Methods and compositions for manipulating DA neuronal cells

Abstract

Novel strategies for the treatment of patients with Parkinson's disease and other secondary Parkinson's disease-like disorders are disclosed. DA neuronal cells modified in vitro by GDNF gene insertion are disclosed. The GDNF coding sequence is inserted under the transcriptional control of a promoter, resulting in the production of a secreted protein after the administered engineered cells have matured into a neuronal mature cell type, which is then taken up by endogenous cells to promote endogenous neuronal survival. Also disclosed are DA neuronal cells modified in vitro by GBA gene insertion, which are hemizygous nulls with respect to SNCA. The GBA coding sequence is inserted under the transcriptional control of a ubiquitous promoter, and the secreted protein is produced immediately after transplantation, which is then taken up by grafts and endogenous cells to promote long-term graft integrity.

Description

Technical Field

[0001] Cross-Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 579,861, entitled "Methods And Compositions for Engineered DA Neuronal Cells", filed on August 31, 2023, the entire content of which is incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing XML which has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The accompanying Sequence Listing has a size of 108 KB, is named "87874_00116.xml", and was created on August 13, 2024.

[0003] Field of the Invention 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.

Background Art

[0004] Background Gene Editing Technology Gene editing technology has proven to be a useful tool in the development of in vitro disease models since its inception. Homologous recombination (HR), which involves the replacement of endogenous genomic sequences with exogenous donor DNA / RNA and the precise insertion of exogenous DNA / RNA at specified mammalian chromosomal locations, was first developed in the 1980s (Smithies et al., 1984) and subsequently applied to genome modification of mouse embryonic stem cells (PSCs) (Hasty et al., 1991). The discovery of the I-SceI yeast meganuclease, which promotes the endogenous HR cellular mechanism and repairs DNA / RNA double-strand breaks (DSBs) in the presence of donor DNA / RNA (Jacquier and Dujon, 1985), led to the establishment of genome editing strategies in mouse cells (Choulika et al., 1995) and PSCs (Cohen-Tannoudji et al., 1998) based on proteins derived from single-celled organisms.

[0005] The emergence of zinc finger nuclease (ZFN) technology has improved the efficiency of genome editing in mammalian cells (Bibikova et al., 2001) and led to the generation of the first knockout rats (Geurts et al., 2009). Following its use in animal and cell models (Petersen and Niemann, 2015), ZFN-based genome editing has been applied to the correction of genetic 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 the insertion of known disease-associated mutations into iPSCs derived from healthy individuals (Verheyen et al., 2018), enabling direct investigation of specific genomic alterations and disease phenotypes. In addition, ZFNs have been applied to generate engineered systems to study cell fate determination and improve iPSC differentiation protocols (Hockemeyer et al., 2009), and to produce cell-type-specific reporter systems for investigating the pathogenic mechanisms of diseases (Zhang et al., 2016).

[0006] Genome editing technology has advanced further with the emergence of activator-like effector nucleases (TALENs), which have demonstrated their efficiency in generating animal models (Tesson et al., 2011). TALENs have also been used in the study of neurological disorders through the introduction of disease-causing mutations in 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 HL et al., 2015, Tanaka et al., 2018, Akiyama et al., 2019), leading to greater confidence in the underlying mechanisms of disease and the development of therapeutic approaches. Furthermore, TALEN technology was used to develop reporter systems for stem cell-based academic research (Cerbini et al., 2015, Pei et al., 2015).

[0007] Following the development of TALEN technology, the clustered, regularly spaced, short palindromic repeats (CRISPR) and CRISPR-related protein (Cas13) system (Gasiunas et al., 2012, Jinek et al., 2012) rapidly demonstrated innovative capabilities for manipulating mammalian cell genomes in cultures (Cong et al., 2013, Mali et al., 2013) and animal models (Wang H. et al., 2013). Similar to ZFNs or TALENs, CRISPR-Cas13 utilizes separate RNA cleavage and binding modules. However, the CRISPR-Cas13 system relies on CRISPR RNA (crRNA) and transactivating RNA (transRNA) to specifically bind to target RNA sequences and activate Cas13, using its own native endonuclease. Therefore, the long and complex process of manipulated nuclease production was rapidly overcome by the plasticity and simplicity that gives rise to different CRISPR-based approaches, which require only the design of RNA that matches a specific target. The extraordinary potency of CRISPR-Cas13, along with its great versatility in generating a wide range of substitutions, duplications, deletions, reversals, and numerous other complex modifications, up to chromosomal rearrangements, has transformed the genome editing field. However, several limitations remained that needed further improvement.Increased efficiency and reduced off-target effects are due to the manipulation of the Cas13 protein (Kleinstiver et al., 2015, 2016, Anders et al., 2016, Slaymaker et al., 2016, Chen et al., 2017, Casini et al., 2018, Hu et al., 2018, Lee et al., 2018, Nishimasu et al., 2018), as well as the design and modification of guide RNA (Jinek et al., 2012, Hsu et al., 2013, Cui et al., 2018, Filippova et al., 2019, Moon et al., 2019), and the discovery and application of Cas proteins with different and specific gene editing properties (Zetsche et al., 2015, Abudayyeh et al., This has been achieved through (Burstein et al., 2016, 2017). CRISPR-based technologies have also been developed to enable transcriptional inhibition (CRISPR interference, CRISPRi) or activation (CRISPR activation, CRISPRa). This CRISPR-based transcriptional modulation has been achieved by a repressor or activator transcription domain fused to a catalytically inactive Cas13 (dCas13), and guide RNA that is directed to the promoter or regulatory region of a specific gene (Gilbert et al., 2013).

[0008] CRISPR-based manipulation techniques have enabled researchers to excise the function of specific gene elements or correct disease-causing mutations. In parallel, CRISPR tools are now being implemented for the active control and modulation of desired messenger RNA (mRNA). This allows for the investigation of transcriptome dynamics and the establishment of causal relationships between observed transcriptional changes and cellular phenotypes. Previously, RNA interference (RNAi) techniques enabled the inhibition of desired transcripts using microRNAs (miRNAs), but this had significant off-target effects and misgetting effects associated with endogenous miRNAs due to cross-reactivity with targets of limited sequence similarity (Flynt and Lai, 2008). Investigations of Cas proteins capable of targeting RNA have led to the development of engineered RNA guides and RNA targeting enzymes (CasRx) (Konermann et al., 2018), which have shown improved efficiency in knockdown at endogenous mRNA levels compared to RNAi techniques and have enabled the easy manipulation of alternative splicing in human cells. Furthermore, Konermann and colleagues successfully modulated the balance of tau isoforms by applying Cas-Rx editing to a patient-derived cortical neuronal model of FTD. Several forms of FTD with parkinsonism are associated with chromosome 17 (FTDP-17), while other tauopathies are caused by mutations in the intron following exon 10 of MAPT (Boeve and Hutton, 2008). These variants disrupt the intron splicing site, increasing the expression of the 4R tau isoform containing more microtubule-binding domains (Kar et al., 2005), inducing pathological changes and activating the progression of neurodegeneration (Schoch et al., 2016). By excluding the CasRx-mediated exon, 4R tau expression was reduced to levels similar to those of unaffected control neurons, suggesting that this technique can be utilized for transcriptional modulation in in vitro models.Interestingly, the small size of CasRx makes it suitable for packaging adeno-associated virus (AAV) for delivery to post-mitotic neurons, promising future clinical applications in the treatment of neurological disorders, and it can be paired with arrays encoding multiple guide RNAs for reduplication. Thus, CasRx technology opens the way for transcriptome manipulation and RNA-targeted therapeutic applications.

[0009] Differentiation into lineage-specific cell populations There is a need for methods to produce DA neuronal cells from pluripotent cells because such cells could be used to identify novel therapeutic agents for the treatment of Parkinson's disease and other and secondary Parkinson's disease-like disorders, including but not limited to idiopathic Parkinson's disease, vascular parkinsonism, drug-induced parkinsonism, and non-idiopathic Parkinson's disease disorders, both in therapeutic and disease models.

[0010] Various efforts have been made to generate midbrain DA neurons from pluripotent cells. For example, methods for generating midbrain DA neurons from pluripotent cells typically require the use of both LDN-193189 (which inhibits ALK 1 / 2 / 3 / 6 and blocks SMAD 1 / 5 / 8), a BMP signaling inhibitor, and SB-431542 (which inhibits ALK 4 / 5 / 7 and blocks SMAD 2 / 3), a TGF-beta signaling inhibitor, as described, for example, in its entirety incorporated herein by reference. Because these methods utilize a combination of two inhibitors of Small Mothers Against Decapetaplegic (SMAD) signaling, they are typically referred to as “dual SMAD inhibition” or “dual SMADi.”

[0011] One method for generating DA neurons using dual SMAD inhibition involves a step of differentiating pluripotent stem cells, and then differentiating multiple pluripotent stem cells with at least one inhibitor of TGFβ / activin-Nodal signaling, at least one inhibitor of bone morphogenetic protein (BMP) signaling, and at least two activators of sonic hedgehog (SHH) signaling, e.g., purmorphamine and SHH The method includes the step of exposing cells to C25II and at least one inhibitor of glycogen synthase kinase 3β (GSK3β) signaling that activates wingless (Wnt) signaling, wherein the exposure of cells to at least one inhibitor of TGFβ / activin-Nodal signaling and at least one inhibitor of BMP signaling begins on day 0, and the cells are exposed to at least one inhibitor of GSK3β signaling in an amount effective to produce a cell population containing at least about 10% differentiated cells expressing both forkheadbox protein A2 (FOXA2) and LIM homeobox transcription factor 1 alpha (LMX1A) from day 3 to day 11 from the initial exposure of the cells to at least one inhibitor of TGFβ / activin-Nodal signaling and at least one inhibitor of BMP signaling. U.S. Patents 10,858,625 and 10,273,452, which are incorporated herein in their entirety by reference, disclose another method for producing DA neurons using a dual SMAD technique. A method for obtaining an enriched population of midbrain dopaminergic (DA) neurons is described in U.S. Patent No. 10,828,335, which is incorporated herein by reference in its entirety. A method for preparing pluripotent stem cells for neural differentiation is described in U.S. Patent No. 9,487,752, which is incorporated herein by reference in its entirety. Other methods include the step of generating midbrain DA neurons from pluripotent cells using a single SMAD inhibitor (single SMADi). See, for example, U.S. Patent No. 10,590,383, which is incorporated herein by reference in its entirety. Generally, a method includes the steps of culturing human pluripotent cells in the presence of the following signaling modulators: (a) a single inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling, (b) at least one activator of Sonic Hedgehog (SHH) signaling, and (c) at least one activator of Wingless (Wnt) signaling; and culturing the cells in the presence of the modulators for a period sufficient to provide a cell composition comprising FOXA2+ / LMX1+ cells, wherein the culturing step does not include culturing the human pluripotent cells in the presence of a second inhibitor of Small Mothers Against Decapentaplegic (SMAD) signaling. GBA deficiency promotes SNCA / α-synuclein accumulation. Loss-of-function mutations in the gene encoding GBA (glucocerebrosidase, β-acid), an enzyme deficient in Gaucher disease (lysosomal storage disorder), increase the risk of Parkinson's disease (PD) characterized by SNCA / α-synuclein misprocessing. Loss of GBA function has been found to result in increased SNCA levels via inhibition of the autophagy pathway in SK-N-SH neuroblastoma cells, primary rat cortical neurons, or rat striatum. Du TT, Wang L, Duan CL, Lu LL, Zhang JL, Gao G, Qiu XB, Wang XM, Yang H. GBA deficiency promotes SNCA / α-synuclein accumulation through autophagic inhibition by inexpressed PPP2A. Autophagy. 2015;11(10):1803-20. doi: 10.1080 / 15548627.2015.1086055. PMID: 26378614; PMCID: PMC4824589. These findings suggest that loss of GBA function may contribute to SNCA accumulation through autophagy inhibition via PPP2A inactivation, thereby providing a mechanistic basis for the increased risk of Parkinson's disease associated with GBA deficiency. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] U.S. Patent No. 10,280,398 [Patent Document 2] U.S. Patent No. 10,858,625 [Patent Document 3] U.S. Patent No. 10,273,452 [Patent Document 4] U.S. Patent No. 10,828,335 [Patent Document 5] U.S. Patent No. 9,487,752 [Patent Document 6] U.S. Patent No. 10,590,383 [Non-patent literature]

[0013] [Non-Patent Document 1] Du TT, Wang L, Duan CL, Lu LL, Zhang JL, Gao G, Qiu XB, Wang XM, Yang H. GBA deficiency promotes SNCA / α-synuclein accumulation through autophagic inhibition by inexpressed PPP2A. Autophagy. 2015;11(10):1803-20. doi: 10.1080 / 15548627.2015.1086055. PMID: 26378614; PMCID: PMC4824589 [Overview of the project] [Means for solving the problem]

[0014] There is a great need in the art for novel compositions and methods for treating and preventing central nervous system degeneration.

[0015] overview DA neuronal cells exhibiting delayed expression of exogenous GDNF In cell transplantation therapy, one major challenge is avoiding cells with poor viability, engraftment, proliferation, migration, innervation, differentiation, or function. This is likely to occur when manipulated genes are partially or completely inexpressed, expressed at the wrong time, or misregulated during expression.

[0016] To avoid these pitfalls, it is desirable to include transcriptional regulation such that genes are not expressed until after the graft is established, i.e., until the graft is integrated into the host tissue. In some embodiments, exogenous genes under the control of an endogenous promoter are not expressed until the transplanted cells (DA neurons) differentiate into more mature cell types in vivo, e.g., immature neurons, mature neurons, or neurons. Delaying the expression of a knock-in gene, e.g., GDNF, is predicted to increase the therapeutic potential of transplanted engineered DA neurons by improving the function of the neuronal mature cell type that the transplanted engineered DA neurons differentiate into in vivo. Delaying the expression of a knock-in gene, e.g., GDNF, is predicted to increase the therapeutic potential of transplanted engineered DA neurons by improving the function of the neuronal mature cell type found in the intracranial putamen environment near the transplanted engineered DA neurons, i.e., the endogenous neuronal mature cell type. Exogenous gene sequences(s) that are expressed or co-expressed after transplanted cells have differentiated into mature neuronal cell types in vivo are introduced by gene-targeted insertion using sequence-specific endonuclease reagents, and as a result, their coding sequences are transcribed under the control of endogenous promoters located at selected loci that are expressed in mature neuronal cell types. Alternatively, loci that are not expressed during neuronal differentiation can be used as "safe harbor loci" for the incorporation of expression cassettes without any adverse consequences for the in vitro differentiation of engineered pluripotent cells into engineered DA neurons, or for the in vivo differentiation of transplanted DA neurons into mature neuronal cell types. In some embodiments, the sites selected for exogenous gene insertion are safe harbor loci, highly expressive loci, temporally expressed loci, or interruption loci.In some embodiments, safe harbor loci are AAVS1, CCR5, hROSA26, collagen, HTRP, H11, beta-2 microglobulin, GAPDH, TCR, or RUNX1, or loci that meet the criteria for a genome safe harbor as defined herein. In some embodiments, loci for interruption include GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA. In some embodiments, safe harbor loci are selected from the list in Table 1.

[0017] These cell manipulation strategies generally tend to enhance the therapeutic potential of transplanted and manipulated DA neurons, particularly by increasing their viability, engraftment, proliferation, migration, innervation, and / or differentiation, or by increasing the function of the neuronal mature cell type that transplanted and manipulated DA neurons differentiate into in vivo and / or the survival of endogenous neurons. This strategy can be performed on patient-derived cells as part of an autologous treatment strategy and on donor-derived cells as part of an allogeneic treatment strategy.

[0018] DA neuronal cells that immediately express exogenous GBA and are hemijunctive nulls with respect to SNCA Immediate expression of a knock-in gene, such as GBA, upon transplantation of modified DA neuronal cells is expected to increase the therapeutic potential of the transplanted manipulated DA neuronal cells by improving long-term graft integrity. Exogenous gene sequences(s) are expressed or co-expressed post-transplant. In some embodiments, exogenous gene sequences(s) are transcribed under the control of endogenous ubiquitous promoters(s). Alternatively, loci identified in advance as not containing genes that are endogenously expressed during DA neuronal cell transplantation may be used as "safe harbor loci" for the incorporation of expression cassettes under the control of ubiquitous promoters.

[0019] Manipulated pluripotent cell population Disclosed is a population of engineered pluripotent cells, wherein the engineered pluripotent cells contain exogenous knock-in polynucleotide sequences encoding wild-type Parkinson's protein 2, E3 ubiquitin protein ligase (PARK2), PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1), leucine-rich repeat kinase 2 (LRRK2), alpha-synuclein (SCNA), proto-oncogene c-Rel (c-Rel), ubiquitin-like modifier-activating enzyme (ATG7), synaptic vesicle amine transporter (VMAT2), glucocerebrosidase (GBA), and / or glial cell-derived neurotrophic factor (GDNF) genes. In some embodiments, the knock-in polynucleotide sequences are under the control of the GDNF promoter (SEQ ID NOs. 21-30). In some embodiments, the knock-in polynucleotide sequences are under the control of the GBA promoter (SEQ ID NOs. 31-40 or 43).

[0020] Manipulated DA neuron cell population A population of engineered DA neurons is disclosed, wherein the engineered DA neurons contain exogenous knock-in PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF genes. In some embodiments, the knock-in polynucleotide sequence is under the control of the GDNF promoter (SEQ ID NOs. 21-30). In some embodiments, the knock-in polynucleotide sequence is under the control of the GBA promoter (SEQ ID NOs. 31-40 or 43).

[0021] Gene editing systems The manipulated pluripotent cells and / or manipulated DA neuronal cells discussed above can be produced by any known gene editing system known to those skilled in the art. Accordingly, the population of manipulated pluripotent cells and / or manipulated DA neuronal cells discussed above may include repair templates comprising nucleases (Cas protein, TALE nuclease, or zinc finger nuclease), PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous null SNCA, and / or the GDNF gene, or functional fragments or variants thereof.

[0022] The manipulated pluripotent cells and / or manipulated DA neuronal cells discussed above can be produced by any known gene editing system known to those skilled in the art. Accordingly, the population of manipulated pluripotent cells and / or manipulated DA neuronal cells discussed above may include repair templates comprising Cas protein, guide RNA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous null SNCA, and / or the GDNF gene, or functional fragments or variants thereof. Therefore, the manipulated pluripotent cells and / or manipulated DA neuronal cell populations discussed above may include a repair template comprising a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease), PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF gene, or a functional fragment or variant thereof, in combination with editing (or disruption) of the SNCA and / or MAPT genes to make the cell population hemizygous null with respect to SNCA and / or MAPT. In some embodiments, the repair template does not include SCNA.

[0023] Recombinant gene vectors Various embodiments of recombinant gene vectors and related methods are disclosed, including PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF genes, or functional fragments or variants thereof. Various embodiments of recombinant gene vectors and related methods are disclosed, including SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or their complements or RNA equivalents.

[0024] method Contacting pluripotent cells with a gene editing system in vitro In some embodiments, a method for producing manipulated DA neuronal cells is disclosed, comprising the step of contacting pluripotent cells with a gene editing system comprising a repair template containing a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease) and a functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF gene, or a functional variant or fragment thereof. In some embodiments, the repair template is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or their complement or RNA equivalent.

[0025] In some embodiments, a method for producing engineered DA neuronal cells is disclosed, comprising the step of contacting pluripotent cells with a gene editing system comprising a repair template comprising a Cas protein or a polynucleotide encoding a Cas protein, guide RNA (gRNA), and a functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF gene, or a functional variant or fragment thereof. In some embodiments, the repair template is a knock-in repair template comprising SEQ ID NOs. 1-10 or selected from the group comprising SEQ ID NOs. 1-10. In some embodiments, the gene editing system can enhance viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or function of the administered engineered DA neuronal cells compared to wild-type neuronal cells. In some embodiments, administered engineered DA neurons differentiate into mature neuronal cell types with enhanced function in vivo, because they originate from administered engineered DA neurons. In some embodiments, administered engineered DA neurons have an effect on endogenous mature neuronal cell types in vivo.

[0026] Contacting nerve cells with gene editing systems ex vivo or in vivo In some embodiments, a method is disclosed for contacting neurons with a gene editing system comprising a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease) and a repair template comprising a functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF gene, or a functional variant or fragment thereof. In some embodiments, the repair template is a knock-in repair template comprising SEQ ID NOs. 1-10 or selected from the group comprising SEQ ID NOs. 1-10.

[0027] In some embodiments, a method is disclosed for contacting neurons with a gene editing system comprising a repair template containing a Cas protein or a polynucleotide encoding a Cas protein, a guide RNA (gRNA), and a functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF gene, or a functional variant or fragment thereof. In some embodiments, the repair template is a knock-in repair template comprising or selected from the group consisting of SEQ ID NOs: 1-10. In some embodiments, the gene editing system can enhance viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or the function of the administered engineered DA neuron cells compared to wild-type neurons. In some embodiments, the neurons are present in the patient's body when contacted with the engineered DA neuron cells. In some embodiments, the neurons are contacted with the gene editing system ex vivo and then transplanted back into the patient's body in vivo after the contact step.

[0028] In vivo, wild-type neurons are brought into contact with DA neurons that have been manipulated. In some embodiments, a method is disclosed for contacting neurons with manipulated DA neuronal cells, the method comprising the step of contacting the pluripotent cells with a gene editing system comprising a repair template containing a nuclease (Cas protein, TALE nuclease, or zinc finger nuclease) and functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF genes, or functional variants or fragments thereof. In some embodiments, the repair template is a knock-in repair template comprising SEQ ID NOs. 1-10 or selected from the group comprising SEQ ID NOs. 1-10.

[0029] In some embodiments, a method is disclosed for contacting neurons with engineered DA neuronal cells, the step of contacting the pluripotent cells with a gene editing system comprising a repair template containing a Cas protein or a polynucleotide encoding the Cas protein, a guide RNA (gRNA), and functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF genes, or functional variants or fragments thereof. In some embodiments, the repair template is a knock-in repair template comprising or selected from the group consisting of SEQ ID NOs: 1-10. In some embodiments, engineered DA neuronal cells can enhance viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, survival of endogenous wild-type neurons, or function of endogenous wild-type neurons compared to uncontacted wild-type neurons. In some embodiments, endogenous wild-type neurons are present in the patient's body when they come into contact with the engineered DA neuronal cells. In some embodiments, endogenous wild-type neurons are brought into contact with ex vivo engineered DA neuronal cells, and then, following the contact step, are implanted back into the patient's body in vivo.

[0030] transplant The engineered DA neurons, recombinant gene vectors, or gene editing systems can be administered in a variety of ways. In some embodiments, the administration step includes systemic, parenteral, intravenous, intrathecal, intracerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular administration. In some embodiments, the administration step includes intravenous, intrathecal, intracerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular injection. In some embodiments, the administration step includes intrathecal injection using the Trendelenburg tilt. In some embodiments, the administration step includes direct injection into the substantia nigra pars compacta of the brain. In some embodiments, the administration step includes introducing the engineered DA neurons, recombinant gene vectors, or gene editing system into the target brain or cerebrospinal fluid (CSF).

[0031] In some embodiments, 1 × 10 per kilogram of the subject's body weight 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the subject. In some embodiments, 1 x 10⁶ per kilogram of the subject's body weight is used. 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the target brain. In some embodiments, 1 x 10⁶ per kilogram of the target's body weight is used. 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the target CSF. In some embodiments, 1 x 10⁶ per kilogram of the target's body weight is used. 7 ~1 × 10 9 A gene therapy vector containing a recombinant gene vector genome (vg / kg) is administered to the target.

