deletions, inversions, and chromosomal rearrangements, has completely transformed the field of genome editing. However, there are some limitations that require further improvement. Increased efficiency and reduced off-target effects are related to the manipulation of Cas13 proteins (Kle