[0032] Treatment results The methods of this disclosure (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) may have a variety of effects. In some embodiments, the administered engineered DA neurons may have increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neurons. In some embodiments, the administered engineered DA neurons may not have any change in action. In some embodiments, the administered engineered DA neurons may not have increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neurons. In some embodiments, only the cells to which the administered engineered DA neurons differentiate (e.g., neuronal mature cell types) may have an increased change in action. In some embodiments, administered engineered DA neuronal cells result in endogenous neuronal mature cell types having increased viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neuronal cells.

[0033] In some embodiments, administered engineered DA neurons having an engineered GBA gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neurons. In some embodiments, administered engineered DA neurons having an engineered GBA gene and a disrupted SNCA gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neurons. In some embodiments, administered engineered DA neurons having an engineered GDNF gene have increased cell viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or increased function of the administered engineered DA neurons. In some embodiments, administration of engineered DA neurons treats or inhibits the development of Parkinson's disease and secondary Parkinson's disease-like disorders in a subject. In some embodiments, administration of engineered DA neurons treats or inhibits the development of bradykinetics. In some embodiments, administration of engineered DA neuronal cells treats or inhibits the development of slow thinking. In some embodiments, administration of engineered DA neuronal cells treats or inhibits the development of neuroscience-related disorders or functional impairments associated with dopamine, acetylcholine, serotonin, and / or norepinephrine signaling disorders.

[0034] array In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF gene (or vector) each contains the nucleic acid sequence described in SEQ ID NOs: 1-10.

[0035] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF proteins each contain the amino acid sequences described in SEQ ID NOs. 11-20.

[0036] In some embodiments, the gene is the GDNF gene, and the wild-type GDNF protein contains the amino acid sequence described in any of Sequence ID No. 10.

[0037] In some embodiments, the gene is the GBA gene, and the wild-type GBA protein contains the amino acid sequence described in any of Sequence ID No. 7.

[0038] In some embodiments, the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity to the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, or GDNF polynucleotide sequences described in SEQ ID NOs. 1-7 and 10, respectively. In some embodiments, the polynucleotide is codon-optimized. In some embodiments, the polynucleotide comprises fewer than 40, fewer than 30, fewer than 20, or 10 or fewer CpG islands. In some embodiments, the polynucleotide comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 CpG islands. In some embodiments, it comprises 5-20 CpG islands. Scalar [ka] [ka] [ka] [ka] [ka] [ka] [ka]

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

change

[0039] [Figure 1] Figure 1 shows different types of neural cells and their markers.

[0040] [Figure 2-1] Figure 2 shows research strategies for identifying genome-safe harbor (GSH) sites suitable for iPSC gene editing and expression in dopaminergic neurons. [Figure 2-2] Same as above.

[0041] [Figure 3]Figure 3 illustrates a stepwise approach to prioritizing GSH sites for therapeutic gene insertion. [Modes for carrying out the invention]

[0042] Detailed explanation Unless specifically defined herein, all technical and scientific terms used herein have the same meaning as those widely understood by those skilled in the art of gene therapy, biochemistry, genetics, and molecular biology.

[0043] All publications, patent applications, patents, and other references mentioned herein are incorporated in their entirety by reference. In the event of any conflict, including definitions, this specification shall prevail. Furthermore, unless otherwise specified, materials, methods, and examples are illustrative and not intended to limit the scope of the text.

[0044] Unless otherwise specified, the gene editing procedures disclosed herein utilize conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the skills of those skilled in the art. Such techniques are described in detail in the literature. For example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA), Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press), Oligonucleotide Synthesis (MJ Gait ed., Nucleic Acid Hybridization (BD Harries & SJ Higgins eds. 1984), Transcription And Translation (BD Hames & SJ Higgins eds. 1984), Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987), Immobilized Cells And Enzymes (IRL Press, 1986), B. Perbal, A Practical Guide To Molecular Cloning (1984), in the series Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.), Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987), Handbook Of Experimental Immunology, Volumes l-IV (DM Weir and CC Blackwell, eds., 1986), and Manipulating the See Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0045] Generally speaking, compositions comprising engineered cells are disclosed herein. Engineered cells can be administered to a subject in a therapeutically effective dose. Administration of engineered cells can produce a therapeutic effect in the subject, where the therapeutic effect is modulated by exogenous genes attached to the cells.

[0046] definition amino acid residues In this specification, amino acid residues within a polypeptide sequence are represented by single-letter codes, for example, Q means Gln or glutamine residue, R means Arg or arginine residue, and D means Asp or aspartic acid residue.

[0047] Amino acid substitutions An amino acid substitution means replacing one amino acid residue with another. For example, replacing an arginine residue with a glutamine residue in a peptide sequence is an amino acid substitution.

[0048] Cutting The term "cleavage" refers to the cleavage of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods, including but not limited to enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand and double-strand breaks are possible, and a double-strand break can result from two different single-strand break events. Cleavage of double-stranded DNA, RNA, or DNA-RNA hybrids can result in the production of either blunt or adherent ends.

[0049] DA neuron cells DA neuronal cells can be differentiated in vitro from pluripotent cells by various methods known in the art, such as the single-SMAD or double-SMAD techniques discussed herein.

[0050] DNA target The terms “DNA target,” “DNA target sequence,” “target DNA sequence,” “nucleic acid target sequence,” “target sequence,” or “processing site” refer to polynucleotide sequences that can be targeted and processed by rare-cutting endonucleases. These terms refer not only to specific DNA locations, preferably genomic locations in cells, but also to parts of the genetic material that may exist independently of the body of the genetic material, such as in non-limiting examples, plasmids, episomes, viruses, transposons, or organelles such as mitochondria. A non-limiting example of an RNA guide target sequence is a genomic sequence that can hybridize an RNA guide endonuclease into a guide RNA that directs it to a desired locus.

[0051] dopaminergic neurons Dopaminergic neurons are cells that express TH, DAT, FOXA2, GIRK2, Nurr1, and LMX1B.

[0052] Manipulated DA neuron cells "Manipulated DA neuron cells" refers to a population of DA neuron cells containing exogenous knock-in PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF genes.

[0053] To enhance therapeutic activity "Enhancing therapeutic activity" means that DA neuronal cells or populations of cells manipulated as described herein are more viable or have improved engraftment, proliferation, migration, innervation, and / or differentiation than unmanipulated cells or populations of cells.

[0054] Endonuclease The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within DNA or RNA molecules, preferably within DNA molecules. Endonucleases do not cleave DNA or RNA molecules indiscriminately, but rather recognize specific polynucleotide sequences where they cleave DNA or RNA molecules; this specific sequence is further referred to as the "target sequence" or "target site." Endonucleases can be classified as low-frequency cleavage endonucleases if they typically have polynucleotide recognition sites longer than 10 base pairs (bp), more preferably 14–55 bp. Low-frequency cleavage endonucleases significantly increase homologous recombination by inducing DNA double-strand breaks (DSBs) at defined loci, thereby enabling gene repair or gene insertion therapy (Pingoud, A. and GH Silva (2007). PreSNCAion genome surgery. Nat. Biotechnol. 25(7): 743-4.).

[0055] Exogenous sequence An “exogenous sequence” refers to any nucleotide or nucleic acid sequence that was not initially present at a selected locus. This sequence may be homologous to or a copy thereof of the genomic sequence, or it may be an exogenous sequence introduced into the cell. In contrast, an “endogenous sequence” refers to the cellular genomic sequence that was initially present at a given locus. The exogenous sequence preferably encodes a polypeptide whose expression yields a therapeutic benefit over that of sister cells in which this exogenous sequence is not incorporated at the locus. An endogenous sequence that is gene-edited by nucleotide or polynucleotide insertions according to methods disclosed for expressing different polypeptides is broadly referred to as an exogenous coding sequence.

[0056] The methods disclosed herein can be associated with other methods of gene transformation, including physical methods such as viral transduction or transfection using nanoparticles, and can be combined with other gene inactivation and / or gene insertion.

[0057] GBA gene The term "GBA gene" encompasses the GBA gene itself, as well as its functional fragments and variants.

[0058] GDNF gene The term "GDNF gene" encompasses the GDNF gene itself, as well as its functional fragments and variants.

[0059] GABAergic neurons GABAergic neurons are cells that express GAT1, GABAB receptor 1, GABAB receptor 2, GAD65, and GAD67.

[0060] gRNA molecule gRNA molecules can be localized to a site containing, consisting of, or essentially derived from, a nucleic acid sequence that is completely or partially complementary to a target domain, such as a gene or a portion of a gene. In certain embodiments, a monomolecule or chimeric gRNA preferably includes, from 5' to 3', a targeting domain complementary to a target domain in a nucleotide of a cell, e.g., a chromosome, a first complementarity domain, a ligation domain, a second complementarity domain (complementary to the first complementarity domain), a proximal domain, and optionally a tail domain.

[0061] Gene editing systems Gene editing systems, or genome editing systems, are a set of technologies that give scientists the ability to alter the DNA of living organisms. These technologies allow for the addition, removal, or modification of genetic material at specific locations within the genome.

[0062] Genome-safe harbor A genome-safe harbor (GSH) is a site within the genome that can tolerate the integration of new genetic material in a manner that ensures the newly inserted gene element (i) functions predictably and (ii) does not cause host genome modifications that pose a risk to host cells or the organism. Table 1 below lists genome-safe harbor loci that can be used as described herein. Table 1 shows the genomic coordinates in the GRCh38 / hg38 human genome assembly. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0063] Gene targeting integration "Genetic integration" means any known site-specific method that enables the insertion, replacement, or modification of a genomic sequence into a living cell. In some embodiments, genetic integration involves homologous recombination at the locus of a targeted gene, resulting in the insertion or replacement of at least one exogenous nucleotide, preferably a sequence of multiple nucleotides (i.e., polynucleotides), and more preferably a coding sequence.

[0064] Glutamate-mediated neurons Glutamatergic neurons are cells that express VGLUT1, VGLUT2, NMDAR1, NMDAR2B, glutaminase, and glutamine synthetase.

[0065] identity "Identity" refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing the positions in each sequence that can be aligned for comparison. If a position in the sequences being compared is occupied by the same base, then these molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Identity between two sequences can be calculated using various alignment algorithms and / or programs, including FASTA or BLAST, which are available as part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.) and can be used, for example, with default settings. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98%, or 99% identity with respect to a particular polypeptide described herein, and preferably substantially identical in function, as well as polynucleotides encoding such polypeptides, are considered.

[0066] Immature neurons Immature neurons are cells that express doublecortin, NeuroD1, TBR1, beta-III tubulin, and stasmin 1. Immature neurons can differentiate into mature neurons.

[0067] Improving treatment potential "Improving therapeutic potential" means that the manipulated DA neuronal cells acquire at least one beneficial property for their use in cell therapy compared to their sister, the unmanipulated DA neuronal cells. The desired therapeutic property may be any measurable property mentioned in the relevant scientific literature and may include, but is not limited to, the viability, engraftment, proliferation, migration, innervation, and / or differentiation of the administered manipulated DA neuronal cells.

[0068] Improvements in therapeutic potential may be reflected more specifically in the resistance of DA neuronal cells to the drug, increased persistence of these cells in vitro or in vivo, or safer / more conventional handling during the manufacture and administration of therapeutic compositions.

[0069] Generally, molecules that improve therapeutic potential are polypeptides, but they may also be nucleic acids, such as interfering RNA or guide RNA, that can induce or repress the expression of other genes. Polypeptides can act directly or indirectly, for example, as signaling factors or transcription regulators.

[0070] gene locus As used herein, the term “locus” refers to a specific physical location of a DNA sequence (e.g., a gene) within the genome. The term “locus” may also refer to a specific physical location of a low-frequency cleavage endonuclease target sequence on a chromosome or on the genomic sequence of an infectious agent. Such loci may include target sequences recognized and / or cleaved by sequence-specific endonucleases. A locus of interest may refer not only to a nucleic acid sequence present in the body of the cellular genetic material (i.e., a chromosome), but also to a portion of the genetic material that may exist independently of the body of the genetic material, such as, in non-limiting examples, plasmids, episomes, viruses, transposons, or organelles such as mitochondria.

[0071] Mature neuron Mature neurons are cells that express NeuN, MAP2, 160kDa neurofilament medium chains, neurofilament heavy chains, synaptophysin, and PSD95. Mature neurons can differentiate into neurons.

[0072] Modified DA neuron cells "Modified DA neuron cells" or "manipulated DA neuron cells" refers to DA neuron cells that are either genetically modified themselves or derived from genetically modified pluripotent cells.

[0073] Modified pluripotent cells "Modified pluripotent cells" or "manipulated pluripotent cells" refers to pluripotent cells that have been genetically modified.

[0074] mutation A "mutation" is defined as the substitution, deletion, or insertion of up to one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, forty, fifty, or more nucleotides / amino acids in a polynucleotide (cDNA, gene) or polypeptide sequence. Mutations can affect the coding sequence or its regulatory sequence of a gene. They can also affect the structure or structure / stability of the genomic sequence of the encoded mRNA.

[0075] neuron Glutamate-mediated neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, and cholinergic neurons are collectively referred to as "neurons."

[0076] nucleotide Nucleotides are denoted as follows: A single-letter code is used to specify the base of the nucleoside, where a is adenine, t is thymine, c is cytosine, and g is guanine. For degenerate nucleotides, r represents g or a (purine nucleotide), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotide), d represents g, a, or t, v represents g, a, or c, b represents g, t, or c, h represents a, t, or c, and n represents g, a, t, or c.

[0077] nucleic acid As used herein, “nucleic acid” or “polynucleotide” refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease activity, and exonuclease activity. Nucleic acid molecules may consist of monomers that are naturally occurring nucleotides (e.g., DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides may have alterations in the sugar moiety and / or pyrimidine or purine base moiety. Examples of sugar modifications include the replacement of one or more hydroxyl groups with halogen, alkyl, amine, and azide groups, or the sugar may be functionalized as an ether or ester. Furthermore, the entire sugar moiety may be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications to the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic alternatives. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids may be single-stranded or double-stranded.

[0078] Nuclease reagent "Nuclease reagent" means a nucleic acid molecule that, either on its own or in a complex, for example, as a subunit of a guide RNA / Cas protein, contributes to a nuclease-catalyzed reaction in a target cell, preferably an endonuclease reaction, and preferably results in the cleavage of a nucleic acid sequence target.

[0079] The nuclease reagents used herein are generally “sequence-specific reagents,” meaning they can induce DNA cleavage in cells at a predetermined gene locus, referred to as a “target gene.” The nucleic acid sequence recognized by the sequence-specific reagent is referred to as the “target sequence.” Target sequences are typically selected to be rare or unique in the cell’s genome, or more broadly, in the human genome, so that they can be determined using software and data available from the human genome database, e.g., http: / / www.ensembl.org / index.html.

[0080] Neuronal mature cell type The term "neuronal mature cell type" refers to immature neurons, mature neurons, or neurons as a whole.

[0081] patient The terms “subject” or “patient” as used herein include all members of the animal kingdom, including non-human primates and humans.

[0082] The promoters for GDNF knock-in include SEQ ID NOs. 21-30 (these sequence listings are for "synthetic constructs"). [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] The promoters for GBA knock-in include the following sequence numbers 31-40 and 43 (these sequence listings are for "synthetic constructs"): [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0083] pluripotent cells Pluripotent cells are cells that can differentiate into cells of all three germ layers. Examples of pluripotent cells include stem cells, such as umbilical cord blood stem cells, precursor cells, bone marrow stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (IPS).

[0084] Low-frequency cleavage endonuclease "Low-frequency cleavage endonucleases" are selected sequence-specific endonuclease reagents, provided that their recognition sequences generally consist of 10 to 50 consecutive base pairs, preferably 12 to 30 bp, and more preferably 14 to 20 bp.

[0085] Serotonergic neurons Serotonergic neurons are cells that express TPH, SERT, and Pet1.

[0086] SNCA-modified DA neuron cells As used herein, the term "SNCA-modified DA neuron cells" refers to DA neuron cells in which SNCA activity is inhibited by any of several strategies, either alone or in combination. For example, SNCA-modified DA neuron cells include SNCA - / - gene that is deleted or modified by means such as gene editing, i.e., SNCA "knockout" DA neuron cells, but are not limited thereto. In some embodiments, only a single allele of the SNCA gene is affected by gene editing, i.e., the cell is SNCA + / - heterozygous; SNCA protein "knockdown" DA neuron cells in which expression of SNCA protein is reduced by use of a gene silencing strategy (e.g., siRNA or RNAi) or expression of a dominant-negative SNCA sequence variant or dominant-negative SNCA fragment; or alternatively, SNCA-modified DA neuron cells are DA neuron cells that have been exposed to an SNCA inhibitor (e.g., a small molecule compound, peptide, or peptidomimetic agent) that inhibits SNCA activity, for example, by inhibiting its binding to a target protein (e.g., α-synuclein). Alternatively, the SNCA inhibitor can be a peptide or fragment derived from SNCA that acts to inhibit SNCA activity in trans. In some embodiments, SNCA is irreversibly inhibited in SNCA-modified DA neuron cells, for example, by genetic modification. In some embodiments, SNCA is reversibly inhibited in SNCA-modified DA neuron cells, such that SNCA inhibition in the cells decreases over time.

[0087] When used herein, CIS inhibition refers to reducing one or more of the following: net SNCA gene expression, net SNCA protein levels, or other similar effects.

[0088] Sequence-specific reagents "Sequence-specific reagent" means any active molecule that has the ability to specifically recognize a selected polynucleotide sequence at a genomic locus, preferably at least 9 bp, more preferably at least 10 bp, and even more preferably at least 12 pb in length, in light of modifying the genomic locus. In some embodiments, the sequence-specific reagent is preferably a sequence-specific nuclease reagent.

[0089] target nucleic acid As used herein, the terms “target nucleic acid” or “target gene” refer to a nucleic acid targeted for modification by the Cas system, Talen, or ZNF system described herein, for example, for the generation of a specific deletion. In certain embodiments, the target nucleic acid comprises one gene. In certain embodiments, the target nucleic acid comprises a portion of one gene. In certain embodiments, the target nucleic acid may comprise one or more genes, for example, two genes, three genes, four genes, or five genes. In one embodiment, the target nucleic acid comprises two strands, a first strand and a second strand.

[0090] target position When used herein, “target site” refers to a site on a target nucleic acid (e.g., a chromosome) that is modified by a nuclease. For example, a target site may be modified by cleavage of the target nucleic acid mediated by a Cas protein molecule and by modification of the target site by a template nucleic acid, e.g., modification. In some embodiments, a target site may be a site between two nucleotides on a nucleic acid, e.g., adjacent nucleotides, to which one or more nucleotides are added. A target site may include one or more nucleotides that are modified, e.g., modified, by a template nucleic acid. In some embodiments, a target site is located within a “target sequence” (e.g., a sequence to which gRNA binds). In some embodiments, a target site is located upstream or downstream of a target sequence (e.g., a sequence to which gRNA binds).

[0091] target location area "Target location region," as used herein, is a region containing a target location. In certain embodiments, the target location is flanked by sequences of the target location region, i.e., the target location is located within the target location region such that the target location region sequences are present at both the 5' and 3' ends of the target location. In certain embodiments, the target location region provides on both sides of the target location (i.e., 5' and 3') sufficient sequences to enable gene transformation of the target location, where the gene transformation uses an endogenous sequence homologous to the target location region as a template.

[0092] target sequence When used herein, "target sequence" refers to a nucleic acid sequence containing the target site of the target gene.

[0093] Targeted domains The “targeting domain” (sometimes referred to as a guide sequence or complementary region) contains, consists of, or is essentially composed of a nucleic acid sequence that is complementary or partially complementary to the target nucleic acid sequence.

[0094] template nucleic acid When this term is used herein, "template nucleic acid" refers to a nucleic acid sequence that can be used with a nuclease to modify the structure of a target site.

[0095] Therapeutic potential "Therapeutic potential" reflects therapeutic activity, as measured through in vitro experiments. Scalar Sequence ID 1:> Human PARK2 Sequence ID 2:> Human PINK1 Sequence ID 3:> Human LRRK2 Sequence ID 4:> Human c-Rel Sequence ID 5:> Human ATG7 Sequence ID 6:> Human VMAT2 Sequence ID 7:> Human GBA Sequence ID 8:> Human SNCA Sequence ID 9:> Human MAPT Sequence ID 10:> Human GDNF Sequence ID 11:> Human PARK2 Sequence ID 12:> Human PINK1 Sequence ID 13:> Human LRRK2 Sequence ID 14:> Human c-Rel Sequence ID 15:> Human ATG7 Sequence ID 16:> Human VMAT2 Sequence ID 17:> Human GBA Sequence ID 18:> Human SNCA Sequence ID 19:> Human MAPT Sequence ID 20:> Human GDNF GDNF Knock-in Promoter Sequence ID 21: Monoamine oxidase B (MAOB) Sequence ID 22:>GTP cyclohydrolase 1 (GCH1) Sequence ID 23:> Nuclear receptor subfamily group 4A member 2 (NR4A2) Sequence ID 24:> Mouse tyrosine hydroxylase (TH) Sequence ID 25:> Solute transporter family 6 member 3 (SLC6A3) Sequence ID 26:> Synapsin 1 (SYN1) Sequence ID 27:> Mouse alpha-calcium-calmodulin-dependent kinase II (Camk2a) Sequence ID 28: Neurofilament Medium Chain (NEFM) Minipromoter Sequence ID 29: Neurofilament Light Chain (NEFL) Minipromoter Sequence ID 30: Neurofilament Heavy Chain (NEFH) Promoter GBA Knock-In Promoter Sequence ID 31:> Mouse Thy-1 cell surface antigen (mTHY1) Sequence ID 32:> Rat neuron-specific enolase (rNSE) promoter Sequence ID 33:> DOPAcarboxylase (DDC) promoter Sequence ID 34: Catechol-O-methyltransferase (COMT) promoter Sequence ID 35: Glucosylceramidase beta-1 promoter 1 (GBA P1) Sequence ID 36: Glucosylceramidase beta-1 promoter 2 (GBA P2) Sequence ID 37:> Human eukaryotic translation elongation factor 1 alpha-1 truncated form (EFS) promoter Sequence ID 38:> Human eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) promoter Sequence ID 39:>Chicken Beta-Actin (CBA) Promoter Sequence ID 40:> Cytomegalovirus (CMV) promoter Protospacer adjacent motif (PAM) Sequence ID 41: NNGRRT Sequence ID 42: NNGRRV

[0096] SNCA gene The term "SNCA gene" encompasses SNCA genes as well as their functional fragments and variants. Hemizygous null SNCA genes can become hemizygous nulls through cellular genetic manipulation or cell selection.

[0097] SNCA-modified DA neuron cells DA neurons with modified SNCA refer to DA neurons that are SNCA+ / -.

[0098] vector A “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. Examples of “vectors” include, but are not limited to, viral vectors, plasmids, RNA vectors, or linear or circular DNA or RNA molecules that may consist of chromosomes, non-chromosomes, semi-synthetic, or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episome vectors) and / or those capable of expressing the nucleic acid to which they are ligated (expression vectors). Numerous suitable vectors are known to those skilled in the art and are commercially available. Examples of viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus (AAV)), coronaviruses, negative-strand RNA viruses (e.g., orthomyxovirus (e.g., influenza virus)), rhabdoviruses (e.g., rabies and varicella stomatitis virus)), paramyxoviruses (e.g., measles and varicella), positive-strand RNA viruses (e.g., picornavirus and alphavirus), and double-strand DNA viruses (e.g., adenovirus, herpesvirus (e.g., herpes simplex virus)). Examples of viruses include pesvirus types 1 and 2, Epstein-Barr virus, cytomegalovirus, and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis viruses. Examples of retroviruses include avian leukemia-sarcoma, mammalian leukemia-sarcoma viruses C, B, and D, HTLV-BLV group, lentivirus, and spumavirus (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0099] composition Disclosed herein are engineered cells comprising at least one exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence can be inserted into an endogenous gene. In some embodiments, the endogenous gene is a safe harbor. In some embodiments, the safe harbor locus is selected from Table 1. In some embodiments, the endogenous gene is knocked out by knock-in of the exogenous nucleic acid sequence. In some embodiments, the endogenous gene comprises a promoter. In some embodiments, the endogenous gene comprises a ubiquitous promoter. In some embodiments, the endogenous gene comprises a promoter that is expressed only after the engineered cell has been transplanted, i.e., not during in vitro differentiation. In some embodiments, the endogenous gene comprises a promoter that is expressed only after the engineered cell has engrafted, i.e., not during in vivo differentiation.

[0100] DA neuronal cells exhibiting delayed expression of GDNF Disclosed herein are engineered DA neurons that, when administered to a patient, exhibit delayed expression of the exogenous GDNF gene in the engineered DA neurons, i.e., the exogenous GDNF gene is downstream of an endogenous promoter that is not expressed after transplantation. In some embodiments, the exogenous GDNF gene is downstream of an endogenous promoter that is inactive during the differentiation of pluripotent cells into DA neurons in vitro. In some embodiments, the exogenous GDNF gene is downstream of an endogenous promoter that is inactive during the differentiation of DA neurons into mature neuronal cells in vivo. In some embodiments, the exogenous GDNF gene is downstream of a GDNF knock-in promoter that includes or is selected from the group consisting of SEQ ID NOs. 21-30. In some embodiments, the exogenous GDNF gene is downstream of a GBA knock-in promoter that includes or is selected from the group consisting of SEQ ID NOs. 31-40 or 43. In some embodiments, delayed expression of the exogenous GDNF gene is delayed until graft function is established. In some embodiments, delayed expression of the exogenous GDNF gene is delayed until the transplanted engineered DA neuronal cells differentiate into the neuronal mature cell type.

[0101] DA neurons that immediately express GDB and are hemizygous nulls with respect to SNCA Disclosed herein are engineered DA neurons that, when administered to a patient, immediately exhibit expression of the exogenous GBA gene and the SCNA+ / - (exogenous or endogenous) gene in the engineered DA neurons upon transplantation; that is, the exogenous GBA gene is downstream of an endogenous promoter that is expressed after transplantation. In some embodiments, expression of the exogenous GBA gene occurs before the engineered DA neurons have graft function. In some embodiments, expression of the exogenous GBA gene occurs before the transplanted engineered DA neurons differentiate into a mature neuronal cell type. In some embodiments, expression of the exogenous GBA gene occurs before and after the transplanted engineered DA neurons differentiate into a mature neuronal cell type. In some embodiments, the GDNF coding sequence is inserted under the transcriptional control of an endogenous gene promoter and is not expressed until the administered engineered DA neurons differentiate into a mature cell type in vivo. In some embodiments, the exogenous GBA gene is located downstream of a GDNF knock-in promoter that includes or is selected from the group consisting of SEQ ID NOs. 21-30. In some embodiments, the exogenous GBA gene is located downstream of a GBA knock-in promoter that includes or is selected from the group consisting of SEQ ID NOs. 31-40 or 43.

[0102] In some embodiments, engineered DA neurons, which are engineered to contain the exogenous GBA gene, are hemizygous nulls with respect to SNCA and / or MAPT. In some embodiments, engineered DA neurons are engineered to be hemizygous nulls with respect to SNCA and / or MAPT, and in some embodiments, they are not engineered to be hemizygous nulls with respect to SNCA and / or MAPT.

[0103] In some embodiments, engineered DA neurons, which are engineered to contain an exogenous GBA gene and are hemizygous null with respect to SNCA and / or MAPT, have an altered GBA protein expression ratio compared to alpha-synuclein and / or MAPT proteins. In some embodiments, the GBA:alpha-synuclein protein expression ratio in the administered engineered DA neurons is higher. In some embodiments, the GBA:alpha-synuclein protein expression ratio in the administered engineered DA neurons is lower.

[0104] Manipulated pluripotent cell population A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells include the exogenous knock-in genes PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF genes, or functional fragments thereof. In some embodiments, the exogenous knock-in genes include or are selected from the group consisting of SEQ ID NOs: 1-10.

[0105] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain the exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF genes, or functional fragments thereof, in a genome-safe harbor. In some embodiments, the exogenous genes include or are selected from the group consisting of SEQ ID NOs: 1-10. In some embodiments, the genome-safe harbor includes or is selected from the group consisting of the safe harbors in Table 1.

[0106] A population of manipulated pluripotent cells is disclosed, wherein the manipulated DA neuronal cells contain the exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF genes, or functional fragments thereof, under the control of an endogenous gene promoter. In some embodiments, the endogenous gene promoter includes or is selected from the group consisting of SEQ ID NOs. 21-30. In some embodiments, the endogenous gene promoter includes or is selected from the group consisting of SEQ ID NOs. 31-40 or 43.

[0107] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain the exogenous GDNF gene or a functional fragment thereof. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain the exogenous SEQ ID NO: 10 or a functional fragment thereof.

[0108] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells include exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SCNA, and / or GDNF genes, as well as endogenous knockout PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA, and / or GDNF genes.

[0109] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous GDNF gene, SEQ ID NO: 10, or a functional fragment thereof, under the control of a promoter that is not expressed until graft function is established in vivo. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous GDNF gene or a functional fragment thereof, under the control of a promoter that is not expressed until the transplanted population of manipulated pluripotent cells differentiates into a neuronal mature cell type in vivo. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous GDNF gene or a functional fragment thereof, under the control of one of SEQ ID NOs: 21-40 or 43.

[0110] A population of manipulated pluripotent cells is disclosed, wherein each manipulated pluripotent cell contains an exogenous knock-in GBA gene or a functional fragment thereof. A population of manipulated pluripotent cells is disclosed, wherein each manipulated pluripotent cell contains an exogenous knock-in SEQ ID NO: 7 or a functional fragment thereof.

[0111] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous GBA gene or a functional fragment thereof under the control of a promoter expressed in vivo after cell transplantation. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous GBA gene or a functional fragment thereof under the control of one of sequence numbers 21-40 or 43.

[0112] Disclosed are engineered pluripotent cells, wherein the engineered pluripotent cells include an exogenous GDNF gene or a functional fragment thereof under the control of a promoter that is not expressed until graft function is established in vivo, and further include an exogenous GBA gene or a functional fragment thereof under the control of a promoter that is expressed after cell transplantation.

[0113] A population of engineered pluripotent cells is disclosed, wherein the engineered pluripotent cells (i) contain the exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, and / or GDNF genes, and (ii) are hemizygous nulls with respect to the SNCA and / or MAPT genes.

[0114] A population of engineered pluripotent cells is disclosed, wherein the engineered pluripotent cells (i) contain an exogenous GDNF gene or a functional fragment thereof, and (ii) are hemizygous nulls with respect to the SNCA and / or MAPT genes.

[0115] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain disrupted SCNA and / or MAPT genes.

[0116] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells include (i) an exogenous GBA gene or a functional fragment thereof, and (ii) a hemizygous null SNCA and / or MAPT gene.

[0117] A population of manipulated pluripotent cells is disclosed, wherein each manipulated pluripotent cell contains (i) an exogenous GBA gene or a functional fragment thereof, and (ii) a hemizygous null SNCA gene.

[0118] In some embodiments, the population of engineered pluripotent cells does not express detectable levels of alpha-synuclein and / or MAPT protein in vivo and / or in vitro.

[0119] In some embodiments, the population of engineered pluripotent cells expresses less alpha-synuclein and / or MAPT protein in vivo and / or in vitro as compared to wild-type pluripotent cells.

[0120] In some embodiments, the population of engineered pluripotent cells expresses less alpha-synuclein and / or MAPT protein in vivo and / or in vitro as compared to engineered pluripotent cells that do not comprise edits to the SNCA and / or MAPT gene.

[0121] Disclosed is a population of engineered pluripotent cells, wherein the engineered pluripotent cells comprise an exogenous SCNA gene.

[0122] Disclosed is a population of engineered pluripotent cells, wherein the engineered pluripotent cells comprise an exogenous SCNA+ / - gene.

[0123] Disclosed is a population of engineered pluripotent cells, wherein the engineered pluripotent cells comprise an exogenous SCNA gene, and the SCNA gene is located in a genomic safe harbor. In some embodiments, the SCNA genotype is SCNA+ / +, SCNA+ / -, or SCNA- / -. In some embodiments, the genomic safe harbor is selected from the group consisting of safe harbors listed in Table 1 or consisting of safe harbors from Table 1.

[0124] In some embodiments, the population of engineered pluripotent cells is human.

[0125] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous hemizygous null SNCA gene. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous hemizygous null SNCA gene under the control of a promoter that is expressed after cell transplantation. A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells contain an exogenous hemizygous null SNCA gene under the control of a promoter that is not expressed until graft function is established in vivo.

[0126] In some embodiments, the manipulated population of pluripotent cells containing a hemizygous null SNCA gene contains a disrupted SCNA gene. In some embodiments, the disrupted SCNA gene contains the nucleotide sequence of SEQ ID NO: 8.

[0127] Manipulated DA neuron cell population A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain the exogenous knock-in genes PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF, or functional fragments thereof. A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain the exogenous knock-in genes that contain SEQ ID NOs. 1-10 or functional fragments thereof, or that are selected from the group consisting of SEQ ID NOs. 1-10 or functional fragments thereof.

[0128] A population of engineered DA neurons is disclosed, wherein the engineered DA neurons contain exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF genes, or functional fragments thereof, in the genome safe harbor. In some embodiments, the genome safe harbor includes or is selected from the group consisting of safe harbors in Table 1.

[0129] A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain the exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / +, SCNA+ / -, SCNA- / -, and / or GDNF genes, or functional fragments thereof, under the control of an endogenous gene promoter. In some embodiments, the endogenous gene promoter is one of SEQ ID NOs. 21-40 or 43.

[0130] A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain an exogenous GDNF gene or a functional fragment thereof.

[0131] A population of manipulated pluripotent cells is disclosed, wherein the manipulated pluripotent cells include exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SCNA, and / or GDNF genes, as well as endogenous knockout PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA, and / or GDNF genes.

[0132] A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain an exogenous GDNF gene or a functional fragment thereof, which is under the control of a promoter that is not expressed until a portion of the transplanted population of DA neurons engrafts in vivo.

[0133] A population of manipulated DA neurons is disclosed, wherein each manipulated DA neuron contains an exogenous GBA gene or a functional fragment thereof. A population of manipulated DA neurons is disclosed, wherein each manipulated DA neuron contains an exogenous SEQ ID NO: 7 or a functional fragment thereof.

[0134] A population of engineered DA neurons is disclosed, wherein the engineered DA neurons contain an exogenous GBA gene or a functional fragment thereof under the control of a promoter expressed after cell transplantation. In some embodiments, the endogenous gene promoter is one of SEQ ID NOs: 21-40 or 43.

[0135] Disclosed are engineered DA neuron cells, wherein the engineered DA neuron cells include an exogenous GDNF gene or a functional fragment thereof under the control of a promoter that is not expressed until graft function is established in vivo, and further include an exogenous GBA gene or a functional fragment thereof under the control of a promoter that is expressed after cell transplantation.

[0136] A population of engineered DA neurons is disclosed, wherein the engineered DA neurons contain (i) exogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, and / or GDNF genes, and (ii) are hemizygous nulls with respect to SNCA and / or MAPT genes.

[0137] A population of manipulated DA neurons is disclosed, wherein each manipulated DA neuron contains (i) an exogenous GDNF gene or a functional fragment thereof, and (ii) a hemizygous null SNCA and / or MAPT gene.

[0138] A population of manipulated DA neurons is disclosed, wherein the manipulated DA neurons contain disrupted SCNA and / or MAPT genes.

[0139] A population of manipulated DA neurons is disclosed, wherein each manipulated DA neuron contains (i) an exogenous GBA gene or a functional fragment thereof, and (ii) a hemizygous null SNCA and / or MAPT gene.

[0140] A population of manipulated DA neurons is disclosed, wherein each manipulated DA neuron contains (i) an exogenous GBA gene or a functional fragment thereof, and (ii) a hemizygous null SNCA gene.

[0141] In some embodiments, the manipulated population of DA neurons does not express detectable levels of alpha-synuclein and / or MAPT protein in vivo and / or in vitro.

[0142] In some embodiments, the population of engineered DA neuronal cells expresses less alpha-synuclein and / or MAPT protein compared to wild-type DA neuronal cells.

[0143] In some embodiments, the population of engineered DA neuronal cells expresses less alpha-synuclein and / or MAPT protein compared to engineered DA neuronal cells that do not have edits to the SNCA and / or MAPT gene in vivo and / or in vitro.

[0144] Disclosed is a population of engineered pluripotent cells, wherein the engineered pluripotent cells comprise an exogenous SCNA gene, and the SCNA gene is in a genomic safe harbor. In some embodiments, the SCNA genotype is SCNA+ / +, SCNA+ / -, or SCNA- / -. In some embodiments, the genomic safe harbor is selected from the group consisting of or comprising the safe harbors listed in Table 1.

[0145] In some embodiments, the population of engineered DA neuronal cells is human. In some embodiments, the population of engineered DA neuronal cells is derived from pluripotent cells.

[0146] Disclosed is a population of engineered DA neuronal cells, wherein the engineered DA neuronal cells comprise an exogenous hemizygous null SNCA gene. Disclosed is a population of engineered DA neuronal cells, wherein the engineered DA neuronal cells comprise an exogenous hemizygous null SNCA gene under the control of a promoter that is expressed after cell transplantation. Disclosed is a population of engineered DA neuronal cells, wherein the engineered DA neuronal cells comprise an exogenous hemizygous null SNCA gene under the control of a promoter that is not expressed until graft function is established in vivo.

[0147] In some embodiments, a population of manipulated DA neurons containing a hemizygous null SNCA gene contains a disrupted SCNA gene. In some embodiments, the disrupted SCNA gene contains the nucleotide sequence of SEQ ID NO: 8.

[0148] In some embodiments, at least 5–95% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 20–75% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 30% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 50% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 70% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 80% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein. In some embodiments, at least 90% of the manipulated DA neurons do not express detectable levels of alpha-synuclein protein.

[0149] In some embodiments, the GBA gene is under the control of a promoter that is expressed immediately upon transplantation of the manipulated cells into the patient. In some embodiments, the SCNA gene is also under the control of a promoter that is expressed immediately upon transplantation of the manipulated cells into the patient, but the SCNA gene promoter is expressed to a lesser extent than the GBA gene promoter.

[0150] DA neurons that promote GDNF expression by site-directed gene editing are disclosed. DA neurons that promote GBA expression by site-directed gene editing are disclosed. DA neurons that promote GBA expression and regulate SNCA expression by site-directed gene editing are disclosed. DA neurons that regulate SNCA expression by site-directed gene editing are disclosed. DA neurons that promote GBA expression by site-directed gene editing and are hemizygous nulls with respect to SNCA are disclosed. DA neurons that promote GBA expression by site-directed gene editing and are hemizygous nulls with respect to SNCA are disclosed.

[0151] Site-directed gene editing is used to promote the expression of GDNF and GBA, resulting in DA neurons that are hemizygous nulls with respect to SNCA. In some embodiments, GDNF expression is delayed until after graft formation is established. In some embodiments, the GBA gene is expressed immediately upon transplantation of the engineered DA neurons. In some embodiments, the GBA gene is expressed immediately upon transplantation of the engineered DA neurons, and SNCA gene expression is regulated, where SNCA protein expression is lower than GBA protein expression. In some embodiments, GDNF expression is promoted after graft formation is established, and the GBA gene is expressed immediately upon transplantation of the engineered DA neurons.

[0152] In some embodiments, the endogenous promoter is a constitutive promoter. In some embodiments, the endogenous promoter is not a constitutive promoter. In some embodiments, the endogenous promoter is a conditional promoter. In some embodiments, the endogenous promoter is not a conditional promoter. In some embodiments, the endogenous gene promoter is one of sequence numbers 21-40 or 43.

[0153] In some embodiments, the exogenous sequence is incorporated at an endogenous locus that is constitutively expressed / constitutively transcribed.

[0154] In some embodiments, it may be beneficial to inactivate the endogenous SNCA coding sequence while simultaneously transcribing an integrated exogenous sequence encoding the GBA, SNCA+ / -, or GDNF gene, or a functional fragment thereof, at this locus.

[0155] In some embodiments, SNCA gene expression in transplanted, engineered DA neuronal cells is prevented or reduced compared to wild-type cells.

[0156] The embodiments disclosed herein can be further understood by the following numbered paragraphs.

[0157] Paragraph 1. The following characteristics: Exogenous sequences incorporated under transcriptional regulation by endogenous gene promoters A population of DA neurons possessing the following characteristics.

[0158] Paragraph 2. Genotype [SNCA] neg [GBA] pos The manipulated DA neuron cells described in paragraph 1, having the characteristics of the manipulated DA neuron cells described in paragraph 1.

[0159] Paragraph 3. Genotype [SNCA] neg [GBA] pos [GDNF] pos The manipulated DA neuron cells described in paragraph 1, having the characteristics of the manipulated DA neuron cells described in paragraph 1.

[0160] Paragraph 4. Genotype [SNCA] + / - [GBA] pos [GDNF] pos The manipulated DA neuron cells described in paragraph 1, having the characteristics of the manipulated DA neuron cells described in paragraph 1.

[0161] Paragraph 5. The following characteristics: Exogenous GDNF, GBA, and / or SNCA sequences incorporated under transcriptional regulation by endogenous gene promoters. A population of DA neurons possessing the following characteristics.

[0162] Paragraph 6. A population of therapeutically effective DA neurons comprising at least 30%, preferably at least 50%, and more preferably at least 80%, of the manipulated DA neurons described in any one of paragraphs 1 to 5.

[0163] Types of exogenous sequences In some embodiments, the exogenous sequence to be attached to the cells to be manipulated (pluripotent or DA neuron cells) is a polynucleic acid. In some embodiments, the polynucleic acid is DNA or RNA. In some embodiments, the RNA may be mRNA. Exogenous polynucleic acid sequences comprising at least one exogenous GDNF, GBA, or SNCA sequence are also disclosed herein. The exogenous polynucleic acid sequence may be complementary to a genomic sequence, which may be a partial or full-length sequence. In some embodiments, the exogenous polynucleic acid sequence includes a coding sequence. In some embodiments, the exogenous polynucleic acid sequence includes a non-coding sequence. In some embodiments, the exogenous polynucleic acid sequence includes one or more genes.

[0164] In some embodiments, the polynucleic acid may be a plasmid vector. The plasmid vector may include a promoter. In some embodiments, the promoter may be constitutive. In some embodiments, the promoter may be inductive. In some embodiments, the promoter may be synapsin 1, DAT, VMAT, TH, AADC, a tamoxifen-inductive promoter, a RU486-inductive promoter, or other promoters that allow temporal or small molecule control. In some embodiments, the promoter may be flanked by exogenous GDNF, GBA, and / or SNCA sequences. In some embodiments, the plasmid vector further includes a splicing acceptor. In some embodiments, the splicing acceptor may be flanked by exogenous GDNF, GBA, and / or SNCA sequences.

[0165] In some embodiments, the plasmid vector further includes a translation initiation codon, e.g., an "ATG" sequence. The translation initiation codon sequence may be adjacent to GDNF, GBA, and / or SNCA sequences. In some embodiments, the GDNF, GBA, and / or SNCA sequences may be in a multicistronic vector. In some embodiments, the polynucleic acid includes an exogenous promoter, an endogenous promoter via splicing, and / or an endogenous promoter via in-frame translation.

[0166] In some embodiments, the plasmid may be modified. Modifications may include demethylation, addition of CpG methylation, removal of bacterial methylation, and addition of mammalian methylation.

[0167] The embodiments can be further understood by the following numbered paragraphs.

[0168] Paragraph 1. Engineered DA neuronal cells containing the exogenous PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene, or a functional fragment thereof.

[0169] Paragraph 2. A composition comprising the manipulated DA neuron cells described in Paragraph 1.

[0170] Paragraph 3. A genome editing system, (a) gRNA molecule, (b) Cas molecules configured to modify the PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF genes. This includes genome editing systems.

[0171] Paragraph 4. A method for inhibiting the degeneration or death of dopaminergic neurons, The process includes the step of bringing the neuron into contact with the manipulated DA neuron cell described in paragraph 1, After contact with the manipulated DA neuron cells, the neurons express PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA, and / or GBA proteins. If necessary, the neuron may have mutations in the PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA, and / or GBA genes. method.

[0172] Paragraph 5. The method according to Paragraph 4, wherein neurons express a reduced amount of alpha-synuclein after contact with the manipulated DA neuronal cells.

[0173] Paragraph 6. The method according to any one of paragraphs 4 to 5, wherein a neuron produces and / or releases an increased amount of dopamine after contact with an engineered DA neuron cell.

[0174] Paragraph 7. The method according to any one of paragraphs 4 to 6, wherein the neurons express a lower amount of alpha-synuclein compared to the amount expressed in neurons not in contact with the manipulated DA neuron cells, and, if necessary, the lower amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in neurons not in contact with the manipulated DA neuron cells.

[0175] Paragraph 8. The method according to any one of paragraphs 4 to 7, wherein the neuron produces and / or releases an increased amount of dopamine compared to the amount of dopamine produced and / or released by the neuron not in contact with the manipulated DA neuron cell, and where applicable, the increased amount is 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%, at least twice, at least three times, at least four times, at least five times, or at least ten times more than the amount produced and / or released by the neuron not in contact with the manipulated DA neuron cell.

[0176] Paragraph 9. The method according to any one of paragraphs 4 to 8, wherein a neuron receives an increased amount of autophagy compared to the amount of autophagy received by a neuron not in contact with the manipulated DA neuron, and where applicable, the increased amount is 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%, at least twice, at least three times, at least four times, at least five times, or at least ten times more than the amount received by a neuron not in contact with the manipulated DA neuron.

[0177] Paragraph 10. The method according to any one of paragraphs 4 to 9, wherein the neuron is a primary tyrosine hydroxylase-positive neuron.

[0178] Paragraph 11. A method for treating or inhibiting the development of Parkinson's disease (PD) in a subject who has or is at risk of having PD, The step includes administering the manipulated DA neuronal cells described in paragraph 1, Administration of manipulated DA neuronal cells treats or inhibits the development of Parkinson's disease in subjects. method.

[0179] Paragraph 12. The method according to claim 11, wherein the subject is an adult or a child.

[0180] Paragraph 13. The method according to any one of paragraphs 11 to 12, wherein the number of dopaminergic neurons in the subject after the administration step is greater than the number of dopaminergic neurons in the subject before the administration step.

[0181] Paragraph 14. The method according to any one of paragraphs 11 to 13, wherein the level of dopamine in the subject after the administration step is higher than the level of dopamine in the subject before the administration step.

[0182] Paragraph 15. A method according to any one of paragraphs 11 to 14, wherein the number of dopaminergic neurons in a subject treated by the method is increased compared to the number of dopaminergic neurons in a subject not treated in the same way.

[0183] Paragraph 16. A method according to any one of paragraphs 11 to 15, wherein the level of dopamine in a subject treated by the method is increased compared to the level of dopamine in a subject not treated in the same way.

[0184] Paragraph 17. A method according to any one of paragraphs 11 to 16, wherein the level of dopamine in the substantia nigra of a subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not treated in the same way.

[0185] Paragraph 18. The method according to any one of paragraphs 11 to 17, wherein the levels of GDNF and / or GBA in the subject's CSF after the administration step are higher than the levels of GDNF and / or GBA in the subject's CSF before the administration step.

[0186] Paragraph 19. The method according to any one of paragraphs 11 to 18, wherein the subject's Unified Parkinson's Disease Rating Scale (UPDRS) score prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step.

[0187] Paragraph 20. A method according to any of paragraphs 11 to 19, wherein the level of PRKN in the CSF of a subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not treated in the same manner.

[0188] Paragraph 21. A method according to any of paragraphs 11 to 19, wherein the UPDRS score of a subject treated by the method is improved compared to the UPDRS score of a subject not treated in the same way.

[0189] Paragraph 22. The method according to any one of paragraphs 11 to 21, wherein the target neuron expresses a reduced amount of alpha-synuclein and / or contains a reduced amount of Lewy bodies after contact with the manipulated DA neuron cell.

[0190] Paragraph 23. A population of DA neurons comprising a recombinant gene vector containing a polynucleotide encoding wild-type PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA gene, ubiquitin-like modifier-activating enzyme (ATG7) gene, synaptic vesicle amine transporter (VMAT2) gene, or glucocerebrosidase (GBA) gene, GDNF gene, SNCA+ / - gene, or a functional variant or fragment thereof, wherein the polynucleotide is operably linked to a promoter active in eukaryotic cells.

[0191] Type of cells to be manipulated In some embodiments, the cells to be manipulated may be primary cells. Primary cells may be neurons, pluripotent cells, precursor cells, or a combination thereof. In some embodiments, precursor cells are DA neurons (see markers for different nervous system cell types in Figure 1). Examples of pluripotent cells include stem cells, e.g., umbilical cord blood stem cells, precursor cells, bone marrow stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (IPS). The manipulated cells may be human cells. The manipulated cells may be animal (non-human cells). The manipulated cells may be enlarged ex vivo. The manipulated cells may be enlarged in vitro. The manipulated cells may be enlarged in vivo. The manipulated cells may be autologous to the target that needs them. The manipulated cells may be non-autologous to the target that needs them. The manipulated cells may be in vitro. The manipulated cells may be in vivo. Manipulated cells may be part of a combination therapy to treat Parkinson's disease and other and secondary Parkinson's disease-like disorders in subjects who require them.

[0192] Endogenous genes are disrupted by exogenous gene sequences. In some embodiments, the endogenous gene is disrupted by the exogenous GDNF, GBA, and / or SNCA gene sequence. In some embodiments, the endogenous gene is GDNF, GBA, and / or SNCA. In some embodiments, the endogenous gene is the wild-type GDNF, GBA, and / or SNCA gene. In some embodiments, the endogenous gene is the mutated GDNF, GBA, and / or SNCA gene. In some embodiments, the endogenous gene is the mutated PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF gene. In some embodiments, the endogenous gene may be under the control of a promoter. In some embodiments, the exogenous gene may be under the control of a promoter selected from the group including synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, and / or other promoters that allow temporal or small molecule control. In some embodiments, endogenous genes may be under the control of a promoter. In some embodiments, exogenous genes may be under the control of a promoter selected from the group including synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, or RU486-inducible promoter.

[0193] Manipulation of more than one exogenous gene sequence In some embodiments, the manipulated cells may contain a single GDNF, GBA, and / or SNCA exogenous sequence. In some embodiments, the manipulated cells may contain multiple GDNF, GBA, and / or SNCA exogenous sequences. In some embodiments, the manipulated cells may contain more than one of the GDNF, GBA, or SNCA exogenous sequences. The GDNF exogenous sequence may include a manipulated GDNF exogenous sequence. The GDNF exogenous sequence may produce a functional GDNF protein. The GBA exogenous sequence may include a manipulated GBA exogenous sequence. The GBA exogenous sequence may produce a functional GBA protein. The SCNA exogenous sequence may include a manipulated SCNA exogenous sequence. The SNCA exogenous sequence may produce a functional SCNA protein.

[0194] Protospacer adjacent motif arrangement Engineered cells containing at least one exogenous GDNF may be disclosed herein. GBA and / or SNCA genes or functional fragments thereof that can be flanked by a protospacer flanking motif sequence of genomic DNA. In some embodiments, the protospacer flanking motif sequence (PAM) may be recognized by a CRISPR endonuclease. The endonuclease may be a Cas protein. Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr 1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi, their homologs, or modified versions thereof may be selected from the list. In some embodiments, the CRISPR endonuclease may be Cas9. The Cas9 disclosed herein may recognize PAM sequences that may be 5' NGG 3'.

[0195] At least one exogenous GDNF capable of disrupting at least one gene may be disclosed herein. The disrupted gene may be any of the genes described in Sequence IDs 1 to 10. At least one exogenous GBA capable of disrupting at least one gene may be disclosed herein. The disrupted gene may be any of the genes described in Sequence IDs 1 to 10. At least one exogenous SNCA capable of disrupting at least one gene may be disclosed herein. The disrupted gene may be any of the genes described in Sequence IDs 1 to 10. In some embodiments, the disrupted may include a protospacer. The protospacer may be disrupted by insertion of an exogenous gene sequence. A GDNF gene sequence may produce functional GDNF. A GBA gene sequence may produce functional GBA. An SNCA gene sequence may produce functional SNCA.

[0196] A composition comprising at least one guide RNA that binds to an endogenous GDNF, GBA, or SNCA gene, and a secondary guide RNA that binds to an endogenous gene selected from the group consisting of any of the genes described in SEQ ID NOs: 1 to 10 or any of the genes described in SEQ ID NOs: 1 to 10 may be disclosed herein.

[0197] Engineered cells having disruption in the endogenous PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene sequence, and at least one secondary disruption in a second endogenous gene other than PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF may be disclosed herein.

[0198] Disclosed herein are genetically modified cells derived from human subjects; polynucleic acid-targeted polynucleic acids engineered to hybridize to a specific region of a target gene in the cell's genome; nucleases capable of associating with polynucleic acid-targeted polynucleic acids to form a nuclear protein complex, wherein the nuclear protein complex may be capable of generating targeted double-strand breaks in the target gene in the cell's genome; and targeted polynucleic acids which may be genomic DNA containing double-strand breaks in the target gene, wherein the double-strand breaks in the target gene result in the disruption of the target gene function, wherein the genetically modified cells may be scaled to produce a clonal population of cells in which the function of the target gene has been altered, and the clonal population of modified cells is suitable for administration to humans in need.

[0199] Gene editing systems The genome editing systems disclosed herein comprise at least two components adapted from naturally occurring CRISPR systems: a guide RNA (gRNA) and an RNA guide nuclease. These two components can associate with specific nucleic acid sequences in a cell to form a complex capable of editing DNA within or around that nucleic acid sequence by producing one or more of, for example, single-strand breaks (SSBs or nicks), double-strand breaks (DSBs), and / or point mutations. In various embodiments, the genome editing system may comprise (a) one or more Cas9 / gRNA complexes, and (b) separate Cas9 molecules and gRNAs that can associate in a cell to form one or more Cas9 / gRNA complexes. The genome editing systems of this disclosure may be encoded by one or more nucleotides (e.g., RNA, DNA) containing coding sequences for Cas9 and / or gRNA that can associate to form Cas9 / gRNA complexes, and one or more nucleotides encoding the gene editing system may be carried by a vector described herein.

[0200] In certain embodiments, the genome editing system targets neuronal genes that include PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF, or are selected from the group consisting of PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF. In certain embodiments, the genome editing system targets neuronal genes that include SEQ ID NOs. 1-10, or are selected from the group consisting of SEQ ID NOs. 1-10.

[0201] This disclosure relates to guide RNA (gRNA) comprising a targeting domain configured to associate with a target nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 (or a portion thereof), or with a complement or RNA equivalent thereof. In certain embodiments, the targeting domain has a length of 3 to 100, 5 to 100, 10 to 100, or 20 to 100 nucleotides, and in certain embodiments of these embodiments, the targeting domain has a length of 3 to 15, 3 to 20, 5 to 20, 10 to 20, 15 to 20, 5 to 50, 10 to 50, or 20 to 50 nucleotides. In certain embodiments, the targeting domain is the length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. Methods for selecting the targeting domain are known in the art (see, for example, Fu et al. (2014) NAT. BIOTECHNOL. 32(3): 279-84 and Sternberg et al. (2014) NATURE 507(7490): 62-67, the entire contents of each of these are expressly incorporated herein by reference). Since the targeting domain is part of a gRNA molecule, it contains the base uracil (U) rather than thymine (T), and in contrast, any DNA molecule encoding a gRNA molecule contains thymine rather than uracil. In a targeting domain / target domain pair, the uracil base in the targeting domain pairs with the adenine base in the target domain. In certain embodiments, the degree of complementarity between the targeting domain and the target domain is sufficient to enable the targeting of the Cas9 molecule to the target nucleic acid. Therefore, the targeting domains or gRNAs described herein include portions of Sequence IDs 1-10, which are 3-100 nucleotides in length, and contain uracil (U) rather than thymine (T).In certain embodiments, the targeting domain is fully complementary to the target domain. In certain embodiments, the targeting domain is fully complementary to sequence numbers 1-10 and contains uracil (U) instead of the base thymine (T). Similarly, if the targeting domain includes a core domain and / or a secondary domain, in certain embodiments, one or both of the core domain and the secondary domain are fully complementary to the corresponding portions of sequence numbers 1-10.

[0202] In other embodiments, the targeting domain is partially complementary to the target domain (SEQ ID NOs: 1-10). In certain embodiments of these embodiments, the nucleic acid sequence of the targeting domain is at least 80, 85, 90, or 95% complementary to the target domain or a corresponding portion of the target domain. In certain embodiments of these embodiments, the targeting domain comprises one or more nucleotides that are not complementary to the target domain or a portion thereof, and in certain embodiments of these embodiments, the targeting domain comprises one, two, three, four, five, six, seven, or eight nucleotides that are not complementary to the target domain. In certain embodiments of these embodiments, the targeting domain comprises one or more nucleotides that are not complementary to the target domain, and one or more of the non-complementary nucleotides are located within five nucleotides from the 5' or 3' end of the targeting domain. In certain embodiments of these embodiments, the targeting domain contains one, two, three, four, or five nucleotides that are not complementary to the targeting domain within five nucleotides from its 5' end, 3' end, or both of its 5' and 3' ends. In certain embodiments, the targeting domain contains two or more nucleotides that are not complementary to the targeting domain, and two or more of the non-complementary nucleotides are adjacent to each other, and in certain embodiments of these embodiments, two or more consecutive non-complementary nucleotides are located within five nucleotides from the 5' or 3' end of the targeting domain. In other embodiments, any two or more consecutive non-complementary nucleotides are located at a distance of more than five nucleotides from the 5' and 3' ends of the targeting domain.

[0203] In certain embodiments, the targeting domain consists of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 consecutive nucleotides) that are complementary or partially complementary to the target domain or a portion thereof, essentially consisting of these nucleotides, or containing these nucleotides, for example, the targeting domain having a length of 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides. In certain embodiments of these embodiments, the targeting domain is complementary to the target domain over its entire length, over its entire length, or both.

[0204] In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein comprises the first 20 Ns (residues 1-20) of the amino acid sequence described in SEQ ID NOs: 1-10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein comprises the second 20 Ns (residues 20-40) of the amino acid sequence described in SEQ ID NOs: 1-10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein comprises the third 20 Ns (residues 40-60) of the amino acid sequence described in SEQ ID NOs: 1-10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein comprises the fourth 20 Ns (residues 60-80) of the amino acid sequence described in SEQ ID NOs: 1-10. In certain embodiments, the monomolecule or chimeric gRNA molecules disclosed herein (including the targeting domain) comprise the fifth 20 N (residues 80-100) of the amino acid sequences described in SEQ ID NOs: 1-10. In some embodiments, the targeting domain is listed as 20 N (residues 1-20), but may range in length from 16 to 26 nucleotides.

[0205] In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein contains the last 20 Ns of the amino acid sequence described in SEQ ID NOs: 1 to 10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein contains the second to last 20 Ns of the amino acid sequence described in SEQ ID NOs: 1 to 10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein contains the third to last 20 Ns of the amino acid sequence described in SEQ ID NOs: 1 to 10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein contains the fourth to last 20 Ns of the amino acid sequence described in SEQ ID NOs: 1 to 10. In certain embodiments, the monomolecule or chimeric gRNA molecule (including the targeting domain) disclosed herein contains the fifth to last 20 Ns of the amino acid sequence described in SEQ ID NOs: 1 to 10.

[0206] In certain embodiments, modifications to one or more nucleotides in the targeting domain or non-complementary nucleotides do not interfere with targeting efficiency, which can be evaluated by testing the candidate modifications using systems known in the art. gRNAs having candidate targeting domains having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated using systems known in the art. Candidate targeting domains may be positioned alone or together with one or more other candidate modifications in a gRNA / Cas9 molecular system known to be functional for a selected target and may be evaluated.

[0207] This disclosure also covers DNA-targeting compositions comprising a first gRNA and a second gRNA. The first gRNA molecule and the second gRNA molecule include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 (or a portion thereof), or include a targeting domain configured to associate with a target nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or its complement. In some embodiments, the first gRNA molecule and the second gRNA molecule include different targeting domains.

[0208] This disclosure also covers isolated polynucleotides comprising the above-mentioned gRNA molecules or the above-mentioned DNA-targeting compositions.

[0209] This disclosure relates to vectors comprising the above-mentioned gRNA, the above-mentioned DNA targeting composition, or the above-mentioned isolated polynucleotide.

[0210] This disclosure also covers vectors containing the DNA targeting compositions described above.

[0211] The present invention also covers a vector encoding (a) a first guide RNA (gRNA) molecule, (b) a second gRNA molecule, and (c) at least one Cas9 molecule that recognizes either NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42). The first and second gRNA molecules include a targeting domain configured to associate with a target nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 (or a portion thereof), or a complement thereof. In some embodiments, the first and second gRNA molecules include different targeting domains.

[0212] This disclosure also covers cells comprising the above-mentioned gRNA, the above-mentioned DNA-targeting composition, the above-mentioned isolated polynucleotide, and / or the above-mentioned vector.

[0213] This disclosure also covers kits, including the above-mentioned gRNA, the above-mentioned DNA targeting system, the above-mentioned isolated polynucleotide, the above-mentioned vector, or the above-mentioned cells, and, if applicable, instructions for use.

[0214] This disclosure also covers methods for modifying variant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA genes in cells. The method comprises administering cells with the above-mentioned gRNA, the above-mentioned DNA targeting system, the above-mentioned isolated polynucleotide, or the above-mentioned vector.

[0215] This disclosure also covers methods for genome editing mutant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA genes in a subject. The method comprises administering a genome editing composition to the subject, comprising the above-mentioned gRNA, the above-mentioned DNA targeting system, the above-mentioned isolated polynucleotide, the above-mentioned vector, or the above-mentioned cells.

[0216] This disclosure also relates to methods for treating subjects that require treatment and have variant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA genes. The methods include administering the subject the above-mentioned gRNA, the above-mentioned DNA targeting system, the above-mentioned isolated polynucleotide, the above-mentioned vector, or the above-mentioned cells.

[0217] This disclosure also covers modified adeno-associated virus vectors for genome editing of variant PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA genes in the subject, comprising a first polynucleotide sequence encoding the above-mentioned gRNA and a second polynucleotide sequence encoding a Cas9 molecule that recognizes either the NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42) protospacer-adjacent motif (PAM).

[0218] This disclosure also covers cells containing the above-mentioned compositions.

[0219] A vector encoding a guide RNA (gRNA) molecule and a Cas9 molecule, wherein the gRNA molecule includes SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10 (or a fragment thereof), or includes a targeting domain configured to associate with a target nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or its complement.

[0220] (a) A gRNA molecule containing a targeting domain configured to associate with a target nucleotide sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or its complement thereof; and (b) A Cas9 molecule comprising a genome editing system.

[0221] Non-restrictive methods for evaluating chromatin accessibility include micrococcal nuclease (MNase)-assisted isolation of nucleosomes sequencing (MNase-seq), DNase I hypersensitive site sequencing (DNase-seq), formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq), and assays for transposase-accessible chromatin using sequencing (ATAC-seq). In some embodiments, chromatin accessibility is determined by an assay for transposase-accessible chromatin using high-throughput sequencing (ATAC-seq). In some embodiments, the disclosed method further includes selecting a locus as a GSH if the locus is located at a distance of up to approximately 10kb, up to approximately 9kb, up to approximately 8kb, up to approximately 7kb, up to approximately 6kb, up to approximately 5kb, up to approximately 4kb, up to approximately 3kb, up to approximately 2kb, or up to approximately 1kb from the ATAC-seq peak, or within the ATAC-seq peak.

[0222] The gene editing systems disclosed herein can be further understood by the following numbered paragraphs.

[0223] Paragraph 1. A gene editing system for cells, a. A Cas protein or a polynucleotide encoding a Cas protein, b. Guide RNA (gRNA), c. Repair templates containing functional PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GDNF, or GBA genes, or their functional variants or fragments. The gene editing system includes the ability to repair endogenous genes in cells or insert functional genes into the cell's genome in vivo or in vitro. Gene editing systems.

[0224] Paragraph 2. The gene editing system described in Paragraph 1, wherein at least one component of the gene editing system is delivered by recombinant AAV.

[0225] Paragraph 3. The gene editing system described in Paragraph 1, wherein the gene editing system is delivered by recombinant AAV.

[0226] Paragraph 4. A gene editing system according to any one of paragraphs 1 to 3, wherein the cells are in vitro pluripotent cells or DA neuron cells.

[0227] Paragraph 5. A gene editing system described in any of paragraphs 1 to 3, wherein the cells are dopaminergic neurons in vivo.

[0228] Recombinant gene vectors Various embodiments of recombinant gene vectors and related methods are disclosed, comprising PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, and / or GBA, or functional fragments or variants thereof. In some embodiments, the vector further includes editing of the alpha-synuclein (SCNA) gene to make the cell population into which the vector is introduced hemizygous null with respect to SNCA. In some embodiments, the recombinant gene vector makes the cells into which it is introduced hemizygous null with respect to SNCA and / or homozygous null with respect to MAPT. In some embodiments, the recombinant gene vector makes the cells into which it is introduced hemizygous null with respect to SNCA and / or homozygous null with respect to MAPT. In some embodiments, the recombinant gene vector makes the cells into which it is introduced hemizygous null with respect to SNCA and / or hemizygous null with respect to MAPT. In some embodiments, the recombinant gene vector does not include SCNA gene editing.

[0229] Various embodiments of recombinant gene vectors comprising the GDNF gene (SEQ ID NO: 10) or a functional fragment or variant thereof, and related methods are disclosed. In some embodiments, the vector further includes editing of the GBA gene. In some embodiments, the vector further includes editing of the SNCA gene.

[0230] Various embodiments of recombinant gene vectors and related methods comprising the GBA gene (SEQ ID NO: 7) or a functional fragment or variant thereof are disclosed. In some embodiments, the vector further includes editing of the SNCA gene.

[0231] Various viral or nonviral vectors can be used. In some embodiments, the recombinant gene vector is recombinant adeno-associated virus (AAV). Any of the known serotypes can be used. In some embodiments, the AAV has serotypes AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant gene vector contains self-complementary AAV. In some embodiments, the recombinant gene vector contains single-stranded AAV. In some embodiments, the AAV is wild-type AAV or modified AAV. In some embodiments, the AAV contains a capsid protein having at least 95% identity to wild-type VP1, VP2, or VP3 capsid protein.

[0232] Recombinant gene vectors may contain gene regulatory elements. In some embodiments, the recombinant gene vector comprises a polynucleotide comprising, in the following 5' to 3' order, a promoter sequence active in eukaryotic cells and a sequence encoding a wild-type protein or a functional fragment or variant thereof. The sequence encoding the wild-type protein or a functional fragment or variant thereof is operably ligated to the promoter sequence active in eukaryotic cells.

[0233] In some embodiments, the vectors and methods disclosed herein include an expression cassette comprising the following, in order from 5' to 3':

[0234] Promoter (GDNF knock-in promoter [e.g., MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [e.g., mTHY1, rNSE, DDC, COMT, GBA P1, GBA P2, EFS, EEF1A1, CBA, CMV, hPGK], synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, or other promoters that allow temporal or small molecule control), gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF),

[0235] Promoters (GDNF knock-in promoters [e.g., MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoters [e.g., mTHY1, rNSE, DDC, COMT, GBA P1, GBA P2, EFS, EEF1A1, CBA, CMV, hPGK], Synapsin 1, DAT, VMAT, TH, AADC, Tamoxifen-inducible promoter, RU486-inducible promoter), Genes (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF),

[0236] Promoter (GDNF knock-in promoter [e.g., MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [e.g., mTHY1, rNSE, DDC, COMT, GBA P1, GBA P2, EFS, EEF1A1, CBA, CMV, hPGK], synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, or other promoters that allow temporal or small molecule control), gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF), and hemizygous null with respect to SNCA and / or MAPT genes,

[0237] Promoter (GDNF knock-in promoter [e.g., MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [e.g., mTHY1, rNSE, DDC, COMT, GBA P1, GBA P2, EFS, EEF1A1, CBA, CMV, hPGK], synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, or other promoters that allow temporal or small molecule control), 5' enhancer, gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF),

[0238] Promoter (GDNF knock-in promoter [e.g., MAOB, GCH1, NR4A2, TH, SLC6A3, SYN1, Camk2a, NEFM, NEFL, or NEFH], GBA knock-in promoter [e.g., mTHY1, rNSE, DDC, COMT, GBA P1, GBA P2, EFS, EEF1A1, CBA, CMV, hPGK], synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, or other promoters that allow temporal or small molecule control), 5' enhancer, gene (PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, and / or GDNF), and hemizygous null with respect to SNCA and / or MAPT genes.

[0239] In some embodiments, the recombinant gene vector optionally includes the promoters hSYN1 (human synapsin), INA (alpha-internexin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (forkheadbox A2), CaMKII (calmodulin-dependent protein kinase II), and NSE (neuron-specific enolase), or includes one or more neuron-specific promoters selected from the group consisting of hSYN1 (human synapsin), INA (alpha-internexin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (forkheadbox A2), CaMKII (calmodulin-dependent protein kinase II), and NSE (neuron-specific enolase).

[0240] In some embodiments, the recombinant gene vector includes CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and a chicken beta-actin promoter, or includes a ubiquitous promoter selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and a chicken beta-actin promoter.

[0241] In some embodiments, the engineered DA neuronal cells transduced with a recombinant gene vector also include inducing mutations in genes associated with Parkinson's disease (PD). The genes to be mutated may be SNCA and / or MAPT. The genes to be mutated may be one of Sequence IDs 1-10 or a combination thereof.

[0242] A vector encoding a guide RNA (gRNA) molecule and one Cas protein molecule is disclosed, wherein the gRNA molecule comprises a targeting domain containing a portion of a nucleotide sequence selected from SEQ ID NOs: 1 to 10. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adeno-associated virus vector. In some embodiments, the vector is a nucleic acid sequence containing an origin of replication. In some embodiments, the vector may be a viral vector, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. In some embodiments, the vector is a DNA or RNA vector. In some embodiments, the vector is a self-replicating extrachromosomal vector, preferably a DNA plasmid. For example, the vector may encode a Cas9 protein and at least one gRNA molecule. In some embodiments, the Cas9 protein may be S. aureus Cas9, e.g., SaCas9.

[0243] (a) a gRNA molecule containing a targeting domain that includes a portion of a nucleotide sequence selected from Sequence IDs 1 to 10, and (b) a genome editing system containing a Cas protein molecule are disclosed.

[0244] In some embodiments, the vector or genome editing system is configured to modify the GDNF, GBA, or SNCA gene. In some embodiments, the vector or genome editing system is configured to modify SEQ ID NO: 10, SEQ ID NO: 7, or SEQ ID NO: 8. In some embodiments, the Cas protein molecule is the S. aureus Cas9 molecule. In some embodiments, the Cas protein molecule recognizes either the NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42) protospacer adjacent motif (PAM).

[0245] The subject matter of this disclosure also provides cells comprising vectors or genome editing systems described herein. In some embodiments, the cells are pluripotent cells or DA neurons or dopaminergic neurons.

[0246] The subject matter of this disclosure further provides a method for enhancing one or more of the following: viability, engraftment, proliferation, migration, innervation, or differentiation, comprising the step of administering to a cell (a) a vector encoding a gRNA molecule and one Cas protein molecule, or (b) a genome editing system comprising a gRNA molecule and a Cas protein molecule, wherein the gRNA molecule comprises a targeting domain comprising a 20-base pair portion of a nucleotide sequence selected from the GDNF (SEQ ID NO: 10), GBA (SEQ ID NO: 7), and / or SNCA (SEQ ID NO: 8) gene or a functional fragment thereof.

[0247] In one embodiment, the present disclosure relates to a method for treating or modifying cells in a subject (e.g., a human subject or an animal subject), comprising the step of administering to the subject nucleic acids encoding first and second guide RNAs (gRNAs) that target the Cas protein and the subject's GDNF (SEQ ID NO: 10), GBA (SEQ ID NO: 7), and / or SNCA (SEQ ID NO: 8) genes. In some embodiments, the first and second gRNAs target one or more target sequences that encompass or are adjacent to 20 target sites of nucleic acids that may be located at the 5', 3', or center of the sequence. The first gRNA may include a targeting domain selected to target first 20 Ns (residues 1-20), while the targeting domain of the second gRNA may be selected to target second 20 Ns (residues 20-40). The first gRNA may contain a targeting domain selected to target a second set of 20 Ns (residues 20-40), while the targeting domain of the second gRNA may be selected to target a third set of 20 Ns (residues 40-60). The first gRNA may contain a targeting domain selected to target a third set of 20 Ns (residues 40-60), while the targeting domain of the second gRNA may be selected to target a fourth set of 20 Ns (residues 60-80). The first gRNA may contain a targeting domain selected to target a second set of 20 Ns (residues 20-40), while the targeting domain of the second gRNA may be selected to target a fifth set of 20 Ns (residues 80-100).

[0248] A Cas protein, which may be a modified Cas protein (e.g., Cas9 manipulated to alter the PAM specificity of Cas9, improve fidelity, or modify or improve another structural or functional aspect), may contain one or more nuclear localization signals (NLS) and / or polyadenylation signals. Certain embodiments are characterized by a Cas protein containing both C-terminal and N-terminal NLS. The Cas protein is optionally driven by a promoter selected from the group including synapsin 1, DAT, VMAT, TH, AADC, tamoxifen-inducible promoter, RU486-inducible promoter, or other promoters that allow for temporal or small molecule control. The nucleic acid also, in various cases, contains first and second terminal inverted repeat sequences (ITRs).

[0249] The embodiments disclosed herein can be further understood by the following numbered paragraphs.

[0250] Paragraph 1. A vector encoding a guide RNA (gRNA) molecule and a Cas protein molecule, wherein the gRNA molecule comprises a targeting domain configured to modify the PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene.

[0251] Paragraph 2. The vector described in Paragraph 1, wherein the Cas molecule is the S. aureus Cas9 molecule.

[0252] Paragraph 3. The vector described in Paragraph 1, wherein the Cas molecule recognizes either the NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42) protospacer adjacent motif (PAM).

[0253] Paragraph 4. The vector is a viral vector, as described in Paragraph 1.

[0254] Paragraph 5. A composition comprising the vector described in Paragraph 1.

[0255] Paragraph 6. A genome editing system, (a) A gRNA molecule comprising a targeting domain configured to modify the nucleotide sequences described in SEQ ID NOs: 1-10 or containing the nucleotide sequences described in SEQ ID NOs: 1-10, (b) Cas molecule and This includes genome editing systems.

[0256] Paragraph 7. The genome editing system described in Paragraph 6, wherein the genome editing system is configured to modify the PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene.

[0257] Paragraph 8. The genome editing system described in Paragraph 6, wherein the Cas molecule is the S. aureus Cas9 molecule.

[0258] Paragraph 9. The genome editing system described in Paragraph 6, wherein the Cas molecule recognizes either NNGRRT (SEQ ID NO: 41) or NNGRRV (SEQ ID NO: 42) PAM.

[0259] Paragraph 10. Isolated cells containing the vector described in Paragraph 1.

[0260] Paragraph 11. Isolated cells containing the genome editing system described in Paragraph 6.

[0261] Paragraph 12. A method for producing gene-edited cells, wherein the cells, (a) A vector encoding a gRNA molecule and a Cas protein molecule, or (b) Genome editing system including gRNA molecules and Cas protein molecules The step includes administering The gRNA molecule includes a targeting domain configured to contain a portion of the nucleotide sequence described in SEQ ID NOs: 1-10 or to modify the nucleotide sequence described in SEQ ID NOs: 1-10. method.

[0262] Paragraph 13. The method according to Paragraph 12, wherein the cells include pluripotent cells and DA neurons, or are selected from the group consisting of pluripotent cells and DA neurons.

[0263] Paragraph 14. A method for inhibiting the degeneration or death of dopaminergic neurons, including mutations in genes associated with Parkinson's disease (PD), The procedure includes the step of contacting a neuron with a recombinant gene therapy vector described in any one of paragraphs 1 to 4, A method for in which neurons express PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GDNF, SNCA+ / -, and / or GBA proteins after contact with a recombinant gene therapy vector.

[0264] Paragraph 15. The method according to Paragraph 14, wherein neurons express a reduced amount of alpha-synuclein after contact with a recombinant gene therapy vector.

[0265] Paragraph 16. The method according to any one of paragraphs 14 to 15, wherein neurons produce and / or release increased amounts of dopamine after contact with a recombinant gene therapy vector.

[0266] Paragraph 17. The method according to any one of paragraphs 14 to 16, wherein the neurons express a lower amount of alpha-synuclein compared to the amount expressed in neurons not in contact with the recombinant gene therapy vector, and where applicable, the lower amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in neurons not in contact with the recombinant gene therapy vector.

[0267] Paragraph 18. The method according to any one of paragraphs 14 to 17, wherein a neuron produces and / or releases an increased amount of dopamine compared to the amount of dopamine produced and / or released by a neuron not in contact with the recombinant gene therapy vector, and where applicable, the increased amount is 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%, at least twice, at least three times, at least four times, at least five times, or at least ten times more than the amount produced and / or released by a neuron not in contact with the recombinant gene therapy vector.

[0268] Paragraph 19. The method according to any one of paragraphs 14 to 18, wherein a neuron receives an increased amount of autophagy compared to the amount of autophagy received by a neuron not in contact with the recombinant gene therapy vector, and where applicable, the increased amount is 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%, at least twice, at least three times, at least four times, at least five times, or at least ten times more than the amount received by a neuron not in contact with the recombinant gene therapy vector.

[0269] Paragraph 20. The method according to any one of paragraphs 14 to 19, wherein the neuron is a primary tyrosine hydroxylase-positive neuron.

[0270] Paragraph 21. A method for treating or inhibiting the development of Parkinson's disease (PD) in a subject who has or is at risk of having PD, The step includes administering a recombinant gene therapy vector as described in any one of paragraphs 1 to 4, Administration of recombinant gene therapy vectors treats or inhibits the development of Parkinson's disease in subjects. method.

[0271] Paragraph 22. The method according to claim 22, wherein the subject is an adult or a child.

[0272] Paragraph 23. The method according to any one of paragraphs 21 to 22, wherein the number of dopaminergic neurons in the subject after the administration step exceeds the number of dopaminergic neurons in the subject before the administration step.

[0273] Paragraph 24. The method according to any one of paragraphs 21 to 23, wherein the level of dopamine in the subject after the administration step is higher than the level of dopamine in the subject before the administration step.

[0274] Paragraph 25. A method according to any one of paragraphs 21 to 24, wherein the number of dopaminergic neurons in a subject treated by the method is increased compared to the number of dopaminergic neurons in a subject not treated in the same way.

[0275] Paragraph 26. A method according to any one of paragraphs 21 to 25, wherein the level of dopamine in a subject treated by the method is increased compared to the level of dopamine in a subject not treated in the same way.

[0276] Paragraph 27. A method according to any one of paragraphs 21 to 26, wherein the level of dopamine in the substantia nigra of a subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not treated in the same way.

[0277] Paragraph 28. The method according to any one of paragraphs 21 to 27, wherein the levels of GDNF and / or GBA in the subject's CSF after the administration step are higher than the levels of GDNF and / or GBA in the subject's CSF before the administration step.

[0278] Paragraph 29. The method according to any one of paragraphs 21 to 28, wherein the Unified Parkinson's Disease Rating Scale (UPDRS) score of the subject prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step.

[0279] Paragraph 30. A method according to any of paragraphs 21 to 29, wherein the level of PRKN in the CSF of a subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not treated in the same manner.

[0280] Paragraph 31. A method according to any one of paragraphs 21 to 29, wherein the UPDRS score of a subject treated by the method is improved compared to the UPDRS score of a subject not treated in the same way.

[0281] Paragraph 32. The method according to any one of paragraphs 21 to 31, wherein the target neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with a recombinant gene therapy vector.

[0282] The embodiments disclosed herein can be further understood by the following numbered paragraphs.

[0283] Paragraph 1. Manipulated pluripotent cells containing an exogenous sequence encoding the GDNF gene, which is incorporated under the transcriptional control of an endogenous gene promoter.

[0284] Paragraph 2. Manipulated DA neuronal cells containing an exogenous sequence encoding the GDNF gene, which is incorporated under the transcriptional control of an endogenous gene promoter.

[0285] Paragraph 3. Manipulated pluripotent cells as described in Paragraph 1, having the genotype SNCA+ / -.

[0286] Paragraph 4. Manipulated DA neuronal cells as described in Paragraph 2, having the genotype SNCA+ / -.

[0287] method Method for creating manipulated cells In some embodiments, a method for producing engineered cells is disclosed, comprising the step of contacting pluripotent cells with a gene editing system comprising a repair template comprising a Cas protein or a polynucleotide encoding a Cas protein, guide RNA (gRNA), and functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF genes, or functional variants or fragments thereof.

[0288] In some embodiments, pluripotent cells are present in vitro. In some embodiments, engineered pluripotent cells differentiate into DA neurons in vitro to form engineered DA neurons. In some embodiments, engineered DA neurons are translated into a patient. In some embodiments, engineered DA neurons can enhance viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or the function of the administered engineered DA neurons compared to wild-type neurons. In some embodiments, the administered engineered DA neurons differentiate into mature neuronal cell types in vivo, and the administered engineered DA neurons enhance the function of mature neuronal cell types compared to wild-type mature neuronal cells. In some embodiments, the administered engineered DA neurons enhance the function of endogenous (unengineered) mature neuronal cell types in the patient.

[0289] In some embodiments, the pluripotent cells and / or subjects to be genetically engineered include mutations in the PARK2 gene, PINK1 gene, LRRK2 gene, c-Rel gene, ATG7 gene, VMAT2, GBA, SNCA, MAPT, and / or GDNF gene. In some embodiments, the pluripotent cells and / or subjects to be genetically engineered include mutations in one or a combination thereof of SEQ ID NOs: 1-10.

[0290] A method for producing engineered cells is disclosed herein, comprising the steps of introducing into cells a guide polynucleotide containing a spacer region complementary to a target nucleic acid in a cellular genomic region, a nuclease guided by the guide polynucleotide, and a polynucleotide encoding exogenous GDNF, GBA, or SNCA; site-specifically cleaving the target nucleic acid within the cell with the nuclease guided by the guide polynucleotide; and inserting the polynucleotide encoding exogenous GDNF, GBA, and / or SNCA into the cellular genomic region at the cleavage site. The nuclease may be Cas9. In some embodiments, the guide polynucleotide may be a single guide polynucleotide. The guide polynucleotide may be RNA. The target nucleic acid may be DNA. The spacer region may be 10 to 30 nucleotides long. The nuclease may induce a double-strand break in the target nucleic acid. The nuclease may induce a single-strand break in the target nucleic acid.

[0291] In some embodiments, the guide polynucleotide may be introduced into cells by electroporation. The guide nucleic acid may be introduced into cells by nucleofection. The nuclease may also be introduced into cells by a delivery vector. The polynucleotide encoding exogenous GDNF, GBA, and / or SNCA may further include a promoter sequence. The promoter sequence may be selected from one of SEQ ID NOs: 21-40 or 43. The exogenous GDNF, GBA, and / or SNCA may be inserted by homologous recombination. The guide polynucleotide and nuclease may form a nuclear protein complex.

[0292] By cleaving the target nucleic acid, genomic nucleic acid sequences can be removed and replaced with polynucleotides encoding exogenous GDNF, GBA, and / or SNCA. The polynucleotides encoding exogenous GDNF, GBA, and / or SNCA may further comprise a first recombinant arm and a second recombinant arm. The first recombinant arm may comprise a first sequence identical to a first portion of the target nucleic acid, and the second recombinant arm may comprise a second sequence identical to a second portion of the target nucleic acid. In some embodiments, the first recombinant arm may comprise a first sequence identical to a first portion adjacent to the target nucleic acid, and the second recombinant arm may comprise a second sequence identical to a second portion adjacent to the target nucleic acid. The target nucleic acid may be located within a gene. The gene may be selected from GDNF, GBA, and / or SNCA.

[0293] In some embodiments, insertion of exogenous GDNF, GBA, and / or SNCA sequences at cleavage sites can result in gene disruption. Target nucleic acids may be located within intergenetic sites. Exogenous GDNF, GBA, and / or SNCA can be expressed in cells. Manipulated cells can be introduced into organisms. Manipulated cells can be expanded ex vivo.

[0294] A method for efficient target gene disruption in DA neuronal cells is disclosed herein, comprising the steps of: contacting pluripotent cells with a Cas protein nuclease and guide RNA, wherein the guide RNA comprises a region of 17 to 22 nucleotides substantially complementary to a region in the target gene; and cleaving the target gene, wherein the target gene is GDNF, GBA, and / or SNCA, wherein when a population of pluripotent cells is contacted with the Cas protein nuclease and guide RNA, an exogenous knockout event occurs in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of the pluripotent cells.

[0295] In some embodiments, the method comprises one or more cells, which are pluripotent cells, DA neuron cells, neurons, or any combination thereof. In some embodiments, the method comprises a first nucleic acid, which is DNA, RNA, or a hybrid thereof. In some embodiments, the method comprises a first nucleic acid, which is single-stranded or double-stranded. In some embodiments, the method comprises a second nucleic acid, which is DNA, RNA, or a hybrid thereof. In some embodiments, the method comprises a second nucleic acid, which is single-stranded or double-stranded. In some embodiments, the method comprises a step of introducing the first nucleic acid, which includes non-viral transfection, biolytics, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method comprises viral transfection, which includes adeno-associated virus. In some embodiments, the method includes a step of introducing a second nucleic acid, the step of introducing a second nucleic acid includes nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection.

[0296] In some embodiments, the method includes a double-strand break, and the creation of the double-strand break includes CRISPR, TALEN, transposon-based, ZFN, meganuclease, or mega-TAL. In some embodiments, the method includes a double-strand break, and the creation of the double-strand break includes CRISPR. In some embodiments, the method includes a double-strand break, and the double-strand break is repaired by insertion of a gene encoding an engineered GDNF, GBA, and / or SNCA gene. In some embodiments, the method includes a second nucleic acid, the second nucleic acid includes a recombinant arm, and the second gene encoding an engineered GDNF, GBA, and / or SNCA gene is flanked by the recombinant arm. In some embodiments, the method includes a recombinant arm, the recombinant arm is at least partially complementary to at least a portion of at least one endogenous GDNF, GBA, and / or SNCA gene. In some embodiments of the methods of this disclosure, increased isogeneity between the recombinant arm and at least one endogenous GDNF, GBA, and / or SNCA gene corresponds to increased efficiency of the insertion of the second gene. In some embodiments, the method includes the insertion of the second gene, and the efficiency of the insertion of the second gene is measured using fluorescence-expressing cell sorting. In some embodiments, the method includes the step of introducing a second nucleic acid, and the step of introducing the second nucleic acid includes nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. In some embodiments, the method includes the insertion of a second gene, and the insertion of the second gene encoding the manipulated GDNF, GBA, and / or SNCA gene includes homology-dependent repair (HDR). In some embodiments, the method includes the insertion of a second gene, and the insertion of the second gene is assisted by a homologous recombination (HR) enhancer. In some embodiments, the method includes an enhancer, which is derived from a viral protein.In some embodiments, the method includes an HR enhancer, which includes E4orf6, E1b55K, E1b55K-H354, E1b55K-H373A, Scr7, L755507, or any combination thereof, or is selected from the group consisting of E4orf6, E1b55K, E1b55K-H354, E1b55K-H373A, Scr7, L755507, or any combination thereof. In some embodiments, the method includes an HR enhancer, which is a chemical inhibitor. In some embodiments, the method includes an HR enhancer, which inhibits ligase IV. In some embodiments, the method includes reducing cytotoxicity, which includes at least one of the following: DNA breakage, cell death, apoptosis, nuclear condensation, cell lysis, necrosis, alteration of cell motility, alteration of cell stiffness, alteration of cytoplasmic protein expression, alteration of membrane protein expression, swelling, loss of membrane integrity, cessation of metabolic activity, decrease in metabolic activity, increase in metabolic activity, increase in reactive oxygen species, cytoplasmic contraction, or any combination thereof. In some embodiments, the method includes a step of measuring viability, which is measured using at least one of the following: fluorescence-expressing cell sorting, trypan blue exclusion, CD4+ cell surface marker, CD8+ cell surface marker, telomere length, or any combination thereof. In some embodiments, the method includes a subject, which is a human subject.

[0297] Method for preparing a therapeutically effective composition In one embodiment, a method for preparing a therapeutically effective composition comprising one or more cells is disclosed. In some embodiments, the method comprises gene editing, which comprises introducing a first nucleic acid into one or more cells. In some embodiments, the method comprises a first nucleic acid, which comprises a first gene encoding the PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF protein. In some embodiments, the method comprises gene editing, which comprises introducing a second nucleic acid into one or more cells. In some embodiments, the method comprises a second nucleic acid, which comprises a second gene encoding the PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF protein, and the second gene and the first gene are different.

[0298] A method for treating or inhibiting the development of Parkinson's disease (PD) in a person who has or is at risk of having PD, A step of administering DA neuronal cells, wherein the DA neuronal cells comprise a recombinant gene vector comprising a polynucleotide sequence encoding a wild-type PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, or GDNF gene, or a functional variant or fragment thereof, wherein the administration of DA neuronal cells comprising the recombinant gene vector treats or inhibits the development of Parkinson's disease in a subject. The method will be disclosed.

[0299] Methods for identifying safe harbor loci A method for identifying safe harbor loci is disclosed. A method for identifying safe harbor loci suitable for editing PSCs and expression in post-mitotic dopaminergic neurons, comprising, or essentially comprising, these steps: a. generating a prioritized genomic safe harbor site from clinically compliant PSC systems and / or dopaminergic neuron precursors using scATAC-seq data; b. comparing the data obtained from step a with publicly available ATAC-seq data obtained from dopaminergic neurons isolated from healthy and Parkinson's diseased human brains; and c. defining regions of chromatin accessibility common between the data in a and the comparison in step b, thereby generating a list of prioritized GSH sites suitable for editing in PSCs and expression in post-mitotic dopaminergic neurons. In some embodiments, the prioritized list of GSH sites is further prioritized based on the distality of the GSH site from the gene, regulatory region, telomere, and centromere to prevent the transgene from interfering with gene expression, division, and function. In some embodiments, GSH sites are selected based on the highest efficiency of transgene insertion, sustained expression after long-term culture, and / or the absence of oncogenic gene expression. In some embodiments, GSH sites are selected based on peak size and / or distribution data generated using processed ATAC-seq data.

[0300] A method for identifying safe harbor loci suitable for editing PSCs and expression in post-mitotic dopaminergic neurons is disclosed, comprising, or essentially comprising, the steps of: a. generating prioritized genomic safe harbor sites from clinically compliant PSC systems and / or dopaminergic neuron precursors using scATAC-seq data; b. comparing the data obtained from step a with publicly available ATAC-seq data obtained from dopaminergic neurons isolated from healthy and Parkinson's diseased human brains; c. defining regions of chromatin accessibility common between the data in a and the comparison in step b; and d. selecting safe harbor loci based on threshold parameters, wherein the safe harbor loci are selected for insertion of at least one sequence encoding a transgene in the cell. In some embodiments, the threshold parameters are based on peak size and / or distribution data generated using processed ATAC-seq data. In some embodiments, threshold parameters are based on peak size data generated using processed ATAC-seq data, distribution data generated using processed ATAC-seq data, the highest efficiency for transgene insertion, sustained expression after long-term culture, and / or the absence of oncogenic gene expression. In some embodiments, chromatin accessibility is defined based on (1) PSCs for optimizing editing efficiency in step a, (2) DA neuron cells for optimizing editing efficiency in step a, and (3) post-mitotic dopaminergic neurons derived from human brain samples. In some embodiments, chromatin accessibility is defined based on (1) PSCs for optimizing editing efficiency in step a, (2) DA neuron cells for optimizing editing efficiency in step a, (3) post-mitotic dopaminergic neurons derived from human brain samples, and (4) neuronal mature cell type cells.

[0301] In some embodiments, a method for targeting a transgene (e.g., GDNF) to a safe harbor locus in PSCs for expression in post-mitotic dopaminergic neurons further includes the steps of selecting an sgRNA having the highest on-target activity and the lowest off-target activity, and empirically defining the best cleavage sgRNA.

[0302] In some embodiments, the step of identifying safe harbor loci in PSCs for expression in post-mitotic dopaminergic neurons further includes defining chromatin accessibility based on a comparison of ATAC-seq peak data in a first cell type (PSC, DA neurons, and / or neuronal mature cell type) with ATAC-seq peak data in a second different cell type (PSC cells, DA neuron cells, neuronal mature cell type, and / or HEK cells). In some embodiments, chromatin accessibility is based on ATAC-seq peak data compared with validated transgene insertions, and / or alignment between ATAC-seq peaks and sgRNA locations. In some embodiments, chromatin accessibility is based on ATAC-seq peak data compared with scATAC-seq data obtained from substantia nigra (SN) cells of healthy controls and Parkinson's disease patients. In some embodiments, chromatin accessibility is based on ATAC-seq peak data compared to scATAC-seq data obtained from healthy controls and Parkinson's disease patients, compared to publicly available ATAC-seq data obtained from human iPSCs and mature dopaminergic neurons. In some embodiments, chromatin accessibility is based on ATAC-seq peak data compared to scATAC-seq data obtained from healthy controls and Parkinson's disease patients, compared to publicly available ATAC-seq data obtained from human iPSCs and mature dopaminergic neurons, compared to scATAC-seq data obtained from healthy controls and Parkinson's disease patients, compared to scATAC-seq data obtained from healthy controls and Parkinson's disease patients, compared to publicly available ATAC-seq data obtained from human iPSCs and mature dopaminergic neurons. In some embodiments, chromatin accessibility is based on candidate GSH sites that are classified as "open" in healthy controls but "closed" in Parkinson's disease patients. In some embodiments, chromatin accessibility is based on candidate GSH sites that are classified as "closed" in healthy controls but as "open" in Parkinson's disease patients.In some embodiments, chromatin accessibility is based on candidate GSH sites that are classified as "open" in healthy controls and also classified as "open" in Parkinson's disease patients. In some embodiments, chromatin accessibility is based on any combination of the factors discussed in this paragraph. In some embodiments, transgene insertion (e.g., GDNF) does not affect differentiation ability, i.e., DA neuronal cells can differentiate into mature neuronal cell types (in vivo and in vitro).

[0303] A method for identifying safe harbor loci is disclosed, comprising, or essentially comprising, the steps of: a) identifying a gene or non-coding region on a chromosome that exceeds a threshold level with respect to chromatin accessibility; b) generating a model that correlates the gene or non-coding region on the chromosome obtained from step (a) with publicly available ATAC-seq data obtained from dopaminergic neurons isolated from the brains of healthy and Parkinson's diseased humans; and c) selecting a safe harbor locus based on a threshold parameter, wherein the safe harbor locus is selected for the insertion of at least one sequence encoding a transgene into the cell. In some embodiments, threshold parameters are based on peak size data generated using processed ATAC-seq data, distribution data generated using processed ATAC-seq data, highest efficiency for transgene insertion, sustained expression after long-term culture, absence of oncogenic gene expression, stable transgene expression, gene knockout conferring benefits to cellular function, no known intracellular function, stable transgene expression in vitro, negligible off-target cleavage detected by iGuide-Seq or CRISPR-Seq, lower off-target cleavage compared to other loci detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cytotoxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, and negligible dysregulation or silencing of neighboring genes. Chromatin accessibility is measured using a transposase-accessible chromatin assay (ATAC-seq) with sequencing. In some embodiments, chromatin accessibility is based on (1) PSCs for optimizing editing efficiency in step a, and / or (2) DA neuronal cells for optimizing editing efficiency in step a, and (3) post-mitotic dopaminergic neurons derived from a human brain sample.In some embodiments, defining chromatin accessibility is based on (1) PSCs for optimizing editing efficiency in step a, (2) DA neuronal cells for optimizing editing efficiency in step a, (3) post-mitotic dopaminergic neurons derived from a human brain sample to the extent that the manipulated cells have matured into neuronal mature cell types and can promote the survival of endogenous neurons, and (4) neuronal mature cell type cells.

[0304] Disclosed is an ex vivo method for obtaining engineered cells or a population thereof, comprising the steps of a) obtaining cells, and b) genetically modifying the cells by inserting at least one sequence encoding a transgene into a safe harbor locus, wherein the safe harbor locus is selected from any one of the target loci in Table 1. The genetic modification step in step (b) comprises contacting the cells with one or more guide ribonucleic acids (gRNAs), at least one sequence, and one or more Cas9 endonucleases, wherein the one or more gRNAs and Cas9 endonucleases facilitate the insertion of at least one sequence into chromosomal DNA within the safe harbor locus. The at least one sequence includes an exogenous promoter, which is operably ligated to the transgene. The transgene comprises, consists of, or essentially consists of, one or more genes selected from the group of functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, hemizygous SNCA, hemizygous MAPT, and / or GDNF genes, or their functional variants or fragments.

[0305] Targeted gene insertion in pluripotent stem cells (PSCs) A method is provided for introducing an exogenous coding sequence under the control of an endogenous promoter, particularly an endogenous promoter of a gene specifically expressed in a particular neuronal lineage or a particular differentiation stage, preferably in a cell at a late differentiation stage, by performing gene-targeted insertion in PSCs. In some embodiments, the PSC may be transduced with a polynucleotide vector (donor template), such as an AAV vector, during the ex-vivo treatment considered herein, while a sequence-specific nuclease reagent is expressed to facilitate the insertion of the coding sequence at a selected locus. In some embodiments, the PSC may be manipulated to produce a double-strand break using CRISPR, TALEN, transposon-based, ZFN, meganuclease, or mega-TAL, and the double-strand break is repaired by insertion of a gene encoding the manipulated GDNF, GBA, and / or SNCA gene. The resulting manipulated PSC may then differentiate into manipulated DA neurons, which may engraft in a patient requiring engraftment for long-term in vivo production of the exogenous coding sequence. Depending on the activity of a selected endogenous promoter, the coding sequence is selectively expressed in a particular strain or in vivo in response to the local environment of DA neuronal cells, thereby providing treatment for Parkinson's disease and other and secondary Parkinson's disease-like disorders.

[0306] In some embodiments, the exogenous coding sequence is under the control of the gene promoter, and its transcriptional activity is specifically induced in cells after graft function has been established, i.e., in cells that are neuronal mature cell types. In some embodiments, the exogenous coding sequence is under the control of the gene promoter, and its transcriptional activity is specifically induced in cells after cell transplantation. In some embodiments, the exogenous coding sequence is under the control of the gene promoter, and its transcriptional activity is not specifically induced in cells differentiating in vitro. In some embodiments, the exogenous coding sequence is under the control of the gene promoter, and its transcriptional activity is not specifically induced in cells expanding in vitro. In some embodiments, the exogenous coding sequence is under the control of the gene promoter, and its transcriptional activity is not specifically induced in cells until after graft function has been established.

[0307] Preferably, the introduction of GDNF, GBA, SCNA+ / -, or exogenous sequences encoding these components into PSCs under the transcriptional control of a promoter of a gene not expressed in pluripotent cells, differentiated cells, or DA neuronal cells is disclosed. In some embodiments, the gene is under the transcriptional control of a gene that is expressed only after transplantation in vivo. In some embodiments, the gene is under the transcriptional control of a gene that is expressed only in neurons produced by the administered engineered ESCs and / or DA neuronal cells. In some embodiments, the gene is under the transcriptional control of a gene that is expressed only in mature neuronal cell types. In some embodiments, the gene is under the transcriptional control of one of SEQ ID NOs. 21-40 or 43.

[0308] In some embodiments, an exogenous sequence encoding GDNF or a component thereof is introduced into an ESC under the transcriptional control of a gene described as being specifically expressed in, preferably only in, mature neuronal cell types. In some embodiments, the GDNF gene is under the transcriptional control of one of sequence numbers 21-40 or 43.

[0309] In some embodiments, the PSC includes an exogenous coding sequence intended to be exclusively expressed in a selected nervous system. More broadly, methods are disclosed for manipulating PSCs by gene-targeted insertion of an exogenous coding sequence intended to be selectively expressed in mature neuronal cells derived from the PSC. In one embodiment, mature neuronal cells produced by the manipulated PSC express the exogenous coding sequence in response to selected environmental factors or in vivo stimuli to improve their therapeutic potential. More broadly, methods are disclosed for manipulating PSCs by gene-targeted insertion of an exogenous coding sequence intended to be selectively expressed in the in vivo environment in which mature neuronal cells are present. In one embodiment, manipulated cells derived from the manipulated PSC express the exogenous coding sequence in response to selected environmental factors or in vivo stimuli to improve the therapeutic potential in mature neuronal cells.

[0310] Combining targeted sequence insertion(s) in DA neuronal cells with inactivation of endogenous genomic sequences. In some embodiments, inactivation of a gene in DA neuronal cells at a particular locus improves the therapeutic potential of cells whose expression is manipulated by incorporating an exogenous coding sequence into that locus.

[0311] In some embodiments, the insertion of a coding sequence has the effect of reducing or preventing the expression of the associated gene.

[0312] Expansion of DA neuron cells Manipulated pluripotent cells and / or DA neurons can be enlarged, either before or after genetic modification. Pluripotent cells and / or DA neurons can be enlarged in vitro or in vivo. In some embodiments, manipulated pluripotent cells and / or DA neurons do not express exogenous genes during enlargement. In some embodiments, manipulated pluripotent cells and / or DA neurons express exogenous genes during enlargement.

[0313] gene editing In some embodiments, - A step of providing a population of pluripotent cells, - In a certain proportion of the population of pluripotent cells, i) Introducing at least one nucleic acid containing an exogenous nucleotide or polynucleotide sequence to thereby produce engineered pluripotent cells (the exogenous nucleotide or polynucleotide is incorporated into a selected endogenous locus to encode at least one molecule for improving the therapeutic potential of the DA neuron cell population derived from the engineered pluripotent cells), ii) Introduce at least one sequence-specific reagent that specifically targets the selected endogenous locus (an exogenous nucleotide or polynucleotide sequence is inserted into the endogenous locus by targeted gene integration, and as a result, the exogenous nucleotide or polynucleotide sequence forms an exogenous coding sequence under the transcriptional control of the endogenous promoter present at that locus). Step A method including the following is disclosed.

[0314] In some embodiments of this method, the sequence-specific reagent is a nuclease, and targeted gene integration is performed by homologous recombination or non-homologous end joining (NHEJ) into pluripotent cells. In some embodiments, NHEJ may be suppressed in cells. Suppressing NHEJ in cells may include inhibiting ligase IV. Suppressing NHEJ in cells may also include introducing a homologous recombination (HR) enhancer. The enhancer may be derived from a viral protein. The enhancer may be E1B55K, E4orf6, Scr7, or L755507. Suppressing NHEJ in cells may promote the insertion of polynucleotides encoding exogenous GDNF, GBA, and / or SNCA at the cleavage site by homologous recombination.

[0315] In some embodiments, the endogenous promoter is selected to be inactive during in vitro cell differentiation and preferably upregulated after graft function is established in vivo. In some embodiments, the endogenous promoter is selected to be inactive during in vitro cell differentiation and preferably upregulated after cell transplantation into the target.

[0316] This method can be further understood by the following (flowing) numbered paragraphs.

[0317] Paragraph 1. A method for treating Parkinson's disease and other and secondary Parkinson's disease-like disorders in a human subject, comprising the step of administering to a human subject an effective amount of a pharmaceutical composition comprising (i) a population of genetically modified human DA neurons comprising genomic disruption that suppresses or eliminates the expression of a protein encoded by an endogenous gene, and (ii) a pharmaceutically acceptable carrier or excipient.

[0318] Paragraph 2. The method according to Paragraph 1, wherein the gene is a GDNF, GBA, and / or SNCA gene.

[0319] Paragraph 3. The method according to Paragraph 1, wherein the said DA neuronal is autologous to the human subject.

[0320] Paragraph 4. The method according to Paragraph 1, wherein the genome disruption is a nucleotide insertion or deletion in the gene.

[0321] Paragraph 5. The method according to Paragraph 1, wherein the genetically modified human DA neuron cells contain the exogenous GDNF, GBA, and / or SNCA genes.

[0322] Paragraph 7. The method according to Paragraph 1, wherein the genetically modified DA neuronal cells are derived from genetically modified pluripotent stem cells.

[0323] Gene editing of hemizygous nulls regarding SNCA In one embodiment, a method for adoptive cell therapy or prophylaxis is provided, comprising the step of administering SNCA-modified DA neuronal cells to a subject suffering from or at risk of suffering from an α-synuclein accumulation condition. In some embodiments, the SNCA-modified DA neuronal cells are autologous. In some embodiments, the SNCA-modified DA neuronal cells are homogeneous.

[0324] In some embodiments, SNCA-modified DA neurons are genetically modified DA neurons that have reduced SNCA expression. In some embodiments, SNCA-modified DA neurons are genetically modified DA neurons that have reduced SNCA expression and increased GBA expression.

[0325] In some embodiments, DA neurons with modified SNCA are derived from pluripotent cells that have been genetically modified to have reduced SNCA expression through gene editing using programmable nucleases or gene silencing (RNA interference). In some embodiments, DA neurons with modified SNCA are derived from SNCA - / - In some embodiments, genetically modified SNCAs are DA neuronal cells that have been modified. + / - In some embodiments, genetically modified SNCAs are DA neuronal cells that have been modified. + / + That is the case.

[0326] SNCA-modified DA neuronal cells can be further understood by the following numbered paragraphs.

[0327] Paragraph 1. A method for obtaining human DA neuronal cells, comprising the step of differentiating human pluripotent stem cells into human DA neuronal cells, wherein the human pluripotent cells are genetically modified cells in which one or both SNCA alleles are not expressed by genetic modification.

[0328] Paragraph 2. The method according to Paragraph 1, wherein both SNCA alleles are in a state of non-expression due to genetic modification.

[0329] Paragraph 3. The method according to Paragraph 1, wherein human pluripotent stem cells are induced pluripotent stem cells.

[0330] Paragraph 4. The method according to Paragraph 3, wherein both SNCA alleles are in a state of non-expression due to genetic modification.

[0331] Paragraph 5. The method according to Paragraph 1, comprising the step of enlarging human DA neuron cells.

[0332] Paragraph 6. The method according to Paragraph 1, wherein human pluripotent cells are genetically modified using CRISPR / Cas protein gene editing.

[0333] Paragraph 7. A population of human cells, including human DA neurons, wherein the human DA neurons are genetically modified human DA neurons in which one or both SNCA alleles are not expressed by genetic modification.

[0334] cytotoxicity The disclosure herein may further include a step of introducing a modifying factor into cells that reduces cytotoxicity. The modifying factor may be the pancaspase inhibitor Z-VADFMK and / or BX795. The cells may be pluripotent cells, DA neurons, or nerve cells. The cells may be mammalian cells. The cells may be human cells.

[0335] CIS and IRES In some embodiments, methods are disclosed that involve gene editing of pluripotent cells (or, in some embodiments, DA neuronal cells) such that the transcription of the incorporated gene is under the control of an endogenous promoter, while maintaining the expression of the native gene through the use of a CIS regulatory element (e.g., a 2A CIS-active hydrolase element) or an internal ribosome entry site (IRES) in the donor template.

[0336] GDNF is upregulated after the completion of DA neuron cell differentiation. In some embodiments, a method is disclosed that includes the step of generating double-strand breaks at loci that are highly transcribed after graft function has been established by expressing a sequence-specific nuclease reagent, such as a TALEN, ZFN, or RNA-guided endonuclease, in the presence of a DNA repair matrix inserted into an AAVS1-based vector, preferably. This DNA donor template generally includes a unique or multiple open reading frame and two homologous arms embedded with regulatory gene elements (stop codons and poly-A sequences).

[0337] Disclosed is a method for expressing GDNF at a selected locus that is upregulated after the in vivo differentiation, engraftment, proliferation, migration, and completion of innervation of DA neurons, i.e., after graft function is established, or after administered engineered DA neurons have differentiated into a mature neuronal cell type. The exogenous sequence(s) encoding the GDNF gene or its functional fragment, and the endogenous gene coding sequence(s) may be co-transcribed by separation, for example, by a CIS regulatory element (e.g., a 2A CIS-acting hydrolase element) or an internal ribosome entry site (IRES). For example, the exogenous sequence encoding the GDNF gene or its functional fragment may be placed under the transcriptional control of the promoter of the endogenous gene expressed by the graft microenvironment.

[0338] GBA is upregulated after the completion of DA neuron cell transplantation. In some embodiments, a method is disclosed which preferably includes the step of generating double-strand breaks at loci that are highly transcribed immediately upon transplantation of the manipulated cells by expressing a sequence-specific nuclease reagent, such as a TALEN, ZFN, or RNA-guided endonuclease, in the presence of a DNA repair matrix inserted into an AAVS1-based vector. This DNA donor template generally includes a unique or multiple open reading frame and two homologous arms embedded with regulatory gene elements (stop codons and poly-A sequences).

[0339] Disclosed is a method for expressing GBA at a selected locus that is upregulated after transplantation of DA neuronal cells, not after the completion of engraftment of DA neuronal cells, i.e., before graft function is established or before the administered DA neuronal cells differentiate into mature neuronal cell types. The exogenous sequence(s) encoding the GBA gene or its functional fragment and the endogenous gene coding sequence(s) can be co-transcribed by separation, for example, by a CIS regulatory element (e.g., a 2A CIS-acting hydrolase element) or an internal ribosome entry site (IRES). For example, the exogenous sequence encoding the GBA gene or its functional fragment can be placed under the transcriptional control of a promoter, which may be a promoter of the endogenous gene expressed by graft transplantation, or a ubiquitous promoter.

[0340] A novel framework for identifying candidate genome-safe harbors ("GSHs") is disclosed in Aznauryan et al., Cell Reports Methods, 2,100154 (2022). Candidate GSHs are selected based on ATAC-seq data showing a favorable chromatin accessibility profile in DA neurons. Fullard et al., Genome Research, 28:1243-1252 (2021) and Corces et al., Nature Genetics, 52:1158-1168 (2020) show candidate GSH loci as evidence of accessible chromatin in the human striatum (nucleus accumbens and putamen).

[0341] In vivo contact between nerve cells and manipulated DA neuron cells. In some embodiments, a method is disclosed for enhancing viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or the function of administered engineered DA neurons, comprising the step of contacting a neuron with a gene editing system comprising a repair template comprising a Cas protein or a polynucleotide encoding the Cas protein, a guide RNA (gRNA), and a functional PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF gene, or a functional variant or fragment thereof, wherein the gene editing system can enhance viability, engraftment, proliferation, migration, innervation, differentiation, long-term graft integrity, endogenous neuron survival, or the function of the administered engineered DA neurons compared to wild-type neurons. In some embodiments, the neuron is in the patient's body when it comes into contact with the engineered DA neurons.

[0342] Disclosed is a method for engrafting, proliferating, or promoting the viability, migration, innervation, differentiation, or function of administered engineered DA neurons, comprising a recombinant gene vector comprising a polynucleotide encoding wild-type PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SCNA+ / -, and / or GDNF genes, or functional variants or fragments thereof, wherein the polynucleotide is operably linked to a promoter active in eukaryotic cells, and the recombinant gene-transfected DA neurons express the wild-type protein or a functional variant or fragment upon implantation in a host / patient. In some embodiments, the recombinant gene-transfected DA neurons express the wild-type protein or a functional variant or fragment upon maturation in the host / patient.

[0343] Nonviral introduction of exogenous genes into cells Also disclosed herein is a method for producing engineered cells, comprising the steps of: a) nonvirally introducing one or more polynucleic acids comprising at least one exogenous GDNF, GBA, and / or SNCA sequence into cells; and b) contacting at least one exogenous GDNF, GBA, and / or SNCA sequence with a double-strand break region comprising at least one gene. The at least one gene may be GDNF, GBA, and / or SNCA. The double-strand break region may be repaired by insertion of at least one exogenous GDNF, GBA, and / or SNCA sequence. Insertion of at least one exogenous GDNF, GBA, and / or SNCA sequence may involve disruption of at least one gene.

[0344] In some embodiments, gene transfer may include nonviral transfection, gene gun, chemical transfection, electroporation, nucleofection, heat shock transfection, lipofection, microinjection, or viral transfection. The polynucleic acid may be co-delivered with at least one modifying factor that alters the cellular response to the polynucleic acid. At least one modifying factor may reduce cytotoxicity. The modifying factor may include the pan(abPan) caspase inhibitor Z-VAD-FMK or BX795.

[0345] Exogenous gene introduction into cells via double-strand break regions Methods for producing manipulated cells, which include the step of introducing a double-strand break region rather than cells, are also disclosed herein. In some embodiments, the double-strand break region may be produced by CRISPR, TALEN, transposon-based, ZFN, meganuclease, or mega-TAL. In some embodiments, the double-strand break region may be produced by CRISPR. In some embodiments, CRISPR may be multiplexed. In some embodiments, multiplexing may be performed by adding at least two guide RNAs. GDNF, GBA, and / or SNCA exogenous sequences may be inserted near the double-strand break region.

[0346] Introduction of exogenous genes into cells using reverse transcriptase. In some embodiments, the cells to be manipulated can be brought into contact with reverse transcriptase (RT). In some embodiments, the cells can be brought into contact with a primer complementary to the polynucleic acid. In some embodiments, RT transcribes mRNA into a first ssDNA template. In some embodiments, RT transcribes the first ssDNA template into a second dsDNA template. In some embodiments, transcription can be performed in situ. The ssDNA or dsDNA may contain at least one exogenous GDNF sequence. In some embodiments, the presence of RT can be determined using a primer sequence. The reverse transcriptase (RT) reporter forward primer can be determined by a method known to those skilled in the art. The reverse transcriptase (RT) reporter forward primer can be determined by being able to (selectively) bind to one of the sequences 1-10. In some embodiments, the forward primer is the length of 10 nucleic acids and can bind to the first 10 Ns (residues 1-10), the second 10 Ns (residues 10-20), the third 10 Ns (residues 20-30), the fourth 10 Ns (residues 30-50), or the fifth 10 Ns (residues 50-60) of the amino acid sequences described in SEQ ID NOs. 1-10.

[0347] Homology-dependent repair Also disclosed herein is a method for promoting homology-dependent repair (HDR), comprising the steps of: a) nonvirally introducing mRNA, reverse transcriptase (RT), enhancer, and primer into a cell; b) reverse transcribing the mRNA into one or more copies of a polynucleic acid; and c) promoting HDR between the cellular genome and the genome of the polynucleic acid. In some embodiments, the method may include a step of generating a double-strand break. In some embodiments, the double-strand break can be generated by CRISPR, TALEN, transposon-based, ZFN, meganuclease, and mega-TAL. In some embodiments, the double-strand break can be generated by CRISPR. In some embodiments, the HDR in c) repairs the double-strand break. In some embodiments, CRISPR can be multiplexed with at least two guide RNAs. In some embodiments, the polynucleic acid may be DNA. In some embodiments, the polynucleic acid may be cDNA. In some embodiments, the polynucleic acid may be single-stranded.

[0348] In some embodiments, the RT transcribes mRNA into a first ssDNA template. In some embodiments, the polynucleic acid may be double-stranded. In some embodiments, the RT transcribes mRNA into a second dsDNA template in situ. The mRNA or polynucleic acid may contain at least one GDNF sequence. In some embodiments, the GDNF sequence contains at least two adjacent recombinant arms having sequences complementary to a genomic region. In some embodiments, the GDNF sequence may be used in the HDR of c). In some embodiments, the GDNF sequence may be used in the HDR of c), further comprising the binding of recombinant arms to a complementary region of the cell's genome. In some embodiments, the GDNF sequence may be used in the HDR of c), further comprising the binding of recombinant arms to a complementary region of the cell's genome, and further comprising the insertion of the GDNF sequence. In some embodiments, the HDR between the cell's genome and the polynucleic acid genome disrupts one or more genes. One or more genes may include XX. In some embodiments, one or more genes contain GDNF. In some embodiments, HDR between the cellular genome and the polynucleic acid genome may be assisted by one or more homologous recombination (HR) enhancers.

[0349] Targeted homologous recombination In some embodiments, an exogenous sequence is introduced into endogenous chromosomal DNA by targeted homologous recombination. Thus, the exogenous nucleic acid introduced into pluripotent cells contains at least one coding sequence, along with a sequence capable of hybridizing with the endogenous chromosomal sequence under physiological conditions. Generally, such homologous sequences exhibit at least 70%, preferably 80%, and more preferably 90% sequence identity with the endogenous gene sequence located at the insertion locus. These homologous sequences may be flanked by coding sequences to improve the accuracy of recombination, as already taught, for example, in U.S. Patent No. 6,528,313 (which is incorporated in its entirety by reference).

[0350] Using available software and online genome databases, vectors containing coding sequences(s) can be designed such that the sequences(s) are introduced into the precise locus under the transcriptional control of at least one endogenous promoter, which is the promoter of the endogenous gene. The exogenous coding sequences(s) are then preferably inserted “in-frame” with the endogenous gene. The sequences obtained by incorporating the exogenous polynucleotide sequences(s) can encode a number of different types of proteins, including fusion proteins, tagged proteins, or mutated proteins. Fusion proteins allow for the addition of novel functional domains to proteins expressed in cells, such as dimerization domains that can be used to activate or deactivate protein activity, e.g., caspase-9 switches. Tagged proteins may be useful for detecting manipulated DA neuronal cells and for following up patients treated with the cells. By introducing mutations into proteins, drug resistance can be conferred, as further described below.

[0351] Low-frequency cleavage endonuclease The sequence-specific reagent used in this method is preferably a low-frequency cleavage endonuclease known to those skilled in the art. Targeted gene integration is generally performed by homologous recombination into pluripotent cells or by NHEJ. The specific endonuclease reagent is preferably selected from RNA or DNA-guided endonucleases, such as Cas9 or Cpf1, RNA or DNA guides, TAL-endonucleases, zinc finger nucleases, homing endonucleases, or any combination thereof.

[0352] TALE-Nuclease In some embodiments, the endonuclease reagent is an "engineered" or "programmable" low-frequency cleavage endonuclease, for example, a homing endonuclease described by Arnould S. et al. (W02004067736), a zinc finger nuclease (ZFN) described by Urnov F., et al. (Highly efficient endogenous human gene correction using designed zinc-finger nucleases (2005) Nature 435:646-651), a TALE nuclease described by Mussolino et al. (A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity (2011) Nucl. Acids Res. 39(21):9283-9293), or a TALE nuclease described by Boissel et al. (MegaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering (2013) Nucleic Acids Research 42 It is a nucleic acid that encodes a megaTAL nuclease, as described in (4):2591-2601).

[0353] In some embodiments, the endonuclease reagent is, in particular, an RNA-guide intended for use with an RNA-guided endonuclease, such as Cas9 or Cpf1, as taught by Doudna, J., and Chapentier, E. (The new frontier of genome engineering with CRISPR-Cas9 (2014) Science 346 (6213): 1077), which is incorporated herein by reference.

[0354] In some embodiments, the endonuclease reagent is transiently expressed in cells; that is, the reagent is not intended to be integrated into the genome or to persist over long periods, as is the case with RNA, more specifically mRNA, proteins, or complexes of proteins and nucleic acids (e.g., ribonucleoproteins).

[0355] Generally, 80% of the endonuclease reagent is degraded up to 30 hours after transfection, preferably up to 24 hours, and more preferably up to 20 hours.

[0356] Endonucleases in mRNA form are preferably synthesized with a cap to enhance their stability, according to techniques well known in the art, for example, as described by Kore AL, et al. (Locked nucleic acid (LNA)-modified dinucleotide mRNA cap analogue: synthesis, enzymatic incorporation, and utilization (2009) J Am Chem Soc. 131 (18):6364-5).

[0357] Generally, the electroporation step used to transfect pluripotent cells is typically performed in a sealed chamber equipped with parallel plate electrodes that provide a substantially uniform pulsed electric field across the treatment volume, with a pulse between the parallel plate electrodes exceeding 100 volts / cm and below 5,000 volts / cm, as described in WO / 2004 / 083379 incorporated by reference. One such electroporation chamber is preferably such that the square of the distance between electrodes (cm²) is equal to the chamber volume (cm²). 3 The geometric factor (cm) is defined by the quotient obtained by dividing by ). 1 ) has, where the geometric factor is 0.1 cm 1The cell and sequence-specific reagent suspension is present in a culture medium adjusted to have a conductivity ranging from 0.01 to 1.0 millisiemens, equal to or below that value. Generally, the cell suspension is subjected to one or more pulsed electric fields. This method allows the treatment volume of the suspension to be expanded or contracted, and the treatment time of the cells in the chamber is substantially uniform.

[0358] Due to its high specificity, TALE nucleases are, for example, Mussolino et a / (TALEN). (R) As reported in "facilitate targeted genome editing in human cells with high specificity and low cytotoxicity (2014) Nucl. Acids Res. 42(10): 6762-6773)", it has been demonstrated to be a sequence-specific nuclease reagent particularly suitable for therapeutic application when acting in heterodimer form, i.e., as a pair of the "right" monomer (also called "5'" or "forward") and the "left" monomer (also called "3'" or "reverse").

[0359] As described above, sequence-specific reagents are preferably in the form of nucleic acids, for example, low-frequency cleavage endonucleases, DNA or RNA encoding their subunits, but they may also be part of a conjugate containing polynucleotides and polypeptides, such as so-called "ribonucleoproteins." Such conjugates can be formed using reagents such as Cas9 or Cpf1 (RNA-guided endonuclease) or Argonaute (DNA-guided endonuclease), as described in recent years by Zetsche, B. et al. (Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System (2015) Cell 163(3): 759-771) and Gao F. et al. (DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (2016) Nature Biotech), and these contain RNA or DNA guides that can form complexes with their respective nucleases.

[0360] AAV Vector Improving the efficiency of gene targeting insertions in DA neurons using AAV vectors

[0361] The donor template is generally a polynucleotide sequence, which can be included in various vectors described in the art to deliver the donor template to the nucleus at the point when the endonuclease reagent becomes active, thereby obtaining site-directed insertion into the genome, typically by NHEJ or homologous recombination.

[0362] A method for inserting an exogenous nucleic acid sequence into an endogenous polynucleotide sequence in a cell, comprising at least the step of transducing a cell with an AAV vector containing a sequence homologous to the exogenous nucleic acid sequence and a target endogenous DNA sequence,

[0363] A method is disclosed that includes the step of inducing the expression of a sequence-specific endonuclease reagent to cleave an endogenous sequence at the insertion locus.

[0364] The insertion of an exogenous nucleic acid sequence allows for the introduction of genetic material, modification of an endogenous sequence, or replacement, more preferably "in-frame" with respect to the endogenous gene sequence at that locus.

[0365] In some embodiments, 10 cells per cell 5 ~10 7 pieces, preferably 10 6 ~10 7 Individual, comfortable, approximately 5.10 6 A viral genome is transduced. In some embodiments, cells may be treated with a proteasome inhibitor, such as bortezomib, to further assist homologous recombination. In some embodiments, the AAV vector used in this method may contain a promoterless exogenous coding sequence to place under the control of an endogenous promoter at one locus selected from those enumerated herein. In some embodiments, the AAV vector used in this method may contain cDNA (without a start codon) that forms an exogenous coding sequence following a 2A peptide cleavage site. Any AAV vector designed to perform the methods described herein, in particular vectors containing sequences homologous to the locus of insertion, are disclosed. Numerous other vectors known in the art, such as plasmids, episomatic vectors, linear DNA matrices, etc., can also be used in accordance with the teachings herein.

[0366] As described above, the DNA vector comprises (1) an exogenous nucleic acid containing an exogenous coding sequence to be inserted by homologous recombination, and (2) a sequence encoding a sequence-specific endonuclease reagent to facilitate insertion. In some embodiments, the exogenous nucleic acid (1) does not contain any promoter sequences, while the sequence (2) has its own promoter. In some embodiments, the nucleic acid (1) includes an internal ribosome entry site (IRES) or a "self-cleaving" 2A peptide, such as T2A, P2A, E2A, or F2A, so that the endogenous gene into which the exogenous coding sequence is inserted is multicistronic. The IRES of the 2A peptide may precede or follow the exogenous coding sequence.

[0367] transplant In some embodiments, GDNF-manipulated DA neurons are transplanted into patients with rapidly progressive and mild Parkinson's disease. In some embodiments, GBA / SNCA-manipulated DA neurons are transplanted into patients to improve cognitive symptoms in Parkinson's disease.

[0368] In some embodiments, the engineered cells may be administered to subjects requiring administration as monotherapy. Engineered DA neurons, recombinant gene vectors, or gene editing systems may be administered in a variety of ways. In some embodiments, the administration step includes systemic, parenteral, intravenous, intrathecal, intraspinal, intracisional, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular administration. In some embodiments, the administration step includes intravenous, intrathecal, intraspinal, intracisional, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular injection. In some embodiments, the administration step includes intrathecal injection using a Trendelenburg tilt. In some embodiments, the administration step includes direct injection into the substantia nigra pars compacta of the brain. In some embodiments, the administration step includes introducing the engineered DA neurons, recombinant gene vectors, or gene editing system into the subject's brain or cerebrospinal fluid (CSF).

[0369] The administration of cells or populations of cells is 10 per kg of body weight. 4 ~10 9 individual cells, preferably 10 5 ~10 6 A dose may consist of a number of cells per kg of body weight, which includes the number of cells in all integer values ​​within these ranges. A dose of cells or a population of cells originates from a single donor or patient sample. 6 ~10 8 This can consist of doses of more than 10, more commonly more than 50, more commonly more than 100, and usually more than 1000, containing individual gene-edited cells.

[0370] In some embodiments, 1 × 10 per kilogram of the subject's body weight 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the subject. In some embodiments, 1 x 10⁶ per kilogram of the subject's body weight is used. 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the target brain. In some embodiments, 1 x 10⁶ per kilogram of the target's body weight is used. 9 ~1 × 10 14 A gene therapy vector containing 1 x 10⁶ recombinant gene vector genomes (vg / kg) is administered to the target CSF. In some embodiments, 1 x 10⁶ per kilogram of the target's body weight is used. 7 ~1 × 10 9 A gene therapy vector containing a recombinant gene vector genome (vg / kg) is administered to the target.

[0371] In some embodiments, a subject requiring it undergoes a treatment comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing the manipulated cells to the subject. In some embodiments, a subject requiring it undergoes a treatment comprising the step of administering a therapeutically effective amount of a pharmaceutical composition containing manipulated cells containing an exogenous GDNF, GBA, or SNCA gene, or a functional fragment thereof. The pharmaceutical composition may be administered intravenously. The pharmaceutical composition may be administered topically. In some embodiments, the method may further include the step of administering one or more additional treatments. One or more additional treatments may include transplantation. One or more additional treatments may include immunotherapy. In some embodiments, the manipulated cells may be autologous to the subject. In some embodiments, the manipulated cells may be homogeneous to the subject.

[0372] Manipulated DA neuronal cells can be administered to an individual in any amount or number that provides a detectable therapeutic or preventive benefit to the individual, e.g., an effective dose. In some embodiments, the dose of manipulated DA neuronal cells to be administered is simply the absolute number of cells, e.g., the individual receives about 1 × 10⁻⁶ cells. 5 Individual cells, 5 x 10 5 Individual cells, 1 × 10 6 Individual cells, 7 × 10 6 Individual cells, 1 × 10 7 individual cells, 6 × 10 7 Individual cells, 2 × 10 8 Individual cells, 5 x 10 8 Individual cells, 1 × 10 9 individual cells, 6 × 10 9 Individual cells, 2 × 10 10 Individual cells, 5 x 10 10 individual cells, or 1 × 10⁶ 11 Individual cells may be administered.

[0373] In some embodiments, the number of manipulated DA neurons is approximately 1 × 10⁶ cells per kilogram of the body weight of the subject to be treated, for example, about 1 × 10⁶ cells per kilogram of the subject to be treated. 5 Individual cells, 5 x 10 5Individual cells, 1 × 10 6 Individual cells, 7 × 10 6 Individual cells, 1 × 10 7 individual cells, 6 × 10 7 Individual cells, 2 × 10 8 Individual cells, 5 x 10 8 Individual cells, 1 × 10 9 individual cells, or 6 × 10 9 Individual cells are administered to the target.

[0374] Cells or populations of cells may be administered in one or more doses. In another embodiment, an effective amount of cells is administered in a single dose. In another embodiment, an effective amount of cells is administered in more than one dose over a period of time. The timing of administration is within the discretion of the attending physician and depends on the patient's clinical condition. Cells or populations of cells can be obtained from any source, e.g., a blood bank or a donor. While individual needs vary, determining the optimal effective dose range for a given cell type for a particular disease or condition is within the scope of the skill of those skilled in the art. An effective dose means the amount that produces a therapeutic or preventive benefit. The dose administered depends on the recipient's age, health condition, and weight, the type of parallel treatment if present, the frequency of treatment, and the nature of the desired effect.

[0375] In another embodiment, an effective amount of cells or a composition containing such cells is administered parenterally. Administration may be intravenous.

[0376] The disclosure also includes means for detecting engineered cells containing desired gene insertions, particularly by performing a step of using a PCR method to detect insertions of exogenous coding sequences at endogenous loci. In some embodiments, allotags are used to monitor the integrity and functionality of grafts.

[0377] The above description provides a manner and process for making and using it, thereby enabling those skilled in the art to make and use it, and this applicability is provided in particular with respect to the subject matter of the appended paragraphs forming part of this specification.

[0378] Combination treatment The modified DA neuron cell composition can also be used in combination with other therapeutically valuable agents in the treatment of PD symptoms. According to one embodiment, the treatment may be administered to patients receiving immunosuppressive treatment. Generally, other agents do not necessarily have to be administered in the same pharmaceutical composition and may be administered, preferably by different routes, due to their different physical and chemical characteristics. The mode of administration and, where possible, the appropriateness of administration in the same pharmaceutical composition are well within the knowledge of a skilled clinician. The initial dose may be administered according to established protocols known in the art, and the dose, mode of administration, and timing of administration may then be modified by a skilled clinician based on the observed effects.

[0379] In some embodiments, treatment of the modified DA neuron cell composition may be used in conjunction with a pharmacopoeia designed to act on the cholinergic system in the brain. Examples of such pharmacopoeias may include, but are not limited to, donepezil (Aricept), rivastigmine (Exelon), and galantamine (Razadyne), or comprise donepezil (Aricept), rivastigmine (Exelon), and galantamine (Razadyne).

[0380] It is known to those skilled in the art that the therapeutically effective dose may vary when a drug is used in combination with other treatments. Methods for experimentally determining the therapeutically effective dose of a drug and other agents for use in combination treatment regimens are described in the literature. For example, the use of regular dosing, i.e., providing lower doses more frequently to minimize toxic side effects, is described in detail in the literature. Combination treatments may further include periodic treatments that are initiated and discontinued at various points in time to assist in the clinical management of a patient.

[0381] Regarding combination therapy, the dosage of co-administered therapeutic agents will naturally vary depending on the type of co-agent used, the specific modified DA neuronal cells, and the symptoms of Parkinson's disease and other secondary Parkinson's disease-like disorders being treated.

[0382] It is understood that dosage regimens for treating, preventing, or alleviating the one or more conditions requiring reduction may be modified in accordance with various factors. These factors include the condition the subject is suffering from, as well as the subject's age, weight, sex, diet, and overall medical condition. Therefore, the dosage regimens actually used may vary widely and thus deviate from the dosage regimens described herein.

[0383] The modified DA neuronal cells and additional therapeutic agents constituting the combination therapies described herein may be in combination dosage forms or in separate dosage forms intended to be administered substantially concurrently. The drugs constituting the combination therapy may also be administered sequentially, and any of the therapeutic compounds may be administered by a regimen requiring two-step administration. A two-step administration regimen may require sequential administration of the active agents or intervald administration of separate active agents. The intervals between multiple administration steps may range from a few minutes to several hours, depending on the characteristics of each drug, e.g., the drug's potency, solubility, bioavailability, plasma half-life, and kinetic profile. Circadian rhythms of various physiological parameters may also be evaluated to determine the optimal administration interval.

[0384] The initial dose may be administered via any practical route, such as intravenous injection, bolus injection, infusion over 5 minutes to approximately 5 hours, pills, capsules, inhaler, injection, transdermal patch, buccal delivery, or a combination thereof. The compound should be administered as early as possible after the onset of the disease or condition is detected or suspected, for the duration necessary to treat or prevent the PD condition.

[0385] Dosage form Useful compositions can be formulated for administration to subjects via any conventional means, including but not limited to oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), buccal, inhalation, intranasal, rectal, or transdermal routes of administration.

[0386] Pharmaceutical compositions comprising modified DA neuronal cells alone or in combination with one or more other therapeutic agents may be formulated into any suitable dosage form, including but not limited to aqueous oral dispersions, liquids, mists, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled-release formulations, rapid-dissolving formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, sugar-coated tablets, capsules, delayed-release formulations, long-release formulations, pulsed-release formulations, multi-particle formulations, and immediate-release and controlled-release mixed formulations, for oral administration by the patient to be treated.

[0387] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipients with one or more compounds, grinding the resulting mixture as needed, adding appropriate adjuvants if desired, and then processing the granular mixture to obtain tablets or sugar-coated tablet cores. Suitable excipients include, for example, fillers, sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, microcrystalline cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc., and polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrants such as cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid, or salts thereof, such as sodium alginate, may be added.

[0388] In another embodiment, the dosage form may include microcapsule-encapsulated formulations. In some embodiments, one or more other compatible materials are present in the microcapsule-encapsulating material. Exemplary materials include, but are not limited to, pH adjusters, erosion accelerators, foam inhibitors, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0389] Microcapsule-encapsulated formulations of DA neuron cell populations can be formulated by methods known to those skilled in the art. Such known methods include, for example, spray drying processes, spin disk solvent processes, hot melt processes, spray cooling methods, fluidized beds, electrostatic deposition, centrifugal extrusion, rotary suspension separation, polymerization at liquid-gas or solid-gas interfaces, pressure extrusion, or spray solvent extraction baths. In addition, several chemical techniques, such as composite coacervation, solvent evaporation, polymer-polymer incompatibility, interfacial polymerization in liquid media, in situ polymerization, drying in liquid, and desolvation in liquid media, may also be used. Furthermore, other methods, such as roller compression, extrusion / spheroidization, coacervation, or nanoparticle coating, may also be used.

[0390] Pharmaceutical solid oral dosage forms, including formulations, can be further formulated to provide controlled release of DA neuronal cell populations. Controlled release refers to the release of one or more active agents over a prolonged period from a dosage form incorporated according to a desired profile. Examples of controlled release profiles include sustained release, long-acting release, pulsed release, and delayed release profiles. In contrast to immediate-release compositions, controlled-release compositions allow for the delivery of a drug to a target over a prolonged period according to a predetermined profile. Such release rates provide therapeutically effective levels of the drug over a prolonged period, thereby providing a longer-lasting pharmacological response compared to conventional rapid-release formulations, while simultaneously minimizing side effects. Such longer-lasting responses offer numerous unique benefits not achieved with corresponding short-acting immediate-release preparations.

[0391] In some embodiments, the solid dosage form may be formulated as an enteric-coated delayed-release oral dosage form, i.e., an oral dosage form of a pharmaceutical composition that utilizes the enteric coating to achieve release in the small intestine of the gastrointestinal tract. The enteric-coated dosage form may be a compressed or molded or extruded tablet / mold (coated or uncoated) comprising granules, powders, pellets, beads, or particles of the active ingredient and / or other compositional components, which are themselves coated or uncoated. The enteric-coated oral dosage form may also be a capsule (coated or uncoated) comprising pellets, beads, or granules of a solid carrier or composition, which are themselves coated or uncoated.

[0392] The term "delayed release," as used herein, refers to delivery such that release can be achieved at some generally predictable location in the intestinal tract distal to where it would be achieved without the modification of delayed release. In some embodiments, the method for delaying release is a coating. Any coating should be applied to a sufficient thickness such that the entire coating does not dissolve in digestive fluids below about 5 pH, but dissolves at pH about 5 and above. Any anionic polymer exhibiting a pH-dependent solubility profile is expected to be used as an enteric coating in this method and composition to achieve delivery to the lower gastrointestinal tract. In some embodiments, the polymer is an anionic carboxylic acid polymer.

[0393] In some embodiments, the coating may contain, or usually contains, a plasticizer, and possibly other coating excipients, such as colorants, talc, and / or magnesium stearate, which are well known to those skilled in the art. Suitable plasticizers include triethyl citrate (Citroflex 2), triacetin (glyceryl triacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400 (polyethylene glycol 400), diethyl phthalate, tributyl citrate, acetylated monoglycerides, glycerol, fatty acid esters, propylene glycol, and dibutyl phthalate. Specifically, anionic carboxylate acrylic polymers typically contain 10 to 25% by weight of a plasticizer, particularly dibutyl phthalate, polyethylene glycol, triethyl citrate, and triacetin. Conventional coating techniques, such as spray or pan coating, are used to apply the coating. The coating thickness must be sufficient to ensure that the oral dosage form remains intact until it reaches the desired site of local delivery in the intestinal tract.

[0394] Liquid formulations for oral administration may be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups.

[0395] Aqueous suspensions and dispersions may remain homogeneous for at least 4 hours, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905). Homogeneity may be determined by a consistent sampling method for determining the homogeneity of the entire composition. In one embodiment, the aqueous suspension may be resuspended in a homogeneous suspension by physical stirring lasting less than 1 minute. In another embodiment, the aqueous suspension may be resuspended in a homogeneous suspension by physical stirring lasting less than 45 seconds. In yet another embodiment, the aqueous suspension may be resuspended in a homogeneous suspension by physical stirring lasting less than 30 seconds. In yet another embodiment, stirring is not required to maintain a homogeneous aqueous dispersion.

[0396] In addition to the additives listed above, liquid formulations may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers. Exemplary emulsifiers include ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, sodium lauryl sulfate, sodium doxate, cholesterol, cholesterol esters, taurocholic acid, phosphotidylcholine, oils, such as cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, fatty acid esters of sorbitan, or mixtures of these substances.

[0397] Injectable preparations Formulations suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders or dusts for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene glycol, glycerol, and cremophor), suitable mixtures thereof, vegetable oils (e.g., olive oil), and organic esters for injection, such as ethyl oleate (including). Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection may also include additives such as preservatives, humectants, emulsifiers, and dispensing agents. Prevention of microbial growth can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may be desirable to include isotonic agents, such as sugars and sodium chloride. Long-term absorption of injectable pharmaceutical forms can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0398] For intravenous injection, the compound may be formulated in an aqueous solution, preferably a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or saline buffer. For transmucosal administration, a permeable substance suitable for the barrier to be penetrated is used in the formulation. Such permeable substances are generally known in the art. For other parenteral injections, a suitable formulation may contain an aqueous solution or a non-aqueous solution, preferably with a physiologically compatible buffer or excipient. Such excipients are generally known in the art.

[0399] Parenteral injection may include bolus injection or continuous infusion. Formulations for injection, along with added preservatives, may be presented in unit dosage forms, e.g., ampoules or containers for multiple doses. Pharmaceutical compositions may be in forms suitable for parenteral injection, such as sterile suspensions, solutions, or emulsions in oily or aqueous vehicles, and may include formulation agents, e.g., suspenders, stabilizers, and / or dispersants. Pharmaceutical formulations for parenteral administration contain aqueous solutions of the active compound in a water-soluble form. In addition, suspensions of the active compound may be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, e.g., sesame oil, or synthetic fatty acid esters, e.g., ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, e.g., sodium carboxymethylcellulose, sorbitol, or dextran. If necessary, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound, enabling the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for use with a suitable vehicle, such as a sterile, pyrogen-free substance.

[0400] Pharmaceutical compositions may be in unit dosage forms suitable for a single dose of a precise amount. In a unit dosage form, the formulation is divided into unit doses containing an appropriate amount of one or more compounds. Unit doses may also be in the form of packages containing different amounts of the formulation. Non-limiting examples include packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions may be packaged in single-dose, non-resealable containers. Alternatively, resealable containers for multiple doses may be used, in which case the composition typically includes a preservative. For example, formulations for parenteral injection may be presented in unit dosage forms, including, but not limited to, ampoules or multi-dose containers with added preservatives.

[0401] Treatment results In some embodiments, the number of mature neurons in a subject after the administration step (administering manipulated DA neurons, recombinant gene vectors, or gene editing systems) exceeds the number of mature neurons in the subject before the administration step. In some embodiments, the number of endogenous mature neurons in a subject after the administration step exceeds the number of endogenous mature neurons in the subject before the administration step.

[0402] In some embodiments, the number of dopaminergic neurons in a subject after the administration step (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) exceeds the number of dopaminergic neurons in the subject before the administration step. In some embodiments, the dopamine level in a subject after the administration step exceeds the dopamine level in the subject before the administration step. In some embodiments, the number of dopaminergic neurons in a subject treated by the method increases compared to the number of dopaminergic neurons in a subject not treated in this way. In some embodiments, the dopamine level in a subject treated by the method disclosed herein (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) increases compared to the dopamine level in a subject not treated in this way. In some embodiments, the dopamine level in the substantia nigra of a subject treated by the method disclosed herein increases compared to the dopamine level in the substantia nigra of a subject not treated in this way. In some embodiments, the level of GDNF in the subject's cerebrospinal fluid (CSF) after the administration step (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) is higher than the level of GDNF in the subject's CSF before the administration step. In some embodiments, the level of GBA in the subject's CSF after the administration step (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) is higher than the level of GBA in the subject's CSF before the administration step. In some embodiments, the ratio of GBA to SNCA in the subject after the administration step (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) is lower than the ratio of GBA to SNCA in the subject before the administration step. In some embodiments, the ratio of GBA to SNCA in the subject after the administration step (administering engineered DA neurons, recombinant gene vectors, or gene editing systems) is higher than the ratio of GBA to SNCA in the subject before the administration step.

[0403] In some embodiments, the ratio of GBA protein to alpha-synuclein protein in the subject after the administration step (administration of engineered DA neurons, recombinant gene vectors, or gene editing systems) is lower than the ratio of GBA protein to alpha-synuclein in the subject before the administration step. In some embodiments, the ratio of GBA protein to alpha-synuclein protein in the subject after the administration step (administration of engineered DA neurons, recombinant gene vectors, or gene editing systems) is higher than the ratio of GBA protein to alpha-synuclein protein in the subject before the administration step.

[0404] In some embodiments, the Unified Parkinson's Disease Rating Scale (UPDRS) score of the subject prior to the administration step (administering the manipulated DA neuron cells, recombinant gene vector, or gene editing system) is improved compared to the UPDRS score of the subject prior to the administration step. In some embodiments, the subject neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after the administration step.

[0405] In some embodiments, the cognitive symptoms of the subject prior to the administration step (administering manipulated DA neurons, recombinant gene vectors, or gene editing systems) are improved compared to the cognitive symptoms of the subject prior to the administration step.

[0406] In some embodiments, the long-term graft integrity of manipulated cells administered to the subject is improved compared to the long-term graft integrity of unmanipulated cells administered to the subject.

[0407] array In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF gene (or vector) each contains the nucleic acid sequence described in SEQ ID NOs: 1-10.

[0408] In some embodiments, the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA, MAPT, or GDNF proteins each contain the amino acid sequences described in SEQ ID NOs. 11-20.

[0409] In some embodiments, the gene is the GDNF gene, and the wild-type GDNF protein contains the amino acid sequence described in any of Sequence ID No. 20.

[0410] In some embodiments, the gene is the GBA gene, and the wild-type GBA protein contains the amino acid sequence described in any of Sequence ID No. 17.

[0411] In some embodiments, the polynucleotides each contain a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity to the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, or GDNF polynucleotide sequences described in SEQ ID NOs.1-10. In some embodiments, the polynucleotides are codon-optimized. In some embodiments, the polynucleotides contain fewer than 40, fewer than 30, fewer than 20, or 10 or fewer CpG islands. In some embodiments, the polynucleotides contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 CpG islands. In some embodiments, it contains 5 to 20 CpG islands.

[0412] The donor template, which includes the vector's polynucleotide sequence, the exogenous coding sequence and / or sequences homologous to the endogenous gene locus, the sequences associated with the resulting engineered cells, and anything that enables the detection of the engineered cells are all part of this disclosure.

[0413] In some embodiments, the transgene encodes a GDNF gene, and the transgene polynucleotide sequence shares at least 95% identity with one of sequence numbers 10 and is located downstream of a promoter selected from sequence numbers 21-30.

[0414] In some embodiments, the transgene encodes a GBA gene, and the transgene polynucleotide sequence shares at least 95% identity with one of sequence numbers 7 and is located downstream of a promoter selected from sequence numbers 31-40 or 43.

[0415] In some embodiments, the transgene encodes an SNCA gene, and the transgene polynucleotide sequence shares at least 95% identity with one of sequence numbers 8 and is located downstream of a promoter selected from sequence numbers 21-40 or 43.

[0416] In some embodiments, the first transgene encodes GBA and the second transgene encodes SNCA, the first transgene is located downstream of a promoter selected from SEQ ID NOs. 31-40 or 43, and the second transgene is located downstream of a promoter selected from SEQ ID NOs. 21-40 or 43.

[0417] Gene sequences encoding GDNF, GBA, and / or SNCA GDNF, GBA, and / or SNCA sequences(or sequences) may be introduced into selected loci, more specifically, under the control of an endogenous promoter, by targeted recombination. In some embodiments, exogenous GDNF is preferably expressed only after the DA neuronal cells have differentiated and graft function has been established, while exogenous GBA and SNCA+ / - are preferably expressed immediately upon transplantation of the engineered cells into the patient.

[0418] According to one embodiment, the exogenous sequence encodes a polypeptide exhibiting at least 80% amino acid sequence identity with PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / -, or GDNF, or a functional variant thereof. These exogenous sequences can be introduced into the genome by deleting or modifying (knock-in knockout) the one or more endogenous coding sequences present at the gene locus, thereby combining gene inactivation with genesis.

[0419] In some embodiments, exogenous sequences can be introduced into the genome without deleting or modifying endogenous coding sequences (or sequences) present at the gene locus. This occurs regardless of whether the endogenous coding sequences (or sequences) are present in vitro in pluripotent cells or in vivo.

[0420] Depending on the targeted gene locus and its involvement in DA neuron cell activity, the targeted endogenous gene may be non-expressed or its original function may be maintained (regardless of whether the targeted gene is in vitro or in vivo). If the targeted gene is essential for DA neuron cell activity or differentiation, this insertion procedure may generate a single (Kl) without gene inactivation. Alternatively, if the targeted gene is thought to be involved in DA neuron cell differentiation, the insertion procedure is designed to prevent the expression of the endogenous gene by knocking out the endogenous sequence, while enabling the expression of the introduced exogenous coding sequence(s).

[0421] In some embodiments, the method relies on targeted integration (with or without disruption of native genes) to upregulate target gene expression in various dynamics during activation of the GDNF / GBA signaling pathway and / or regulation of the SNCA signaling pathway at specific loci, for example, in non-limiting examples, SEQ ID NOs: 21-40 or 43.

[0422] In some embodiments, manipulated DA neurons, preferably DA neurons for injection into a patient, are disclosed, comprising exogenous sequences encoding PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / -, or GDNF polypeptides(y), which are incorporated into the PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / -, or GDNF endogenous loci for their expression under the control of endogenous promoters present at these loci. In some embodiments, the endogenous promoters comprise or consist of SEQ ID NOs. 21-40 or 43.

[0423] Manipulated DA neuron cells are used in SNCA depending on the treatment indication and recipient patient. + / + SCNA - / - , or SCNA + / - It is possible. In some embodiments, the manipulated DA neuron cells are further used for allogeneic transplantation, including T cell receptors. negatlve To be made to.

[0424] A gene editing step incorporating an exogenous sequence encoding PARK2, PINK1, LRRK2, c-Rel, ATG7, VMAT2, GBA, SNCA+ / -, or GDNF can be combined with any other step that contributes to enhancing the potency or safety of the manipulated DA neuronal cells.

[0425] According to one embodiment, the method relies on introducing a donor template containing a sequence-specific endonuclease reagent and / or sequences homologous to the gene of interest and target genes by transfecting ssDNA (in non-limiting examples, oligonucleotides), dsDNA (in non-limiting examples, plasmid DNA), and more specifically, in non-limiting examples, adeno-associated virus (AAV).

[0426] kit Kits for transfection of pluripotent cells and / or DA neuronal cells are disclosed, comprising a sequence-specific endonuclease reagent and a polynucleotide encoding a donor sequence designed to incorporate an exogenous sequence into a locus targeted by the reagent. Examples of such kits include a kit comprising an AAV vector containing an exogenous sequence encoding GDNF and mRNA encoding a low-frequency cleavage endonuclease targeting synapsin-1, DAT, VMAT, TH, AADC, a tamoxifen-inducible promoter, a RU486-inducible promoter, or other promoters that enable temporal or small molecule control of a locus; and a kit comprising an AAV vector containing an exogenous sequence encoding GBA and mRNA encoding a low-frequency cleavage endonuclease targeting synapsin-1, DAT, VMAT, TH, AADC, a tamoxifen-inducible promoter, a RU486-inducible promoter, or other promoters that enable temporal or small molecule control of a locus.

[0427] A kit for transfection of pluripotent cells and / or DA neuron cells comprising a polynucleotide encoding a sequence-specific endonuclease reagent, and, in some embodiments, an exogenous polynucleotide sequence contained in an AAV vector, wherein the exogenous sequence is GBA, GBA, and / or SCNA + / - A kit is disclosed that includes a sequence encoding a functional fragment or variant thereof. [Examples]

[0428] Examples

[0429] (Example 1)

[0430] The applicants identified genome-safe harbor (GSH) sites suitable for iPSC gene editing and expression in dopaminergic neurons, enabling the development of novel GNDF-secreting stem cell replacement therapies for Parkinson's disease. See Figure 2 for GSH site identification based on computer studies and additional predictive and validation modeling. Based on these results, GDNF can be inserted into GSH in iPSCs and novel cells, potentially identifying gene therapies for the treatment of Parkinson's disease.

[0431] scATAC-seq data were generated from clinically compliant iPSC systems and the resulting dopaminergic neuron precursor drug product RNDP-001. This data was compared with publicly available ATAC-seq data obtained from dopaminergic neurons isolated from healthy and Parkinson's diseased human brains to define common chromatin-accessible regions based on catalog data of GSH site suitability. Firstly, the applicant defined 1,875 novel GSH candidates identified based on their distality to genes, regulatory regions, telomeres, and centromeres to prevent transgenes from interfering with gene expression, division, and function. A key challenge in precisely defining where transgenes should be targeted is that these 1,875 putative GSH regions span over 142 million nucleotides in the genomic space, requiring further concretization of criteria for prioritization.

[0432] The predicted top sgRNAs are selected for editing by using publicly available tools to predict the sgRNA with the highest on-target activity and the lowest off-target activity, and by empirically defining the sgRNA that cleaves best. The viable sgRNAs can then be inserted into a identified genome-safe harbor (GSH) using a DNA donor template for hGDNF. Single-cell clones can also be isolated and tested for safety and GSH accessibility.

[0433] Selected clones can then be differentiated into dopaminergic precursors and postmittal neurons, and GDNF secretion can be measured. Existing release criteria, including flow cytometry panels and ddPCR assays, are used to establish whether the edited iPSCs have the ability to correctly identify the dopaminergic precursor fate. RNA-seq profiling can also be performed to assess the effect of GDNF insertion on differentiation ability.

[0434] Genome accessibility can be used as a criterion to narrow down the optimal GSH locus for inserting GDNF into iPSCs, ultimately targeting expression in post-mitotic dopaminergic neurons.

[0435] Figure 3 shows an example of a starting point for GSH selection, which can be further refined based on publicly available criteria, including micro-regional selectivity.

[0436] This disclosure can be better understood by the following numbered paragraphs.

[0437] 1. An engineered cell comprising at least one sequence encoding a transgene, wherein at least one sequence is inserted into a safe harbor locus, and the safe harbor locus is located in one or more of the loci provided in Table 1.

[0438] 2. Manipulated cells comprising at least one sequence encoding a transgene, wherein at least one sequence is inserted into a safe harbor locus, the safe harbor locus is located in one or more loci provided in Table 1, and the expression of at least one sequence encoding a transgene is operably linked to an endogenous or exogenous promoter.

[0439] 3. Manipulated cells comprising at least one sequence encoding a transgene, wherein at least one sequence is inserted into a safe harbor locus, the safe harbor locus is located at any one target locus in Table 1, the expression of at least one sequence encoding the transgene is operably linked to an endogenous or exogenous promoter, and the manipulated cells are undifferentiated.

[0440] 4. The target gene 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: Manipulated cells as described in paragraph 1, 2, or 3, selected from 68894085-68895495, chrX: 20530099-20531285, chrX: 40996550-40997770, chrX: 20527023-20528155, chrX: 94058277-94059471, or chrX: 138127607-138128791.

[0441] 5. The engineered cells described in paragraphs 1-4, wherein the endogenous promoter is one of sequence numbers 21-40 or 43.

[0442] 6. Manipulated cells described in any one of paragraphs 2-5, wherein the endogenous promoter is expressed in the neuronal mature cell type.

[0443] 7. The manipulated cells described in any one of paragraphs 1-6, wherein the manipulated cells are stem cells, pluripotent cells, iPSCs, human cells, primary cells, DA neurons, DA neuron precursors, or a combination thereof.

[0444] 8. Manipulated cells as described in any one of paragraphs 1-7, wherein the manipulated cells are undifferentiated.

[0445] 9. Manipulated cells as described in any one of paragraphs 1 to 8, wherein the manipulated cells are capable of differentiating into DA neuronal cells.

[0446] 10. Manipulated cells as described in any one of paragraphs 1 to 9, wherein the transgene comprises GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof; or consists of GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof; or encodes a gene selected from the group essentially consisting of GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0447] 11. A composition comprising the manipulated cells described in any one of paragraphs 1 to 10 and a pharmaceutical excipient.

[0448] 12. Guide ribonucleic acid (gRNA) for editing cells at safe harbor loci located in Table 1.

[0449] 13. A method for editing a cell having chromosomal DNA, comprising the step of inserting at least one sequence encoding a transgene into a safe harbor locus in the cell's chromosomal DNA, wherein the safe harbor locus is one or more of the target loci provided in Table 1.

[0450] 14. A method for editing pluripotent cells, comprising the step of contacting the pluripotent cells with one or more guide ribonucleic acids (gRNAs), at least one sequence encoding a transgene, and one or more Cas9 endonucleases, wherein the one or more gRNAs and Cas9 endonucleases facilitate the insertion of at least one sequence into chromosomal DNA within a safe harbor locus, and the safe harbor locus is selected from any one of the target loci in Table 1.

[0451] 15. Target gene 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: The method described in paragraph 13 or 14, selected from 68894085-68895495, chrX: 20530099-20531285, chrX: 40996550-40997770, chrX: 20527023-20528155, chrX: 94058277-94059471, or chrX: 138127607-138128791.

[0452] 16. The method according to any one of paragraphs 13-15, comprising an exogenous promoter in which at least one sequence is one of sequence numbers 21-40 or 43.

[0453] 17. The method according to any one of paragraphs 13-16, wherein the cells are stem cells, pluripotent cells, iPSCs, human cells, primary cells, DA neuron cells, DA neuron cell precursors, or a combination thereof.

[0454] 18. The method according to any one of paragraphs 13-17, wherein the manipulated cells are undifferentiated.

[0455] 19. The method according to any one of paragraphs 13-18, wherein at least one sequence is inserted using homology-dependent repair.

[0456] 20. The method according to any one of paragraphs 13-19, wherein at least one sequence is inserted using homology-independent targeted insertion.

[0457] 21. The method according to any one of paragraphs 13-20, wherein at least one sequence is inserted using one or more guide ribonucleic acids (gRNAs) and one or more Cas9 endonucleases.

[0458] 22. The method according to any one of paragraphs 13-21, wherein the transgene is selected from the group including GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0459] 23. An ex vivo method for obtaining manipulated cells or a population thereof, comprising the steps of a. obtaining cells and b. genetically modifying the cells by inserting at least one sequence encoding an introduced gene into a safe harbor locus, wherein the safe harbor locus is selected from any one of the target loci in Table 1.

[0460] 24. The method according to paragraph 23, wherein the step of obtaining cells comprises (i) taking a tissue sample from a subject, (ii) isolating cells from the tissue sample, and (iii) culturing the cells in vitro.

[0461] 25. The method according to paragraph 23, wherein the tissue sample is a blood sample.

[0462] 26. The method according to any one of paragraphs 23-25, wherein the cells are stem cells, human cells, primary cells, hematopoietic cells, adoptive immune cells, innate immune cells, T cells, or T cell precursors, or a combination thereof.

[0463] 27. The method according to any one of paragraphs 23-26, wherein the manipulated cells are undifferentiated.

[0464] 28. The method according to any one of paragraphs 23-27, wherein at least one sequence is selected from the group comprising GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0465] 29. The method according to any one of paragraphs 23-28, wherein at least one sequence is inserted using homology-dependent repair.

[0466] 30. The method according to any one of paragraphs 23-29, wherein at least one sequence is inserted using homology-independent targeted insertion.

[0467] 31. The method according to any one of paragraphs 23-30, wherein the gene modification step in step (b) comprises contacting a cell with one or more guide ribonucleic acids (gRNAs), at least one sequence, and one or more Cas9 endonucleases, the one or more gRNAs and Cas9 endonucleases facilitate the insertion of at least one sequence into chromosomal DNA within a safe harbor locus.

[0468] 32. The method according to any one of paragraphs 23-31, wherein the transgene encodes a gene selected from the group including GDNF, GBA, PARK2, PINK1, DJ-1, LRRK2, c-Rel, ATG7, VMAT2, or SNCA, and combinations thereof.

[0469] 33. The method according to any one of paragraphs 23-32, wherein at least one sequence comprises an exogenous promoter, the exogenous promoter being operably ligated to the transgene.

[0470] 34. The method according to any one of paragraphs 23 to 33, wherein the exogenous promoter is one of sequence numbers 21 to 40 or 43.

[0471] 35. A method for treating a subject that has or is at risk of having a disease, comprising the step of administering to the subject an effective amount of cells, populations thereof, or compositions described in any one of paragraphs 13 to 24 or paragraphs 1 to 12.

[0472] 36. A method for treating a subject having or at risk of having a disease, comprising the steps of a. carrying out a method according to any one of paragraphs 13 to 35, and b. administering to the subject a composition comprising an effective amount of cells or a population thereof.

[0473] 37. The method according to paragraph 35 or 36, wherein the composition is administered to a subject by injection.

[0474] 38. The method described in paragraphs 35-37, wherein the disease is Parkinson's disease.

[0475] 39. The method according to any one of paragraphs 35-38, wherein the disease is a disorder associated with central nervous system degeneration.

[0476] 40. A method for treating a subject that has or is at risk of having a disease, comprising the step of administering to the subject an effective amount of cells, populations thereof, or compositions described in any one of paragraphs 1 to 11 or paragraph 12.

[0477] 41. A method for treating a subject having or at risk of having a disease, comprising the steps of c. performing a method according to any one of paragraphs 13 to 34, and d. administering to the subject a composition comprising an effective amount of cells or a population thereof.

[0478] 42. The method according to paragraphs 40-41, wherein the composition is administered to a subject by injection.

[0479] 43. The method described in paragraphs 40-42, wherein the disease is Parkinson's disease.

[0480] 44. A method for identifying safe harbor loci, comprising: a. identifying a gene or non-coding region on a chromosome that exceeds a threshold level with respect to chromatin accessibility; b. generating a model that correlates the gene or non-coding region on the chromosome obtained from step (a) with publicly available ATAC-seq data obtained from dopaminergic neurons isolated from the brains of healthy and Parkinson's diseased humans; and c. selecting a safe harbor locus based on a threshold parameter, wherein the safe harbor locus is selected for the insertion of at least one sequence encoding a transgene into the cell.

[0481] 45. The method according to paragraph 44, wherein the threshold parameters include one or more of the following: stable expression of the transgene, gene knockout conferring a benefit to cellular function, no known function in the cell, stable transgene expression in vitro, negligible off-target cleavage detected by iGuide-Seq or CRISPR-Seq, low off-target cleavage compared to other loci detected by iGuide-Seq or CRISPR-Seq, negligible transgene-independent cytotoxicity, negligible transgene-independent cytokine expression, negligible transgene-independent chimeric antigen receptor expression, negligible dysregulation or silencing of neighboring genes, peak size data generated using processed ATAC-seq data, distribution data generated using processed ATAC-seq data, highest efficiency for transgene insertion, sustained expression after long-term culture, and absence of oncogenic gene expression.

[0482] 46. ​​The method according to any one of paragraphs 44-45, wherein chromatin accessibility is measured using a transposase-accessible chromatin assay (ATAC-seq) with sequencing.

[0483] 47. The method according to any one of paragraphs 44-46, wherein the manipulated cells are stem cells, pluripotent cells, iPSCs, human cells, primary cells, DA neuron cells, DA neuron cell precursors, or a combination thereof.

[0484] 48. An engineered cell, composition, or method according to any one of the preceding paragraphs, wherein an insertion within the safe harbor gene locus increases GDNF secretory protein, which is produced after the administered engineered cells have matured into a neuronal mature cell type and is taken up by endogenous cells.

[0485] 49. The manipulated cells, composition, or method described in any of the preceding paragraphs, wherein the knock-in efficiency at any one of the safe harbor loci in Table 1 is increased compared to other locations along the chromosome.

Claims

1. A population of manipulated cells, wherein each manipulated cell contains at least one exogenous GBA gene or a functional fragment thereof.

2. The cell population according to claim 1, wherein the manipulated cells are human DA neuron cells.

3. The cell population according to claim 1, wherein the human DA neuron cells are derived from pluripotent cells.

4. The population of cells according to claim 1, wherein the manipulated cells are manipulated to become hemijunctive nulls with respect to SNCA.

5. The population of cells according to claim 1, wherein the exogenous GBA gene or its functional fragment is located within or near the safeharbour locus.

6. The population of cells according to claim 5, wherein the safe harbor locus is selected from the list in Table 1.

7. The aforementioned 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, hr5: 123875580-123876580, hr5: 71932489-71933717, hr5: 34474174-34475918, hr5: 18113286-18115121, hr5: 103808080-103809225, hr5: 144598204-144599350, hr5: 108521605-1085222526, hr5: 113692453-113694380, hr5 101678253-101679223, y ... 113678518-113679693, hr5: 88089287-88091255, hr5: 121797421-121798482, hr5 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, hr6: 90890535 - 90891762, hr7: 152925055 - 152926297, hr7: 12111174-12112347, yy: 42493270-42494306, yy: 31192574-31193687, yy: 22000950-22002150chr7: 96785947-967873280chr8: 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: A population of cells according to claim 5, selected from 68894085-68895495, chrX: 20530099-20531285, chrX: 40996550-40997770, chrX: 20527023-20528155, chrX: 94058277-94059471, or chrX: 138127607-138128791.

8. A population of manipulated cells, wherein each manipulated cell contains at least one exogenous GDNF gene or a functional fragment thereof.

9. The cell population according to claim 8, wherein the manipulated cells are human DA neuron cells.

10. The cell population according to claim 8, wherein the human DA neuron cells are derived from pluripotent cells.

11. A method for preparing manipulated pluripotent cells, - A step of providing a population of cells including pluripotent cells, - A certain proportion of the pluripotent cells contain, i) at least one nucleic acid comprising an exogenous polynucleotide sequence intended to be incorporated into a selected endogenous locus to encode at least one GDNF gene, ii) Step of introducing at least one sequence-specific reagent that specifically targets the selected endogenous gene locus. Includes, The exogenous polynucleotide sequence is inserted into the endogenous gene locus by targeted gene integration. method.

12. The method according to claim 11, wherein the sequence-specific reagent is a nuclease.

13. The method according to claim 11 or 12, wherein the targeted gene insertion is carried out by homologous recombination or NHEJ into the pluripotent cells.

14. The method according to any one of claims 11 to 13, wherein the exogenous polynucleotide sequence is incorporated under the transcriptional control of an endogenous promoter located at the gene locus.

15. The method according to any one of claims 11 to 14, wherein the manipulated pluripotent cells differentiate into DA neuron cells to form manipulated DA neuron cells.

16. The method according to claim 15, wherein the manipulated DA neuron cells are transplanted into a human in order to form the manipulated DA neuron cells.

17. The method according to claim 16, wherein the transplanted manipulated DA neuronal cells do not initially express GDNF.

18. The method according to claim 16, wherein the transplanted manipulated DA neuron cells do not express GDNF until they differentiate into mature neuron cell types.

19. A method for preparing manipulated pluripotent cells, - A step of providing a population of cells including pluripotent cells, - In a certain proportion of the aforementioned pluripotent cells, i) at least one nucleic acid comprising an exogenous polynucleotide sequence to be incorporated into a selected endogenous locus in order to encode at least one GBA gene and at least one hemizygous null SCNA gene, ii) Step of introducing at least one sequence-specific reagent that specifically targets the selected endogenous gene locus. Includes, The exogenous polynucleotide sequence is inserted into the endogenous gene locus by targeted gene integration. method.

20. The method according to claim 19, wherein the manipulated pluripotent cells differentiate into DA neuron cells to form manipulated DA neuron cells.

21. The method according to claim 20, wherein the manipulated DA neuron cells are transplanted into a human.

22. The method according to claim 21, wherein the transplanted manipulated DA neuronal cells express GBA and SCNA after transplantation.

23. The method according to claim 21, wherein the transplanted manipulated DA neuronal cells do not show α-synuclein accumulation.

Images